Introduction

Transcription

Introduction
Introduction
1|
General introduction
chapter 1
general introduction
Outline introduction
1. General introduction
1 General introduction
1.1 Historical perspective of Chlamydia trachomatis
1.2 Taxonomy of Chlamydiales
1.3 Chlamydia trachomatis developmental cycle
1.4 Chlamydia trachomatis plasmid and genes
1.4.1 Endogenous plasmid
1.4.2 MOMP and OmpA gene
1.4.3 Polymorphic Membrane Protein and pmp genes
2Chlamydia trachomatis epidemiology, serovar distribution and clinical
manifestation
2.1 Epidemiology of urogenital Ct infections
2.2 Distribution of Ct serovars
2.3 Clinical manifestation and complications
2.4 Anogential serovars & symptoms
2.5 Lymphogranuloma venereum
3 Chlamydia trachomatis diagnostic methods
4 Chlamydia trachomatis typing
4.1 Chlamydia serotyping
4.2 Chlamydia genotyping
5 Chlamydia trachomatis and the development of cervical cancer
5.1 Cervical cancer
5.2 High-risk HPV types
5.3 Chlamydia trachomatis and cervical cancer
6 Thesis Outline
1.1 Historical perspective of Chlamydia trachomatis
Chlamydia trachomatis (Ct) comprises a collection of serological ­d istinguishable
Gram-negative intracellular bacteria. Ct can colonize different types of tissue,
like the conjunctiva, cervix, urethra, oropharynx, proctum and lymph nodes,
and leads to a variety of human diseases depending on the anatomical site of
infection (i.e., trachoma, lymphogranuloma venereum, pelvic inflammatory
diseases, reactive arthritis). Using current highly sensitive methods, viable Ct
is also detected in peripheral blood mononuclear cells in skin lesions and
synovial fluid (14-15).
Although trachoma and lymphogranuloma venereum have been known for
centuries, the etiology remained mysterious (128). More insight in the disease
trachoma was obtained in 1907 when Halberstädter and von Prowazek
inoculated the eyes of Orang-utans with conjunctival swabs of trachoma
patients (36) (figure 1). In Giemsa-stained epithelial cells from the Orang
utan’s conjunctiva they discovered intracytoplasmic vacuoles containing
numerous particles. Some small particles, now recognized as Ct elementary
bodies, were also detected outside the epithelial cells. The intracytoplasmic
vacuoles contained larger particles, known as reticulate bodies. Professor
Tang isolated Ct in 1957 for the first time and initially recognized it as a viral
infection (126). In the 1960s, the use of tissue culture techniques and electron
microscopy revealed evidence for bacterial, ribosomes and cell wall structures,
suggesting that Ct is a bacterium. During the sexual revolution in the late 1960s
it became clear that Ct can be sexually transmitted (78) and in the late 1970s
it was even recognized as the most prevalent sexually transmitted bacterial
infection (100, 154). Currently, the total of infections is estimated over 92 million
cases each year, which is supposed to be an underestimation (153).
In 1963, Wang and Grayston described for the first time the existence of
different Ct serotypes (146). They inoculated egg yolk sacs with Ct and
subsequently injected the crude extracts into mice to trigger the mice’s
immune response. In some of these mice, a protective mechanism against the
Ct infection was observed, while other mice were only partly protected,
apparently by cross-protection. This observation suggested the existence of
different Ct serotypes. Because this method was time-consuming and complex,
more easier tests were needed for larger scale epidemiological studies, leading
to the development of polyclonal microimmunofluorescence tests (147) and
subsequently to the more specific monoclonal microimmunofluorescence
tests, targeting the major outer membrane protein (MOMP) (150). While
­m icroimmunofluorescence tests contributed to more knowledge on the etiology
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of the different Ct serovars in the early days of Ct research, the technique was
replaced at the end of previous century by more sensitive genotyping methods
targeting the gene coding for the MOMP (OmpA gene). Currently, different
types of genotyping methods are used in sexual network studies, phylogenetic
studies and serovar distribution studies resulting in a better understanding of
the epidemiology and etiology of the different Ct serovars.
Figure 1 Halberstädter and von Prowazek inoculating the eyes of an
Orang-utan (1906).
the former family Chlamydiacea into two genera and add three new non-­
Chlamydiacea families. Thus, according the new taxonomy, the species
Chlamydia trachomatis, together with the species Chlamydia suis and Chlamydia
muridarum, belong to the genus Chlamydia, the family Chlamydiaceae and
the order Chlamydiales while the species C.pneumoniae and C.psittaci belong
to the new genus Chlamydophila (figure 2). The new taxonomy is better
structured than the previous taxonomy, although not commonly accepted by
all chlamydiologist (103).
1.3 Chlamydia trachomatis developmental cycle
Ct has a biphasic developmental cycle characterized by alternating between
two morphological forms, the electron dense round elementary body (EB) and
the reticulate body (RB) (Table 1) (73). EBs are small (300nm), metabolically
inactive, extracellular forms, that are responsible for dissemination of Ct and
are able to attach to and invade susceptible cells.
Table 1 Review of within normal limits cervical smears within five years
Property
Adapted from: Pospischil A. (2009). Drug. Today. 45(suppl. B): 141-146
1.2 Taxonomy of Chlamydiales
All members of the order Chlamydiales are gram-negative, non-motile bacteria
with a biphasic developmental cycle. At the end of the 20 th century, a proposal
for revision of the order Chlamydiales was developed, due to the discovery of a
number of Chlamydia-like organisms such as the Simkania strain and
Parachlamydia acanthamoehae (26). The revision is based on 16S rRNA
sequence similarity and analysis of full-length 16S and 23S rDNAs. Members of
the order Chlamydiales share at least 80% sequence identity for the genes
encoding the 16S rRNA and 23S rRNA. The new Chlamydiales taxonomy splits
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before the diagnosis of cervical cancer.
Size
Rigid cell Wall
Extracellular stability
Infective
Induces phagocytosis
Inhibits phago-lysosomal fusion
Metabolic active
Replication
Elementary body
Reticulate body
0.2-0.4 µm
Yes
Yes
Yes
Yes
Yes
No
No
0.6-1.0 µm
No
No
No
No
No
Yes
Yes
The EBs interact with the host cell membrane in a two-stage process. First,
initial attachment occurs through reversible electrostatic interactions of the
Ct MOMP with heparin sulfate containing glycosaminoglycans present on the
surface of epithelial cells (118, 124-125, 127). The second, irreversible binding
is possibly associated with protein disulfide isomerase, a component of the
estrogen receptor complex (20). This irreversible binding step is immediately
followed by a type three secretion system (TTSS) mediated injection of the
translocated actin-recruiting phosphoprotein (TARP) into the host cells
cytoplasma (figure 3). Inside the host cell, TARP is rapidly phosphorylated,
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Figure 3 T he three steps seems necessary for Elementary Body uptake
Adapted from: Bush, R.M. and Everett, K.D.E. (2001). Int. J. Syst. Evol. Microbiol. 51: 203 - 220
Figure 2 Taxonomy diagram of the old and new Chlamydiales classification.
triggering phagocytosis of the EB attached to the host membrane (19). Once
entered into the cell by phagocytosis, the EB starts an immediate differentiation to the larger (1μm) metabolically active non infectious RB. The RB
expresses directly early genes, associated with inclusion body modification
(i.e., inc-like genes CT228, CT229 and EEA1-like gene CT147). The modification
of the inclusion body prevents the inclusion body from entry into the endocytic
pathway leading to lysosomal fusion. Six to 14 hours after phagocytosis, the
RB starts replicating, resulting in extensive growth of the inclusion body,
containing up to 10.000 Chlamydia particles. Approximately 30 hours after
phagocytosis, the RBs differentiate back to EBs and are released by exocytosis
or host cell lysis (Figure 4) (1).
in the host cell. First, a reversible electrostatic binding between
MOMP and heparin-sulfate occurs, followed by an irreversible
binding involving protein disulfide isomerase. Finally, Tarp
injection occurs causing endocytose.
Adapted from: Y.M.AbdelRahman et al. FEMS Microbiology Reviews 2005 29;949-959
1.4 Chlamydia trachomatis endogenous plasmid and genomic genes
The first complete Ct genome (serovar D) was sequenced during the late 1990
as part of the Chlamydia genome project (117). This provides valuable
information about the genome organization and allowed further research in
protein expression and gene regulation. The Ct genome comprises 1.042.519
basepairs with 894 open reading frames encoding for predicted proteins.
The endogenous plasmid, consisting of 7.493 basepairs, was also sequenced.
Subsequently, the endogenous plasmid, the chromosomal OmpA gene and the
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Figure 4 Developmental cycle of Chlamydia trachomatis.
Adapted from Gerald J Stine 1992, the biology of sexually transmitted diseases Wm. C. Brown, USA.
chromosomal pmp genes will be discussed in detail, since those genes are the
most frequent DNA targets for diagnostic Ct assays.
1.4.1 The endogenous plasmid
Almost all Ct isolates carry a small plasmid which contains 8 open reading
frames. Although the exact function of the endogenous plasmid and its proteins
remain unclear, it is supposed that the plasmid genes plays an important role
in transcriptional regulation of chromosomal genes and contributes to the
virulence of the Ct strain (13). Naturally occurring plasmid-free strains of
Ct have been reported, but are exceedingly rare and do not spread into the
population, possibly due to low virulence (27, 123). Plasmid-free Ct isolates
are also unable to accumulate glycogen in their inclusion body, which is a
typical characteristic of Ct. For the viability of Ct, the plasmid seems important
but not necessary (87).
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Recently, a new variant of Ct has been discovered in Sweden (designated as the
Swedish variant). This Swedish variant carries a plasmid showing a 377
basepairs deletion within coding sequence 1 (93). Although the Swedish Ct
variant seems normally transmittable, the infection seems to remain more
frequently asymptomatic than regular Ct strains (7). Importantly, most nucleic
acid amplification Ct detection tests are targeting the endogenous plasmid,
because the plasmid copy number is approximately 10 times higher than
chromosomal Ct DNA and therefore permits more sensitive Ct detection.
The 377bp deletion found in the Swedish strain overlaps the target region for
2 frequently used nucleic acid amplification tests ( Roche Diagnostics and
Abbott Laboratories). This results in false-negative Ct tests results by these
assays for the Swedish strain (61), causing a major underestimation of the Ct
prevalence in Sweden (113). The underestimation has a further impact on the
Swedish healthcare system, since infected individuals remain untreated,
which leads to persistent Ct infections with possible severe late complications.
Infected and untreated individuals have also more time to transmit the
bacteria to other persons. Immediately after identification of the Swedish
strain, Roche and Abbott developed next generation dual target assays,
targeting the endogenous plasmid as well as chromosomal DNA (OmpA gene)
(35). The proportion of the Swedish variant has declined significantly, since
the introduction of the upgraded detection assays, possibly due to faster
treatment (48).
1.4.2 MOMP and the OmpA gene
The Major Outer Membrane Protein (MOMP) is the most studied Ct protein and
is encoded by the chromosomal OmpA gene. The protein structure comprises
5 transmembrane constant domains and 4 variable domains outside the
membrane (figure 5).
The differences in MOMP variable epitopes permits classification of the Ct
strains into 3 major serogroups and 19 different serovars, designated
serogroup B (comprising serovars B, Ba, D, Da, E, L2 and L2a), serogroup C
(comprising serovars A, C, H, I, Ia, J, K, L1 and L3) and the intermediate
serogroup (comprising serovars F, G, Ga) (52, 79).
Serovars A, B and C are mainly detected in conjunctival samples, while the
serovars D-K are mostly found in the anogenital tract as common STD confined
to the mucosal layer. In contrast, the serovars L1, L2 and L3 invade the
submucosal layer and lymph nodes causing the disease lymphogranuloma
venereum (106). It seems that the disease type is not determined by MOMP but
other proteins, although it is possible to cluster the serovars into the 3 disease
groups (biovars).
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Figure 5 T he Major Outer Membrane Protein and the OmpA gene
(V= variable, C=constant, D=domain, R=region, OM= outer
membrane).
complete Ct genome revealed a gene family, consisting of 9 genes that encode
for autotransporter proteins (pmp genes), (117). While most pmp genes are
highly conserved among the different Ct serovars, one pmp gene (pmpH) can
be clustered into three clades that are compatible with the biovars (ie ocular
trachoma clade, STD clade and LGV clade; Table 2) (122).
Table 2 T he three biovars (pmpH clade) and the relation with serovars
and disease type.
Biovar
(pmpH clade)
Serovars
(OmpA)
Trachoma Clade
A, B, Ba, C
STD Clade
Lymphogranuloma
Venereum Clade
Figure was kindly provided by SA Morré.
Since MOMP is the most commonly available protein on the membrane of the
elementary body, several vaccine developing studies are performed targeting
MOMP. (56, 82). However, so far now no adequate Ct vaccine has been
developed.
The OmpA gene, encoding MOMP, comprises of 4 variable regions and 5
conserved regions, encoding the different protein domains. At present, DNA
technology has replaced the conventional sero-determination and dozens of
new genovariants have been identified, like genovariant Ja and L2b, due to an
increased resolution (24, 114). Although OmpA genotyping has replaced MOMP
serotyping, most literature still refers to serovars instead of genovariants,
because it is a clear and stable nomenclature reflecting clinical relevance.
1.4.3 Polymorphic Membrane Protein and pmp genes
Like MOMP, other surface-exposed proteins are considered important
mediators in interaction between Ct and host cells. Sequence analysis of the
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Major infection
site
Conjunctivae,
urogenital tract
(rare)
D, Da, E, F, G, Ga, Anogenital tract
H, I, Ia, J, K
L1, L2, L2a, L3
Inguinal lymph
nodes, proctum
Sequelae
Trachoma, blindness,
reactive arthritis
PID in women, ectopic
pregnancy, infertility,
reactive arthritis
Genital Ulcer Disease,
Anal fistulas, elephantiasis,
frozen pelvic syndrome
The compatibility of the 3 clades with disease suggests that this protein might
play a role in pathogenesis, virulence an tropism. Nevertheless, there is still an
incomplete understanding of the virulence and host immune response to these
proteins, despite the potential importance of pmp in chlamydial biology.
2. Chlamydia trachomatis epidemiology, serovar
distribution and clinical manifestation
2.1 Epidemiology of urogenital Ct infections
Genital Ct infections are the most common bacterial sexually transmittable
disease worldwide, with between the 90 and 92 million new infections each
year(32). Over the past decade, the detection rate of Ct has increased, due to
the application of more sensitive Ct detection assays and the rise of Ct screening
programs. Infection may occur at any age, but peaks in the 16-25 year age
groups, since these persons show high-risk behavior. Important risk groups
are female sex workers, homosexual men, unmarried young adults and
swingers (25, 141, 159). Risk factors for a Ct infection are high frequency of
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partner change, and unprotected sexual intercourse (60). In the USA, more
than a million cases of chlamydial infections were reported in 2007, while the
estimated amount of infected individuals was around 3 million (16, 152). In
the past decade, the incidence of Ct infections in Europe has also increased. In
2005, over 200.000 cases were reported in 17 European countries, which is
certainly an underestimation, since in the Netherlands alone 60.000 Ct
infection occur annually (94, 130). This underestimation might be due to the
asymptomatic course in the majority of Ct infections and the lack of extensive
diagnosis in other countries.
2.2 Distribution of Ct serovars
Throughout the world many serovar-distribution studies are performed. Most
distribution studies focus at Ct infections retrieved from the urogenital tract
(serovars D-K). In general, similar serovar distributions are observed in
different countries. Although some regional differences are observed, serovar
E is the most prevalent serovar worldwide. The serovar E prevalence varies
between the 30-60% of the Ct positive samples in the countries USA (Alabama),
Australia, China, Costa Rica, France, Iceland, Iran, Korea, Russia, Sweden,
Taiwan and the Netherlands (6, 28-29, 41, 44, 46, 53, 89, 95, 108, 116, 155). In
contrast, serovar E was far less prevalent in Uganda, Thailand, India and
Columbia (3, 67, 91, 107). This variation might be due to the different Ct
genotyping methods (Thailand) or a small isolated study population (Uganda,
India). Overall, serovars D, E and F contribute for over 70% of all typed
urogenital serovars, making it difficult to study an association with symptoms
(23, 71, 132). In specific subgroups, like men who have sex with men (MSM), a
different serovar distribution is observed. The majority of anogenital Ct
infections in MSM are caused by serovars G, D and J (4, 31, 49, 55, 120, 143).
The difference in serovar distributions between MSM and the general
population can be network related, like observed among heterosexual
individuals living in an isolated town in Canada (11). Apart from network-associated factors like sexual prevalence, tissue tropism could explain the
difference in serovar distribution. In a recent study, an association between
rectal tropism and polymorphisms of open reading frames within genomic
DNA of serovar G is observed (43). Genovariants from the serovars G, D and J
might be more affected by the polymorphisms associated with rectal tropism
than other serovars, although those polymorphisms are not located on the
OmpA gene. So for a better understanding of tissue tropism we should focus on
other Ct genes, independent of the OmpA gene.
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2.3 Clinical manifestation and complications
An urogenital Ct infection can cause urethritis in men and vaginitis, cervicitis
and urethritis in women. Since most infections are asymptomatic (women:70%,
men: 50%), the great majority of infections remain unnoticed and thus
untreated (131). Symptoms in men are dysuria and a clear urethral discharge,
both occurring 7-21 days after primary infection. Complications in men can be
epididymitis, prostatitis and reactive arthritis (85). At this moment, the role
of Ct in the development of male infertility is not fully understood, since
contradicting results are published (17, 21). It is suggested that the infertility
is caused by strictures of the Vas deferens, due to previous inflammation.
Reactive arthritis (previously called Reiter’s syndrome) can be triggered by a
urogenital Ct infection and is more frequently observed in men (39). Besides
the triad of urethritis, conjunctivitis and arthritis, a reactive arthritis can also
give rise to mucocutaneaous lesions on the penis, mouth, hands and feet (90).
The majority of women have no complaints, but symptoms can be a muco­purulent
vaginal discharge or post-coital bleeding. A cervical Ct infection might induce
an ascending infection causing endometritis, salpingitis, pelvic inflammatory
disease (PID) and perihepatitis (Fitz-Hugh-Curtis syndrome). Although high
percentages of progression have been given for these complications, it appears
that most of the complications as described are the result of symptomatic Ct
infections (72). In Europe, Ct infections are responsible for at least 60% of the
PID cases (98). PID can lead to late severe reproductive complications later in
life due to tubal adhesions. It is estimated that two-third of women with tubal
factor infertility and one-third of the women with ectopic pregnancies have Ct
antibodies detectable in the serum, indicating a Ct infection earlier in life (81, 85).
The course and duration of an untreated, asymptomatic Ct infection is not totally
clear. It would be relatively easy to investigate the course and duration of an
untreated Ct infection, but ethical considerations, regarding the prominent health
risk for untreated individuals, do not allow those prospective cohort studies and
restrict the investigations to retrospective studies. However, the primary time
point at which a person had been initially infected is hard to determine in
retrospective studies. Also, discrimination between a recurrent or persistent Ct
infection is not possible, although Ct genotyping might give an indication. Still,
various studies have investigated the natural course of Ct infections in minimal
prospective studies and larger retrospective studies (30, 45, 62, 68, 72, 84, 97,
105, 134, 140). In general, approximately 50% of the asymptomatic Ct infections
were cleared spontaneously within the first year and no complications occurred.
After 5 years, 95% of the women had resolved their Ct infection spontaneous
without receiving antibiotic treatment. Those results indicate a currently
overrated fear for complications due to a Ct infection.
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2.4 Anogential serovars & symptoms
The serovars D-K are most prevalent in the anogenital tract, although other
serovars are sporadically observed. A number of reports have been published
regarding clinical manifestation and the D-K serotypes (Table 3) (2, 5, 23-24,
28, 71, 132, 139). In men, serovars F and G are associated with less
severe symptoms compared to other serovars and the serovars H and J are
associated with urethral discharge (132, 139). In women, the serovars F and G
are associated with lower abdominal pain, while serovar K frequently causes
vaginal discharge (71, 139). Also, the serogroup C serovars are associated
with persistence of the Ct infection (24). Serovar E has an asymptomatic
course of infection, that possibly contributes to a higher prevalence and
transmission rate, since asymptomatic infections remain more frequently
unnoticed and thus untreated (23).
Table 3 T he main outcome of studies that observed a correlation between
serovars and clinical manifestation.
Investigators
Sample Main outcome
numbers
Duynhoven et al.
310
Dean et al.
56
Van de Laar et al.
275
Batteiger et
al.
124
Morre et al.
221
Gao et al.
240
Antilla et al.
530.000
(cohort)
Dean et al.
24
552
Urethral discharge associated with serovars H and J
in men. Lower abdominal pain was associated with
serovars F and G in women.
Serovar F associated with severe endometrial disease
and serovar E associated with asymptomatic milder
­infections.
Serovar F and G were associated with less severe
symptoms in men, while in women no associations were
observed.
Serovars F en G were associated with more polymorpho­
nuclear leukocytes in smears from men. Overall no
­association with symptoms was observed
Serovar G was associated with lower abdominal pain
and serovar F with infection at a younger age.
Serovar Ga was associated with symptoms in men,
­especially dysuria. Serovar K was associated with vaginal
discharge.
Serovar G, D and I antibodies were associated with
­cervical squamous cell carcinoma.
Serovars belonging to serogroup C are associated with
persistence.
However, it remains important to realize that most of those associations are
observed in single studies that cannot be reproduced. Several other studies do
not confirm the associations between serovars and symptoms (57, 65, 76, 86,
121). So more studies will be required to confirm or reject the association
between symptoms and serovars.
2.5 Lymphogranuloma venereum
Lymphogranuloma venereum (LGV) is a sexually transmitted disease, caused
by Ct serovars L1, L2, L2a or L3. Originally, LGV was known as an endemic
disease in Africa, Southeast Asia, Central/South America and the Caribbean.
Until 2003, LGV was only diagnosed sporadically in the Netherlands as a
traveler’s disease imported from foreign countries (142). The disease has an
aggressive nature with serious late sequelae. The disease LGV comprises 3
stages. In the first stage, a painless erosion/ulcer in the anogenital region
appears 3-30 days after infection. This erosion frequently remains unnoticed
and is observed in only 4% of the LGV patients (102). A visible erosion or ulcus
is difficult to differentiate from a trauma or other ulcerative diseases, like
primary Syphilis and Herpes simplex infections. The second disease stage
occurs 1-4 weeks after infection and is characterized by lymphadenopathy in
the so-called bubonic form of the inguinal lymph nodes in men or retro-peritoneal lymph nodes in women (Figure 6).
However, other lymph nodes (e.g., femoral) in the lower abdomen can also be
infected, depending on the infection site. Over time, the infected lymph nodes
become abscesses and may rupture, leading to fistulae. If LGV infections
remains untreated a tertiary (final) stage will develop, due to progressive
spread and tissue destruction. At this stage, irreversible damage to the lymph
nodes will lead to an impaired lymph drainage and possibly elephantiasis.
Also signs of more cachexic illness can be observed (135).
Since 2003, LGV proctitis (the anorectal variant) is observed in developed
countries among men who have sex with men (MSM), caused by the genovariant
L2b (37, 77, 114). The majority of the infected MSM (80%) are co-infected with
blood borne diseases like HIV and hepatitis C (133). First it was thought that
LGV proctitis in MSM was a new epidemic, but retrospective studies performed
in San Francisco revealed that LGV was already present in rectal swabs from
MSM in 1981, indicating an old unnoticed epidemic (115, 135).
Differentiation between an LGV and non-LGV Ct infection is important, since
an LGV infection need prolonged antibiotic treatment (22). Several in-house
PCRs are available for differentiation between a LGV serovar and an anogenital
(D-K) serovar (18, 37, 70).
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Figure 6 E xample of a bubonic inguinal lymph node in a men.
The picture was kindly provided by Henry de Vries.
3. Chlamydia trachomatis diagnostic methods
In the 1970s Ct culture was the standard reference method for diagnosing Ct
infections. However, the restrictive specimen collection, transport and storage
conditions limited the availability of the test for clinical purposes. During the
1980s, two antigen detection methods were developed. The first assay was a
direct immunofluorescence assay (DFA), utilizing a fluorescein-tagged
monoclonal antibody to allow Ct elementary bodies to be microscopically
visualized in cellular smears collected from the endocervix and conjunctiva
(MicroTrak, Trinity Biotech, Ireland) (129). The second detection method was
a solid phase enzyme-linked immunosorbent assay (EIA), using polyclonal anti-chlamydial antibodies to capture Ct antigen onto beads (Chlamydiazyme,
Abbott Laboratories, USA) (88). Both assays generate a fare more rapid result
(2-3 hours) as compared to Ct culture (3-7 days), but had a slightly lower
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sensitivity. A limitation of DFA is the requirement of expert microscopists,
making it laborious and thus less suitable for high-throughput studies
compared to the EIA. Currently, other Ct detection methods are available like
point of care (POC) rapid assays, DNA probe assays and highly sensitive nucleic
acid amplification tests (NAATs). The three POC assays most commonly used
are the Clearview (Unipath, UK), Biostar (Thermo Electron Corp, USA) and the
QuickVue (Quidel, USA), all based on direct detection of Ct antigens(136). The
POC assays give a result within 30-40 minutes ensuring that patients can
immediately receive antibiotic treatment. Like HIV rapid tests, the assays have
a very high specificity, but a low to moderate sensitivity indicating that a
second more sensitive assay is needed to confirm the obtained test result.
The main utility of those POC tests are in applications where return for
treatment is unlikely or in population screening programs (59, 99). The two
most commonly used DNA probe assays are the PACE2 (Gen-Probe Inc, USA)
and the Hybrid Capture 2 (HC2) Ct assay (Qiagen, USA) (66, 137). The PACE2
system uses a labeled DNA probe that binds to Ct rRNA, generating a DNA/RNA
hybrid. No amplification is required, due to the high copy number of Ct rRNA.
Although this assay has a very good specificity, the test is being replaced by
the cheaper and more sensitive HC2 assay. The HC2 targets both the genomic
OmpA gene and the endogenous plasmid DNA by using a labeled RNA probe.
The HC2 does not need DNA amplification, due to the use of signal amplification
with multiple labeled antibodies specific for DNA/RNA hybrids. The use of signal
amplification instead of DNA amplification prevents the HC2 assay against
carry-over contamination. The HC2 system is a good alternative for NAATs,
although the sensitivity of DNA probe assays is slightly lower (table 4) (8).
Table 4 O verview of the mean costs, mean throughput and mean
sensitivity of the 3 major assay types.
Assay type
Costs
Throughput
Sensitivity
EIA
DNA-Probe
NAATs
Low
Moderate
High
Very good
Very good
Very good
Moderate/Good
Very good
Excellent
NAAT: Nucleic Acid Amplification Test
EIA: Enzyme Immuno Assay
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NAATs are in general the best diagnostic tools for Ct detection, although they
are relatively expensive (Table 4) (8, 40). However, NAATs are nowadays the
gold standard for Ct detection. Different types of commercially available
NAATs like the rRNA based Aptima Combo 2 assay (Gen-probe), the COBAS
Amplicor (Roche Diagnostics), the COBAS TaqMan (Roche Diagnostics) and the
Ct-DT DEIA (Labo Biomedical Products BV) are used in routine diagnostics.
Most assays have different targets (i.e., endogenous plasmid, chromosomal
DNA or rRNA), but have more or less the same sensitivity.
4. Chlamydia trachomatis typing
4.1 Chlamydia serotyping
During the sixties, the first experiments involving Ct serotyping were
performed by Wang and Grayston, who developed the mouse toxicity prevention
test (MTPT) (146). Briefly, they inoculated egg yolk sacs with “trachoma-inclusion conjunctivitis organisms”. Extracts were subsequently intravenously
injected into mice that subsequently triggered an immune response. A
protective mechanism in the mice was described when the mice were
challenged for the second time with the same isolates of Chlamydia. When the
mice were challenged with other isolates, the protective mechanism was less
pronounced indicating cross-protection, but more important, the existence of
different serovars. The MTPT method was very time-consuming, since it took
7 years to classify 80 Ct strains into 6 serotypes (150). Therefore, a more rapid
microimmunofluorescence (MIF) test was developed which was based on
culturing of elementary bodies of known serotypes in yolk sacs and the
production of polyvalent antiserum in mice (147, 149). The combination of the
MIF test and the ability of culture of cells with Ct inclusion bodies facilitated
the discovery of 15 different Ct strains (A-L3) based on variability in the major
outer membrane protein (MOMP) (51, 145, 148). Later, other subserovars like
Ba, Da and Ga, were discovered. During the 1980s the relatively aspecific
polyclonal antibodies were replaced by more specific monoclonal antibodies
(119). The above-described principle was the basis for the development of
several immunoassays that were used until the early 1990 when DNA
sequencing of the OmpA gene became available (138). A disadvantage of
immunotyping was the requirement of Ct culture which is time-consuming,
expensive and requires expert staff. Also, the sensitivity of culture is low
compared to DNA analysis.
28
general introduction
4.2 Chlamydia genotyping
During the 1990s serotyping was replaced by molecular genotyping of the
OmpA gene, encoding MOMP. The first genotyping method consists of a PCR
followed by restriction fragment length polymorphism (RFLP) and pattern
analyses on a polyacrylamide gel. With this method it was possible to
differentiate between 13 genotypes (96). The patterns observed by RFLP were
compatible with the results obtained by serotyping, so the name “serovar”
could be maintained. The strength of the PCR-RFLP methods is the ability to
detect double serovar infections (156). At this moment, RFLP is still performed
to differentiate between an LGV and anogenital serovar, even with other
alternatives available (38, 54).
With the availability of fluorophore-marked nucleotides enabling DNA
sequence analyses, OmpA sequencing became an alternative for RFLP.
Sequencing has a higher resolution than RFLP and serotyping, leading to the
discovery of new genovariants (69). A drawback with sequencing is the
inability to detect double serovar infections, because a multiple serovar
infection will lead to a mixed and therefore non-interpretable sequence
result.
Currently, different reverse line blot assays are available that can identify the
different serovars (6, 155, 158). Those assays are sensitive simple technology
reverse hybridization methods, targeting the variable regions in the OmpA
gene. The method is highly suitable for epidemiological studies due to its
ability to detect multiple serovar infections, the relatively fast results and the
ability to perform the assay manually with the use of simple technology (ie.,
PCR). However a drawback of the reverse line blot is the lower discriminating
power compared to OmpA sequencing. At this moment, new high throughput
microsphere suspension assays are developed, based on reverse hybridization
technology for the Luminex 100 platform, which can be used in large-scale
serovar distribution studies (42). We can consider those microsphere
suspension assays as second generation hybridization methods.
At STD clinics, Real-Time PCRs are used for discrimination between a LGV
infection and a non-LGV Ct infection (12, 33). The probe target for those Real
Time PCRs is a L-specific gap in the polymorphic protein H (pmpH). Those PCRs
are all in-house validated methods, since no commercial pmpH Real-Time PCRs
are available. Although this method belongs to the Ct genotyping methods, it
cannot identify the different serovars and is thus not used in epidiological
studies.
An upcoming genotyping method is the high-resolution typing of Ct strains by
multi locus sequence typing (MLST) that uses several other genes as targets
(47). This method has a higher discriminatory power then OmpA sequencing,
29
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chapter 1
but uses another nomenclature. This makes it difficult to compare the results
obtained with previous studies. However, a new nomenclature may lead to further
understanding of the natural history of Ct and its relation to symptoms.
In general, most Ct genotyping is now performed in research settings. The only
clinical application for genotyping is the differentiation between LGV serovars
and non-LGV serovars. New typing methods may lead to a better understanding
of the natural history and might reveal more clinical relevance for Ct
genotyping. Whether this goal will be reached with the highly established
serovar nomenclature or a new nomenclature is unknown.
5. Chlamydia trachomatis and the development of
cervical cancer
5.1 Cervical cancer
Cervical cancer is the second most common cancer worldwide, with annually
over 500.000 cases (145). The majority of cases (>80%) occur in developing
countries, due to poor screening programs for precancerous lesions (104).
Since the introduction of population based screening programs in developed
countries, in which high grade precursor lesions of cervical cancer are
diagnosed and treated, a reduction of approximately 75% of the cervical cancer
cases in the past half century have been observed.
It is commonly accepted that infections with specific types of Human
Papillomavirus (HPV) are a necessary cause for the development of squamous
cell- and adenocarcinoma of the cervix. During life, 80% of all sexually active
women get infected with HPV, although the great majority of these infected
women (90-95%) clear the HPV infection without any symptoms. The remaining
5-10% of the women with a persistent HPV infection are at a higher risk of
developing (pre)cancerous lesions. The most common histological types of
cervical cancers are squamous cell carcinoma (80%) and endocervical
adenocarcinoma (20%) (10, 109, 151). Precancerous lesions can be categorized
into the low-grade cervical intraepithelial neoplasia 1 (CIN1) and high-grade
CIN2 and 3 lesions. Most of the CIN1 lesions will regress, making progression
to cervical cancer a rare event(34, 80). Even the majority of high grade CIN
lesions will spontaneously regress (63). Thus, only a small proportion (<1%) of
all women infected with a high risk HPV infection will finally develop cancer.
5.2 High-risk HPV types
As mentioned above, HPV has been recognized as a necessary cause for the
development of cervical cancer. HPV types can be divided into low risk (lr)
30
general introduction
HPV types, associated with genital warts and high risk (hr) HPV types,
associated with cervical cancer. Besides cervical cancer, HPV is also associated
with other carcinomas (e.g., anus, vulva/vagina, penis and oropharynx) (83).
Worldwide, approximately 53% of the cervical cancers are caused by hr-HPV
type 16 and 17.2% by hr-HPV type 18 (111). Taken together, 94.6% of the
cervical cancers are caused by the hr-HPV types 16, 18, 45, 31, 33, 52, 58, 35,
59, 56, 51, 39, 68, 73 and 82 (74). The remaining 5.4% are caused by unknown
HPV types.
5.3 Chlamydia trachomatis and cervical cancer
Several co-factors, like smoking, oral anticonceptive usage, and other sexually
transmitted infections (e.g., HIV infection, Herpes Simplex 2 infection, Ct
infection) have been suggested to facilitate the persistence of an HPV infection
or even the development of cancer(9). The first report of an association
between Ct infections and cervical cancer was already published during the
1970s (101). Multiple, but not all, studies determined an association between
Ct antibodies in the serum and squamous cell carcinoma of the Cervix (SCC) or
persistence of carcinogenic HPV types (2, 50, 75, 110, 112). However, no
association between Ct antibodies or Ct DNA and adenocarcinomas of the
cervix were observed, while the columnar cells of the endocervix are the major
Ct infection site (58, 92, 157). So based on the pathogenesis of Ct we would
expect an association with adenocarcinomas instead of SCCs.
Most studies observed only an association between Ct antibodies and SCC, but
one study also observed Ct DNA in cervical tumors (144). Serovar G antibodies
were even associated with a higher risk for SCC (2). Overall, these results
suggest a role for Ct as a co-factor in cervical carcinogenesis for SCC either by
facilitating a persistent HPV infection via interactions with the immune system
or by facilitating HPV induced SCC itself due to the inflammatory status of
(parts of ) the genital tract (64).
While the association between Ct antibodies and SCC has been interpreted by
some groups as a causal link, others think it might be caused by residual
confounding of other factors that are related to an HPV-positive status. Ct and
HPV are both common sexually transmitted infection that share the same risk
factors, such as sex at young age or multiple sexual partners. This strong
relationship between HPV and Ct can result in inadequate adjustment for HPV,
especially when low sensitive HPV serology is used as a marker for an HPV
infection. So further studies, restricted to HPV positive cases and controls
only, should be performed to overcome the issue of residual confounding.
31
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chapter 1
Thesis outline
The current thesis comprises of three sections, In the first section (Chapter
2-5) comprises a technical part, in which several Ct detection and genotyping
methods are evaluated. The second section (Chapter 6-9) comprises several
studies regarding the Ct serovar distribution and the relation with clinical
signs and symptoms. In the third part (Chapter 10, 11), the association
between Ct and the development of cervical cancer is investigated. Briefly, the
aim of the specific chapters are mentioned below.
Section 1
Chapter 2 describes the evaluation of a sensitive Ct amplification, Ct detection
and Ct genotyping method (Ct-DT assay) that allows detection and identification
of the 14 major Ct serovars (A-L3). Also, the serovar distribution was
determined in samples obtained from the Netherlands and Uganda.
Chapter 3 evaluates the Ct amplification (PCR) and detection step (DEIA) in
clinical samples from Costa Rican women by a comparison with Hybrid Capture
2 Ct detection assay results. Also the Ct serovar distribution among Costa
Rican women was determined with the Ct genotyping step.
In Chapter 4 a comparison between the Ct-DT RHA (genotyping) and 2 other
Ct genotyping methods (ie., OmpA sequencing and LGV/non-LGV differentiation by a pmpH real time PCR) was made.
Chapter 5 describes the evaluation of a new high-throughput microsphere
suspension (MS) assay for simultaneously detection and identification of the
14 serovars on the Luminex platform.
general introduction
infections were determined in LGV positive MSM. Besides the serovar
distribution, this study serves as an evaluation of the LGV/non-LGV differentiation test used at the STI-outpatient clinic in Amsterdam.
Chapter 9 investigates the correlation between the different serogroups and
the level of Ct IgG antibodies in the serum.
Section 3
In Chapter 10 the Ct co-factor role in the development of CIN2+ lesions among
Costa Rican women was investigated on Ct antibody level and Ct DNA level.
Chapter 11 investigates whether Ct DNA was detected in cervical adenocarcinomas. Also, HPV genotyping was performed for all adenocarcinomas.
Chapter 12 consists of a general discussion and conclusion.
Section 2
Chapter 6 contains a description of the first Russian serovar distribution
study (St Petersburg). The obtained Russian distribution results were
compared with previous performed distribution studies in the Netherlands.
Chapter 7 describes the incidence of concurrent serovar infections at Ct
genotyping level. Also, the incidence of multiple anatomical sites (i.e., vagina/
cervix, urethra, rectum and pharynx) on detection and genotyping level were
investigated.
In Chapter 8 the D-K serovar distribution was determined among MSM
infected with non-LGV serovars. Also the percentage of mixed LGV/non-LGV
32
33
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general introduction
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