Detecting extrapulmonary tuberculosis using immunohistochemistry with anti-Mycobacterium tuberculosis antibody ab905; a cross-sectional study at Mbarara Regional Referral Hospital

Detecting extrapulmonary tuberculosis using immunohistochemistry with anti-Mycobacterium tuberculosis antibody ab905; a cross-sectional study at Mbarara Regional Referral Hospital | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Detecting extrapulmonary tuberculosis using immunohistochemistry with anti-Mycobacterium tuberculosis antibody ab905; a cross-sectional study at Mbarara Regional Referral Hospital Lawrence Amadile, Abraham Birungi, Hassan Wasswa, Saphurah Nabaasa, and 8 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8496524/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background: Diagnosing extrapulmonary tuberculosis (EPTB) poses challenges such as delayed diagnosis, missed diagnosis, and misdiagnosis of EPTB. Immunohistochemistry is believed to improve the accuracy of EPTB diagnosis in tissues fixed in formalin and embedded in paraffin. Patients and methods: This was a cross-sectional study, and data were collected between June 2023 and November 2023, involving 87 archived tissue specimens from patients with clinical suspicion of EPTB. Demographic data were collected from pathology registers, and Hematoxylin and Eosin (H&E) and Ziehl Neelsen (ZN) staining and microscopy techniques were performed on sections from the tissue blocks to confirm the diagnosis of EPTB. A concentrated anti- Mycobacterium tuberculosis antibody (ab905, Abcam, Cambridge, UK), diluted (1:100) for immunohistochemistry (IHC) was used. Indirect manual IHC technique was performed. ZN and/or histopathology were used as the component composite reference standard tests. Results: The study population comprised of 87 tissue blocks. Lymph nodes constituted the majority (50/87, 57%) of specimens. Histopathologically, 52.9% (46/87) of the specimens had granulomatous inflammation suggestive of EPTB, whereas 47.1% (41/87) did not. Of the 87 specimens analyzed, 17 (19.5%) were positive using the ZN technique and 70 (80.5%) were negative. The sensitivity, specificity, PPV, and NPV of anti -Mycobacterium tuberculosis antibody staining were 74.5%, 80.0%, 81.4%, and 72.7%, respectively. Conclusion: Based on the findings of our study, anti -Mycobacterium tuberculosis antibody ab905 staining is a valuable method for excluding or diagnosing EPTB in tissues. Prospective data collection using a monoclonal ab905 anti- Mycobacterial tuberculosis antibody and a more sensitive gold standard would negate the limitations of this study. Sensitivity Specificity Formalin-fixed paraffin-embedded Hematoxylin & Eosin Immunohistochemistry Ziehl-Neelsen Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Background Tuberculosis (TB) is a communicable and contagious disease caused by Mycobacterium tuberculosis that majorly cause pulmonary tuberculosis but has the capacity to cause disease in other body organs or sites than the lungs. Globally, extrapulmonary tuberculosis (EPTB) contributes an appreciable fraction of tuberculosis cases accounting for nearly 20–30% of total active TB patients, and attacks majorly children (≤ 15 years of age) and immune-compromised adults 1 . TB and Human Immunodeficiency Virus (HIV) infection disproportionately affects Africa. In a ten-year retrospective TB prevalence and patient clinical profiling study conducted in Nigeria, EPTB accounted for 12.9% of all TB cases. 2 In South Africa, the prevalence of EPTB was found to be 25.0% among adults. 3 However, in Larache, Morocco, EPTB prevalence of 43.5% was found in a study conducted in 2020. 4 A significantly high proportion of extrapulmonary tuberculosis cases are missed, misdiagnosed or underdiagnosed because of the below par sensitivity of available diagnostic tests. 5 Being diverse clinically and having few bacilli in specimens microbiologically, there are usually several conditions that mimic EPTB leading to delayed/missed diagnosis. Uganda is one of the 30 countries that contribute 80% of the world’s tuberculosis burden, with a reported EPTB prevalence of 11%. 6 Uganda, also being in the category of the world’s 30 high TB/HIV burden countries, missed or misdiagnosis of EPTB exacerbates the spread of TB in the public with HIV-infected persons at more risk. Although TB culture is taken as the benchmark for the diagnosis of EPTB, its major shortcoming is the significantly extended time to obtain a result. It also requires unfixed tissue specimen. 7 Furthermore, the sensitivity of culture varies from (0–80%), 8 this heterogeneity is not indicative of a good diagnostic test. Polymerase chain reaction (PCR) based assays are rapid and specific but they are not readily available in routine practice, 9 yet the bacilli are not evenly dispersed throughout the biopsy. 10 ZN staining and microscopy of tissue sections that are formalin fixed and paraffin embedded is employed to confirm EPTB infection; however, its major disadvantage is the low sensitivity due to paucity of bacilli in EPTB tissue biopsies. 11 Sensitivity of the ZN technique decreases acutely after formalin fixation and/or xylene treatment. 12 Histopathological analysis of tissue specimen for extra pulmonary tuberculosis diagnosis has remained the most reliable method at present and it is mostly arrived at by identifying classical granuloma formation with caseous necrosis. 13 This study was therefore aimed at determining the sensitivity, specificity, positive predictive and negative predictive values of ab905 immunohistochemistry for the detection of Mycobacterial antigen in archived tissue specimens at Mbarara Regional Referral Hospital. Methods Aim The primary objective of this study was to determine the sensitivity and specificity of anti- Mycobacterium tuberculosis antibody staining for diagnosing EPTB in tissue specimens. Study design We conducted a cross-sectional laboratory-based study including archived tissue blocks of patients with suspicion of EPTB. Study setting This study was carried out at the pathology department of Mbarara Regional Referral Hospital (MRRH), between June 2023 and November 2023. MRRH serves as a regional referral center for the entire south western Uganda and it is located in Mbarara city, approximately 260km from the capital city, Kampala. The laboratory receives about 10 biopsies requiring TB testing per month. It receives patients from more than 13 districts in Western Uganda and the neighbouring countries that include Tanzania, Rwanda and Congo (DRC). Sampling procedure We retrospectively selected the tissue blocks. We used consecutive sampling to select the tissue blocks. Sample size determination Sample size at the required absolute precision level for sensitivity and specificity was calculated using Buderer’s formula 14 at 95% confidence level. A 68.29% sensitivity and 96.5% specificity of IHC on FFPE blocks was reported using TB culture or PCR as reference standard in previous studies. 15 Prevalence of extrapulmonary tuberculosis based on tissue diagnosis in Uganda is not clearly known, therefore we used an arbitrary prevalence value of 50% (i.e., p=0.5). This gave a total of 78 patients represented by 78 blocks. After factoring in the possible 15% attrition, the consequential sample size was at least 90 patients represented by 90 blocks. Data collection Demographic data collection Demographic information was gathered using data collection tool by the PI and research assistants during the register review process. The collected data encompassed information such as age, gender and tissue type. Collection of tissue blocks and microtomy Each FFPE tissue block retrieved was trimmed and 5 microns thick sections were cut using a rotary microtome. From each tissue block, three sets of serial sections were cut for each of the three staining protocols. Hematoxylin and Eosin staining and interpretation The sections were stained using progressive H&E staining method. In brief, we used xylene to dewax the sections, sections were taken to water and stained with hematoxylin. Bluing was done using tap water followed by staining in eosin, the sections were rinsed in ethanol, air dried and mounted in Dibutylphthalate Polystyrene Xylene (DPX). Histological features were evaluated under the microscope under low power (4x, 10x and 20x) and confirmed at high power (40x). Appearance of classical granulomas, caseous necrosis, epithelioid histiocytes and Langhans giant cells were used as features suggestive of EPTB. Ziehl Neelsen (ZN) and interpretation We used routine AFB kit for histology to stain the sections, slides were flooded with filtered Carbol fuchsin solution for 15 minutes and washed using running tap water. This was followed by differentiation in 1% acid alcohol until sections were pale pink, washed using tap water for 2 minutes and dipped in deionized water for 1 minute. We used methylene blue to counterstain sections for 30 seconds. Slides were rinsed in tap water and dipped once in deionized water. They were dehydrated using ascending grades of ethanol, dipped in xylene for clearing and cover slipped using DPX. We used 10x, 40x and 100x objectives to examine the slides and findings were reported as positive or negative for AFB when red staining AFB in clusters or singular AFB were present or absent respectively. Immunohistochemistry (IHC) Sections were mounted on positively charged slides (Bio-Optica Milano Spa, Milano MI, Italy). Immunohistochemistry (IHC) was done on FFPE specimens using concentrated anti- Mycobacterium tuberculosis antibody (ab905, Abcam, Cambridge, UK) diluted (1:100). Indirect manual IHC technique with one-step envision method (HRP-Streptavidin-biotin method, Dako, Germany) with diaminobenzidine (DAB) as chromogen was used. Positive (ZN positive lymph node tissue) and negative control (section from a tissue that did not have TB) were run together with test tissues under similar conditions. Slides were baked in 50-52 o C oven overnight. Sections were deparaffinized by dipping in xylene three times with every dip lasting three minutes and taken to water where descending concentrations of ethanol were used i.e., 100%, 100%, 95%, 95% for two minutes each followed by 5 dips in 80% alcohol and 30 seconds in deionized water. Slides were incubated at 37 o C for 15 minutes in Proteinase K. Sections were allowed to cool for 10 minutes at room temperature. Slides were then rinsed three times in distilled water. Tissue sections were circled with a pap pen to contain fluids. Slides were placed in diluted Tris-buffer and rinsed well (three times in coplin jar). Endogenous peroxidase activity was blocked by covering slides with 1% aqueous hydrogen peroxide and incubated for 10 minutes; rinsed with Tris buffer and covered sections with DAKO Serum-free protein block and incubated for 10 minutes. Slides were carefully rinsed in Tris buffer for three minutes in a coplin jar. We thereafter shook off excess Tris buffer around tissue and placed slides back on tray. We placed two drops (200 µl; enough to cover tissue) of the primary antibody (ab905) on the tissue section; closed the lid and incubated for 30 minutes. Slides were carefully rinsed in Tris buffer three times and then carefully shook off excess Tris buffer around tissue and placed slides back on tray. Two drops of “Labelled polymer-HRP” anti-rabbit was placed on the tissue section and incubated for 30 minutes. Slides were carefully rinsed in Tris buffer three times; carefully shook off excess Tris buffer around tissue and placed slides back on tray. Made the DAB chromogen solution (for the Dako kit, added a drop (20µl) of DAB chromogen on every 1 ml of DAB substrate used (e.g., for 7 slides, used 2 ml DAB substrate buffer and 2 drops DAB chromogen)). Enough DAB chromogen to cover the tissue was applied and incubated for 10 minutes; collected DAB chromogen waste in a hazardous container and rinsed slides in distilled water three times. Counterstained with hematoxylin, dehydrated sections in two changes of 95% ethanol and absolute ethanol, and cleared in xylene thereafter slides were cover slipped using DPX (dibutyl phthalate xylene), which acts as an indefinite mounting medium. Anti- Mycobacterium tuberculosis antibody (ab905) immunohistochemistry staining interpretation The PI together with an experienced pathologist examined and reviewed the slides under 20x and 40x objective lenses. Brown granular staining in the cytoplasm or around the inflammatory cells was regarded positive (ab905 insert). Data handling, analysis and interpretation The PI and research assistants gathered the raw data which was then entered into an excel spread sheet (Microsoft Office Professional Plus 2013, version 15.0.4675.1003, Microsoft Inc, USA). It was then imported into STATA 17 (StataCorp LLC, College Station, Texas, United States) software for all analyses. Descriptive statistics were used to characterize the population using frequencies, median values and interquartile ranges (IQRs). Clinical, H&E and ZN data were made available to the pathologists since they used these both at optimisation and slide reading steps. The variables analyzed included; H&E results, ZN results and IHC results, tissue type; diagnostic accuracy, predictive values of anti- Mycobacterium tuberculosis antibody staining calculated using histopathology and/or ZN staining as reference standard since TB culture could not be used on archived specimens. Sensitivity, specificity, positive predictive and negative predictive values of anti- Mycobacterium tuberculosis antibody staining were reported as proportions with 95% confidence intervals. Receiver Operating Characteristic curve was used to determine the predictive performance of IHC for EPTB. Eligibility criteria We included formalin-fixed paraffin-embedded tissue blocks of patients with symptoms of EPTB archived between January 2009 and August 2023. See study flow (Fig.1). Results Flow of participants The flow of participants through enrolment, eligibility and outcomes of the study was as shown on the STARD flow diagram in Figure 1. Study participant characteristics In this cross-sectional study, we retrieved 87 tissue specimens (Table 1), 51/87 (59%) were from males. The median age of the study participants was 29 (interquartile range: 17-49) years. There was no notable difference in the distribution of tissue specimens among the different age categories. Different tissue types were analyzed; lymph nodes 50/87 (57%) constituted the majority of the specimens. Table 1 Demographic characteristics of the patients whose specimens were analyzed. Variable Description Frequency (n) Percentage (%) Age Median=29 IQR= (17-49) <15 18 21 15-24 16 18 25-34 13 15 35-44 13 15 45-54 10 21 55+ 17 20 Sex Female 36 41 Male 51 59 Tissue type Lymph node 50 57 Bone 4 5 Prostate 4 5 Spine 2 2 Skin 2 2 Other 23 26 IQR, interquartile range Test results H&E staining photomicrographs Under H&E, three inflammatory patterns were observed namely non-classical granuloma, classical granuloma and no granuloma (Fig.2). More specimens for patients in the 54 age group 10/46 (21.7%) as shown in Table 2. Immunohistochemistry results Using the anti- Mycobacterium tuberculosis antibody, 43/87 (49.4%) of the specimens stained positive while 44/87 (50.6%) were negative (Fig.3). While 41/43 (95.3%) of the tissues stained strongly positive, 3/43 (6.97%) stained weakly positive as shown in Fig.4. More lymph node tissues 27/43 (62.7%) stained positive than non-lymph node tissues 16/43 (37.2%). In terms of the predictive performance of Anti-Mycobacterium tuberculosis antibody staining, Fig.5 shows that Anti- Mycobacterium tuberculosis antibody staining was a good predictor for EPTB in tissues sections. It significantly predicted EPTB, area under the ROC curve 0.7723 (0.6834-0.8613). The predictive performance of Anti- Mycobacterium tuberculosis antibody staining by tissue type did not show significant difference between lymph nodes and non-lymph node tissues with the ROC area of 0.7833 and 0.7529 respectively, p value=0.78486 as shown in Figure 6 below. Ziehl Neelsen staining photomicrographs ZN negative and positive staining patterns indicated in (Fig.7) below and further results indicated in Table 2). A comparison of positive IHC and ZN staining is shown in Figure 8. Table 2: Test results for H&E, ZN and Anti-Mycobacterium tuberculosis antibody immunohistochemistry by Age, Sex, Tissue type and inflammatory pattern Variable Description H&E ZN Ab905 immunostaining Positive f (%) n=46 Negative f (%) n=41 Positive f (%) n=17 Negative f (%) n=70 Positive f (%) n=43 Negative f (%) n=44 Sex Female 22 (48) 14 (34) 5 (29) 31 (44) 21 (49) 15 (34) Male 24 (52) 27 (66) 12 (71) 39 (56) 22 (51) 29 (66) Age <15 11 (24) 7 (17) 4 (24) 14 (20) 13 (30) 5 (11) 15-24 7 (15) 9 (22) 3 (18) 13 (19) 7 (16) 9 (20) 25-34 7 (15) 6 (15) 5 (29) 8 (11) 8 (19) 5 (11) 35-44 6 (13) 7 (17) 2 (12) 11 (16) 5 (12) 8 (18) 45-54 5 (11) 5 (12) 0 (0) 10 (14) 3 (7) 7 (16) 55+ 10 (22) 7 (17) 3 (18) 14 (20) 7 (16) 10 (23) Tissue type Lymph node 30 (65) 20 (49) 11 (65) 39 (56) 27 (63) 23 (52) Non-Lymph nodes 16 (35) 21 (51) 6 (35) 31 (44) 16 (37) 21 (48) Inflammatory pattern Classical granuloma 45 (98) 0 (0) 16 (94) 29 (41) 35 (81) 10 (23) Non-classical granuloma 0 (0) 9 (22) 0 (0) 9 (13) 3 (7) 6 (14) No granuloma 1 (2) 32 (78) 1 (6) 32 (46) 5 (12) 28 (64) Diagnostic utility of anti-Mycobacterium tuberculosis antibody staining using ZN and H&E as a composite reference standard. As calculated from the 2x2 table (Table 3), the overall sensitivity, specificity, positive and negative predictive values for Anti- Mycobacterium tuberculosis antibody staining are shown in Table 4. Table 3: Cross-tabulation of anti- Mycobacterium tuberculosis antibody staining and H&E and/or ZN (composite reference standard) results. Anti-Mycobacterium tuberculosis antibody immunohistochemistry Composite reference standard Total f (%) Negative f (%) Positive f (%) Negative 32 (80.00%) 12 (25.53%) 44 (50.57%) Positive 8 (20.00%) 35 (74.47%) 43 (49.43%) Total 40 (100%) 47 (100%) 87 (100%) Table 4: Diagnostic utility of anti- Mycobacterium tuberculosis antibody staining Parameter Measure 95% CI Sensitivity 74.5% 65.31-83.63% Specificity 80.0% 71.59-88.41% Positive predictive value 81.4% 73.22-89.57% Negative predictive value 72.7% 63.37-82.09% Discussion We present a relatively high and balanced sensitivity, specificity, positive predictive and negative predictive values of ab905 IHC (74.5%, 80.0%, 81.4% and 72.7% respectively) for EPTB in tissue sections. Our data indicate that ab905 anti- Mycobacterium tuberculosis antibody staining is a useful tool in diagnosis or exclusion of EPTB in tissue sections. This was applicable to the total study population (maximum AUC 0.77), lymph nodes tissues (maximum AUC 0.78) and non-lymph node tissues (maximum AUC 0.75). The sensitivity of anti- Mycobacterium tuberculosis antibody (ab905) staining in this study was 74.5%. This meant that ab905 IHC correctly identified 74.5% of individuals who actually had EPTB. However, sensitivity of 74.5% also implies that about 25.5% of individuals with EPTB might not be identified by ab905 IHC because of technical factors affecting the sensitivity of the test such as effect of storage on the archived tissue specimens. The sensitivity of 74.5% found in this study is comparable to those found by previous authors who assessed sensitivity of IHC for the diagnosis of EPTB in tissue biopsies. 15 – 17 The comparability could be attributed to the same specimen type used being biopsies in the current study and the previous studies, same antigen preservation technique (FFPE sections), and the use of composite reference standard. However, there has been reported variability in the sensitivity of IHC for EPTB in tissue sections across different studies. Many studies reported sensitivity which were non-comparable to the one obtained in this study. 7 , 18 – 21 The plausible reason for the higher sensitivity obtained above could be the use of superior gold standards such as PCR, composite reference standard with more component tests, and use of primary antibody which is Mycobacterium tuberculosis complex (MTBC-specific). Some other factors such as sample type; such as the use of archived/FFPE in this study could have affected antigen preservation and distribution in tissue sections. Storage effect on specimens; and staining protocols where we used manual IHC technique in this study, which could have led to slight variations in reaction temperatures compared to automated and closed systems used in other studies. 15 The specificity of anti- Mycobacterium tuberculosis antibody staining in this study was 80.0% (95% CI: 71.59–88.41%). This meant that 80% of subjects without EPTB were correctly identified as negative by this test. It however, also implied that about 20% of participants without EPTB might have been incorrectly identified as positive. This finding is comparable to those obtained by other studies. A study conducted in India found specificity of 81.9% 16 with bronchoalveolar and bronchial washings as specimens. Similarly, 72.5% and 88.0% specificity was obtained in previous studies. 7 , 20 The comparability could be due to use of polyclonal primary antibody in these studies. However, some studies obtained specificity values which were non-comparable to what we obtained in this study. 22 – 24 The high specificity could be a result of using composite diagnostic criterion with more than two component tests as opposed to two in the current study, and these studies used anti-MPT64 and anti-BCG which are MTBC-specific. The low specificity obtained by the other studies could have resulted from antigen distribution in the granuloma, clinical stage of disease, duration of anti-TB treatment prior to biopsy collection. Other factors impacting specificity include cross-reactivity and staining interpretation. Sensitivity and specificity are the most pertinent statistical measures for evaluating the diagnostic potential of a diagnostic assay. However, in clinical work, instead of ascertaining the percentage of diseased patients who will test positive (or non-diseased patients who will test negative), it is preferable to prognosticate whether a particular individual will truly have the disease based on a positive or negative test result. Hence, positive predictive value reflects the proportion of subjects with a positive test result who truly have the disease while negative predictive value reflects the proportion of subjects with a negative test result who truly do not have the disease. In this study, the positive predictive value of ab905 IHC was 81.4%. A PPV of 81.4% meant that of all the positive test results, the probability that a specimen with a positive result was actually positive is 0.81, the remaining percentage might be false positives. A study in Ethiopia found comparable PPV of 82.2%. 25 Similar studies found varying PPV values compared to the one we obtained in our study. 15 , 18 , 19 , 24 This difference could be a result of sample differences, whereas these studies utilized mixed samples such as biopsies and effusions, bronchoalveolar lavage we used archived FFPE specimens with longer storage duration which could have had effect on specimen and antigen preservation. The NPV obtained in the present study was 72.7%, which meant that the probability that a participant with a negative ab905 IHC test did not have EPTB was 0.724. This however, also implied that a significant percentage (27.3%) could have been false negative. This finding is comparable to NPV of 75% obtained in Zanzibar. 22 However, the NPV obtained in the present study was not comparable to values obtained in other studies. 15 , 18 , 19 These differences could have arisen because we used archived specimens and the current study suffered from the use of an imperfect reference standard (H&E and ZN Composite reference standard). In summary, in contrast to sensitivity and specificity, which are universally considered intrinsically stable for a given diagnostic assay, positive and negative predictive values are highly dependent on pre-test probability, wherein positive predictive values increase with increased disease prevalence, and negative predictive values increase with decreased disease prevalence. 26 The strength of our study was the retrospective design which allowed us to collect real-world data and negate potential biases of the index test on clinician’s decision making. The cross-sectional study was designed with adequate power to perform a verification study. We used a polyclonal antibody (ab905) for the immunohistochemical staining with known caveats of cross-reactivity, this could have affected the validity of our findings. Effect of storage on tissue blocks could not be independently verified, which could have impacted on the antigen preservation hence some specimens could have been wrongly assigned as negative by ab905 IHC test. The composite reference standard used (ZN and Histopathology) have low sensitivity for EPTB hence could have affected our downstream analyses. Conclusion From the findings of our study, anti -Mycobacterium tuberculosis antibody ab905 staining is a useful method for the exclusion or diagnosis of EPTB in tissues. Going forward, prospective data collection with use of a monoclonal ab905 anti- Mycobacterial tuberculosis antibody and more sensitive gold standard would negate the limitations this study encountered. Abbreviations Ab905 IHC, Anti-Mycobacterium tuberculosis antibody immunohistochemistry ; AIDS, acquired immunodeficiency syndrome; EPTB, Extrapulmonary tuberculosis; FFPE, Formalin Fixed Paraffin Embedded; H&E, Hematoxylin and eosin; HIV, Human immunodeficiency virus; IHC, immunohistochemistry; MTB, Mycobacterium tuberculosis ; MTBC, Mycobacterium tuberculosis complex; PPD, Purified Protein Derivative; WHO, World Health Organisation; ZN, Ziehl Neelsen Declarations Contribution: Our study provides insights into the potential role of immunohistochemistry in the diagnosis of EPTB. Ethics approval and consent to participate We obtained waiver of informed consent from Mbarara University Research Ethics Committee. This study was conducted in accordance with the World Medical Association Declaration of Helsinki and ethical approval was granted by the Mbarara University of Science and Technology Research Ethics Committee (MUST-2023-899) and the study was registered by Uganda National Council for Science and Technology (HS3031ES). Consent for publication Not Applicable Availability of data and materials The datasets used and/or analysed during the study that support the conclusions of this article are available from the corresponding author upon reasonable request. Competing interests The authors declare that no personal or financial relationships unseemly suborned them while writing this article. Funding This study received no specific grant from any commercial, private-not-for profit or public sector funding agency. Financial support was secured by the principal investigator for this project. Author’s contributions A.L. developed the research question, wrote the protocol, facilitated the data collection. F.S., A.B., R.A., Y.M., H.W., J.L.N., G.M., R.K., and L.T., assisted in the supervision of the protocol, data collection, and writing of the manuscript. A.L., F.S., A.B., C.N.B, S.N., assisted with methodological planning, statistical analysis, and data interpretation. All authors reviewed the manuscript. Acknowledgements We are grateful to the following organizations and persons: The Uganda Cancer Institute Pathology Laboratory and Mbarara University Histopathology Laboratory that optimized the manual ab905 anti-Mycobacterium tuberculosis antibody staining protocol. Mr. Rugera Simon Peter, Head of the Medical Laboratory Sciences Department, Mbarara University, supervised the general team. Author information Lawrence Amadile: [email protected] , https://orcid.org/0009-0008-2928-4672 Saphurah Nabaasa: [email protected] , https://orcid.org/0009-0000-0221-9741 Jolly Lydia Ninsiima: [email protected] , https://orcid.org/0009-0004-2282-3777 Abraham Birungi: [email protected] , https://orcid.org/0009-0002-4141-8900 Raymond Atwine: [email protected] , https://orcid.org/0000-0001-7888-7818 Hassan Wasswa: [email protected] , https://orcid.org/0009-0002-5438-9173 Geoffrey Mutale [email protected] https://orcid.org/0009-0000-9526-9675 Richard Kasadha: [email protected] , https://orcid.org/0009-0007-8995-5119 Charles Nkubi Bagenda: [email protected] , https://orcid.org/0009-0006-4235-1003 Tibenderana Lauben: [email protected] , https://orcid.org/0009-0000-8697-3732 Yekosani Mitala [email protected] https://orcid.org/0000-0001-9344-8967 Frank Ssedyabane: [email protected] , https://orcid.org/0000-0002-6836-549 Disclaimer The authors are solely responsible for the opinions and views expressed in this article. 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Rapid and specific diagnosis of tuberculous pleuritis with immunohistochemistry by detecting Mycobacterium tuberculosis complex specific antigen MPT64 in patients from a HIV endemic area. Applied Immunohistochemistry & Molecular Morphology. 2008;16(6):554-561. Kohli R, Punia RS, Kaushik R, Kundu R, Mohan H. Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections. Indian Journal of Pathology and Microbiology. 2014;57(4):574. Tadele A, Beyene D, Hussein J, et al. Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis. BMC infectious diseases. 2014;14:1-9. Monaghan TF, Rahman SN, Agudelo CW, et al. Foundational statistical principles in medical research: sensitivity, specificity, positive predictive value, and negative predictive value. Medicina. 2021;57(5):503. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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3","display":"","copyAsset":false,"role":"figure","size":157637,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eShowing negative ab905 anti-Mycobacterium tuberculosis immunostaining; \u003c/strong\u003eA: negative staining with background staining, B: negative staining (Total Magnification: 100x)\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8496524/v1/040442473ff678fdd608ab86.jpeg"},{"id":100362348,"identity":"e2d88045-989a-4d88-9174-a3d4a49bde5e","added_by":"auto","created_at":"2026-01-16 07:46:35","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":177615,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eShows different ab905 anti-Mycobacterium tuberculosis antibody staining intensities: \u003c/strong\u003e\u003c/em\u003e\u003cem\u003eA; weak staining pattern; B: moderate staining pattern; C: strong staining 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400x)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8496524/v1/1582425a9736842fb6edd974.jpeg"},{"id":100022733,"identity":"5443ddb4-f468-4dd0-8226-902d37a8f4d1","added_by":"auto","created_at":"2026-01-12 08:10:10","extension":"jpeg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":234517,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003e\u003cstrong\u003eShowing a comparison of multibacillary positive ZN staining (A) and very strong intensity IHC staining (B) \u003c/strong\u003e\u003c/em\u003e\u003cem\u003e(Total Magnification: 400x)\u003c/em\u003e\u003c/p\u003e","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8496524/v1/48f1b98b3583ecd990a816c6.jpeg"},{"id":101745894,"identity":"b5a94f53-d37f-4264-a08e-e9537691af8a","added_by":"auto","created_at":"2026-02-03 09:12:38","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3450442,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8496524/v1/04763055-b96a-42cc-9c53-7b8d03f234b8.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Detecting extrapulmonary tuberculosis using immunohistochemistry with anti-Mycobacterium tuberculosis antibody ab905; a cross-sectional study at Mbarara Regional Referral Hospital","fulltext":[{"header":"Background","content":"\u003cp\u003eTuberculosis (TB) is a communicable and contagious disease caused by \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e that majorly cause pulmonary tuberculosis but has the capacity to cause disease in other body organs or sites than the lungs. Globally, extrapulmonary tuberculosis (EPTB) contributes an appreciable fraction of tuberculosis cases accounting for nearly 20\u0026ndash;30% of total active TB patients, and attacks majorly children (\u0026le;\u0026thinsp;15 years of age) and immune-compromised adults\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e \u003cp\u003eTB and Human Immunodeficiency Virus (HIV) infection disproportionately affects Africa. In a ten-year retrospective TB prevalence and patient clinical profiling study conducted in Nigeria, EPTB accounted for 12.9% of all TB cases.\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e In South Africa, the prevalence of EPTB was found to be 25.0% among adults.\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e However, in Larache, Morocco, EPTB prevalence of 43.5% was found in a study conducted in 2020.\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eA significantly high proportion of extrapulmonary tuberculosis cases are missed, misdiagnosed or underdiagnosed because of the below par sensitivity of available diagnostic tests.\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e Being diverse clinically and having few bacilli in specimens microbiologically, there are usually several conditions that mimic EPTB leading to delayed/missed diagnosis. Uganda is one of the 30 countries that contribute 80% of the world\u0026rsquo;s tuberculosis burden, with a reported EPTB prevalence of 11%.\u003csup\u003e6\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eUganda, also being in the category of the world\u0026rsquo;s 30 high TB/HIV burden countries, missed or misdiagnosis of EPTB exacerbates the spread of TB in the public with HIV-infected persons at more risk.\u003c/p\u003e \u003cp\u003eAlthough TB culture is taken as the benchmark for the diagnosis of EPTB, its major shortcoming is the significantly extended time to obtain a result. It also requires unfixed tissue specimen.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Furthermore, the sensitivity of culture varies from (0\u0026ndash;80%),\u003csup\u003e8\u003c/sup\u003e this heterogeneity is not indicative of a good diagnostic test. Polymerase chain reaction (PCR) based assays are rapid and specific but they are not readily available in routine practice,\u003csup\u003e9\u003c/sup\u003e yet the bacilli are not evenly dispersed throughout the biopsy.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eZN staining and microscopy of tissue sections that are formalin fixed and paraffin embedded is employed to confirm EPTB infection; however, its major disadvantage is the low sensitivity due to paucity of bacilli in EPTB tissue biopsies.\u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Sensitivity of the ZN technique decreases acutely after formalin fixation and/or xylene treatment.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eHistopathological analysis of tissue specimen for extra pulmonary tuberculosis diagnosis has remained the most reliable method at present and it is mostly arrived at by identifying classical granuloma formation with caseous necrosis.\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThis study was therefore aimed at determining the sensitivity, specificity, positive predictive and negative predictive values of ab905 immunohistochemistry for the detection of Mycobacterial antigen in archived tissue specimens at Mbarara Regional Referral Hospital.\u003c/p\u003e"},{"header":"Methods","content":"\u003ch2\u003eAim\u003c/h2\u003e\n\u003cp\u003eThe primary objective of this study was to determine the sensitivity and specificity of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining for diagnosing EPTB in tissue specimens.\u003c/p\u003e\n\u003ch2\u003eStudy design\u003c/h2\u003e\n\u003cp\u003eWe conducted a cross-sectional laboratory-based study including archived tissue blocks of patients with suspicion of EPTB.\u003c/p\u003e\n\u003ch2\u003eStudy setting\u003c/h2\u003e\n\u003cp\u003eThis study was carried out at the pathology department of Mbarara Regional Referral Hospital (MRRH), between June 2023 and November 2023. MRRH serves as a regional referral center for the entire south western Uganda and it is located in Mbarara city, approximately 260km from the capital city, Kampala. The laboratory receives about 10 biopsies requiring TB testing per month. It receives patients from more than 13 districts in Western Uganda and the neighbouring countries that include Tanzania, Rwanda and Congo (DRC). \u003c/p\u003e\n\u003ch2\u003eSampling procedure\u003c/h2\u003e\n\u003cp\u003eWe retrospectively selected the tissue blocks. We used consecutive sampling to select the tissue blocks.\u003c/p\u003e\n\u003ch2\u003eSample size determination\u003c/h2\u003e\n\u003cp\u003eSample size at the required absolute precision level for sensitivity and specificity was calculated using Buderer\u0026rsquo;s formula\u003csup\u003e14\u003c/sup\u003e at 95% confidence level. A 68.29% sensitivity and 96.5% specificity of IHC on FFPE blocks was reported using TB culture or PCR as reference standard in previous studies.\u003csup\u003e15\u003c/sup\u003e \u003c/p\u003e\n\u003cp\u003ePrevalence of extrapulmonary tuberculosis based on tissue diagnosis in Uganda is not clearly known, therefore we used an arbitrary prevalence value of 50% (i.e., p=0.5).\u003c/p\u003e\n\u003cp\u003eThis gave a total of 78 patients represented by 78 blocks. After factoring in the possible 15% attrition, the consequential sample size was at least 90 patients represented by 90 blocks.\u003c/p\u003e\n\u003ch2\u003eData collection\u003c/h2\u003e\n\u003ch3\u003eDemographic data collection \u003c/h3\u003e\n\u003cp\u003eDemographic information was gathered using data collection tool by the PI and research assistants during the register review process. The collected data encompassed information such as age, gender and tissue type. \u003c/p\u003e\n\u003ch3\u003eCollection of tissue blocks and microtomy\u003c/h3\u003e\n\u003cp\u003eEach FFPE tissue block retrieved was trimmed and 5 microns thick sections were cut using a rotary microtome. From each tissue block, three sets of serial sections were cut for each of the three staining protocols.\u003c/p\u003e\n\u003ch3\u003eHematoxylin and Eosin staining and interpretation\u003c/h3\u003e\n\u003cp\u003eThe sections were stained using progressive H\u0026amp;E staining method. In brief, we used xylene to dewax the sections, sections were taken to water and stained with hematoxylin. Bluing was done using tap water followed by staining in eosin, the sections were rinsed in ethanol, air dried and mounted in Dibutylphthalate Polystyrene Xylene (DPX). \u003c/p\u003e\n\u003cp\u003eHistological features were evaluated under the microscope under low power (4x, 10x and 20x) and confirmed at high power (40x). Appearance of classical granulomas, caseous necrosis, epithelioid histiocytes and Langhans giant cells were used as features suggestive of EPTB.\u003c/p\u003e\n\u003ch3\u003eZiehl Neelsen (ZN) and interpretation\u003c/h3\u003e\n\u003cp\u003eWe used routine AFB kit for histology to stain the sections, slides were flooded with filtered Carbol fuchsin solution for 15 minutes and washed using running tap water. This was followed by differentiation in 1% acid alcohol until sections were pale pink, washed using tap water for 2 minutes and dipped in deionized water for 1 minute. We used methylene blue to counterstain sections for 30 seconds. Slides were rinsed in tap water and dipped once in deionized water. They were dehydrated using ascending grades of ethanol, dipped in xylene for clearing and cover slipped using DPX.\u003c/p\u003e\n\u003cp\u003eWe used 10x, 40x and 100x objectives to examine the slides and findings were reported as positive or negative for AFB when red staining AFB in clusters or singular AFB were present or absent respectively.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry (IHC)\u003c/h3\u003e\n\u003cp\u003eSections were mounted on positively charged slides (Bio-Optica Milano Spa, Milano MI, Italy). Immunohistochemistry (IHC) was done on FFPE specimens using concentrated anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody (ab905, Abcam, Cambridge, UK) diluted (1:100). Indirect manual IHC technique with one-step envision method (HRP-Streptavidin-biotin method, Dako, Germany) with diaminobenzidine (DAB) as chromogen was used. Positive (ZN positive lymph node tissue) and negative control (section from a tissue that did not have TB) were run together with test tissues under similar conditions.\u003c/p\u003e\n\u003cp\u003eSlides were baked in 50-52\u003csup\u003eo\u003c/sup\u003eC oven overnight. Sections were deparaffinized by dipping in xylene three times with every dip lasting three minutes and taken to water where descending concentrations of ethanol were used i.e., 100%, 100%, 95%, 95% for two minutes each followed by 5 dips in 80% alcohol and 30 seconds in deionized water. \u003c/p\u003e\n\u003cp\u003eSlides were incubated at 37\u003csup\u003eo\u003c/sup\u003eC for 15 minutes in Proteinase K. Sections were allowed to cool for 10 minutes at room temperature. Slides were then rinsed three times in distilled water. Tissue sections were circled with a pap pen to contain fluids. Slides were placed in diluted Tris-buffer and rinsed well (three times in coplin jar). Endogenous peroxidase activity was blocked by covering slides with 1% aqueous hydrogen peroxide and incubated for 10 minutes; rinsed with Tris buffer and covered sections with DAKO Serum-free protein block and incubated for 10 minutes. \u003c/p\u003e\n\u003cp\u003eSlides were carefully rinsed in Tris buffer for three minutes in a coplin jar. We thereafter shook off excess Tris buffer around tissue and placed slides back on tray. We placed two drops (200 \u0026micro;l; enough to cover tissue) of the primary antibody (ab905) on the tissue section; closed the lid and incubated for 30 minutes. Slides were carefully rinsed in Tris buffer three times and then carefully shook off excess Tris buffer around tissue and placed slides back on tray.\u003c/p\u003e\n\u003cp\u003eTwo drops of \u0026ldquo;Labelled polymer-HRP\u0026rdquo; anti-rabbit was placed on the tissue section and incubated for 30 minutes. Slides were carefully rinsed in Tris buffer three times; carefully shook off excess Tris buffer around tissue and placed slides back on tray. Made the DAB chromogen solution (for the Dako kit, added a drop (20\u0026micro;l) of DAB chromogen on every 1 ml of DAB substrate used (e.g., for 7 slides, used 2 ml DAB substrate buffer and 2 drops DAB chromogen)).\u003c/p\u003e\n\u003cp\u003eEnough DAB chromogen to cover the tissue was applied and incubated for 10 minutes; collected DAB chromogen waste in a hazardous container and rinsed slides in distilled water three times. Counterstained with hematoxylin, dehydrated sections in two changes of 95% ethanol and absolute ethanol, and cleared in xylene thereafter slides were cover slipped using DPX (dibutyl phthalate xylene), which acts as an indefinite mounting medium.\u003c/p\u003e\n\u003ch3\u003e Anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody (ab905) immunohistochemistry staining interpretation\u003c/h3\u003e\n\u003cp\u003eThe PI together with an experienced pathologist examined and reviewed the slides under 20x and 40x objective lenses. Brown granular staining in the cytoplasm or around the inflammatory cells was regarded positive (ab905 insert).\u003c/p\u003e\n\u003ch2\u003e Data handling, analysis and interpretation\u003c/h2\u003e\n\u003cp\u003eThe PI and research assistants gathered the raw data which was then entered into an excel spread sheet (Microsoft Office Professional Plus 2013, version 15.0.4675.1003, Microsoft Inc, USA). It was then imported into STATA 17 (StataCorp LLC, College Station, Texas, United States) software for all analyses. Descriptive statistics were used to characterize the population using frequencies, median values and interquartile ranges (IQRs). \u003c/p\u003e\n\u003cp\u003eClinical, H\u0026amp;E and ZN data were made available to the pathologists since they used these both at optimisation and slide reading steps. The variables analyzed included; H\u0026amp;E results, ZN results and IHC results, tissue type; diagnostic accuracy, predictive values of anti-\u003cem\u003eMycobacterium tuberculosis \u003c/em\u003eantibody staining calculated using histopathology and/or ZN staining as reference standard since TB culture could not be used on archived specimens. \u003c/p\u003e\n\u003cp\u003eSensitivity, specificity, positive predictive and negative predictive values of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining were reported as proportions with 95% confidence intervals. Receiver Operating Characteristic curve was used to determine the predictive performance of IHC for EPTB.\u003c/p\u003e\n\u003ch2\u003eEligibility criteria\u003c/h2\u003e\n\u003cp\u003eWe included formalin-fixed paraffin-embedded tissue blocks of patients with symptoms of EPTB archived between January 2009 and August 2023. See study flow (Fig.1).\u003c/p\u003e"},{"header":"Results","content":"\u003ch2\u003eFlow of participants\u003c/h2\u003e\n\u003cp\u003eThe flow of participants through enrolment, eligibility and outcomes of the study was as shown on the STARD flow diagram in Figure 1.\u003c/p\u003e\n\u003ch2\u003eStudy participant characteristics\u003c/h2\u003e\n\u003cp\u003eIn this cross-sectional study, we retrieved 87 tissue specimens (Table 1), 51/87 (59%) were from males. The median age of the study participants was 29 (interquartile range: 17-49) years. There was no notable difference in the distribution of tissue specimens among the different age categories. \u0026nbsp;Different tissue types were analyzed; lymph nodes 50/87 (57%) constituted the majority of the specimens.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 1\u003c/strong\u003e \u003cstrong\u003eDemographic characteristics of the patients whose specimens were analyzed.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eFrequency (n)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003ePercentage (%)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMedian=29\u0026nbsp;\u003c/p\u003e\n \u003cp\u003eIQR= (17-49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026lt;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e16\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e25-34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e35-44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e13\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e45-54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e21\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e55+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e20\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eFemale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e36\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e41\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eMale\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e51\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e59\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"4\" valign=\"top\" style=\"width: 596px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTissue type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eLymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e57\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eBone\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eProstate\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e5\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSpine\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eSkin\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003eOther\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e23\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\"\u003e\n \u003cp\u003e26\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003eIQR, interquartile range\u003c/p\u003e\n\u003ch2\u003eTest results\u003c/h2\u003e\n\u003ch3\u003eH\u0026amp;E staining photomicrographs\u003c/h3\u003e\n\u003cp\u003eUnder H\u0026amp;E, three inflammatory patterns were observed namely non-classical granuloma, classical granuloma and no granuloma (Fig.2). More specimens for patients in the \u0026lt;15 age group had histopathological feature of classical granuloma 11/46 (23.9%) followed by \u0026gt;54 age group 10/46 (21.7%) as shown in Table 2.\u003c/p\u003e\n\u003ch3\u003eImmunohistochemistry results\u003c/h3\u003e\n\u003cp\u003eUsing the anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody, 43/87 (49.4%) of the specimens stained positive while 44/87 (50.6%) were negative (Fig.3). While 41/43 (95.3%) of the tissues stained strongly positive, 3/43 (6.97%) stained weakly positive as shown in Fig.4. More lymph node tissues 27/43 (62.7%) stained positive than non-lymph node tissues 16/43 (37.2%).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eIn terms of the predictive performance of \u003cem\u003eAnti-Mycobacterium tuberculosis\u0026nbsp;\u003c/em\u003eantibody staining, Fig.5 shows that Anti-\u003cem\u003eMycobacterium tuberculosis\u0026nbsp;\u003c/em\u003eantibody staining was a good predictor for EPTB in tissues sections. It significantly predicted EPTB, area under the ROC curve 0.7723 (0.6834-0.8613).\u003c/p\u003e\n\u003cp\u003eThe predictive performance of Anti-\u003cem\u003eMycobacterium tuberculosis\u0026nbsp;\u003c/em\u003eantibody staining by tissue type did not show significant difference between lymph nodes and non-lymph node tissues with the ROC area of 0.7833 and 0.7529 respectively, \u003cstrong\u003ep value=0.78486\u0026nbsp;\u003c/strong\u003eas shown in Figure 6 below.\u003c/p\u003e\n\u003ch3\u003eZiehl Neelsen staining photomicrographs\u003c/h3\u003e\n\u003cp\u003eZN negative and positive staining patterns indicated in (Fig.7) below and further results indicated in Table 2). A comparison of positive IHC and ZN staining is shown in Figure 8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 2: Test results for H\u0026amp;E, ZN and Anti-Mycobacterium tuberculosis antibody immunohistochemistry by Age, Sex, Tissue type and inflammatory pattern\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"595\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eVariable\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eDescription\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 133px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eH\u0026amp;E\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 136px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eZN\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 130px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAb905 immunostaining\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=46\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=41\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=17\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=70\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003ePositive\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=43\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003cp\u003en=44\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eSex\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eFemale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e22 (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e14 (34)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e5 (29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e31 (44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e21 (49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e15 (34)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eMale\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e24 (52)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e27 (66)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e12 (71)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e39 (56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e22 (51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e29 (66)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 595px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eAge\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e\u0026lt;15\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e11 (24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e7 (17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e4 (24)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e14 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e13 (30)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e15-24\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e9 (22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e3 (18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e13 (19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7 (16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e9 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e25-34\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e6 (15)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e5 (29)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e8 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e8 (19)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e5 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e35-44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e6 (13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e7 (17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e2 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e11 (16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e5 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e8 (18)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e45-54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e5 (11)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e5 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e10 (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e3 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e7 (16)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003e55+\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e10 (22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e7 (17)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e3 (18)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e14 (20)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e7 (16)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e10 (23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 595px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eTissue type\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eLymph node\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e30 (65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e20 (49)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e11 (65)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e39 (56)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e27 (63)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e23 (52)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eNon-Lymph nodes\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e16 (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e21 (51)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e6 (35)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e31 (44)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e16 (37)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e21 (48)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"8\" valign=\"top\" style=\"width: 595px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eInflammatory pattern\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eClassical granuloma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e45 (98)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e16 (94)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e29 (41)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e35 (81)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e10 (23)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eNon-classical granuloma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e9 (22)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e0 (0)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e9 (13)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e3 (7)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e6 (14)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 100px;\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 97px;\"\u003e\n \u003cp\u003eNo granuloma\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e1 (2)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 70px;\"\u003e\n \u003cp\u003e32 (78)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 64px;\"\u003e\n \u003cp\u003e1 (6)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 72px;\"\u003e\n \u003cp\u003e32 (46)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 63px;\"\u003e\n \u003cp\u003e5 (12)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 67px;\"\u003e\n \u003cp\u003e28 (64)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003ch2\u003eDiagnostic utility of anti-Mycobacterium tuberculosis antibody staining using ZN and H\u0026amp;E as a composite reference standard.\u003c/h2\u003e\n\u003cp\u003eAs calculated from the 2x2 table (Table 3), the overall sensitivity, specificity, positive and negative predictive values for Anti-\u003cem\u003eMycobacterium tuberculosis\u0026nbsp;\u003c/em\u003eantibody staining are shown in Table 4.\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 3: Cross-tabulation of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining and H\u0026amp;E and/or ZN (composite reference standard) results.\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e\u003cem\u003eAnti-Mycobacterium tuberculosis antibody immunohistochemistry\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd colspan=\"2\" valign=\"top\" style=\"width: 301px;\"\u003e\n \u003cp\u003eComposite reference standard\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd rowspan=\"2\" valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003cp\u003ef (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eNegative f (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003ePositive f (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eNegative\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e32 (80.00%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e12 (25.53%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e44 (50.57%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003ePositive\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e8 (20.00%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e35 (74.47%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e43 (49.43%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003eTotal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e40 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e47 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 150px;\"\u003e\n \u003cp\u003e87 (100%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTable 4: Diagnostic utility of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining\u003c/strong\u003e\u003c/p\u003e\n\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eParameter\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003eMeasure\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e\u003cstrong\u003e95% CI\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003eSensitivity\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e74.5%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e65.31-83.63%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003eSpecificity\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e80.0%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e71.59-88.41%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003ePositive predictive value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e81.4%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e73.22-89.57%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003eNegative predictive value\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e72.7%\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" style=\"width: 200px;\"\u003e\n \u003cp\u003e63.37-82.09%\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eWe present a relatively high and balanced sensitivity, specificity, positive predictive and negative predictive values of ab905 IHC (74.5%, 80.0%, 81.4% and 72.7% respectively) for EPTB in tissue sections. Our data indicate that ab905 anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining is a useful tool in diagnosis or exclusion of EPTB in tissue sections. This was applicable to the total study population (maximum AUC 0.77), lymph nodes tissues (maximum AUC 0.78) and non-lymph node tissues (maximum AUC 0.75).\u003c/p\u003e \u003cp\u003eThe sensitivity of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody (ab905) staining in this study was 74.5%. This meant that ab905 IHC correctly identified 74.5% of individuals who actually had EPTB. However, sensitivity of 74.5% also implies that about 25.5% of individuals with EPTB might not be identified by ab905 IHC because of technical factors affecting the sensitivity of the test such as effect of storage on the archived tissue specimens.\u003c/p\u003e \u003cp\u003eThe sensitivity of 74.5% found in this study is comparable to those found by previous authors who assessed sensitivity of IHC for the diagnosis of EPTB in tissue biopsies.\u003csup\u003e\u003cspan additionalcitationids=\"CR16\" citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e The comparability could be attributed to the same specimen type used being biopsies in the current study and the previous studies, same antigen preservation technique (FFPE sections), and the use of composite reference standard.\u003c/p\u003e \u003cp\u003eHowever, there has been reported variability in the sensitivity of IHC for EPTB in tissue sections across different studies. Many studies reported sensitivity which were non-comparable to the one obtained in this study.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan additionalcitationids=\"CR19 CR20\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe plausible reason for the higher sensitivity obtained above could be the use of superior gold standards such as PCR, composite reference standard with more component tests, and use of primary antibody which is Mycobacterium tuberculosis complex (MTBC-specific). Some other factors such as sample type; such as the use of archived/FFPE in this study could have affected antigen preservation and distribution in tissue sections. Storage effect on specimens; and staining protocols where we used manual IHC technique in this study, which could have led to slight variations in reaction temperatures compared to automated and closed systems used in other studies.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe specificity of anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody staining in this study was 80.0% (95% CI: 71.59\u0026ndash;88.41%). This meant that 80% of subjects without EPTB were correctly identified as negative by this test. It however, also implied that about 20% of participants without EPTB might have been incorrectly identified as positive. This finding is comparable to those obtained by other studies. A study conducted in India found specificity of 81.9%\u003csup\u003e16\u003c/sup\u003e with bronchoalveolar and bronchial washings as specimens. Similarly, 72.5% and 88.0% specificity was obtained in previous studies.\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e The comparability could be due to use of polyclonal primary antibody in these studies.\u003c/p\u003e \u003cp\u003eHowever, some studies obtained specificity values which were non-comparable to what we obtained in this study.\u003csup\u003e\u003cspan additionalcitationids=\"CR23\" citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe high specificity could be a result of using composite diagnostic criterion with more than two component tests as opposed to two in the current study, and these studies used anti-MPT64 and anti-BCG which are MTBC-specific. The low specificity obtained by the other studies could have resulted from antigen distribution in the granuloma, clinical stage of disease, duration of anti-TB treatment prior to biopsy collection. Other factors impacting specificity include cross-reactivity and staining interpretation.\u003c/p\u003e \u003cp\u003eSensitivity and specificity are the most pertinent statistical measures for evaluating the diagnostic potential of a diagnostic assay. However, in clinical work, instead of ascertaining the percentage of diseased patients who will test positive (or non-diseased patients who will test negative), it is preferable to prognosticate whether a particular individual will truly have the disease based on a positive or negative test result.\u003c/p\u003e \u003cp\u003eHence, positive predictive value reflects the proportion of subjects with a positive test result who truly have the disease while negative predictive value reflects the proportion of subjects with a negative test result who truly do not have the disease.\u003c/p\u003e \u003cp\u003eIn this study, the positive predictive value of ab905 IHC was 81.4%. A PPV of 81.4% meant that of all the positive test results, the probability that a specimen with a positive result was actually positive is 0.81, the remaining percentage might be false positives. A study in Ethiopia found comparable PPV of 82.2%.\u003csup\u003e25\u003c/sup\u003e Similar studies found varying PPV values compared to the one we obtained in our study.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e This difference could be a result of sample differences, whereas these studies utilized mixed samples such as biopsies and effusions, bronchoalveolar lavage we used archived FFPE specimens with longer storage duration which could have had effect on specimen and antigen preservation.\u003c/p\u003e \u003cp\u003eThe NPV obtained in the present study was 72.7%, which meant that the probability that a participant with a negative ab905 IHC test did not have EPTB was 0.724. This however, also implied that a significant percentage (27.3%) could have been false negative. This finding is comparable to NPV of 75% obtained in Zanzibar.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e However, the NPV obtained in the present study was not comparable to values obtained in other studies.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e,\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e These differences could have arisen because we used archived specimens and the current study suffered from the use of an imperfect reference standard (H\u0026amp;E and ZN Composite reference standard).\u003c/p\u003e \u003cp\u003eIn summary, in contrast to sensitivity and specificity, which are universally considered intrinsically stable for a given diagnostic assay, positive and negative predictive values are highly dependent on pre-test probability, wherein positive predictive values increase with increased disease prevalence, and negative predictive values increase with decreased disease prevalence.\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e \u003cp\u003eThe strength of our study was the retrospective design which allowed us to collect real-world data and negate potential biases of the index test on clinician\u0026rsquo;s decision making. The cross-sectional study was designed with adequate power to perform a verification study. We used a polyclonal antibody (ab905) for the immunohistochemical staining with known caveats of cross-reactivity, this could have affected the validity of our findings. Effect of storage on tissue blocks could not be independently verified, which could have impacted on the antigen preservation hence some specimens could have been wrongly assigned as negative by ab905 IHC test. The composite reference standard used (ZN and Histopathology) have low sensitivity for EPTB hence could have affected our downstream analyses.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eFrom the findings of our study, anti\u003cem\u003e-Mycobacterium tuberculosis\u003c/em\u003e antibody ab905 staining is a useful method for the exclusion or diagnosis of EPTB in tissues.\u003c/p\u003e \u003cp\u003eGoing forward, prospective data collection with use of a monoclonal ab905 anti-\u003cem\u003eMycobacterial tuberculosis\u003c/em\u003e antibody and more sensitive gold standard would negate the limitations this study encountered.\u003c/p\u003e "},{"header":"Abbreviations","content":"\u003cp\u003eAb905 IHC, \u003cem\u003eAnti-Mycobacterium tuberculosis antibody immunohistochemistry\u003c/em\u003e; AIDS, acquired immunodeficiency syndrome; EPTB, Extrapulmonary tuberculosis; FFPE, Formalin Fixed Paraffin Embedded; H\u0026amp;E, Hematoxylin and eosin; HIV, Human immunodeficiency virus; IHC, immunohistochemistry; MTB, \u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e; MTBC, Mycobacterium tuberculosis complex; PPD, Purified Protein Derivative; WHO, World Health Organisation; ZN, Ziehl Neelsen\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003e\u003cstrong\u003eContribution:\u003c/strong\u003e\u0026nbsp;\u003c/h2\u003e\n\u003cp\u003eOur study provides insights into the potential role of immunohistochemistry in the diagnosis of EPTB.\u003c/p\u003e\n\u003ch2\u003eEthics approval and consent to participate\u003c/h2\u003e\n\u003cp\u003eWe obtained waiver of informed consent from Mbarara University Research Ethics Committee. This study was conducted in accordance with the World Medical Association Declaration of Helsinki and ethical approval was granted by the Mbarara University of Science and Technology Research Ethics Committee (MUST-2023-899) and the study was registered by Uganda National Council for Science and Technology (HS3031ES).\u003c/p\u003e\n\u003ch2\u003eConsent for publication\u003c/h2\u003e\n\u003cp\u003eNot Applicable\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eThe datasets used and/or analysed during the study that support the conclusions of this article are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eCompeting interests\u003c/h2\u003e\n\u003cp\u003eThe authors declare that no personal or financial relationships unseemly suborned them while writing this article.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThis study received no specific grant from any commercial, private-not-for profit or public sector funding agency. Financial support was secured by the principal investigator for this project.\u003c/p\u003e\n\u003ch2\u003eAuthor\u0026rsquo;s contributions\u003c/h2\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eA.L. developed the research question, wrote the protocol, facilitated the data collection.\u003c/li\u003e\n \u003cli\u003eF.S., A.B., R.A., Y.M., H.W., J.L.N., G.M., R.K., and L.T., assisted in the supervision of the protocol, data collection, and writing of the manuscript.\u003c/li\u003e\n \u003cli\u003eA.L., F.S., A.B., C.N.B, S.N., assisted with methodological planning, statistical analysis, and data interpretation.\u003c/li\u003e\n \u003cli\u003eAll authors reviewed the manuscript.\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch2\u003eAcknowledgements\u003c/h2\u003e\n\u003cp\u003eWe are grateful to the following organizations and persons:\u003c/p\u003e\n\u003cp\u003eThe Uganda Cancer Institute Pathology Laboratory and Mbarara University Histopathology Laboratory that optimized the manual ab905 anti-Mycobacterium tuberculosis antibody staining protocol.\u003c/p\u003e\n\u003cp\u003eMr. Rugera Simon Peter, Head of the Medical Laboratory Sciences Department, Mbarara University, supervised the general team.\u003c/p\u003e\n\u003ch2\u003eAuthor information\u003c/h2\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eLawrence Amadile: [email protected] , https://orcid.org/0009-0008-2928-4672\u003c/li\u003e\n \u003cli\u003eSaphurah Nabaasa: [email protected] , https://orcid.org/0009-0000-0221-9741\u003c/li\u003e\n \u003cli\u003eJolly Lydia Ninsiima: [email protected] , https://orcid.org/0009-0004-2282-3777\u003c/li\u003e\n \u003cli\u003eAbraham Birungi: [email protected] , https://orcid.org/0009-0002-4141-8900\u003c/li\u003e\n \u003cli\u003eRaymond Atwine: [email protected] , https://orcid.org/0000-0001-7888-7818\u003c/li\u003e\n \u003cli\u003eHassan Wasswa: [email protected] , https://orcid.org/0009-0002-5438-9173\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul\u003e\n \u003cli\u003eGeoffrey Mutale [email protected] https://orcid.org/0009-0000-9526-9675\u003c/li\u003e\n\u003c/ul\u003e\n\u003cul type=\"disc\"\u003e\n \u003cli\u003eRichard Kasadha: [email protected] , https://orcid.org/0009-0007-8995-5119\u003c/li\u003e\n \u003cli\u003eCharles Nkubi Bagenda: [email protected] , https://orcid.org/0009-0006-4235-1003\u003c/li\u003e\n \u003cli\u003eTibenderana Lauben: [email protected] , https://orcid.org/0009-0000-8697-3732\u003c/li\u003e\n \u003cli\u003eYekosani Mitala [email protected] https://orcid.org/0000-0001-9344-8967\u003c/li\u003e\n \u003cli\u003eFrank Ssedyabane: [email protected] , https://orcid.org/0000-0002-6836-549\u003c/li\u003e\n\u003c/ul\u003e\n\u003ch2\u003eDisclaimer\u003c/h2\u003e\n\u003cp\u003eThe authors are solely responsible for the opinions and views expressed in this article. These do not mirror the official policy or position of any affiliated agency/institutions of the authors.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGopalaswamy R, Dusthackeer VA, Kannayan S, Subbian S. Extrapulmonary tuberculosis\u0026mdash;an update on the diagnosis, treatment and drug resistance. \u003cem\u003eJournal of Respiration. \u003c/em\u003e2021;1(2):141-164.\u003c/li\u003e\n\u003cli\u003eEmorinken A, Ugheoke AJ, Agbadaola OR, et al. Prevalence and Clinical Profile of Tuberculosis Patients in a Rural Teaching Hospital in South-South Nigeria: A Ten-Year Retrospective Study. \u003cem\u003eInternational Journal of TROPICAL DISEASE \u0026amp; Health. \u003c/em\u003e2023;44(8):33-42.\u003c/li\u003e\n\u003cli\u003eKarstaedt AS. Extrapulmonary tuberculosis among adults: experience at Chris Hani Baragwanath academic hospital, Johannesburg, South Africa. \u003cem\u003eSouth African Medical Journal. \u003c/em\u003e2014;104(1):22-24.\u003c/li\u003e\n\u003cli\u003eSbayi A, Arfaoui A, Janah H, Koraichi SE, Quyou A. Epidemiological characteristics and some risk factors of extrapulmonary tuberculosis in Larache, Morocco. \u003cem\u003ePan African Medical Journal. \u003c/em\u003e2020;36(1).\u003c/li\u003e\n\u003cli\u003eWang B, Gao W, Hao D. Current study of the detection and treatment targets of spinal tuberculosis. \u003cem\u003eCurrent Drug Targets. \u003c/em\u003e2020;21(4):320-327.\u003c/li\u003e\n\u003cli\u003eOhene S-A, Bakker MI, Ojo J, Toonstra A, Awudi D, Klatser P. Extra-pulmonary tuberculosis: a retrospective study of patients in Accra, Ghana. \u003cem\u003ePloS one. \u003c/em\u003e2019;14(1):e0209650.\u003c/li\u003e\n\u003cli\u003eAddo SO, Abrahams AOD, Mensah GI, et al. Utility of anti-Mycobacterium tuberculosis antibody (ab905) for detection of mycobacterial antigens in formalin-fixed paraffin-embedded tissues from clinically and histologically suggestive extrapulmonary tuberculosis cases. \u003cem\u003eHeliyon. \u003c/em\u003e2022:e12370.\u003c/li\u003e\n\u003cli\u003eSayed S, Njau AN, Moloo Z, Gakinya SM. Xpert\u0026reg; MTB/RIF assay on formalin-fixed paraffin-embedded tissues in the diagnosis of extrapulmonary tuberculosis. \u003cem\u003eAfrican journal of laboratory medicine. \u003c/em\u003e2019;8(1):1-5.\u003c/li\u003e\n\u003cli\u003eOyero S, Onwuliri F, Itelima J, et al. A Comparative Diagnosis of Breast Tuberculosis using Ziehl Neelson Stain and GeneXpert\u0026reg; Assay in North Central Nigeria.\u003c/li\u003e\n\u003cli\u003e Li JY, Lo ST, Ng C-S. Molecular detection of Mycobacterium tuberculosis in tissues showing granulomatous inflammation without demonstrable acid-fast bacilli. \u003cem\u003eDiagnostic Molecular Pathology. \u003c/em\u003e2000;9(2):67-74.\u003c/li\u003e\n\u003cli\u003e VerMa D, Soni G, raShMi SharMa PK. Diagnostic utility of fluorescent microscopy vis-a-vis GeneXpert MTB/RIF in extra pulmonary tuberculosis: a retrospective analysis. \u003cem\u003eNatl J Lab Med. \u003c/em\u003e2019;8:MO08-MO12.\u003c/li\u003e\n\u003cli\u003e Fukunaga H, Murakami T, Gondo T, Sugi K, Ishihara T. Sensitivity of acid-fast staining for Mycobacterium tuberculosis in formalin-fixed tissue. \u003cem\u003eAmerican journal of respiratory and critical care medicine. \u003c/em\u003e2002;166(7):994-997.\u003c/li\u003e\n\u003cli\u003e Tahseen S, Ambreen A, Ishtiaq S, et al. The value of histological examination in the diagnosis of tuberculous lymphadenitis in the era of rapid molecular diagnosis. \u003cem\u003eScientific Reports. \u003c/em\u003e2022;12(1):1-11.\u003c/li\u003e\n\u003cli\u003e Buderer NMF. Statistical methodology: I. Incorporating the prevalence of disease into the sample size calculation for sensitivity and specificity. \u003cem\u003eAcademic Emergency Medicine. \u003c/em\u003e1996;3(9):895-900.\u003c/li\u003e\n\u003cli\u003e Rao PD, Devi DG, Gouri SM, Arjun A, Krishnappa L, Azeem A. Evaluation of immunohistochemistry technique for diagnosis of extrapulmonary tuberculosis in biopsy tissue specimen as compared to composite diagnostic criteria. \u003cem\u003eJournal of Global Infectious Diseases. \u003c/em\u003e2022;14(4):136.\u003c/li\u003e\n\u003cli\u003e Nasare AS, Swain M, Roa R. The utility of immunohistochemistry for detecting mycobacterial infections in bronchoalveolar lavage \u0026amp; bronchial washings. \u003cem\u003eThe Indian journal of medical research. \u003c/em\u003e2023;157(1):81.\u003c/li\u003e\n\u003cli\u003e Mukherjee A, Kalra N, Beena K. Immuno-histochemical detection of mycobacterial antigen in tuberculous lymphadenitis. \u003cem\u003eIndian Journal of Tuberculosis. \u003c/em\u003e2002;49(4):213-216.\u003c/li\u003e\n\u003cli\u003e Ulain N, Ali A, Khan M, et al. Improving diagnosis of tuberculous lymphadenitis by combination of cytomorphology and MPT64 immunostaining on cell blocks from the fine needle aspirates. \u003cem\u003ePlos one. \u003c/em\u003e2022;17(10):e0276064.\u003c/li\u003e\n\u003cli\u003e Purohit MR, Sviland L, Wiker H, Mustafa T. Rapid and specific diagnosis of extrapulmonary tuberculosis by immunostaining of tissues and aspirates with anti-MPT64. \u003cem\u003eApplied immunohistochemistry \u0026amp; molecular morphology. \u003c/em\u003e2017;25(4):282-288.\u003c/li\u003e\n\u003cli\u003e Purohit MR, Mustafa T, Wiker HG, Sviland L. Rapid diagnosis of tuberculosis in aspirate, effusions, and cerebrospinal fluid by immunocytochemical detection of Mycobacterium tuberculosis complex specific antigen MPT64. \u003cem\u003eDiagnostic cytopathology. \u003c/em\u003e2012;40(9):782-791.\u003c/li\u003e\n\u003cli\u003e Hoel IM, Sviland L, Syre H, et al. Diagnosis of extrapulmonary tuberculosis using the MPT64 antigen detection test in a high-income low tuberculosis prevalence setting. \u003cem\u003eBMC Infectious Diseases. \u003c/em\u003e2020;20(1):1-11.\u003c/li\u003e\n\u003cli\u003e J\u0026oslash;rstad MD, Marijani M, Dyrhol-Riise AM, Sviland L, Mustafa T. MPT64 antigen detection test improves routine diagnosis of extrapulmonary tuberculosis in a low-resource setting: A study from the tertiary care hospital in Zanzibar. \u003cem\u003ePloS one. \u003c/em\u003e2018;13(5):e0196723.\u003c/li\u003e\n\u003cli\u003e Baba K, Dyrhol-Riise AM, Sviland L, et al. Rapid and specific diagnosis of tuberculous pleuritis with immunohistochemistry by detecting Mycobacterium tuberculosis complex specific antigen MPT64 in patients from a HIV endemic area. \u003cem\u003eApplied Immunohistochemistry \u0026amp; Molecular Morphology. \u003c/em\u003e2008;16(6):554-561.\u003c/li\u003e\n\u003cli\u003e Kohli R, Punia RS, Kaushik R, Kundu R, Mohan H. Relative value of immunohistochemistry in detection of mycobacterial antigen in suspected cases of tuberculosis in tissue sections. \u003cem\u003eIndian Journal of Pathology and Microbiology. \u003c/em\u003e2014;57(4):574.\u003c/li\u003e\n\u003cli\u003e Tadele A, Beyene D, Hussein J, et al. Immunocytochemical detection of Mycobacterium Tuberculosis complex specific antigen, MPT64, improves diagnosis of tuberculous lymphadenitis and tuberculous pleuritis. \u003cem\u003eBMC infectious diseases. \u003c/em\u003e2014;14:1-9.\u003c/li\u003e\n\u003cli\u003e Monaghan TF, Rahman SN, Agudelo CW, et al. Foundational statistical principles in medical research: sensitivity, specificity, positive predictive value, and negative predictive value. \u003cem\u003eMedicina. \u003c/em\u003e2021;57(5):503. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":true,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Sensitivity, Specificity, Formalin-fixed paraffin-embedded, Hematoxylin \u0026 Eosin, Immunohistochemistry, Ziehl-Neelsen","lastPublishedDoi":"10.21203/rs.3.rs-8496524/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8496524/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eDiagnosing extrapulmonary tuberculosis (EPTB) poses challenges such as delayed diagnosis, missed diagnosis, and misdiagnosis of EPTB. Immunohistochemistry is believed to improve the accuracy of EPTB diagnosis in tissues fixed in formalin and embedded in paraffin.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePatients and methods:\u003c/strong\u003e This was a cross-sectional study, and data were collected between June 2023 and November 2023, involving 87 archived tissue specimens from patients with clinical suspicion of EPTB.\u003c/p\u003e\n\u003cp\u003eDemographic data were collected from pathology registers, and Hematoxylin and Eosin (H\u0026amp;E) and Ziehl Neelsen (ZN) staining and microscopy techniques were performed on sections from the tissue blocks to confirm the diagnosis of EPTB. A concentrated anti-\u003cem\u003eMycobacterium tuberculosis\u003c/em\u003e antibody (ab905, Abcam, Cambridge, UK), diluted (1:100) for immunohistochemistry (IHC) was used. Indirect manual IHC technique was performed. ZN and/or histopathology were used as the component composite reference standard tests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003eThe study population comprised of 87 tissue blocks. Lymph nodes constituted the majority (50/87, 57%) of specimens. Histopathologically, 52.9% (46/87) of the specimens had granulomatous inflammation suggestive of EPTB, whereas 47.1% (41/87) did not. Of the 87 specimens analyzed, 17 (19.5%) were positive using the ZN technique and 70 (80.5%) were negative. The sensitivity, specificity, PPV, and NPV of anti\u003cem\u003e-Mycobacterium tuberculosis \u003c/em\u003eantibody staining were 74.5%, 80.0%, 81.4%, and 72.7%, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusion: \u003c/strong\u003eBased on the findings of our study, anti\u003cem\u003e-Mycobacterium tuberculosis \u003c/em\u003eantibody ab905 staining is a valuable method for excluding or diagnosing EPTB in tissues.\u003c/p\u003e\n\u003cp\u003eProspective data collection using a monoclonal ab905 anti-\u003cem\u003eMycobacterial tuberculosis \u003c/em\u003eantibody and a more sensitive gold standard would negate the limitations of this study.\u003c/p\u003e","manuscriptTitle":"Detecting extrapulmonary tuberculosis using immunohistochemistry with anti-Mycobacterium tuberculosis antibody ab905; a cross-sectional study at Mbarara Regional Referral Hospital","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 08:08:16","doi":"10.21203/rs.3.rs-8496524/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"315556a5-5afb-4fd5-96a3-31f249b28500","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-02-03T09:12:10+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 08:08:16","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8496524","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8496524","identity":"rs-8496524","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}