Diagnostic Utility of Polymerase Chain Reaction, Immunohistochemistry and Ziehl-Neelsen Stain in Detection of Mycobacterium Tuberculosis in Histological Sections

Abstract

Background: Tuberculosis remains a major global health problem. Lymph nodes represent the most common site for extra pulmonary tuberculosis. Several techniques have been employed for the diagnosis of extrapulmonaryTuberculosis (EPTB) including; conventional Hematoxylin and Eosin stain, the Ziehl-Neelsen(ZN) stain, Immunohistochemistry(IHC) and Polymerase chain reaction (PCR). Each of these techniques has advantages and limitations. Aim: To evaluate the diagnostic utility of PCR, IHC and ZN stain in detection of Mycobacterium tuberculosis in histological sections. Materials and methods: This is descriptive analytical cross sectional study conducted in Khartoum State during the period from July 2012 to July 2015. In this study 161lymph nodes tissue biopsies were used in this study. These 161 samples were reinvestigated by H and E. The specific monoclonal anti 38KD was used to detect mycobacterium tuberculosis (MTB) in histological section by IHC, and IS6110 sequence was used to detect MTB by PCR. IS6110 PCR assay was performed in comparison with the H&E, ZN stain and IHC. Results: In this study, 161 enlarged lymph nodes were diagnosed as having lymph nodetuberculosis by histopathology. The minimum age of study population was 4 years and the maximum was 85 years with a mean age of 51 years. The study population was divided in to two group pediatric 42 (26%) and adult 119 (74%). The male female ratio was 0.89:1.11.The study population subsequently subdivided in to other groups started from >8 years up to 51+. The great majority of the specimens were obtained from cervical lymph node which representing 100 (62%), followed by axillary lymph node representing 17 (11%).The other sites include mediastinal, mesenteric, inguinal, and submandibular, constituting 10 (6%), 7 (4%), 7 (4%), and 4 (3%) respectively. The study populations were further divided into two groups according to the presences of strong and weak tuberculosishistopathological evidences. Accordingly, of the 161 cases, 118 (73.3%) were categorized as having strong evidences and the remaining 43(26.7) were detected with weaker evidences, cases. In this study, of 161 studied lymph nodes, only 4 (2.5%) were positive with ZN and 157(97.5%) were negative. These 4(2.5%) ZN positive cases were previously found as strong evidence, while the remaining 114 strong evidences cases were negative with ZN stain. Statistically, no significant association between TB histopathologicalevidences and ZN stain, P-value=0.221. IHC monoclonal anti 38-KD was positive in 129 (80%) of cases, the remaining 32 (20%) were negative with IHC. Of the entire 129 IHC positive cases, 100 (62%) cases were identified as strong evidences cases and the remaining 29 (18%) were at weak level. Statistically, IHC stain is significantly associated with TB histopathologicalevidences P-value = 0.015. In this study, of 161 studied lymph nodes, 135 (84%) were positive for IS1160 PCR and the remaining were negative, from the entire 135 (84%) PCR positive cases, 106 cases were previously found as strong evidence and 29 was found weak evidence. Of remaining 26 (16%) PCR negative cases, only 12 specimens were showed strong evidence for TB, and 14 were weak evidence, statistically, PCR is significantly associated with TB histopathologicalevidences, P-value=0.001. On the other hand PCR was used as a gold standard for comparing the other variables, accordingly the sensitivity and specificity of histopathology diagnosis & ZN stain were 78.5%, 46.1% and 3.0%, 100% respectively. In contrast the sensitivity and specificity of anti 38KD IHC was 95.5%, 100% respectively. Conclusion: IHC with monoclonal anti 38KD and PCR with IS6110 oligonucleotides are rapid, sensitive, and specific methods for establishing the diagnosis of tuberculosis in histologic specimens. Immunohistochemistry has the advantages over PCR of being robust, quicker, and cheaper, and it can be used in high-endemic countries.