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Extra-pulmonary TB (EPTB) is difficult to diagnose due to paucibacillary nature of disease. Current study evaluated accuracy of Truenat MTB and MTB-Rif Dx (TN), for detection of  Mycobacterium tuberculosis  and resistance to rifampicin. Samples were collected from 2103 treatment naive adults with presumptive EPTB, and tested by smear microscopy, liquid culture (LC) (MGIT-960) and GeneXpert MTB/RIF (GX) (Microbiological Reference Standards, MRS). TN results were compared to MRS and Composite Reference Standards (CRS, Microbiology, histopathology, radiology, clinical features prompting decision to treat, response to treatment). CRS grouped patients into 551 confirmed, 1096 unconfirmed, and 409 as unlikely TB. TN sensitivity and specificity was 73.7% and 90.4% against GX. Against LC, Overall sensitivity of GX was 67.6%, while that of TN was 62.3%. Highest sensitivity by TN was observed in pus samples (89%) and highest specificity (92%) in CSF samples, similar to GX. TN sensitivity was better in fluid and biopsy samples and slightly inferior for lymph node aspirates compared to GX. TN sensitivity for RIF resistance detection was slightly superior to GX. TN and GX results were further compared to Clinical Reference Standards. TN detected 170 TB patients initiated on treatment missed by GX, while GX detected 113 such patients missed by TN. Of 124 samples with RIF resistance discordance between GX and TN, GX reported 103/124 as sensitive, 3/124 as indeterminate and 18 as resistant (13/18 samples had low/very low DNA load) while TN reported RIF resistance indeterminate in 103/111 low/very low DNA load samples. Due to paucibacillary nature of EPTB samples, culture yield was poor and phenotypic drug susceptibility testing failed to resolve the discordance. The study establishes TN at par with GX and can be utilized for quick and accurate diagnosis of EPTB.

Pleural tuberculosis (pTB) is a grave form of extrapulmonary tuberculosis. Microbiological tests are usually found to be inadequate for pTB diagnosis. The absence of a uniform 'composite reference standard' is challenging; therefore, diagnosis is usually performed using a combination of diversified criteria. Nucleic acid tests vary in diagnostic accuracy and have not yet been integrated into clinical decision making. This review assesses the varied criteria used for pTB classification and the challenges afflicting pleural fluid-based DNA diagnostic tests, namely, PCR and Xpert MTB/RIF. In the 58 studies (PCR: n = 33; Xpert: n = 25) analyzed, reference standards were heterogeneous and PCR/Xpert pooled sensitivity values (range: 0-100%) were inadequate. However, the consistent high specificity of Xpert (range: 90-100%) indicated its utility as a 'rule-in' test. There is an urgent need to evaluate existing and new molecular tests in well-designed studies to accurately assess their utility for pTB diagnosis. To conclude, rapid and accurate tests are warranted for pTB diagnosis.

Introduction: Soil-transmitted helminthiasis (STH) remains a major public health problem in school children in Ethiopia. Although direct wet mount microscopy (DWMM) is the means to diagnose parasitic diseases in health care facilities in Ethiopia, it remains unclear what its diagnostic performance is for STH. Methodology: A cross-sectional study was performed in Jimma Town (Ethiopia) and included 600 children from 10 primary schools. The diagnostic sensitivity of DWMM was compared to a composite reference standard (CRS) consisting of Kato-Katz, McMaster and Mini-FLOTAC. We also explored the impact of intensity of infection (the highest faecal egg counts (FECs; expressed as eggs per gram of stool (EPG)) across the CRS) on the diagnostic sensitivity of DWMM. Results: Based on the CRS, there were 210 Ascaris (35.0%), 312 Trichuris (52.0%) and 102 hookworm cases (17.0%). The median intensity of infections equalled 2,057 EPG for Ascaris, 200 EPG for Trichuris and 110 EPG for hookworms. The sensitivity of DWMM was 73.8% for Ascaris, but was around 17% for both Trichuris and hookworms. The sensitivity significantly increased with intensity of STH. For Ascaris, the odds for detecting an infection intensity of 1,000 EPG was 6.2 times higher than detecting an infection of 100 EPG. For Trichuris and hookworms, these odds ratios were 7.1 and 14. Conclusions: The diagnostic sensitivity of DWMM is low for STH, but it is able to detect those subjects that are in the highest need of treatment, and hence contributes to the global goal to eliminate STH as a public health problem.

Background Control of the global burden of tuberculosis is obstructed due to lack of simple, rapid and cost effective diagnostic techniques that can be used in resource poor-settings. To facilitate the early diagnosis of TB directly from clinical specimens, we have standardized and validated the use of nested multiplex PCR, targeting gene fragments IS6110 , MTP40 and 32kD α -antigen encoding genes specific for Mycobacterium tuberculosis complex and non-tubercular mycobacteria (NTM), in comparison to smear microscopy, solid culture and single step multiplex PCR. The results were evaluated in comparison to a composite reference standard (CRS) comprising of microbiological results (smear and culture), clinical, radiological and cytopathological findings, clinical treatment and response to anti-tubercular therapy. Methods The nested multiplex PCR (nMPCR) assay was evaluated to test its utility in 600 (535 pulmonary and 65 extra-pulmonary specimens) clinically suspected TB cases. All specimens were processed for smear, culture, single step multiplex PCR and nested multiplex PCR testing. Results Out of 535 screened pulmonary and 65 extra-pulmonary specimens, 329 (61.5 %) and 19 (29.2 %) cases were culture positive for M. tuberculosis . Based on CRS, 450 patients had “clinical TB” (definitive-TB, probable-TB and possible-TB). Remaining 150 were confirmed “non-TB” cases. For culture, the sensitivity was low, 79.3 % for pulmonary and 54.3 % for extra-pulmonary cases. The sensitivity and specificity results for nMPCR test were evaluated taken composite reference standard as a gold standard. The sensitivity of the nMPCR assay was 97.1 % for pulmonary and 91.4 % for extra-pulmonary TB cases with specificity of 100 % and 93.3 % respectively. Conclusion Nested multiplex PCR using three gene primers is a rapid, reliable and highly sensitive and specific diagnostic technique for the detection and differentiation of M. tuberculosis complex from NTM genome and will be useful in diagnosing paucibacillary samples. Nested multiplex PCR assay was found to be better than single step multiplex PCR for assessing the diagnosis of TB.

Objective: The objective was to study the sensitivity, specificity, and diagnostic accuracy of various computed tomography (CT) chest findings in diagnosing recurrence among pulmonary tuberculosis (PTB) suspects. Materials and Methods: A prospective observational study was conducted in a tertiary care hospital in New Delhi. A total of 130 suspects with a past history of treatment for PTB, who presented with any of the symptoms suggestive of recurrence were included. Sputum-positive, HIV-positive patients, pregnant females, and patients aged <18 years were excluded. Patients underwent CT chest followed by bronchoalveolar lavage (BAL). Results: A total of 62 patients were there in the final analysis. The median age of the patients with recurrent PTB was 27.5 years. Cough was the universal symptom in all these patients (>90%). Hemoptysis was the predominant symptom among patients with chronic pulmonary aspergillosis (66.6%). Necrotic mediastinal lymph nodes had good diagnostic accuracy of 88.71% with area under the curve of 0.806, P < 0.001 in diagnosing recurrent TB. BAL GeneXpert and mycobacteria growth indicator tube had good sensitivity (83.33% and 84.62%, respectively), specificity (100% for both), and excellent diagnostic accuracy (95.16% and 96.36%, respectively) for diagnosing recurrence in sputum negative and sputum scarce patient, (P < 0.001) when compared with composite reference standard. For culture-positive cases, BAL GeneXpert MTB/RIF had 100% sensitivity and 97.73% specificity in diagnosing recurrent PTB patients. Conclusion: The presence of mediastinal necrotic lymph node is the most accurate CT finding that can differentiate recurrent TB from post-TB sequelae. No other single chest CT scan finding had reliable diagnostic accuracy in comparison to microbiological tools in diagnosing recurrence among sputum negative or scarce previously treated PTB suspects.

Background Tuberculosis (TB) remains a major public health concern in many low-income countries accounting for approximately two-thirds of deaths in people living with human immunodeficiency virus (HIV) infection. With prompt, accurate and appropriate treatment, almost all TB disease can be cured. The present study was to evaluate the diagnostic performance of an in-house duplex PCR (D-PCR) using IS1610 and rpoB specific primers in sputum samples from TB suspected patients. Methods A hospital-based cross-sectional study was conducted at the Limbe and Buea Regional Hospitals of the South West Region of Cameroon from June 2016 to April 2017. Sputum samples, decontaminated with hypertonic saline/sodium hydroxide solution were centrifuged and pellets processed for smear microscopy, culture and DNA extraction. Suspected inhibition was resolved by serial dilution of genomic DNA. Results were compared to culture as gold standard as well as a Composite Reference Standard (CRS). Results A total of 129 participants aged between 5 to 82 years were enrolled in to the study. The median age of the participants was 37 years (interquartile range, IQR: 27–50 years), with 54.3% being male. Forty-seven samples (36.4%) were positive by direct sputum microscopy, 49 (38%) by microscopy after concentration, 51 (39.5%) by culture and 62 (40.1%) by D-PCR. PCR inhibition was resolved in 85.7% (18/21) of the samples that had inhibition. The overall sensitivity, specificity, positive and negative predictive values, positive and negative likelihood ratios and area under the curve AUC) of the D-PCR was 93.5, 94, 94, 94%, 15.6, 0.005 and 89.0% respectively using CRS as reference. The sensitivities of D-PCR observed among different sample categories were 95.7, 87.5 and 87.5% for smear-and culture-positives, smear-negative/culture-positive, and clinically diagnosed cases respectively. Conclusion IS1610 and rpoB duplex PCR using relatively cheap decontamination and DNA extraction methods in addition to simple serial dilutions to resolve PCR inhibition shows high sensitivity in the diagnosis of paucibacillary tuberculosis.

Background: Female genital tuberculosis (FGTB) is a common problem in developing countries causing significant morbidity, especially infertility. Radiological imaging, especially ultrasound, can help in diagnosis of FGTB with tubo-ovarian masses. Aims: The present study was performed to evaluate the role of ultrasound in diagnosis of FGTB and to see various findings of FGTB on ultrasound. Study Setting and Design: It was a prospective cross-sectional study over 4-year period between August 2015 and August 2019 in a tertiary referral center. Subjects and Methods: One hundred and seventy-five patients of infertility diagnosed to have FGTB on composite reference standard (CRS) of positive acid-fast bacilli on microscopy or culture of endometrial biopsy, positive polymerase chain reaction, positive GeneXpert, epithelioid granuloma on histology of endometrial biopsy, or definite or probable finding of FGTB on laparoscopy were subjected to transvaginal ultrasound by an experienced sonographer for various findings of FGTB. Statistical Analysis: Data analysis was carried out using STATA software 12.0. Comparison of categorical values was tested using Chi-square Fisher's exact test, with P < 0.05 being taken as significant. Results: Mean age, body mass index, parity, and duration of infertility were 28.9 years, 22.9 kg/m2, 0.26, and 6.06 years, respectively. Menstrual dysfunction was common (44%). Diagnosis of FGTB was made by CRS. Ultrasound was normal in 112 (64%) cases and was abnormal in 63 (36%) cases. Various ultrasound findings were ovarian cyst (23.42%), tubo-ovarian masses (15.42%), unilateral or bilateral hydrosalpinx (13.71%), pyosalpinx (0.57%), adhesion (1.14%), adnexal fixity (6.28%), thin endometrium (24.57%), endometrial fluid (12.57%), endometrial calcification (1.7%), endometrial synechiae (4.57%), cornual synechiae (2.28%), impaired endometrial vascularity (21.71%), ascites (6.85%), and peritoneal or omental thickening (1.75%). Conclusion: Carefully performed ultrasound is a useful modality in diagnosis of FGTB, especially in adnexal masses.

Diagnosing extrapulmonary tuberculosis (EPTB) is challenging. Immunohistochemistry or immunocytochemistry has been used to diagnose tuberculosis (TB) by detection of MPT64 antigen from various extrapulmonary specimens and has shown good diagnostic performance in our previous studies. The test can distinguish between disease caused by Mycobacterium tuberculosis (Mtb) complex and nontuberculous mycobacteria and can be applied on formalin‐fixed paraffin‐embedded tissue. As the antibodies previously used were in limited supply, a new batch of polyclonal antibodies was developed for scale‐up and evaluated for the first time in this study. Our aim was to assess the diagnostic accuracy of the MPT64 test with reproduced antibodies in the high burden settings of Pakistan and India. Patients were enrolled prospectively. Samples from suspected sites of infection were collected and subjected to histopathologic and/or cytologic evaluation, routine TB diagnostics, GeneXpert MTB/RIF (Xpert), and the MPT64 antigen detection test. Patients were followed until the end of treatment. Based on a composite reference standard (CRS), 556 patients were categorized as TB cases and 175 as non‐TB cases. The MPT64 test performed well on biopsies with a sensitivity and specificity of 94% and 75%, respectively, against a CRS. For cytology samples, the sensitivity was low (36%), whereas the specificity was 81%. Overall, the MPT64 test showed higher sensitivity (73%) than Xpert (38%) and Mtb culture (33%). The test performed equally well in adults and children. We found an additive diagnostic value of the MPT64 test in conjunction with histology and molecular tests, increasing the yield for EPTB. In conclusion, immunochemical staining with MPT64 antibodies improves the diagnosis of EPTB in high burden settings and could be a valuable addition to routine diagnostics.

Background: Respiratory infections are a major contributor to hospital admissions. Identification of respiratory pathogens by means of conventional culture and serology methods remains challenging. Multiplex molecular assays are an appealing alternative that endeavours to be rapid, more accurate and less arduous.Objective: The study aimed to compare the clinical performance of three commercial multiplex molecular assays for respiratory viruses.Methods: Forty-eight respiratory specimens obtained from patients at Tygerberg Hospital in the Western Cape province of South Africa were studied. These specimens were collected between May 2020 and August 2020. The results of the Seegene Anyplex™ II RV16, FilmArray® Respiratory 2.1 plus Panel (FARP), and QIAstat-Dx® Respiratory SARS-CoV-2 Panel (QRP) were analysed based on the overlapping targets. A composite reference standard was applied to provide a standard reference for comparison.Results: The overall sensitivity of the Seegene Anyplex™ II RV16 was 96.6% (57/59), the FARP 98.2% (56/57) and the QRP 80.7% (46/57). The overall specificities were 99.8% (660/661), 99.0% (704/711) and 99.7% (709/711), respectively. The QRP failed to detect coronaviruses and parainfluenza viruses in 41.7% (5/12) and 28.6% (4/14) of positive specimens, respectively, while the FARP produced the lowest target specificity of 88.4% (38/43) for rhinovirus/enterovirus.Conclusion: The overall specificity of all three platforms was comparable; however, the sensitivity of the QRP was inferior to that of the ARV and FARP.What this study adds: This study adds to the body of performance characteristics described for respiratory multiplex panels, especially in the African context where molecular diagnostics for infectious diseases are gaining momentum.

Background: Dengue virus (DENV) infection remains a global public health concern. Enzyme-linked immunosorbent assays (ELISAs), which detect antibodies targeting the envelope (E) protein of DENV, serve as the front-line serological test for presumptive dengue diagnosis. Very few studies have determined the serostatus by detecting antibodies targeting the nonstructural protein 1 (NS1), which can function as diagnostic biomarkers to distinguish natural immunity from vaccine-induced immunity. Methods: We used community-acquired human serum specimens, with the serostatus confirmed by focus reduction microneutralization test (FRμNT), to evaluate the diagnostic performances of two NS1-based ELISA methods, namely, immunoglobulin G antibody-capture ELISA (NS1 GAC–ELISA) and indirect NS1 IgG ELISA, and compared the results with an E-based virus-like particle (VLP) GAC–ELISA. Results: NS1-based methods had comparable accuracies as VLP GAC–ELISA. Although the sensitivity in detecting anti-NS1 IgM was poor, indirect NS1 IgG ELISA showed similar limits of detection (~1–2 ng/mL) as NS1 GAC–ELISA in detecting anti-NS1 IgG. Combining the results from two or more tests as a composite reference standard can determine the DENV serostatus with a specificity reaching 100%. Conclusion: NS1-based ELISAs have comparable accuracies as VLP GAC–ELISA in determining dengue serostatus, which could effectively assist clinicians during assessments of vaccine eligibility.