Medicinski Glasnik - ljekarska komora zeničko

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Medicinski Glasnik
Official Publication of the Medical Association of
Zenica-Doboj Canton
Bosnia and Herzegovina
Editor-in-chief
Selma Uzunović
Zenica, Bosnia and Herzegovina
MANAGING EDITOR
Tarik Kapidžić
Zenica, Bosnia and Herzegovina
Editors
Adem Balić, Tuzla, Bosnia and Herzegovina
Dubravka Bartolek, Zagreb, Croatia
Branka Bedenić, Zagreb, Croatia
Asja Čelebić, Zagreb, Croatia
Josip Čulig, Zagreb, Croatia
Filip Čulo, Mostar, Bosnia and Herzegovina
Jordan Dimanovski, Zagreb, Croatia
Branko Dmitrović, Osijek, Croatia
Ines Drenjančević, Osijek, Croatia
Harun Drljević, Zenica, Bosnia and Herzegovina
Davorin Đanić, Slavonski Brod, Croatia
Lejla Ibrahimagić-Šeper, Zenica, Bosnia and Herzegovina
Tatjana Ille, Belgrade, Serbia
Vjekoslav Jerolimov, Zagreb, Croatia
Mirko Šamija, Zagreb, Croatia
Sven Kurbel, Osijek, Croatia
Snježana Pejičić, Banja Luka, Bosnia and Herzegovina
Belma Pojskić, Zenica, Bosnia and Herzegovina
Besim Prnjavorac, Tešanj, Bosnia and Herzegovina
Asja Prohić, Sarajevo, Bosnia and Herzegovina
Velimir Profozić, Zagreb, Croatia
Radivoje Radić, Osijek, Croatia
Amira Redžić, Sarajevo, Bosnia and Herzegovina
Suad Sivić, Zenica, Bosnia and Herzegovina
Sonja Smole-Možina, Ljubljana, Slovenia
Vladimir Šimunović, Mostar, Bosnia and Herzegovina
Adrijana Vince, Zagreb, Croatia
Jasmina Vraneš, Zagreb, Croatia
Živojin Žagar, Zagreb, Croatia
Secretary: Tatjana Žilo;
Proofreaders: Aras Borić (Bosnian, Croatian, Serbian),
Glorija Alić (English)
MEDICINSKI GLASNIK
Official Publication of the Medical Association of Zenica-Doboj Canton, Bosnia and Herzegovina
Volume 12, Number 2, August 2016
Free full-text online at: www.ljkzedo.com.ba, and www.doaj.org (DOAJ, Directory of Open Access
Journals)
Original
article
108 Effects of post-sampling analysis time, type of blood samples and collection tubes on values of blood
gas testing
Jasmina Smajić, Damira Kadić, Sabaheta Hasić, Nafija Serdarević
113 Association of LPIN1 gene variations with markers of metabolic syndrome in population from
Tamer Bego, Tanja Dujić, Barbara Mlinar, Sabina Semiz, Maja Malenica, Besim Prnjavorac, Barbara
Ostanek, Janja Marc, Anida Čaušević-Ramoševac, Adlija Čaušević
122 Alpha-lipoic acid reduces body weight and regulates triglycerides in obese patients with diabetes
mellitus
Azra Okanović, Besim Prnjavorac, Edin Jusufović, Rifat Sejdinović
128 Positive correlation between uric acid and C-reactive protein serum level in healthy individuals and
patients with acute coronary syndrome
Emina Spahić1, Sabaheta Hasić2, Emina Kiseljaković2, Halima Resić3, Mehmed Kulić4
133 Role of echocardiography in diagnosis and management of complete papillary muscle rupture
caused by myocardial infarction
Josip Vincelj, Stanko Biočić, Mario Udovičić, Mario Sičaja, Sandra Jakšić Jurinjak
139 Impact of reperfusion therapy and infarct localization on frequency of premature ventricular beats
in acute myocardial infarction
Davor Horvat, Josip Vincelj
144 Possibilities of differentiation of solitary focal liver lesions by computed tomography perfusion
Irmina Sefić Pašić, Anes Pašić, Spomenka Kristić, Adnan Beganović, Aladin Čarovac, Amra Džananović,
Lidija Lincender, Sandra Vegar Zubović
151 Familial autoimmune thyroid disease and PTPN-22
Gabriel Conzuelo Rodríguez, Hugo Mendieta Zerón
157 Methicillin-resistant S. aureus (MRSA), extended-spectrum (ESBL)- and plasmid-mediated AmpC
ß-lactamase -producing Gram-negative bacteria associated with skin and soft tissue infections in
hospital and community settings
Selma Uzunović, Branka Bedenić, Ana Budimir, Amir Ibrahimagić, Farah Kamberović, Zlatko Fiolić,
Michelle I. A. Rijnders, Ellen E. Stobberingh
169 Emergence of extensive drug-resistant (XDR) Acinetobacter baumanniiin the Clinical Center
University of Sarajevo, Bosnia and Herzegovina
Amela Dedeić-Ljubović, Ðana Granov, Mirsada Hukić
177 Brucellosis in children in Bosnia and Herzegovina in the period 2000 - 2013
Sead Ahmetagić, Humera Porobić-Jahić, Nada Koluder, Lejla Čalkić, Snježana Mehanić, Eldira Hadžić,
Nevzeta Ibrahimpašić, Svjetlana Grgić, Mirela Zirić, Jelena Bajić, Denis Žepić
183 Differences in newborn umbilical cord care
Sanja Kanisek, Nada Prlić, Ivana Barać, Lorna Dubac Nemet
190 Effects of perioperative statin treatment on postoperative atrial fibrillation and cardiac mortality in
patients undergoing coronary artery bypass grafting: a propensity score analysis
Ayşegül Kunt, Sedat Özcan, Aslihan Küçüker, Dolunay Odabaşi, Alper Sami Kunt
196 Efficacy of chronic statin therapy on major cardiac events after coronary artery bypass grafting:
low-dose versus high-dose
Ayşegül Kunt, Sedat Özcan, Aslihan Küçüker, Dolunay Odabaşi, Alper Sami Kunt
202 Incidence of endophthalmitis after intravitreal application of anti VEGF therapy at the University
Clinical Center in Tuzla, Bosnia and Herzegovina
Svjetlana Terzić, Adisa Pilavdžić, Amra Nadarević Vodenčarević
206 Therapeutic efficacy and toxicity of bolus application of chemotherapy protocol in the treatment of
metastatic colorectal cancer
Ibrahim Šišić, Belma Pojskić, Alma Mekić-Abazović, Vladimir Kovčin
212 Differences in body mass index and height factors between men with and without varicocele
Hamid Shafi, Mouloud Agajani Delavar
216 Genetic polymorphisms variants in interleukin-6 and interleukin-1beta patients with obstructive
sleep apnea syndrome in East Northern Turkey
Ilhami Gok, Nergiz Huseyinoglu, Dogan Ilhan
Medicinski Glasnik is indexed by MEDLINE, EMBASE (Exerpta Medica), EBSCO, Scopus, and Directory of Research Journals Indexing (DRJI)
ORIGINAL ARTICLE
Effects of post-sampling analysis time, type of blood samples
and collection tubes on values of blood gas testing
Jasmina Smajić¹, Damira Kadić², Sabaheta Hasić³, Nafija Serdarević¹
¹Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo, Sarajevo, ²Department of Laboratory Diagnostics, Cantonal Hospital Zenica, Zenica, ³Department of Medical Biochemistry, School of Medicine, University of Sarajevo, Sarajevo; Bosnia and
Herzegovina
ABSTRACT
Aim To investigate effects of post-sampling analysis time, a type
of blood samples and collection tubes on blood gas testing.
Corresponding author:
Jasmina Smajić
Clinical Chemistry and Biochemistry,
Clinical Centre University of Sarajevo
Bolnička 25, 71 000 Sarajevo, Bosnia and
Herzegovina
Phone: +387 33 297 501;
Fax: +387 33 297 844;
E-mail: [email protected]
Original submission:
27 May 2015;
Revised submission:
06 July 2015;
Accepted:
08 July 2015.
doi: 10.17392/823-15
Med Glas (Zenica) 2015; 12(2): 108-112
108
Methods This study included 100 patients at the Clinic for Pulmonary Diseases, Clinical Centre Sarajevo. The partial pressure of
oxygen (pO2) and carbon dioxide (pCO2), and the oxygen saturation level of hemoglobin (sO2) were analyzed in the arterial blood
samples (ABS) and capillary blood samples (CBS) by a potentiometric method using a blood gas analyzer ABL 555 (Radiometer,
Copenhagen, Denmark). Paired measurements of ABS were performed within 15 minutes and after 60 minutes from sampling and
compared. The results of CBS obtained within 15 minutes were
compared with matching ABS results, as well as the results obtained from CBS within 15 minutes taken into glass and plastic tubes.
Results pO2 and sO2 values were significantly lower after 60 minutes compared to those within 15 minutes in ABS (9.20±1.89 vs.
9.51±1.95 and 91.25±5.03 vs. 92.40±4.5; p<0.01, respectively).
Values of pO2 and sO2 in CBS were significantly lower than values obtained in ABS (8.92±2.07 vs. 9.51±1.95 and 91.25±4.86 vs.
92.40±4.50; p<0.01, respectively). Obtained pO2 and sO2 values in
CBS in the plastic tubes were higher than those in the glass tubes
(8.50±1.98 vs. 7.89±2.0 and 89.66±11.04 vs. 88.23±11.22, p<0.01
respectively). pCO2 blood values were not influenced significantly
(p>0.05).
Conclusion The length of post-sampling analysis time, a type of
blood samples and collection tubes have significant impact on
blood oxygen parameters. Analysis within 15 minutes after blood
sampling is considered as appropriate.
Key words: blood gas analysis, capillary tubing, syringes, glass,
plastics.
Smajić et al. The influences on blood gas testing
INTRODUCTION
Blood gas testing is a common analysis in critically ill patients that provides important information for their treatment. It comprises an analysis
of partial pressures of oxygen (pO2) and carbon
dioxide (pCO2), blood pH and oxygen saturation
level of haemoglobin (sO2) in arterial, venous or
capillary blood, depending on a clinical question.
The interval between sampling and analysis, as
well as a type of blood samples and collection
tubes are important pre-analytical factors that can
affect blood gas testing (1).
The gold standard samples for blood gas analysis
are arterial blood samples (ABS) collected from
arterial catheter or by arterial puncture. Properly
arterial blood sampling is difficult and painful for
the patient, but a clinician is able to get a true
picture about oxygen supply. Venous blood sampling is less painful and risky, but venous blood samples are not an adequate replacement for
ABS in routine blood gas testing (2). Capillary
blood samples (CBS), least painful and easiest
to obtain, from the fingertip, heel or earlobe, can
also be used in routine practice (3,4). Obtaining
CBS is less invasive and can be performed by
various healthcare professionals, while arterial
blood sampling requires specially trained medical personnel (5).
Proper time processing of blood samples is a critical pre-analytical step in the integrity of laboratory results because of continued metabolism in
the blood that can alter blood gas values during
the time between sampling and analysis or by
diffusion of gases through the wall of the collection device (6,7).
A type of collection tubes can also affect blood gas analysis (8). While glass tubes were
common collection devices in the past, today
plastic devices are mainly used because of safety concerns (1).
The study aim was to investigate the effects of
time interval between sampling and analysis, a
type of blood samples and collection tubes on
blood gas testing.
PATIENTS AND METHODS
The study included analysis of blood samples
obtained from 100 hospitalized patients and outpatients at the Clinic for Pulmonary Diseases,
Clinical Centre University of Sarajevo during a
six-month period in 2007. From 50 patients, two
types of blood samples, arterial and capillary,
were collected at the same time. Arterial blood
samples were collected by arterial puncture of radial artery into the heparinized plastic syringes,
while CBS were collected by skin puncture of
fingertip into heparinized capillary plastic tubes.
From other 50 patients, CBS were collected both
in glass and plastic heparinized capillary tubes at
the same time. Samples were thoroughly mixed
with metal stirrer and magnet to obtain proper
anticoagulation, and to avoid contamination
by clots. Care was taken that no air entered the
collection devices.
Arterial blood samples were analyzed within 15
minutes, and after 60 minutes from sampling.
Capillary blood samples were analyzed within
15 minutes from sampling. Blood gas analyses of
pO2, pCO2 and sO2 were performed at the room
temperature. All determinations were performed
at the Clinical Chemistry and Biochemistry, Clinical Centre University of Sarajevo by potentiometric method using a blood gas analyzer ABL
555 (Radiometer, Copenhagen, Denmark), according to the manufacturer’s instructions. Tubes
and syringes for blood sampling were purchased
from Radiometer.
The research was done respecting ethical standards of the Declaration of Helsinki. Ethics approval was obtained from the Ethics Committee of
School of Medicine, University of Sarajevo.
Variables are presented as a mean ± standard deviation. Comparison of means between two groups was analyzed by the Student t-test for independent samples. The paired-samples t-test has
been used to compare the means between two related groups on the same continuous, dependent variable. Values of p<0.05 were considered
as statistically significant.
RESULTS
One hundred arterial blood determinations from
50 patients were performed, e. g., 50 measurements for each of the two experimental conditions, within 15 minutes and after 60 minutes from
sampling. Values of pO2 and sO2 in ABS after 60
minutes were significantly lower compared to
those analyzed within 15 minutes (p=0.007 and
p=0.0001, respectively), while no statistically si-
109
Medicinski Glasnik, Volume 12, Number 2, August 2015
gnificant differences were found for pCO2 values
(p=0.17) (Table 1).
Table 1. Arterial blood gas testing during two post-sampling
time points
Parameters
0-15 min
after 60 min
p
pO2 (kPa)
sO2 (%)
pCO2 (kPa)
9.51±1.95
92.40±4.5
5.36± 1.99
9.20 ± 1.89
91.25 ± 5.03
5.77 ± 2.03
0.007
0.0001
0.17
pO2, partial pressure of oxygen; sO2 , oxygen saturation level of
hemoglobin; pCO2, partial pressure of carbon dioxide
Blood gas testing in CBS within 15 minutes from
blood sampling were performed for the same patients in order to investigate the influence of a
sample type on blood gas values. Results showed
significantly lower values of pO 2 and sO2 in CBS
compared to ABS (p=0.009 and p=0.0001, respectively), while no statistically significant differences were found for pCO2 values (p=0.348)
(Table 2).
Table 2. Arterial and capillary blood gas testing within 15
minutes of post-sampling period
Parameters
ABS
CBS
p
pO2 (kPa)
sO2 (%)
pCO2 (kPa)
9.51 ± 1.95
92.40 ± 4.50
5.36 ± 1.99
8.92 ± 2.07
91.25 ± 4.86
5.16 ± 1.02
0.009
0.0001
0.348
ABS, arterial blood samples; CBS, capillary blood samples; pO2, partial pressure of oxygen; sO2 , oxygen saturation level of hemoglobin;
pCO2, partial pressure of carbon dioxide
For other 50 patients, blood gas analyses of CBS
in glass and plastic tubes were performed, also
within 15 minutes, in order to investigate the
impact of a type of sampling tubes on blood gas
values. Results showed significantly higher values of pO 2 and sO2 in CBS in the plastic tubes
compared to CBS in the glass tubes (p=0.0001
and p=0.002, respectively), while no statistically
significant differences were found for pCO2 values (p=0.278) (Table 3).
Table 3. Capillary blood gas testing in different tube types
Parameters
pO2 (kPa)
sO2 (%)
pCO2 (kPa)
Glass tube
Plastic tube
7.89 ± 2.0
8.50 ± 1.98
88.23 ± 11.22 89.66 ± 11.04
5.13 ± 1.36
5.00 ± 1.45
p
0.0001
0.002
0.278
pO2, partial pressure of oxygen; sO2 , oxygen saturation level of
hemoglobin; pCO2, partial pressure of carbon dioxides.
DISCUSSION
The results of our study showed that arterial blood
gas testing were affected by the length of postsampling time, but only pO2 and sO2 blood values
showed statistically significant changes. Similar
110
to our findings, previous studies described the
influence of time between sampling and analysis
on blood gas values. Beaulieu at al. concluded
that pO2, in blood samples stored on ice, should
be analyzed within 30 minutes compared to pCO2
which showed no clinically significant changes
within 60 minutes (7). Knowles at al. found significantly higher pO2 values in the samples kept for
30 minutes compared to those analyzed immediately (8). Observed results can be explained by the
oxygen diffusion through the wall of plastic syringes. Although insignificantly, Mohammadhoseini
at al. showed that pO2 values in ABS stored for
60 minutes at 22°C were lower compared with
those analyzed immediately. They also found statistically significant higher values of pCO2 in those samples but those changes were not clinically
significant (9). Our study results are mainly consistent with the previous finding that post-sampling
time significantly affects pO2 and sO2, so we have
to analyze these parameters immediately, or as
soon as possible. Statistically significant decrease
of pO2 and sO2 that we found in ABS analyzed
after 60 minutes at room temperature indicates
that metabolic consumption of oxygen exceeds its
diffusion through the wall of plastic syringes.
Values of pO2 and sO2 in CBS in our study were
significantly lower in relation to those measured
in ABS within 15 minutes. Statistically significant differences were not found for pCO2 values.
Zavorski et al. showed that fingertip blood sample analysis can predict arterial pCO2, but not pO2
and sO2 (10). Murphy at al. found that agreement
between measurements of arterial and capillary
blood gases were good for pCO2, but poor for
pO2 and that continuous pulse oximetry is more
suitable for ensuring adequate and controlled
oxygenation of the patient (11). Zavorsky et al.
considered that CBS may be an appropriate replacement for ABS when analyzing pO2, except
residual standard error of 6 mmHg is required for
precision, and that capillary pCO2 accurately reflects arterial pCO2 over a wide range of values
(12). Lower values of pO2 and sO2 in CBS are
expected due to the fact that capillary blood is a
mixture of capillary, arteriolar and venial blood
as well as interstitial and intracellular fluid. Values of pCO2 have no significant differences in the
ABS and CBS because of lower arteriovenous
gradient (1).
Smajić et al. The influences on blood gas testing
Comparison of our findings for sO2 and pO2 values in CBS within 15 minutes from sampling,
between samples in the glass and plastic tubes
showed that values obtained in the plastic tubes
were higher compared to those in the glass tubes (p<0.01). Statistically significant differences
were not found for pCO2 blood values. The plastic containers for blood gas testing are partially
gas permeable with increasing oxygen permeability at lower temperature (7,8,13,14). Our findings confirmed that pO₂ values in the plastic tubes
at room temperature increase within 15 minutes,
despite ongoing cellular metabolism which is
dominant in the glass tube, because of the differences in gas partial pressures between blood and
ambient. Historically implemented practice of
keeping glass syringes on ice is not recommended for plastic syringes any more (1,9).
Finally, we conclude that getting an accurate result for blood gas testing, special attention should be devoted to many pre-analytical activities.
The length of post-sampling analysis interval has
significant impact on blood oxygen parameters.
Although there is no doubt that blood samples
should be analyzed as soon as possible, analysis
within 15 minutes of blood sampling is also con-
sidered as appropriate. Even though the gold standard samples for blood gas testing are ABS, when
the circumstances do not permit taking such samples, CBS can be taken as an alternative sample.
On such occasions, pO 2 and sO2 results should
be taken with caution. For the patients requiring
continuous monitoring of blood gases it is best
to analyze ABS, but also it can be extremely beneficial to analyze CBS in combination with pulse oximetry. Since type of collection tubes also
affect the oxygen parameters significantly, samples should be taken into glass tubes if we are not
able to analyze it in appropriate time.
ACKNOWLEDGEMENT
The authors would like to thank Professor Hasan Žutić and all medical staff at the Clinic for
Pulmonary Diseases and Clinical Chemistry and
Biochemistry, Clinical Centre University of Sarajevo, who participated in the study.
FUNDING
No specific funding was received for this study.
TRANSPARENCY DECLARATIONS
Competing interests: none to declare.
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J, Fiddler C, Craig M, Robertson C. Capillary blood gases in acute exacerbations of COPD. Respir
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Učinak vremena proteklog od uzorkovanja do analize, vrste uzoraka
krvi i cjevčica za uzorkovanje na analiziranje gasova u krvi
Jasmina Smajić¹, Damira Kadić², Sabaheta Hasić³, Nafija Serdarević¹
¹Klinička hemija i biohemija, Klinički centar Univerziteta u Sarajevu, Sarajevo, ²Služba za laboratorijsku dijagnostiku, Kantonalna bolnica
Zenica, Zenica, ³Institut za medicinsku biohemiju, Medicinski fakultet Univerziteta u Sarajevu, Sarajevo; Bosna i Hercegovina
SAŽETAK
Cilj Istražiti učinke vremena od uzorkovanja krvi do analiziranja, vrste uzoraka krvi i cjevčica za uzorkovanje na analiziranje gasova u krvi.
Metode Ispitivanje je uključivalo 100 pacijenata Klinike za plućne bolesti Kliničkog centra Univerziteta u Sarajevu. Parcijalni pritisak kiseonika (pO2) i ugljen dioksida (pCO2), te nivo saturacije hemoglobina kiseonikom (sO2) analizirani su u arterijskim i kapilarnim uzorcima krvi potenciometrijskom metodom na analizatoru ABL 555 (Radiometer, Kopenhagen, Danska). Upoređivana su uparena mjerenja
uzoraka arterijske krvi izvođena unutar 15 minuta i nakon 60 minuta od uzorkovanja. Rezultati dobijeni
iz kapilarnih uzoraka unutar 15 minuta upoređivani su s odgovarajućim rezultatima arterijskih uzoraka,
kao i rezultati dobijeni iz kapilarnih uzoraka uzetih u roku 15 minuta u staklene i plastične cjevčice.
Rezultati Vrijednosti pO2 i sO2 nakon 60 minuta bile su signifikantno niže u poređenju s rezultatima
dobijenim unutar 15 minuta u arterijskim uzorcima krvi (pO2-9.20±1.89 vs. 9.51±1.95; sO2- 91.25±5.03
vs. 92.40±4.5; p<0.01). Vrijednosti pO2 i sO2 u kapilarnim uzorcima također su bile signifikantno niže
od vrijednosti dobijenih iz arterijskih uzoraka (pO2-8.92±2.07 vs. 9.51±1.95; sO2- 91.25±4.86 vs.
92.40±4.50; p<0.01). Dobijene vrijednosti pO2 i sO2 u kapilarnim uzorcima u plastičnim kapilarnim
cjevčicama bile su veće od onih u staklenim cjevčicama (pO2-8.50±1.98 vs. 7.89±2.0; sO2-89.66±11.04
vs. 88.23±11.22, p<0.01). Utjecaj na vrijednosti pCO2 u krvi nije bio signifikantan (p>0.05).
Zaključak Vrijeme od uzimanja uzorka krvi do analiziranja, vrsta uzoraka i cjevčica za uzorkovanje
imaju signifikantan učinak na parametre kiseonika u krvi. Analiza unutar 15 minuta od uzorkovanja
smatra se prihvatljivom.
Ključne riječi: analiza gasova u krvi, kapilarne cjevčice, šprice, staklo, plastika
112
ORIGINAL ARTICLE
Association of LPIN1 gene variations with markers of metabolic
syndrome in population from Bosnia and Herzegovina
Tamer Bego1, Tanja Dujić1, Barbara Mlinar2, Sabina Semiz1,4, Maja Malenica1, Besim Prnjavorac3,6,
Barbara Ostanek2, Janja Marc2, Anida Čaušević-Ramoševac5, Adlija Čaušević1
Department of Biochemistry and Clinical Analysis, Faculty of Pharmacy, University of Sarajevo, Sarajevo, Bosnia and Herzegovina,
Department of Clinical Biochemistry, Faculty of Pharmacy, University of Ljubljana, Ljubljana, Slovenia, 3General Hospital of Tešanj,
Tešanj, 4Faculty of Engineering and Natural Sciences, International University of Sarajevo, Sarajevo, 5Bosnalijek, Pharmaceutical and
Chemical Industry, Joint Stock Company, Sarajevo, 6Department of Pathophysiology, Faculty of Pharmacy; University Sarajevo, Sarajevo; Bosnia and Herzegovina
1
2
ABSTRACT
Aim To investigate association of two LPIN1 gene variations with
main traits of metabolic syndrome (MS) (waist circumference,
body mass index, blood pressure, triglycerides, HDL-cholesterol
and fasting glucose levels) in population from Bosnia and Herzegovina.
Tamer Bego
Department for Biochemistry and Clinical
Analysis, Faculty of Pharmacy, University
of Sarajevo
Zmaja od Bosne 8, 71000 Sarajevo,
Phone: +387 33 586 188;
Fax: +387 33 586 188;
E.mail: [email protected]
Methods This study included 43 patients with metabolic syndrome and 43 healthy controls from General Hospital in Tešanj, Bosnia and Herzegovina. Subjects were genotyped for two LPIN1
gene variations (rs11693809: C>T and rs2716610: C>T) by real
time PCR method.
Results In control subjects LPIN1 polymorphism, rs2716610:
C>T, was significantly associated with a lower body mass index
(BMI) (p=0.008) and waist circumference (p=0.008). The second
analyzed rs11693809: C>T polymorphism was associated with
lower blood HbA1c levels (p=0.048) in a group of MS patients.
Conclusion Results of our study suggest that rs2716610: C>T
polymorphism of LPIN1 gene could have a protective effect against development of metabolic syndrome, while rs11693809: C>T
might affect a glucose control in patients with MS.
Keywords: metabolic syndrome, LPIN1 gene, markers, gene variations.
28 November 2015;
Revised submission:
03 February 2015;
Accepted: 21 March 2015.
doi: 10.17392/795-15
Med Glas (Zenica) 2015; 12(2): 113-121
113
INTRODUCTION
The metabolic syndrome (MS) is also known as
syndrome X(1), the insulin resistance syndrome
(2) and the deadly quartet (3). Metabolic syndrome is a combination of medical disorders that,
when occurring together, increase the risk of developing cardiovascular disease and diabetes (4).
The pathogenesis of the metabolic syndrome is
thought to involve a complex interaction of multiple factors, including obesity and abnormal fat
distribution, insulin resistance, hepatic, vascular, and immunologic factors, as well as lifestyle
and genetic contributions (4). The available evidence indicates that between 20% and 30% of
the world’s adult population have MS (5). New
harmonized definition and criteria for MS was
accepted finally in 2009, which included measurement of waist circumference, blood pressure,
triglycerides, HDL-cholesterol and serum glucose (6). Insulin resistance and abdominal obesity
appear to be predominant underlying risk factors
of MS. Beside this, other associated conditions
for this syndrome can be genetic factors, physical
inactivity, aging, and hormonal imbalance (7).
A newly discovered gene for lipin 1 (LPIN1) resides in the 2p25 region, and codes for phosphatidic acid phosphatase, a key enzyme in triglyceride (TG) biosynthesis (8). There is evidence that
chromosome region 2p25 is in linkage disequilibrium with several obesity related phenotypes,
such as body mass index (BMI), waist circumference, skin-fold thickness, and percentage of body
fat (9). The lipin protein family consists of three
members, lipin-1, lipin-2, and lipin-3 (10). Lipin 1
is a newly discovered multifunctional protein that
participates in the metabolism of lipids in different
ways (11). Lipin-1 is abundantly expressed in adipose tissue and skeletal muscle, and lipin-1 protein localizes to either the cytosol or the nucleus,
which may be related to its two known functions
(12). Namely, in the cytosol, lipin-1 acts as a phosphatidate phosphatase (PAP) enzyme converting
phosphatidate to diacylglycerol during triglyceride
biosynthesis (13-14), while in the nucleus of adipocytes and hepatocytes lipin-1 acts as a transcriptional coactivator that interacts with the nuclear receptor peroxisome proliferator-activated receptor
α (PPARα) and PPARγ (PPARG) coactivator 1α
(PPARGC1α) in a complex that modulates fatty
acid oxidation gene expression (10, 15-16).
114
Null mutations in the murine Lpin1 gene result
in a severe defect in adipose tissue development,
which is related to insulin resistance and fatty liver
dystrophy (fld mice) (10). Based upon the comprehensive biological knowledge of lipin-1 role in
energy metabolism, human lipin-1 has been considered as an obvious biological candidate to explain some of the inter-individual variations in the
common metabolic phenotypes. In addition, variants in LPIN1 have been associated with the fasting
serum insulin levels, body mass index (BMI), waist circumference, and obesity development (1719). Previous data from four meta-analysis studies
found an association of LPIN1 variants with increased BMI (20), while another study found an association with hypertension (21). Other studies have
shown correlation of expression of LPIN1 gene
in adipose tissue with BMI and insulin resistance
in humans (14, 16). Recently published German
population study (n=1674), showed an interesting
association of LPIN1 gene variants with metabolic
phenotype (22). They identified three associated
three-marker haplotypes, one common haplotype
that increased the risk for metabolic syndrome,
while other two were associated with lower blood
pressure levels, lower BMI, waist circumference,
and HbA1C levels (22).
In this study, for the first time in population from Bosnia and Herzegovina, it was
analyzed whether LPIN1 gene polymorphisms
(rs11693809: C>T and rs2716610: C>T), including haplotype analysis, were associated with
the traits of metabolic syndrome. An association
between biochemical parameters including, but
not limited to, glucose, HbA1c, insulin levels,
HDL and LDL cholesterol, triglycerides, serum
proteins levels, and activity of liver enzymes
and these two polymorphisms in LPIN1 gene
was analyzed in patients with metabolic syndrome and healthy controls.
Study participants
The study included 43 patients with metabolic
syndrome and 43 healthy controls from General
Hospital in Tešanj, Zenica-Doboj Canton, Bosnia
and Herzegovina. Investigation was done in accordance with ethical recommendations and practices
of the General Hospital Tešanj, and with ethical
Bego et al. LPIN1 variations and metabolic syndrome
principles outlined in the World Medical Association Declaration of Helsinki – Ethical Principles
of Medical Research Involving Human Subjects
(initiated in June 1964, last amendment in October 2000). Each subject in the study signed written
informed consent. Metabolic syndrome was diagnosed according to new harmonized definition
and criteria for MS from 2009. According to this
new definition, MS is diagnosed when any three of
the following five criteria are met: increased waist
circumference (recommended waist circumference thresholds for Euripides) ≥ 94 cm for men, and
≥ 80 cm for women), triglycerides ≥ 1.7 mmol/L,
HDL-cholesterol < 1.0 mmol/L in males and <
1.3 mmol/L in females, blood pressure ≥130/85
mmHg and fasting glucose ≥ 5.6 mmol/L (6). Patients treated with insulin and patients with acute
infection and / or inflammation and endocrine disorders were excluded from the study. All patients
included in the study were using heterogeneous
therapy (74% of all patients received antihypertensive therapy, 58% were treated with glucose-lowering drugs, and 47% were treated with lipid-lowering drugs). Healthy control group consisted of 43
non-obese, age-matched subjects, who had less
than three features of MS. They were not taking
any medication during the course of the study.
Biochemical and anthropometrical measurements
Waist circumference, height, weight, systolic and
diastolic blood pressure were measured in all
participants. BMI was calculated as weight (kg)/
(height (m))2. Serum levels of fasting glucose,
triglycerides, total cholesterol, HDL-cholesterol,
LDL-cholesterol, albumin, globulin, bilirubin,
creatinine, urea, urate, HbA1c and C-reactive
protein (CRP), as well as activities of aspartate
aminotransferase, alanine aminotransferase, and
γ-glutamyltransferase were determined by using
the VITROS auto analyzer 350 Chemistry System
(Ortho-Clinical Diagnostics, Rochester, New York,
USA). Serum insulin levels were measured by the
Abbott AxSYM (Abbott Diagnostics, North Chicago, Illinois, USA) analyzer. HOMA IR index was
calculated by using following formula: fasting insulin (mU/L) x fasting glucose (mmol/L)/22.5 (23).
Genotyping analysis
The Miller extraction protocol was used for the
DNA extraction (24). Genotyping analysis for
rs11693809 (IVS1 +3341C > T, denoted intron 1
SNP) and rs2716610 (IVS17-228C > T, denoted
intron 17 SNP) polymorphisms was performed
with real-time PCR allelic discrimination on ABI
PRISM with C_2096848_10 and C_16280532_10
assays, respectively (Applied Biosystems, Foster
City, CA, USA). We double-genotyped twenty percent of all samples with 100% concordant results.
Statistical analysis
Chi-square (χ2) and Fisher’s exact tests (in the
case where frequencies were less or equal to 5)
were applied to examine differences in allele
frequencies and genotype distributions between
healthy controls and patients with MS. Significance of difference of biochemical and anthropometrical measurements according to genotypes
of analyzed polymorphisms, sex and age were
estimated by linear regression Corrections for
Table 1. Characteristics of the study participants
Parameter*
MS patients (n=43) Controls (n=43)
Age (years)
49 (40-56)
45 (41-51)
BMI (kg/m2)
33.0 (29.2-35.5)
24.7 (22.2-27.4)
Waist circumfe110 (96-120)
83 (78-90)
rence (cm)
Systolic BP
143 (130-158)
120 (110-125)
(mm Hg)
Diastolic BP
90 (80-100)
78 (70-80)
(mm Hg)
Fasting insulin
10.6 (8.0-13.9)
7.3 (6.4-10.1)
(mU/L)
Fasting glucose
8.4 (5.5-11.7)
5.0 (4.7-5.2)
(mmol/L)
HOMA-IR
4.1 (2.7-6.1)
1.6 (1.4-2.3)
Blood HbA1c (%)
6.1 (5.5-7.2)
5.6 (4.8-6.0)
Total cholesterol
5.6 (5.1-6.3)
5.8 (5.2-6.5)
(mmol/L)
LDL-cholesterol
3.20 (2.60-4.01)
3.37 (2.87-4.19)
(mmol/L)
HDL-cholesterol
1.07 (0.88-1.30)
1.67 (1.38-1.87)
(mmol/L)
Triglycerides
2.26 (1.77-3.27)
1.17 (0.76-1.45)
(mmol/L)
CRP (mg/L)
5.0 (3.0-6.0)
1.3 (0.8-4.0)
Creatinine
82.5 ( 72.5-94.5)
96.0 (59.0-78.0)
(mmol/L)
Urea (mmol/L)
5.05 (4.15-6.12)
4.30 (3.70-5.40)
Urate (mmol/L) 297.0 (239.0-333.0) 272.0 (229.0-323.0)
Albumin (g/L)
49.4 (43.0-52.7)
44.0 (42.0-48.1)
Globulin (g/L)
24.6 (21.4-30.7)
32.0 (28.0-33.0)
Bilirubin
12.9 (10.7-14.7)
11.5 (9.7-14.6)
(mmol/L)
AST (IU/L)
26.0 (18.2-31.0)
26.0 (21.0-31.0)
ALT (IU/L)
27.0 (22.2-42.2)
23.0 (16.0-30.0)
GGT (IU/L)
23.5 (17.7-38.0)
17.5 (14.0-26.5)
p†
0.206
<0.001
<0.001
<0.001
<0.001
0.010
<0.001
<0.001
<0.001
0.385
0.133
<0.001
<0.001
<0.001
<0.001
0.036
0.018
0.012
<0.001
0.302
0.593
0.017
0.298
*Values represent medians (lower-upper quartile); †significance of
difference in Mann-Whitney test.
BMI, body mass index; BP, blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density
lipoprotein; HDL , high-density lipoprotein; hsCRP, high-sensitivity
C-reactive protein; AST, aspartate aminotransferase; ALT, alanine
aminotransferase; GGT – γ, glutamyl transferase γ
115
multiple testing were performed by using Bonferroni correction for a total of two SNPs. A p value ≤ 0.05 was considered statistically significant.
Linkage disequilibrium was calculated by using
the 2LD program (25). Haplotype reconstruction
was done with the PHASE program (26).
Table 2. Allele and genotype frequencies for LPIN1 gene
polymorphisms*
Polymorphism
MS
CC 16 (40.0%)
Intron
CT 19 (47.5%)
1SNP
(rs11693809) TT 5 (12.5%)
Total
40
p
0.985
CC 26 (65.0%)
Intron
17 SNP
CT 12 (30.0%)
(rs2716610) TT 2 (5.0%)
Total
40
p‡
0.925
RESULTS
All analyzed parameters were significantly
different between patients and control groups
except total cholesterol, LDL cholesterol, and
bilirubin levels, as well as aspartate aminotransferase (AST) and γ-glutamyltransferase activity (GGT) (Table 1).
T-allele
T-allele
frequ- Controls frequ- p†
ency
ency
14 (33.3%)
0.36 23 (54.8%) 0,39 0.792
5 (11.9%)
42
0.631
32 (76.2%)
0.20
9 (21.4%) 0.13 0.513
1 (2.4%)
42
0.931
*rs11693809: C>T and rs2716610: C>T; †Significance of χ2 /
Fisher’s exact test for comparison of genotype frequencies between
healthy controls and MS patients; ‡p value for Hardy-Weinberg
equilibrium.
Table 3. Effects of LPIN1 SNPs on biochemical and anthropometrical parameters in controls
Parameter*
BMI
(kg/m2)
Waist circumference
(cm)
Systolic BP
(mm Hg)
Diastolic BP
(mm Hg)
Fasting insulin
(mU/L)
Fasting glucose
(mmol/L)
HOMA-IR
Blood HbA1c
(%)
Total cholesterol
(mmol/L)
LDL-cholesterol
(mmol/L)
HDL-cholesterol
(mmol/L)
Triglycerides
(mmol/L)
hsCRP
(mg/L)
Creatinine
(mmol/L)
Urea
(mmol/L)
Urate
(mmol/L)
Albumin
(g/L)
Globulin
(g/L)
Bilirubin
(mmol/L)
AST
(IU/L)
ALT
(IU/L)
GGT
(IU/L)
C/C
(n=14)
23.3
(20.5-25.4)
81
(72-85)
120
(101-137)
75
(66-87)
7.3
(6.2-14.7)
5.1
(4.5-5.6)
1.6
(1.3-3.1)
5.6
(5.0-6.0)
6.2
(5.3-7.0)
3.81
(2.97-4.85)
1.72
(1.48-2.03)
1.15
(0.92-1.67)
1.0
(0.5-1.4)
63.0
(56.0-71.0)
4.5
(3.6-5.5)
251.5
(223-320)
44.5
(42.7-49.1)
30.0
(25.4-33.0)
9.6
(6.9-11.4)
22.0
(20.5-30.0)
21.0
(11.7-29.0)
16.0
(13.7-28.7)
rs11693809 C>T
C/T + T/T
B (95% CI) †
(n=24)
25.1
2.156
(22.2-28.7) (-0.758, 5.071)
88
5.168
(78-93)
(-1.405, 11.740)
120
3.803
(120-125) (-8.272, 15.878)
80
2.255
(75-80)
(-5.21, -9.728)
6.9
-3.646
(5.8-7.7)
(-7.582, 0.290)
4.9
-0.089
(4.7-5.1)
(-0.392, 0.213)
1.5
-0.880
(1.3-1.7)
(-1.905, 0.144)
5.6
0.161
(4.4-5.9)
(-0.289, 0.611)
5.7
-0.334
(5.1-6.4)
(-1-059, 0.392)
3.35
-0.154
(2.93-4.18) (-0.904, 0.596)
1.66
-0.205
(1.37-1.86) (-0.474, 0.064)
1.12
-0.073
(0.67-1.44) (-0.439, 0.294)
1.7
1.154
(0.9-4.7)
(-0.582, 2.889
70.0
4.348
(59.0-80.5) (-3.075, 11.771)
4.4
0.188
(3.8-5.4)
(-0.70, -1.082)
289.5
3.283
(228-326) (-38.087, 44,634)
44.0
-1.690
(42.2-47.4) (-4.365-0.985)
32.0
2.900
(28.7-34.5) (-0.689-6.488)
12.5
2.488
(10.5-16.2) (-0.389-5.365)
26.0
1.358
(21.5-30.7) (-2.611-5.326)
24.0
0.343
(16.0-29.7) (-7.313-8.000)
-5.483
18.0
(15.0-26.0) (-22.380-11.414)
p†
pB
0.141 0.282
0.119 0.238
0.524 1.000
0.541 1.000
0.068 0.136
0.553 1.000
0.089 0.178
0.472 0.944
0.357 0.714
0.679 1.000
0.130 0.260
0.690 1.000
0.185 0.370
0.242 0.484
0.671 1.000
0.873 1.000
0.208 0.416
0.110 0.220
0.088 0.176
0.491 0.982
0.928 1.000
0.514 1.000
C/C
(n=29)
25.4
(22.9-28/.7)
86
(80-93)
120
(120-130)
80
(70-80)
6.9
(6.1-9.9)
5.0
(4.8-5.2)
1.5
(1.4-2.3)
5.6
(4.5-6.0)
5.9
(5.1-6.4)
3.34
(2.88-4.46)
1.69
(1.37-1.87)
1.13
(0.77-1.47)
1.5
(0.8-3.8)
70.0
(60.5-80.0)
4.7
(4.0-5.4)
285
(229-323)
44.0
(42.0-47.8)
32.0
(28.8-34.0)
11.7
(9.6-15.6)
26.0
(21.5-30.5)
23.0
(14.0-28.5)
17.0
(15.0-25.5)
rs2716610 C>T
C/T + T/T
B (95% CI)†
(n=10)
21.0
-4.272
(20.2-24.0) (-7.104, -1.441)
78
-9.637
(72-81) (-16-032, -3.243)
120
-5.752
(102-122) (-18.509, 7.004)
75
-0.974
(67-82)
(-8.974, 7.026)
7.3
-1.695
(6.0-9.6)
(-6.112, 2.722)
4.6
-0.298
(4.3-5.3)
(-0.606, 0.010)
1.5
-0.566
(1.2-2.2)
(-1.697, 0.564)
5.6
0.065
(5.0-5.8)
(-0.420, 0.549)
6.0
0.199
(5.5-6.8)
(-0.584, 0.981)
3.69
0.123
(2.98-4.54) (-0.679, 0.926)
1.68
0.014
(1.48-1.92) (-0.283, 0.312)
1.29
-0.008
(0.63-1.49) (-0.401, 0.385)
1.1
-1.018
(0.5-2.0)
(-2.890, 0.853)
56.0
-8.440
(51.5-70.0) (-15.985, -0.894)
3.8
-0.809
(3.3-4.8)
(-1.724, 0.106)
244
-2.706
(213-330) (-46.896, 41.485)
44.5
0.619
(42.7-47.4) (-2.302, 3.540)
31.5
-0.075
(27.4-34.0) (-4.062, 3.913)
10.6
-1.401
(7.7-11.9) (-4.578, 1.777)
23.5
-1.147
(19.5-30.0) (-5.399, 3.105)
22.0
4.157
(16.7-35.2) (-3.894, 12.208)
18.5
15.814
(13.7-35.5) (-1.500, 33.127)
p†
pB
0.004
0.008
0.004
0.008
0.364
0.728
0.805
1.000
0.438
0.876
0.057
0.114
0.313
0.626
0.788
1.000
0.609
1.000
0.757
1.000
0.922
1.000
0.968
1.000
0.277
0.554
0.029
0.058
0.081
0.162
0.902
1.000
0.669
1.000
0.970
1.000
0.377
0.754
0.587
1.000
0.301
0.602
0.072
0.144
*Values represent medians (lower-upper quartile); †Effects (unstandardized coefficients B) and p values were assessed using multiple linear regression adjusted for age and gender, under dominant genetic model; pB, adjusted by using Bonferroni correction for two SNPs;
BMI, body mass index; BP; blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density lipoprotein;
HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase;
GGT-γ; glutamyltransferase;
116
Table 4. Effects of LPIN1 SNPs on biochemical and anthropometrical parameters in the patients
Parameter*
BMI
(kg/m2)
Waist circumference
(cm)
Systolic BP
(mm Hg)
Diastolic BP
(mm Hg)
Fasting insulin
(mU/L)
Fasting glucose
(mmol/L)
HOMA-IR
Blood HbA1c
(%)
Total cholesterol
(mmol/L)
LDL-cholesterol
(mmol/L)
HDL-cholesterol
(mmol/L)
Triglycerides
(mmol/L)
hsCRP
(mg/L)
Creatinine
(mmol/L)
Urea
(mmol/L)
Urate
(mmol/L)
Albumin
(g/L)
Globulin
(g/L)
Bilirubin
(mmol/L)
AST
(IU/L)
ALT
(IU/L)
GGT
(IU/L)
C/C
(n=16)
33.5
(29.3-36.4)
111
(103-117)
145
(140-160)
95
(81-100)
10.3
(7.3-12.6)
11.0
(7.2-14.0)
4.3
(3.6-5.6)
6.9
(5.9-7.7)
5.6
(5.1-6.0)
3.02
(2.56-3.28)
1.11
(0.83-1.42)
2.38
(1.69-3.21)
5.0
(3.2-6.0)
83.0
(78.7-88.5)
4.9
(3.9-5.8)
275
(232-336)
49.9
(45.1-51.8)
26.0
(21.6-30.3)
13.3
(12.4-15.6)
22.0
(17.0-28.0)
25.0
(22.0-35.0)
15.0
(8.75-27.5)
rs11693809 C>T
C/T + T/T
B (95% CI)†
(n=24)
33.0
0.280
(26.9-35.3) (-3.845, 4.404)
111
-5.482
(96-123) (-17.623, 6.659)
140
-6.002
(130-155) (-20.321, 8.317)
90
-4.814
(80-97)
(-14.276, 4.647)
11.5
0.381
(8.9-16.4) (-4.951, 5.712)
6.2
-2.315
(5.4-9.8)
(-5.305, 0.675)
4.3
0.088
(2.6-7.7)
(-1.909, 2.084)
5.9
-1.082
(5.2-6.6)
(-2.010, -0.154)
5.6
0.142
(5.0-6.4)
(-0.629, 0.913)
3.31
0.309
(2.46-4.13) (-0.515, 1.134)
1.00
0.043
(0.88-1.32) (-0.181, 0.266)
2.24
-0.595
(1.88-3.69) (-1.473, 0.283)
5.0
-0.103
(2.2-6.7)
(-1.946, 1.740)
83.0
2.708
(70.0-110.0) (-12.677, 18.093)
5.3
0.444
(4.66.2)
(-0.703, 1.590)
325
38.578
(294-338) (-19.443, 96.600)
48.0
-0.799
(43.0-53.3) (-4.794, 3.195)
23.5
-1.053
(21.4-32.0) (-4.852, 2.746)
12.0
-2.408
(9.1-14.0) (-5.738, 0.921)
26.0
3.968
(19.2-31.7) (-4.941, 12.876)
27.0
-0.843
(22.2-48.0) (-12.511, 10.826)
-1.335
25.0
(16.0-38.0) (-18.571, 15.900)
p†
pB
0.891 1.000
0.365 0.730
0.398 0.796
0.307 0.614
0.885 1.000
0.125 0.250
0.929 1.000
0.024 0.048
0.711 1.000
0.451 0.902
0.701 1.000
0.177 0.354
0.910 1.000
0.723 1.000
0.437 0.874
0.185 0.370
0.686 1.000
0.576 1.000
0.151 0.302
0.372 0.744
0.884 1.000
0.876 1.000
C/C
(n=26)
33.5
(28.9-36.9)
112
(106-120)
145
(140-160)
92
(81-100)
10.4
(7.4-12.9)
9.0
(5.8-11.9)
4.3
(3.3-8.0)
6.4
(5.5-7.4)
5.7
(5.1-6.2)
3.16
(2.77-3.90)
1.20
(0.93-1.46)
2.22
(1.85-3.15)
5.0
(3.0-6.0)
82.0
(74.2-89.5)
5.0
(4.3-6.0)
295
(246-337)
50.5
(43.7-52.9)
24.6
(21.5-30.2)
13.3
(10.9-15-9)
26.0
(19.5-31.5)
33.0
(23.0-50.5)
23.0
(12.0-38.0)
rs2716610 C>T
C/T + T/T
B (95% CI)†
(n=14)
33.0
-1.825
(25.7-35.1) (-5.904, 2.253)
110
-6.339
(87-122)
(-18.423, 5.746)
140
-7.799
(130-150) (-21.990, 6.392)
85
-6.985
(70-97)
(.16.248, 2.277)
11.3
-0.869
(9.0-16.0)
(-6.191, 4.454)
6.1
-2.118
(5.5-10.3)
(-5.124, 0.888)
3.6
-1.047
(2.3-5.6)
(-3.006, 0.912)
5.9
-0.223
(5.5-6.5)
(-1.222, 0.775)
5.5
-0.228
(4.5-6.3)
(-0.996, 0.549)
3.05
-0.016
(2.01-4.18) (-0.848, 0.815)
0.91
-0.228
(0.83-1.13) (-0.438, -0.019)
2.37
-0.112
(1.69-4.03) (-1.013, 0.788)
5.0
0.233
(2.0-7.2)
(-1.608, 2.074)
89.0
-0.377
(67.5-105.5) (-15.788, 15.034)
5.0
-0.110
(3.9-6.5)
(-1.266, 1.047)
316
-0.178
(278-337) (-59.775, 59.419)
46.4
-2.595
(41.7-51.3) (-6.488, 1.299)
24.6
-0.464
(19.7-33.2) (-4.277, 3.350)
12.0
-2.690
(10.0-13.6) (-5.993, 0.613)
22.5
-6.080
(18.0-30.2) (-14.845, 2.686)
23.5
-15.079
(20.0-33.0) (-25.539, -4.619)
22.5
-11.987
(9.5-31.2) (-28.694, 4.721)
p†
pB
0.370 0.740
0.293 0.586
0.270 0.540
0.134 0.268
0.741 1.000
0.162 0.324
0.284 0.568
0.653 1.000
0.551 1.000
0.969 1.000
0.033 0.066
0.801 1.000
0.798 1.000
0.961 1.000
0.848 1.000
0.995 1.000
0.184 0.368
0.806 1.000
0.107 0.214
0.168 0.336
0.006 0.012
0.154 0.308
*Values represent medians (lower-upper quartile);
†Effects (unstandardized coefficients B) and p values were assessed using multiple linear regression adjusted for age and gender, under dominant
genetic model; pB adjusted by using Bonferroni correction for two SNPs;
BMI, body mass index; BP; blood pressure; HOMA-IR, homeostasis model assessment insulin resistance index; LDL, low-density lipoprotein;
HDL, high-density lipoprotein; hsCRP, high-sensitivity C-reactive protein; AST, aspartate aminotransferase; ALT, alanine aminotransferase;
GGT-γ; glutamyltransferase;
Table 5. Haplotype frequencies and diplotypes of LPIN1 gene
in MS patients and healthy controls
Diplotype
No. of
Haplotype
Frequency
No
subjects
(intron 1
Diplotype
SNP-intron MS
ConMS pa- Con17 SNP)* patients trols
tients trols
CC
CT
TC
TT
0.548
0.102
0.252
0.098
0.505
0.116
0.344
0.035
CC/CC
CC/CT
CC/TC
CC/TT
CT/CT
CT/TC
CT/TT
TC/TC
TC/TT
1
2
3
4
5
6
7
8
9
14
3
9
8
2
3
2
*corresponding to the rs11693809: C>T (intron SNP1) and
rs2716610: C>T (intron SNP17) polymorphism
8
4
12
1
3
4
1
Allele frequencies were in Hardy-Weinberg equilibrium for both, patients and control subjects
(p>0.05). However, no significant differences
in analyzed genotype frequencies were found
between patients and healthy controls (Table 2).
In a control group, after Bonferroni correction,
rs11693809: C>T polymorphism did not show significant association with any biochemical or anthropometrical measurements. However, the carriers
of T allele (CT + TT) of another analyzed polymorphism of LPIN1 gene, rs2716610: C>T, had significantly lower BMI (p=0,008), waist circumference
(p=0.008), and tendency of association with lower
117
creatinine levels (p=0.058), as compared to the
carriers of a wild type allele (CC) (Table 3).
In group of MS patients, the carriers of T allele (CT + TT) of rs11693809: C>T polymorphism had significantly lower blood HbA1c (%)
(p=0.048), as compared to the carriers of a wild
type allele (CC). The carriers of T allele (CT +
TT) of rs2716610: C>T polymorphism had significant lower ALT activity (p=0.012) and tendency of association with lower HDL cholesterol
levels (p=0.066), as compared to the carriers of
a wild type allele (CC). As shown in Table 4, no
association was found for both analyzed LPIN1
gene polymorphisms with anthropometrical measurements (body mass index (BMI) and waist
circumference).
The selected LPIN1 variants, rs11693809: C>T
and rs2716610: C>T were in a weak linkage disequilibrium (D’=0.261). No significant differences in distribution of haplotype frequencies
between patients with MS and control subjects
were demonstrated. Furthermore, an association of LPIN1 haplotypes with biochemical and
anthropometrical parameters was also tested. In
control group, the carriers of CC haplotype had
significantly higher plasma insulin (p=0.024),
higher glucose levels (p=0.036) and higher
HOMA IR index (p=0.024) as compared to the
carriers of CT haplotype. No significant associations of LPIN1 haplotypes with traits of MS were
found in patients group (Table 5).
DISCUSSION
Members of the lipin protein family have a
newly discovered enzymatic role in triglyceride
and phospholipid biosynthesis as a phosphatidate
phosphatase, and act also as inducible transcriptional coactivators in conjunction with peroxisome proliferator-activated receptor c (PPARc)
coactivator-1a and PPARa (13). Through these
activities, the founding member of the family,
lipin-1, influences lipid metabolism and glucose
homeostasis (13).
Results of our study showed significant association of rs2716610: C>T genetic variant with body
mass index (BMI) and waist circumference. No
significant associations between disease-associated traits and rs11693809: C>T were found.
Since the majority of patients participating in this
study were using medications (antihypertensive,
118
glucose-lowering or lipid-lowering drugs), the
influence of LPIN1 gene polymorphisms on the
most of selected biochemical parameters in MS
patients should be interpreted cautiously. However, it was reasonable to analyze the effects of
LPIN1 gene variations on the BMI and waist circumference in these patients.
The selected genotype-phenotype analysis
showed that, the mutant T allele of rs11693809:
C>T polymorphism was associated with lower
blood HbA1 in a group of MS patients. T allele
of another analyzed polymorphism in our study,
rs2716610: C>T, was associated with lower BMI
and waist circumference, and creatinine levels in
controls, and lower HDL cholesterol levels and
lower ALT activity in a group of MS patients.
Only a few studies analyzed association of
LPIN1 gene polymorphisms (rs11693809: C>T
and rs2716610: C>T) with metabolic syndrome (17,22,27). Suviolahti et al. analyzed seven
polymorphisms in the LPIN1 gene. They found
an association of rs11693809: C>T polymorphism with the insulin levels. In addition,
rs11693809: C>T and rs2716610: C>T polymorphism were associated with BMI in lean males.
Since an association of LPIN1 variants with BMI
in the lean or obese females was not found, the
association of LPIN1 alleles with BMI appeared to be sex specific (17). Mlinar et al., in their
study tested an association of LPIN1 polymorphisms (rs11693809: C>T and rs2716610: C>T)
with polycystic ovary syndrome (PCOS) (28).
Their results showed that mutated T allele of
rs11693809: C>T polymorphism was associated with lower plasma LDL-cholesterol levels in
controls, with lower glucose levels after OGTT
in the PCOS patients, and with lower insulin levels and HOMA-IR in nonobese PCOS patients.
These results suggest a protective role of mutated
T allele of rs11693809: C>T polymorphism against development of IR and dyslipidemia. Mutated allele T of another analyzed polymorphism
in this study (28), rs2716610: C>T, showed an
association with higher triglyceride levels in control subjects, suggesting a negative effect of this
polymorphism on the metabolic phenotype. Wiedmann at al. in their study analyzed an association of 15 genetic variants of LPIN1 gene, including rs2716610: C>T, with metabolic phenotype.
Their results showed the borderline significant
association of this polymorphism (rs2716610:
C>T) with higher plasma triglyceride levels (22).
In our recent study we have also tested an association of LPIN1 and PPARG variants with biochemical and anthropometrical parameters in patients with MS and type 2 diabetes. Interestingly,
results of this study showed that mutated T allele
of rs11693809: C>T polymorphism was associated with higher insulin levels in patients with MS
and type 2 diabetes (27). Neither of these studies
analyzed an association of two LPIN1 variants
(rs11693809: C>T and rs2716610: C>T) with
the biochemical parameters, including albumin,
globulin, creatinine, urea and uric acid levels, as
well as ALT, AST and GGT activity that we tested in the current study. The mechanism of an
association of LPIN1 polymorphisms with above
biochemical parameters is not completely understood. However, many previous studies demonstrated an association of selected biochemical parameters, which are known as possible markers,
with increased risk of development Type 2 diabetes (T2D) (29-33). Recently published studies
found a positive correlation between higher creatinine and urea levels with increased risk for T2D
development (32-33). Furthermore, other studies found a correlation of higher activity of liver
enzymes (AST, ALT or GGT) with increased risk
for MS and T2D development, or their association with obesity and insulin resistance (29-31).
Effects of two polymorphisms of LPIN1 gene
(rs11693809: C>T and rs2716610: C>T) on
metabolic phenotype was further tested by the
haplotype analyses. An association of rs2716610:
C>T polymorphism with the lower BMI and
waist circumference, and protective role of this
polymorphism were confirmed by this analysis.
Another analyzed polymorphism, rs11693809:
C>T, did not show any association disease-associated traits, that is also confirmed by haplotype analysis. In control subjects, carriers of CT
haplotype had significant lower insulin levels,
glucose levels and HOMA IR levels as compa-
red to the carriers of CC haplotype. Thus, these
findings suggested that non-mutated first locus,
and mutated second locus (CT haplotype) decreased metabolic risk, while non-mutated first and
second locus (CC haplotype) increased metabolic
risk. These positive findings have to be replicated
in a larger cohort of subjects.
In conclusion, the reason why we have chosen
these two SNPs of LPIN1 gene is because previous studies have shown association with metabolic
phenotype, but with opposite results, prompting
us to examine impact of these polymorphisms of
LPIN1 gene in development of metabolic syndrome in the population of Bosnia and Herzegovina. These two polymorphisms did not cover the
role of all variation of LPIN1 gene, which is a
limitation of our study.
Results of our study showed that rs2716610: C>T
decrease the risk of MS, while rs11693809: C>T
did not show any association with risk factors of
MS. An association of rs2716610: C>T polymorphism with lower BMI and waist circumference
suggest that this genetic variant of LPIN1 gene could have a protective role against development of
metabolic syndrome. Future investigations including larger cohorts of subjects, genes and larger
number of SNPs, as well as studies on other populations, are needed to substantiate these findings. Illuminating the mechanism and pathogenesis
of this complex disorder, might lead to effective
treatment, and also to predict individual’s risk of
developing of metabolic syndrome.
ACKNOWLEDGEMENTS
We greatly appreciate the technical assistance of
Mr. Nermin Kotoric, Mr. Hajrudin Mujic and Ms.
Elma Topić.
FUNDING
TRANSPARENCY DECLARATION
Competing interests: None to declare
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Povezanost varijacija LPIN1 gena s markerima metaboličkog
sindroma u populaciji Bosne i Hercegovine
Tamer Bego1, Tanja Dujić1, Barbara Mlinar2, Sabina Semiz1,4, Maja Malenica1, Besim Prnjavorac3,6,
Barbara Ostanek2, Janja Marc2, Anida Čaušević-Ramoševac5, Adlija Čaušević1
1
Katedra za biohemiju i kliničke analize, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo, Bosna i Hercegovina; 2Katedra za
kliničku biohemiju, Farmaceutski fakultet, Univerzitet u Ljubljani, Ljubljana, Slovenija; 3Opća bolnica u Tešnju, Tešanj, 4Fakultet za
inženjering i prirodne nauke, Internacionalni univerzitet u Sarajevu, Sarajevo, 5Bosnalijek d. d, Farmaceutska i hemijska industrija,
Sarajevo, 6Katedra za patofiziologiju, Farmaceutski fakultet, Univerzitet u Sarajevu, Sarajevo; Bosna i Hercegovina
SAŽETAK
Cilj Istražiti povezanost dvije varijante LPIN1 gena s glavnim karakteristikama metaboličkog sindroma
(MS) (opseg struka, indeks tjelesne mase, krvni pritisak, trigliceridi, HDL holesterol i glukoza) u populaciji Bosne i Hercegovine.
Metode Studija je uključila 43 pacijenta s metaboličkim sindromom i 43 zdrava ispitanika (kontrole) iz
Opće bolnice u Tešnju. Varijante LPN1 gena (rs11693809: C>T i rs2716610: C>T) analizirane su PCR
metodom (real time PCR).
Rezultati Kod kontrolnih ispitanika, polimorfizam LPIN1 gena, rs2716610: C>T, sa statistički značajnom razlikom bio je povezan s nižim vrijednostima indeksa tjelesne mase (ITM) (p=0.008) i opsega
struka (p=0.008). Drugi analizirani genski polimorfizam, rs11693809: C>T, pokazao je povezanost s
nižim vrijednostima HbA1c (p=0.048) u skupini pacijenata s MS-om.
Zaključak Rezultati studije sugerišu da bi rs2716610: C>T polimorfizam LPIN1 gena mogao imati
zaštitnu ulogu u razvoju metaboličkog sindroma, dok bi polimorfizam rs11693809: C>T mogao imati
ulogu u kontroli glukoze kod pacijenata s MS-om.
Ključne riječi: metabolički sindrom, LPIN1 gen, marker, varijacije gena
121
ORIGINAL ARTICLE
Alpha-lipoic acid reduces body weight and regulates
triglycerides in obese patients with diabetes mellitus
Azra Okanović1, Besim Prnjavorac2,3, Edin Jusufović4,5, Rifat Sejdinović2, 6
1
Health Centre Tešanj, 2Department for Internal and Lung Diseases of General Hospital Tešanj, 3Pharmaceutical Faculty of University
in Sarajevo, 4Polyclinic for Pulmonary Diseases of Health and Educational Institution “Dr. Mustafa Šehović” Tuzla, 5Medical School of
University in Tuzla, 6Faculty of Health Care, University in Zenica; Bosnia and Herzegovina
ABSTRACT
Aim To determine an influence of alpha-lipoic acid to reduction
of body weight and regulation of total cholesterol concentration,
triglycerides and glucose serum levels in obese patients with diabetes mellitus type 2.
Besim Prnjavorac
Department of Internal and Lung disease,
General Hospital Tešanj
Braće Pobrića 17, 74260 Tešanj, Bosnia
and Herzegovina
Phone: +387 32 650 662;
Fax: +387 32 650 605;
Methods A prospective study includes two groups of obese patients with diabetes mellitus and signs of peripheral polyneuropathia: examined group (30 patients; 15 females and 15 males), and
control group (30 patients; 12 females and 18 males). All were treated with metformin (850-1700 mg/day). Examined patients were
additionally treated with alpha-lipoic acid 600 mg/day during 20
weeks. Body mass index and concentrations of total cholesterol,
triglycerides and glucose in serum were compared before and after
the treatment.
Results The group treated with 600 mg alpha-lipoic acid lost significantly more weight, and had lower triglyceride level than the
control group. There were no significant differences in total cholesterol and glucose serum levels between the groups.
Conclusion Alpha-lipoic acid of 600 mg/day treatment have influenced weight and triglycerides loss in obese patients with diabetes
mellitus type 2. It should be considered as an important additive
therapy in obese patients with diabetes mellitus type 2.
Key words: body mass index, serum glucose, lipid status.
28 November 2014;
Revised submission:
06 July 2015;
Accepted:
09 July 2015.
doi: 10.17392/798-15
Med Glas (Zenica) 2015; 12(2): 122-127
122
Okanović et al. Alpha-lipoic acid in treatment of diabetes mellitus
INTRODUCTION
Most patients with diabetes mellitus type 2 suffer
from disorders of lipoprotein metabolism as well
as obesity (1). Diabetic dyslipoproteinemia is
characterised with increased levels of total cholesterol, low density lipoproteins (LDL) and
triglycerides, as well as decreased level of high
density lipoproteins (HDL) (1-3). Lipid metabolic changes and obesity are both strong and intensive risk factors for developing complications,
first of all microvascular ones (1,4). Therefore, it
is very important to correct properly metabolism
disorders of lipoprotein and obesity in diabetes
mellitus type 2 patients (1,3).
Alpha lipoic acid is established in many studies as
a protector against oxidative damage cells, which
is described in diabetes mellitus patients (5) and
positively influences the regulation level of glucose (6) and reduces blood lipids (total cholesterol,
LDL, and triglycerides (7). It has been shown
that that alpha-lipoic acid markedly reduces body
weight gain in rodents (8), but also in humans (911). Also, alpha-lipoic acid has shown to be effective in reducing symptoms of diabetic polyneuropathy without serious adverse effects (12,13).
The usual daily intake of alpha lipoic acid by foods (muscles, heart, kidney, liver) is quite low in
order to achieve a therapeutic effect in conditions of increased needs for this substance. (10).
Therefore, the potential health implications of
alpha lipoic acid have been investigated in clinical practice in countries such as Germany and
Korea (14) with multicentric trials currently ongoing in Europe and North America (15). These
studies have included products containing alpha
lipoic acid in a very wide range of doses, ranging
50-1800 mg/day (2,6, 9-13, 16-19).
The aim of this study was to examine the effect
of alpha lipoic acid in reducing body weight, the
regulation of lipid status, as well as the regulation
of glucose in blood in obese patients with diabetes mellitus type 2.
This prospective study has been done in Public
Health Centre Tešanj, Bosnia and Herzegovina, in
the period from May to September 2013. Sixteen
obese patients with diabetes mellitus type 2 and
signs of peripheral neuropathy were included. All
were treated with metformin (850 to 1700 mg/day)
and divided into 2 groups: examined (30; 25 females and 5 males) and control (30; 22 females and 8
males). Control patients were additionally treated
with alpha-lipoic acid of 600 mg/day during 20
weeks. Body mass index and serum concentrations of glucose, cholesterol and triglycerides were
measured and compared before and after the treatment, as well as between examined and control
group. Body mass index was calculated from body
weight and height according to the formula:
BMI (kg/m2) = Weight (kg) / Height (m)2.
Referral values of observed parameters were:
body mass index 18.50-24.99, concentration of
glucose 4.4-6.1 mmol/L, concentration of cholesterol 3.1-5.7 mmol/L and concentration of triglycerides 0.34-2.3 mmol/L.
The distribution of values was

determined by
D’Agostino test. Mean values were shown as
mean ± standard deviation. Student’s t-test,
Mann-Whitney test, Fisher’s test and χ2 test, with
double and single orientation, are used for calculating the difference between the groups.
ANOVA test was used to calculate relative differences of variance of the distribution between the
variables. Statistical hypotheses were tested at the
level of α=0.05, and the difference between the
groups was considered significant if p<0.05 or less.
RESULTS
Sex distribution was similar in both groups
(p=0.6042).
Age distribution was similar among patients
between the groups for both genders, as well as after stratification to male and female patients. Also,
age distribution was similar among male and female patients in both groups (experimental group:
p=0.1691; control group: p=0.4541) (Table 1).
Table 1. Age and sex distribution in experimental and control
group
Gender
Mean age ± standard
deviation
p
ExperiControl
Control
mental
18 (60) 64.40±1.887
61.0±1.7
0.0955
12 (40) 61.53±2.255 61.33±2.294 0.4755
30 (100) 62.97±1.469 61.13±1.347 0.1808
No (%) of patients
Experimental
Males
15 (50)
Females 15 (50)
Total
30 (100)
Before the treatment, body mass index in both
groups was similar (p>0.05).
123
Body mass index was significantly lower in experimental and control groups after the treatment
(p<0.001 and p=0.01, respectively).
Body mass index was significantly lower in the
group treated with metformin and alpha-lipoic
than in the group treated with metformin only
(p<0.05) (Figure 1).
Despite lowering of serum concentration of total
cholesterol after the treatment, differences were
not significant before and after the treatment in
any of the two groups (p>0.5 ).
Also, despite lower concentration of total cholesterol after the treatment in the group treated with
metformin and alpha-lipoic acid than in the group treated with metformin only, this difference
was not significant (p>0.05)
Before, as well as after the treatment, serum concentration of glucose was similar in both groups
(p>0.05).
After the treatment serum concentration of
glucose was significantly lower compared to
concentration before the treatment in both groups
(p<0.001) (Figure 3).
Figure 1. Body mass index in experimental and control group
before and after the treatment
Serum concentration of triglycerides was similar
in both groups before the treatment (p>0.05).
After the serum concentration of triglycerides
was significantly lower in both groups (experimental group: p<0.01; control group: p<0.05).
After the treatment, serum concentration of triglycerides was significantly lower in the group treated with metformin and alpha-lipoic than the group treated with metformin only (p<0.5) (Figure 2).
Figure 2. Serum concentration of triglycerides in experimental
and control group before and after the observed period
Before and after the treatment, serum concentration of total cholesterol was similar in both groups (p>0.5).
124
Figure 3. Serum concentration of glucose in experimental and
control group before and after the observed period
Achievement referral values of body mass index,
concentration of tryglicerides, as well as glucose, before and after the treatment were similar
(p=0.0562, p=0.0602 and p=0.5, respectively)
(Figure 4).
Figure 4. Achievement referral values (Normalisation) of body
mass index (A), serum concentration of triglycerides (B) and
serum concentration of glucose (C) after the treatment in experimental and control group.
DISCUSSION
There has been little research that would evaluate
an effect of oral treatment of humans with diabetes mellitus type 2 with alpha lipoic acid so far
(2,9,10). Limited evidence from human studies
suggests that alpha lipoic acid may be an effective body weight or lipid-lowering compound (20).
To our knowledge, this is the first study in Bosnia and Herzegovina showing that alpha-lipoic
acid treatment could lead to a significant weight
reduction and regulate lipid status in obese patients with diabetes mellitus type 2. As a dietary
supplement, alpha lipoic acid appears to have
broad molecular specificity with an impressive
array of metabolic benefits including protection
against weight gain (10), diet-induced dyslipidemia (4,7), arterial lesion formation (5,18,19), and
insulin resistance (21).
Our research included patients in experimental
and control group with similar sex and age distribution, which led to a minimization of impact of
these factors on metabolic and oxidative changes
that could be possibly linked to sex differences
or aging.
Several studies showed body weight loss and reduction of triglycerides serum concentration in
obese patients with diabetes mellitus type 2 treated with alpha lipoic acid (4,9, 10-12). But, some
of the studies showed that oral dose of 600 mg/
day of alpha lipoic acid did not influence weight
lost or normalization of lipid status (9). However,
the dose of 600 mg/day applied intravenously
led to a significant reduction in plasma free fatty
acids, triglycerides, total cholesterol, LDL-cholesterol, oxidized LDL-cholesterol, and VLDLcholesterol in obese patients treated for 2 weeks
(21). Therefore, in most studies the patients were
treated with 1200 mg/day or higher orally (2,6,
9-12, 16,18). In the randomized double-blind and
placebo-controlled study during 20 weeks 360
obese individuals (body mass index 27-30 kg/m2
plus hypertension, diabetes mellitus, or hypercholesterolemia) were randomized to alpha-lipoic acid 1200 or 1800 mg/day or placebo. Reduction in body mass index was significantly greater
in the 1800 mg/day alpha-lipoic acid group than
in the placebo group, as was the percentage of
patients who achieved a 5% reduction in baseline body weight (21.6% vs 10.0%) (9). In our
research, patients who completed 20 weeks treatment with 600 mg/day of alpha-lipoic acid
orally showed modest but significant reduction in
body mass index and serum triglycerides concentration in comparison with the patients that were
not treated with alpha lipoic acid. Hence our results are opposite to literature results with regard
to orally administrated alpha-lipoic acid dose.
However, 20 weeks treatment in our research was
longer than in other studies and this might result
in regulation of body weight and triglycerides.
At the same time, after the observed period there
were no differences in frequencies of normalized
weight or triglyceride between the group treated
with and group not treated with alpha-lipoic acid
in our research. Considering literature studies
(2,9,12,13,18) and results of our study, doses of
alpha-lipoic acid higher than 600 mg/day could
have more benefit in obese patients with diabetes
mellitus type 2. Although the maximum tolerated
dose of alpha-lipoic acid in human patients has
not been well defined, some studies have suggested that humans can tolerate several grams per
day of oral alpha-lipoic acid (9). Thus, this could
be a part of another prospective research based
on tolerance of alpha-lipoic acid.
In the majority of studies alpha-lipoic acid led
to significant reduction of serum total cholesterol concentration in obese patients (3,4, 6-9). In
our research the cholesterol concentration did
not differ after the treatment with alpha-lipoic
acid in comparison with baseline level at the
beginning of the treatment. Possible reasons for
this could be that the patients in our study were
treated with 600 mg/day, but in majority of other
studies this dose was pretty higher (9,10, 12-14,
18,21). Also considering low dose of alpha-lipoic acid, the treatment period (20 weeks) was
relatively short in our study.
Although alpha lipoic acid regulates glucose
concentration (2,6,8) in our study there were no
differences between groups in glucose concentration after the treatment with alpha-lipoic acid.
This could be explained with the fact that metformin, given in both groups, leads to a significant
reduction of glucose levels in obese patients (22),
as well as relatively low dose (600 mg/day) of
alpha-lipoic acid; probable reason is a relatively
short treatment period.
This research has several limitations. The duration of observed period (20 weeks) was relatively
short. Furthermore, outpatients were not monitored intensively with regard to individual conducting of hypocaloric diet, which was prescribed in
a similar way to all patients.
125
In conclusion, this study showed that 600 mg/day
of oral alpha-lipoic acid was effective in achieving significant regulation of weight loss and
serum triglycerides in obese patients with diabetes mellitus type 2, but without difference in
frequencies of normalization in patients with and
without alpha-lipoic acid. Alpha-lipoic acid may
be effective as an additional treatment in obese
patients with diabetes mellitus type 2, but further
studies are required to determine an adequate
dosage of alpha-lipoic acid as well as long-term
safety and efficacy.
ACKNOWLEDGEMENT
Authors would like to thank all colleagues from
Health Centre Tešanj, Bosnia and Herzegovina who
supported us and took part in collecting all necessary materials needed for this study. Also, thanks
to all patients who accepted to be part of the study.
FUNDING
Competing interests: None to declare.
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Alfa-lipoična kiselina smanjuje tjelesnu masu i regulira
koncentraciju triglicerida u gojaznih pacijenata sa šećernom
bolešću
Azra Okanović1, Besim Prnjavorac2,3, Edin Jusufović 4,5, Rifat Sejdinović 2,6
Dom zdravlja Tešanj, 2Odjeljenje za interne i plućne bolesti Opće bolnice Tešanj, 3Farmacijski fakultet Univerziteta u Sarajevu, 4Poliklinika za plućne bolesti Zdravstveno-nastavne ustanove “Dr. Mustafa Šehović” Tuzla, 5Medicinski fakultet Univerziteta u Tuzli, 6Univerzitet u
Zenici; Bosna i Hercegovina
1
SAŽETAK
Cilj Utvrditi utjecaj alfa-liponske kiseline na smanjenje tjelesne mase i regulaciju koncentracije ukupnog holesterola, triglicerida i glukoze u gojaznih osoba sa šećernom bolešću tipa 2.
Metode Prospektivno istraživanje uključilo je dvije grupe gojaznih osoba sa šećernom bolešću i znakovima periferne polineuropatije: ispitivana grupa (30 pacijenata, odnosno 15 žena i 15 muškaraca) i
kontrolna grupa (30 pacijenata, odnosno 12 žena i 18 muškaraca). Svi su bili tretirani metforminom
(850-1700 mg/dan). Ispitanici u ispitivanoj grupi bili su dodatno tretirani alfa-liponskom kiselinom,
600 mg/dan tokom 20 sedmica. Indeks tjelesne mase i koncentracije ukupnog holesterola, triglicerida i
glukoze u serumu upoređivani su prije i poslije tretmana.
Rezultati Ispitanici tretirani alfa-liponskom kiselinom, u dozi od 600 mg/dan, imali su značajniji gubitak indeksa tjelesne mase, kao i triglicerida u poređenju s kontrolnom grupom. Nije bilo značajne
razlike u koncentraciji ukupnog holesterola i glukoze u serumu između grupa.
Zaključak Tretman alfa-liponskom kiselinom, u dozi od 600 mg/dan, utječe na gubitak indeksa tjelesne mase i dovodi do snižavanja koncentracije triglicerida u gojaznih osoba sa šećernom bolešću tipa
2. Alfa-liponska kiselina treba biti uzeta u obzir kao važna dodatna terapija u gojaznih bolesnika sa
šećernom bolešću tipa 2.
Ključne riječi: indeks tjelesne mase, serumska glukoza, lipidni status
127
ORIGINAL ARTICLE
Positive correlation between uric acid and C-reactive protein
serum level in healthy individuals and patients with acute
coronary syndrome
Emina Spahić1, Sabaheta Hasić2, Emina Kiseljaković2, Halima Resić3, Mehmed Kulić4
Primary Health Center Zenica, 2Department of Medical Biochemistry, Faculty of Medicine University of Sarajevo, 3Clinic for Hemodialysis, Clinical Center University of Sarajevo, 4Cardiology Clinic, Clinical Centre University of Sarajevo; Bosnia and Herzegovina
1
ABSTRACT
Aim To assess serum levels and correlation between uric acid
(UA) and C-reactive protein (CRP) in acute coronary syndrome
(ACS) and apparently healthy individuals.
Methods The cross-sectional study included 116 examinees of
age 44 to 83 years, distributed in two groups: 80 ACS patients
including 40 with acute myocardial infarction (AMI), and 40 with
unstable angina pectoris (UAP), and 36 apparently healthy (control group) individuals. Patients with ACS were hospitalized at the
Cardiology Clinic, Clinical Centre Sarajevo in the period OctoberDecember 2012. Laboratory analyses were conducted by standard
methods. The accepted statistical significance level was p<0.05.
Emina Spahić
Primary Health Center Zenica
Fra Ivana Jukića 2, 72000 Zenica, Bosnia
and Herzegovina
Phone +387 32 403 418;
Fax; +387 32 242 113;
14 May 2015;
Revised submission:
20 June 2015;
Accepted:
29 June 2015.
doi: 10.17392/821-15
Med Glas (Zenica) 2015; 12(2): 128-132
128
Results Serum levels of CRP and UA were higher in patients with
ACS as compared to control group (p<0.01). The median serum
UA was insignificantly lower, and CRP was significantly higher
in patients with AMI compared to UAP (p=0.118 and p=0.001,
respectively). CRP and UA correlated positively in both ACS and
control groups (rho=0.246; p=0.028 and rho=0.374; p=0.027). A
positive correlation between serum CRP and UA was noted in patients with AMI, but negative in patients with UAP (p>0.05).
Conclusion The correlation between CRP and UA in the patients with ACS indicates the association of oxidative stress and
inflammation intensity in damaged cardiomyocytes. Correlation
between UA and CRP in apparently healthy individuals indicates a
possible role of UA as a marker of low-grade inflammation and its
potential in risk assessment in cardiovascular diseases.
Key words: angina, unstable, inflammation, myocardial infarction, oxidative stress
Spahić et al. Uric acid and C - reactive protein correlation
INTRODUCTION
Inflammation plays a significant role in all stages of atherosclerosis from initiation through
progression, but also in pathogenesis of acute
cardiovascular event (1). Acute inflammatory
response is a major characteristic of cardiovascular occlusive event. Significant increase of
serum C-reactive protein (CRP) occurs in inflammatory processes of different etiology, as
a part of or independently of cardiac etiology
(1,2). In healthy individuals, CRP circulates in
very low concentrations (2), and individuals
who are at the risk of development of cardiovascular diseases show systemic inflammatory
response registered by increased serum CRP
level. Apart from being a marker of inflammation, CRP contributes to development of atherosclerotic disease, and it is considered as the
most powerful predictor of myocardial infarction and stroke (3). The elevated uric acid (UA)
levels are related to arterial hypertension, systemic inflammation, and cardiovascular diseases
through endothelial dysfunction and pathological remodeling of blood vessels, but physiological UA levels act as a powerful antioxidant
(4). One of the mechanisms of elevated level
of serum UA that contributes to systemic inflammation is induction of CRP expression in
endothelial and smooth muscle cells of blood
vessels, which facilitates its increased synthesis and elevated level in circulation (5). The
CRP induces proinflammatory cytokine release
and forms terminal complement complexes in
the intima of the early atherosclerotic lesion
which contribute to plaque instability (6).
The study objective was to assess a correlation
between the serum UA and CRP levels in apparently healthy individuals and acute coronary
syndrome (ACS) patients.
EXAMINEES AND METHODS
The cross-sectional study included 116 examinees of both genders (59 males and 57 females), aged 44 to 83. Examinees were distributed into two groups: acute coronary syndrome
group (ACS), consisted of 40 acute myocardial
infarction patients (AMI) and 40 unstable angina pectoris (UAP), and group consisted of
apparently healthy individuals as control group
(CG). The patients with ACS were hospitalized
at the Intensive Care Unit of Cardiology Clinic,
Clinical Centre of Sarajevo in the period October- December 2012. Diagnosis of acute coronary syndrome was established on the basis
of electrocardiogram changes (ECG), clinical
symptoms, and elevated levels of serum cardiac troponin (cTnI). The control group included apparently healthy individuals, who were
without signs of diseases and who underwent a
routine laboratory check.
Results of biochemical analysis obtained within
48 hours of hospital admission were collected
from patients’ medical histories. Stable angina
pectoris patients and those with malignant, liver
and kidneys diseases, acute or chronic systematic inflammatory diseases, infectious or septic states, patients treated with allopurinol and
chronic alcohol consumption were excluded
from the study. The data of control group are
drawn from a database from individuals whose laboratory values were within physiological
range during routine check..
Biochemical analyses were conducted at the
Clinic of Chemistry and Biochemistry, Clinical
Centre of Sarajevo. The serum cTnI was measured by AxSYM Troponin-I ADV Immunoassay
(reference range 0-0.04 ng/mL) using AxSym
analyzer (Abbott Laboratories, Abbott Park, IL,
USA). Immunoturbidimetric method of CRP (reference range 0-5 mg/L) and spectrophotometric
method of uric acid (155-428 μmol/L) were used
for analysis on Dimension Xpand Plus (Siemens,
Munich, Germany).
The study was performed according to the principles outlined in the Declaration of Helsinki.
This investigation was approved by Faculty of
Medicine, University of Sarajevo, Bosnia and
Herzegovina.
Normality of distribution of variables analyzed
by Shapiro-Wilk test was not satisfied, so the numeric variables were presented by median with
interquartile range (25-75 percentiles). The difference between groups was analyzed by MannWithney U, non-parametric test. The degree of
correlation was examined by the test according
to Spearman. Levels p<0.05 were considered as
statistically significant.
129
RESULTS
DISCUSSION
The patients with acute coronary syndrome were
slightly older in the comparison to the control
group (p>0.05) (Table 1).
The development of atherosclerosis depends on
the balance between proinflammatory stimuli,
anti-inflammatory and anti-oxidative defense
mechanisms. The range of CRP values in blood of healthy volunteers indicates a low grade
inflammation (CRP > 3 mg/L). According to
American Heart Association, this category of
apparently healthy individuals is at the risk of
development of cardiovascular diseases (6,7).
Yamada et al. have found that in 94% of the apparently healthy individuals CRP was lower than 2
mg/L with median of 0.12 mg/L (8). In Koenig
et al. study, 55-80 % of the individuals had CRP
lower than 2 mg/L, which is considered as a level for detection of active inflammation, infection, or tissue damage (9). Investigation of serum
UA concentration and its role and importance as
an independent risk factor for the development
of cardiovascular diseases is complicated by the
fact that elevated levels of UA combine with
other factors to increase the cardiovascular risk
(10). The physiological range of uric acid concentration has been noted in healthy individuals
included in the present study. As one of the most
powerful antioxidants, uric acid eliminates free
radicals from the body, and serum UA elevation could represent a compensatory mechanism
against free radicals (10). Uric acid functions
in early stages of atherosclerosis as an antioxidant, but it becomes prooxidant in the cell due
to the increased rate of xanthine oxidase activity
(11). In prospective cohort study which included
middle- aged Finish men without cardiovascular
diseases, cancer or diabetes, serum UA levels in
the lower third of reference range were associated with greater risk of cardiovascular death than
those with concentrations of UA in upper third.
(12). Consistent with Jalal at al. study (13), we
observed a positive association between CRP and
UA in the control group. De Carvalho Vidigal
study suggested that uric acid is suitable biochemical indicator for detecting changes in hs-CRP
and can also predict higher C-reactive protein
levels in apparently healthy men improving the
assessment of cardiovascular risk (14).
Table 1. Demographic characteristics of patients with acute
coronary syndrome and controls
Variables
CG (n=36) ACS (n=80) AMI (n=40) UAP (n=40)
Age (years)
64 (44-81)* 67 (50-83) 66 (53-81) 67 (50-83)
No (%) of males 17 (47.2)
40 (50)
20 (50)
20 (50)
*NS (not significant) compared to ACS patients (p>0.05) CG, control
group; ACS, acute coronary syndrome; AMI, acute myocardial
infarction; UAP, unstable angina pectoris;
Median level of serum cTnI in the ACS patients
was 0.33 (0.06-18.84) ng/mL and AMI patients
had significantly higher cTnI level in the comparison to UAP patients (p<0.01). Levels of CRP
and UA were significantly higher in the ACS
compared to the control group (p<0.01). Levels
of CRP in AMI patients were higher in comparison to UAP group (p=0.001), but UA was lower
compared to UAP (p=0.118) (Table 2).
Table 2. Blood levels of cardiac troponin I, C-reactive protein
and uric acid in acute coronary syndrome patients and controls
Patient’s group
ACS
CG
Variables
cTnI (ng/mL) CRP (mg/L)
0.33
21.70
(0.06-18.84) (10.40-63.42)*
1.90
/
(1.0-4.8)
AMI
16.22
(4.38-29.97)†
UAP
0.06
(0.027-0.12)
UA (µmol/L)
364
(312-437.75)*
263
(216-320)
357.50
47.5O
(323.25(15.60-74.75)†
498.25)‡
13.35
379
(8.02-31.0) (290.75-414.50)
*p<0.01 between ACS and control group; †p<0.01 between AMI and
UAP groups; ‡ no significant difference between and AMI and UAP
groups; ACS, acute coronary syndrome; CG, control group; AMI,
acute myocardial infarction; UAP, unstable angina pectoris; cTnI,
cardiac troponin I; CRP, C reactive protein; UA, serum uric acid
A moderate positive relationship between serum
CRP and UA levels (rho=0.374; p=0.027) was
observed in the CG patients (Table 3), while weaker relationship between the two observed parameters was noticed in ACS group (rho=0.246,
p=0.028). Although the association between CRP
and UA was positive in AMI and negative in UAP
group, both of them were no significant (p>0.05).
Table 3. Spearman’s correlation of serum C-reactive protein
and uric acid in patients with acute coronary syndrome and
in control group
Variables
CRP / UA
rho
p
Patient groups
CG
ACS
AMI
0.374
0.246
0.244
0.027*
0.028*
0.129
UAP
- 0.150
0.355
*p<0.05; CRP, C-reactive protein; UA, serum uric acid; CG, control
group; ACS, acute coronary syndrome; AMI, acute myocardial
infarction; UAP, unstable angina pectoris; rho-Spearman correlation
coefficient;
130
Serum levels of CRP in the present study were
significantly higher in ACS subjects compared to
the controls, which is in accordance with Baruah
et al. and Kushner et al. studies (15,16). Myocar-
Spahić et al. Uric acid and C - reactive protein correlation
dial necrosis leads to inflammatory response,
cytokines activation and consequential increase
of CRP synthesis. Binding of CRP to necrotic
myocardial cells and consecutive complement
activation is considered responsible for a further
myocardial necrosis expansion (17).
Higher level of serum CRP concentration in the
patients with AMI indicates greater myocardial
inflammatory response as the consequence of
more severe myocardial lesion in AMI than in
UAP patients (18). In Munir et al. study, significant increase of serum CRP within 12 to 24 hours
has been shown in patients with UAP, myocardial infarction without elevation of ST-segment
(NSTEMI), and myocardial infarction with ST
elevation (STEMI) (19). Besides the CRP localization in atherosclerotic lesions, it localizes also
in ischemic myocardium and promotes complement activation (20). Results of this study have
shown that levels of UA in ACS group were statistically significantly higher in comparison with
the control group, and insignificantly lower in the
AMI group compared to UAP (p=0.118). The similar results were published in Gur et al. study
who found increased UA values in AMI and UAP
patients compared with controls, but uric acid level was not in relation to the severity of coronary
artery disease (21). Higher levels of uric acid in-
dicate greater intensity of oxidative stress in ACS
than in control group. Under local ischemia condition and increased synthesis of oxygen radicals,
UA becomes prooxidant (22-24).
We have notified that levels of CRP and UA positively correlated in ACS patients. In Baruah et
al. study, the similar kinetic of CRP and UA have
been registered in post-infarction period. Both
markers peak on the 3rd day, and then return to
baseline level (15). In the present study a positive
correlation between CRP and UA in the serum
was established in AMI, and a negative correlation in UAP group, but it was not statistically significant. We consider that a positive correlation
between uric acid and C-reactive protein in serum
of healthy individuals indicates the importance of
serum levels of uric acid in monitoring the intensity of low-grade inflammation. The correlation
between the two observed parameters in acute
coronary syndrome indicates correlation between
intensity of oxidative stress and inflammation in
myocardial tissue.
FUNDING
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9. Koenig W, Sund M, Frohlich M, Fischer HG, Löwel
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12. Niskanen LK, Laaksonen DE, Nyyssonen K, Alfthan
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13. Jalal D, Jablonski K, McFann K, Choncholand M,
Seals D. Vascular Endothelial Function Is Not Related to Serum Uric Acid in Healthy Adults. Am J
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14. Vidigal F, Rosado L, Rosado GP, Ribeiro C. The
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T, Sukiennik A, Navarese EP, Odrowaz-Sypniewska
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Pozitivna korelacija između mokraćne kiseline i C-reaktivnog proteina u
serumu zdravih osoba i pacijenata s akutnim koronarnim sindromom
Emina Spahić1, Sabaheta Hasić2, Emina Kiseljaković2, Halima Resić 3, Mehmed Kulić4
1
Dom zdravlja Zenica, 2Katedra za medicinsku biohemiju, Medicinski fakultet, Univerzitet u Sarajevu, 3Klinika za hemodijalizu, 4Klinika za
srce i reumatizam; Klinički centar Univerziteta u Sarajevu; Bosna i Hercegovina
SAŽETAK
Cilj Ispitati serumske vrijednosti i odnos između mokraćne kiseline (MK) i C-reaktivnog proteina
(CRP) kod naizgled zdravih osoba i kod pacijenata s akutnim koronarnim sindromom (AKS).
Metode U presječnu studiju bilo je uključeno 116 ispitanika, starosne dobi između 44 i 83 godine,
distribuiranih u dvije grupe: 80 ispitanika s AKS-om i to 40 s akutnim infarktom miokarda (AIM) i 40
s nestabilnom anginom pektoris (NAP), te 36 naizgled zdravih ispitanika (kontrolna grupa, KG). Ispitanici s AKS-om bili su hospitalizirani na Klinici za bolesti srca i reumatizam Univerzitetskog kliničkog
centra u Sarajevu u periodu od oktobra do decembra 2012. godine. Laboratorijske analize su urađene
korištenjem standardnih metoda. Prihvaćeni nivo statističke značajnosti iznosio je p<0.05.
Rezultati Vrijednosti CRP-a i MK-a u serumu bile su više u ispitanika s AKS-om u odnosu na KG
(p<0.01). Vrijednost MK-a u serumu nije bila značajno niža, dok je vrijednost CRP-a bila značajno
viša kod pacijenata s AIM-om u poređenju s pacijenatima s nestabilnom anginom pektoris (p=0.118;
p=0.001). Pozitivna, značajna korelacija je utvrđena između serumskih vrijednosti CRP-a i MK-a i kod
ispitanika s AKS-om i kod KG-a. (rho=0.246; p=0.028 i rho=0.374; p=0.027). Utvrđen je pozitivan odnos CRP-a i MK-a u serumu ispitanika s AIM-om, a negativan u NAP-u, ali nije bio statistički značajan
(p>0.05).
Zaključak Odnos CRP-a i MK-e u serumu oboljelih od AKS-a ukazuje na povezanost intenziteta oksidativnog stresa s intenzitetom inflamacije zahvaćenog dijela kardiomiocita. Povezanost vrijednosti
MK-a i CRP-a u serumu naizgled zdravih ispitanika ukazuje na moguću ulogu MK-a kao markera
intenziteta hronične inflamacije niskog stepena i ima potencijal u procjeni rizika za razvoj kardiovaskularnih bolesti.
132
ORIGINAL ARTICLE
Role of echocardiography in diagnosis and management of
complete papillary muscle rupture caused by myocardial
infarction
Josip Vincelj1,2,3, Stanko Biočić1, Mario Udovičić1, Mario Sičaja1, Sandra Jakšić Jurinjak1
Institute of Cardiovascular Diseases, Dubrava University Hospital, Zagreb, 2University of Applied Health Studies; Zagreb, 3School of
Medicine, University J. J. Strossmayer, Osijek; Croatia
1
ABSTRACT
Aim To evaluate the usefulness of echocardiography in the diagnosis of complete rupture of papillary muscle.
Methods Transthoracic (TTE) and transesophageal echocardiography (TEE) was performed with the ATL 3000 HDI Ultrasound
Inc (Bothell, WA, USA) with a 2.5 MHz transducer and 5-7 MHz
multiplane phased array transducer. We are reporting about two
patients (a 45 and a 51-year old male) with complete ruptures of
papillary muscle following acute myocardial infarction (AMI).
Josip Vincelj
Dubrava University Hospital,
Institute of Cardiovascular Diseases
Av. G. Šuška 6, 10000 Zagreb, Croatia
Phone:+ 385 1 2902 444;
Fax: +385 12863695;
Results Both patients were previously treated with fibrinolysis in
their local hospitals, 400 and 300 km, respectively, away from our
hospital. Massive mitral regurgitation developed in both followed
by rapid deterioration of hemodynamic state and severe heart failure, because of which both were transferred by helicopter to the
Coronary Care Unit of our clinic. The diagnosis of complete papillary muscle rupture was confirmed in both patients by TTE and
TEE. Due to the significant deterioration in their hemodynamic
state, vasoactive drugs and intra-aortic balloon pump support were
applied. Both patients then underwent mitral valve replacement,
accompanied by concomitant coronary artery bypass grafting in
one case.
Conclusion Transesophageal echocardiography is a more accurate
and rapid diagnostic method in patients with mechanical complications of AMI than TTE.
19 November 2014;
Keywords: mitral valve, replacement, coronary artery, bypass
grafting
Revised submission:
15 January 2015;
Accepted:
27 April 2015.
doi: 10.17392/793-15
Med Glas (Zenica) 2015; 12(2): 133-138
133
INTRODUCTION
Papillary muscle rupture is a rare, but often fatal mechanical complication of acute myocardial infarction (AMI); it occurs in 1% to 5%
of patients with AMI (1). Echocardiography is
the imaging technique of choice for detecting
mechanical complications of AMI including
myocardial free wall rupture, acute ventricular
septal defect and mitral regurgitation secondary
to papillary muscle rupture or ischemia (2,3).
Transesophageal echocardiography is more
sensitive than TTE, and useful for providing
more detailed and/or unique anatomic information in patients with papillary muscle rupture
than TTE (3,4).
Minami et al (5) have reported on six patients
(from 1986 to 2002) with posterior (n=4) and
anterior (n=2) papillary muscle rupture; all patients underwent mitral valve replacement, concomitant coronary artery bypass grafting (CABG)
was performed in five of six patients, and the
perioperative mortality rate was 33%. Tavakoli
et al (6) reviewed 21 consecutive patients (from
1988 to 1998) with a perioperative mortality of
19%. Transthoracic (TTE) and/or transesophageal echocardiography (TEE) established the diagnosis of papillary muscle rupture in 14 patients,
and in others the diagnosis was suspected on the
basis of the presence of flail mitral leaflets (3).
Emergency surgery, even as a salvage procedure
for acute postinfarction mitral papillary muscle
rupture is justified (6).
The aim of this study was to evaluate the usefulness of echocardiography in the diagnosis of
complete rupture of papillary muscle, based on
a presentation of two male patients with massive mitral regurgitation and severe heart failure
due to complete mitral papillary muscle rupture
following acute myocardial infarction.
Dubrava University Hospital provides acute care
for about 220000 inhabitants from the east Zagreb
area including Sesvete, DugoSelo, Vrbovec and
Sv. Ivan Zelina. Our hospital is also one of the
primary percutaneous coronary intervention(PCI)
centers included in primary-PCI network of Croatia which provides care for patients with acute
coronary syndrome for Međimurje County, Ko-
134
privnica - Križevci County, Bjelovar - Bilogora
County and east part of the Zagreb County. About
750 patients with acute coronary syndrome are admitted to our hospital per year.
Diagnosis of AMI included clinical symptoms,
electrocardiogram, cardiac biomarkers (troponin I, normal limit is 0.04 ug/mL), echocardiography and coronary angiography. Definite
diagnosis of AMI requires cardiac biomarker
troponin with at least one value above the 99th
percentile of the upper reference limit and with
at least one of the following: symptoms of ischemia, new or presumably new significant
ST-T changes or new left bundle branch block
(LBBB), development of pathologic Q waves
in the ECG, imaging evidence of new loss of
viable myocardium and identification of an intracoronary thrombus by angiography or autopsy (2). The diagnosis of heart failure was based
on typical symptoms, typical signs and relevant
structural heart disease. Patients’ symptomatic
limitation is graded using the New York Heart
Association (NYHA) functional classification
(class I - IV). This classification assigns patients
to one of four class depending on the degree
of effort needed to elicit symptoms of angina,
fatigue, dyspnea or palpitation. Patients with
NYHA IV class presenting with symptoms at
rest and unable to carry out any physical activity without discomfort.
Transthoracic (TTE) and transesophageal
echocardiography (TEE) was performed with
the ATL 3000 HDI Ultrasound Inc (Bothell,
WA, USA) with a 2.5 MHz transducer and 5-7
MHz multiplane phased array transducer.The
presence and grade of mitral regurgitation (MR)
were screened by color-flow imaging with the
MR jet area to left atrium area ratio; a ratio of
>20%, 20-40% and >40% represents mild, moderate and severe MR, respectively. In patients
with more than trace regurgitation, the regurgitant orifice area was calculated by the proximal
isovelocity surface area (PISA) method and the
degree of MR was graded as mild (regurgitant
orifice area <0.2cm2 ), moderate (regurgitant
orifice area 0.2 to 0.4 cm2 ), or severe (regurgitant orifice area >0.4cm2 ). Classically a vena
contracta width <3mm indicates mild MR, whereas a vena contracta width ≥7 mm indicates severe MR (7).
Vincelj et al. Echocardiography and papillary muscle rupture
RESULTS
Case 1
A 45-year-old man was first admitted to a local hospital for chest pain, faintness, dyspnea and waning
exercise tolerance. This hospital is approximately
400 km away from our clinic. Treatment consisted
of fibrinolytic therapy with streptokinase, along
with other standard therapeutic regimens. Transthoracic echocardiography showed mitral regurgitation (MR) grade 3, and in spite of aggressive
therapy, the patient began developing severe hemodynamic instability. After a week-long clinical
deterioration he was transferred to the coronary
care unit (CCU) of our clinic. He presented with
clinical signs of severe heart failure,NYHAclass
IV, while physical examination revealed bilateral
basal pulmonary rales without jugular venous distension. On careful auscultation soft heart sounds
and a holosystolic heart murmur with a point of
maximum intensity at the apex grade 4/6 were
detected. Blood pressure was 140/80 mm Hg, accompanied by tachycardia 133 beats/min. Electrocardiogram (ECG) revealed sinus tachycardia of
133 beats/min and Q-waves of the left ventricular
inferoposterior wall. Chest radiography disclosed
cardiac enlargement on the left side and pulmonary
plethora. In laboratory findings there was increased
aspartate aminotransferase 55 U/L, alanine aminotransferase 51 U/L, lactate dehydrogenase 765
U/L, uric acid 698 U/L, total plasma cholesterol
6.09 mmol/L, LDL-cholesterol 4.36 mmol/L, and
fibrinogen 7.8 g/L. Transthoracic echocardiography and TEE discovered mitral regurgitation grade
4. There was a mobile solid mass of 1.6x0.6 cm
in size on the posterior mitral leaflet, of the same
echo-dense structure as the myocardium and suggestive of complete rupture of the head of the posteromedial papillary muscle. The ruptured head of
the papillary muscle was detected in the left ventricle during diastole accompanied by its systolic
displacement displaced to the left atrium during
systole (Figure 1). Echocardiography showed
hypokinesis of the left ventricular inferoposterior
wall with normal ejection fraction (LVEF=65%).
Ventriculography confirmed the diagnosis of severe MR with normal left ventricular function.
Coronary angiography detected significant stenosis of the circumflex, the first obtuse marginal and
of the right coronary artery, as well as occlusion
Figure 1. Transesophageal echocardiogram showing complete
rupture of the head of posteromedial papillary muscle prolapsing into the left atrium (arrow) (Vincelj J, 2004)
LA, left atrium; LV, left ventricle; AO, aorta.
of the second obtuse marginal artery. Due to the
significant fluctuations in the hemodynamic state
of the patient, vasoactive drugs and the intra-aortic
balloon pump (IABP) were applied. Ten days after
AMI, the patient underwent mitral valve replacement with a Carbomedics mechanical valve and
concomitant coronary artery bypass grafting of the
circumflex artery. During surgery an intraoperative TEE also confirmed the diagnosis of complete
posteromedial papillary muscle rupture (Figure 2).
The patient was weaned from cardiopulmonary
bypass without hemodynamic disturbances and 24
hours later he was also successfully weaned from
both mechanical ventilation and IABP. Transthoracic echocardiography performed in the early
postoperative course diclosed normal function of
mechanical mitral valve and normal left ventricular function. Histopathological analysis found
ischemic necrosis of the posteromedial papillary
muscle and fibroelastic thickness of the endocardium. A part of the mitral leaflet showed fibroelastic thickness to some extent. Microbiology results
showed a sterile papillary muscle and leaflet.
Figure 2. Specimen of the resected ruptured posteromedial
papillary muscle (Vincelj J, 2004)
135
Case 2
A 51-year-old man was treated for acute ST-segment elevation myocardial infarction (STEMI)
of inferior localization in his local hospital, 300
km away from our clinic. Treatment consisted of
fibrinolytic therapy with streptokinase, along with
other standard therapy. During the first week of
hospitalization, acute heart failure emerged and
the patient started exhibiting signs of rapid hemodynamic deterioration. On day 12, he was transferred to the CCU of our hospital. He presented
with clinical signs of severe heart failure, NYHA
class IV. Physical examination showed bilateral
basal pulmonary rales without jugular venous distension. Careful auscultation revealed calm heart
sounds and holosystolic heart murmur grade 3/6.
Blood pressure was 105/65 mm Hg, accompanied
by sinus tachycardia 120 beats/min. ECG revealed
sinus tachycardia of 120 beats/min and Q-waves
of the left ventricular inferoposterior wall. Chest
radiography disclosed cardiac enlargement on the
left side and pulmonary plethora. His routine laboratorytest revealed increased creatine kinase 254
U/L, aspartate aminotransferase 36 U/L, alanine
aminotransferase 74 U/L, lactate dehydrogenase
775 U/L, uric acid 452 U/L, LDL-cholesterol 3.74
mmol/L, and fibrinogen 10.4 g/L. Transesophageal
echocardiography exposed a completely ruptured
head of the anterolateral papillary muscle prolapsing into the left atrium with massive MR. There
was a display of chaotic movement of mitral valve. Excessive amount of pericardial effusion (20
mm) was seen, along with a collapse of the right
atrium in telediastole, as sign of impending tamponade (Figure 3). Coronary angiography detected
Figure 3. Transesophageal echocardiogram showing complete
rupture of the head of anterolateral papillary muscle (P) prolapsing into the left atrium (Vincelj J, 2004)
LA, left atrium; LV, left ventricle; RA, right atrium; PE, pericardial
effusion.
136
a subtotal stenosis of first obtuse marginal artery
along with a non-significant ostial stenosis of hypoplastic right coronary artery. The patient was referred for urgent cardiac surgery. Being unsuitable
for surgical revascularization, he only underwent
mitral valve replacement with a Carbomedics
mechanical valve 24 days after AMI.An intraoperative TEE was also performed. Postoperative
course of the patient (with known prior medical
history of obstructive lung disease) was complicated by a respiratory infection and two episodes
of bronchospasm, which were both successfully
treated. Rapid postoperative improvement of cardiac function and regression of congestion in pulmonary circulation ensued, and echocardiography
performed during postoperative course revealed
normal function of the mechanical mitral valve
and no impairment of left ventricular systolic function. Histopathological analysis showed ischemic
necrosis of the anterolateral papillary muscle. The
patient was discharged, fully recovered, 14 days
after surgery. Both patients fully recovered and
were discharged from hospital 8 and 14 days, respectively, after surgery.
DISCUSSION
Papillary muscle rupture is a rare but often fatal mechanical complication of AMI with hemodynamic instability (4,8). The involvement
of the posteromedial papillary muscle is 6-12
times more common than that of the anterolateral (4,8). Indeed, posteromedial papillary muscle
vascularization is provided only by the interventricular posterior coronary artery originating
even from the right coronary artery or from the
circumflex coronary artery, and it may aggravate
infarction heralded by occlusion in such vessels
(8). Echocardiography is the imaging technique
of choice for detecting mechanical complications of AMI including myocardial free wall
rupture, acute ventricular septal defect, and mitral regurgitation secondary to papillary muscle
rupture or ischemia (2). Transthoracic echocardiography is able to identify a papillary muscle
rupture with a diagnostic sensitivity of 65-85%.
Transesophageal echocardiography is more sensitive than TTE (4,9). Recently, TEE has been
reported as a valuable adjunct to conventional
echocardiography, by providing more detailed
and/or unique anatomic information in patients
Vincelj et al. Echocardiography and papillary muscle rupture
with papillary muscle rupture involving either
mitral or tricuspid valve apparatus, to either ischemic or traumatic damage.When the head of
the papillary muscle is ruptured, it can often be
imaged prolapsing into the left atrium. In up to
30% of patients, however, the ruptured head may
not prolapse into the left atrium. In this case the
diagnosis is made by noting chaotic movement
of the ruptured head in the left ventricle (10).
Complete papillary muscle rupture may cause
acute MR with pulmonary edema or cardiogenic
shock (8). These events depend on the infarction
severity and papillary muscle morphology. There
are morphology differences in papillary muscle
attachment to the left ventricular wall. The main
part of the posteromedial papillary muscle may
be attached to the left ventricular wall with one,
two or three heads, whereby the papillary muscle
heads maybe attached separately or jointly (3).
Furthermore, the cordae tendineae of one papillary muscle may be attached to both mitral leaflets. In papillary muscle rupture, the grade of
mitral regurgitation depends on rupture severity
(complete or incomplete) and number of papillary muscle heads. The regurgitation jet will be
smaller if the papillary muscle has two or three
heads, only one of them being involved by rupture. Severe mitral regurgitation develops upon
complete rupture of a papillary muscle which has
only one or two heads, or complete rupture of all
three muscle heads, depending on differences in
the muscle morphology (11). In our patients, the
posteromedial and anterolateral papillary muscle
was attached to the muscle wall with one head,
while chordae tendineae were attached to the anterolateral leaflet, thus additionally contributing
to MR. In this case mitral valve repair was impossible for two reasons. First, myocardial necrosis caused complete posteromedial papillary
muscle rupture; and second, chordae tendineae of
the posteromedial papillary muscle were in part
attached to the anterolateral leaflet (A2 scallop)
thus also participating in severe MR.
Our patients have some distinctive features related to prior published data. Distinctive features of
our cases are that both patients were being treated
for AMI in hospitals 400 and 300 km away from
our hospital. Initially, in both cases the diagnosis of papillary muscle rupture was suspected,
and as rapid hemodynamic destabilization became evident, both patients were transported, as
safely and as fast as possible. Optimal cooperation and coordination were crucial for successful
outcome. This relates to interregional and regional cooperation, but also to collaborative work
between cardiologists and cardiac surgeons, so
called “hybrid strategy”, which included early reperfusion, when possible, and subsequent mitral
valve surgery (12).
Fibrinolytic therapy has positive effect on mechanical complications in AMI.The study of Gueret
et al. demonstrated a dramatic reduction in the
incidence of mechanical complications of AMI in
the reperfusion era when compared with the non
reperfusion era. Results of study documented beneficial effect of thrombolysis on some of these
complications, whereas similar information after
primary coronary angioplasty is scarce (13).
Transesophageal echocardiography as an accurate and rapid diagnostic method is highly important in patients with papillary muscle rupture in
AMI. The role of coronary angiography is mandatory before making any therapeutic decision.
Surgery, which should be ensured without delay,
was performed after three days of treatment, with
a satisfactory result. In our patients, urgent transportation, accurate and rapid diagnostics, hemodynamic stabilization and timely surgery were
all crucial for successful outcome.
FUNDING
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Minami H, Mukohara N, Obo H, Yoshida M, Nakagiri K, Hanada T, Maruo A, Matsuhisa H, Morimoto
N, Shida T. Papillary muscle rupture following acute
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2004;52:367-71.
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Turina M.Surgical management of acute mitral valve
regurgitation due to post-infarction papillary muscle
rupture. J Heart Valve Dis 2002;11:22-6.
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Connery C, Hochman JS.Cardiogenic shock due to
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Uloga ehokardiografije u dijagnostici i zbrinjavanju kompletne
rupture papilarnog mišića uzrokovane infarktom miokarda
Josip Vincelj1,2,3, Stanko Biočić1, Mario Udovičić1, Mario Sičaja1, Sandra Jakšić Jurinjak1
Zavod za bolesti srca i krvnih žila, Klinička bolnica Dubrava Zagreb, 2Zdravstveno veleučilište; Zagreb, 3Sveučilište J. J. Strossmayera,
Osijek, Medicinski fakultet, Osijek; Hrvatska
1
SAŽETAK
Cilj Procijeniti korisnost ehokardiografije u dijagnostici kompletne rupture papilarnog mišića.
Metode Za transtorakalnu (TTE) i transezofagusnu ehokardiografiju (TEE) korišten je aparat ATL
3000 HDI Ultrasound Inc. (Bothell, WA, USA), sondama od 2,5 MHz i od 5 do 7 MHz. Opisana su dva
muška bolesnika u dobi 45 i 51 godine s kompletnom rupturom papilarnog mišića u akutnom infarktu
miokarda (AIM).
Rezultati Oba bolesnika liječena su trombolitičkom terapijom zbog AIM-a u dvije lokalne bolnice udaljene 400, odnosno 300 km. Zbog masivne mitralne regurgitacije koja je uzrokovala hemodinamsku nestabilnost i zatajivanje srca teškog stupnja bolesnici su helikopterom transportirani u koronarnu jedinicu
naše Klinike. Dijagnozu kompletne rupture papilarnog mišića potvrdili smo u oba bolesnika pomoću
TTE-a i TEE-a. Zbog teškog općeg stanja i hemodinamske nestabilnosti bolesnici su primali vazoaktivu
terapiju i priključeni su na intraaortalnu balon-crpku. U oba bolesnika implantirana je umjetna mitralna
valvula, a aortokoronarno premoštenje učinjeno je u jednog bolesnika.
Zaključak Transezofagusna ehokardiografija u odnosu na TTE je točna i brza dijagnostička metoda u
bolesnika s mehaničkim komplikacijama u akutnom infarktu miokarda.
Ključne riječi: mitralna valvula, implantacija, koronarne arterije, premoštenje
138
ORIGINAL ARTICLE
Impact of reperfusion therapy and infarct localization on frequency
of premature ventricular beats in acute myocardial infarction
Davor Horvat1, Josip Vincelj2,3,4
Internal Disease Service, General Hospital Karlovac, Karlovac, 2Institute of Cardiovascular Diseases, Dubrava University Hospital,
Zagreb, 3 University of Applied Health Studies, Zagreb, 4School of Medicine Osijek, University J.J. Strossmayer Osijek; Croatia
1
ABSTRACT
Aim To determine the impact of infarct localization and types of
reperfusion therapy on the frequency of ventricular premature beats (VPBs) in patients with acute myocardial infarction (AMI) and
reduced left ventricular ejection fraction (LVEF).
Methods A total of 705 patients with acute ST elevation myocardial infarction (STEMI) were divided according to the infarct
localization (anteroseptal, anterolateral, inferior and posterior)
and reperfusion therapy (fibrinolysis or percutaneous coronary
intervention with stenting) into two groups: LVEF<45% was an
experimental group and LVEF>45% was a control group. The
occurrence of VPBs<10 per hour was defined as a non-significant,
and the occurrence of VPBs>10 per hour defined as a significant.
Davor Horvat
Department of Cardiology, General
Hospital Karlovac
Andrija Štampar 3, 47000 Karlovac,
Croatia
Phone: +385 47 608 007;
Fax: +385 47 431 337;
Results In patients with fibrinolysis therapy and LVEF<45% significant number of VPBs were in anteroseptal (p=0.017), anterolateral (p<0.001) and posterior AMI (p<0.001), but in patients with
percutaneous coronary intervention (PCI) and LVEF<45% significant number of VPBs were only in anteroseptal AMI (p=0.001)
localization.
Conclusion In patients with reduced ejection fraction in AMI, treatment with PCI method has a better antiarrhythmic effect compared to fibrinolysis treatment.
Key words: fibrinolysis, percutaneous coronary
ventricular arrhythmias
intervention,
11 May 2015;
Revised submission:
09 June 2015;
Accepted:
01 July 2015.
doi: 10.17392/820-15
Med Glas (Zenica) 2015; 12(2): 139-143
139
INTRODUCTION
Ventricular premature beats (VPB) represent a
premature ventricular contraction (1). The association between VPBs and poor patient prognosis has been well documented (2). Ventricular
premature beats in the early phase of an acute
myocardial infarction (AMI) are not considered
in the high mortality rate (3,4). However, after
the first 48 hours, VPBs are a problem with longterm prognostic significance (5).
The background might be an unsynchronized
myocardial repolarization on the grounds of ischemia, lesion or myocardial necrosis (6). Patients
with frequent premature ventricular contractions or
certain patterns of premature ventricular contractions may be at increased risk of developing heart rhythm problems (arrhythmias) or weakening
of the heart muscle (cardiomyopathy) (7-9). The
difference in conduction velocity between injured and uninjured tissue can trigger re-entry or a
feedback loop that is believed to be the cause of
many lethal arrhythmias (10). The most serious of
arrhythmias is ventricular fibrillation, an extremely
fast and chaotic heart rhythm that is the leading
cause of sudden cardiac death. Another life-threatening arrhythmia is ventricular tachycardia, which
can cause sudden cardiac death. Rarely, when
accompanied by underlying heart disease, frequent
premature contractions can lead to chaotic, dangerous heart rhythms and possibly sudden cardiac
death (11,12). Post-AMI VPBs, particularly if
frequent (more than 10 per hour) or complex (repetitive forms, primarily nonsustained ventricular
tachycardia), appear to be associated with a worse
prognosis in patients with a prior AMI (10).
The relationship among reduced left ventricular
ejection fraction (LVEF) and the size of affected
myocardial area might be also important (13).
Therefore, reperfusion therapy as a percutaneous
coronary intervention (PCI) or fibrinolytic therapy
should be attempted as soon as possible (14).
The impact of AMI localization on VPBs is also
unclear. In general, large studies analyzed the
occurrence of VPBs in the post infarction period
by anterior or inferior localization but, the results
are contradictory. Bluazite had more VPBs in inferior (15), while Stone concluded that VPBs are
more frequent in anterior AMI (16) localization.
Our aim was to determine the impact of AMI loca-
140
lization and type of reperfusion therapy on the
frequency of VPBs in patients with reduced LVEF.
Patients
This retrospective research was conducted from
the 2000 to 2012. Among the total of 705 patients with acute ST elevation myocardial infarction (STEMI) (the average age 62.5 years), 484
(68.7%) were males and 221 (31.3%) were females. They were hospitalized in the Coronary Unit
of the General Hospital in Karlovac, Croatia.
Methods
All patients were divided according to the reperfusion therapy to patients with fibrinolysis
and the PCI. According to the AMI localization,
the patients were divided into four groups (anteroseptal, anterolateral, inferior and posterior
localization). Because LVEF<50% represents
a reduced value of LVEF, we established the
experimental group of patients with LVEF<45%
and the control group with better LVEF (>45%).
According to the number of VPBs, we established the group with less VPBs (<10 per hour) and
the group with more VPBs (>10 per hour).
The exclusion criteria were cardiomyopathy, left
ventricular hypertrophy, hyperkalemia, hyperthyroidism, mitral valve prolapse, digitalis therapy, history of VPBs, and previous myocardial
infarctions.
The AMI diagnostic criteria were chest pain (the
first six hours). The ECG diagnosis of the ST segment elevation type of acute myocardial infarction require at least 1 mm (0.1 mV). These elevations must be present in anatomically contiguous
leads (I, aVL, V3-V6 correspond to the anterolateral wall; V1-V4 correspond to the anteroseptal
wall; II, III, aVF correspond to the inferior wall
and ST-denivelation in V3-4 or R-wave enlargement in V1-V3 leads for posterior AMI). The
cardiac enzyme troponin I was >1.00 ug/L.
Streptokinase was used as a fibrinolytic agent,
and the dose was 1.5 million international units.
Each patient was treated with beta-blockers,
acetylsalicylic acid (ASA), angiotensin converting enzyme inhibitor, statin and in the PCI method with clopidogrel. The PCI treatment had to
be with stenting.
Horvat et al. VPBs in myocardial infarction
A coronary angiogram in the PCI treatment had
the criteria of post angioplasty blood flow of the
TIMI grade III (17). Ventricular premature beats
had been verified between the sixth and tenth day
of hospitalization with a 24 hour Holter ECG
monitoring. Left ventricular ejection fraction was
assessed with the heart ultrasound and the Simpson biplane technique in the apical projection.
RESULTS
The study evaluated a total of 705 patients. The
patients’ average age was 62.4 years, 484 (68.7%)
were males and 221 (31.3%) were females.
A significant number of VPBs were identified in
194 (27.4%) patients. A total of 155 (22.0%) patients had the LVEF<45%. A total of 166 (23.5%)
patients had anteroseptal, 106 (15.0%) anterolateral, 159 (22.6%) inferior and 274 (38.9%) posterior infarct localization (Table 1).
Table 1. Distribution of myocardial infarction localization
Localization
N (%) of patients
Anteroseptal
Anterolateral
Inferior
Posterior
Total
166 (23.5)
106 (15.0)
159 (22.6)
274 (38.9)
705 (100)
There were 522 patients with fibrinolysis and 183
patients with PCI treatments (Table 2).
Table 2. Distribution of reperfusion therapy
Reperfusion therapy
Fibrinolysis
Percutaneous coronary intervention
Total
N (%) of patients
522 (74)
183 (26)
705 (100)
In the fibrinolysis and LVEF<45% group, a significant number of VPBs were in anteroseptal
(p=0.017), anterolateral (p<0.001) and posterior
localization (p<0.001) (Table 3).
Table 3. Myocardial infarction and ventricular premature
beats in fibrinolysis
No (%) of patients
Localization Ventricular
with left ventricular
of myocardial premature
95% CI
p
ejection fraction
infarction
beat/h
<45%
>45%
<10
28 (5.4) 72 (13.8) 50.57-54.91
Anteroseptal
>10
18 (3.4) 18 (3.4) 44.44-52.62 0.017
<10
14 (2.7) 52 (10.0) 48.28-52.78
Anterolateral
>10
16 (3.1) 8 (1.5) 38.56-47.27 <0.001
<10
12 (2.3) 88 (16.9) 56.38-59.94
Inferior
>10
8 (1.5) 22 (4.2) 49.64-57.89 0.080
<10
10 (1.9) 120 (23.0) 54.63-57.66
Posterior
>10
14 (2.7) 22 (4.2) 45.76-51.80 <0.001
In the PCI treatment significant number of VPBs
were in anteroseptal localization (p=0.001)
(Table 4).
Table 4. Myocardial infarction and ventricular premature
beats in percutaneous coronary intervention
No (%) of patients
Localization Ventricular
with left ventricular
of myocardial premature
ejection fraction
infarction
beat/h
<45%
>45%
<10
2 (1.1) 20 (10.9)
Anteroseptal
>10
6 (3.3)
2 (1.1)
<10
2 (1.1)
8 (4.4)
Anterolateral
>10
4 (2.2)
2 (1.1)
<10
4 (2.2)
14 (7.7)
Inferior
>10
2 (1.1)
9 (4.9)
<10
6 (3.3) 60 (32.8)
Posterior
>10
9 (4.9) 33 (18.0)
95% CI
50.90-59.10
31.75-53.00
41.50-56.90
26.95-57.38
49.22-62.23
51.65-66.17
53.86-58.47
51.65-57.88
p
0.001
0.118
0.999
0.071
DISCUSSION
This study established several VPBs in patients
with fibrinolysis than PCI treatment in acute
STEMI.
In relevant literature sources primary PCI was
better than thrombolytic therapy (18). Primary
PCI is more effective than thrombolytic therapy
for the treatment of acute STEMI because it reduces overall short-term death (7% vs 9%), death
excluding the shock (5% vs 7%), non-fatal reinfarction (3% vs 7%), stroke (1% vs 2%) and the
combined endpoint of death, non-fatal reinfarction, and stroke (8% vs 14%) (19).
However, there are scarce data about VPBs
in patients with fibrinolysis comparing to PCI
treatment in acute STEMI. Our results can be
compared with a limited number of relevant literature sources. A higher VPBs frequency in anterior than inferior infarctions (70.2 vs 58.9%)
has been described by Stone (16). Breithardt
described a higher frequency of tardive ventricular potentials in anterior AMI compared to the
inferior AMI (20). However, Bluzaite (15) had
higher occurrence in the inferior than anterior infarction. Pascale also found a significantly more
VPBs in the inferior infarction (21). Future researches with an additional electrophysiological
testing would be of interest. Although the cardiac
conductive system is mainly in the septum (22),
the distal area like His-Purkinje system is also a
source of ectopic impulses (23).
An early spontaneous reperfusion in AMI could
be a protective factor for VPBs (24,25). The impact of collateral circulation is also important. In
case of collateral circulation, the infarction affec-
141
ted area will be smaller than the area with occluded artery (26).
Our results had the link among the residual
LVEF<45% and the significant number of VPBs.
These data are in correlation with results of low
LVEF and several VPBs (27). Solomon demonstrated the rise in the mortality rate within 30
post-MI days in LVEF<30% (13).
Our analysis verified good results in the PCI treatment. The Croatian PCI network is very important for this process. This is important because
the previous large trials such as DANAMI-2 study did not verify less VPBs in the PCI than the
fibrinolysis (28).
In conclusion, the AMI cum small LVEF and
PCI therapy has less VPBs than the fibrinolysis
therapy. In the fibrinolysis therapy more VPBs
were in anteroseptal, anterolateral and posterior localization but in the PCI treatment only in
anteroseptal localization. In patients with reduced ejection fraction in AMI, treatment with PCI
method has a better antiarrhythmic effect compared to fibrinolysis treatment. For this reason,
the PCI treatment reduces the possibility of the
occurrence of malignant ventricular arrhythmias
and sudden death of patients.
FUNDING
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Utjecaj reperfuzijske terapije i lokalizacije infarkta na učestalost
ventrikulskih ekstrasistola u akutnom infarktu miokarda
Davor Horvat1, Josip Vincelj2,3,4
1
3
Služba za internu medicinu, Opća bolnica Karlovac, Karlovac, 2Zavod za bolesti srca i krvnih žila, Klinička bolnica Dubrava, Zagreb
Zdravstveno veleučilište, Zagreb, 4Medicinski fakultet Osijek, Sveučilište J. J. Strossmayera, Osijek; Hrvatska
SAŽETAK
Cilj Odrediti utjecaj lokalizacije infarkta i reperfuzijske terapije na učestalost ventrikulskih ekstrasistola (VES) kod pacijenata s akutnim infarktom miokarda (AIM) i reduciranom ejekcijskom frakcijom
lijevog ventrikla (EFLV).
Metode Ukupno je 705 bolesnika s akutnim infarktom miokarda i ST elevacijom (STEMI) podijeljeno
prema lokalizaciji infarkta (anteroseptalni, anterolateralni, inferiorni i posteriorni) i reperfuzijskoj terapiji (fibrinoliza ili perkutana koronarna intervencija sa stentom) u dvije grupe: EFLV<45% kao ispitivana grupa, te EFLV>45% kao kontrolna grupa. Pojava VES<10/h bila je nesignifikantna, a pojava
VES>10/h signifikantna.
Rezultati U pacijenata s fibrinolizom i EFLV<45% značajan broj VES-a bio je u anteroseptalnom
(p=0,017), anterolateralnom (p<0,001) i posteriornom AIM-u (p<0,001), a u pacijenata s perkutanom
koronarnom intervencijom (PCI) i EFLV<45% značajan broj VES-a bio je samo u anteroseptalnoj AIM
(p=0,001) lokalizaciji.
Zaključak U pacijenata sa smanjenom ejekcijskom frakcijom u AIM-u, tretman s PCI metodom imao
je bolji antiaritmički efekat u odnosu na fibrinolitički tretman.
Ključne riječi: fibrinoliza, perkutana koronarna intervencija, ventrikulska aritmija
143
ORIGINAL ARTICLE
Possibilities of differentiation of solitary focal liver lesions by
computed tomography perfusion
Irmina Sefić Pašić1, Anes Pašić2, Spomenka Kristić1, Adnan Beganović1, Aladin Čarovac1, Amra
Dzananovic1, Lidija Lincender3, Sandra Vegar Zubović1
1
Radiology Clinic, 2 Oncology Clinic; Clinical Center of Sarajevo University, 3Academy of Sciences and Arts of Bosnia and Herzegovina;
Sarajevo, Bosnia and Herzegovina
ABSTRACT
Aim To evaluate possibilities of computed tomography (CT) perfusion in differentiation of solitary focal liver lesions based on
their characteristic vascularization through perfusion parameters
analysis.
Irmina Sefic Pašić
Radiology Clinic, Clinical Center of
University of Sarajevo
Bolnička 25, 71000 Sarajevo, Bosnia and
Herzegovina
Phone : +387 33 297 731;
Fax : +387 33 297 811;
E-mail : [email protected]
22 May 2015;
Revised submission:
06 July 2015;
Accepted:
08 July 2015.
doi: 10.17392/822-15
Med Glas (Zenica) 2015; 12(2): 144-150
144
Methods Prospective study was conducted on 50 patients in the
period 2009-2012. Patients were divided in two groups: benign
and malignant lesions. The following CT perfusion parameters
were analyzed: blood flow (BF), blood volume (BV), mean transit
time (MTT), capillary permeability surface area product (PS), hepatic arterial fraction (HAF), and impulse residual function (IRF).
During the study another perfusion parameter was analyzed: hepatic perfusion index (HPI). All patients were examined on Multidetector 64-slice CT machine (GE) with application of perfusion
protocol for liver with i.v. administration of contrast agent.
Results In both groups an increase of vascularization and arterial blood flow was noticed, but there was no significant statistical
difference between any of 6 analyzed parameters. Hepatic perfusion index values were increased in all lesions in comparison with
normal liver parenchyma.
Conclusion Computed tomography perfusion in our study did not
allow differentiation of benign and malignant liver lesions based
on analysis of functional perfusion parameters. Hepatic perfusion
index should be investigated in further studies as a parameter for
detection of possible presence of micro-metastases in visually homogeneous liver in cases with no lesions found during standard
CT protocol
Key words: CT protocol, contrast media, hepatic perfusion index
Sefić Pašić et al. Computed tomography perfusion in liver
INTRODUCTION
The liver is the most common site of metastases
from gastrointestinal tumors (1,2). High blood
flow (about 25% of cardiac output), favorable
microscopic anatomy (liver sinusoids and gaps
in subendothelial basement membrane), and rich
biochemical environment favor the rapid growth
of metastatic deposits in the liver (1,2). In malignant diseases diagnosis of the extent of the
primary tumor and staging of a potential spread of the disease have fundamental importance.
Without this information, an appropriate therapy
is not possible (1,2). A significant problem for
all diagnostic imaging methods in the staging
of malignant disease is a relatively high incidence of benign lesions (1,3). For this reason high
diagnostic specificity is a major requirement in
order to distinguish various benign lesions that
may affect the therapeutic decisions in case of
misinterpretation (1,3).
In general, detection of metastases with diagnostic methods is based on micro and macrostructure changes that distinguish tumor tissue from
normal liver tissue (1,2).
Because many pathological conditions of the liver leading to changes in regional or whole blood
flow, perfusion imaging of the liver proved to be
a method with high sensitivity and specificity in
differentiating of liver lesions (3,4).
Kinkel at al. analyzed in meta-analysis a sensitivity of different diagnostic methods in diagnosing metastasis and showed that in liver tumors
over 1 cm (sensitivity 55-90%), or for lesions
less than 1 cm sensitivity is much lower (below
50%), and microscopic lesions remain occult (3).
Smith et al. pointed that CT perfusion is one of
the last achievements in the field of physiological
imaging, which can provide new opportunities
for the use of imaging as a biomarker (5).
Since it was first described by Miles et al. (6), CT
perfusion has been successfully applied in a variety of clinical conditions including assessment
of liver cirrhosis (7), characterization of liver tumors (8,9), and evaluation of therapy response in
liver diseases (9-11)
The aim of this study was to analyze CT perfusion parameters (blood flow, blood volume, hepatic arterial fraction, mean transit time, capillary
permeability surface area product, impulse resi-
dual function) and to determine whether one or
more of the six parameters significantly stand out
in differentiating pathological lesions to benign
and malignant. The purpose of the study was to
evaluate the possibility of the application of CT
perfusion imaging in the differentiation of focal
hepatic lesions based on the perfusion analysis.
The prospective study included 50 patients in
the period 2009-2012 at the Radiology Clinic of
Clinical Center of Sarajevo University. All patients were examined on Multidetector 64-slice
CT machine (Light Speed VCT) (GE Medical
Systems, Milwaukee, WI, USA) with application
of CT perfusion protocol for liver with i.v. administration of contrast agent.
Solitary liver characteristics changes and their division into benign and malignant were confirmed
by at least two radiological methods (ultrasound,
computerized tomography or magnetic resonance)
with follow up of focal lesion over a certain period of time, and based on clinical parameters. All
patients had previously performed an ultrasound
examination of the abdomen with a special focus
on the liver to verify solitary focal lesion in liver.
Based on the differences in perfusion parameter
results, further categorization of tumors or secondary deposits (based on histological diagnosis)
into subgroups of solitary liver lesions was made.
Tube voltage 120 kV, power tubes 60mA, exposure time 50 sec, thickness 0.5 cm, and the beam
width 4 cm were used as an examination protocol. The amount of contrast agent that is administered was 0.5 mL/kg body weight of the patient
at a flow rate 4 mL/sec.
Before administration of contrast material all patients signed a consent for the application, and
were informed about adverse reactions.
The study was approved by the Ethics Committee
of the Clinical Center of University of Sarajevo
and all patients signed informed consents for
inclusion in study.
Six perfusion parameters were analyzed (12).
Blood flow (BF) (mL/min/100 g tissue), which
was carried out both in arterial and in the portal
phase, is the volume of blood flow through blood
vessels including large collecting blood vessels,
arteries, arterioles, venules, veins and sinuses.
145
Mean transit time (MTT) is measured in seconds.
Blood moves through the blood vessels at different speed so that there is no universally defined
time flow of blood from the arterial network in
the vein. Distribution of flow time and MMT
represent median time of that distribution.
Capillary permeability surface area product (PS) (mL/min/100 g tissue) is the flow
of the contrast medium through the capillary endothelium in interstitial space.
Hepatic arterial fraction (HAF) (%) is the percentage of blood that supplies hepatic arteries in relation to the portal vein in the liver.
Impulse residual function (IRF) (mL/min/100 g)
is the ratio of arterial and interstitial concentration of the contrast medium.
Functional perfusion parameters were analyzed
by Deconvolution (13,14) method and compartmental model (15,16).
The balance between the arterial and portal inputs was expressed by the hepatic perfusion index
(HPI), which represents the ratio of arterial blood
flow (Fa) and total hepatic flow Ft (Fa + Fp).
Statistical analysis was done by using chi-square test, Student’s t-th test, analysis of variance
(ANOVA), and to determine the degree of mutual dependence (correlation) of certain parameters Spearman’s rank correlation coefficient was
used. Testing the sensitivity of some parameters
was carried out by analyzing the area under the
ROC curve (receiver operator curve). For statistical analysis of hepatic perfusion index, control
group was introduced referring to the values of
normal liver parenchyma, and it was compared
with the values obtained in pathologic lesions (it
only applies on HPI parameter)
RESULTS
A total of 30 women and 20 men was included
in the study.
Analysis by gender revealed that women were
more frequently represented in the group of benign lesions, 18 (69.2%), than men, eight (30.8%),
while in the group with other lesions the same
number of men and women was recorded, 25 in
each (50%) (p>0.05).
The patients with benign lesions were (on average) slightly older (60.7 ± 9.6 years; range 44-80
yr.) than the patients with other lesions (57.8 ±
13.5 years; range 27-83 years) (p> 0.05).
146
Of the 24 malignancies 10 had histological diagnosis, be it a primary tumor or a secondary deposit.
Analysis of blood flow showed that patients
with malignant lesions had a little higher value,
but without significant statistical difference (p>
0.05), with emphasis that the significant difference was not shown among the subgroups, e.g.,
metastases vs. metastases with patohistological
diagnosis (phd).
There was no statistically significant difference
found in blood volume or arterial or in the portal
series (p>0.05).
Values for the MTT, HAF, SF and IRF (including
both arterial and portal phase), showed no statistically significant differences.
Analysis of the parameters of aortic blood flow
showed that the highest sensitivity in differentiating between benign and malignant lesions has
shown surface permeability (53.8%), and the
lowest one hepatic arterial fraction (42.9%) with
no statistical differences (Figure 1).
Figure 1. Specificity and sensitivity of aortic blood flow in both
groups of patients (benign and malignant lesions) Analysis of the parameters of the portal system
showed that the highest sensitivity in differentiating between benign and malignant lesions has
shown permeability surface (56.8%), and the
lowest one volume hepatic blood volume (42.3%)
without statistically significant difference between individually observed parameters (Figure 2).
A statistically significant correlation between the
groups, e.g., benign and malignant lesions, and
six perfusion parameters tested in both arterial
and portal phase was not observed in any case.
The highest correlation between perfusion parameters in differentiating benign from malignant lesions showed blood flow in the aortic
Figure 2. Sensitivity and specificity of portal blood flow in both
groups of patients (benign and malignant lesions)
blood stream (Ro = 0.137) followed by hepatic
arterial fraction aortic (Ro = 0.133) and blood volume – portal (Ro = 0.133), then the permeability surface - portal (Ro = -0.118), and
hepatic arterial fraction – portal (Ro = 0.108)
followed by other parameters with less than 10%
effect on the differentiation of groups (Table 1).
The patients with benign lesions had an average HPI slightly higher (55.7 ± 5.1; range 50 to
65.5%) than the patients with other lesions (54.6
± 3.95; range 50 to 66.9 %) with no statistically
significant differences (p> 0.05).
Table 1. Correlations between groups (benign and malignant
lesions) and between each of six perfusion parameters
Correlation coefficient
Benign Malignant
Blood flow (mL/min/100g) - aortal
Blood volume (mL/100g) - aortal
Mean transit time (sec) - aortal
Hepatic arterial fraction - aortal
Permeability surface (mL/min/100g) - aortal
Impulse residual function - aortal
Blood flow (mL/min/100g) - portal
Blood volume (mL/100g) - portal
Mean transit time (sec) - portal
Hepatic arterial fraction - portal
Permeability surface (mL/min/100g) - portal
Impulse residual function - portal
0.137
0.011
0.009
0.133
-0.073
0.082
0.069
0.133
0.019
0.108
-0.118
0.05
0.342
0.939
0.951
0.357
0.613
0.573
0.632
0.357
0.894
0.455
0.415
0.73
The analysis of HPI showed that sensitivity in
differentiating benign lesions from other lesions
on the basis of this parameter was 8.4% (Figure 3).
A comparison of HPI values in focal liver lesions
(benign and malignant) with values of normal
liver parenchyma has shown statistically significant difference (p <0.05).
The Spearman’s correlation coefficient indicated
that with 82.6% certainty the patients with normal values of hepatic perfusion index in liver
parenchyma belonged to the control group and
Figure 3. Sensitivity and specificity of hepatic perfusion index
in hypervascular benign lesions
patients with elevated values belonged to the group with benign or malignant lesions.
DISCUSSION
Quantitative measurement of perfusion CT provides information about the processes that affect
the structure and function of the tissue. The concept is based on monitoring the first pass bolus of
iodinated contrast agents through blood vessels
of a certain tissue. This method allows non-invasive monitoring of changes in malignant process,
as well as the results of treatment, and considering that CT perfusion provides data on angiogenesis activity may be useful in monitoring the
treatment of angiogenesis inhibitors (17).
In patients with known metastatic disease, an elevated arterial perfusion was noticed with values
of about 40-50 mL/min/100 mL m versus 17-19
mL/min/100 mL in the healthy control group
(values of normal liver parenchyma). Therefore,
HPI was significantly higher in patients with metastatic disease, which was proven by Miles et al.
(18-20) and Blomley et al. (21).
In our study, the value of HPI in solitary lesions
ranged between 50 and 60%, with no significant
differences by type of lesion. Also, benign and
malignant lesions had the same value of HPI,
considering that the benign lesions in this study
represent only hemangiomas, which are basically
hypervascular lesions. The results of this study
indicated that CT perfusion is not the method
of choice in the diagnosis of hemangioma, be-
147
cause they can be diagnosed with the standard
protocol that is used during the CT examination.
Certainly, HPI parameter can be used to prove
the presence of micrometastases in visually homogeneous liver, where a standard way of CT
protocols showing no enhancement after contrast
administration (4,18). Any increase in the value
of HPI favors of the changes with intense vascularization, which comes from the hepatic artery,
but without the possibility of characterization
of these lesions, so that this parameter remains
highly sensitive, but not specific enough (4,18).
In rats, Cuenod et al. used deconvolution technique and found colon cancer metastasis in the
liver with increased HPI and reduction in hepatic
perfusion due to the reduction of portal perfusion. They also observed decrease in distribution
volume and increase in MTT (18).
Further sub-analyses of the two groups (benign
and malignant lesions) in the present study revealed that patients with histologically verified malignant lesions had no significant differences in
perfusion parameters values compared to patients
with malignant change without histopathologic
verification.
Modification of hepatic perfusion can be found not
only in patients with visible liver metastases, but
also in patients with occult metastases that develop
liver metastases at follow-up examinations (21).
Leggett et al. described changes in hepatic perfusion in patients with visible metastases, reduction of
portal perfusion and increased HPI in patients with
occult metastases in whom the disease is detected
at follow-up (4). Routine CT and MRI are insensitive to discover occult and early stage hepatic
micrometastasis of tumors (22). Hemangioma is
one of the common benign liver tumors; however,
it is sometimes misdiagnosed as a malignant tumor
(23). Although there is no apparent abnormality
in morphology, computed tomography perfusion
can display changes in hemodynamics through its
functional imaging (24). An increase in both HAP
and HPI can declare the possibility of liver micrometastasis (15). Cuenod et al. (13-17) used the deconvolution method to study liver hemodynamic
changes caused by occult hepatic micrometastasis
in rats and found micrometastases in normal liver
leading to 34% decrease in portal blood flow and
25% increase in MTT, suggesting that resistance is
increased in sinusoidal capillaries.
148
Limitation of CT perfusion arises from the large
number of parameters that require dual model
of hepatic microcirculation. The liver perfusion models, measurements are taken during the
first passing of the contrast (25). In cases of the
existence of liver nodules or in chronic liver disease, where there is a modification of a sinusoidal permeability and interstitial volume, it is
required to have more complex models (26,27).
Little attention is devoted to biomarkers resulting from radiological examinations. CT
perfusion is one of the recent developments in
the field of physiological imaging, which can
provide new opportunities for the use of imaging as a biomarker (28). Preliminary evidence
suggests that measurement of liver perfusion
can be connected with the survival of the patients with visible metastasis and patients with
micrometastasis, where conventional CT protocol did not detect changes in liver parenchyma.
Many cancer patients undergo CT examinations
of the liver, and consequently, recurrent tumors
are identified after a primary treatment. For colorectal carcinomas, intensified follow-up in this
way is associated with decreased mortality (29)
and the American Association of Clinical Oncologists now recommends annual CT examinations of the lungs and abdomen in the first three
years after the primary therapy in patients with
high risk of recurrent disease (30). CT perfusion could be incorporated into such programs.
CT perfusion is especially suitable for the assessment of the response to biological therapy,
which affects the tumor blood vessels, giving
quantitative information, and more importantly,
studies have shown that obtained perfusion parameters correlate with histological measurements of angiogenesis (31-34). The possibility
of identifying high risk of liver metastases in
common diseases such as colorectal cancer can
help with decisions on adjuvant chemotherapy,
but also can avoid unnecessary treatment of patients who are at low risk of developing liver
metastases (9,34).
Computed tomography is the most common
modality for evaluating cancer patients. In our
study CT perfusion did not allow differentiation
between benign and malignant focal liver lesions.
However, it is possible that larger patient population should be studied. CT perfusion can be ea-
sily included as a part of standard CT protocol in
order to provide functional information about the
solitary change. It is an available method, easy
to perform, allows repeated examinations and
applicable to all organic systems.
FUNDING
No specific funding was received for this study
Competing interests : None to declare
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Mogućnost diferencijacije solitarnih fokalnih jetrenih lezija
putem perfuzije kompjuteriziranom tomografijom
Irmina Sefić Pašić1, Anes Pašić2, Spomenka Kristić1, Adnan Beganović1, Aladin Čarovac1, Amra
Džananovic1, Lidija Lincender3, Sandra Vegar Zubović1
Klinika za radiologiju; Univerzitetski klinički centar Sarajevo, 2Klinika za onkologiju; Univerzitetski klinički centar Sarajevo, 3Akademija
nauka i umjetnosti Bosne i Hercegovine; Sarajevo, Bosna i Hercegovina
1
SAŽETAK
Cilj Utvrditi mogućnosti perfuzije kompjuteriziranom tomografijom u diferencijaciji solitarnih fokalnih
lezija jetre na osnovu njihove karakteristične vaskularizacije, a putem analize parametara perfuzije.
Metode Prospektivna studija je obuhvatila 50 pacijenata koji su pregledani u periodu od 2009. do 2012.
godine. Pacijenti su podijeljeni u dvije grupe, odnosno u grupu s benignim i malignim lezijama jetre.
Analizirano je šest parametara CT perfuzije: protok krvi (BF), volumen krvi (BV), produkt kapilarne
površne permeabilnosti (PS), jetrena arterijska frakcija (HAF) i impulsna rezidualna frakcija (IRF).
Tokom studije analiziran je dodatni parametar perfuzije, jetreni perfuzioni indeks (HPI). Svi pacijenti
pregledani su na multidetektorskom 64-slojnom CT aparatu (GE) uz primjenu protokola za perfuziju
jetre i uz i.v. aplikaciju kontrastnog sredstva.
Rezultati Kod obje grupe pacijenata dokazana je povećana vaskularizacija i povišen arterijski protok,
ali nije utvrđena signifikantna razlika između analiziranih šest parametara perfuzije. Vrijednosti HPI-a
bile su povišene kod svih lezija u komparaciji s normalnim jetrenim parenhimom.
Zaključak Perfuzija kompjuteriziranom tomografijom u našoj studiji nije omogućila diferencijaciju
benignih i malignih lezija na osnovu analize funkcionalnih parametara perfuzije. Jetreni perfuzioni
indeks trebao bi se u budućim studijama ispitati kao parametar za otkrivanje potencijalnog prisustva
mikrometastaza u vizuelno homogenoj jetri u slučajevima kada se lezije nisu otkrile tokom pregleda
standardnim CT-protokolom.
Ključne riječi: CT-protokol, kontrastno sredstvo, jetreni perfuzioni indeks
150
ORIGINAL ARTICLE
Familial autoimmune thyroid disease and PTPN-22
Gabriel Conzuelo Rodríguez1, Hugo Mendieta Zerón2
Faculty of Medicine, Autonomous University of the State of Mexico (UAEMex), 2Asociación Científica Latina (ASCILA) and Ciprés
Grupo Médico (CGM); Toluca, Estado de México, México.
1
ABSTRACT
Aim Autoimmune thyroid disease (AITD) is a multifactorial disease with a genetic predisposition. The protein tyrosine phosphatase-22 (PTPN-22) gene is a powerful inhibitor of T-cell activation.
The aim of this study was to compare messenger RNA (mRNA)
PTPN22 expression between healthy persons and patients with
hypothyroidism and with their affected relatives.
Hugo Mendieta Zerón
Felipe Villanueva Sur 1209 Col. Rancho
Dolores CP. 50170 Toluca, Estado de
México, México
Phone/fax: +52 722 219 4122;
E- mail: [email protected]
29 December 2014;
Revised submission:
Methods This was a cross-sectional, prospective and descriptive
study. DNA was extracted from leukocytes (4,000-10,000 cells)
using the Magna Pure LC 2.0 Instrument and MagNA Pure LC
RNA Isolation Kit I (Roche, Germany). A real-time polymerase
reaction (qPCR) was performed utilizing the primer sets specific
for the PTPN-22 gene, and the succinate dehydrogenase complex,
the subunit A, Flavoprotein (Fp) (SDHA) constitutive gene. All reactions were performed with the 7500 Fast Real Time PCR System
(Applied Biosystems, Applera International, Inc. Cheshire, UK)
employing the SYBR Advantage qPCR Premix Kit (Clontech,
USA).
Results Twenty five patients with AITD (hypothyroidism), all females (mean age 39.6 ± 11.8 years) and 23 control subjects (mean
age 24.4 ± 4.2 years) were included in the study. There was no
statistical difference between both groups in PTPN-22 mRNA
expression (p = 0.125).
Conclusion There is no clear difference in mRNA PTPN-22
expression. The ideal genes for a systematic screening for familial
AITD are yet to be found.
Key words: genetic screening, hypothyroidism, qPCR
08 June 2015;
Accepted:
02 July 2015.
doi: 10.17392/803-15
Med Glas (Zenica) 2015; 12(2): 151-156
151
INTRODUCTION
Autoimmune thyroid disease (AITD) is the
most common autoimmune condition, affecting
approximately 2% of the female population and
0.2% of the male population (1). Although the
exact etiology is not yet known, AITD is multifactorial in that a genetic predisposition combines with environmental risk factors to promote
disease (2,3).
Up to 90% of patients with AITD-related
hypothyroidism are anti-thyroid peroxidase
(TPO) antibody-positive. It should be noted that
10–15% of the general population are positive
for anti-TPO antibodies and that low titers are
less specific for AITD (2).
Symptoms of hypothyroidism may be subtle,
even with marked biochemical derangement, but
as the disease progresses, subclinical and then
clinical hypothyroidism appears. In the early stages of hypothyroidism, the thyroid stimulating
hormone (TSH) may be normal and anti-thyroperoxidase (TPO) antibodies may be positive with
or without goiter. Later, TSH elevation becomes
modest (5–10 IU/ml) with a normal FT4 (biochemical or subclinical hypothyroidism).
Some studies have reported that siblings of persons affected by Graves disease or hypothyroidism have a 33% chance of developing the
disease (4). In the casuistry of Ciprés Grupo
Médico (CGM), Toluca, Mexico, the percentage
of mothers with AITD and with an affected daughter is 13% (5).
The first gene found to be associated with both
Graves disease and hypothyroidism was HLADR3. Since this discovery, significant progress
has been made in the genetic contributions and
the mechanisms underlying thyroid autoimmunity. To date, several loci have been associated
with AITD. In addition to HLA-DR subtypes, these include two groups of the non-major histocompatibility complex (MHC) genes, e. g., immunoregulatory genes (CD40, CTLA-4, PTPN-22,
FOXP3, and CD25), and thyroid-specific genes
(Tg and TSHR) (6,7). Polymorphic variations of
all these genes have been identified and linked
with AITD susceptibility, but the existing studies
have often given inconsistent results, with some
showing associations and others not (8,9). One
of the many unexpected findings of these gene-
152
tic studies is that the majority of the genes identified exert very minor effects (10). Indeed, with
the exception of the DRb1-Arg74 HLA variant,
which resulted in an Odds ratio (OR) for Graves’
disease of >5, all of the remaining AITD genes
gave very low OR of <1.5 (11); on the other hand,
family history is positive in about 50% of patients
with AITD. It is usually supposed that a strong
genetic effect in the disease is related with the inheritance of many genes with small effect (12).
Lymphoid tyrosine phosphatase (Lyp) encoded
by protein tyrosine phosphatase-22 (PTPN-22)
gene locus on chromosome 1p13.3–13.1, such
as CTLA-4, is a powerful inhibitor of T-cell activation (13). A single nucleotide polymorphism
in PTPN-22 has been reported as an autoimmune
susceptibility locus that associates with type 1
diabetes (14), rheumatoid arthritis (15), systemic
lupus erythematosus (16), Hashimoto’s thyroiditis (17), Graves’ disease (18), Addison’s disease (19), Myasthenia gravis (20), vitiligo (21),
systemic sclerosis (22), and juvenile idiopathic
arthritis (23), suggesting that allelic variants of
PTPN-22 could predispose to a more general autoimmune diathesis.
Protein tyrosine phosphatase and protein tyrosine kinases (PTKs) are enzymes that specifically catalyze the reversible addition or release
of phosphate groups from tyrosine residues on
signaling intermediates. Broadly speaking, PTK
amplify signals, while the mode, tempo and duration of the signal are governed by PTP. Protein
tyrosine phosphatase and PTK are divided into
two groups: receptor (membrane bound-RPTP or
RPTK) or non-receptor (cytoplasmic-NRPTP or
NRPTK) (24).
Aim of this study was to compare mRNA PTPN22 expression between healthy persons and patients with AITD.
Study population
This was a cross-sectional prospective and descriptive study conducted at the Medical Sciences
Research Center (CICMED), Autonomous University of the State of Mexico (UAEMex) and at
the Ciprés Grupo Médico (CGM), both in Toluca,
Mexico, from August 2013 to July 2014. Diagnosis of thyroidopathy was made based on the
Conzuelo Rodríguez et al. Familial thyroidopathy and PTPN-22
presence of a thyroid profile with TSH ≥ 10 IU
together with a significant titer of autoantibodies
(anti-thyroglobulin > 40 IU or anti-TPO > 35 IU).
The study was approved by the Institutional Review Board of the Medical Sciences Research
Center (CICMED), UAEMex (25/09/13) and
was performed according to the ethical standards
of the Helsinki Declaration (Fortaleza, Brazil).
Written informed consents were obtained from
all patients and their relatives who participated
in this project.
Sample calculation
Accepting an alpha risk of 0.05 and a beta risk of
0.2 in a two-sided test, 25 subjects per group were
required for the recognition as statistically significant of a difference ± 4 relative units (RU). The
common standard deviation was assumed to be 5.
Clinical measurements
Weight (kg), height (m) (Seca, GmbH, Germany)
and waist circumference (cm) were measured in all
participants. Body mass index (BMI) was calculated as weight (kg) divided by height (m) squared.
RNA extraction
A blood sample (vacutainer tubes) was taken
from each patient. Leukocytes were obtained
according to the ACK-lysing buffer (LONZA)
protocol. Briefly, a peripheral blood sample was
placed in an EDTA tube and then centrifuged at
2,500 rpm for 10 min. All samples were maintained at -80°C until further analysis.
The RNA was extracted from leukocytes (4,00010,000 cells) in the Magna Pure LC 2.0 Instrument
using the MagNA Pure LC RNA Isolation Kit I
(Roche, Germany). After extraction, the RNA
was quantified using a Nano Photometer (Implen
GmbH, Germany), reporting concentration (in μg/
mL) and purity (as 260/280 absorbance).
Gene expression
A total of 200–400 ng total mRNA was reversetranscribed into complementary RNA (cDNA)
utilizing a Transcriptor High Fidelity cDNA
Synthesis Kit (Roche Applied Science). The
amount of extracted RNA was quantified by measuring the absorbance at 260 nm. The purity of
the RNA was assessed according to the ratio of
the absorbance values at 260 and 280 nm; purity
ranged between 1.8 and 2.1, demonstrating a
high RNA quality. The samples were measured
with a NanoPhotometer (Implen GmbH, Germany), and the extracts were then adjusted to
a concentration of 20 μg RNA L−1 for the PCR
reaction. A real-time Polymerase chain reaction
(qPCR) was performed using the primer sets
specific for the PTPN-22 (NM_001193431.1)
as
follows:
forward:
5’-AGGCAGACAAAACCTATCCTACA-3’,
reverse:
5’-TGGGTGGCAATATAAGCCTTG-3’
and
the succinate dehydrogenase complex, subunit A, flavoprotein (Fp) (SDHA) constitutive gene (NM_004168) as follows:, forward:
5’-AGAGGGAGGCATTCTCATTAAC-3’, reverse: 5’-ACCGAGACACCACATCTCTA-3’.
All reactions were performed with the 7500 Fast
Real Time PCR System (Applied Biosystems,
Applera International, Inc., Cheshire, UK)
using the SYBR Advantage qPCR Premix Kit
(Clontech). The expression levels of the genes
were examined by placing 4 μL of the reverse
transcription mix for each PCR reaction in a total volume of 20 μL. The thermal cycling conditions were as follows: 10 min at 95°C followed
by 45 cycles of denaturation at 95°C for 15 sec
and annealing/extension at 60°C for 1 min. The
comparative threshold cycle (CT) method was
used to calculate fold amplification, as specified
by the manufacturer.
The Taguchi method was employed to set the
best conditions for primer amplification. The fold
change in PTPN-22 was normalized against the
constitutively expressed reference gene and then
compared with the controls (healthy volunteers)
as follows: 2−ΔΔCT, where ΔΔCT=(CT-target −
CT-reference) treated-sample − (CT-target − CTreference) calibrator-sample. Calibrator-sample
refers to the expression level (1×) of the target
gene normalized to the constitutive gene. The calibrator was chosen from healthy volunteers and
was given a relative expression value of 1.
Statistical analysis was performed using the
Mann–Whitney U-test after performing the Levene test. The normality hypothesis was tested
using the Kolmogorov-–Smirnov test. Statistical
significance was tested at the p ≤0.05 level.
153
RESULTS
Twenty five patients with AITD (hypothyroidism), all females (mean age, 39.6 ± 11.8 years) and
23 control subjects (mean age, 24.4 ± 4.2 years)
were included in the study. In Table 1, we depict
the affected relatives of the patients, which included one case for a grandmother, nine cases for a
mother, one for a father, seven for a sister, one for
a brother, four for an aunt, one for an uncle, and
six for a cousin. Average time of disease evolution was 18.4 ± 24.2 months prior to participation
in this study with a mean Levothyroxine dose of
66.2 ± 23.3 μg per day.
Table 1. Characteristics of thirty three affected relatives in 25
patients with autoimmune hypothyroidism
Case
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
GrandMother Father Sister Brother Aunt Uncle Cousin
mother
X*
X*
X*
X
X
X*,†
X† (3)
X
X
X
X
X
X
X*
X†
X†
X
X*
X
X† X† (2)
X
X
X
X
X
X
X
X
X*
*maternal, †paternal
Anthropometrically, while the patients showed a
BMI of 24.7 ± 2.4, the control group showed one
of 25.1 ± 1.7.
According to the place of residence there were
12 patients from Toluca, five from Mexico City,
four from Metepec, and one patient from each
Otzolotepec, Naucalpan, Zinacantepec, and Guadalajara.
In relation to antibodies, six patients were positive for anti-thyroglobulin, five for anti-TPO, and
14 for both.
In the qPCR analysis there was no statistically significant difference in PTPN-22 expression between both groups (p=0.125). In the subgroup of pati-
154
ents with affected relatives, the 50th percentile of
PTPN-22 expression was 1.13 RU (Figure 1).
Figure 1.
DISCUSSION
The comparable prevalence and incidence of
AITD in geographically different populations
suggest a significant genetic effect (19,20). In
this regard, the purpose of this study was not to
associate a geographical location with AITD, due
to lack of information related with this topic in
Latin America. In another line, obesity has been
postulated as a factor linked to the cause of AITD
(21). Although this is a strong argument, in our
sample population this possible link does not
play a critical role as the patients maintained a
normal BMI.
In all patients with associated diseases, AITD is
usually detected in its initial phase when thyroid
function is preserved, with normal or only slightly elevated TSH levels. At this stage, signs
and symptoms of thyroid disease are usually
absent, but because worsening of thyroid function is a possibility, early recognition of thyroid
dysfunction is necessary to prevent the negative
effects of hypothyroidism on growth and metabolic function (16). In our study, we evaluated
patients who had been already diagnosed.
Subclinical thyroid disease is a common clinical
problem, and because the majority of patients are
asymptomatic, screening is the only way to detect the condition in most patients (22).
In our study, we performed an initial familial survey without finding a clear difference in PTPN22 mRNA expression. This is in accordance with
Inoue et al. whose authors only found an association of CD40 and FCRL3 gene polymorphisms
with Graves’ disease intractability, and a ZFAT
Conzuelo Rodríguez et al. Familial thyroidopathy and PTPN-22
polymorphism association with Hashimoto’s disease severity, but neither PTPN-22 -1123C/G
nor PTPN22 SNP37 were associated with any
option (23). Contrariwise, Ichimura et al. found
an association of the SNP37 of the PTPN-22
gene with susceptibility to Graves’ disease in a
Japanese population (24).
In an initial attempt, a difference in PTPN-22
expression between familiar hypothyroidism and
healthy subjects cannot be concluded from our
results. In contrast, several other genes have been
tested without a definitive conclusion (25-26) but
of these genes taken together probably do not
explain more than about 10% of the heritability
of AITD (27).
We cannot exclude two kinds of bias in our study: recall bias and ‘lead-time’ bias. In the first
case, we registered four patients with affected
relatives who initially denied knowing another
relative suffering from hypothyroidism until a
second clinical interview. In the second type of
bias, it is possible that, with a different evolution
time with biochemical hypothyroidism, PTPN22 expression could exhibit different patterns.
Unfortunately, our sample size limits us to perform a stratified analysis. Expecting a lower RU
difference between healthy subjects and families
affected with AITD implies a higher number of
persons per group to be analyzed.
The question pertaining to how to explain the familial heritability may be more straightforward.
First, the female preponderance is explained
partially by fetal microchimerism and X-chromosome inactivation (27). Second, imprinted genes and epistasis, the modification of expression
of one gene by one or several other genes, are
believed to be important genetic contributors to
complex diseases. We could not forget that in
addition to the gene–gene interactions, the clinical phenotype of affected individuals is also influenced by gene–environment interactions (28).
In summary, the results of our study indicate that
the ideal genes for systematic AITD screening
continue to be missing, but these must be defined
in order to be carried out in relatives of patients
with AITD, including the offspring of these patients. PTPN-22 remains a possible gene candidate for further study including its promiscuous
association with familial AITD, but it would be
better to study a set of genes. Such a determination would have huge implications in our current
screening strategies for diagnosing earlier novel
thyroidopathies.
Admittedly, the sample size of our family collection remained insufficiently large for conclusion of
and exclusion of the participation of PTPN-22 in
the genesis of AITDs in Mexican population, but
we must take into account that studies in different
geographic regions revealed ethnic differences in
associations most probably due to founder effects
and/or to the presence or absence of certain variants in specific ethnic groups (4-8). Further studies
including proteomic analyses are required.
ACKNOWLEDGMENTS
Authors are grateful for the collaboration of José
Meneses-Calderón, MD, for his support in sending
three patients for genetic evaluation, and Maggie
Brunner, MA, for the English style correction.
FUNDING
This work was partially funded by Ciprés Grupo
Médico (CGM).
Conflict of interest: none to declare.
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ORIGINAL ARTICLE
Methicillin-resistant S. aureus (MRSA), extended-spectrum
(ESBL)- and plasmid-mediated AmpC ß-lactamase -producing
Gram-negative bacteria associated with skin and soft tissue
infections in hospital and community settings
Selma Uzunović1, Branka Bedenić2,3, Ana Budimir3, Amir Ibrahimagić1, Farah Kamberović4, Zlatko
Fiolić5, Michelle I. A. Rijnders6, Ellen E. Stobberingh6
Department of Laboratory Diagnostics, Cantonal Public Health Institute of Zenica-Doboj Canton, Bosnia and Herzegovina; 2School of
Medicine, University of Zagreb, 3Department of Molecular Microbiology, Clinical Hospital Center Zagreb; Croatia, 4Microbiology Department, Biotechnical Faculty, University of Ljubljana, Slovenia, 5Department of Surgery, Clinical Hospital Center Zagreb, Croatia, 6Department of Medical Microbiology, School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center, Mastricht,
The Netherlands
1
ABSTRACT
Aim To investigate the characteristics of meticillin-resistant S. aureus (MRSA), extended-spectrum (ESBL), and plasmid-mediated
AmpC beta-lactamase producing Gram-negative bacteria causing
skin and soft tissue infections (SSTIs) in hospital and outpatient
settings of Zenica-Doboj Canton, Bosnia and Herzegovina.
Selma Uzunović
Department of Laboratory Diagnostics,
Cantonal Public Health Institute of ZenicaDoboj Canton
Fra Ivana Jukića 2, 72000 Zenica, Bosnia
and Herzegovina
Phone: +387 32 443 580;
Fax: +387 32 443 530;
28 April 2015;
Revised submission:
25 May 2015;
Accepted:
19 June 2015.
doi: 10.17392/816-15
Methods Antibiotic susceptibility was determined by disc-diffusion and broth microdillution methods according to CLSI guidelines. MecA gene was detected by PCR, and genetic characterization of MRSA was performed using spa-typing and the algorithm
based upon repeat patterns (BURP). Double-disk-synergy test was
used to screen for ESBLs. PCR was used to detect blaESBL alleles.
Genetic relatedness of the strains was tested by PFGE.
Results Seventeen in-patients with MRSA, 13 with ESBL-producing Gram-negative bacteria and three patients co-infected with
both, were detected. Five MRSA and 16 ESBL-producing Gramnegative bacteria were found in outpatient samples. Klebsiella
spp. was isolated in 11 in- and seven outpatients. MLST CC152
was the most prevalent MRSA. Seven (38.9%) Klebsiella spp. yielded amplicons with primers specific for SHV, TEM-1 and CTXM group 1 β-lactamases. Eight K. pneumonia (44.4%) and 16
(64%) MRSA (including the in- and outpatient) strains were clonally related.
Conclusion The presence of MRSA and ESBL-producing organisms causing SSTIs in the community poses a substantial concern,
due to the high morbidity and mortality associated with possible
consequent hospital infections.
Key words: surgical wound infections, CTX-M beta-lactamase,
MLST CC152, antibiotic resistance
Med Glas (Zenica) 2015; 12(2): 157-168
157
INTRODUCTION
According to Edelsberg’s classification skin and
soft tissue infections (SSTIs) include eighteen
types of infections (1). With regard to predominanted (microbial etiology) pathogens and risk of
mortality (severity of local and systemic signs)
there are superficial SSTIs caused by Staphylococcus aureus or Streptococcus pyogenes, deeper or healthcare-associated infections caused by
anaerobic or gram-negative organisms, and gangrenous or necrotizing infections (or “often fatal
infections”) (1,2). The practice guidelines of the
Infectious Diseases Society of America (IDSA)
for the diagnosis and management of skin and
soft tissue infections (3) classifies SSTIs into five
categories, including superficial, uncomplicated
infection (impetigo, erysipelas and cellulitis), necrotizing infection, infections associated with bites and animal contact, surgical site infections and
infections in the immunocompromised host. The
purpose of all SSTI definitions is to develop the
useful guidelines for the clinical management and
treatment options for patients with SSTIs (1,3).
The annual frequency of visits to physicians’ offices for SSTIs have an increasing trend (4). The
predominant pathogens associated with SSTIs in
hospitalized patients include S. aureus (ranked first
in all geographical regions), Pseudomonas aeruginosa, Escherichia coli and Enterococcus spp. (5).
Methicillin-resistant
Staphylococcus
aureus (MRSA) causes many infections, but most
frequently SSTIs, such as cutaneous abscesses,
furuncles and cellulitis. Thus, the prevalence of
these infections has increased dramatically (6).
Risk factors for MRSA SSTIs include the presence of an abscess, previous MRSA colonization/
infection, antibiotic prescriptions within 8 weeks,
diabetes mellitus and hospital admission within
the preceding year (6).
Extended-spectrum beta-lactamase (ESBL) production is one of the main mechanisms of resistance to beta-lactam antibiotics in Enterobacteriaceae, so the therapeutic choices in infections
caused by such strains are limited (7,8). Most
ESBLs belong to SHV and TEM family, but
recently a new family of ESBLs with predominant activity against cefotaxime (CTX-M
β-lactamase) has been reported (8). In contrast to
TEM or SHV-ESBLs, CTX-M β-lactamases are
158
native ESBLs and are derived from the chromosomal β-lactamases of the genus Kluyvera (9).
In many countries CTX-M β-lactamases are the
most prevalent type of ESBLs (9,10). Plasmidmediated AmpC β-lactamases are derived from
chromosomal ampC genes of the family Enterobacteriaceae. AmpC enzymes encoded by both
chromosomal and plasmid genes are also evolving to hydrolyze broad-spectrum cephalosporins more efficiently (11).
Since most skin and soft tissue infections in
outpatient settings are treated with empiric antimicrobial therapy, it is very important to estimate the prevalence of causative agents associated with skin and soft tissue infections, as well
as their antimicrobial resistance patterns and
mechanisms (3-5) .
The aim of this study was to determine prevalence and molecular epidemiology of SSTIs caused
by MRSA, ESBL- and plasmid-mediated AmpCproducing β-lactamase Gram-negative bacteria
in the in- and outpatient settings in Zenica-Doboj
Canton, Bosnia and Herzegovina (B&H).
MATERIALS AND METHODS
Setting, bacterial isolates and study design
The Cantonal Hospital Zenica, B&H, is a 849bed tertiary level hospital admitting about 25.000
patients/year, with 240.000 patient days, and covers a population of 331.229 in Zenica-Doboj
Canton, B&H.
All consecutive, non-duplicate strains identified
as MRSA and/or ESBL- or plasmid-mediated
AmpC β-lactamase-producing Gram-negative
bacteria obtained from SSTIs of the in- and outpatients during the period December 2009–May
2010 were analyzed. The SSTIs comprise surgical wound infections (SWIs) (postoperation
and postraumatic wound infection) and ‘’other
SSTIs’’ (oSSTIs) (including furuncles/abscesses,
cellulitis, folliculitis) documented by the clinical
provider/physician).
Clinical and epidemiological data recorded for
the patients involved in the study included: age,
gender, occupation, place of residence at admission to the hospital (e.g. at home, other hospital, nursing home), contact with person(s) with
history of hospitalization in the past 12 months,
hospital department, antibiotic usage in the past
Uzunović et al. MRSA and ESBL skin and soft tissue infections
four months, isolated causative agent (MRSA
and/or ESBL or plasmid-mediated AmpC
β-lactamase-producing Gram–negative bacteria).
An institutional review board approval had been
obtained from the Ethics Committee in the Cantonal Hospital of Zenica prior to the initiation of the
study, and all the participants read and signed informed consents about the purpose of the study (participation was voluntary and anonymous) as well.
Identification of MRSA and susceptibility testing
Staphylococcus aureus isolates were identified
according to standard microbiological methods.
The strains were tested for oxacillin and cefoxitin
sensitivity/resistance by disk-diffusion method at
Mueller-Hinton (MH) agar (Oxoid, Basingstoke,
UK) (growth zone inhibition around 1 µg and 30
µg oxacillin and cefoxitin disk, respectively) in
accordance with CLSI (Clinical Laboratory Standards) guidelines (12).
All S. aureus isolates were analyzed for the presence of the S. aureus-specific femA gene as well
as the MRSA-specific mecA gene using a multiplex real-time PCR assay (13).
The disc diffusion method using Mueller-Hinton
agar (Oxoid, Besingstoke, UK) was used to test
susceptibility to 11 antimicrobials (Oxoid, Basingstoke, UK): mupirocin, MUP (200 µg), imipenem, IPM (10 µg), erythromycin, ERY (15 µg),
vancomycin, VAN (30 µg), gentamicin, GEN (10
µg), amikacin, AMK (30 µg), ciprofloxacin, CIP
(5 µg), clindamycin, CLI (2 µg), trimethoprim/
sulfamethoxazole, SXT (25 µg), chloramphenicol, CHL (30 µg), and rifampicin, RIF (5 µg).
The susceptibility testing results were interpreted
according to CLSI (12). Staphylococcus aureus
ATCC 25923 control strain was used for quality
control. Multidrug resistance (MDR) was defined
as resistance to three or more groups of antibiotics.
Susceptibility testing of ESBL and AmpC producing bacteria
The susceptibility testing to cefuroxime (CXM),
ceftazidime (CAZ), ceftriaxone (CRO), cefotaxime (CTX), cefoxitin (FOX), tazobactam (TZP),
cefepime (FEP), gentamicin (FEP), ciprofloxacin
(CIP), and piperacillin (PIP) was performed by
a twofold microdilution technique according to
CLSI standard procedures (12). Susceptibility
to imipenem (10 μg), meropenem (10 μg), tetracycline (30 μg), chloramphenicol (30 μg) and
sulphametoxazole /trimethoprim (23.75 /1.25 μg)
was performed by disk diffusion test (12).
Phenotypic detection of ESBLs and plasmidmediated AmpC ß-lactamases
A double-disk-synergy test (DDST) using the
combination of amoxycillin/clavulanate with
cefotaxime, ceftriaxone, ceftazidime, and cefepime was performed to detect the production of
ESBLs. Distortion of the inhibition zones around
cephalosporin and aztreonam disks towards central disk was considered as a positive result (14).
Production of ESBLs was confirmed by CLSI
combined disk test.
Production
of
plasmid-mediated AmpC
β-lactamase was tested in E. coli, Klebsiella spp.
and P. mirabilis by combined disk test using
3-amino phenyl boronic acid. An overnight Mueller-Hinton (MH) broth culture of the strains
was adjusted and swabbed on MH agar and disks
of cefotaxime, ceftriaxone, ceftazidime and cefepime were placed on the surface of the agar plate.
10 µl of 3-amino phenyl boronic acid (20 mg/
mL) was dropped on the disks containing cefotaxime (30 µg), ceftriaxone (30 µg), ceftazidime
(30 µg) and cefepime (30 µg). Control plate contained disks of the same cephalosporins without
phenyl boronic acid. Augmentation of the inhibition zones around cephalosporin disks for ≥ 5
mm in the presence of boronic acid was indicative for production of AmpC β-lactamases (15).
Transfer of resistance determinants
The transferability of cefotaxime resistance was
tested by conjugation (broth mating method).
Enterobacteriaceae were investigated for the
transferability of their resistance determinants.
Conjugation experiments were set up employing
plasmid-free and rifampin-resistant E. coli A15
R- recipient strain (15). Transconjugants were
selected on the combined plates containing cefotaxime (1 mg/L) and rifampicin (256 mg/L). The
frequency of conjugation was expressed relatively to the number of donor cells.
Typing of the spa locus of MRSA isolates
Real-time amplification of the spa locus
followed by sequencing was performed as des-
159
cribed above (16). The spa types were clustered into spa CCs (clonal complexes) using the
algorithm based upon repeat pattern (BURP)
with the Ridom Staph Type, version 1.5,
software package (http://www.ridom.de) (17).
The default settings recommended by the manufacturer were used. Since it has been shown
that spa typing, together with the algorithm
BURP, yields results consistent with typing
results obtained by MLST (17-19), the associated CCs, as determined with MLST, were
allocated through the Ridom SpaServer (http://
spaserver.ridom.de).
Molecular characterization of ESBL and plasmidmediated AmpC ß-lactamases
PCR was used to detect alleles encoding ESBL
enzymes. Extended-spectrum β-lactamases were
characterized at the molecular genetic level. The
presence of blaTEM, blaSHV, blaCTX-M, ESBL genes
was investigated by polymerase chain reaction
(PCR) using primers and conditions as described previously (9,20,21). Template DNA was
extracted by boiling method. PCR mix (50 µl)
contained 25 µl of master mix (Roche), 20 µl of
ultrapure water, 1 µl of each primer (10 pmol)
and 3 µl of template DNA. Strains positive for
CTX-M beta-lactamases were further tested by
multiplex PCR with primers specific for CTXM groups 1, 2, 8, 9 and 25 (22). Amplicons were
column-purified (Quiagen DNA purification kit)
and sequenced directly using ABI PRISM 377
Genetic Analyzer (Applied Biosystems). Sequences were analyzed using BioEdit v.7.0.9. (Ibis
Biosciences) program. Designation of bla genes
based on identified mutations was done according to Bush and Jacoby (23).
Primers IS26F (5’-GCG-GTA-AAT-CGT-GGAGTG-AT-3) and IS26R (5’-ATT-CGG-CAAGTT-TTT-GCT-GT-3’) were used to amplify
400 bp fragment spanning the link between
IS26 insertion sequence and blaCTX-M gene in
CTX-M producing isolates (22). Primers ISEcp1L1 (CAGCTTTTATGACTCG) and ALA-5
(CCTAAATTCCACGTGTGT) were applied to
amplify the ISEcpI insertion sequence (10).
Multiplex PCR with primers specific for MOX,
CMY, DHA, and FOX β-lactamases was used
to detect plasmid-mediated Amp β-lactamases
in E. coli, Klebsiella spp. and P. mirabilis stra-
160
ins resistant to cefoxitin and β-lactam/inhibitor
combinations (11).
Typing by pulsed-field gel electrophoresis (PFGE)
of bacterial DNA
Isolation of genomic DNA, its digestion with
the XbaI restriction enzyme (Invitrogen) and
PFGE of the resulting fragments was performed as described by Kaufman et al. (24,25).
The electrophoresis was carried out with a
CHEF-DRII apparatus (Bio-Rad Laboratories,
Hercules, CA). The PFGE patterns were compared following the criteria of Tenover et al.
(26) and analyzed by the GelComparII software (Applied Maths, St Martens, Belgium). The
patterns obtained were compared by clustering
methods (unweighted pair group methods with
arithmetic averages) using the Dice coefficient.
The optimization of 0.5% and position tolerance of 3% were applied.
RESULTS
During the period December 2009 – May 2010,
33 hospitalized patients with SSTIs caused by
MRSA or/and ESBL-producing Gram-negative
bacteria were identified: 17 patients had infection
caused by MRSA, 13 patients had infection caused by ESBL-producing Gram-negative bacteria,
and three patients had co-infection with MRSA
and ESBL-producers.
MRSA infections
Among 20 in-patients infected with MRSA, six
(30%) had surgical wound infections (SWI) and
14 (60%) had oSSTIs. Three patients with MRSA
(two were spa-type t355) infection had co-infection with MSSA (one patient with oSST at Dermatology, and two patients with SWI at surgery
and orthopaedic department, respectively). Five
(25%) in-patient and two (out of five) outpatient
isolates were susceptible to all antibiotic tested,
respectively. None of the isolates were multidrugresistant. Most in-patient isolates have shown
gentamicin resistance phenotype, 13 (65.0%).
In outpatient settings five SSTIs were noted, all
were oSSTIs; one was MRSA MLST CC152
(newborn).
Among 14 oSSTIs, all but one MRSA belonged
to spa-clonal-complex (CC) 355/595 associated
with MLST CC152. Among six MRSA isolated
from SWIs, MLST CC152 was found in three cases (Table 1).
Most in-patients with MRSA SSTIs were admitted to the hospital from home, with an exception of five patients who were transferred from
other hospital or from nursing home (four MRSA
belonged to MLST CC152) (Table 1). All hospitalized patients with SSTIs had contact with
persons with positive history of hospitalization.
A history of β-lactam antibiotics usage in combination with glycosides was positive in 16 patients
(data not shown).
Table 1. Characteristics of patients with MRSA infections and susceptibility /resistance to antibiotics
Protocol
Gender
No
Isolate
origin
Age
HospItal
(years) department
3196
F
oSSTI
42
Dermatology
21441†
M
oSSTI
40
Dermatology
245
F
SWI
54
ICU
13549
M
SWI
60
Internal
13476 †
F
SWI
45
Orthopedics
4357
F
2236
M
oSSTI
01
Pediatrics
17304
F
oSSTI
01
Pediatrics
2822
F
oSSTI
01
Pediatrics
33733
M
oSSTI
01
Pediatrics
4189
M
oSSTI
<01
Pediatrics
39027
M
oSSTI
<01
Pediatrics
8723
M
oSSTI
<01
Pediatrics
oSSTI (um<01
bilicus)
Pediatrics
oSSTI (um<01
bilicus)
Pediatrics
5928
M
9522
F
oSSTI
<01
Pediatrics
25621
F
oSSTI
02
Pediatrics
16578
M
oSSTI
<01
Pediatrics
26267 †
F
SWI
01
Surgery
32913
M
SWI
<01
Surgery
129/U
M
SWI
7559
M
oSSTI
01
Outpatient
13802
F
oSSTI (umbilicus)
01
Outpatient
20733
F
oSSTI
<01
Outpatient
33005
M
oSSTI
29
Outpatient
16548
M
oSSTI
01
Urology
Spa-type
(Spa CC)
(MLST CC)
Residance
before
hospitalization
Susceptibility/resistance to antimicrobial agents*
IMP ERY VAN GEN AMK
t355
Home
S
S
S
R
S
(355/595) (152)
t355
Home
NT S
S
R
NT
(355/595) (152)
t355
Other
S
S
S
R
S
(355/595) (152) hospital
t1855
Home
NT S
S
S
NT
(singleton)
t041
Other
NT S
S
S
NT
(002) (005)
hospital
t355
Health care
S
S
S
S
S
(355/595) (152) center
t355
Health care
S
S
S
R
S
(355/595) (152) center
t355
Home
NT S
S
S
NT
(355/595) (152)
t355
Home
S
S
S
R
S
(355/595) (152)
t355
Home
NT S
S
R
(355/595) (152)
t355
Home
S
S
S
R
S
(355/595) (152)
t355
Home
NT S
S
R
NT
(355/595) (152)
t355
Home
S
S
S
R
S
(355/595) (152)
t355
Home
S
S
S
R
S
(355/595) (152)
t355
Home
S
S
S
R
S
(355/595) (152)
t595
Home
NT S
S
R
NT
(355/595) (152)
t919
Home
NT S
S
S
NT
(008/024) (008)
t355
Home
NT S
S
R
NT
(355/595) (152)
t355
Health care
NT S
S
R
NT
(355/595) (152) center
t7250
Home
S
S
S
S
S
(singleton)
t728
DM
S
S
S
S
S
(015) (045)
t355
DM
NT S
S
S
NT
(355/595) (152)
Not typeable
t451
(008/024) (008)
Outpatient
Not typeable
DM
DM
CIP CLI SXT CHL RA MUP
S
S
NT
NT
S
S
S
S
S
S
S
S
S
S
NT
NT
S
S
S
S
S
NT
S
S
S
S
S
NT
S
S
S
S
NT
NT
S
S
S
S
NT
NT
S
S
S
S
R
S
S
S
R
S
NT
NT
S
S
S
S
S
S
S
S
S
S
NT
NT
S
S
S
S
S
S
S
S
S
S
NT
NT
S
S
S
S
NT
NT
S
S
S
S
NT
NT
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
NT
S
S
S
NT
R
S
R
NT
R
S
R
S
S
S
NT
R
S
R
NT
S
S
R
S
S
S
NT
S
S
R
NT
S
S
S
S
S
S
*IMP, imipenem (10 µg); ERY, erythromycin (15 µg); VAN, vancomycin (30 µg); GEN, gentamicin (10 µg), AMK, amikacin (30 µg); CIP,
ciprofloxacin (5 µg); CLI, clindamycin (2 µg); SXT, trimethoprim/sulfamethoxazole (25 µg); CHL, chloramphenicol (30 µg); RIF,. rifampicin (5
µg); MUP, mupirocin (100 µg); †Patients with MRSA and ESBL-producing Gram-negative bacteria coinfection Spa- CC, spa clonal complex;
MLST CC, MLST clonal complex; SWI, surgical wound infection; oSSTI, other skin and soft tissue infection (other than SWI); NT, not tested;
DM, data missing;
161
Among 23 MRSA strains analyzed by PFGE, 15
(65.2%) belonged to the same clonal complex
(Figure 1).
ESBL infections
Among 16 in-patients with infection caused by
ESBL-producing Gram-negative bacteria two
(12.5%) had SWI and 14 (87.5%) had oSSTI.
Most oSSTI hospital infections were registered
at pediatric department, nine (56.3%) (all were
newborns). ESBL-producing Klebsiella spp. was
the most frequently isolated Gram-negative bacteria, in 12 (75%) in-patients of which K. pneumonia was isolated in 11 cases.
The total number of 18 outpatients were infected
with ESBL-producing Gram-negative bacteria,
of which 17 (94.4%) had SWI and one had oSSTI. Two outpatients had co-infection with two
Gram-negative bacteria (K. pneumoniae in both
cases with K. pneumoniae and Pseudomonas aeruginosa, respectively). Klebsiella spp. was the
most isolated, in seven (38.9%) cases. The four
in-patients were transferred from another hospital. Eleven (61.1%) outpatients were ≥60 years of
age (Table 2).
Almost all in-patient ESBL-producing Klebsiella
spp. isolates were resistant to gentamicin. Resistance to ciprofloxacin was noted in six Klebsiella spp. from in-patients, and in four outpatients.
Two E. coli isolates have shown high-level resistance to almost all tested antibiotics. From one
of these, MRSA was isolated too. All but one
(Pseudomonas) isolates remained susceptible to
carbapenems (Table 2).
Eight strains transferred cefotaxime resistance to
E. coli recipient strains with frequency ranging
from 10-7 to 10-4. Resistance to gentamicin, tetracycline, chloramphenicol and cotrimoxazole
was cotransferred alongside with cefotaxime resistance in four strains (data not shown).
Seven (out of 18, 38.9%) K. pneumoniae isolates
(all were from the in-patients) yielded amplicons
with primers specific for all three SHV, TEM-1
and CTX-M group 1 β-lactamases, one of which
Figure 1. Dendogram showing the genetic relatedness of the 23 MRSA isolates. Two groups were identified by PFGE typing (A and
B) by using 80% similarity. Group A consisted of two, group B of 15 isolates and they appeared to be clonally related S, singleton;
oSSTI, other skin and soft tissue infections; SWI, surgical wound infections; NT, non-typeable;
162
M
M
M
M
M
F
M
M
F
M
33369
51978
52055
21441‡
22222§
2721‡
22367
6627
84874
52158
28
68
65
67
45
60
<01
40
47
64
SWI
SWI
SWI
SWI
oSSTI
SWI
oSSTI (umbilicus)
oSSTI
SWI
SWI (Coxarthrosis)
60
SWI
53
SWI
72
SSTI
71
SWI
73
SWI
85
SWI
<01
oSSTI (umbilicus)
46
SWI
39
oSSTI
63 oSSTI (combustio, gangrena)
01
oSSTI (umbilicus)
01
oSSTI (umbilicus)
<01
Other SSTI (umbilicus)
<01
oSSTI (umbilicus)
28
SWI
47
SWI
<01
oSSTI (umbilicus)
01
oSSTI (umbilicus)
74
oSSTI
82
SWI
76
SWI
<01
oSSTI (umbilicus)
<01
Other SSTI (umbilicus)
82
SWI
Isolate origin/ diagnosis
outpatient
outpatient
outpatient
outpatient
ICU
outpatient
Pediatrics
outpatient
Internal
Surgery
Pediatrics
Pediatrics
Pediatrics
Pediatrics
outpatient
outpatient
Pediatrics
Pediatrics
Internal
outpatient
outpatient
Pediatrics
Pediatrics
outpatient
Ortopedics/
traumatology
outpatient
outpatient
Dermatology
outpatient
Orthopedics/
traumatology
outpatient
outpatient
outpatient
outpatient
Hospital
department
Residance
before hospitalization
P. mirabilis
P. mirabilis
P. vulgaris
Proteus vulgaris
P. mirabilis
K. pneumoniae
K. pneumoniae
P. aeruginosa
P. aeruginosa
K. pneumoniae
Home
Home
Home
Home
≥256 128 16
≥256 >256 1
≥256 >256 16
≥256 64 16
8
≥256 >256 8
≥256 >256 64
≥256 128 16
≥256 4
4
≥256 >256 64
Other hospital ≥256 >256
Home
Home
Home
Home
Home
>256
CZ
NT
>256
>256
4
>128
>128
>128
>128
>256
NT
>256
>256
>256
>256
>256
>256
>256
<0.12
>256
>256
>256
>256
>256
>256
128
CXM
>256
>256
>256
>256
>256
32
>256
16
>256
NT
>256
64
>256
>256
64
>256
>256
>256
NT
>256
>256
>256
>256
>256
<0.12
CAZ
32
>256
1
16
8
128
>256
128
>256
NT
128
8
>256
>256
64
16
>256
>256
NT
<0,12
>256
>256
>256
>256
64
CTX
128
64
1
32
32
32
>256
32
16
NT
128
128
16
>256
128
64
128
32
NT
<0,12
64
256
128
128
16
32
FOX FEP GM
>256 16
64
256
64 256
256 0,25 64
256 128 32
>128
8 >256
128
16
16
>128 64 >256
128
16
16
>128 16
16
NT
NT NT
>128
8
128
>128 32 >256
>128
2
4
128
32 0,5
128
64 >256
256 128 128
>128
8
64
>128 32
32
NT
NT NT
4
<0,12 16
128
8 >256
64
8
32
>128 16
16
>256 16
32
<0,12 >256
CRO
32
256
<0.12
128
32
4
>256
8
256
NT
128
128
32
>256
16
128
>256
64
NT
4
32
>256
>256
128
Antibiotic (MIC in µg/mL)*
16
16
1
16
16
2
256
>128
32
>256 256
>256 32 >256 >256 128 128 >256 >128
>256 <0.12 <0,12 <0,12
1
<0.12 8 0.25
>256 256 128 256 >256 16
32
16
>256
4
32
64
4
16
32 0.5
>256 >256 >256 256
>256
8
>256
>256
Type of ß-lactamase†
+
TEM
+
CTX-M, OXA1
+
SHV-1, TEM-1
SHV-1, CTX-M
VIM
+
SHV-1, TEM-1, CTX-M 15, OXA1
CIP
1
TEM
8
+
16
TEM, CTX-M
2
TEM
2
+
1
AmpC
256 SHV-1, TEM-1, CTX-M 15, OXA1
1
TEM, CTX-M, OXA1
16
SHV-1, TEM, CMY-2
NT
SHV-1, TEM, CTX-M 1
4
SHV-1, TEM-1, CTX-M 15, OXA1
32
SHV-1
≤0.12
SHV-1, TEM
64
SHV-1, TEM-1, CTX-M 15
128
SHV-1
2
SHV-1
1
SHV-1, TEM, CTX-M 1
4
SHV-1
NT
16
SHV-1
>128
SHV-1
1
2
SHV-1, CTX-M 1, OXA1
2
SHV-1
8 >256 >256 16 <0,12 <0.12
4
<0,12 32 128
4
256
32
32
32
16
1
32
8 ≤0.12
16 >256 >256 >256 64
64
>128 16 >256 256
NT NT >256
8
16
4
>256
8
64
2
16
AMX PIP TZP AMC
E. coli
Home
≥256 32 16 NT
E. coli
Home
≥256 >256 16 16
E.coli
Home
≥256 16
8
4
E.coli
Home
≥256 64 64
4
Enterobacter cloacae Other hospital ≥256 >256 8 >128
Enterobacter cloacae
Home
≥256 8
2 >128
Home
≥256 >256 64 >128
Home
≥256 4
2 >128
K. oxytoca
Home
≥256 128 32 16
K. pneumoniae
Home
≥256 NT NT NT
K. pneumoniae
Home
≥256 >256 16 16
K. pneumoniae
Home
≥256 >256 64 16
K. pneumoniae
Other hospital ≥256 128 1
16
K. pneumoniae
Home
≥256 >256 64 16
K. pneumoniae
Home
≥256 16 16 16
K. pneumoniae
Home
≥256 64 64 16
K. pneumoniae
Home
≥256 128 16 16
K. pneumoniae
Home
≥256 128 128 4
K. pneumoniae
Other hospital ≥256 NT NT 8
K. pneumoniae
Home
≥256 32 32 16
K. pneumoniae
Home
>256 >256 16 16
K. pneumoniae
Home
≥256 >256 16
8
K. pneumoniae
Home
≥256 >256 16
2
K. pneumoniae
Home
≥256 4
4
16
Causative agent
isolated
*AMX, amoxycillin; PIP, piperacilin; TZB tazobactam; AMC, amoxycillin+clavulanic acid; CZ, cefazolin; CXM, cefuroxime; CAZ, ceftazidime; CRO, ceftriaxone; CTX, cefotaxime; FOX, cefoxitin; FEP, cefepime; GEN, gentamicin; CIP, ciprofloxacin; NT, non-tested; †Type of ß-lactamase or AmpC, or positive phenotypic test for ESBL; ‡Patients with MRSA-ESBL coinfection; §Patient with two ESBL-producing strains coinfection;
M
M
M
F
F
F
F
M
M
F
M
F
F
F
M
M
F
F
F
M
F
F
M
M
11284
30047
11511
22853
8851
13819
22040‡
28268
14754
32049
1360
2671
4357
5139
9474
21438§
21534
22050
22063
24805§
24848
30396
30398
33014§
Protocol
Age
gender
No
(years)
Table 2. Characteristics of patients with ß-lactamase producing Gram-negative bacteria causing skin and soft tissue infections and antibiotic susceptibility
163
additionally produced plasmid-mediated AmpC
β-lactamase. One of four E. coli (all from outpatients) coproduced both TEM and CTX-M
β-lactamase. In one Pseudomonas aeruginosa
isolate VIM β-lactamase was found. CTX-M beta-lactamases were most prevalent with 13 positive isolates (K. pneumoniae, E. coli, E. cloacae,
Proteus vulgaris), and in five cases they were
accompanied by both TEM-1 and OXA-1 betalactamase (Table 2).
Insertion sequence IS26 was located upstream of
blaCTX-M gene in two Enterobacter cloacae strains
(data not shown).
PFGE typing of K. pneumoniae using the 80%
breakpoint for clonal relatedness revealed dominant cluster A which contained two subclusters:
the clone A comprised 4 outpatient and the clone
B two outpatient K. pneumoniae strains; three
strains from pediatric and one from surgery hospital units were allocated in the dominant cluster
A (Figure 2 A).
Two E. coli strains were clonally related with
90% similarity of their banding patterns and assi-
gned to cluster A, and one strain was singleton
(Figure 2 B).
MRSA/ESBL coinfection
Among 17 and 16 in-patients with MRSA and
ESBL infections, respectively, three patients
were coinfected with both (Table 3).
DISCUSSION
S. aureus is a causative agent of large number
of ambulatory healthcare visits for skin and soft
tissue infections each year (6). The prevalence of
MRSA-positive SSTIs has increasing trend, and
up to 46-72% prevalence was noted (4,6,27,28).
Of the SSTI cultures negative for MRSA, almost
half are usually caused by methicillin-sensitive
S. aureus (MSSA), 6% by gram-negative organisms, and 3% infections are polymicrobial (6).
The Gram-negative ESBL producing bacteria
were identified more commonly as a causative
agent of post-surgical than other skin and soft
tissue infections (7), and both Escherichia coli
and Klebsiella spp. are among the most frequent
Figure 2. Dendogram showing the genetic relatedness of the K. pneumoniae and Escherichia coli strains by PFGE typing. A) The clone
A comprised four K. pneumoniae outpatient strains and the clone B comprised two outpatient strains using the 80% similarity; three
strains from pediatric and one from surgery hospital units were allocated in dominant cluster A, which contained two subclusters;
B) Two E. coli strains were clonally related with 90% similarity of their banding patterns and assigned to cluster A; one strain was
singleton. SSTI, skin and soft tissue infection; SWI, surgical wound infection;
164
Table 3. Patients with MRSA and ESBL-producing Gram-negative bacteria coinfection
Patient
Protocol Age
Hospital
Isolate origin
No
(years)
department
Patient 1
13476
2721
Patient 2
26267
45
SWI
<01
22040
Patient 3
21441
21441
SWI
Other SSTI
40
Other SSTI
(umbilicus)
Causative agent
Residance
before hospitalization
ATB used
Fluoroquinolones
Orthopedics MRSA (spa-type t041; spa-CC 002; MLST CC5)
Other hospital
Orthopedics
Proteus mirabilis (ESBL+)
Glycosides
MRSA (spa-type t355; spa-CC 355/595; MLST
Beta lactam+beta
Surgery
CC152)
lactamase inhibitors
Home
Enterobacter cloacae (TEM-1, CTX-M-15,
Pediatrics
Glycosides, penicillins
SHV-1)
Other SSTI Dermatology
MRSA (spa-type t355; spa-CC 355/595; MLST
CC152)
P. aeruginosa (VIM)
Home
Fluoroquinolones
* Patient with MRSA-ESBL coinfection; SWI, surgical wound infection; Other SSTI, skin and soft tissue infection other than SWI;
enterobacteria producing ESBLs in these infections (7). Almost equal proportion of both SSTI
caused by MRSA and MSSA was noted in this
study (among 43 SSTIs identified during the study period caused by Staphylococcus spp., 39.5%
were infected with MRSA, 41.9% with MSSA,
9.3% with S. epidermidis, and 9.3% of patients
had co-infection with MRSA and MSSA) (Uzunović S, unpublished data).
According to the results of this study ESBLproducing Gram-negative bacteria in outpatients
were more frequently causative agents of SWIs,
in contrast to the in-patients in which they more
frequently caused other SSTIs. K. pneumoniae
was dominant ESBL-producing pathogen of the
in-patient SSTIs in this study, while the causative agents obtained from outpatient SSTIs were
much more heterogeneous. A co-infection in
SSTIs occurred frequently, mainly with Pseudomonas aeruginosa and MRSA (7), as demonstrated in this study. Moreover, two patients had
co-infection with two ESBL-producing Gramnegative bacteria. Chronic infections, especially
in patients previously treated with antibiotics,
tend to be polymicrobial. Such mixed infections
additionally complicate the antibiotic treatment
and the outcome (29).
Community-associated methicillin-resistant S.
aureus (CA-MRSA) is the most common cause
of SSTIs, especially in closed populations with
frequent skin-to-skin contact (4,21). It is well
known that skin infections occurred predominantly in children and young adults without risk
factors, where family members can serve as a reservoir of CA-MRSA, so, the epidemic MRSA
clone might be propagated in the community
(30). As reported previously, an outbreak of CAMRSA infections in a neonatal intensive care
unit was initiated by a mother with CA-MRSA
wound infection and mastitis (31). In the present
study, it was not possible to differentiate between hospital-associated MRSA (HA-MRSA) and
CA-MRSA. However, we found that all patients
with MRSA infection in this study had contacts
with persons recently hospitalized and who used
antibiotics in the previous 4 months, which were
identified as risk factors for HA-MRSA (6). Based on antimicrobial susceptibility testing, MRSA
in this study was susceptible to a wide range of
antibiotics, which is typical for CA-MRSA (27).
From the genotyping results in this study, a MLST
CC152 MRSA strains (Balkan clone) found in the
in-patients (at multiple hospital departments), as
well as in the outpatients, suggesting clonal spread by cross-transmission following introduction
in the hospital (32,33), which was previously
described in other reports (31,34). Indeed, PFGE
results have shown that 80% of the MRSA strains
belonged to one clone. Similar to MRSA, ESBLproducing K. pneumoniae were also clonally
related indicating a common source. TEM- and
SHV-type β-lactamases, mainly produced by K.
pneumoniae, have spread throughout hospital
settings, while CTX-M enzymes, mainly produced by E. coli, have become predominant in the
community (35,36). ESBL-producing organisms
are increasingly prevalent worldwide, and represent an emerging infectious threat, thus indicating that ESBL-producing organisms may be an
emerging problem not only in hospital, but also
in outpatient settings (35-37). The gut colonization of in-patients was identified as a risk factor
for developing self and cross infections due to
ESBL-producers, and further dissemination of
ESBL-producing clones was a consequence of the
transfer of patients between various units of the
same hospital, but also between hospitals in the
same countries, as well as across the borders (38).
165
MRSA colonization of patients or their household members represents an important risk for subsequent MRSA infection (38). Nares and umbilicus were the two most common sites of MRSA
colonization, and sampling of these two sites is
necessary and might be adequate for surveillance
cultures (24). Some authors suggest MRSA screening of family members, but others recommend
the screening only for some categories of patients
(40). Our study confirmed the importance of umbilicus as a possible site of the future infection
and screening of mothers as possible sources of
the future infection.
The three most commonly described groups of
SSTIs are “other cellulitis or abscess”, “decubitus ulcer” and “post-operation wound infection”
(superficial infections), and they accounted for
76.5% of all hospitalized cases (41), which is
confirmed in this study.
The presence of MRSA and ESBL-producing organisms in outpatients is a substantial concern,
due to the high morbidity and mortality associated
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167
Meticilin-rezistentni S. aureus (MRSA) i gram-negativne
bakterije koje proizvode ß-laktamaze proširenog spektra (ESBL)
i plazmidom-posredovane AmpC ß-laktamaze kao uzročnici
bolničkih i vanbolničkih infekcija kože i mekih tkiva
Selma Uzunović1, Branka Bedenić2,3, Ana Budimir3, Amir Ibrahimagić1, Farah Kamberović4, Zlatko
Fiolić5, Michelle I. A. Rijnders6, Ellen E. Stobberingh6
Služba za laboratorijsku dijagnostiku, Kantonalni zavod za javno zdravstvo Zeničko-dobojskog kantona, Zenica, Bosna i Hercegovina;
Medicinski fakultet, Sveučilište u Zagrebu, 3Laboratorij za molekularnu mikrobiologiju, Klinički centar Zagreb; Hrvatska; 4Mikrobiologija,
Biotehniška fakulteta, Univerza v Ljubljani, Slovenia; 5Kirurški odjel, Klinički centar Zagreb, Hrvatska; 6Department of Medical Microbiology, The School for Public Health and Primary Care (CAPHRI), Maastricht University Medical Center, Mastricht, The Netherlands
1
2
ABSTRACT
Cilj Istražiti meticilin-rezistentni S. aureus (MRSA) i gram-negativne bakterije koje proizvode ß-laktamaze proširenog spektra (ESBL) i plazmidom-posredovane AmpC ß-laktamaze kao uzročnike bolničkih i vanbolničkih infekcija kože i mekih tkiva (SSTI).
Metode Osjetljivost na antibiotike određivana je disk-difuzijskom i mikrodilucijskom metodom u skladu s CLSI. MecA gen je određivan pomoću PCR-a, a genetička karakterizacija MRSA-e pomoću spatipizacije i BURP-a (algorithm based upon repeat patterns). Dvostruki sinergistički disk-test korišten je
za probir ESBLs. blaESBL aleli su detektirani pomoću PCR-a. Genetska srodnost između sojeva testirana
je pomoću PFGE-a.
Rezultati Kod bolničkih pacijenata izolirano je 17 MRSA, 13 ESBL-producirajućih gram-negativnih
bakterija, kod tri pacijenta zabilježena je koinfekcija obje bakterije, a kod vanbolničkih pacijenata pet
MRSA i 16 ESBL-producirajućih gram-negativnih bakterija. Klebsiella spp. izolirana je u 11 bolničkih
i sedam vanbolničkih pacijenata. MLST CC152 bio je najprevalentniji MRSA. Kod sedam (38,9%)
Klebsiella spp. detektirani su amplikoni s početnicama specifičnim za SHV, TEM-1 i CTX-M grupu 1
β-laktamaza. Osam (44,4%) sojeva K. pneumonia i 16 (64%) MRSA (bolničkih i vanbolničkih) pripadali su klonovima.
Zaključak MRSA i ESBL-producirajuće gram-negativne bakterije koje uzrokuju infekcije kože i mekih tkiva vrijedne su pažnje zbog toga što usljed visokog morbiditeta i mortaliteta predstavljaju rizik
za nastanak bolničkih infekcija.
Ključne riječi: infekcije kirurških rada, CTX-M beta-laktamaze, MLST CC152, otpornost na antibiotike
168
ORIGINAL ARTICLE
Emergence of extensive drug-resistant (XDR) Acinetobacter
baumanniiin the Clinical Center University of Sarajevo, Bosnia
and Herzegovina
Amela Dedeić-Ljubović1, Ðana Granov1, Mirsada Hukić2,3
Department of Clinical microbiology, Clinical Centre University of Sarajevo, 2Department of Medical Science, Academy of Sciences
and Arts of Bosnia and Herzegovina, 3International Burch University; Sarajevo, Bosnia and Herzegovina 1
ABSTRACT
Aim Recently increased attention and interest for Acinetobacterbaumannii are the result of the occurrence of multidrug resistant
(MDR), extensive drug resistant (XDR) and pandrug resistant
(PDR) isolates around the world. The aim of this study was to examine the resistance of A. baumannii isolates to antimicrobials in
Clinical Centre University of Sarajevo, Bosnia and Herzegovina.
Methods Two hundred and fifty-seven A.baumannii isolates were
collected between July 2011 and June 2012 in different wards and
from different clinical samples. Multidrug resistant, XDR and
PDR were defined according to international expert proposal for
interim standard definitions for acquired resistance.
Amela Dedeić-Ljubović
Department of Clinical microbiology,
Clinical Centre University of Sarajevo
71 000 Sarajevo, Bolnička 25,
Phone: +387 63 51 25 60;
Fax: +387 33 29 85 25;
23 February 2015;
Revised submission:
Results A total of 257 A. baumannii isolates showed eleven different patterns of resistance, of which ten patterns corresponded to
MDR and one corresponded to XDR (sensitive only to colistin).
Multidrug resistant and XDR strains were the most common at
Intensive Care Units and surgical departments. The largest numbers of isolates were found in wound swabs, blood and bronchial
aspirate.
Conclusion This is the first report of XDR A. baumannii in the
2000-bed Clinical Centre University of Sarajevo, Bosnia-Herzegovina. Although XDR strains have been detected, the resistance
to colistin has not. The elevated prevalence of these strains indicates that local antibiotic prescription policies should be revised and
infection prevention and control should be improved.
Key words: antimicrobials, nosocomial infections, intensive care
unit
21 April 2015;
Accepted:
27 April 2015.
doi: 10.17392/809-15
Med Glas (Zenica) 2015; 12(2): 169-176
169
INTRODUCTION
Acinetobacter baumannii is a nonfermentative,
gram-negative, nonmotile, oxidase-negative bacillus, whose natural reservoir still remains to be
determined (1). Its ability to survive in a hospital
milieu and its ability to persist for extended periods of time on surfaces makes it a frequent cause
for healthcare-associated infections that include
pneumonia, bacteremia, meningitis, urinary tract
infection, wound infection and it has led to multiple outbreaks (2).
Acinetobacter spp. can develop antibiotic resistance extremely rapidly what is in contrast
to other clinical bacteria, which require greater
time to acquire resistance, usually in response
to therapeutic strategies (3). The emergence of
antimicrobial-resistant Acinetobacter species
is due both to the selective pressure exerted by
the use of broad-spectrum antimicrobials and
transmission of strains among patients, although
the relative contributions of these mechanisms
are not yet known (3). Antimicrobial resistance
greatly limits the therapeutic options for patients
who are infected with this organism, especially if
isolates are resistant to the carbapenem class of
antimicrobial agents (3).
Definitions of multidrug-resistant Acinetobacter
species vary and different terms like multidrug
resistant (MDR), extensive drug resistant (XDR),
and pandrug resistant (PDR) have been used to
describe the extent of antimicrobial resistance
among Acinetobacter spp. (4). A group of international experts came together through a joint
initiative by the European Centre for Disease
Prevention and Control (ECDC) and the Centers
for Disease Control and Prevention (CDC), to
create a standardized international terminology
with which to describe acquired resistance profiles in all bacteria often responsible for healthcare-associated infections and prone to multidrug
resistance(4). In the current review MDR Acinetobacter spp. is defined as the isolate resistant
to at a least one agent in three or more antimicrobial categories, XDR Acinetobacter spp. as
an isolate that is resistant to at least one agent in
all but two or fewer antimicrobial categories (i.e.
bacterial isolates remain susceptible to only one
or two categories), and PDR Acinetobacter spp.
as the XDR Acinetobacter spp. that is resistant to
all agents in all antimicrobial categories (4).
170
To date, there has been no record about extensive
drug resistance (XDR) Acinetobacter baumanniiisolates in Bosnia and Herzegovina. Given the
scarcity of data the aim of the present study was
to examine the occurrence of resistant isolates of
A. baumannii in the Clinical Centre University
of Sarajevo.
MATERIALS AND METHODS
In this retrospective study which was conducted
in Clinical Centre University of Sarajevo resistance to antimicrobials was observed in 257 Acinetobacter baumannii isolates in the period from
July 2011 to June 2012. Isolates were detected
from different clinical samples including urine,
wound swab, blood, bronchial aspirate and other
samples which were collected from patients admitted to various hospital wards.
Identification of A. baumannii isolates was done
on the basis of morphological, cultural and biochemical characteristics (5). In addition, automated VITEK 2 Compact system (bioMérieux,
Marcy l’Étoile, France) was used to help with
the confirmation of the A. baumannii and for antimicrobial susceptibility testing. Antimicrobial
resistance profile was determined by disk-diffusion method for: amoxicillin/clavulanic acid,
piperacillin/tazobactam, ceftriaxone, ceftazidime, cefepime, amikacin, gentamicin, tobramycin, imipenem, meropenem, ciprofloxacin,
trimethroprim-sulphamethoxazole, colistin, minocycline. P.aeruginosa ATCC 27853 was used
as quality control strain. Results were interpreted
according EUCAST breakpoints (5).
Multidrug resistant, XDR and PDR were defined
according to international expert proposal for interim standard definitions for acquired resistance (4).
RESULTS
The survey contained 257 primo isolates of
A.baumannii isolated from different samples and
clinical departments in Clinical Centre University of Sarajevo from July 2011 to June 2012.
Results of testing for antimicrobial resistance
showed the presence of eleven (I-XI) different
patterns of resistance (Table 1). The pattern VII
belonged to XDR.
Resistotype III (sensitive to colistin, tobramycin
and minocycline) was the most frequent, 84 (33%)
Dedeić-Ljubović et al. Emergence of extensive drug resistant A. baumannii
Table 1. Different patterns of resistance of A. baumannii isolates
Pattern of resistance
I
II
III
IV
V
VI
VII
VIII
IX
X
XI
Antimicrobial agent*
AN
R
R
R
R
R
S
R
S
S
R
S
GA MEM
S
R
R
S
R
R
S
R
S
R
R
R
R
R
S
R
S
S
R
R
R
R
IM
R
S
R
R
R
R
R
R
S
R
R
FEP CAZ
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
PIP AMC COL TOB
R
R
S
R
R
R
S
S
R
R
S
S
R
R
S
R
R
R
S
S
R
R
S
S
R
R
S
R
R
R
S
S
R
R
S
R
R
R
S
S
R
R
S
R
TS
R
R
R
R
R
R
R
R
R
R
R
CIP CRO MYN
R
R
S
R
R
S
R
R
S
R
R
S
R
R
S
R
R
S
R
R
R
R
R
S
R
R
S
R
R
S
R
R
S
MDR/XDR
MDR
MDR
MDR
MDR
MDR
MDR
XDR
MDR
MDR
MDR
MDR
R, resistant; S, sensitive; *AN, amikacin; GA, gentamycin MEM, meropenem; IM, imipenem; FEP, cefepime CAZ, ceftazidime; PIP,
piperacillin+tazobactam; AMC, amoxicillin+clavulanate; COL, colistin; TOB, tobramycin; TS, trimethroprim- sulfamethoxasole; CIP, ciprofloksacin; CRO, ceftriakson; MYN, minocycline
strains, followed by the resistotype I (sensitive to
colistin, gentamycin and minocycline), 59 (23%),
resistotype VII (sensitive only to colistin), 37
(14.4%) and resistotype V(sensitive to colistin,
gentamycin, tobramycin and minocycline), 21.8
(8.5%) isolates. From the total number of isolates
of A.baumannii, 220 (85.6%) were MDR strains,
while 37 (14.4%) were XDR strains (Figure 1).
Multidrug resistant strains were most commonly
obtained from Anesthesiology and Reanimation
Unit, 39 (18.3%), Intensive Internal Therapy Unit,
33 (15.5%), and from Neurosurgery Intensive Care
Unit (ICU), 22 (10.3%), while XDR strains were
most commonly obtained from Plastic Surgery Unit,
nine (24.3%), following with Anesthesiology and
Reanimation Unit and Intensive Internal Therapy
Unit, seven (18.9%) strains in each (Figure 3).
Figure 1. Prevalence of different resistance patternsof 257 A.
baumannii isolates
The examination of time of the occurrence of
XDR isolates showed that first strains were recorded in July 2011 (six isolates). The highest
number of isolates were recorded in March 2012
(nine isolates). Multidrug resistant strains were
isolated in the largest number in September 2011,
May and April 2012, with 25 and 29 isolates, respectively (Figure 2).
Figure 2. Distribution 257 A. baumannii isolates in the period
2011-2012
Figure 3. Multidrug resistant (MDR) and extensive drug resistant (XDR) Acinetobacter baumannii according to hospital
departments
ICU, Intesive Care Unit; IITU, Intensive internal Therapy Unit;
ARU, Anestesiology and Reanimation Unit; FR Fiziatric and Rehabilitation Unit
Analysis of isolates distribution according to the samples showed that the largest number of isolates were
from wound swabs, 81 (31.5%), blood 48 (18.6%),
and bronchial aspirate, 39 (15.1%) (Figure 4).
Figure 4. Multidrug resistant (MDR) and extensive drug resistant (XDR) A.baumannii according to the samples origin
CVC, central venous catheter
171
Monitoring of total resistance of isolates showed
that there was no resistance to colistin, while resistance to minocycline was 14.4%. Resistance to
aminoglycosides varied among isolates, to amikacin was the highest (87.9%), while the lowest
was to tobramycin (45.2%). Resistance to other
antimicrobials was higher than 90% (Figure 5).
Figure 5. Overall resistance of A. baumannii to antimicrobials
PIP, piperacillin+tazobactam; AMC, amoxicillin+clavulanate; TS,
trimethroprim-sulfamethoxasole; S, Sensitive; R, Resistant
DISCUSSION
A. baumannii once considered opportunistic species and insignificant clinical pathogen today is one
of the most important Gram-negative bacteria (6).
It is responsible for various serious nosocomial infections particularly in intensive care units (7). A.
baumannii can complicate the primary disease in
severely ill patients and to increase the cost of the
treatment (7). Studies has shown that the costs of
treating patients infected with A. baumannii increased by an average of $ 60,916, and the hospital
stay is longer by 13 days compared to patients who
are not infected with this agent (7).
Beside the ability of expressing resistance to
many antibiotics, MDR, XDR and PDR strains
have the ability for long-lasting survival in the
inanimate surfaces, as well as the tendency for
epidemic spread (8).
Results of this study have shown 11 different
resistance patterns among 257 A.baumannii isolates;85.6% isolates had 10 MDR patterns and
14.4% of isolates had one XDR pattern (sensitive only to colistin). Resistotypes III (sensitive
only to colistin, tobramycin and minocycline)
was the most frequent (33%). First XDR strains
were recorded in July 2011 (6 isolates). In
March 2012 9 isolates were recorded. In September 2011, May and April 2012 increased
172
number of MDR strains were recorded with 25
and 29 of isolates, respectively.
Multi-drug resistant and extensive resistant isolates of A. baumannii are increasingly reported
all over the world, with the incidence range from
75% (Spain) (9), up to 100% (Italy, Greece,
Turkey, Bulgaria) (10-12).In the neighboring countries there was also a high percentage of multi-drug resistant isolates of A. baumanniiand it
ranges from 96.1% in Serbia to 100% in Croatia
(13).Similar data were observed in distant parts
of the world (USA, Taiwan, Iran, Jordan, China)
where multidrug resistance in this microorganism ranged 65-100% (14-18).
Study of Kuo et al. (19) showed that the prevalence of XDR A. baumannii increased significantly
from 1.3% in 2002 to 41.0% in 2010. Tertiary
Hospital in Central Part of Iran, also recorded
elevated prevalence of XDR strains over a sixmonth period (20).
A. baumannii isolates are common among patients
in intensive care units and various surgical departments, primarily at the department for burns treatment. Incidence in theintensive care units ranges
from 22% in Tunisia, 47-49% in China and Spain
to 58.8% in Turkey, while in the departments of
surgery from 30% to 62% (9, 21-23). Our data
showed that A. baumannii isolates were commonly
isolated among patients at the Anesthesiology and
Reanimation Unit, Neurosurgery ICU, Intensive
Internal Therapy Unit and Plastic Surgery Unit. It
could be because patients in these sections were
all in critical condition, they had extended hospitalization, lower immune defense, suffering from
severe underlying diseases or frequent invasive
procedures like tracheotomy. Risk factors for colonization or infection with multidrug-resistant
Acinetobacter species include prolonged length of
hospital stay, hospital size (over 500 beds), exposure to ICU, receipt of mechanical ventilation,
colonization pressure, exposure to antimicrobial
agents, recent surgery, invasive procedures, and
underlying severity of illness (24-26).
Wilkes et al. reported a recent outbreak of multidrug-resistant Acinetobacter infection, caused by
environmental contamination (curtains, laryngoscope blades, patient lifting equipment, door handles,
mops, and keyboards) (27). Medical equipment has
been implicated, emphasizing the need for special
attention to disinfection of shared items and extra
caution with respiratory care and wound care procedures. One or more epidemic Acinetobacter clones often coexist with endemic strains making it
difficult to detect and control transmission (3).
In the present study, nearly 32% of A. baumannii recovered from clinical specimens were
from wounds, blood, bronchial aspirate, and urine.
A. baumannii causes a wide range of nosocomial
infections. The most common infections are pneumonia in patients on artificial ventilation, bacteremia, urinary tract infections, wound infections, and
less frequently meningitis. Very often these infections are associated with a high mortality rate (28).
A. baumannii bacteremia caused a significant
problem in hospitals worldwide. They are usually
the result of pneumonia or catheter use and it is
the most common cause of mortality in intensive
care units (14). Studies from various parts of the
world show different frequency of A. baumannii
bacteremia. High prevalence of 92.5% is found
in Korea (29). Slightly lower, but still high percentage of the A. baumannii bacteremia is found
in studies from Brazil (78%) and Iran (60.5%)
(30,14),while the incidence was significantly
lower in China, Iran, Turkey and England and
ranged from 15% to 52% (18,28,31,32).
In our study over the period 2011-2012 the isolates
of A. baumannii exhibited high rate of resistance to
all antibiotics tested. Resistance to carbapenems,
cephalosporins (third and fourth generation), as
well as to piperacillin-tazobactam, quinolones,
and cotrimoxasole was over 95%. Among aminoglycosides resistance was lowest on tobramycin
(below 50%).There was no resistance to colistin.
The results of monitoring hospital infections caused by A. baumannii show that in recent years there has been a significant increase in its resistance
to the most commonly used group of antibiotics
(33). Except still good sensitivity to colistin A. baumannii is in high percentage (over 80%) resistant
to cephalosporins (ceftazidime and cefepime), and
the combination of piperacillin/tazobactam(34-36).
The frequency of resistance to ciprofloxacin ranges
from 85%, and even up to 100% (34-36). Among
aminoglycosides data are different (34-36). The incidence of isolates resistant to gentamicin and amikacin ranges from 80-90%, while for tobramycin
is below 75% (34-36). The percentage of carbapenem resistant varies from one country to another
and ranges from 45% to over 90% (34-36).
Development of multiresistance is contributed by
previous usage of carbapenems, cephalosporins
III generation, as well as fluoroquinolones and
aminoglycosides (33). Certainly the most important is its ability to acquire resistance genes.
Isolates of A. baumannii usually contain a set of
genes encoding resistance to different groups of
antibiotics at the same time (14).
Of all multidrug resistant organisms (MDROs),
carbapenemase-producing Acinetobacter spp.
require special attention; this organism can be resistant to all currently available antimicrobial agents
or remain susceptible only to older, potentially
more toxic agents such as the polymyxins, leaving
limited and suboptimal options for treatment (37).
The problem of increasing resistance of A. baumannii is even more threatening when considering the very limited number of new antimicrobial agents that are in development (38,39).
Management of Acinetobacter spp. infections is a
great challenge for physicians and clinical microbiologists (40). Often colistin or tigecycline are the
only available treatments for A. baumannii infections(41). Unfortunately, resistance to colistin has
recently emerged in Europe. The European arm of
the SENTRY surveillance program identified 2.7%
of polymyxin B-resistant A. baumannii isolates
collected during 2001 – 2004 (41). In a recent surveillance study from Greece, among 100 A. baumannii strains derived from ICU patients, 3% were
colistin resistant, whereas the minimum inhibitory
concentration (MIC) levels of tigecycline ranged
between 0.12- 4 μg/mL (42). A surveillance study
performed in 34 centers across UK, during 2000,
reported a 2% resistance rate to colistin among 443
A. baumannii tested, while tigecycline MICs ranged
from < 0.032-16 μg/mL (43).These data suggest
that an antibiotic therapy should always be guided
by in vitro susceptibility profile of the organism.
The selective pressure caused by indiscriminate
usage of broad-spectrum antibiotics in empirical
therapy of hospital infections and environmental
contamination is the main reason for such increased number of colonization and infection due to
this highly resistant pathogen (20).
In conclusion, A. baumannii are rapidly spreading
with emergence of extended resistance to even
newer antimicrobials. In this study we confirmed
emergence of XDR A. baumannii strains in the
most compromised patients in the ICUs, where all
173
patients are in critical conditions, mostly intubated
with long stay in hospital. Although we detected
XDR strains, resistance to colistin wasnot detected.
Carbapenem resistance was high, while somewhat
lower resistance to aminoglycosides was recorded.
The elevated prevalence of these strains indicates
that local antibiotic prescription policies should be
revised and infection control should be improved.
FUNDING
Competing interests: none to declare.
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175
Pojava ekstremno rezistentnih (XDR) sojeva Acinetobacter
baumannii u Kliničkom centru Univerziteta u Sarajevu (Bosna i
Hercegovina)
Amela Dedeić-Ljubović1, Ðana Granov1, Mirsada Hukić2,3
1
Klinički centar Univerziteta u Sarajevu, 2Odjel za medicinske nauke, Akademija nauka i umjetnosti Bosne i Hercegovine, 3Internacionalni
Burch univerzitet; Sarajevo, Bosna i Hercegovina
SAŽETAK
Cilj Nedavno povećana pažnja i interesovanje za Acinetobacter baumannii rezultat je pojave multipli
otpornih (MDR), ekstremno otpornih (XDR) i potpuno otpornih (PDR) sojeva širom svijeta. Cilj ovog
istraživanja bio je ispitati otpornost A. baumannii izolata na antimikrobna sredstva u Kliničkom centru
Univerziteta u Sarajevu (Bosna i Hercegovina).
Metode Dvije stotine i pedeset sedam izolata A. baumannii prikupljeno je u periodu od jula 2011. do
juna 2012. godine na različitim odjelima i iz različitih kliničkih uzoraka. Multiplootporni (MDR), XDR
i PDR izolati definirani su u skladu s prijedlogom međunarodnih stručnjaka za donošenje privremene
standardne definicije stečene rezistencije.
Rezultati Kod 257 izolata A. baumannii otkriveno je jedanaest različitih tipova otpornosti od kojih
deset odgovara MDR, a jedan XDR sojevima (osjetljiv samo na kolistin). Multipli otporni i XDR sojevi
najčešće su izolirani u jedinicama intenzivne njege i hirurškim odjelima. Najveći broj izolata izolirano
je iz brisa rane, krvi i aspirata bronha.
Zaključak Ovom studijom su dokazani prvi izolati ekstremno otpornih sojeva (XDR) A. baumannii
u Kliničkom centru Univerziteta u Sarajevu (Bosna i Hercegovina) koji raspolaže s 2.000 bolesničkih
kreveta. Iako su dokazani XDR sojevi, otpornost na kolistin nije otkrivena. Povišena prevalenca ovih
sojeva pokazuje da se lokalna antibiotska politika treba revidirati, a kontrola i prevencija infekcija unaprijediti.
Ključne riječi: antimikrobna sredstva, bolničke infekcije, jedinice intenzivne njege
176
ORIGINAL ARTICLE
Brucellosis in children in Bosnia and Herzegovina in the period
2000 - 2013
Sead Ahmetagić¹, Humera Porobić-Jahić¹, Nada Koluder2, Lejla Čalkić3, Snježana Mehanić2, Eldira
Hadžić3, Nevzeta Ibrahimpašić4, Svjetlana Grgić5, Mirela Zirić4, Jelena Bajić6, Denis Žepić¹
Clinic for Infectious Diseases, University Clinical Centre Tuzla, 2Clinic for Infectious Diseases, University Clinical Centre Sarajevo,
Department for Infectious Diseases, Cantonal Hospital Zenica, 4Department for Infectious Diseases, Cantonal Hospital Bihać, 5Clinic for
Infectious Diseases, Clinical Hospital Mostar, 6Clinic for Infectious Diseases, Clinical Center Banja Luka; Bosnia and Herzegovina
1
3
ABSTRACT
Aim To analyse clinical, laboratory and epidemiological characteristics of brucellosis in children in Bosnia and Herzegovina.
Methods The study included 246 children aged 0-18 years, who
were hospitalized in Clinics and Departments for Infectious Diseases in Tuzla, Sarajevo, Banja Luka, Zenica and Bihać in the
period 2000-2013, in whom the diagnosis of brucellosis was established based on anamnestic data, clinical features and positive
results from blood culture and/or positive results from one of the
serological tests.
Humera Porobić-Jahić
Clinic for Infectious Diseases,
University Clinical Center Tuzla
Trnovac bb, 75000 Tuzla,
Phone: +38735303326;
Fax: +38735303480;
03 March 2015;
Revised submission:
21 April 2015;
Results In this period, a total of 2630 patients, 246 (9.35%) of
whom were children, were treated from brucellosis at the Clinics and Departments in Bosnia and Herzegovina. In the majority
of cases, the children were from rural parts of the country, 226
(91.87%);214 (87.04%) cases had direct contact with sick animals,
sick family member or consumption of unpasteurized dairy products from farms where brucellosis had been already established.
Male children predominated, 157 (63.82%). The most frequent
clinical features in affected children were fever, 194 (78.86%)
and joint pain, 158 (64.22%). The average duration of antimicrobial treatment was 42.85 ± 10.67 days. A total of 228 (92.68%)
children were completely cured, while relapses occurred in 18
(7.32%) children.
Conclusion Since brucellosis is an endemic disease in Bosnia and
Herzegovina, it is important that physicians in their daily practice
consider brucellosis and establish proper diagnosis and therapy in
children with prolonged fever, arthralgia, leukopenia and positive
epidemiological data, especially in rural parts of the country.
Key words: clinical features, epidemiological characteristics, diagnosis, treatment
Accepted:
26 April 2015.
doi: 10.17392/811-15
Med Glas (Zenica) 2015; 12(2): 177-182
177
INTRODUCTION
Brucellosis, also known as ‘’undulant fever’’,
‘’Mediterranean fever’’ or ‘’Malta fever’’ is a zoonosis, and the infection is almost invariably transmitted by direct or indirect contact with infected
animals or their products (1,2). The major reservoirs of the disease include goats and sheep (Brucella melitensis), swine (Brucella suis), cattle (Brucella abortus) and dogs (Brucella canis) (3,4). It
is an important infection of humans in many parts of the world such as Latin America, Southern
Europe, Africa and Asia. In endemic, rural parts
of the country the infection frequently affects all
family members, including children regardless of
their gender (5-8). The disease usually starts after
consumption of unpasteurized milk and dairy products, and through contact with infected animals
(9). Earlier, brucellosis in children was regarded a
mild and rare disease, but today it is well known
that brucellosis affects all age groups, especially in
endemic countries (10-12). Brucellosis in endemic
regions appears on average in 3% to 10% of children (4). Acute form of brucellosis is very frequent
in children, with many nonspecific symptoms, and
also clinical forms which affect musculoskeletal, gastrointestinal, genitourinary, hematopoetic,
cardiovascular, respiratory and central nervous
systems (11,12). Clinical manifestations in children are not significantly different from manifestations in adults (2,13,14). Bosnia and Herzegovina
(B&H) was free from brucellosis from 1980 until
2000. Since then, the number of infected people in
the country has rapidly increased, and infections
have been recorded in almost the entire territory.
Brucellosis reached its peak in 2008 for the observed period 2000-2013, with 778 patients recorded (15). Published papers from different centers
in B&H indicated that brucellosis has become a
public health problem in the country (16-18). The
aim of this study was to analyze the clinical, laboratory and epidemiological characteristics of
brucellosis in patients younger than 18 years, who
were hospitalized in Clinics and Departments for
Infectious Diseases in B&H, in the period from
2000 to 2013.
The study included 246 children aged 0-18 years,
in whom the diagnosis of brucellosis was established according to anamnestic data, epidemi-
178
ological data, clinical features and in correlation
with positive results of blood cultures and/or
with one of the relevant serological tests. Patients
were hospitalized in six Clinics and Departments
for Infectious Diseases in B&H, Tuzla, Sarajevo,
Mostar, Banja Luka, Zenica and Bihać in the period 2000-2013.
A retrospective analysis was conducted of the clinical, laboratory and epidemiological data on brucellosis, collected from medical records of patients
younger than 18 years of age who were treated in
Clinics and Departments for Infectious Diseases
in B&H. Analyzed anamnestic data included:
age, gender, place of living (urban or rural region), month of disease onset, contact with animals,
consumption of raw milk or cheese, family history
of brucellosis, animal farming on small rural households. Clinical symptoms and signs, laboratory
findings, course and outcome of the disease were
particularly analyzed. The diagnosis of brucellosis was established on the basis of anamnestic
and epidemiological data, clinical features, and
positive results from blood culture and/or one of
the relevant serological tests (Rose-Bengal, CFT,
Wright agglutination test, ELISA test).
The study had been approved by the Research
Ethics Committee of the University Clinical
Centre Tuzla.
RESULTS
In the period 2000-2013, a total of 2630 patients
with brucellosis, of whom 246 (9.35%) were
children, were treated in six selected Infectious
Disease Clinics and Departments in B&H (Table
1). In this period there were no declared outbreaks of brucellosis, because brucellosis is endemic
in B&H and the frequency of cases is counted
cumulatively, year by year.
The largest number of infected children, 87 (out
of 246, 35.36%) was registered in 2008, and the
Table 1. Children with brucellosis in B&H in the period 20002013
No (%) of patients with brucellosis
City
Tuzla
Zenica
Sarajevo
Bihać
Mostar
Banja Luka
Total
Total
Children (0-18 years)
196 (7.45)
885 (33.65)
672 (25.55)
589 (22.40)
64 (2.43)
224 (8.52)
2630
12 (6.12)
104 (11.75)
58 (8.63)
54 (9.17)
8 (12.50)
10 (4.46)
246 (9.35)
Ahmetagić et al. Brucellosis in children
most cases were from Tuzla Canton, Una-Sana Canton and Central Bosnia Canton of B&H.
The number of affected male children was 157
(63.82%), and female children 89 (36.18%). The
average age of children was 10.76 years ± 5.19.
The youngest child was a one- month old baby,
and the oldest was 18 years old (Table 2).
Table 2. Distribution of 246 children with brucellosis in B&H
in the period 2000-2013 according to age groups
Age groups
0-2
3-6
7-10
11-14
15-18
Total
No (%) of patients
22 (8.94)
38 (15.45)
42 (17.07)
71 (28.86)
73 (29.68)
246 (100)
The majority of affected children came from rural regions, 226 (91.87%), of whom 214 (87.04%)
had positive epidemiological data and/or confirmed direct contact with an infected animal, an
infected family member or consumption of unpasteurized milk and dairy products from households
in which brucellosis was already established. The
consumption of raw milk or cheese was recorded
in 26 (48.14%) patients. The most frequent clinical manifestations in affected children included
fever in 194 (78.86%), joint pain in 158 (64.22%),
malaise and tiredness in 125 (50.81%) and night
sweating in 109 (44.30%) patients (Table 3).
Table 3. Anamnestic data in 246 children (0-18 years) with
brucellosis
Anamnestic data
No (%) of
patients
Contact with animal
Sheep, goat or cow farming on a small rural household
Shepherd
Veterinarian
Veterinarian technician
Consumption of raw milk or cheese
Unknown
Family history of brucellosis
197 (80.08)
116 (47.15)
52 (21.13)
0 (00.00)
0 (00.00)
155 (63.00)
33 (13.41)
142 (57.72)
Other clinical symptoms and signs in infected
children are shown in Table 4.
Elevated erythrocyte sedimentation rate (ESR) was
recorded in 140 (56.81%) patients, increased values of CRP in 133 (54.06%), leucocytosis in twelve
(04.87%), increased aspartate aminotransferase
(AST) levels in 114 (46.34%), and increased alanine aminotransferase (ALT) levels in 40 (16.26%)
patients. Decreased values of erythrocytes were
registered in 58 (23.57%), thrombocytopenia in
Table 4. Symptoms and signs in 246 children (0-18 years)
with brucellosis
Symptoms and signs
No (%) of patients
Fever
Night sweating
Headache
Weakness
Anorexia
Weight loss
Rash
Cough
Vomiting
Diarrhea
Stomach pain
Frequent urination
Dysuria
Arthralgia
One or more swollen joints
Myalgia
Hepatomegaly
Splenomegaly
Hepatosplenomegaly
Testicular swelling
Scrotal redness
Scrotal pain
Lymphadenitis
194 (78.86)
109 (44.30)
54 (21.95)
125 (50.81)
56 (22.76)
43 (17.48)
11 (4.47)
41 (16.66)
27 (10.97)
18 (7.31)
42 (17.07)
6 (2.44)
7 (2.84)
158 (64.22)
39 (15.85)
55 (22.35)
27 (10.97)
15 (6.09)
58 (23.57)
5 (2.03)
5 (2.03)
6 (2.44)
21 (8.53)
28 (11.38%) and decreased values of haemoglobin
in 216 (87.80%) patients (Table 5).
The diagnosis of brucellosis was established on
the basis of positive blood cultures, Rose Bengal agglutination test, Elisa test and complement
fixation test (CFT) (Table 5).
Table 5. Laboratory findings in 246 pediatric patients (0-18
years) with brucellosis
Laboratory finding
Erythrocyte sedimentation rate
C-reactive protein
Leukocytes
Neutrophils
Lymphocytes
Monocytes
Erythrocytes
Hemoglobin
Thrombocytes
Aspartate aminotransferase
Alanine aminotransferase
Reference ranges
No (%) of
patients
≤20 mm/hours
0.0-3.3 mg/L
3.4-9.7 x109/L
44.0-72.0%
20.0-46.0%
2.0-12.0%
4.34-5.72%
138-175 g/L
158-424x109/L
15-37 U/L
30-65 U/L
140 (56.91)
133 (54.06)
12 (4.87)
5 (2.03)
151 (61.38)
9 (3.65)
58 (23.57)
216 (87.80)
28 (11.38)
114 (46.34)
40 (16.26)
The number of patients with positive blood culture was 63 (25.61%). Brucella mellitensis was isolated from blood cultures in 20 (8.13%), Brucella
abortus in 2 (0.81%), and Brucella species in 41
(16.66%) patients. The diagnosis of brucellosis
was established only on the basis of serological tests in 183 (74.39%) patients. The number
of patients with positive Rose Bengal (RB) test
was 85 (34.55%), with positive ELISA test 35
(14.22%), and with positive RB and ELISA test
179
119 (48.37%), while 7 patients had positive CFT
and BAB reaction (rapid agglutination for brucella).
Complications occurred in 64 (26.01%) patients
as follows: monoarthritis in 15 (6.09%), polyarthritis in 16 (6.50%), synovitis in 8 (3.25%),
spondylitis in 2 (0.81%), sacroileitis in 8 (3.25%),
spondylodiscitis in 3 (1.22%), endocarditis in
1 (0.40%), pneumonia in 9 (3.66%), orchiepididymitis in 5 (2.03%) and splenic abscess in 5
(2.03%) patients.
All hospitalized children received symptomatic
antimicrobial therapy according to standard protocols. The average duration of treatment with
antibiotics was 42.85 ± 10.67 days. The average hospital stay of infected children was 26.66
± 10.63 days. Tetracycline in combination with
rifampicin was used in 85 (34.56%), aminoglycosides in combination with tetracycline in 70
(28.46%), gentamycin + rifampicin in 7 (2.48%),
gentamycin + trimetoprim-sulfamethoxasol
(TMP-SMX) in 61 (24.79%) and triple therapy:
gentamycin + rifampicin + TMP-SMX in 14
(5.69%) patients. Tetracyclines were used according to standard protocol in children older than
8 years of age. No fatal cases were registered or
chronic forms of the disease. Relapses were registered in 18 (7.32%) cases.
DISCUSSION
Brucellosis was first diagnosed in B&H in 2000,
and thereafter the number of patients constantly
increased until 2008 (15), but from 2009, the
number of patients has been decreasing. Until
now the clinical, epidemiological and laboratory
characteristics of brucellosis in children in B&H
have not been systematically analyzed. Brucellosis in children was registered in 9.35% of the
total number of cases of brucellosis in B&H in
the observed period, which is very similar to the
reported data on brucellosis in children in endemic regions like Mediterranean, Middle East and
Latin America, were the incidence of brucella cases ranges from 3% to 10% (4). Bosilkovski et al
(2010) reported that in the Republic of Macedonia in the period from 1998 to 2007, out of 550
registered cases of brucellosis, 86 (16%) were
patients aged 0-14 years (19).
Human brucellosis, as shown in our study, affected people living in rural regions, and was asso-
180
ciated with the consumption of unpasteurized
milk and dairy products. Similar data have also
been reported by the majority of authors coming from other international endemic regions
(9,14,20). Iranian authors have reported more
frequent incidence of brucellosis in children
coming from urban population (21). Shen from
the USA (2014) has reported that controlling
the disease in animals and humans significantly
reduces the incidence of brucellosis in children
in non-endemic countries (22). The fact that the
majority of affected children (35.26%) were registered in 2008 is similar to reports from other
endemic areas for brucellosis (19,23). Numerous
studies have registered more frequent presence of
brucellosis in boys than in girls (12,14,20). Data
from our analysis that 58.54% of affected children were older than 10 years is also found in
other reports from around the world (14,20,24).
Brucellosis is a systemic disease that can involve any organ or organ system in the human
body. The majority of our patients had clinical
symptoms which were described by other authors as well (2,7,25). In our patients, the disease
affected mostly bones and joints in the form of
monoarthritis and polyarthritis, which has also
been described by authors from Iran, Greece and
B&H (6,21,26). Clinical manifestations in children were not significantly different from those
in adults (1,4,27).
In majority of published papers, the diagnosis of
brucellosis was usually confirmed by positive blood cultures (24,25,28). Positive blood culture was
recorded in 25.61% of our patients, which corresponds to data from around the world, where the
percentage of positive blood culture ranges from
15% to 70% (5). Brucella melitensis was the most
frequently isolated pathogen from blood in our country and in some other parts of the world as well
(4,25). In all patients the disease was confirmed
by positive blood cultures and/or serological Rose
Bengal agglutination test, Elisa test, CFT.
The clinical picture of brucellosis varies from
very mild to severe. Antimicrobial treatment of
six weeks or longer proved successful in 92.5 %
of infected children (29). According to the World
Health Organization (WHO) recommendations,
the choice of antimicrobials for the treatment of
brucellosis in children older than 8 years of age
is the same as in adults. Since tetracyclines are
Ahmetagić et al. Brucellosis in children
contraindicated in pregnant women and children
younger than 8 years of age, alternative medicines are recommended in these groups of patients
(4,30). In our study, all children were cured, and
relapse occurred in 7.32% of cases. The most
commonly used combination of antibiotics in
children older than 8 years of age was doxycycline + rifampicin in 34.56% patients. To date many
clinical studies have been published about the
use of various antibiotics in the treatment of brucellosis in children (6,24,27). Impressive results
were obtained from a prospective study of 1100
children with brucellosis, where the treatment
scheme included a 3-week combination of TMPSMX + streptomycin, gentamicin, or rifampicin
(31). In another study in which a triple therapy
was administered, relapses were not registered
(24). In our study, a triple therapy was administered in a total of 23 (9.35 %) patients, and no
relapses were recorded either in these patients.
Our study suggests that in our country, which is
considered endemic for brucellosis, it is necessary to harmonize the views and approach to the
treatment of brucellosis patients as recommended
by the WHO (4).
In conclusion, brucellosis in children in Bosnia
and Herzegovina is a disease that cannot be ignored. Considering its endemic nature, it is important that physicians in their daily practice consider brucellosis, and establish proper diagnosis
and therapy in children with prolonged fever, arthralgia, leukopenia and positive epidemiological
data, especially in rural parts of the country. Public health education is one of the most important
methods for brucellosis prevention.
FUNDING
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Bruceloza kod djece u Bosni i Hercegovini u periodu od 2000. do
2013. godine
Sead Ahmetagić¹, Humera Porobić-Jahić¹, Nada Koluder2, Lejla Čalkić3, Snježana Mehanić2, Eldira
Hadžić3, Nevzeta Ibrahimpašić4, Svjetlana Grgić5, Mirela Zirić4, Jelena Bajić6, Denis Žepić¹
Klinika za infektivne bolesti, Univerzitetski klinički centar Tuzla, Tuzla, 2Klinika za infektivne bolesti, Klinički centar Univerziteta u
Sarajevu, Sarajevo, 3Odjel za infektivne bolesti, Kantonalna bolnica Zenica, Zenica, 4Odjel za infektivne bolesti, Kantonalna bolnica Bihać,
Bihać, 5Klinika za infektivne bolesti, Klinička bolnica Mostar, Mostar, 6Klinika za infektivne bolesti, Klinički centar Banja Luka, Banja Luka;
Bosna i Hercegovina
1
SAŽETAK
Cilj Ispitati kliničke, laboratorijske i epidemiološke karakteristike kod djece oboljele od bruceloze u
Bosni i Hercegovini.
Metode U ispitivanje je bilo uključeno 246 djece, u dobi do 18 godina, koja su bila hospitalizirana u
klinikama i odjelima za infektivne bolesti u Tuzli, Sarajevu, Banja Luci, Zenici i Bihaću, u periodu od
2000. do 2013. godine, a kod kojih je bruceloza dijagnosticirana na osnovu anamnestičkih podataka,
kliničke slike i pozitivnih rezultata hemokulture i/ili pozitivnih rezultata jednog od seroloških testova.
Rezultati Od ukupno 2630 pacijenata liječenih od bruceloze u klinikama i odjelima u Bosni i Hercegovini, 246 (9,35%) su bila djeca. U većini slučajeva djeca su bila iz ruralnih područja, 226 (91,87%),
a u 214 (87,04%) slučajeva imala su pozitivnu epidemiološku anamnezu o direktnom kontaktu s bolesnom životinjom, bolesnim članom porodice ili konzumacijom nepasteriziranih mliječnih proizvoda s
farmi gdje je bruceloza već registrirana. Muška djeca su bila u većini, 157 (63,82%) slučajeva. Najčešći
klinički simptom kod oboljele djece bila je povišena temperatura, 226 (78,86%), te bolovi u zglobovima, 158 (64,22%). Prosječno trajanje antimikrobne terapije bilo je 42,85 ± 10,67 dana. Ukupno 228
(92,68%) pacijenata bilo je potpuno izliječeno, dok se relaps pojavio kod 18 (7,32%) djece.
Zaključak S obzirom da je bruceloza endemska bolest u Bosni i Hercegovini važno je da liječnici u
svom svakodnevnom radu imaju na umu ovu bolest i postave pravu dijagnozu, te odgovarajući tretman
djeci sa simptomima u vidu produžene temperature, artralgijama, leukopenijom i pozitivnom epidemiološkom dijagnozom, posebno u ruralnim dijelovima zemlje.
Ključne riječi: klinički simptomi, epidemiološke karakteristike, dijagnoza, tretman
182
ORIGINAL ARTICLE
Differences in newborn umbilical cord care
Sanja Kanisek1, Nada Prlić2, Ivana Barać2, Lorna Dubac Nemet2
Community-Health Nursing Services, Community Health Centre Osijek, 2University Study of Nursing, Faculty of Medicine, J. J. Strossmayer University of Osijek; Osijek, Croatia
1
ABSTRACT
Aim To investigate the frequency of different cord care practices as
well recommendations to parents on cord care, along with the need
to identify as well as reach the consensus on best cord care practices and other procedures in newborn care among health workers.
Methods The study was conducted among 110 health care workers
at the nursery departments in two general hospitals, six community-health nursing services and 16 pediatric practices in Eastern
Croatia. The questionnaire created for this research has evaluated
different cord care practices and recommendations to parents, a
need to identify, as well as reach the consensus on best practices in
cord care and other procedures in newborn care.
Sanja Kanisek
Community-Health Nursing Services,
Community Health Centre Osijek
Park kralja P. Krešimira IV/6, 31000
Osijek, Croatia
Phone: +385 31 399 628;
Fax: +385 31 225 340;
Results Statistically significant differences have been found
among respondent groups in three “dry“ cord practices (p=0.000,
p=0.002, and p=0.004, respectively) and three “wet“ cord practices (p=0.000, p=0.001, and p=0.000, respectively). Significant
differences were determined in three types of recommendations
to parents about the care of ”dry” cord (p=0.000, p=0.000, and
p=0.002, respectively) and two recommendations for ”wet” cord
(p =0.000, p=0.000, respectively). The majority of respondents
stressed the need for publishing guidelines on cord care, 104
(94.5%), and for other procedures in newborn care, 108 (98.2%).
More than a half of respondents, 63 (57.3%), declared the need to
reach a national agreement on guidelines for umbilical cord care.
Conclusion Healthcare workers employ, as well as recommend,
different umbilical cord care practices. It is necessary to prepare
and reach a national agreement on written guidelines for umbilical
cord care as well as for other procedures in newborn care.
Key words: guidelines, health care workers
05 November 2014;
Accepted:
02 February 2015.
doi: 10.17392/792-15
Med Glas (Zenica) 2015; 12(2): 183-189
183
INTRODUCTION
Umbilical cord stump and wound in newborns
represent the point of entrance and development
of systemic infections (1). This infection risk
was the most frequent cause of changes in trends of newborn umbilical cord care practices over
many years (2). The diversity in newborn cord
care practices (3,4), suggests uncertainty about
the most efficient care practice and was therefore
the subject of many studies whose purpose was
to demonstrate the most efficient newborn cord
care regime (3). Based on the findings of many
studies mostly from the developed countries, the
World Health Organization (WHO) has published general guidelines on newborn umbilical
cord care, recommending further, larger and longer-term studies (3). The principle of the WHO
guideline is to keep the cord stump dry and clean;
to apply antimicrobial agents in a hospital setting
and in underdeveloped countries (3,5). Further
studies acknowledge that there is not enough evidence to support the use of antimicrobial agents
over keeping the cord stump dry and clean in developed countries, with the exception of preterm
babies and newborns in Intensive Care Units (6).
Croatian authors (1,7) recommend application of
antimicrobial agents (70% isopropyl alcohol) in
cord care, in contrast to WHO Guidelines which
declared them not efficient enough in reduction
of bacterial colonization of umbilical cord, as
well as prolonging the cord stump healing (3).
Differences in conducting cord care practices,
which are often present within the same or different institutions (4,8,9), create the feeling of insecurity among healthcare workers (9). Healthcare
workers’ inconsistence in conducting cord care
practices as well as giving recommendations on
cord care in a home setting (4,9,10), increases
the already present maternal fear during the period of stump healing (9,10).
Another important aspect, although not explicit,
is the use of resources, whether it is the case of
multiple visits of community-health nurse (11) or
the amount of medical supplies used (11,12 ). Parents are additionally facing confusing diversity of
recommendations on newborn cord care practices
produced by healthcare workers such as midwives, nurses and pediatricians, based on beliefs
rather than on scientific research findings (13).
184
It is questionable whether there are any written guidelines on nursing interventions in newborn health care during stump healing phase, such as umbilical cord care, maintaining personal hygiene of
a newborn or umbilical granuloma treatment with
silver nitrate, in Croatian healthcare institutions.
Moreover, it is arguable, whether the application
methods of the mentioned procedures are evidence-based or simply relicts from the past, based on
tradition, deeply rooted in the practice.
The objectives of this study were to investigate the
frequency of different cord care practices among
healthcare workers, as well as their recommendations to parents on cord care during its healing
related to healthcare worker’s place of work, along
with the need to identify as well as reach the consensus on best practices in cord care and other
procedures in newborn care, (umbilical granuloma
treatment with silver nitrate, personal hygiene).
EXAMINEES AND METHODS
Prior to the beginning of this study, the consent was
issued by hospital managements and the Ethical
Committee of the University Hospital Centre Osijek, General County Hospital Našice, Community
Health Centers in Osijek, Našice, Valpovo, Donji
iholjac, Beli Manastir and Đakovo.
Participation in this study was completely voluntary and anonymous, and respondents were given
both written and oral information on research.
Written consent was obtained from all respondents.
The study was conducted in April 2013 among
healthcare workers involved in umbilical cord
care in Eastern Croatia, Osijek-Baranja County.
The study was conducted at the University Hospital Centre Osijek, Newborns Department, General County Hospital Našice, Newborns Department, community-health nursing services and
pediatric primary care practices of the Community Health Centers in Osijek, Našice, Valpovo,
Donji Miholjac, Beli Manastir and Đakovo.
Anonymous questionnaire created for the purpose
of this research was used as a research instrument,
previously pretested and piloted on 15 health workers of different workplaces (hospital, community-health nursing services, pediatric primary care
practice) and qualifications (nurse, bachelor of
nursing, medical doctor). The questionnaire comprises socio-demographic data (age, gender, profession, workplace, years of service with current em-
Kanisek et al. Differences in umbilical cord care
ployer), as well as questions on conduction of cord
care practices and recommendations to parents on
cord care during its healing (“dry“ umbilicus – dry
cord stump; “wet“ umbilicus – wet cord stump,
wet wound), questions on availability of written
guidelines and instructions on umbilical cord care
for parents, questions on policies and rationales on
maintaining hygiene in newborns during stump
healing, questions on the necessity of creating guidelines on newborn cord care practices, reasons
and levels, as well as questions on need to create
and reach a mutual consent on other procedures in
newborn care. Multiple response questions were
introduced when questioning the necessity of creating guidelines on cord care practices.
Respondents were asked to hand in a copy of
written guidelines on umbilical cord practices and
recommendations for parents, if available. There
was a total of 35 open-ended and closed-ended
questions. Before data processing, the respondents were divided into three groups according
to their workplace (hospitals, community-health
nursing services and pediatric primary care practices). Categorical data were presented in absolute and relative frequencies. Numerical data were
described by arithmetic average and standard
deviation. Differences between categorical variables were tested by χ² test and Fischer’s Exact
test. Distribution of numerical variables was tested by Kolmogorov-Smirnov test. The level of
significance was set at α=0.05.
RESULTS
Out of 110 respondents who have participated in
this study there were 22 (20%) hospital employees,
55 (50%) community-health nursing service employees and 33 (30%) pediatric primary care practice employees. In relation to their qualifications,
there were 35 (32%) nurses, 57 (52%) bachelors of
nursing and 18 (16%) medical doctors.
The majority of the respondents were females,
108 (98.2%). The average age of the respondent
was 46.46 years, ranging from 20.0 to 64.0 years
(SD; 12.295). The average number of years of
service with current employer was 15.93 years,
ranging from 1.0 to 45.0 years, (SD; 12.786).
Healthcare workers stated that they conducted a
total of 11 “dry“ cord care practices, whereby
statistically significant differences were found
according to workplace in three care practices:
alcohol-free disinfectant, antibiotic powder and
sterile gauze (p=0.000); alcohol-free disinfectant
and sterile gauze (p=0.002) and antibiotic powder
and sterile gauze (p=0.004).
Healthcare workers stated that they conducted a
total of six “wet“ cord care practices, whereby statistically significant differences were also found
according to workplace in three care practices:
sterile gauze (p=0.000); 70% isopropyl alcohol,
antibiotic powder and sterile gauze (p=0.001) and
3% hydrogen peroxide, saline solution, antibiotic
powder and sterile gauze (p=0.000) (Table 1).
Healthcare workers stated a total of nine types
of recommendations for parents on “dry“ umbilical cord care in a domestic setting. Statistically
significant differences among the respondents
of different professions were found in three
recommended cord care practices: antibiotic
Table 1. “Dry“ (cord stump) and “wet“ (cord stump/wound) umbilical cord care practices used by healthcare workers
UC
care practices
Alcohol-free disinfectant, antibiotic powder, gauze
70% isopropyl alcohol, gauze
70% isopropyl alcohol, antibiotic powder, gauze
Alcohol-free disinfectant, gauze
3% hydrogen peroxide, saline solution, antibiotic powder,
gauze
3% hydrogen peroxide, antibiotic powder, gauze
Antibiotic powder, gauze
Saline solution, antibiotic powder, gauze
3% hydrogen peroxide, saline solution, gauze
Saline solution, gauze
Gauze
UC healing
phases
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Number (%) of respondents
H (n=22) C-HNS (n=55) PPCP (n=33) Total (n=110)
0
25 (45.6)
6 (18.2)
31 (28.2)
0
27 (49.1)
8 (24.2)
35 (31.8)
5 (22.7)
7 (12.7)
3 (9.1)
15 (13.7)
4 (18.2)
4 (7.3)
2 (6.1)
10 (9.1)
0
11 (20.0)
2 (6.0)
13 (11.8)
0
19 (34.5)
4 (12.1)
23 (20.9)
0
1 (1.8)
7 (21.2)
8 (7.3)
0
0
3 (9.1)
3 (2.7)
0
0
2 (6.1)
2 (1.8)
18 (81.8)
4 (7.3)
10 (30.3)
32 (29.1)
1 (4.5)
0
1 (3.0)
2 (1.8)
0
1 (1.8)
6 (18.2)
7 (6.4)
10 (45.6)
8 (14.5)
4 (12.1)
22 (20.0)
5 (22.7)
1 (1.8)
2 (6.1)
8 (7.3)
0
0
3 (9.1)
3 (2.7)
0
1 (1.8)
2 (6.1)
3 (2.7)
1 (4.5)
1 (1.8)
1 (3)
3 (2.7)
p
0.000
0.000
0.385
0.150
0.024
0.001
0.002
0.032
0.127
0.000
0.248
0.009
0.004
0.008
0.008
0.437
0.773
UC, umbilical cord; H, hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices
185
Table 2. Recommendations for parents on “dry“(cord stump) and “wet“(cord stump/wound) newborn cord care practices in a
domestic setting
Recommendations on UC care practices in a domestic setting
UC healing
phases H (n=22) C-HNS (n=55) PPCP (n=33) Total (n=110)
Alcohol-free disinfectant, antibiotic powder, gauze
Antibiotic powder, gauze
70% isopropyl alcohol, antibiotic powder, gauze
Alcohol-free disinfectant, gauze
70% isopropyl alcohol, gauze
3% hydrogen peroxide, antibiotic powder, gauze
Gauze
Saline solution, antibiotic powder, gauze
Pure water, drying, gauze
3% hydrogen peroxide, saline solution, antibiotic powder, gauze
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
Dry
Wet
0
0
13 (59.1)
0
0
0
0
0
5 (22.7)
4 (18.2)
0
1 (4.5)
0
4 (18.2)
0
17 (77.3)
18 (32.7)
31 (56.3)
15 (27.3)
4 (7.3)
10 (18.2)
15 (27.3)
1 (1.8)
0
4 (7.3)
4 (7.3)
0
1 (1.8)
5 (9.1)
1 (1.8)
1 (1.8)
0
6 (18.2)
11 (33.3)
2 (6.1)
2 (6.1)
4 (12.1)
5 (15.2)
11 (33.3)
3 (9.1)
2 (6.1)
1 (3.0)
5 (15.1)
6 (18.2)
1 (3.0)
0
2 (6.1)
5 (15.1)
24 (21.8)
42 (38.1)
30 (27.3)
6 (5.5)
14 (12.8)
20 (18.2)
12 (10.9)
3 (2.7)
11 (10.0)
9 (8.2)
5 (4.5)
8 (7.3)
6 (5.5)
5 (4.5)
3 (2.7)
22 (20.0)
p
0.006
0.000
0.000
0.564
0.090
0.017
0.000
0.032
0.114
0.150
0.002
0.017
0.330
0.007
0.437
0.000
UC, umbilical cord; H,hospitals; C-HNS, community-health nursing services; PPCP, pediatric primary care practices
powder and sterile gauze (p=0.000); alcohol-freedisinfectant and sterile gauze (p=0.000) and 3%
hydrogen peroxide, antibiotic powder and sterile
gauze (p=0.002). Respondents listed a total of
seven types of recommendations for parents on
methods of “wet” newborn cord care in a domestic setting. Statistically significant differences
among respondents of different professions were
found in two recommended cord care practices:
sterile gauze (p=0.000); and 3% hydrogen peroxide, saline solution, antibiotic powder and sterile gauze (p=0.000) (Table 2).
For the majority of respondents the maintenance of personal hygiene in newborns during the
umbilical stump healing time period (with the
avoidance of wetting) had reduced the risk of infection, 88 (80.0%). Just17 (15.5%) of them considered that newborn cord care practices had no
influence on risk of developing an umbilical cord
infection , whereas five respondents (4.5%) were
not familiar with it (p=0.870).
As the most frequent method of and/or recommendation on personal hygiene maintenance
in newborns during umbilical cord stump healing
the healthcare workers highlighted the method
of cleaning the baby on the changing mat with
avoidance of wetting, 50 (45.5%), whereby statistically significant difference was found among
groups (p=0.000) (Table 3).
The availability of written guidelines on newborn
umbilical cord care for healthcare workers at healthcare institutional level was acknowledged
by only a small number of hospital respondents,
186
17 (15.5%). The majority of respondents, 89
(80.9%) stated that no written guidelines were
available, just a verbal agreement, whereby four
respondents (3.6%) were not familiar with the
existence of written guidelines at institutional level. Statistically significant difference was found
among groups of respondents (p=0.000).
Only a small number of respondents, nine of them
(8.2%), confirmed the existence of written recommendations for parents at institutional level.
The majority of respondents, 99 (90%), stated the
lack of written recommendations for parents, yet
confirmed verbal instructions. In contrast, two
respondents (1.8%) were not familiar with the
existence of such recommendations. In view of
that, statistically significant difference was also
found among groups of respondents (p=0.000).
Table 3. Methods of and/or healthcare workers’ recommendations on personal hygiene maintenance in newborns during
umbilical cord stump healing
Methods and/or recommendations
H C-HNS PPCP Total
(n=22) (n=55) (n=33) (n=110)
While cleaning the baby
30
20
50
on a changing mat avoid
0
(54.5) (60.6) (45.5)
wetting the UC stump
While washing the baby in
8
25
11
44
a bath tub avoid wetting the
(36.4) (45.5) (33.3) (40.0)
UC stump
While washing the baby
1
15
14
under running water avoid
0
(63.6)
(3.0) (13.6)
wetting the UC stump
While washing the baby in
1
1
a bath tub the UC stump
0
0
(3.0) (0.9)
may get wet
p
0.000
0.605
0.000
0.500
UC, umbilical cord; H,hospitals; C-HNS, community-health nursing
services; PPCP, pediatric primary care practices
The majority of respondents, 104 (94.5%), agreed
that there was a necessity to create written guidelines on newborn umbilical cord care, whereby
six of them (5.5%) were of different opinion
(p=0.004).
As the most frequent reason for creating written
guidelines on newborn umbilical cord care the
respondents, 87 (76.4%), reported decrease and/
or elimination of parental confusion regarding
differences in umbilical cord care practices
among healthcare workers of different healthcare
institutions, (p=0.668) (Table 4).
Table 4. The reasons for the necessity of creating written
guidelines on newborn cord care practices
Reasons
It would decrease and/or
eliminate parental confusion regarding differences in
UC care practices among
healthcare workers of different healthcare institutions
It would increase the
sense of confidence and job
satisfaction of healthcare
workers (when I know
precisely what to do/ how
to do it)
It would decrease and/or
eliminate healthcare workers confusion regarding
differences in UC care
practices
It would reduce the risk
of newborn UC stump
infection
It would rationalize the
resources used during UC
stump healing (number of
community-health nurse
visits, medical supplies for
UC care)
H C-HNS PPCP Total
(n=22) (n=55) (n=33) (n=110)
p
16
41
27
84
0.668
(72.7) (74.5) (81.8) (76.4)
7
33
26
66
0.003
(31.8) (60.0) (78.8) (60.0)
13
27
22
62
0.263
(59.1) (49.1) (66.7) (56.4)
13
18
18
49
0.042
(59.1) (32.7) (54.5) (44.5)
6
16
16
38
0.130
(27.3) (29.1) (48.5) (34.5)
UC, umbilical cord; H,hospitals; C-HNS, community-health nursing
services; PPCP, pediatric primary care practices
More than half of the respondents, 63 (57.3%),
declared that the above-mentioned guidelines should be coordinated at the national level,
(p=0.000) (Table 5).
Table 5. The necessary level of coordination concerning the
written guidelines on newborn umbilical cord care
Level
H C-HNS PPCP Total
(n=22) (n=55) (n=33) (n=110)
National level
3
31
29
63
0.000
(13.6) (56.4) (87.9) (57.3)
Local level (H with satellite
C-HNS and PPCP)
18
24
(81.8) (43.6)
Level of particular healthca1
re institution
(4.5)
0
p
3
45
0.000
(9.1) (40.9)
1
(3.0)
2
0.248
(1.8)
H,hospitals; C-HNS, community-health nursing services; PPCP,
pediatric primary care practices
The majority of respondents, 108 of them
(98.2%), declared that there was a necessity of
creating and coordinating the guidelines for other
newborn care practices (p=0.718).
DISCUSSION
The limitation of the study is that it was done only
in one area of the Republic of Croatia. However,
this kind of research in this area of nursing practice has no one tested so far and this paper is a
good indicator of diversity among health professionals involved in the care of newborns’ navel. Healthcare workers involved in conducting
umbilical cord care practices in Osijek-Baranja
County, employ and recommend more different
newborn cord care practices, depending on the
healing phase. Recommendations to parents on
cord care practices are not in line with the WHO
guidelines on umbilical cord care in a non-hospital setting, which recommend keeping the cord
stump/wound dry and clean, without application
of any topical agents (3,5).
The majority of our respondents conduct and/
or recommend maintaining of newborn personal hygiene without wetting the umbilical cord
stump/wound because they consider it would reduce risk of cord stump/wound infection, which
is not in line with the research results that contradict that point of view (5, 13-16).
The availability of written guidelines for healthcare workers on newborn umbilical cord care at the
institutional level is unanimously confirmed only
by a small number of respondents coming from
one and the same hospital, by enclosing a copy of
their written guidelines. The results are similar
to the research conducted in Scotland where the
larger hospital departments share a tendency of
having written guidelines (9). Ireland et al. stress
that the non-existence of guidelines for abovementioned practices is a disappointing fact in the
age of evidence-based practice (9).
The research results show that almost half of the
mothers after being discharged from the hospital are not able to recollect on verbal instructions
on umbilical cord care practices in a domestic
setting, i.e. only a small number of mothers claim
that they have never got the aforementioned recommendations (10). The availability of written
recommendations for parents on umbilical cord
care in a domestic setting is confirmed by only a
187
small number of respondents in this research, i.e.
respondents coming from one pediatric primary
care practice and only a half of the employees
of the above mentioned hospital. However, the
availability of written recommendations for parents at the hospital level is disputable for many
reasons: aside from the fact that the copy of the
recommendations was not enclosed, the question remains how it is possible that more than half
of those respondents had no knowledge of the
existence of the aforementioned recommendations. The listed results are the same as the ones
obtained in a research conducted in Scotland, in
which also a significant number of respondents
from the same department claim existence/nonexistence of written recommendations (9).
As the most frequent reason for creating written
guidelines on newborn cord care practices, the
respondents highlighted the decrease and/or elimination of parental confusion regarding differences
in umbilical cord care practices among healthcare
workers. The obtained results confirm the research
results of Ford and Ritchie, who stress the fact that
different recommendations on cord care practices
presented by healthcare workers impose an additional source of parental concern (10).
The majority of respondents in this research
agree that there is a necessity for creating written
guidelines on umbilical cord care and other
newborn care practices (umbilical granuloma treatment with silver nitrate, methods and frequency
of bathing, as well as the use of personal hygiene
products), whereby more than a half of respondents believe that guidelines on umbilical cord
care should be coordinated at the national level,
which is in accordance with the research conducted in Scotland (9).
With the availability of evidence-based clinical
guidelines providing specific recommendations
for nursing practice and consequently – multiple
benefits for patient and healthcare workers, it is
to be expected that nursing based on experience,
tradition or autonomy of authority is far behind
us. However, the presented research results show
that our nursing practice still lives in the past.
Among healthcare workers involved in conducting umbilical cord care practices in Osijek-Baranja County there are statistically significant differences in methods of cord care conduction and
recommendations to parents on cord care practices during stump/wound healing, in relation to
the workplace (hospitals, community-health nursing service, pediatric primary care practices).
The results emphasize the necessity of creating
written guidelines on umbilical cord care and
other newborn care practices, whereby the guidelines on umbilical cord care must be coordinated
at the national level.
FUNDING
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Razlike u njezi pupka novorođenčadi
Sanja Kanisek 1, Nada Prlić2, Ivana Barać2, Lorna Dubac Nemet2
Patronažna služba, Dom zdravlja Osijek, 2Studij sestrinstva, Medicinski fakultet, Sveučilište Josipa Jurja Strossmayera u Osijeku;
Osijek, Hrvatska
1
SAŽETAK
Cilj Ispitati pojavnost različitih načina provedbe i preporuka o njezi pupka te potrebu oblikovanja i
usuglašavanja smjernica o njezi pupka i drugih postupaka u njezi novorođenčadi.
Metode U istraživanju je sudjelovalo 110 zdravstvenih djelatnika na odjelima za novorođenčad u dvije
opće bolnice, 6 patronažnih službi i 16 pedijatrijskih ordinacija u istočnoj Hrvatskoj. Upitnikom koji je
kreiran za potrebe ovog istraživanja ispitivana je različitost načina provedbe i preporuka roditeljima o
njezi pupka, te potreba oblikovanja i usuglašavanja smjernica o njezi pupka i drugih postupaka u njezi
novorođenčadi.
Rezultati Utvrđene su statistički značajne razlike među skupinama ispitanika u tri načina njege „suhog“
(p=0.000, p=0.002 i p=0.004) i tri načina njege „vlažnog“ pupka (p=0.000, p=0.001 i p=0.000). Također su utvrđene statistički značajne razlike u tri vrste preporuke roditeljima o njezi „suhog“ (p=0.000,
p=0.000, i p=0.002) i dvije vrste preporuke o načinu njege „vlažnog“ pupka (p=0.000, p=0.000).
Većina ispitanika, 104 (94.5%), suglasna je u potrebi oblikovanja pisanih smjernica o njezi pupka, kao
i drugih postupaka u njezi novorođenčadi, 108 (98.2%). Više od polovice ispitanika, 63 (57.3%), izjasnilo se kako je smjernice o njezi pupka potrebno usuglasiti na nacionalnoj razini.
Zaključak Zdravstveni djelatnici u istočnoj Hrvatskoj na različite načine provode i preporučuju provedbu njege pupka novorođenčadi. Potrebno je oblikovati i usuglasiti smjernice o njezi pupka na nacionalnoj razini, kao i smjernice za druge postupke u njezi novorođenčadi.
Ključne riječi: smjernice, zdravstveni djelatnici
189
ORIGINAL ARTICLE
Effects of perioperative statin treatment on postoperative atrial
fibrillation and cardiac mortality in patients undergoing coronary
artery bypass grafting: a propensity score analysis
Ayşegül Kunt1, Sedat Özcan2, Aslihan Küçüker3, Dolunay Odabaşi1, Alper Sami Kunt1
Department of Cardiovascular Surgery, School of Medicine, 100.Yil University, Van, 2Department of Cardiovascular Surgery, School of
Medicine, 18 Mart University, Çanakkale, 3Department of Cardiac Surgery, Ataturk Education and Research Hospital, Ankara; Turkey
1
ABSTRACT
Aim To evaluate the effect of perioperative statin treatment on postoperative atrial fibrillation and cardiac mortality in patients undergoing coronary artery bypass grafting.
Methods A total of 1890 patients who underwent isolated coronary artery bypass were analyzed retrospectively, of which 425
patients (22.4%) older than 70 were included in the study. The
demographic properties, preoperative, operative and postoperative
data and other medications of these patients were recorded. Continuous preoperative and postoperative atorvastatin therapy were
received by 124 (29.17%) patients; 301 (70.82%) patients were
matched to a control group (no-statin group). The two groups were
matched by propensity score analysis in terms of atrial fibrillation
development and cardiac mortality.
Sedat Özcan
Department of Cardiovascular Surgery,
School of Medicine, Canakkale 18 Mart
University Çanakkale, Turkey
Phone: +90 286 2620105
Fax: +90 286 2635956
20 January 2015;
Revised submission:
13 March 2015;
Accepted:
16 March 2015.
doi: 10.17392/796-15
Med Glas (Zenica) 2015; 12(2): 190-195
190
Results Medical history, medical treatment, cardiovascular history, and operative characteristics demonstrated significant heterogeneity in both groups. Postoperative atrial fibrillation was similar
in both groups. Before propensity score matching, the percentages
of patients in postoperative atrial fibrillation with respect to Atorvastain-group and No-statin-group were 13.71 and 10.3 respectively; however, those were 13.71 and 14.51 after matching. In
a multivariate regression analysis, five-vessel bypass (odds ratio
OR, 2.354; 95% confidence interval CI, 0.99 to 5.57) was an independent predictor of postoperative atrial fibrillation in patients
undergoing coronary artery bypass grafting. In-hospital mortality
was higher in the Atorvastatin-group compared with the No-statingroup: 124 (8.9%) versus 301 (3.7%), respectively; p=0.027).
Conclusion Perioperative atorvastatin treatment is not found to be
associated with reduced postoperative atrial fibrillation and cardiac mortality in patients undergoing isolated coronary artery bypass
grafting above the age of seventy years.
Key words: preoperative statin therapy, coronary artery bypass
grafting, postoperative atrial fibrillation, cardiac mortality, propensity score analysis
Kunt et al. Preoperative statin treatment
INTRODUCTION
Atrial fibrillation (AF) occurs in 16% to 33% of
patients undergoing coronary artery bypass grafting (CABG) (1,2). Patient age is one of the most
important risk factors postoperative AF, more
than 50% for patients older than 80 years undergoing CABG [3]. Moreover, a variety of pharmacological agents (4, 5) are thus commonly used
to prevent AF after CABG. It is suggested that
statins are associated with reduced risk of postoperative atrial fibrillation (6). In Atorvastatin
for Reduction of Myocardial Dysrhythmia After
Cardiac Surgery (ARMYDA-3 ) trial, preoperative atorvastatin treatment 7 days before CABG
resulted in 61% decrease for the new-onset atrial
fibrillation compared with placebo (35% vs 57%,
respectively; p=0.003) (7). Postoperative AF increases hospital mortality (1, 8). Almassi and his
colleagues first reported that 6-month survival
after cardiac surgery decreased in patients affected by postoperative AF compared with patients
without it (9.4% vs 4.2%, respectively) (9).
We retrospectively evaluated whether perioperative atorvastatin treatment was associated with
effective prevention of postoperative atrial fibrillation and cardiac mortality in patients aged over
seventy years after CABG, as well as two different doses and duration of atorvastatin treatment
in this surgical cohort.
Data collection and patient selection
We retrospectively evaluated consecutive 1890
patients (above the age of 70) attended to the Ankara Ataturk Education and Research Hospital,
Cardiovascular Surgery Department from June
2004 to April 2012 and who underwent CABG.
A total of 425 eligible patients were selected for
the study . All patients who would have the coronary bypass surgery and no AF before surgery
were included in this study. Patients with documented preoperative AF and associated cardiac
surgery were excluded from the study. Routine
electrocardiograms were obtained before the operation, on admission to the intensive care unit, for
the first 72 hours after the surgery and every day
thereafter until hospital discharge.
Patients were divided into two groups: Atorvastatin Group (n=124, 29.17%) and No-statin Group
(n=301, 70.82%). Patients in the Atorvastatin gro-
up received two different doses (20 and 40 mg) of
atorvastatin starting within several weeks before
the operation and throughout the postoperative period. The two groups were matched by propensity
score analysis in terms of atrial fibrillation development and cardiac mortality.
Primary and secondary end points
The primary end points were the development
of postoperative AF and cardiac mortality during the hospital stay. Postoperative AF was indicated as lasting for more than 10 minutes with
symptoms. Cardiac mortality was defined as inhospital-mortality. Secondary end points were
dose and duration of atorvastatin. Dose table for
usage of atorvastatin was classified as four different categories: 10 mg, 20 mg, 40 mg and 80 mg.
Time table for atorvastatin was analyzed according to the duration of the statin usage: ≤ 1 week,
1-2 weeks and > 2 week.
In our institution, we prefer to use amiodarone in
the management of postoperative AF after coronary
surgery, electrical cardioversion being reserved
only in those patients unresponsive to amiodarone
treatment or having hemodynamic compromise.
Considering embolic events, our routine practice
was to start anticoagulation treatment with enoxaparin in all patients postoperatively. Operative procedure
All patients underwent isolated CABG surgery
and performed using conventional procedures.
Induction and maintenance of anesthesia were
similar for all patients and consisted of fentanyl,
midazolam and pancuronium bromide. All operations were done with a median sternotomy incision. Cardiopulmonary bypass was performed
in a standard fashion with the use of a hollow
fiber membrane oxygenator (Dideco; Sorin Group, Mirandola, Italy) and a roller pump (Stöckert; Sorin Group, Mirandola, Italy), with high
ascending aortic cannulation added to right atrium cannulation. Cardioplegic arrest was achieved with cold blood cardioplegia infused into
the ascending aorta. Moderate temperature was
about 32 ºC. Distal anastomosis was performed
under the cross-clamp and proximal anastomosis
was completed under the side-clamp. Intra-aortic ballon counterpulsation was inserted into the
patients who had hemodynamic instability in the
perioperative period.
191
Variables with a signiﬁcance level of less than
0.2 were entered into a multivariable logit regression model and predictors of atorvastatin-group membership were identiﬁed. The propensity
score matching approach is an alternative approach to address the potential selection bias (endogeneity) in the treatment effects. Predictors for
inclusion in the atorvastatin-group, as identified
by multivariate regression analysis of co-variables, followed by logistic regression, were used
to create the propensity score model to adjust
outcomes. Univariate analysis of all patients
was also done for the development (or not) of
postoperative atrial fibrilation (PAF). Variables
that were found to have a value of p<0.20 in the
univariate analysis were examined by multivariate logistic regression to determine predictors of
PAF. Logistic regression with forward elimination determined the most important denominators
for the development of PAF or not.
Student t-test requires that the continuous variable should be normally distributed, but MannWhitney U test does not assume that the contiTable 1. Demographic variables for atorvastatin and no statin
groups
Variables*
Age
Females
Diabetes Mellitus
Hypertension
Chronic obstructive pulmonary
disease
Creatinine
Other medications
ß-blockers
Angiotension converting
enzyme inhibitors
Calcium channel blockers
Cardiac status
Class III-IV angina
New York Heart Association
class III-IV
Previous myocardial infarction
Ejection fraction <0.50
Body surface area
Emergency
Cross-clamp time (minutes)
Cardiopulmonary bypass time
(minutes)
Inotropic support
Intra-aortic balloon counterpulsation
Exitus
Atorvastatin-Group
(n=124)
No-statinGroup
(n=301)
p
74.4±3.2
86 (69.4%)
42 (33.9%)
80 (64.5%)
73.99±3.78
189 (62.8%)
110 (36.5%)
174 (57.8%)
0.056
0.198
0.601
0.200
30 (24.2%)
69 (22.9%) 0.778
1.21±0.7
1.39±2.9
0.331
104 (83.9%) 210 (69.8%) 0.003
66 (53.2%)
93 (30.9%) <0.001
26 (21%)
32 (10.6%) 0.005
69 (55.6%)
77 (25.6%) <0.001
21 (16.9%)
64 (21.2%)
13 (10.5%)
34 (27.4%)
1.76±0.19
3 (2.4%)
48.23±12.8
51 (16.9%) 0.091
50 (16.6%) 0.011
1.73±0.17 0.138
2 (0.7%) 0.151
45.7±14.1 0.020
0.311
82.1±30.8
77.2±41.2
0.003
80 (64.5%)
256 (85%) <0.001
25 (20.2%)
24 (8%)
<0.001
11 (8.9%)
11 (3.7%)
0.027
*Categorical data are numbers (percentage); continuous data are
means ± standard deviation.
192
nuous variable is normally distributed. It only
assumes that the variable is at least ordinal. Continuous and/or ordinal variables in our data do
not satisfy the normality. Then, Mann-Whitney
U test was used for continuous and/or ordinal
variables to compare two groups. Chi-square test
assumes the expected value of each cell is five
or higher, but the Fisher’s exact test has no such
assumption and can be used regardless of how
small the expected frequency is. Then, chi-square test was used for categorical variables if the
expected value of each cell is five or higher in the
data. In addition, Fisher’s exact test was conducted in the case of violations of this assumption for
some categorical variables in our data. Values of
p<0.05 were considered statistically significant.
RESULTS
Patients’ characteristics
The mean patient age was 74.4±3.2 years in Atorvastatin-Group versus 73.99±3.78 years in Nostatin-Group at the time of surgery (p=0.056). Females surprisingly were higher in both groups; 86
(69.4%) patients in Atorvastatin-Group versus 189
Table 2. Demographic variables for all patients with and
without atrial fibrillation (AF)
Variables*
AF (n=48) no-AF (377)
Age
Females
Diabetes Mellitus
Hypertension
Chronic obstructive pulmonary
disease
Creatinine
Other medications
ß-blockers
Angiotension converting enzyme
inhibitors
Calcium channel blockers
Cardiac status
Class III-IV angina
New York Heart Association class
III-IV
Previous myocardial infarction
Body surface area
Emergency
74.67±3.77
30 (62.5%)
11 (22.9%)
32 (66.7%)
Cross-clamp time (minutes)
Cardiopulmonary bypass time
(minutes)
Inotropic support
Intra-aortic balloon counterpulsation
Exitus
74.04±3.60
245 (65%)
141 (37.4%)
222 (58.9%)
p
0.209
0.734
0.049
0.301
13 (27.1%) 86 (22.8%)
0.510
1.29±0.77
0.963
0.872
35 (72.9%)
1.35±2.57
279 (74%)
18 (37.5%) 141(37.4%) 0.989
9 (18.8%)
49 (13%) 0.274
21 (43.7%) 125 (33.2%) 0.145
10 (20.8%) 75 (19.9%)
0.878
7 (14.6%)
12 (25%)
1.73±0.16
1 (2.1%)
49.57
(11.14%)
0.922
0.334
0.479
0.452
57 (15.1%)
72 (19.1%)
1.74±0.18
4 (1.1%)
46.06
(13.99%)
0.052
80.23±17.57 78.42±40.4 0.054
38 (79.2%) 298 (79.1%) 0.984
8 (16.7%)
41 (10.9%)
0.237
1 (2.1%)
11(2.9%)
0.082
*Categorical data are numbers (percentage); continuous data are
means ± standard deviation
Table 3. Propensity score models for atorvastatin and atrial
fibrillation: multivariate logistic regression, stepwise forward
elimination
Variables*
Age
Females
Hypertension
Previous cerebrovascular accident
Previous Urea
ß-blockers
Angiotension converting enzyme inhibitors
Calcium channel
blockers
Class III-IV angina
Previous myocardial
infarction
Three vessel disease
Bilateral carotid
stenosis
Constant
ACE inhibitors
ß-blockers
Calcium channel
blockers
Class III-IV angina
Bilateral carotid
stenosis
Constant
Five-vessel bypass
Constant
ExpoLogit
Std. nential
coeffici95 % CI
p
error† coefficient
ent
For atorvastatin
0.024 0.031 1.025 0.97-1.09 0.424
0.445 0.261 1.561 0.94-2.60 0.087
0.031 0.253 1.032 0.63-1.69 0.902
0.421
0.487
1.524 0.59-3.96 0.387
0.001
0.927
0.005
0.316
1.001 0.99-1.01 0.855
2.527 1.36-4.69 0.003
0.878
0.247
2.407 1.48-3.91 <0.001
0.84
0.334
2.321 1.20-4.47 0.012
1.305
0.249
3.688 2.26-6.01 <0.001
-0.235
0.374
0.791 0.38-1.65 0.531
0.156
-1.576
0.295
0.574
1.169 0.66-2.08 0.598
0.207 0.07-0.64 0.006
0.864
0.437
2.373 1.01-5.58 0.048
-4.793
0.876
0.820
2.343
0.243
0.309
0.008
0.041
2.402 1.49-3.87 <0.001
2.271 1.24-4.16 0.008
No-statin-Group at the time of surgery (p=0.209)
(Table 3).
Propensity score analysis for atorvastatin and for
AF has shown that three-vessel disease (Logit coefficient: -1.553, Exponential coefficient: 0.212,
95 % confidence interval: 0.05-0.90, p=0.035)
and five-vessel bypass (Logit coefficient: 0.856,
Exponential coefficient: 2.354, 95 % confidence
interval: 0.99-5.57, p=0.050) were found as an
independent predictor for the development of AF
(Table 4).
Table 4. Dose and time of atorvastatin for Atorvastatin group
AtorvastatinGroup (n=124)
AF
Adjusted
OR*
CI*
p*
0.780
0.311
2.182 1.19-4.02 0.012
Dose
10
20
40
80
Time
≤ 1 week
1-2 weeks
> 2 weeks
1.356
-1.757
0.248
0.552
3.882 2.39-6.31 <0.001
0.173 0.59-0.51 <0.001
*adjusted for propensity score; AF, atrial fibrillation; CI, confidence
interval
0.978
0.414
2.660 1.18-5.99 0.018
Cardiac mortality
-2.497 0.347 0.0823
<0.001
For atrial fibrillation
-1.553 0.738 0.212 0.05-0.90 0.035
0.856 0.439 2.354 0.99-5.57 0.050
-2.006 0.172 0.135
<0.001
*
†Robust standard errors.
(62.8%) patients in No-statin-Group (p =0.198)
Atorvastatin-40 mg was used in over half of the
patients, used in 68 (54.83%) patients who took
atorvastatin. Of the patients, postoperative AF occurred in 9 (13.2%) patients (Adjusted OR=0.82;
confidence interval=0.32-2.06; p=0.669). Nearly
half of the patients (n=58, 46.77%) used atorvastatin between 1-2 weeks before surgery. Of the
patients, postoperative AF occurred in 6 (10.3%)
patients in this period (Adjusted OR=0.53; confidence interval=0.18-1.56; p=0.238) (Table 2).
Postoperative atrial fibrillation
Patient characteristics, procedural variables, and
postoperative characteristics for patients with
and without were similar. Postoperative AF occurred in 48 (11.29%) patients in all patients. The
mean patient age for AF was 74.67±3.77 years
in Atorvastatin-Group versus 74.04±3.60 years in
23 (18.54%)
31 (25%)
68 (54.83%)
2 (1.61%)
3 (10.3%)
4 (12.9%)
9 (13.2%)
1(50%)
0.89
0.90
0.82
4.00
0.29 - 2.79
0.23 – 3.52
0.32 - 2.06
0.21 - 74.89
42 (33.87%)
58 (46.77%)
24 (19.35%)
6(14.3%) 0.87
0.34-2.20
6 (10.3%) 0.53
0.18-1.56
5 (20.8%) Şub.66 0.78-9.13
0.854
0.884
0.669
0.316
0.765
0.238
0.106
Cardiac death occurred in 22 (5.2%) patients.
Although mortality was statistically significant
in the no-statin-group comparing to statin group,
the reasons of mortality were independent from
the statin therapy. The reasons of the mortality
were low cardiac output (1.4%), unable to wean
cardiopulmonary bypass (0.7%), respiratory failure (0.7%), major cerebrovascular event (0.47%),
gastrointestinal bleeding (0.47%), intestinal ischemia (0.47%), failure of left internal thoracic artery
(0.47%), ventricular fibrillation (0.23%), and bleeding from the mediastinal space (0.23%).
DISCUSSION
Meta-analyses have demonstrated that some
pharmacological agents and biatrial pacing reduced postoperative AF (10,11). Only amiodarone
and beta-blockers have shown to be effective for
the management of postoperative AF as advised
recently by the American College of Cardiology/
American Heart Association/European Society of
Cardiology guidelines. Subsequently, some studies reported that there is a relationship between
preoperative statin use and reduced postoperative
AF (12-14).
193
Relationship between preoperative statin use and
rates of reduced postoperative was first reported
by ARMYDA-3 trial (7). Two hundred patients
who underwent coronary bypass were randomized
to either atorvastatin (40 mg/d, n=101) or placebo
(n=99) starting 7 days preoperatively. There was a
61% reduction in postoperative AF in patients who
received statins; AF occurred in 35% of patients
with statins and 57% of patients with placebo
(p=.017). However, some authors have suggested
that a dose–response relationship with statins and
postoperative AF may also exist (15-17). Lertsburapa et al (15) conducted a nested case–control
study with data from the randomized, controlled
Atrial Fibrillation Suppression Trials I, II, and III
and found that higher statin doses (atorvastatin
40 mg/d) were associated with greater reduction
in postoperative AF than were lower doses. This
finding was later corroborated by observational
studies conducted by Kourliouros (16) and Mathani (17) et al. One of the findings of our study was
that preoperative statin treatment was not associated with the reduction of postoperative AF in
the patients. The other one was that there was no
relationship between dose and duration of statin
therapy for the development of postoperative AF
in this specific surgical cohort.
As it is known, advanced age is a major risk factor for the development postoperative AF after
cardiac surgery (3,18,19). Levy and colleagues
(20) suggested that age is a very powerful predictor of postoperative AF. Actually, there is limited
data whether statin treatment has beneficial effects in advanced age on postoperative AF after
the coronary surgery. The Multicenter Study of
Perioperative Ischemia Research Group and investigators of the Ischemia Research and Education Foundation have published the prospective
study performed in 70 hospitals on 4 continents
(1) including more than 5,000 patients undergoing CABG operations with or without valve surgery on cardiopulmonary bypass: patients with
postoperative AF were significantly older (67.8
years versus 61.8 years), and a significantly larger
number had a history of AF (14.6% versus 6.0%).
The incidence of postoperative AF was 11.29%
in the present study population after coronary artery bypass grafting.
Postoperative AF is thought to be mostly benign
(21,22), it increases late mortality after isolated
194
coronary surgery only (23). Kalavrouziotis and coworkers (24) concluded the same in a large study on
postoperative AF in cardiac surgery patients after
multivariate analysis and propensity score matching. When considering early cardiac mortality, it is
really hard to combine the effects of these two clinical positions on cardiac death: preoperative statin
therapy and postoperative AF. In the study of Villareal and colleagues (25), postoperative AF was a
significant predictor of early death after adjusting
risk factors. A large number of studies (3,8,9,22,24)
show significantly higher incidence of early death
in patients with postoperative AF after coronary
bypass surgery or cardiac operations, but none
of these studies identified postoperative AF as an
independent predictor of early mortality. In the
present study we have reported in-hospital cardiac
mortality of 8.9% in the Atorvastatin-Group versus
3.7% in the No-statin-Group (p=0.027); cardiac
death in patients with and without AF occurred at
approximately 2.1% in the Atorvastatin-Group versus 2.9% in the No-statin-Group.
Despite a small number of patients in the present
study, we cannot rule out widespread use of statins in patients undergoing coronary artery bypass
grafting to prevent postoperative atrial fibrillation. We cannot exclude the possibility that ß-blockers and ACE inhibitors attenuated the benefits in
our study or that the results were due to chance
or population differences. Two groups were not
homogenous. No-statin-Group had higher patient
population compared to Atorvastatin-Group.
Over a half of the patients were females in contrast to standard population of coronary surgery.
Postoperative atrial fibrillation is a frequent complication of cardiac operations and may result in
serious cardiac adverse events including cardiac
mortality. Although further work is necessary
before any definitive recommendation, omitting
statin drugs is not found to be associated with
reduced postoperative atrial fibrillation and cardiac mortality in patients undergoing isolated
coronary artery bypass grafting above the age of
seventy years in the perioperative period.
FUNDING
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195
ORIGINAL ARTICLE
Efficacy of chronic statin therapy on major cardiac events after
coronary artery bypass grafting: low-dose versus high-dose
Ayşegül Kunt1, Sedat Özcan2, Aslihan Küçüker3, Dolunay Odabaşi1, Alper Sami Kunt1
Department of Cardiovascular Surgery, School of Medicine, 100.Yil University, Van, 2Department of Cardiovascular Surgery, School of
Medicine, 18 Mart University, Çanakkale, 3Department of Cardiac Surgery, Ataturk Education and Research Hospital, Ankara; Turkey
1
ABSTRACT
Aim To investigate whether chronic statin treatment after coronary artery bypass grafting (CABG) protects patients from major
cardiac events and provides percutaneous coronary intervention
(PCI) free survival.
Methods A total of 232 patients with previous CABG and chronic
statin therapy were selected retrospectively and were divided into
two groups according to a dosage of atorvastatin per day, e. g., 20
mg or 40 mg. Groups were compared for the major cardiac events
and freedom from PCI by Kaplan Meier analysis as the primary
end point. Patency of grafts including left internal thoracic artery
(LITA) and saphenous vein (SVG) and progression of non-grafted
native vessel disease were also evaluated as secondary end points.
Sedat Özcan
Department of Cardiovascular Surgery,
School of Medicine, Canakkale On Sekiz
Mart University
Kepez, 17100 Çanakkale, Turkey
Phone: +90 286 262 0105;
Fax: +90 286 263 5956;
21 January 2015;
Revised submission:
13 March 2015;
Accepted:
18 March 2015.
doi: 10.17392/795-15
Med Glas (Zenica) 2015; 12(2): 196-201
196
Results Cardiac mortality, periprocedural myocardial infarction (MI), target vessel revascularization and percutaneous coronary intervention free survival were as follows: 2.9% versus
2.1% (p=1.000); 16.1% versus 21.1% (p=0.331); 56.93% versus
52.63% (p>0.005); 58.4% versus 63.2% (log-rank test; p= 0.347)
in atorvastatin 20 mg and atorvastatin 40 mg groups, respectively.
However, these results were not statistically significant between
two groups (p>0.005). Patency of openness of grafts including
LITA and SVG and progression of non-grafted native vessel disease were similar between groups (p=0.112, p=0.779, p=0.379 and
p=0.663, respectively).
Conclusion Low-dose long-term statin treatment had similar
outcomes on major cardiac events and identical rate of freedom
from percutaneous coronary intervention after coronary artery
bypass grafting compared with high-dose long-term statin treatment. It is better to start from low dose statin treatment after
surgical interventions.
Key words: chronic statin treatment, major cardiac events, coronary bypass grafting
Kunt et al. Major cardiac events after coronary bypass
INTRODUCTION
Percutaneous coronary intervention
Coronary artery bypass grafting (CABG) is a
therapeutic choice for advanced coronary artery
disease. However, patients are still at significant
risk for postoperative major cardiac events (1).
Although statins protect patients from subsequent coronary ischemic events, optimal selection
of drug and ideal dosage are still open to debate (2-3). Moreover, it is not clear whether longterm statin therapy would have improved clinical
outcomes in patients after CABG (3).
Percutaneous coronary interventions were performed with the standard technique; patients
were pre-treated with aspirin (100 mg/day) and
clopidogrel (600-mg loading dose at least 3 h before the procedure) (1-3). Following PCI, aspirin
(100 mg/day) was continued indefinitely, whereas clopidogrel (75 mg/day) was administered for
at least 1 month (12 months in patients treated
for acute coronary syndrome or receiving stents).
The aim of this study is to show low-dose versus
high dose of non-stop atorvastatin therapy after
CABG would keep the patients away from major cardiac events and provide PCI-free survival.
Cardiac death, periprocedural myocardial infarctus (MI), target vessel revascularization and
percutaneous coronary intervention (PCI), free
survival were considered as primary end points.
Patency of grafts including left internal thoracic
artery (LITA) and saphenous vein graft (SVG)
and progression of coronary ischemic disease in
native coronary arteries after CABG were also
evaluated.
Patient selection
This study was performed retrospectively. A total number of 13,558 patients who underwent
conventional coronary angiography at Ankara
Ataturk Education and Research Hospital between 2009 and 2012 (inclusion criteria were stable
angina and indication to coronary angiography,
ST and non–ST-segment elevation acute MI;
exclusion criteria were bypass of left anterior
descending artery other than LITA, free LITA)
were initially reviewed for patients with previous
CABG. Patients were then reviewed for postoperative PCI. A total number of 289 patients fulfilling the inclusion criteria were re-evaluated; 57
patients (19.7%) met the exclusion criteria: not
on statin therapy (n=35, 12.1%), for free LITA
(n=13), bypass of left anterior descending artery
other than LITA (n=9). Finally, 232 patients with
previous CABG and postoperative PCI and chronic statin therapy represented the study population. These eligible patients were divided into two
groups according to the statin dose: as 20 mg or
40 mg atorvastatin per day
Primary end points
Cardiac death was defined as in-hospital 30-day
mortality. Periprocedural MI was evaluated as
elevated cardiac biomarkers (troponin or CKMB) and electrocardiographic findings. Target
vessel revascularization was defined as PCI,
which was divided into 3 separate periods and
target vessels. Target vessel non-revascularization was described as PCI-free survival.
Secondary end points
Stenosis of over 80% of LITA and over 60% of
SVG were evaluated as occluded. Progression of
the coronary ischemic disease in native coronary
arteries was classified according to the degree of
the stenosis. Stenosis was classified as over and
above 50%. These native coronary arteries had
not been bypassed earlier.
Univariate analysis was performed to examine
differences in variables between patients receiving atorvastatin 20 and 40 mg groups. Categorical variables between groups were evaluated by
using the chi-square test or Fisher’s exact test.
Continuous variables were conducted by using
Mann-Whitney U test. Value of p <0.05 was considered statistically significant. Moreover, PCIfree survival analysis was performed by KaplanMeier analysis and compared by the log rank test
in both groups. Stent insertion was considered as
the failure event (hazard) and no-stent insertion
(PCI-free) was considered as the survival.
RESULTS
Patients’ characteristics
Both groups were similar at the time of PCI,
except for recent ex-smoker and using clopido-
197
grel. The group of atorvastatin-40 mg had higher
recent ex-smoker (patients who used to smoke but quitted recently) and taking clopidogrel
(p<0.001 and p=0.004 respectively) (Table 1).
Operative characteristics, follow-up, patency
of grafts and stenosis of non-grafted native coronary arteries and PCI of target vessel(s) were
similar for both groups (Tables 3-5 )
Table 1. Demographic characteristics and other medical
therapy of atorvastatin-20 mg and atorvastatin-40 mg groups
During the follow-up, conventional coronary
angiography was performed in all patients at the
first hospitalization, in 43 patients at the second
hospitalization and in five patients at the third
hospitalization (Table 3).
Variables
Age
Females
Diabetes mellitus
Hypertension
Chronic obstructive
pulmonary disease
Peripheral arterial
disease
Dialysis
Total cholesterol
Triglyceride
High density lipoprotein
Low density lipoprotein
Cigarette smoker
Recent ex-smoker
Ex-smoker
ß-blocker
Angiotension converting enzyme
Ca+2 channel
blocker
Nitrate
Clopidogrel
No (%) of patients
Atorvastatin-20 mg Atorvastatin-40
group (n=137)
mg group (n=95)
p
62.69 ± 9.75
33 (24.1%)
54 (39.4%)
72 (52.6%)
62.99 ± 9.09
20 (21.1 %)
30 (31.6 %)
44 (46.3 %)
0.822
0.588
0.222
0.350
10 (7.3%)
4 (4.2 %)
0.409
6 (4.4%)
6 (6.3 %)
0.513
3 (2.2%)
145.91 ± 80.46
154.93 ± 149.82
1 (1.1%)
145.79 ± 79.31
138.39 ± 97.13
0.646
0.963
0.681
31.35 ± 16.7
32.44 ± 16.94
0.664
85.69 ± 53.93
93.54 ± 57.79
0.219
19 (13.9%)
10 (7.3%)
108 (78.8%)
119 (86.9%)
12 (12.6%)
23 (24.2%)
60 (63.2%)
83 (87.4%)
0.785
<0.001
0.009
0.910
97 (70.8%)
77 (81.1%)
0.076
43 (31.4%)
30 (31.6%)
0.975
132 (96.4 %)
68 (49.6%)
91 (95.8%)
65 (68.4%)
0.828
0.004
Cardiovascular profiles of both groups were also
similar, related with the Canadian Cardiovascular
Society classification and unstable angina pectoris (USAP). The group of Atorvastatin-40 mg had
higher Canadian Cardiovascular Society classification and USAP (p=0.005 and p<0.001 respectively) (Table 2).
Variables*
Atorvasta- Atorvastatin-20 Group tin-40 Group
(n=137)
(n=95)
p
Number of distal anastomosis 2.32 ± 0.94
2.4 ± 1.1 0.693
Sequential bypass
18 (13.1%)
15 (15.8%) 0.570
Number of venous graft
1.24 ± 0.94 1.38 ± 1.07 0.388
Number of arterial graft
1.08 ± 0.34 1.04 ± 0.25 0.492
Distal insertion site other than left anterior descending artery
(No (%) of patients)
Diagonal artery
30 (21.9%)
20 (21.1%) 0.878
Optional diagonal artery
1 (0.7%)
3 (3.2%)
0.308
Circumflex artery
82 (59.9%)
61 (64.2%) 0.502
Right coronary artery
69 (50.4%)
45 (47.4%) 0.653
RCAPDA
13 (9.5%)
7 (7.4%)
0.571
RCAPL
1 (0.7%)
1 (1.1%)
1.000
*Categorical data are numbers (percentage), continuous data are
means ± standard deviation. RCAPD, right coronary artery posterior
descending artery; RCAPL, right coronary artery posterolateral artery
Cardiac death occurred in four (2.9%) patients
in the group of atorvastatin-20 mg and in two
(2.1%) patients in the group of atorvastatin-40
mg (p=1.000) (Table 4).
Periprocedural MI happened in 22 (16.1%) patients in the group of atorvastatin-20 mg and in 20
(21.1%) patients in the group of atorvastatin-40
mg (p=0.331) (Table 4).
Table 2. Cardiovascular profile of atorvastatin-20 mg and
atorvastatin-40 mg groups
Table 4. Follow-up of atorvastatin-20 mg and atorvastatin-40
mg groups
No (%) of patients
Atorvasta- AtorvastaVariables*
tin-20 Group tin-40 Gro(n=137)
up (n=95)
CCS III/IV
86 (62.8 %)
76 (80%)
NYHA III/IV
26 (19%)
13 (13.7%)
Chronic heart failure
13 (9.5%)
4 (4.2%)
Cardiogenic shock
5 (3.6%)
0
Cardiopulmonary resuscitation
5 (3.6%)
0
Unstable angina pectoris
44 (32.1 %) 53 (55.8 %)
Stable angina pectoris
64 (46.7 %) 27 (28.4%)
Previous myocardial infarction 18 (13.1%) 22 (23.2%)
Previous PCI
7 (5.1%)
4 (4.2%)
Ejection fraction < 50
62 (45.3%) 43 (45.3%)
Left main coronary artery
18 (13.1%) 13 (13.7%)
Variables*
p
0.005
0.289
0.199
0.080
0.080
<0.001
0.005
0.047
1.000
0.999
0.897
*Categorical data are numbers (percentage); continuous data are means
± standard deviation. CCS, Canadian Cardiovascular Society; NYHA,
New York Heart Association; PCI, percutanous coronary intervention.
198
Table 3. Operative characteristics of atorvastatin-20 mg and
atorvastatin-40 mg groups
Atorvastatin-20 Atorvastatin-40
Group (n=137) Group (n=95)
Number of outpatient
11.03 ± 10.68
control after CABG
Number of hospitalization
1.88 ± 1.44
Time interval
6.49 ± 4.47
Periprocedural myocardial
22 (16.1%)
infarction
Mortality
4 (2.9%)
p
10.48 ± 11.63 0.312
1.79 ± 1.35
6.33 ± 4.58
0.720
0.722
20 (21.1%)
0.331
2 (2.1%)
1.000
*Categorical data are numbers (percentage), continuous data are
means ± standard deviation. CABG, coronary artery bypass grafting
Time interval between CABG and last coronary
angiography was 6.49 ± 4.47 years and 6.33 ±
4.58 years in the group of atorvastatin-20 mg and
40 mg, respectively. The total number of PCI was
56.93% in 78 patients in the group of atorvasta-
tin-20 mg and 52.63% in 50 patients in the group
of atorvastatin-40 mg (p>0.005)
Primary end points
The total number of intervention was 61 (44.52%)
versus 36 (37.89%) during the first procedure, 12
(8.75%) versus 10 (10.52%) during the second procedure and 5 (3.64%) versus 4 (4.21%) during the
third procedure in the group of atorvastatin 20 and
in the group of atorvastatin 40 mg respectively.
PCI-free survival was 58.4% in the group of atorvastatin-20 mg and 63.2% in the group of atorvastatin-40 mg in this period (p=0.347)
Secondary end points
Patent and occluded LITA were observed in 200
(86.2%) and in 32 (13.79%) out of 232 patients
in both groups (atorvastatin-20 mg and atorvastatin-40 mg), respectively (p=0.112). Patent SVG
was visualized in 186 (80.17%) patients of both
groups (p=0.779). Stenosis of circumflex artery
over 50% obstruction was seen in 38 (33.5%) patients (p=0.379) in both groups. Stenosis of right
coronary artery over 50% was seen in 60 (52.2%)
patients (Table 5).
Table 5. Patency of grafts and stenosis of native coronary
arteries of atorvastatin-20 mg and atorvastatin-40 mg groups
Variables*
LITA-patent
LITA-occluded
SVG-patent
Cx >50% stenosis
RCA >50% stenosis
Atorvastatin-20 Atorvastatin-40
Group (n=137) Group (n=95)
114 (83.2%)
23 (16.8%)
109 (79.6%)
20 (14.6%)
34 (24.8%)
86 (90.5%)
9 (9.5%)
77 (81.1%)
18 (18.9 %)
26 (27.4 %)
p
0.112
0.112
0.779
0.379
0.663
*Categorical data are numbers (percentage), continuous data are means
± standard deviation. Cx, circumflex artery; LITA, left internal thoracic
artery; RCA, right coronary artery; SVG, saphenous vein graft
DISCUSSION
Coronary artery bypass grafting is an effective
treatment for patients with coronary artery disease (4,5). However, hyperlipidemia can cause
recurrent ischemic cardiac events in this patient
population in the postoperative period (6). Guidelines by the American College of Cardiology
and American Heart Association recommended
statins for CABG patients with LDL concentrations greater than 100 mg/dL (7). Although statins
reduce major cardiac events after CABG, there is
no available data for PCI-free survival in patients
with long-term statin intake in the postoperative
period. We also sought primarily the influence of
different doses of atorvastatin on major cardiac
events and secondary patency of LITA and SVG
and progression of native vessel disease in longterm period after CABG.
The Post Coronary Artery Bypass Graft Trial was
designed to compare the effects of 2 lipid-lowering regimens in patients who had CABG (8-10).
The primary endpoint had been planned as cardiac death or nonfatal acute MI and found 15.1%
in the aggressive strategy group and 20.3% in the
moderate strategy group. The investigators could
not have shown significant difference between
two strategies in the occurrence of death. We could not show any difference between groups in
the occurrence of cardiac mortality in patients
after CABG either.
The CARE (In the Cholesterol and Recurrent
Events) trial included 1,091 patients (pravastatin
(n=527) and placebo (n=564)) who had previously undergone CABG for a mean period of 5 years
(11). In our study, follow-up period was: 6.49 ±
4.47 and 6.33 ± 4.58 in the Group of Atorvastatin
20 and 40 mg respectively. The authors demonstrated that pravastatin produced a statistically
significant reduction (24%) in the relative risk
of a composite endpoint of fatal coronary event
or nonfatal acute MI relative risk of a composite endpoint of fatal coronary heart disease death
(33%) or nonfatal acute MI (absolute risk 9.1%
versus 12.9% in the placebo group), by 41% of
the incidence of coronary death (4.6% versus
7.8%), and by 35% total mortality (8% versus
12.4%). In the trial, the authors divided MI as
fatal and nonfatal; we reported periprocedural
MI together in our study. The rates of total and
cardiac mortality had been given separately in
the trial. We excluded the death from non-cardiac
reasons in order not to affect the cardiac results.
Some clinical data indicated that pretreatment with
statins may significantly reduce periprocedural
complications and major adverse cardiac events in
patients undergoing PCI (13-15). Patti et al. performed a collaborative meta-analysis using data from
13 randomized studies in which 3.341 patients
received either high-dose statin (n=1.692) or no
statin/low-dose statin (n=1.649) before PCI, with
all patients receiving statin therapy after intervention (16). Our patient population had prior CABG,
which means that patients received statin before
PCI. The authors found that periprocedural MI was
199
lower in the high-dose statin versus control group,
which corresponds to a 44% risk reduction in the
active-treatment arm. They also demonstrated that
major adverse cardiac events within 30 days was
significantly lower in the high-dose statin group ,
and 1-month major adverse cardiac events, excluding periprocedural events, were also reduced: the
results were much more reliable as expected. In
our study, we evaluated not only PCI but also PCIfree survival after surgery; both groups were similar for PCI and PCI-free survival under the statin
treatment after CABG . We thought that PCI-free
survival was higher in Atorvastatin-40 mg, because
this group of patient had a higher incidence of unstable angina pectoris (USAP)
Carrier et al analyzed the benefit of statin treatment on single and bilateral ITA grafts on longterm survival after CABG (17). The paper included 6.655 patients. The authors reporteded that
the patients with bilateral ITA grafts had an average 10-year-survival rate of 83% compared with
67% in patients with single ITA grafts. The authors also demonstrated that statin treatment caused a significant decrease in the long-term risk of
death among patients who underwent single ITA
grafting, but not in those with bilateral ITA grafting. However, they demonstrated that survival of
statin-treated patients with single ITA grafts was
similar to bilateral ITA patients. In our study, we
only evaluated patency of LITA grafting in two
groups separately, showing that patency of ITA
grafting is appropriate with the literature in each
group because over 90% of LITAs remain patent
within 10 years after surgery.
The CASCADE (Clopidogrel After Surgery for
Coronary Artery Disease) trial was designed to
evaluate the addition of clopidogrel (75 mg) to
aspirin (162 mg) on the development of saphenous vein graft disease after CABG. The patients
received statin therapy after operation achieved
an LDL level less than 100 mg/dL (18-20) and underwent angiography of the bypass grafts and the
native coronary arteries; statin therapy was independently associated with improved graft patency.
In our study, patients reached LDL level less than
100 mg/dL in both atorvastatin groups, but without
improved graft patency of SVG between the groups. Our result of patency of SVG is in accordance
with the literature in each group because within 10
years after surgery, only 60% of the SVGs remain
patent and half of those that are patent have clinically important stenosis (5,21,22 ).
CLAS (The Cholesterol Lowering Arteriosclerosis Study) showed that aggressive cholesterol
reduction for a period of 2 to 4 years significantly
reduces new lesion formation in native vessels
(23). In our study, comparing old clinical reports
of coronary angiograms with the last ones, and
evaluating only the previously non-grafted vessels we could not find any difference between the
atorvastatin groups for the stenosis over 50%.
The major limitation of the present study is its retrospective and nonrandomized design. Furthermore, we are unable to obtain the third group of
patients who had not received statins. Of course,
there could be changes in the dosage of statin
throughout the study.
Essentially all patients should be prescribed
long-term statin therapy independent from the
dose to reduce cardiac events after CABG. This
confirms earlier studies within a contemporary
surgical population and supports the current clinical guidelines.
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ORIGINAL ARTICLE
Incidence of endophthalmitis after intravitreal application of anti
VEGF therapy at the University Clinical Center in Tuzla, Bosnia
and Herzegovina
Eye Clinic, University Clinical Centre Tuzla, Tuzla, Bosnia and Herzegovina
ABSTRACT
Aim To report the incidence of endophthalmitis following the use
of intravitreal injection of anti- vascular endothelial growth factor
(anti VEGF) therapy.
Methods In this retrospective study a total of 986 intravitreal
bevacizumab injections were applied between January 2008 and
April 2015 at the University Clinical Center Tuzla, Bosnia and
Herzegovina (B&H). Since January 2012, a total of 55 intravitreal
ranibizumab injections were applied and since October 2014, 60
intravitreal aflibercept injections were applied to patients.
Amra Nadarević Vodenčarević
Eye Clinic, University Clinical Center Tuzla
Trnovac b.b., 75000 Tuzla,
Phone: +387 35 226 416;
Results Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab or
aflibercept. The overall incidence of clinical endophtahlmitis was
0.2%.
Conclusion The results suggest that a low rate of endophthalmitis
can be achieved by means of a protocol. This is a very important
study as it is the first of this kind in B&H that documents the incidence of endophthalmitis after intravitreal application. Currently,
bevacizumab in B&H is most frequently used intravitreal anti-vascular endothelial growth factor due to very low price.
Key words: bevacizumab, aflibercept, ranibizumab, complications of intravitreal application
07 May 2015;
Revised submission:
14 July 2015;
Accepted:
18 July 2015.
doi: 10.17392/818-15
Med Glas (Zenica) 2015; 12(2): 202-205
202
Terzić et al. Endophthalmitis after VEGF intravitreal application
INTRODUCTION
Endophthalmitis is uncommon, but very severe ocular inflammatory process that can lead to
blindness (1). During this process, inflammation
affects vitreous cavity along with the retinal and
uveal components of the eye. After eye surgery or
intravitreal application of anti vascular endothelial growth factor (anti VEGF) post-operative endophtalmitis is possible to occur, and it can have
two forms, either sterile or infectious (2). Since its beginning in 2004, intravitreal injections
of anti VEGF in whole world have had a huge
impact on treatment of variety of ocular conditions (3). The number of intravitreal injections is
increasing in Bosnia and Herzegovina (B&H).
Currently, at the University Clinic Center (UCC)
in Tuzla, B&H, intravitreal injections have become a common route of administration of medications. At UCC Tuzla currently we are applying
intravitreal injections bevacizumab, ranibizumab and aflibercept. This intravitreal injections
are applied for a variety of conditions including
complications of diabetic retinopathy, such as
diabetic macular edema (DME) and as well other
retinal-vascular disorders, such as branch retinal
vein occlusion, central retinal vein occlusion and
as well age related macular degeneration (AMD)
(4-6). At this moment only ranibizumab and aflibercept are labeled for intravitreal use, while
bevacizumab is currently also being used “offlabel” for the treatment of ocular disease. This
off-label intravitreal injections of bevacizumab
have been given for the treatment of neovascular and exudative ocular diseases since May 2005
(7). To our knowledge until today, from February
2004 bevacizumab has been approved by the US
Food and Drug Administration (FDA) for treating patients with metastatic colorectal cancer
(8). All over the world between 1997 and 2001,
fewer than 5,000 intravitreal injections were performed annually, while more than 800,000 were
performed in 2007 (9).
To minimize any complication or infections after
intravitreal application of anti VEGF several protocols have been proposed (10-11). Recent surveys
have suggested that 40% of retina specialists use
topical antibiotics before anti–vascular endothelial
growth factor intravitreal injections, and 86% use
topical antibiotics after anti–vascular endothelial
growth factor intravitreal injections (12).
The aim of this study was to investigate the incidence of endophthalmitis following the use of
intravitreal injection of anti-vascular endothelial
growth factor (anti VEGF) therapy.
University Clinic Center of Tuzla is a single health
institution equipped for the intravitreal application of anti VEGF therapy in north-eastern part of
B&H. This is a single-center retrospective analysis
of all intravitreal application at the UCC Tuzla.
All patients who received intravitreal injections of
bevacizumab, ranibizumab and aflibercept were
included in this study. Patients receiving other intravitreal injections (including corticosteroids or
antibiotics) were excluded. The indications for intravitreal injection included exudative age-related
macular degeneration (AMD), choroidal neovascular membranes secondary to myopic degeneration, cystoid or diffuse macular edema from central
and branch vein occlusions, and as well edema
due to a diabetic complication. All intravitreal
injections were recorded in doctor and nursing
logbooks. In this study provisions of the Helsinki
Declaration were followed.
Before application all the patients who were treated with 0.05 ml injection containing 1.25 mg of
bevacizumab, 0.3 mg /0.05 mL ranibizumab or
2.0 mg/0.05 mL aflibercept, underwent complete
eye examination. The complete eye examination
included determination of best-corrected visual
acuity, slit-lamp examination, intraocular pressure
measurement and retinal thickness measurement
by optical coherence tomography. After written informed consents were obtained, all patients were
treated. The informed consent was given to all patients due the risks of intravitreal injection which
included pain, bleeding, retinal detachment, cataract, increased transient IOP, infections and sterile
endophthalmitis. Injections were performed by
retinal specialist in the operation room. The medications were given under aseptic conditions. Lids
and conjunctiva were prepared with 5% povidone
ioidine, followed by the placement of an eyelid
speculum. Intravitreal injections were injected with
a needle in infero-temporal quadrant through pars
plana (3,5 – 4,0 mm from limbus) into the vitreous
cavity. Patients were instructed to administer topical Maxitrol (Alcon) 3 times daily for 5 days. All
applications were performed as an outpatient pro-
203
cedure. Patients were told to return to the hospital
immediately if visual disturbance, pain, or redness
of the eyes were noticed. Generally all the patients
following intravitreal injections were followed up
in the clinic usually at 1-3 monthly intervals.
Clinical diagnosis of endophthalmitis was made
on the basis of presence of anterior chamber reaction, keratitic precipitates, hypopion, fibrin and/
or posterior synechia. Ultrasound examination
was performed with (10MHz transducer, UltraScan, ALCON Inc, Fort Worth, TX, USA).
RESULTS
In a period of 7 years a total of 986 patients with
intravitreal bevacizumab injection, 1.25 mg/0.05
mL was treated. Since 2012 fifty-five patients had
been treated with intravitreal ranibizumab injection 0.3 mg /0.05 mL and since October 2014
sixty patients had been treated with intravitreal
aflibercept injection 2.0 mg/0.05 (Table1).
Table 1. Total number of intravitreal injections of anti VEGF
at UCC Tuzla and the number of endophthalmitis cases that
occurred after intravitreal injection
Type of anti VEGF
RANIBIZUMAB
BEVACIZUMAB
AFLIBERCEPT
Total number
of intravitreal
injections
Number of endophthalmitis cases after intravitreal
injections
55
986
60
0
2
0
The overall incidence of clinical endophtahlmitis was 0.2%. Injections were administered in an
operating room. Patients presented with decreased vision, pain and red eye. Two cases of endophthalmitis following intravitreal injection of bevaciuzumab occurred and none after ranibizumab
or aflibercept. Both patients with endophthalmitis
were males. The mean interval between intravitreal injections and onset of symptoms was 3 days.
DISCUSSION
The results suggest that a low rate of endophthalmitis can be achieved by means of a protocol that
includes the use of topical povidone-iodine, a
sterile lid speculum, and topical anesthetic and
postoperative application of topical antibiotics
for seven days. It should be emphasized that this
is the first study in B&H that documents the incidence of endophthalmitis after intravitreal application. It is known that bevacizumab in B&H is
the most frequently used intravitreal anti-vascular endothelial growth factor due to very low price. In the last few months many efforts have been
made to include ranibizumab and aflibercept to
hospital list. A review of literature identified a
number of reports of endophthalmitis rates after
large series of intravitreal injections. The frequency of endophthalmitis after intravitreal anti
VEGF reported in available scientific literature
varies from 0.01% to 1.6% (13-18).
We expect that with the increasing number of
applications of anti VEGF, the overall number of
endophthalmitis will increase as well in B&H in
the near future. Despite the growing number of
indications and agents for intravitreal injections,
there is no consensus on peri-injection guidelines
for the prophylaxis of endophthalmitis (19). Although endophthalmitis cannot be prevented in all
cases, certain strategies must be proposed on the
global level. In conclusion, endophthalmitis remains very a severe complication of intravitreal
application of anti VEGF but infrequent.
ACKNOWLEDGMENT
The authors are grateful to patients who agreed
to participate in this study. Also, authors thank
professor Vahid Jusufović, Chief of Medical Retina Halida Basić and the entire team of Medical
Retina at the University Clinical Center in Tuzla,
Bosnia and Herzegovina.
FUNDING
TRANSPARENCY DECELARATIONS
Competing interest: none to declare.
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Incidenca endoftalmitisa nakon intravitrealne primjene antiVEGF terapije na Univerzitetsko kliničkom centru Tuzla
Univerzitetsko klinički centar Tuzla, Klinika za očne bolesti, Tuzla, Bosna i Hercegovina
SAŽETAK
Cilj Utvrditi incidencu endoftalmitisa nakon intravitrealne aplikacije antivaskularnog endotelnog faktora rasta (anti-VEGF) na Univerzitetsko kliničkom centru u Tuzli.
Metode U ovoj retrospektivnoj studiji ukupno 986 intravitrealnih aplikacija bevacizumaba aplicirano
je u periodu od januara 2008. do aprila 2015. godine na Univerzitetsko kliničkom centru u Tuzli (UKC
Tuzla). Od januara 2012. godine ukupno je aplicirano 55 ranibizumaba dok je od oktobra 2014. broj
apliciranih aflibercepta iznosio 60.
Rezultati Tokom ovog perioda desila su se dva slučaja endoftalmitisa nakon aplikacije bevacizumaba,
a nije se desio ni jedan slučaj nakon aplikacije ranibizumaba ili aflibercepta. Incidenca endoftalmitisa
iznosila je 0,2%.
Zaključak Rezultati indiciraju da se uz određene protokole može smanjiti incidenca endoftalmitisa.
Ova studija je od velikog značaja jer se radi o prvoj studiji ovog tipa u Bosni i Hercegovini (BiH). Studijom su analizirani svi slučajevi endoftalmitisa nakon intravitrealne aplikacije anti-VEGF terapije. U
BiH je bevacizumab najčešće korišten anti-VEGF zbog niske cijene.
Ključne riječi: bevacizumab, aflibercept, ranibizumab, komplikacije nakon intravitrealne aplikacije
205
ORIGINAL ARTICLE
Therapeutic efficacy and toxicity of bolus application of
chemotherapy protocol in the treatment of metastatic colorectal
cancer
Ibrahim Šišić1, Belma Pojskić2, Alma Mekić-Abazović1, Vladimir Kovčin3
1
Department of Oncology, Hematology and Radiotherapy, 2Department of Internal Diseases, Cantonal Hospital Zenica; Bosnia and
Herzegovina, 3Department of Oncology, Clinical Hospital Centre Bežanijska kosa Beograd, Serbia
ABSTRACT
Aim To compare efficacy and toxicity of bolus application of chemotherapy protocol, oxaliplatin, fluorouracil (bolus), leucovorin
(folfox) between two groups of patients in the therapy of metastatic colorectal carcinoma (mCRC).
Šišić Ibrahim
Department of Oncology, Hematology and
Radiotherapy, Cantonal Hospital Zenica
Crkvice 67, 7200 Zenica, Bosnia and
Herzegovina
Phone: +387 32 201 680;
Fax: +387 32 202 681;
29 December 2014;
Revised submission:
10 February 2015;
Accepted:
16 March 2015.
doi: 10.17392/802-15
Med Glas (Zenica) 2015; 12(2): 206-211
206
Methods A total of 63 patients were treated for mCRC in the period January 2009 – January 2010 at the Department of Oncology
of the Cantonal Hospital Zenica, Bosnia and Herzegovina (first
group, 30 patients) and at the Department of Oncology of the Clinical Hospital Centre Bežanijska kosa in Belgrade, Serbia, in the
period January 2005 – January 2006 (second group, 33 patients).
The patients were treated according the same protocol, i.v. bolus
infusion, but in different day intervals (D), 1, 8, 15/28 days or D1D5/28 days, respectively. In all patients the following factors were
analyzed: tumor response, overall survival (OS), progression free
survival, hematological and non-hematological toxicity .
Results Colon was the primary localization in almost two thirds of
patients. There was no statistically significant difference between
the groups according to the age, hematological and non-hematological toxicity, as well as in achieved OS. Progression free survival
expressed in months was in average 5 months though with a large
range between minimal and maximal survival time.
Conclusion Both groups have shown equivalent efficacy to applied chemotherapy protocols. Overall survival in the two groups
matched data from the literature. Further research should confirm
success of the combination of chemotherapy protocols and their
combination with the biological therapy.
Key words: oxaliplatin, therapeutic response, overall survival,
toxicity
Šišić et al. Efficacy and toxicity of bolus application
INTRODUCTION
It is estimated that every year colorectal cancer
(CRC) affects about 1.2 million people and around 609,000 die as a consequence of CRC (1).
The incidence increases with age (2,3). Elevated
rates of incidence were estimated in European
countries - Bosnia and Herzegovina (30 in men,
19 in women). Geographical patterns of mortality
partially follow incidence. Estimated age-standardized rates (European standard) of cancer mortality by sex, cancer site and country 2012 in Bosnia
and Herzegovina were 19.8 in men; 11.7 in women (4). Based on the data of the Cancer Register
for Central Serbia it can be estimated that every
year 4000 persons are affected by CRC in Serbia.
Standardized incidence rate in Central Serbia is
33 per 100,000 in men and 19 per 100.000 in women (5). Approximately 60% of diagnosed CRC
cases develop metastatic disease. In the disease
etiology three groups of risk factors can be mentioned: family history, life style and colon diseases
(6). Among others, it is particularly important to
mention familial adenomatous polyposis (FAP)
in high-risk patients (7,8). It is believed that most
colon cancers occur in a process of several levels
or malignant transformation of adenoma through
the process of activation of oncogenes and inactivation of tumor-suppressor genes, adenoma-carcinoma sequence (9). There are some opinions
which negate so called malignant transformation
of benign polyps, and it is believed that in most
cases those are cancers from the very beginning
-”wolves in sheep’s clothing” (10). In cancer prevention stool tests performed once a year allow for
early detection of cancer in 18-33%, sigmoidoscopy every five years in 34-55%, colonoscopy every
10 years in 75% of persons (11,12).
Surgical treatment is a basis for the treatment
of malignant diseases of the lower part of gastrointestinal system. A type of surgical treatment depends on tumor location (13,14 ).
Possibilities of chemotherapy in patients with
metastatic colorectal cancer (mCRC) are today
promising thanks to oxaliplatin, irinotecan, capecitabine (5-fluorouracil+oxaliplatin, folfox, and
5-fluorouracil+irinotecan, folfiri) (15). Advantages
of the selection of one of the these two protocols
have been examined in a study by Tourgand (CERCOR study) according to which there is no significant difference in the overall survival regardless
of selected therapy. However, there is a clear difference in the profile of toxicity, which means that
the expected undesired differences are adjusted to
age and potential comorbidities. Irinotecan proved
to be safer in patients of older age (16,17). Based
on results of OPTIMOX 1 study, suspension of the
treatment is recommended in patients whose response to the treatment has been achieved or there
is a stable disease, and after 6 or more cycles of the
first-line treatment with FOLFOX protocol.
In such patients a maintenance approach with
Capecitabine or “stop and go” is advocated for,
i.e. absence of therapy until metastases reach the
previous size (OPTIMOX 2 study) (18).
Studies examining three medicaments were published: combination of 5 FU, irinotecan and
oxaliplatin (folfoxiri), which had some promising
results though in certain younger populations of
patients (19). Oral fluoropyrimidine (capecitabine) proved to be efficient and similar to 5FU/LV
(5 fluorouracil /leucovorin), which is administered
in a long-lasting iv and contributes to better quality of life (20). In the last ten years significant
achievement has been made in the treatment of
mCRC applying biological medicines. Target
therapy needs to ensure simultaneous increase in
efficiency and reduced toxicity of chemotherapy
(21-23). In addition to numerous therapeutic protocols it is obvious that there is still no standardized therapy (24). Therefore, when it comes to the
treatment of different subpopulations of patients
with chemotherapy, it is necessary to select them
according to numerous factors in order to achieve
the highest possible number of patients to undergo
curative R0 (clear margins post metastasectomy)
liver resections or whose life will be prolonged to
the maximum with significantly improved quality
of life (24,25). The aim of this paper was primarily to compare efficacy of chemotherapy protocol
of oxaliplatin, 5-fluorouracil (bolus), leucovorin
(folfox) as a “modified protocol” in a three-week
regimen (days), 1, 8, 15/every 28 days at the Oncology Department of the Cantonal Hospital of
Zenica, with data of “modified folfox protocol”
applied in five-day regimen of administering every
4 weeks at the Clinical Hospital Centre Bežanijska kosa in Belgrade. The secondary aim was to
compare toxicity (hematological and non-hematological) of these two modes of bolus application
of chemotherapy protocol (folfox protocol) in the
207
treatment of metastatic colorectal cancer (mCRC).
The parameters followed in both groups of patients were: overall therapeutic response, time to
progression of the disease, overall survival (OS),
and toxicity per number of chemotherapy cycles.
This retrospective study included 63 patients: first
(research) group (30), and second (control) group
(33) in the period of one year. The study was conducted at the Cantonal Hospital Zenica in the period January 2009 – January 2010 (30 patients, research group), and at the Clinical Hospital Centre
Bežanijska kosa in Belgrade in the period between
January 2005 and January 2006 (33 patients, control group). The study included patients who had a
verified diagnosis of metastatic colorectal cancer,
histologically identified as invasive adenocarcinoma. The research group was treated at the Oncology Department of the Cantonal Hospital Zenica. The control group consisted of patients with
same pathohistological diagnosis, e.g. metastatic
colorectal carcinoma with good performance status, who were treated at the Oncology Department
of the Clinical Hospital Centre Bežanijska kosa in
Belgrade, with the same chemotherapy protocol
(folfox protocol) as bolus infusion, but with different regimens, i.e. administration time intervals.
In all patients the survival was calculated from
the date of the first chemotherapy cycle until the
date of death as a result of any cause, and if this
information was not available, until the date of
the last control examination.
All collected data were analyzed applying methods of descriptive and analytical statistics: χ2 test,
T-test, U test, normal distribution test, Wilcoxon
Signed Ranks test, Mann-Whitney, Spearman rank
correlation were used for statistical analysis. The
T-test was used to access the average patients’ age,
χ2 test was used to analyze the distribution of patients to subsets according to therapeutic toxicity,
Mann-Whitney to analyze the distribution of patients to subsets according to therapeutic response,
time to progression, and overall survival.
RESULTS
The study included 63 patients of average age of
60 years. The youngest patient was 34 years old,
while the oldest one was 74 years old. There is
no statistically significant difference between the
208
groups according to the age (p=0.269). Average
difference between groups was 2.6 years (Table
1). Colon was the primary localization in almost
two thirds of patients. Susceptibility to colon
localization was noticed in 42 (67.7%), while 20
(32.3%) patients had rectal cancer. As far as gender is concerned, there was no significant difference in the distribution of primary localization
of cancer (p=1.000)
Table 1. Average age, median and variability of years of age
in research and control group of patients
Group
Research
Control
Total
No (%) of Arithmetic
Mini- MaxiSD Median
patients
mean
mum mum
30 (48)
61.57
9.035 63.00
44
79
33 (52)
58.97
9.392 62.00
34
73
63 (100)
60.21
9.243 63.00
34
79
Analyzing distribution of patients per groups in
relation to toxicity by dividing them to those who
had or had no therapy-related toxicity, there was
no statistically significant difference between the
groups (p=0.424) (Table 2).
Table 2. Distribution of patients per groups in relation to
toxicity of the chemotherapy
No (%) of patients
Toxicity of therapy
Group of patients
Research
Control
Total
NO
YES
11 (36.7)
9 (27.3)
20 (31.7)
19 (63.3)
24 (72.7)
43 (68.3)
Total
30 (100)
33 (100)
63 (100)
In terms of therapeutic response, out of the total
of 63 patients for whom data is available, only
nine (14.8%) patients had partial remission (PR),
(five patients in the examined and four patients
in the control group). One patient in the examined group had complete remission, while the
highest number of patients, 40 (65.6%; 22 in the
examined and 18 in the control group) had the
progression of the disease (PD) mainly after the
third cycle. There was no statistically significant
difference between the groups according to therapeutic response (p=0.431) (Table 3).
Table 3. Distribution of patients per groups in relation to
therapeutic response
No (%) of patients
Group of patients
Research
Control
Total
Therapeutic response
PD
SD
PR
22 (73.3) 2 (6.7) 5 (16.7)
18 (58.1) 9 (29) 4 (12.9)
40 (65.6) 11 (18.0) 9 (14.8)
CR
1 (3.3)
0 (0)
1 (1.6)
Total
30 (100)
31 (100)
61 (100)
PD, disease progression; SD, stable disease; PR, partial regression;
CR, complete response
The applied therapy protocol assured stable disease, in terms of response, only to 11 (18%) patients (p=0.431).
Duration of the response was 5 months in average, with a large range between the minimum and
maximum. There is no statistically significant
difference between the groups according to time
to progression (p=0.880) (Table 4).
Table 4. Arithmetic mean, SD, median and variability of time
to disease progression
Group of
patients
Research
Control
Total
Mini- MaxiSD Median
patients
mean
mum mum
30 (52)
6.00
5.699
5.00
3
33
28 (48)
5.21
2.455
5.50
2
9
58 (100)
5.62
4.420
5.00
2
33
The overall survival of patients in this study was
23 months (in average with standard deviation
of 14.5). Due to high standard deviation, the
best indicator of overall survival is median survival and it was 20 months (Table 5). Three patients had overall survival more than five years.
There was no statistically significant difference
in achieved overall survival (OS) in the two groups (p=0.840). Time to progression (TTP) was
5 months in average, though with a large range
between minimal and maximal survival time.
Table 5. Arithmetic mean, SD, median and variability of
patients’ overall survival
Group of
patients
Research
Control
Total
Mini- MaxiSD Median
patients
mean
mum mum
30 (49)
21.30
10.668 21.50
3
40
31(51)
23.94
17.588 18.00
8
76
61 (100)
22.64
14.541 20.00
3
76
DISCUSSION
Chemotherapy protocols in the treatment of CRC
are selected according to the National Comprehensive Cancer Network (NCCN) guidelines (26,27).
With introduction of biological therapy (bevacizumab, cetuximab and panitumumab) with the
chemotherapy protocols median survival higher
than two years was achieved. Without treatment
patients with metastatic colorectal cancer (mCRC)
live in average for five to six months (15).
Usual protocols for the treatment of mCRC are
given in bolus. Current standard protocol includes administration of continuous infusion in the
period of 48 hours. Such application achieves
better therapeutic effect (22:14 %), longer median survival (12.1:11.3 months; p=0.04) and reduction of myelotoxicity (4:31%) (15).
The patients of the first group, in the Cantonal
Hospital of Zenica, received folfox bolus protocol in the three-week regimen: day 1 (D1);
day 8 (D8); day 15 (D15) / every 28 days, and
the patients of the second group at the Clinical Hospital Centre Bežanijska Kosa, Belgrade,
received folfox-bolus protocol in the five-day
regimen, D1-D5 /every 28 days. Protocol modification, which means chemotherapy administration via bolus in different time frames (D1D5 e.g., D1, 8, 15, every 28 days) in analyzed
groups vs standardized continuous protocols
over 48 hours administration time, was applied
to enable the application administration in the
conditions of daily hospital with patients going
home every day after the therapy, ambulatory
patients or daily clinic patients, and it is very
comfortable for patients.
Analyzing groups examined with stage IV disease (metastatic disease) it is crucial to set the
implementation of systemic measures of primary
and secondary prevention as a basic task of our
health care system (28,29).
The patients in the examined sample received 4
cycles of chemotherapy in average.
Like any other chemotherapy, folfox therapeutic
protocol also causes various side-effects.
The study results indicate that the majority of patients had therapeutic response within 6 months,
which is statistically significant. Comparing the
data from the literature it could be concluded
that they match the overall survival in this study. Multidisciplinary decision and individualized
approach are the main principles when treating
this heterogeneous group of patients.
The study supports administration of both protocols in clinical practice, but when taking into
consideration lowing of the costs and less patients’ visits, three-day administration regimen,
can be considered preferred.
It is the major conclusion of the study that there is a crucial need for implementing prevention methods (primary and secondary) for early
screening and detection of colorectal carcinoma, which would bring a long term therapeutic
effect when treating colorectal carcinoma, but
would also lower the costs.
Further research would need to be directed
towards combining chemotherapy medications
209
and different protocols with biological medicines, which could result in even higher overall
survival and decrease in undesired effects.
FUNDING
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Terapijska efikasnost i toksičnost bolusnih primjena
hemioterapijskog protokola u terapiji metastatskog kolorektalnog
karcinoma
Ibrahim Šišić1, Belma Pojskić2, Alma Mekić-Abazović1, Vladimir Kovčin3
1
3
Služba za onkologiju, hematologiju i radioterapiju, 2Služba za unutrašnje bolesti; Kantonalna bolnica Zenica, Bosna i Hercegovina;
Odeljenje za onkologiju, Kliničko-bolnički centar Bežanijska kosa, Beograd, Srbija.
SAŽETAK
Cilj Uporediti terapijsku efikasnost i toksičnost bolusnih primjena hemioterapijskog protokola, oxaliplatine, fluorouracil (bolus), leukovorin (folfox), između dvije grupe bolesnika u terapiji metastatskog
kolorektalnog karcinoma.
Metode Ukupno 63 bolesnika liječena su od metastatskog kolorektalnog karcinoma, u periodu od
januara 2009. do januara 2010. godine, na Onkološkom odjeljenju Kantonalne bolnice Zenica, Bosna
i Hercegovina (prva grupa od 30 pacijenata ) i na Onkološkom odjeljenju Kliničko-bolničkog centra
Bežanijska kosa u Beogradu, Srbija, u periodu od januara 2005. do januara 2006. godine (druga grupa
od 33 pacijenta). Pacijenti su bili tretirani istim hemioterapijskim protokolom, i.v. bolus infuzije, ali u
različitim vremenskim intervalima, D1, 8, 15/28, odnosno D1-D5/28 dana. Kod svih pacijenata analizirani su terapijski odgovor, ukupno preživljavanje (OS), vrijeme do progresije bolesti, kao i toksičnost.
Rezultati Primarna lokalizacija u skoro dvije trećine pacijenata bio je kolon. Nisu ustanovljene statistički značajne razlike između skupina prema dobi, u hematološkoj i nehematološkoj toksičnosti, kao ni
u ukupnom preživljavanju. Vrijeme do progresije bolesti u mjesecima bilo je u prosjeku pet mjeseci, ali
s velikim rasponom između minimalnog i maksimalnog.
Zaključak Ustanovljena je podjednaka efikasnost hemioterapije u obje grupe bolesnika. Ukupno preživljavanje u dvije grupe bilo je podudarno s podacima iz literature. Dalja istraživanja trebala bi potvrditi uspješnost kombinacije hemioterapijskih protokola i njihove kombinacije s biološkom terapijom.
Ključne riječi: oxaliplatin, terapijski odgovor, ukupno preživljavanje, toksičnost
211
ORIGINAL ARTICLE
Differences in body mass index and height factors between men
with and without varicocele
Hamid Shafi1, Mouloud Agajani Delavar2
1
Department of Surgery, 2Department of Midwifery; Fatemezahra Infertility and Reproductive Health Research Center, Babol University
of Medical Sciences, Babol, Islamic Republic of Iran
ABSTRACT
Aim Despite many studies published in recent years concerning
the relationship between demographic factors and varicocele, this
issue remains controversial. The aim of this study was to identify a
possible influence of body mass index (BMI) and height on occurrence varicocele in men.
Methods In a case-control study 153 patients aged 18-40 years
from 2004 to 20014, with moderate and sever varicocele were studied. The BMI and height of the 153 patients with varicocele were
compared with 250 men who had no varicocele as a control group.
Mouloud Agajani Delavar
Department of Midwifery, Infertility and
Reproductive Health Research Center of
Babol, University of Medical Sciences,
P.O. Box: 47135-547, Babol, Mazandaran,
Iran
Tel: +98 11 322 748 812;
Fax: +98 11 322 748 80;
Email: [email protected]
14 October 2014;
Revised submission:
09 February 2015;
Accepted:
01 June 2015.
doi: 10.17392/788-15
Med Glas (Zenica) 2015; 12(2): 212-215
212
Results After the adjustment for socio-demographic factors, the
risk of varicocele for obese men was lower than for overweight
and normal men (OR= 0.38, 95% CI= 0.17, 0.85). The adjusted
OR for varicocele in taller men was higher than in those with
low height (OR= 3.42, 95% CI= 1.34, 8.72), and moderate height
(OR=2.68, 95% CI= 1.12, 6.46).
Conclusion The results of this study indicated that tall men and
non-obese men may be at higher risk of varicocele, therefore counseling and evaluation of the men at high risk of varicocele may
be of benefit for reduced infertility.
Key words: pampiniform plexus, spermatic vein, anthropometric
parameters
Shafi et al. BMI, height, and varicocele
INTRODUCTION
Varicocele is a dilatation of the testicular veins and swelling of network of veins from the
testicles within spermatic cord (1). It is likely
that varicocele can cause infertility if it associated with abnormal semen analysis (2,3).
Estimated prevalence varies considerably in
general population and infertile men (4). It is
important to note that the prevalence of varicocele has increased tremendously over the past
decades (5,6).
The exact mechanism of varicocele is unclear.
However, the interaction of increased pressure
in the veins of the testicles and its venous drainage, as well as genetic factors suggested to be
the factors that contribute to its development (6,
7). Several studies have shown that varicocele
may be decreased in overweight and obese men
and increased with taller men (5, 7-9). Similarly, obesity may play a role in detection of varicocele in obese men (10). In contrast, a study
showed that the risk of varicocele is associated
not only with low body mass index (BMI), but
also with high BMI (11).
Varicocele is the most common cause of seminal abnormalities (12). However, varicocele is
not associated with infertility despite seminal
abnormalities (13), but progressive infertility as
varicocele increases surgical repairs in Iranian
population. It is critical to identify association
between varicocele with several parameters such
as BMI and height in men.
The study design was a case-control study. It was
performed on 153 patients with moderate and severe varicocele who were referred to the Outpatient Urology Department for varicocele repair in
the period 2004 - 20014. Two hundred and fifty
patients who had no varicocele were randomly
selected, matched to cases based on age as the
control group.
The men with clinical unilateral or bilateral varicocele were detected in the outpatient clinic by
the same investigator during physical examination in the upright position, and confirmed by
Doppler ultrasongraphy of the scrotum in a warm
room (>23 °C). The men with a history of vari-
cocelectomy and / or inguinal hernia surgery, hydrocele, absence of any testicular for any reason,
any musculoskeletal disease or deformity were
excluded from the study.
Weight was recorded using digital scales, with
the subjects minimally without shoes and with a
tape measure. The body mass index was calculated using the formula: BMI=weight (kg)/ height
2
(m). According to the National Institute of Health and Clinical Excellence (14), a patient is placed in one of the three BMI categories (normal
weight - less than 25 kg/m2), overweight (25-29.9
kg/m2), and obese (≥30 kg/m2).
Height was measured with a tape measure. The
height of all subjects was categorized by tertiles.
The frequency of varicocele in each tertile category was compared.
This study was approved by the Ethical Committee of Babol University of Medical Sciences.
Informed written consent was obtained from all
eligible subjects (>18 years).
Descriptive statistics were used to describe baseline demographics. To determine association
between categorical BMI and height with varicocele, varicocele was considered as a dependent variable for logistic factors included height,
education, BMI, residency and occupation of the
men were adjusted as confounders. p< 0.05 was
considered statistically significant.
RESULTS
The mean age of the men with varicocele was
26.7±4.9, and for that one without varicocele it
was 26.1±6.0 years, ranged between 18-40 years
(p=0.309).
All participants had mean height and mean weight
174.4±7.7 cm and 74.1±12.3 kg, respectively.
The mean value for height and weight of the men
with varicocele was 175.5±6.5 cm and 73.3±12.1
kg, respectively. The mean of height and weight
of these without varicocele was 173.8±8.2 cm
and 74.5±12.3 kg, respectively.
The mean BMI of the all participants was
24.3±3.6 kg/m2. The patients with varicocele had
significantly lower BMI (0.014) than the men
without varicocele (Table 1).
213
Table 1. Characteristics of subjects according to varicocele
Variables
Age (Mean±SD)
Height (Mean±SD)
Weight (Mean±SD)
Body mass index
(Mean±SD)
Education (No/%)
Illiterate (No/%)
<12
12
≥12
Residency (No/%)
Urban
Rural
Occupations (No/%)
Office employees
Industrial/ construction
workers
Drivers
Farmers
Business
Patients
Patients
without
with
Total
p
(n=403) varicocele varicocele
(n=250)
(n=153)
26.4±5.6 26.1±6.0
26.6±4.9 0.309
174.4±7.7 173.8±8.2 175.5±6.5 0.027
74.1±12.3 74.5±12.3 73.3±12.1 0.344
24.3±3.6
23.8±3.3
24.7±3.8
9 (2.2)
167 (41.4)
136 (33.7)
91 (22.6)
7 (2.8)
60 (39.2)
41 (26.8)
50 (32.7)
2 (1.3)
107 (42.8)
95 (38.0)
41 (16.4)
274 (68.0) 182 (27.8)
129 (32.0) 68 (27.2)
92 (60.1)
61 (39.9)
0.014
0.001
0.008
0.0001
30 (7.4)
25 (10.0)
5 (3.3)
105 (26.1)
31 (12.4)
74 (48.4)
20 (5.0)
14 (5.6)
26 (6.5)
10 (4.0)
155 (38.5) 129 (51.6)
6 (3.9)
16 (10.5)
26 (17.0)
The mean of height and weight according to each
varicocele category is shown in Table 2. Statistically significant differences between BMI with
each grade of varicocele have been found. There
was no statistically significant difference between height and severity of varicocele.
Table 2. The mean values of body mass index (BMI) and
height according to each varicocele category
Variables
Grade III
Grade I/II Non-varicocele
BMI (kg/m2)
Height (cm)
23.5±3.3
176.5±6.0
24.2±3.2
175.1±6.7
24.7±3.8
173.8±8.21
p
<0.05
>0.05
The adjusted OR for varicocele in taller men was
significantly higher than in men with low height
(OR= 3.42; 95% CI= 1.34, 8.72), and moderate
height (OR=2.68; 95% CI= 1.12, 6.46). The risk
of varicocele for obese men was found to be lower
than in man with overweight and normal BMI
(OR= 0.38; 95% CI= 0.167, 0.852) (Table 3).
Table 3. Adjusted ratio (OR) for varicocele according to body
mass index (BMI) and height of subjects
Variables
BMI (kg/m2)
Obese†
Overweigh‡
Normal
Height (cm)
≥180
165-179
<165
95% Confidence
Interval
BMI (kg/m2)
0.38
0.71
1.00
0.167, 0.852
0.456, 1.09
.019
0.119
3.42
2.68
1.00
1.34, 8.72
1.12, 6.46
0.010
0.028
Adjusted OR*
*Adjusted for confounder were height, BMI, education, occupation and
residency; †Obese: BMI ≥ 30 kg/m2; ‡Overweight: BMI=25-29.9 kg/m2
214
DISCUSSION
Numerous researchers have assessed the relationship between varicocele and BMI. It is suggested that in obese men excess fat around the
renal vein provides a cushion protecting against
the nutcracker phenomenon (15-17). But yet, relationship between varicocele and BMI is controversial. The occurrence of varicocele may be
decreased with increasing BMI (5,10,15,18). The
results of this study support these findings. In
contrast, some researchers have shown no significant differences between varicocele and BMI
(19-20). In addition, Baek et al. (2011) showed
the men with varicocele had a lower BMI (11).
Also, in our study, men with severe varicocele
had the highest mean of BMI compared with non
varicocele men, which is in agreement with two
studies, where authors reported that severity of
varicocele inversely correlated with obesity (8,
16). Since it is well known that palpation of
scrotum in obese men is difficult, it may lead to
decreased diagnosis of varicocele (11). It could
not be discounted that low grade varicocele may
have been missed on physical examination (21).
More research is needed to define a role of BMI
and pathogenesis of varicocele.
Our finding showed that men with varicocele were
significantly taller than the men without varicocele. This finding is consistent with several studies
(7, 22). Some studies emphasized that the greater
height increases hydrostatic pressure in the spermatic vein, which may have a role in the valve
mechanisms in the veins and lead to development
of varicocele (9, 16). Several studies reported that
higher height was not associated with higher risk
of varicocele (22,1). Results of our study support
this concept that height directly affected the prevalence of varicocele. Nevertheless, there is no direct
evidence to show that valve mechanisms effect is
related to the presence of varicocele. Larger control studies may possibly clarify the role of height
in the pathogenesis of varicocele.
A limitation of this study is that the study population was selected from only one outpatient clinic,
Although there is another urology clinic in Babol,
involving of all urology clinics in this study was
difficult to achieve. In addition, all men in the control group were selected in the same outpatient clinic, and therefore, the results of the study cannot
be considered as a representative of the general Ira-
Shafi et al. BMI, height, and varicocele
nian male population. Another limitation is in the
number of obese patients because a convenience
sample was used instead of population based random sample. Another drawback of this study was
related to data collection; unavailability of some
information that can affect varicocele in men.
The findings were nearly comparable with the
findings in other countries. Data from the present
study have shown that males with varicocele had
lower BMI and higher height than men without
varicocele. Counseling and evaluation of men
at high risk of varicocele, especially for couples
attended to infertility center, could be beneficial.
ACKNOWLEDGEMENTS
The authors acknowledge the assistance of Iranian men for their participation in this study, and
the assistance Maryam Rahimi in the sampling.
FUNDING
This research was supported by the Infertility and
Reproductive Health Research center Babol University of Medical Sciences.
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215
ORIGINAL ARTICLE
Genetic polymorphisms variants in interleukin-6 and interleukin1beta patients with obstructive sleep apnea syndrome in East
Northern Turkey
Ilhami Gok1, Nergiz Huseyinoglu2, Dogan Ilhan3
Department of Bioengineering, Faculty of Engineering and Architecture, 2Departments of Neurology, School of Medicine, 3Departments
of Molecular Biology and Genetics, Faculty of Science and Literature; Kafkas University, Kars, Turkey
1
ABSTRACT
Aim To investigate the relationship of IL-1β and IL-6 cytokine
gene polymorphisms with obstructive sleep apnea syndrome
(OSAS) in 61 patients admitted to the neurology clinic in Kafkas
University Hospital with insomnia problem who were diagnosed
with OSAS in sleeping labs, and 80 healthy subjects not associated
with the syndrome.
Ilhami Gök
Department of Bioengineering, Faculty
of Engineering and Architecture, Kafkas
University 36100, Kars, Turkey
Phone: +90 474 225 1279;
Fax: +90 474 225 1282;
E.mail: [email protected]
Results Polymorphic changes were observed for IL-1β gene in
26 of 62 patients (41.9%), and 16 of the 80 (25.8%) in the control
group. The incidence of polymorphic changes in IL-6 gene was
in seen in seven (of the 62 patients) (11.3%), and in the 16 (20%)
controls.
12 January 2015;
Conclusion The findings on the genomic level in OSAS may provide an important contribution to diagnosis of obstructive sleep
apnea syndrome in clinical practice, as well as it helps to obtain
the results easily about environmental and genetic interaction of
OSAS patients.
Revised submission:
Key words: OSAS, cytokine genes, RFLP, Turkey
13 March 2015;
Accepted:
23 March 2015.
doi: 10.17392/804-15
Med Glas (Zenica) 2015; 12(2): 216-222
216
Methods Blood samples were taken to isolate DNA from patients diagnosed with OSAS based on polysomnography results and
healthy controls. DNA amplification of the genes was performed
with PCR. Amplification products were cut with the restriction
enzymes in order to determine IL-1 gene (TaqI) and IL-6 gene
(Lwel) polymorphisms. The cut DNA fragments were carried out
in agarose gel electrophoresis, and RFLP analysis was performed
by utilizing the images with gel imaging system. PCR products
were sequenced with an Applied Biosystems Automated Sequencer.
Gok et al. Gene polymorphisms and sleep apnoea syndrome
INTRODUCTION
Sleep is an essential part of health and sleepbreathing disorders can cause serious health
problems, economic losses both social and personal. The most common type of sleep apnea is
defined as obstructive disorders (1). Obstructive sleep apnea syndrome (OSAS) disease constitutes a risk in people with certain anatomical
features such as obesity, advanced age, gender
and short neck (2). Daytime sleepiness, snoring,
airway obstruction are the main findings of apnea
(3). Traffic accident rate is high with these patients because of excessive daytime sleepiness
(2). Sleep apnea is a risk for the development of
many systemic diseases and is involved in the
etiopathogenesis of various cardiovascular and
disease progression (3). Obstructive sleep apnea
syndrome and obesity are disorders common in
individuals (2-3). Disorders associated with obesity have become some of the most serious social
problems that triggered the formation of OSAS in
recent years. Obstructive sleep apnea syndrome
is a disease characterized in the upper respiratory
tract in adults and in middle-aged individuals; it
often leads to hypoxia and affects the sympathetic nervous system, metabolic reactions may also
lead to an increase in blood pressure (3-4). As
the obesity is a multifactorial disease, a significant relationship between genetic factors, obesity
and OSAS has been defined (5). In OSAS, the
reason of the airway obstruction is that the soft
tissue slumps down the trachea during sleep. In
each apnea event, enough oxygen does not get
to the brain and sleep of persons with syndrome
is broken down due to suffocation, and sleep is
of poor quality and fragmented (6). Obstructive
sleep apnea syndrome, in general, is common in
overweight adults, in patients with diabetes. Risk
factors include male gender, obesity, and age.
Sleep apnea may be seen at any age, or even in
children (6-7).
Cytokine genes of interleukin-1β (IL-1beta) are
shown physiological insomnia roles (6). IL-1
concentration in the cells plasma of healthy human was assumed to be the highest during awakenings and infusion and at the beginning of sleep
(7). The most stable IL-1β levels in plasma with
cytokine cycle variation can affect patients with
narcolepsy, and interleukin-6 (IL-6) and hypersomnia (3-4). The significant increases in cyto-
kine concentrations can be measured by OSAS
and narcolepsy, and average sleep having correlation with the intensity of sleep, sleep can be
measured as latency. It is associated with IL-6
levels and body mass index (BMI) (8-10). Obesity is one of the serious consequences leading to
uneven course of sleep. This interaction can lead
to increases in inflammatory cytokines (9). IL1β and IL-6 genes can alter the levels of protein
synthesis. IL-6 functions are known as lipid metabolism and energy expenditure (11). Polymorphism in the promoter region of the IL-6 gene influences the level of 174 (G174C) interleukin 6.
Also in the arrangement of body mass interleukin
- 1β is known to be effective (12). The gold standard method in the diagnosis of sleep disordered
breathing is the polysomnography method (10).
Currently, the connection of OSAS syndromes
with genetics is investigated (12). In our study,
the genomic aspects of IL-1β and IL-6 gene
polymorphisms from cytokine genes in patients
with obstructive sleep syndrome were examined.
The most common form of polymorphism is the
single nucleotide polymorphism consisting of a
single base pair change in genomic DNA. Allelic and genotypic distribution analysis of cytokine genes were evaluated in individuals with
obesity and individuals with sleep apnea syndromes visiting outpatient clinics. The aim of
the study was to evaluate the clinical importance
of cytokine genes which have been found to be
linked with OSAS. In this sense, genotypic studies polymerase chain reaction (PCR), restriction
fragment length polymorphism (RFLP) method
was used. PCR products were sequenced with an
Applied Biosystems Automated Sequencer.
Patients and controls
During three years (2011-2014) 1000 people
having problems of sleepiness disease reported to the sleep laboratory in Kafkas University, Turkey. Patients diagnosed with OSAS
were selected for the research by the results of
polysomnography (PSG), and were studied at
the sleep laboratory at Kafkas University Medicine Faculty Research and Application Hospital
(apnoea hypopnoea index, AHA of < 5-30). A
control group of 80 voluntarily subjects with an
217
apnoea hypopnoea index (AHI) <5 were selected randomly (general population without any
classification). The population in this region
shows trends toward obesity resulting from poor
nutrition habits and the long winter season. In
addition, sleep disease diagnoses in the region
exceed the country’s average (p<0.05). For the
patients and controls an ethical approval was received from the Medical Ethics Committee of
Ataturk University (Erzurum).
Polysomnography
Full-night polysomnographic recording was
performed using an Embla N7000 system
(Medcare; Reykjavik, Iceland). The following
parameters were recorded: electroencephalography, electrocardiography, electrooculography, submental and anterior tibialis muscle
electromyography, nasal pressure, oronasal airflow by thermal sensor, snoring, oxygen saturation by finger oxymeter, and respiratory effort by
thoracic and abdominal inductance plethysmography (13). Sleep disordered breathing events
were scored manually by the same investigator,
according to the American Academy of Sleep
Medicine criteria (14). Obstructive apnea was
defined as a drop in the peak oronasal thermal
sensor excursion by ≥90% from baseline for
at least 10 s. Hypopnea was defined as at least
a 50% drop in airflow for at least 10 s despite
respiratory efforts and at least a 3% decrease
in oxyhemoglobin saturation. Patients were diagnosed with OSAS if the AHI was ≥5. Grading was conducted according to mild OSAS
with an AHI of 5-14, moderate OSAS with an
AHI of 15-29, and severe OSAS with an AHI
≤30 (13-14). The lowest O saturation value was
2
measured throughout the night for each patient.
Polysomnographic results of the patients and
controls were recorded in the hospital sleep laboratory using the 24-hour duration as a baseline with the laboratory PSG system. The heights
of the patients and controls were measured in
cm, while weights and body mass index (BMI)
values were calculated in kg/m2.
Determination of genotypes the Interleukin- 6
and Interleukin-1
For genotyping 10 mL blood samples were
taken from each patient and the control gro-
218
up into tubes with EDTA. DNA was extracted
using commercially available Invitrogen genomic DNA extraction mini kits (CS11010,
London, UK) DNA purification kit according
to the manufacturer’s instructions. The isolated
DNA samples were measured at the Nanodrop
Spectrophotometer (Thermo ND1000 Wilmington, USA) and kept at -20 °C.
The primers used to identify Interleukin- 6
(IL-6) were F: 5’-TGA CTT CAG CTT TAC
TCT TTG T-3 and R: 5’-CTC AGG TGT CCT
CGAAGAAAT CAAA-3’ the Interleukin-1β
genes (IL-1β) specific primers were: F 5’GCT TTT TTG CTG TGA GTC CCG-3’ and
R 5’-CTC AGG TGT CCT CGAAGAAAT
CAAA-3’ (15). A final volume 25 μL PCR
protocol that included 2.5 μL 10X Taq polymerase buffer solution, 2,5μL magnesium chloride (2 mM), 2μL dNTP mix (0.2 mM), 1 μL
forward primer (10 pmol), 1 μL reverse primer
(10 pmol), 2μL genomic DNA (100ng/μL), 1 μl
DNA taq polymerase enzyme (5u/μL), and 13
μL distilled water; a total volume of 25 μL was
used, PCR conditions were as follows: an initial
denaturation for 5 min at 94 °C , then 35 cycles
at 94°C for 45 s, at 63°C for 45 s, at 72°C for
55 s, and a final extension at 1 cycle 72°C for 7
min. The PCR products were detected by agarose gel electrophoresis (at 90V, 300 A for 1.5
h) on 2% agarose gel containing ethidium bromide, and the fluorescent intensity of each band
was evaluated with a UV transilluminator (Gel
Logic Pro 2200, Montreal,Canada) (16) For the
Interleukin-1β polymorphism, the PCR amplification bands were observed as 195 bp, and the
Interleukin- 6 amplification bands as 198bp .
Amplified products were digested: Interleukin1β with 5U Thermus aquaticus (TaqI), and
Interleukin- 6 was digested with 5U Listeria
welshimeri (LweI) (New England Biolabs, INC
UK) (17). Digestion products were visualized,
and resulting fragments were separated on 2.5%
agarose gel and with ethidium bromide staining under ultraviolet illumination (Gel Logic
Pro 2200, Canada). The single amplicon of 195
bp as a result of the section of the Interleukin1β polymorphism with the restriction enzyme
(TaqI) was separated into two DNA fragments
as 110 bp and 85 bp. As a result of the section of
Interleukin- 6 with enzyme (LweI), the 198 bp
bands were observed as having 110 bp + 88bp.
The PCR products were then isolated using agarose gel electrophoresis (15-18).
All of the genomic analyses were conducted in
the laboratory of molecular genetics in Department of Bioengineering, Kafkas University. In
addition, Bigdye Cycle Sequencing kit v.3.1,
Applied Biosystems and approximately 5 μL
true of PCR products were sequenced with an
Applied Biosystems Automated Sequencer
(ABI 3130 XL Genetic Analyzer, Foster City,
CA 94404 USA). Restrictions mapping and
SNP bioinformatic analysis was done as Vector
NTI Software (Life Technologies).
Table 2. Distribution of patients with obstructive sleep apnea
syndrome (OSAS) and control group according to body mass
index (BMI) and apnoea hypopnoea index (AHI)
No (%) of patients
OSAS patients (BMI)
Control group (BMI )
(N = 62)
(N = 80)
BMI
Range Females Males Range Females
Males
p
28 -30
8 (25.8) 13 (41.9) >24-25 13 (35.2) 21 (48.8) 0.168
22 (51.2)
>30
23 (74.1) 18 (58.0) >30 24 (64.8)
0.198
Total
31 (50)
31 (50)
AHI
Range
Females
Males
5-14
7 (22.58)
0
(mild)
15-29
(medi- 10 (32.25) 13 (41.93)
um)
>30
14 (45.16) 18 (58.07)
(heavy)
Total
31 (50)
31 (50)
For each polymorphism, deviation of the genotype frequencies in the controls from those expected
under Hardy-Weinberg equilibrium was assessed
using the standard χ2 test. Genotype frequencies
in cases and controls were compared by χ2 tests. The genotypic-specific risks were estimated
as odds ratios (ORs) with associated 95% intervals (CIs) by unconditional logistic regression.
p<0.05 was considered to be significant.
There were some similarities between males
and females in terms of frequency of gene
polymorphism. An increase of BMI resulted in more IL-1β gene polymorphism change. Genotype of IL-1β gene polymorphism
showed CC genotype in nine (14.5%), CT in 26
(41.9%), TT in 27(43.5%) in OSAS patients,
and CC in eight (10%), CT in 16 (20%) and TT
genotype in 56 (70%) controls.
RESULTS
In patients IL-1β polymorphic change rate was
observed in 26 of 62 patients (41.9%), and the
same polymorphic change rate was observed
in 16 of 80 (25.8%) controls. The prevalence of polymorphic changes in IL-6 gene was
11.3% (seven of the 62) patients, and 20% (16
of 80) controls (p<0.05 for IL-1B C / T and
IL-6 G174C polymorphic regions of cytokines) (Table 3).
The patients diagnosed with the OSAS disease
from Kafkas University, School of Medicine
Neurology Clinic, between August 2011- July
2014, were examined according to polysomnography data (Table 1).
Table 1. Demographic characteristic of obstructive sleep
apnea syndrome (OSAS) patients and control group
No (%) of patients
OSAS patients (AHI 5-30)
Control group (AHI <5)
(n = 62)
(n = 80)
Age
Females Males
SD Age Females Males SD
25–40
3 (9.6) 5 (16.1) 1.41 25–40 20 (54) 30 (70) 7.07
41–60 10 (32.) 10 (32.6) 0.00 41–60 17 (46) 13 (30) 2.83
>61
18 (59.) 16 (58.1) 1.41 >61
0
0
0.00
Total
31 (50) 31 (50) 0.00 Total 37 (46) 43 (54) 4.24
The percentage of OSAS patients having BMI
more than 30 kg/m2 was 66% (41 out of 62),
23 (74.1%) females and 18 (58%) males. In
the control group, 42.5% (34 out of 80) of examinees had BMI less than 25 kg/m2. Patients
with OSAS mostly had AHI >30, 32 (51.6%),
of which 14 (45.16%) were females and 18
(58.07%) males (Table 2).
Total
37 (46.3)
Range
Females
>5
37 (100)
>15-30
0
>30
0
Total
37 (46.3)
43 (53.7)
Males
p
43 (100) 0.234
0
0
0.000
0.000
43 (53.7)
Table 3. Genotypic and allelic results of IL-1β and IL-6 gene
polymorphisms in the obstructive sleep apnea syndrome
(OSAS) patients and controls
Interleukin-1β
Genotypes
Frequencies of
genotypes
Alleles
Frequencies of
alleles
Interleukin-6
Genotypes
Frequencies of
genotypes
Alleles
Frequencies of
Alleles
No (%) of patients
OSAS (n=62)
Control Group (n=80)
CC TT CT
p
CC TT CT
p
9
27
26
8 56 16
0.180
0.0036
(14.5) (43.5) (41.9)
(10) (70) (20)
C
T
C T
22
40
16 64
(0.3) (0.6)
(0.2) (0.8)
GG CC GC
p
GG CC GC
p
48
7
7
56 8 16
0.0036
0.325
(77.4) (11,3) (11,3)
(70) (10) (20)
G
C
G C
51
11
64 16 (82.3) (17.7) (80) (20) 219
Figure 1. Sequencing IL-1β gene; A) the restriction enzyme cleavage sites are provided in control individuals (TaqI); B restriction
enzyme cleavage site is provided in obstructive sleep apnea syndrome (OSAS) patients as sequencing results (MvaI) (Gok I, 2014)
The most important result of DNA sequence of
IL-1beta gene proved restriction enzymes (TaqI)
cleavage sites for both patients and control group
(Figure 1).
The most important result of DNA sequence of
IL-6 gene was shown as base pair’s changes from
GG in the control group to GC and CC in the
OSAS patients, in other words SNP. Furthermore, IL-6 gene restriction enzymes (Lwe I) cleavage sites were proven for both patients and control
group after sequence resulting. In the SNP regions and restriction enzyme (Lwe I) cleavage site
were detected and are given in Figure 2.
Figure 2. In the IL-6 gene sequencing results: A) SNP region is shown normal (T/T) in control individuals; B) and C) sequencing
result of obstructive sleep apnea syndrome (OSAS) patients (SNP) region the change (G/T) is provided (Gok I, 2014)
220
DISCUSSION
In various countries around the world, OSAS
is different in terms of clinical signs and the
prevalence of incidence is increasing between
40-65 years. Generally, the disease in populations was found to be 4% in males and 2% in
females (5,19). There is sleep apnea syndrome
in approximately 0.9-1.9% of Turkey’s population (20-21). Yang et al. in 2013 in China gave
researches importance of its genetic aspects; until
now many genes and polymorphism region related to the disease have been identified (22). The
diversity of genes which are responsible for the
formation of respiratory-related diseases and of
polymorphic region located in this gene was expected to be made a criterion for the diagnosis of
OSAS and genomic studies were carried out. In
addition, an increase of BMI results in a change
in cytokine gene polymorphisms (17,23). In this
study, according to polysomnography results in
the population of Northern Anatolia, IL-1β, IL-6
polymorphism genotypes distribution was investigated in individuals diagnosed with OSAS:
87% of patients with OSAS included in the study
were over 40 years of age, and 66% of the cases
with BMI >30 were aged above 30 years, and our
results support this relationship.
In the study by Popko et al. in 2008 in Poland
it was found that IL-1β polymorphism was a
risk factor for obesity, IL-6 polymorphism was
associated with interleukin receptor function in
carrying the C allele, but there was no significant relationship with obesity in a group of 140
women, 50% of which had BMI <30 kg/m2 (others had MBI >25 kg/m2(15). Our research is not
in compliance with the research of Popko et al.
regarding IL-6 polymorphism, however it correlates with IL-1β polymorphism.
Riha et al. in 2009 in Germany and the UK, investigated IL-1 polymorphism in normal, overweight
and obese man and women, and they found that
polymorphisms of IL-1β can be effective on the
development of obesity in European society (8).
In other study, Arnardottir et al. in 2012 in Iceland,
confirmed that the BMI increase resulted in IL-6
gene polymorphism change, and obesity and other
respiratory disease triggers of the OSAS (23).
In this study we observed an increase in body
mass index of those bearing the genotypes of
IL-6 and alleles of IL-1β; BMI values of 41 of
62 individuals (66%) with OSAS whose two
polymorphisms in the IL-6 and IL-1β gene were
examined in our study were observed of greater
than 30. Our findings are similar to the results of
the Mannarino et al. (14). In a study conducted by
Buck and colleagues in 2010 for the IL-6 gene in
Northern Europe, authors have found no relationship between IL-6 genotype and obesity (12). In
this research that was carried out for North East
Anatolia region, the IL-6 gene was not associated
with obesity, which is in line with the findings
of Buck et al. In research conducted in various
European countries in a similar manner, the effect of IL-6 gene was found to be associated with
metabolism and energy consumption (24-25).
In our study, we suggested that IL-6 gene polymorphism had no direct relationship with OSAS,
but if the BMI is greater than 30 in patients with
OSAS and controls, it may increase the risk of
obesity. A changing of the balance of IL-1β gene
polymorphism could induce metabolism, and
environmental interactions of genes may trigger
the formation of OSAS (15). There is a number
of genetic variants of this gene polymorphism
affecting the OSAS progress of obesity or vice
versa, as a cause there are or there will be probably susceptibility variants affecting the development of obesity of OSAS but also affecting
pleiotropism. Identification of such genes and
understanding their function will contribute to
new perspectives in the partners pathogenesis of
these diseases. Finally, saying that genetic polymorphisms may influence susceptibility to each
disease may not be very accurate; the possibility
should not be ignored. Similarly, the use of such
a model with information about the genetic aspects of obesity will be able to allow for a more
comprehensive way to understand how OSAS
can affect the obesity.
FUNDING
We are grateful to the Kafkas University Scientific Research Project Unit (Grant No: FEF:
2011-47 Kars, Turkey) for financial support of
this study.
221
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