Journal of Diagnostic Techniques and Biomedical

Yoshioka et al., J Diagnos Tech Biomed Anal 2014, 2:1
http://dx.doi.org/10.4172/jdtba.1000108
Journal of Diagnostic
Techniques and
Biomedical Analysis
Research Article
A SCITECHNOL JOURNAL
Bacillus Calmette-Guérin (BCG)
Vaccine-Induced Disease in
Healthy Infants: Identification of
BCG Gene from Formalin-Fixed
Paraffin-Embedded Tissue
Takako Yoshioka1, Junichiro Nishi2, Kiyofumi Ohkusu3, Kazuhito
Hatanaka1, Sohsuke Yamada4, Kazuhiko Nakame5, Yoshifumi
Kawano2, Tatsuru Kaji5 and Akihide Tanimoto1*
1Department
of Molecular and Cellular Pathology, Kagoshima University Graduate
School of Medical and Dental Sciences, Kagoshima, Japan
2Microbiology
Kagoshima University Graduate School of Medical and Dental
Sciences, Kagoshima, Japan
3Department
of Microbiology, Gifu University Graduate School of Medicine, Gifu,
Japan
4Department
of Pathology and Cell Biology, School of Medicine, University of
Occupational and Environmental Health, kitakyushu, Japan
5Pediatric Surgery, Kagoshima University Graduate School of Medical and Dental
Sciences, Kagoshima, Japan
*Corresponding author: Akihide Tanimoto, MD, PhD, Department of Tumor
Pathology, Kagoshima University of Graduate School of Medical and Dental
Sciences, 8-35-1 Sakuragaoka, Kagoshima 890-8544, Japan, Tel:
81-99-275-5263; Fax: 81-99-264-6348; E-mail: [email protected]
u.ac.jp
Rec date: Feb 18, 2014 Acc date: April 07, 2014 Pub date: April 11, 2014
Keywords: Tuberculosis;
Osteomyelitis
BCG;
Infant;
PCR;
Granuloma;
Abbreviations:
BCG: Bacillus Calmette-Guérin; PCR: Polymerase Chain Reaction;
H&E: Hematoxylin and Eosin; PAS: Periodic Acid-Schiff; FFPE:
Formalin-Fixed Paraffin-Embedding
Introduction
Bacillus Calmette-Guérin (BCG) vaccination has been widely
employed in the world to prevent M. tuberculosis infection. The BCG
vaccine contains live attenuated strain of M. bovis and it rarely causes
systemic complication but only localized and weak skin irritation at
the site of inoculation in healthy infants. In contrast,
immunocompromised human immunodeficiency virus-infected
infants have a high prevalence of BCG-induced complication [1,2].
Even in healthy immunocompetent infants, however, BCG diseases
such as lymphadenitis and regional abscess formation have been
reported [3]. In addition, it has also been reported that BCG diseases
may occur as a consequence of immunotherapy for bladder cancer
(intravesical installation of BCG) [4]. Herein we report three cases of
BCG vaccination-induced osteomyelitis (1 case) and dermal abscess (2
cases) in healthy children more than 6 months after the BCG
vaccination.
Materials and Methods
Histological examination
Abstract
Background: Bacillus Calmette-Guérin (BCG) vaccineinduced systemic complication is rare but localized skin lesion
develops in the site of inoculation in healthy infants.
Methods: In this report, we described three cases of BCG
vaccine-induced skin lesions in healthy immunocompetent
infants and a method of identification of BCG gene by
polymerase chain reaction (PCR) form fresh frozen of formalinfixed and paraffin-embedded tissue samples.
Results: These cases had a history of BCG vaccination but no
family history of tuberculosis and no contact with patients of
tuberculosis. Two cases developed cutaneous granuloma in
the axilla of ipsilateral side of BCG inoculation. Another case
showed granulomatous osteomyelitis of the left 6th rib.
Histological examination demonstrated typical pathologic
findings for epithelioid granuloma with caseation necrosis,
indicating mycobacterium infection. PCR analysis from fresh
frozen or formalin-fixed paraffin-embedded tissue rapidly
confirmed the infection of Mycobacterium bovis BCG but not M.
tuberculosis.
Conclusion: BCG infection should be considered even in
healthy infants with a history of BCG vaccination and histology
of epithelioid granuloma with caseation necrosis. In such
cases, BCG gene identification by PCR would be useful even
from formalin-fixed and paraffin embedded tissue samples.
The resected materials were fixed with phosphate buffered (pH 7.4)
20% formalin and routinely processed for preparation of paraffinembedded tissue. The 5µM thick sections were stained with
hematoxylin and eosin (H&E), Ziehl-Neelsen, Grocott and periodic
acid-Schiff (PAS) stains.
Polymerase chain reaction and sequencing of BCG gene
DNAs from fresh frozen tissue (Case 1) or formalin-fixed paraffinembedding (FFPE) tissue sections (Cases 2 and 3) were extracted with
proteinase K buffer for PCR amplification of BCG gene. A primer set
targeting the M. bovis BCG-specific region of difference 1 (RD1) was
designed according to the genomic DNA sequence as 5’CAAGCACTGGCGGTCAACCT-3’
and
5’-CGACGGGCAGC
TATGCCAGA-3’ (GenBank accession number AM408590.1) [5]. The
PCR was performed in a volume of 50µL with the template DNA. The
thermal profile was 48 cycles at 95°C (30 secs), 66.5°C (30 secs) and
74°C (30 secs). The PCR products (167 bp) were then electrophoresed
on a 3% agarose gel and the sequence was confirmed. No PCR product
was amplified with a primer set (ET2 and ET3) [5] for detection of M.
tuberculosis.
Results
Case 1
A 15-month-old male was admitted to the hospital with a swelling
of the left anterior chest wall. Under a diagnosis of bone tumor of the
left 6th rib, a biopsy was undertaken and histological examination
All articles published in Journal of Diagnostic Techniques and Biomedical Analysis are the property of SciTechnol and is
protected by copyright laws. Copyright © 2015, SciTechnol, All Rights Reserved.
Citation:
YoshiokaT, Nishi J, Ohkusu K, Hatanaka K, Yamada S, et al. (2014) Bacillus Calmette-Guérin (BCG) Vaccine-Induced Disease in Healthy Infants:
Identification of BCG Gene from Formalin-Fixed Paraffin-Embedded Tissue. J Diagnos Tech Biomed Anal 2:1.
doi:http://dx.doi.org/10.4172/jdtba.1000108
showed findings of osteomyelitis by epithelioid granuloma with
caseation necrosis. His family had no history of tuberculosis and he
and his family had no contact with patients of tuberculosis. He was
vaccinated with BCG (Tokyo strain) by an inoculation in the left upper
arm at his 4 months of age. Computed tomography (CT) showed a soft
tissue density mass around the left 6th rib (Figure 1A). The
tuberculosis-specific interferon-γ (Quantiferon, Cellestis Limited,
Australia) was not detected. After chemotherapy, the lesion including
the rib was surgically removed. No immunological abnormality was
demonstrated by clinical tests.
Macroscopic and microscopic examination of the resected lesion
demonstrated osteomyelitis featured by osteolytic bone tissue with
epithelioid granulomas and caseation necrosis (Figure 1B-D). The
adjacent connective tissue and skeletal muscles were involved by the
granuloma. PCR analysis using DNA extracted from the fresh frozen
tissue to amplify the M. bovis BCG-specific region of difference 1
(RD1) confirmed the diagnosis of BCG-induced granuloma (Figure 3).
Figure 2: Pathological presentation of Cases 2 and 3 (A) Gross
appearance of the resected lesion of left axillary subcutaneous mass
of Case 2; (B) Epithelioid granuloma with caseation necrosis and
calcification is noted in the histological section of Case 2 (H&E
stain). (C) Gross feature of the left axillary skin of Case 3 (D)
Microscopic appearance of the resected lesion. Epithelioid
granuloma with giant cells and caseation necrosis is observed in
Case 3(H&E stain).
Figure 1: Clinical and pathological presentation of Case 1 (A) CT
shows a soft tissue density mass around the left 6th rib (arrow); (B)
Gross appearance of resected lesion; (C) Low power view of the
granuloma in the bone with obvious acellular central necrosis; (D)
Epithelioid granuloma with caseation necrosis and giant cells is
noted in the histological section (H & E stain).
Case 2
A 3-year and 7-month-old male was admitted to the hospital due to
a subcutaneous mass in the left axilla. Lymphangioma was suspected
and the lesion was surgically resected. Chest CT showed no abnormal
findings. His family had no history of tuberculosis and he and his
family had no contact with the patients of tuberculosis. He was
vaccinated with BCG (Tokyo strain) by an inoculation in the left upper
arm at his 8 months of age. No immunological abnormality was
demonstrated by clinical tests.
Macroscopic and microscopic examination of the cutaneous mass
demonstrated epithelioid granulomas with caseation necrosis and
calcification (Figure 2A & B). PCR analysis using DNA extracted from
the FFPE tissue section to amplify the M. bovis BCG-specific RD1
region confirmed the diagnosis of BCG-induced granuloma (Figure 3).
Volume 2 • Issue 1 • 1000109
Figure 3: PCR detection of BCG-specific genome. A 167bp band is
amplified from the fresh frozen tissue (Case 1) or FFPE tissue
(Cases 2 and 3) in each case. M: molecular size marker; N: negative
control; P: positive control of BCG Tokyo strain DNA.
• Page 2 of 4 •
Citation:
YoshiokaT, Nishi J, Ohkusu K, Hatanaka K, Yamada S, et al. (2014) Bacillus Calmette-Guérin (BCG) Vaccine-Induced Disease in Healthy Infants:
Identification of BCG Gene from Formalin-Fixed Paraffin-Embedded Tissue. J Diagnos Tech Biomed Anal 2:1.
doi:http://dx.doi.org/10.4172/jdtba.1000108
Case 3
A 23-month-old male was hospitalized by a subcutaneous mass at
the left axilla (Figure 2C), which was suspected to be lymphangioma
and the lesion was surgically resected. Chest CT disclosed no
significant findings. His family had no history of tuberculosis and he
and his family had no contact with the patients of tuberculosis. He was
vaccinated with BCG (Tokyo strain) by an inoculation in the left upper
arm at his 4 months of age. No immunological abnormality was
demonstrated by clinical tests.
Microscopic examination of the cutaneous mass demonstrated
epithelioid granulomas with caseation necrosis and calcification
(Figure 2D). PCR analysis using the FFPE tissue section did not detect
the genes of M. tuberculosis but BCG-specific RD1 region of M. bovis
was confirmed (Figure 3).
Discussion
In general, the complications following BCG vaccination are
estimated to be 0.01% to 3.6% in frequency and most BCG
complication diseases are local or regional diseases manifested by such
as local skin ulceration at the site of inoculation and ipsilateral
lymphadenitis, respectively [1,3,6]. However, distant or disseminated
BCG-related disease occurs even in immunocompetent infants and
children [3,7-9]. Recent classification of BCG disease includes five
categories: (1) local disease, (2) regional disease, (3) distant disease, (4)
disseminated disease, and (5) other BCG syndromes [1]. The local
disease is manifested by abscess or ulcer formation at the site of
vaccination. The regional disease includes lymph node involvement
beyond the vaccination site such as ipsilateral axillary, supraclavicular
and cervical lymph nodes. These two conditions accounted for 88% of
the total BCG-related diseases. The distant disease involves at least one
distant site beyond local or regional ipsilateral process. This includes
any of following: osteomyelitis, distant skin lesions, and pulmonary,
gastric or urinary tract lesions. The disseminated disease is shown to
have more than one lesion as described under the distant disease.
Thirty two % of cases presented with distant or disseminated disease;
20 % of cases had both local and distant or disseminated disease.
According to this classification, the cutaneous granuloma observed in
the Case 2 and Case 3 may be classified into regional disease. In
contrast, the granulomatous osteomyelitis of the Case 1 would be
categorized into distant disease group.
A high prevalence of BCG complications, containing local to more
critical
disseminated
diseases,
has
been
known
in
immunocompromised children [1,2,10,11]. The BCG-induced
diseases, however, have also been reported in immunocompetent
children. These include local or regional diseases and even distant
osteomyelitis [12-15] and disseminated military granulomas [7-9],
indicating a presence of unknown or very subtle form of
immunodeficiency [3]. In the present cases, at least, no major
immunodeficiency diseases such as HIV infection and severe
combined immunodeficiency disease, was detected.
The pathological findings in the present cases revealed
granulomatous lesions consisted of epithelioid cells and Langerhans
giant cells with central caseous necrosis, which are essentially identical
to those described in the case of M. tuberculosis and other
mycobacterial infection. Since we could not find acid-fast bacilli in the
histological sections, the PCR detection of M. bovis BCG-specific gene
was quite useful for the final diagnosis of BCG-related diseases. By the
appropriate design of primer set for PCR to target the RD1 region, we
Volume 2 • Issue 1 • 1000109
could show the specific 167bp PCR products using DNA extracted
even from the FFPE tissue in 2 cases. Since the genomic RD1 region
includes specifically deleted sequence in the M. bovis [5,16], we could
differentiate M. bovis from M. tuberculosis. The conventional bacterial
culture takes long time before mycobacterial growth to extract the
bacterial DNA and is unable to distinguish BCG and other
mycobacterial strains. Thus, FFPE tissue-based PCR would be still very
beneficial as a diagnostic tool for early detection of M.bovis BCG, even
though the sensitivity of PCR detection from FFPE tissue would be
lower than that from fresh tissue or cultured colonies. In the
previously reported cases, the PCR recognition of M. bovis BCG strain
was achieved using the DNA extracted from colony isolates [12,13,15].
In contrast, one autopsy case of disseminated disease has been
reported that M. tuberculosis complex genome but not specific for
BCG strain was detected from FFPE tissue section [9]. To our
knowledge, however, demonstration of M. bovis BCG-specific genome
from FFPE tissue has rarely been reported.
The diagnosis of BCG diseases must be considered even in healthy
infants who manifest the histology of granulomas with caseous
necrosis and history of BCG vaccination. In addition to microscopic
examination, molecular identification of M. bovis BCG genome by
PCR using DNA extracted from FFPE sections would be available for
rapid and confirmative diagnosis, especially when the diagnosis of
tuberculosis is not suspected and all the specimens are fixed with
formalin for routine histological examination.
Conclusion
BCG infection should be considered even in healthy infants with a
history of BCG vaccination and histology of epithelioid granuloma
with caseation necrosis. In such cases, BCG gene identification by PCR
would be useful even from formalin-fixed and paraffin embedded
tissue samples.
Acknowledgements
This work was supported in part by the Kodama Memorial fund
Medical Research.
References
1.
2.
3.
4.
5.
Hesseling AC, Rabie H, Marais BJ, Manders M, Lips N, et al.
(2006) Bacile Calmette-Guérin vaccine-induced disease in HIVinfected and HIV-uninfected children. Clin Infect Dis 42:
548-558.
Nuttall JJ, Davies M, Hussey GD, Eley BS (2008) Bacillus
Calmette-Guérin (BCG) vaccine-induced complications in
children treated with highly active antiretroviral therapy. Int J
Infect Dis 12: e99-e105.
Tajima Y, Takagi R, Nakajima T, Kominato Y (2008) An infant
with asymptomatic hepatic granuloma probably caused by
bacillus Calmette-Guérin (BCG) vaccination found incidentally
at autopsy: a case report. Cases J 1: 337.
Smith RL, Alexander RF, Aranda CP (1993) Pulmonary
granulomata. A complication of intravesical administration of
bacillus Calmette-Guérin for superficial bladder carcinoma.
Cancer 71: 1846-1847.
Talbot EA, Williams DL, Frothingham R (1997) PCR
identification of Mycobacterium bovis BCG. J Clin Microbiol 35:
566-569.
• Page 3 of 4 •
Citation:
YoshiokaT, Nishi J, Ohkusu K, Hatanaka K, Yamada S, et al. (2014) Bacillus Calmette-Guérin (BCG) Vaccine-Induced Disease in Healthy Infants:
Identification of BCG Gene from Formalin-Fixed Paraffin-Embedded Tissue. J Diagnos Tech Biomed Anal 2:1.
doi:http://dx.doi.org/10.4172/jdtba.1000108
6.
7.
8.
9.
10.
11.
Hengster P, Solder B, Fille M, Menardi G (1997) Surgical
treatment of Bacillus Calmette-Guérin lymphadenitis. World J
Surg 21: 520-523.
Abramowsky C, Gonzalez B, Sorensen RU (1993) Disseminated
bacillus Calmette-Guérin infections in patients with primary
immunodeficiencies. Am J Clin Pathol 100: 52-56.
Casanova JL, Jouanguy E, Lamhamedi S, Blanche S, Fischer A
(1995) Immunological conditions of children with BCG
disseminated infection. Lancet 346: 581.
Drut R, Quijano G (1998) Disseminated bacillus CalmetteGuérin, military type: autopsy findings and diagnosis using
polymerase chain reaction. Pediatr Dev Pathol 1: 143-148.
Antaya RJ, Gardner ES, Bettencourt MS, Daines M, Denise Y, et
al. (2001) Cutaneous complications of BCG vaccination in
infants with immune disorders: Two cases and a review of the
literature. Pediatr Dermatol 18: 205-209.
Fehon R, Mehr S, Hei EL, Isaacs D, Wong M (2008) Two-yearold boy with cervical and liver abscesses. J Pediatr Child Health
44: 670-672.
Volume 2 • Issue 1 • 1000109
12.
13.
14.
15.
16.
Funato M, Kaneko H, Matsui E, Teramoto T, Kato Z, et al.
(2007) Refractory osteomyelitis caused by bacilli CalmetteGuérin vaccination: a case report. Diagn Microbiol Infect Dis 59:
89-91.
Kim SH, Kim SY, Eun BW, Yoo WJ, Park KU, et al. (2008) BCG
osteomyelitis caused by the BCG Tokyo strain and confirmed by
molecular method. Vaccine 26: 4379-4381.
Lin WJ, Lu JJ, Chu CC, Chang TY, Wang CC (2007) CalmetteGuérin bacillus sterna osteomyelitis diagnosed by DNA
sequencing analysis of PNC A. Pediatr Infect Dis J 23: 784-786.
Nishi J, Kamenosono A, Sarker KP, Yoshino S, Ikei J, et al. (2007)
Bacilli Calmette-Guérin osteomyelitis. Pediatr Infect Dis J 16:
332-333.
Bedwell J, Kairo SK, Behr MA, Bygraves JA (2001) Identification
of substrains of BCG vaccine using multiplex PCR. Vaccine 19:
2146-2151.
• Page 4 of 4 •