Genital tuberculosis (GTB) is an important cause of severe tubal disease leading to infertility and is often secondary to extra-pulmonary sites. The female genital tract is involved mainly at the tubes, endometrium, and ovaries with an unknown incidence chiefly because of silent asymptomatic cases1. Asymptomatic infection of the female genital organs is, however, frequent in both extra-pulmonary tuberculosis (EPTB)–positive and EPTB-negative women. Because clinical findings are often non-specific, a rapid, sensitive, and accurate diagnostic modality for GTB is required. Currently available conventional diagnostic methods, such as microscopy of smear and culture, have limited sensitivity, and time required for results. Polymerase chain reaction (PCR) offers a relatively faster, more sensitive, and widely helpful confirmatory tool for diagnosing GTB and is able to detect more such cases than by microscopy or culture. Even with these merits, PCR still shows false-negative and false-positive results and is therefore not considered an absolutely reliable method for definitive diagnosis. 2,3,4,5.

Feminine genital tubercular (FGTB) infection has an enormous preponderance of TB cases among women of reproductive age. FGTB has become a leading generator of infertility because of its high infectivity, asymptomatic manifestation, and inadequacy of existent diagnostic facilities to detect the event immediately. Histopathology, GeneXpert, and culture are widely used modalities for FGTB detection. GeneXpert is a cutting-edge, rapid diagnostic test that detects the genetic material of a Mycobacterium tuberculosis-complex (MTB) cell presumptively and simultaneously finds the rifampicin-resistance gene (rpoB) concurrently. The underling concern with this experiment is to evaluate the relative diagnostic accuracy of histopathology, GeneXpert, and culture for FGTB. The sample size encompassed 15,660 women presumed to acquire infertility because past illicit abdominal distress and 1586 women on antecedent sterilization like Endometrial Tuberculosis (ETB). The systematic analysis was executed on seven manuscripts to validate the mode of action and symmetrical comparison of the three modalities available. All seven documents count up to 3298, those detected by culture 1370 depicting 41.5%, whereas, by GeneXpert, 1397 indicating 42.4% and with histopathology, unquestionably 1692 illustrating 51.3%. The histopathology chronicles represent that the epitomes of case retrospectively verify against tubercular, consequently symbolizing its authority on the narrative. 6,7 Endometrial specimens were comprehensively acquired for cultured tubercular candidates. Consequently, the society witnesses that among histopathology, GeneXpert, and culture, such as selected based on the State-of-the-Art Reports (SAR) from attaining research devoid of interjecting each group sanctioning criteria. These display the retrieving Proportion (SFP), consequently when evaluated under the criterion of supply of file demonstrating overall precision of the system where histopathology offers a solution of 5.73 indicative the higher edge over GeneXpert presenting 3.14 and culture following furnishing 2.63.8,9 The tactical course of tracking FGTB inflicts a crucial demand. Furthermore, spotting such inflicted personnel is essential to safeguard the predicaments of feminine reproductive health. FGTB commonly stays unattended owing to the incapacity of available diagnostic facilities. The enhancement of services to an elevated state technologically supports the accomplishment. However, False Dearth remain applicable where establishment chase continues. 10 2.1. Study design and eligibility criteria Genital tuberculosis is an important cause of infertility among women in developed and developing countries. An accurate diagnosis of female genital tuberculosis is very difficult using a standard diagnostic approach with clinical and radiological evaluations. On histopathological examination of genital tuberculosis, the suggested common histopathological features are chronic nonspecific endometritis, multifocal caseating granuloma, and tuberculous salpingitis. A systematic review and meta-analysis of the literature was conducted to compare the diagnostic accuracy of histopathology, culture, and GeneXpert for female genital tuberculosis. The study is reported according to the PRISMA-DTA guidelines.11,4 The systematic review included studies conducted on human samples to determine the diagnostic accuracy of histopathology, GeneXpert, and culture for genital tuberculosis in women, using any reference method as the standard. Studies conducted on laboratory animals were excluded. There were no restrictions concerning the year of publication or the location of the study. The diagnostic value of histopathology was examined. 2.2. Search strategy and study selection Google Scholar and PubMed were searched for studies published until July 2023 that reported on the diagnostic performance of histopathology, GeneXpert, and culture for FGTB in human samples. The following search strategy was employed on PubMed and adapted appropriately for Google Scholar: “genital tuberculosis” OR “tuberculosis of the female genital tract” OR “female genital tuberculosis” OR “genital TB” OR “tuberculosis of the female genital tract” OR “female genital TB” OR “tuberculous salpingitis” OR “tuberculous oophoritis” OR “tuberculous cervicitis” OR “tuberculous endometritis” OR “endometrial tuberculosis” OR “tuberculous endosalpingeal” OR “iso-genital” AND “GeneXpert” OR “Gene-Xpert” OR “Xpert” OR “MTB/RIF” OR “Xpert MTB/RIF” OR “Xpert PCR” OR “Xpert MTB/RIF PCR” AND “PCR” OR “polymerase chain reaction” OR “nucleic acid amplification” OR “amplification” OR “amplified” AND “histopathology” OR “histopathological” OR “pathology” AND “culture” OR “culture technique” OR “culture procedure”; the following string was also tested: “Mycobacterium Tuberculosis” OR “Mycobacterium” OR “M.Tuberculosis” AND “tuberculosis” OR “MTB” OR “TB” OR “tubercle bacillus” OR “Mycobacterium TB” OR “mycobacterial” OR “acid-fast bacilli” AND IS “human”. Study selection was performed independently by two investigators (K.S., S.K.) to identify all potentially relevant studies. Discrepancies were discussed with a third investigator (K.S.) until consensus was reached. 2.3. Data extraction and quality assessment A reference management software products like BibTeX or EndNote would be useful. Data extraction and quality assessment of the comparative diagnostic accuracy studies of histopathology, GeneXpert, and culture were performed in 2 steps. First, data were extracted independently using an Excel sheet-based data extraction form by the 2 reviewers. Evaluation of the detection of Mycobacterium tuberculosis was regarded as the main study parameter. The following data were extracted from each of the studies: authors, year of publication, country where the study was conducted, study design (cross-sectional/prospective), and sample size. For histopathological techniques, the following data were extracted: number of positive cases, method of tissue processing, type of staining, and duration of staining. For the GeneXpert technique, the number of positive cases and the form of the sample (fine-needle aspiration, fluid, or tissue) were noted. For the culture technique, the number of positive cases, type of media used, and duration for obtaining results were collected. Second, the quality of each included study was checked based on the quality assessment of diagnostic accuracy studies 2 (QUADAS-2) checklist through 4 key domains with signalling questions. 12 2.4. Outcome measures and statistical analysis Female genital tuberculosis is a common manifestation of extrapulmonary tuberculosis, but it remains underdiagnosed owing to the limitations of the current diagnostic modalities. Histopathology, GeneXpert, and culture are frequently used diagnostic tests; however, a comparative analysis of their diagnostic accuracy is lacking. Therefore, a systematic review and meta-analysis was conducted to compare the diagnostic accuracy of histopathology, GeneXpert, and culture for female genital tuberculosis. A search was done on scientific databases such as PubMed and Embase to find studies reporting 2×2 contingency tables of histopathology, GeneXpert, and culture. Studies that reported the diagnostic accuracy of these tests were included, while studies on male genital tuberculosis were excluded. Diagnostic accuracy measures, such as sensitivity, specificity, diagnostic odds ratio, and area under the receiver operator characteristic curve were computed. Calibration plots were constructed to illustrate the agreement between observed and predicted probabilities of genital tuberculosis. The random-effects model was used to compute pooled accuracy measures of the diagnostic tests. Data were analysed using the Metadisc software. 13,14

Pelvic tuberculosis is a major cause of morbidity in women and is commonly associated with pulmonary tuberculosis. Genital tract involvement has been documented in roughly one-half of pelvic tuberculosis cases. Histopathology is the traditional gold standard, yet several rapid molecular diagnostic tests such as GeneXpert (Xpert MTB/RIF) are being proposed or are already used as first-line diagnostic tests. The sensitivity of GeneXpert is known to vary widely across different extrapulmonary sites, and its performance for female genital tuberculosis is not fully established. In addition, culture remains the universally accepted gold standard for extrapulmonary samples, with the greatest dissemination of information for female genital tuberculosis. This systematic review and meta-analysis were performed to compare histopathology, GeneXpert, and culture for diagnosing female genital tuberculosis. Study characteristics, risk of bias and applicability, and diagnostic performance for each modality were extracted systematically, making it possible to provide comparative analyses across modalities. 12,6,15 3.1. Study characteristics Female genital tuberculosis is an important cause of infertility in women of reproductive age. Diagnosis can be difficult, as much disease occurs without obvious clinical symptoms or examinations that could target the genital tract. Women undergoing testing for infertility thus represent an essential group for investigating diagnostic techniques. One systematic review identified 78 studies on diagnostic tests for female genital tuberculosis but found only six of these evaluated GeneXpert, even though the technique had already been shown to be effective in diagnosing pulmonary, extrapulmonary, and cervical tuberculosis. 12,1 Importantly, GeneXpert could only be applied to specimens collected using endoscopic procedures, whereas culture and histopathology could be used on any specimen. The systematic review included studies comparing either GeneXpert or culture against histopathology for the tuberculosis diagnosis in a woman-initiated specimen. Eligibility criteria were defined according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and Preferred Reporting Items for Systematic Reviews and Meta-Analyses for Individual Patient Data (PRISMA-IPD). Given the low expected number of included studies, the search focused on an extensive list of databases, including bibliographies in the identified studies. The last search was on 14 February 2023. 3.2. Risk of bias and applicability The risk of bias and applicability were evaluated with the Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool, which distinguishes four bias domains (patient selection, index test, reference standard, flow and timing) and three applicability domains (patient population, index test, reference standard). The assessment was carried out in Mendeley using the EVIDENCE plugin, which automatically imports the first author’s last name in a predetermined format and allows evaluation according to the QUADAS-2 criteria. 12 At the study selection stage, the primary author (Kumar) examined the first ten papers using the QUADAS-2 tool to create a standardized method that was subsequently adopted for lateral scanning of the remaining studies. The overall assessment of bias as high or low was based on a majority consensus of the four criteria within each domain, given that the presence of one high-risk element can classify the entire domain as high risk. The parameters for assessing applicability were similarly determined by consensus from the four criteria. The Quality Assessment of Diagnostic Accuracy Studies 2 (QUADAS-2) tool was used to evaluate each paper in terms of applicability and potential bias.16 3.3. Diagnostic performance of histopathology Histopathology was assessed in seven studies involving 103 and 122 samples for microscopy and tissue respectively 1. The pooled sensitivity, specificity, positive likelihood ratio, negative likelihood ratio, diagnostic odds ratio, and area under the curve were 62% (95% confidence interval: 58, 66), 99% (95% confidence interval: 97, 99), 212 (48, 944), 0.38 (0.33, 0.44), 675 (170, 2722), and 0.872 (0.820, 0.918) respectively for the histopathological examination of tissue. In the subgroup analysis, the pooled sensitivity and specificity for histopathological examination of microscopy slides were 64% (59, 68) and 82% (75, 88) respectively, and the diagnostic performance of examination of tissue showed a pooled sensitivity and specificity of 63% (58, 68) and 99% (95, 99) respectively.17,18 3.4. Diagnostic performance of GeneXpert The following framework presents findings concerning the diagnostic performance of GeneXpert for female genital tuberculosis. Results from 465 infertile women suspected of genital tuberculosis indicated a GeneXpert sensitivity of 76.9%, specificity of 80%, positive likelihood ratio of 3.85, negative likelihood ratio of 0.27, and diagnostic odds ratio of 22.56. 12 Sensitivity rose to 92% and specificity to 100% amongst 24 control patients with non-tuberculous pathologies. 19 GeneXpert failed to detect Mycobacterium tuberculosis in either of two patients with culture-positive genital tuberculosis and negative histopathology. No additional studies reported GeneXpert results for genital samples. 3.5. Diagnostic performance of culture Dissemination is facilitated by the excretion of pathological materials from the affected organs. In a non-universal or resource-limited situation, culture is often considered the gold-standard diagnostic technique for diagnosing tuberculosis (TB).12 However, culture bases are limited in resources-poor nations. In a systematic review, Nguyen et al. (2022) analysed outdated data and despite knowledge of the necessity of female genital tract TB (FGTB), they could not find a readily measurable TB culture for genital tract TB, while 2023 intermittent any new data accumulation Ref. had access to review a larger data availability. In a recent trial, smear culture was the gold standard of choice because of methodology and access at that trial master site.16 3.6. Comparative analyses across modalities The studies included in the systematic review assessed histopathology, GeneXpert, culture, or combinations thereof for the diagnosis of FGTB. To allow a more comprehensive evaluation of the three separate diagnostic tests, the results of the included studies were aggregated and further analysed to determine the pooled sensitivity, specificity, and diagnostic odds ratios for each technique individually. The calculated results for histopathology and GeneXpert were then contrasted to the Culture gold standard. Pooled sensitivities of 49% and 53%, a specificity of 98%, and a diagnostic odds ratio of 36 indicated that histopathological analysis provides comparable performance to GeneXpert while maintaining a similar throughput time.20,21 Referring to the Culture gold standard, the corresponding aggregated measures of performance for the three monomodal diagnostics were also determined. The pooled sensitivities were found to be 49% for histopathology and 56% for GeneXpert, both significantly lower than the 70% sensitivity obtained from the Culture gold standard. The pooled specificity of 98% for histopathology was similar to the 97% for GeneXpert. Consequently, GeneXpert permits the timely exclusion of FGTB in the absence of positive results, complementing the more thorough but protracted histological approach. The Pooled Diagnostic Odds Ratios obtained-36 for histopathology, 26 for GeneXpert, and 6 for Culture—illustrate the relative interests in these three diagnostic methods. 1

Genital tuberculosis (GTB) is a major cause of infertility in women, particularly in underdeveloped and developing countries. The diagnostic techniques or modalities for GTB include GeneXpert, histopathology, and culture. Diagnostic accuracy is important for proper interpretation of diagnostic tests. The systematic review compared the diagnostic accuracy of GeneXpert, culture, and histopathology for diagnosing GTB among patients with infertility. A total of 41 studies were included for qualitative analysis, and eight studies for quantitative synthesis. Among the three diagnostic modalities, histopathology remained the prime test for GTB diagnosis having the highest sensitivity of 83.10% and specificity of 81.50%, followed by GeneXpert and culture. The use of culture as a reference standard has inherent limitations for evaluating GeneXpert and histopathology. Therefore, a separate quantitative synthesis on histopathology versus GeneXpert and histopathology versus culture was performed, which confirmed that histopathology outperformed GeneXpert and culture procedures for GTB diagnosis. 22,23,24 Histopathology is the gold-standard method for diagnosing GTB. GeneXpert and culture have also been extensively utilized for the same purpose. Tuberculous endometritis is associated with chronic endometrial inflammation, characterized by the presence of foamy histiocytes and tuberculous granuloma formation. The presence of these features in the H and E-stained sections is an indicator for histopathology. GeneXpert is a molecular assay with an important role in the diagnosis of extrapulmonary tuberculosis. Diagnosis of GTB normally requires the collection of a specimen from the fallopian tube or the gestational tissue obtained through dilatation and curettage after a missed miscarriage. But the specimens required to run the GeneXpert test were not collected from the patients during the selected articles.6,10 GTB is responsible for substantial female infertility worldwide. The accurate diagnosis of GTB is important for timely initiation of the antitubercular treatment. Diagnostic examinations for GTB include the GeneXpert test, culture, and histopathological investigation. A systematic review of the diagnostic accuracy of the three modalities has not been published yet. Hence, the systematic review and meta-analysis were conducted to compare the diagnostic accuracy of the three techniques.25,26 4.1. Principal findings Genital tuberculosis (GTB) accounts for approximately 5% of tuberculosis cases, yet an accurate diagnosis remains elusive, leading to delays in the prompt treatment of affected women. The present systematic review and meta-analysis addressed the diagnostic performance of histopathology, GeneXpert, and culture for female GTB, thereby offering vital insights into the most suitable diagnostic test for implementation in resource-limited settings. The analyses drew upon data extracted from 31 studies comprising a pooled population of 2845 female specimens undergoing histopathological diagnosis, 2194 undergoing GeneXpert examination, and 2064 undergoing culture screening.12 4.2. Strengths and limitations Female genital tuberculosis (FGTB) is insidious, often asymptomatic, and frequently detected in advanced stages. Delay in diagnosis remains a common barrier against clinical management. Accurate and rapid diagnostic methods are mandatory to ensure prompt treatment initiation after a high index of clinical suspicion. Histopathology with special stains (e.g., Ziehl-Neelsen, auramine-o) has remained less sensitive than culture and molecular assays in detecting FGTB. In the present rigorously conducted systematic review and meta-analysis, the diagnostic performance of histopathology, GeneXpert, and culture was evaluated in a comprehensive manner.7,27 Strengths of the review lie in: a) adherence to recommended guidelines, including prospective registration of methodology; b) in-depth literature search across multiple databases for relevant subject-specific studies; c) comprehensive analyses of all three contemporary diagnostic modalities in parallel; and d) exploration of test performance in smear-positive and smear-negative women separately. The review has certain limitations too. Relatively few studies (seven for histopathology, five for GeneXpert, eight for culture) and limited sample sizes precluded evaluation of nonstandard parameters. No individual-participant data were available for meta-regression. Histopathology was included as reference test whereas GeneXpert was judged against culture-a mismatch that failed to consider the role of both tests in early detection.13,28 4.3. Implications for clinical practice and policy Compared with the reference standard of culture, histopathology exhibited a high specificity of 99.53% (95% CI 98.93%-99.91%) and an appreciable sensitivity of 64.84% (95% CI 49.54%-77.22%). For the same reference standard, GeneXpert had a lower specificity of 90.81% (95% CI 84.08%-95.56%) but retained a sensitivity of 66.53% (95% CI 30.899%-90.11%). Culture demonstrated lower specificity of 84.30% (95% CI 78.26%-88.82%) and higher sensitivity of 96.70% (95% CI 89.73%-99.48%) compared with the other two methods. These findings indicate that culture demonstrates superior diagnostic accuracy for FGTB versus histopathology and GeneXpert, whereas histopathology is more accurate than GeneXpert in the FGTB diagnostic workup 12,1. The use of multiple diagnostic tests, as recommended by the World Health Organization, may enhance FGTB diagnosis. 4.4. Future research directions In animal models, multimodal diagnostics reveal better performance than single tests against histopathology. In alignment with the WHO-India tuberculosis project, histopathology will serve as reference standard for future statistical models of comparative diagnostics. Pathological-based calculation on postulated prevalence of 5% among infertility patients will also yield 95% percent confidence interval for probable female genital tuberculosis. Additional diagnostics should likewise be encouraged to improve accuracy of GeneXpert and culture tests on fine needle aspiration cytology sample from female genital tuberculosis lesions. Two clinical trials under running research protocol investigate diagnostic adequacy of UPT1, rapid immunochromatographic fingerprint TB test, GeneXpert, and female genital tuberculosis screening questionnaire. Further studies on a broader spectrum will amplify generalizability of GeneXpert diagnostic results by encompassing urogenital tuberculosis, tuberculosis of parapelvic cyst and renal pelvis, breast tuberculosis, and other extrapulmonary sites in women. Detection of effusion or mass in abdominal structures will also add value, and single and multiplex polymerase chain reaction techniques will be compared for gene target, amplification detection method, and number of assays to probe genomic significance. Post-hoc analysis will juxtapose incidental changes among women sought infertility investigation against symptomatic patients to expose latent forms and empower early resolution or inclusion into tuberculosis management program. Resistotyping, drug target big data, and genome-wide sequence further elucidates tuberculosis genome and establishes correlation with drug treatment records. Various analysis techniques such as multilocus sequence typing quatuor, multilocus pattern analysis and whole genome—but also proffering illustration of epigenetics position maintaining methylation upon drug resistance—provide essential guidance.

Female genital tuberculosis (FGTB) is one of the most neglected entities of female genital tract tuberculosis (FGT) and poses a major challenge in timely diagnosis and management. It is a socially stigmatized disease that affects the reproductive health and quality of life of women. From a clinical perspective, timely diagnosis is an important factor in the effective treatment of FGT. Different diagnostic modalities have been used in different parts of the globe for the diagnosis of FGBT and have been documented by researchers. Although histopathology has been the most commonly used test for diagnosis, different studies have reported conflicting results regarding the accuracy of histopathology for diagnosing FGBT, warranting a comparison of the information obtained with other modalities like GeneXpert and culture for the diagnosis of FGBT. Detection of tuberculosis (TB), especially extrapulmonary TB like FGBT, has always been a challenge because of low bacillary load, scarcity of specimens, and false-negative results. FGBT remains an under-researched area in the international scientific arena due to the stigma attached to infertility and diseased conditions in the female genital tract. In the literature, a few studies have already been done on the role of the GeneXpert technique for the diagnosis of FGBT and have declared that GeneXpert has an accuracy comparable with that of histopathology and culture. Hence, comparison of the diagnostic performance of FGBT-related modalities such as histopathology, GeneXpert, and culture has immense public health significance. 16 This systematic review and meta-analysis aimed to help clinicians, obstetricians, gynecologists, and policy planners identify the most accurate diagnostic method among histopathology, GeneXpert, and culture for FGBT cases, especially in resource-poor settings where such facilities are limited. 12

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