Extra-pulmonary Tuberculosis Samples Research Articles

Diagnostic performance of Xpert MTB/RIF ultra in detecting Mycobacterium tuberculosis and Rifampicin Resistance in AFB Smear-negative Pulmonary and Extrapulmonary Tuberculosis samples in Malaysia

Rapid and highly accurate diagnostic tools are critically needed to diagnose Mycobacterium tuberculosis and rifampicin resistance in AFB smear-negative samples. In this study, we evaluated the diagnostic performance of Xpert MTB/RIF Ultra (Ultra) as a rapid test to diagnose tuberculosis in smear-negative cases in Malaysia. A retrospective study of 1960 smear-negative pulmonary and extrapulmonary samples obtained from patients was conducted. Culture was used as the reference standard for the study. The overall sensitivity and specificity of Ultra on the tested samples were 88.7 % and 77.2 %, respectively, while the PPV was 32.3 % and the NPV was 98.2 %. Ultra showed slightly higher sensitivity in pulmonary (89.9 %) compared to extrapulmonary samples (86.1 %). The overall accuracy of Ultra was 78.5 % (kappa=0.37; 95 %CI: 0.32,0.42). Ultra showed good diagnostic accuracy for detecting MTB and rifampicin resistance in various AFB smear-negative samples. Ultra also had excellent capability in rifampicin resistance detection.

Tuberculosis Diagnosis and Detection of Drug Resistance: A Comprehensive Updated Review

Tuberculosis (TB) is a significant public health challenge, especially in developing nations. Developing a TB eradication strategy is hampered by the global health concern of drug-resistant (DR) TB. Effective patient treatment, preventing TB transfer and avoiding the upsurge of DR strains depend primarily on the timely and accurate identification of DR TB. Due to inadequate sensitivity, the necessity of trained laboratory personnel, the sluggish growth pattern of Mycobacterium bacilli in culture, and the small number of bacilli that are usually found in extrapulmonary TB samples, TB diagnosis is still tricky in clinical practice. Although mycobacterial culture is the gold standard to identify TB and determine drug resistance, it takes 2 to 8 weeks to develop. Despite their high cost, nucleic acid amplification tests (NAATs) and whole-genome sequencing (WGS) are the commonly employed molecular-based methods for diagnosing and identifying TB. The WHO suggested the GeneXpert MTB/RIF to identify TB and detect resistance to rifampicin. In comparison, numerous molecular techniques were developed, including allele-specific PCR (MAS-PCR), solid-phase hybridization, real-time PCR (RT-PCR) and droplet digital PCR-based technique (DDPCR). This manuscript is intended to overview the current approaches for the phenotypic and genotypic diagnosis of TB disease and identifying resistance to antitubercular drugs depending on recently published articles, WHO and CDC reports, and commercially available diagnostic tools.

Utility of GeneXpert MTB/RIF in the Rapid Diagnosis of Extra Pulmonary Tuberculosis

Introduction: The diagnosis of extrapulmonary tuberculosis (EPTB) is a challenge. World Health Organization (WHO) recommends the use of GeneXpert MTB/RIF assay [Cepheid, United States of America (USA)], using a heminested real-time polymerase chain reaction (PCR) to amplify a Mycobacterium tuberculosis-specific sequence of the rpoB gene for the rapid and simultaneous detection of M. tb complex (MTBC) and resistance to rifampicin from a clinical specimen. The purpose of this study is to evaluate the performance of the GeneXpert MTB/RIF test with conventional mycobacterial culture in EPTB specimens. Materials and methods: This prospective study (February–October 2017, 11 months) includes data on 287 EPTB specimens that were processed by conventional culture on Lowenstein–Jensen (LJ) medium and the rapid molecular-based GeneXpert MTB/RIF assay system. Results: Among the 287 EPTB samples tested, GeneXpert detected the deoxyribonucleic acid of MTBC in 51 samples (17.8%). Standard bacteriological assays, including acid-fast bacilli microscopy and culture, were positive in 26 (9.1%) and 35 (12.1%) specimens respectively. The performance of GeneXpert results was evaluated against culture as a gold standard. The overall sensitivity and specificity of the Xpert assay were calculated to be 94.6 and 94.4%, respectively. The sensitivity of the Xpert assay with tissue specimens was 84.6 and 80.7% specificity, while there was 86.6% sensitivity and 98.1% specificity with the body fluids. Conclusion: GeneXpert had high performance than culture for the EPTB specimen. It can be a useful tool for the early diagnosis of patients with high clinical suspicion of EPTB. The other major advantage of GeneXpert is that it simultaneously detects rifampicin resistance within 2 hours.

Diagnostic and quantitative performance of the GeneXpert Mycobacterium tuberculosis/resistance to rifampin assay on pulmonary and extrapulmonary tuberculosis samples

INTRODUCTION: No study has simultaneously assessed the diagnostic and quantitative performance of the GeneXpert Mycobacterium tuberculosis/resistance to rifampicin (MTB/RIF) assay on pulmonary and extrapulmonary samples processed in an Indian laboratory. We examined this to assess if GeneXpert can complement existing diagnostic facilities.METHODS: The performance of GeneXpert and acid-fast microscopy were evaluated against culture in 455 pulmonary and 69 extrapulmonary specimens. Their sensitivity, specificity, predictive values and area under the curve (AUC) were estimated. GeneXpert cycle threshold (Ct) cut-offs were also computed to assess their utility in predicting smear positivity and time to culture positivity (TTCP).RESULTS: GeneXpert was significantly more sensitive (100% vs. 84.4%; P < 0.001) and nearly as specific (94.8% vs. 96.7%; P = 0.344), with a greater AUC (0.97 vs. 0.91; P = 0.002) than acid-fast microscopy. It was sensitive and specific for pulmonary (100%; 94.1%) and extrapulmonary (100%; 96.5%) samples, and 100% sensitive for sputum, bronchoalveolar lavage (BAL), lymph node aspirates, pleural fluid, pus and urine. It detected smear-negative specimens with 100% sensitivity. GeneXpert Ct was inversely correlated with sputum smear grading (ρ = −0.66) and positively with TTCP (ρ =0.70). Mean Ct cut-offs of 21.4, 20.1 and 24.6 predicted smear positivity with maximum sensitivity and specificity in sputum, BAL and extrapulmonary samples, respectively. Rifampicin resistance was seen in 17% of samples.CONCLUSIONS: GeneXpert can be a reliable first-line diagnostic for both pulmonary and extrapulmonary samples. It can effectively detect smear-negative and paucibacillary patients, and estimate smear positivity and bacillary burden. This can help Indian laboratories to detect cases early and interrupt community transmission.

Isoniazid resistance in patients with Extrapulmonary Tuberculosis using line probe assay in a private multispecialty hospital in Bangalore, India

We aimed to study the rate of isoniazid (INH) resistance in Extrapulmonary Tuberculosis samples from a private care setting.A Line probe assay was performed on 74 culture isolates of Mycobacterium tuberculosis or directly on extrapulmonary samples received in our laboratory from 2018 to 2021.The INH mono-resistance among these extrapulmonary samples was 6.7%. (5 among 74) (95% CI: 1.04%–12.48%) Resistance to rifampicin was not detected.Increasing the availability and leveraging public private partnerships in hospitals for universal testing for INH resistance may increase detection of INH monoresistance in EP-TB and improve the strategy for TB elimination.

Molecular studies on extra-pulmonary tuberculosis among patients attending Aminu Kano Teaching Hospital, Kano, Kano State, Nigeria

In 2010 World Health Organization reported that, one third of the World population had tuberculosis, and it is among the infectious disease with significant morbidity and mortality. Mycobacterium tuberculosis is a successful pathogen that has evolved several mechanisms to manipulate the host immune response. The purpose of this study was to determine the incidence of extra-pulmonary TB in patient that visited DOTs clinic, Aminu Kano Teaching Hospital, Kano Nigeria for treatment. A prospective cross-sectional laboratory based study that involved the use of questionnaire and consent/assent form prior to sample collection. Extra pulmonary tuberculosis samples were tested by MTB/RIF assay using GeneXpert. Descriptive statistics was used to analyzed the data obtained and results were given in charts and tables. Out of 71 samples analyzed along with positive control (H37RV) and molecular grade water as a negative control, 3 (4.2 %) samples had extra-pulmonary TB, among which 2 (2.8 %) were from urine samples and 1 (1.4 %) from acetic fluid. This study shows the existence of extra-pulmonary tuberculosis in the study area; and the tuberculosis was sensitive Mycobacterium tuberculosis. It is therefore important to make screening of patients with sign and symptoms of extra pulmonary tuberculosis using Xpert MTB/RIF assay as regular method for extra pulmonary tuberculosis diagnosis. This will be achieved through enlightenment of the clinicians in our health facilities, our States and local governments should strengthen the laboratory capacity for diagnosis and make the services available and reachable to the patients who need them.

Tracing TB: Are there predictors for active TB disease in patients with Xpert Ultra trace results?

ObjectivesThe Xpert MTB/Rif Ultra (Xpert Ultra; Cepheid, USA) has increased sensitivity compared with its predecessor (Gene Xpert), due to the addition of multicopy amplification targets and a novel trace call. The World Health Organization suggests that tuberculosis (TB) treatment should be initiated in HIV, paediatric, and extra-pulmonary TB patients with trace results. However, other factors such as previous TB disease may complicate the interpretation of trace results in high-burden TB settings. This study aimed to clarify the positive predictors for active TB disease in patients with trace results and to investigate if previous TB disease influences TB culture positivity. MethodsA retrospective descriptive study was performed on 290 patients with trace results, to determine what the positive predictors for active TB are by comparing clinical factors to TB culture. ResultsThe key findings of this study were that extra-pulmonary TB samples (OR, 2.7; p=0.012), no previous TB disease (OR, 4.5; p=0.001) and symptoms suggestive of TB (OR, 6.4; p<0.001) are independent predictors for active TB disease. ConclusionThis study found readily available clinical predictors that can aid clinicians with TB management decisions in patients with trace results.

Study of drug susceptibility pattern of mycobacterium tuberculosis samples from known cases of extrapulmonary tuberculosis samples in Mumbai city

Study of drug susceptibility pattern of mycobacterium tuberculosis samples from known cases of extrapulmonary tuberculosis samples in Mumbai city

1633. Improving the diagnosis of extra-pulmonary tuberculosis (EPTB): experience from north India

BackgroundRapid and accurate diagnosis of extra-pulmonary tuberculosis (EPTB) is imperative for early treatment and better patient outcome. Loop-mediated Isothermal Amplification (LAMP) is a promising nucleic-acid amplification assay. LAMP assay could be carried out in simple water bath under isothermal conditions in 60 minutes, and can be performed in any laboratory even in rural setting in resource poor endemic countries. We evaluated LAMP assay using two different target regions LAMP primers specific for Mycobacterium tuberculosis complex for the diagnosis of EPTB.MethodsLAMP assay using 6 primers (each for IS6110 and IS1081) specific for Mycobacterium tuberculosis complex were performed on patients suspected of EPTB on various EPTB samples(CSF, Synovial fluid, Lymaphnode and tissue biopsies and various other samples) of 150 patients (50 confirmed, 100 suspected) Clinically suspected of EPTB and 100 non-TB control subjects.ResultsOverall LAMP test (using any of the two targets) had sensitivity and specificity of 96% and 100% for confirmed (50 culture positive) EPTB cases. In 100 clinically suspected but unconfirmed EPTB cases, LAMP was positive in 87 out of 100 cases (87%). Sensitivity of IS6110 LAMP, 1S1081 LAMP and IS6110 PCR for clinically suspected cases was 78 (78%), 84 (84%) and 70 (70%), respectively. In total 150 EPTB patients, the overall sensitivity of microscopy, culture, IS6110 PCR, IS6110 LAMP, 1081 LAMP and the LAMP test (if any of the two targets were used) were 4%, 33.3%, 74.6%, 82.66%, 87% and 92%, respectively. Specificity of all the tests was 100%. There were 8 cases which were missed by IS6110 LAMP and 2 cases by 1081 LAMP.ConclusionLAMP assay using two targets is a promising technique for rapid diagnosis of EPTB in 60 minutes especially in a resource poor setting who are still battling with this deadly disease.Disclosures All Authors: No reported disclosures

Evaluation of genotype MTBDRplus V2 and genotype MTBDRsl V2 for the diagnosis of extrapulmonary tuberculosis in India

Microbiological diagnosis of extra-pulmonary tuberculosis (EPTB) has been one of the most difficult aspects of tuberculosis (TB) management. Availability of better imaging and diagnostic modalities has led to an increase in the number of diagnosed cases. The current upsurge in multidrug-resistant tuberculosis warrants routine testing of EPTB samples for resistance at baseline with shorter turn-around time.A total of 369 EPTB specimens were subjected to Ziehl-Neelsen (ZN) stain, liquid culture (LC) with phenotypic drug susceptibility testing, MTBDRplus V.2 and MTBDRsl V.2. The molecular categorisation of resistant specimens was further reconfirmed with sequencing.The sensitivity and specificity of MTBDRplus V.2 to detect Mycobacterium tuberculosis (MTB) when compared to ZN stain was 97.9% and 89.2%, respectively while it was 73.4% and 83.8%, respectively when compared to LC. Similarly, for MTBDRsl V.2, the sensitivity and specificity for detection of MTB when compared with ZN was 95.6% and 91.9%, respectively and 75% and 89.2%, respectively when compared to LC. In smear-positive specimens, 94% (141/150) and 86% (129/150) valid results were observed in MTBDRplus V.2 and MTBDRsl V.2, respectively.The utilisation of both MTBDRplus V.2 and MTBDRsl V.2 for the diagnosis of smear-positive EPTB specimens would be useful in programmatic management of TB in high-burden settings.

Laboratory and clinico-demographic profile of patients investigated for tuberculosis in the National Referral Hospital of Bhutan

Tuberculosis (TB), caused by the bacterium Mycobacterium tuberculosis, is an important public health problem in Bhutan. Microscopy is the primary method of diagnosis of TB in developing countries including Bhutan. Performance of microscopy in the Jigme Dorji Wangchuck National Referral Hospital (JDWNRH), has never been assessed. A retrospective review of laboratory records for three years (2014–2016) was performed to determine the laboratory profile of patients investigated for different types of TB at the JDWNRH.A total of 10,821 sputum and 3,495 non-sputum samples were examined for pulmonary TB (PTB) and extrapulmonary TB (EPTB) respectively. The commonest EPTB samples were Fine Needle Aspiration Cytology (FNAC), urine and sterile fluids. About 6% (127/2163), 5 % (130/2390) and 5% (289/5310) were positive for PTB in 2014, 2015 and 2016 respectively and EPTB positivity was about 7% in all years. During follow-up a significant number of patients remained sputum positive. Sputum sample satisfactory rate (quality) varied between 51 % to 79% in the primary samples. Sample completeness (number) ranged between 62.3% to 94.6% but dropped sequentially in the follow-up cases. Sample completeness of urine samples for EPTB ranged between 75-90%. EPTB positivity rate was highest in FNAC, followed by urine, pleural fluid and ascitic fluid samples.Higher number of patients were investigated for TB in subsequent years from 2014 to 2016. TB positivity rates for PTB and EPTB remained consistent over three years at about 5–6% and 7% respectively. There was a significant variation in sputum sample adequateness (by quality and number). Sputum conversion in the follow-up cases was lower than other countries. Educating the patients on the importance of providing adequate samples can improve TB diagnosis, enhance early treatment, reduce transmission and contribute significantly towards TB elimination.

Primary drug resistance in extra-pulmonary tuberculosis: a hospital-based prospective study from Pakistan.

SETTING: Tertiary level specialised tuberculosis (TB) hospital.OBJECTIVE: To determine the prevalence of antimicrobial resistance in new extra-pulmonary tuberculosis (EPTB) cases.DESIGN: Prospective cross-sectional study. Presumptive EPTB patients with enlarged lymph nodes or pleural effusion having no history of TB treatment were enrolled. Specimens were tested for smear, Xpert® MTB/RIF and culture. Indirect drug susceptibility testing (DST) was performed using MGIT 960 and line-probe assays (LPA).RESULTS: Among 671 cases, 255 were bacteriologically confirmed and 185 DSTs were performed. Multidrug resistance (MDR-TB) was reported in 2.2% (95%CI 0.6-5.4), any resistance to rifampicin (RMP) in 2.7% (95%CI 0.9-6.2), isoniazid (INH) in 7.6% (95%CI 4.1-12.4), ethambutol in 1.1% (95%CI 0.1-3.9), pyrazinamide in 2.2% (95%CI 0.9-5.5) and fluoroquinolones (FQ) in 6.0% (95%CI 3.0-10.4). The sensitivity and specificity of LPA-DST was 100% and >98.8% respectively for RMP, INH and FQ. Among 82 cases with RMP of the results available for all three methods used, five were reported to be resistant on Xpert but all five were susceptible on MGIT 960 and four on MTBDRplus.CONCLUSION: Prevalence of RMP resistance in new EPTB cases is 2.7% (95%CI 0.9-6.2). Caution is warranted for RMP resistance detected using Xpert in EPTB samples with a very low bacterial load.

Proteomic profile of pulmonary and extra pulmonary TB samples

Introduction: Tuberculosis (TB) is a major health problem in India, so early diagnosis and treatment of Mycobacterium tuberculosis (M.tb.) infection can prevent deaths from this pathogen. The secretion of proteins by M.tb. is important in diagnostic purposes for generation of therapeutic drugs and vaccines candidates for TB. The objective of this study was to identify the protein expression (biomarkers) in TB and Tuberculosis meningitis (TBM) using proteomic approach.&#x0D; Methods: In this study, using Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), we analyzed the secretory proteins of M.tb. in serum, cerebrospinal fluid (CSF) samples. The identified proteins were determined by Total Lab- 100 Quantity One densitometry software.&#x0D; Results: Our study showed that protein bands expressed in CSF samples reveals the presence of 72kD, 70kD, 44kD, 40kD &amp; 16kD predominantly in TBM patients compared to healthy individuals. The electrophoretogram identified 97kD, 72kD, 44kD, 38kD, 29kD &amp; 16kD predominant proteins in serum samples of TB patients.&#x0D; Conclusion: The detection of secretory proteins in serum and CSF samples of M.tb. in TB and TBM patients gives reliable and early diagnosis of TB and TBM. The secretory proteins can be useful as immunodiagnostic and vaccine targets which can serve as important biomarkers

Evaluation of GeneXpert MTB/RIF assay in diagnosis of extrapulmonary tuberculosis and rifampicin resistance

Background: The prevalence of tuberculosis is still on the rise particularly extrapulmonary cases. There is an urgent need for rapid diagnosis of these cases for prompt initiation of treatment. Aim: To evaluate the role of GeneXpert MTB/RIF Assay in the diagnosis of clinically suspected extrapulmonary tuberculosis (EPTB) and to detect rifampicin resistance in these cases. Materials and methods: A total of 241 samples from April 2016 to July 2018, from all clinically suspected EPTB patients with support of either laboratory or radiological evidence were included in study. 132 pleural fluid, 59 lymph node aspirate, 21 CSF, 17 pus, 9 ascitic fluid and 3 synovial fluid samples were screened for presence of acid fast bacilli (AFB) by conventional Zeihl-Neelsen (ZN) technique. The same samples were also used for testing by GeneXpert MTB/RIF Assay. Results: Out of 241 EPTB samples tested, none were positive for AFB by ZN staining. Overall 9.54 % (23 out of 241) samples were positive for MTB by GeneXpert with a positivity rate of 23.80%, 13.55%, 11.76% & 6.06% respectively for CSF, lymph node aspirate, pus and pleural fluid. MTB was not detected in any of ascitic fluid & synovial fluid samples. Of the 23 MTB positive samples detected by GeneXpert, all were sensitive to rifampicin. Conclusions: GeneXpert was found to be simple, effective and useful diagnostic method for detection of EPTB due to its rapidity & simultaneous detection of rifampicin resistance, thus reducing the time taken for initiation of treatment.

Evaluation of line-probe assay for molecular analysis and drug susceptibility of extra-pulmonary tuberculosis.

Most epidemiological studies on Mycobacterium tuberculosis focus on pulmonary tuberculosis (TB), whereas extra-pulmonary TB (EPTB) remains poorly explored. To study the rate of resistant EPTB cases among individuals with suspected EPTB using a commercial line-probe assay (LPA), polymerase chain reaction (PCR) and reverse hybridisation test. We also examined the molecular profile of the EPTB isolates obtained at the Himalayan Institute of Medical Sciences, Dehradun, India. EPTB samples were collected from 249 patients with clinical and radiological suspicion of EPTB and subjected to automated liquid culture, PCR and GenoType MDRTBplus according to the manufacturers' instructions. A diagnostic yield of 15% was observed among individuals with suspected EPTB using MGIT™ (Mycobacterium Growth Indicator Tubes), which increased to 38% on LPA and PCR. LPA results had 100% concordance with MGIT, with all culture-positive samples also being positive on LPA. However, 70.2% of LPA-positive samples did not grow Mycobacterium tuberculosis in liquid culture. Two (2.1%) of the culture-negative EPTB PCR-positive samples were multidrug-resistant, 20 (21.2%) were rifampicin-monoresistant and 12 (12.7%) isoniazid-monoresistant on LPA. Given the paucibacillary nature of EPTB, we demonstrated that PCR and LPA can have a vital role in establishing TB diagnosis in extra-pulmonary tissues.

Role of line probe assay in detection of extra-pulmonary tuberculosis: Experience from a tertiary care hospital in western Maharashtra

BackgroundDiagnosis of extra pulmonary tuberculosis (EPTB) is challenging due to its atypical clinical presentation and frequently results in a delay or deprivation of treatment. Apart from rapid case detection, early determination of MDR status is imperative in such situations. The commercially available Geno Type MTBDRplus assay version 2.0 (Hain Lifescience, Nehren, Germany) detects both the presence of Mycobacterium tuberculosis (MTB) complex as well as the presence of INH and Rifampcin resistance. We aim to evaluate the role of this test in diagnosis and detection of resistance by comparing its performance against gold standard i.e. culture and against the composite reference standards (CRS) in the diagnosis of EPTB. Material and methodsThe data of 130 EPTB samples processed form January 2014 till May 2017 at Poona Hospital and Research centre were selected for the study. All the samples were processed for Ziehl–Neelsen stain, Geno Type MTBDRplus assay (LPA) and liquid automated culture (BacT/Alert) simultaneously. Geno Type MTBDRplus assay (LPA) was performed directly on the samples. The 24 samples giving positive results on LPA and grown M. tuberculosis on culture were subjected to anti mycobacterial susceptibility testing for 1st line anti-tubercular drugs by BACTEC MGIT 320 system. ResultsOut of 130 samples, 7 samples grew atypical mycobacterium and all the 7 samples turned negative on Line Probe Assay. Direct LPA on processed samples yielded 48/130 (36.9%) positivity. Geno Type MTBDRplus assay was positive for M. tuberculosis in (72.09%) 31/43 culture positive cases and (21%) 17/80 of culture negative cases. Geno Type MTBDRplus assay sensitivity and specificity results were assessed in comparison to CRS made up of culture results and clinical, radiological and histological findings. The overall sensitivity of Geno Type MTBDRplus assay was 45.19% (47/104) and specificity was 94.73 (18/19). Out of 24 samples which were compared for results between LPA and culture, Geno Type MTBDRplus assay accurately identified 3 of 3 of Rifampcin resistant strains and 20 of 21 Rifampcin sensitive strains. Geno Type MTBDRplus assay identified 4 of 4 INH resistant strains and 19 of 20 INH sensitive strains and MDR was obtained for 3 of 3 strains. ConclusionsGeno Type MTBDRplus assay can give early diagnosis and sensitivity for both INH and Rifampcin in extra pulmonary samples. More number of studies is further required to establish Geno Type MTBDRplus assay as an important tool for obtaining diagnosis and resistance to first line drugs in extra pulmonary samples.

Real-time PCR followed by high-resolution melting curve analysis: A rapid and pragmatic approach for screening of multidrug-resistant extrapulmonary tuberculosis

IntroductionMultidrug resistance (MDR) in extrapulmonary tuberculosis (EPTB) is a diagnostic challenge in an endemic country like India. Timely detection of MDR-TB can contribute to a better patient outcome. ObjectiveTo perform real-time PCR (qPCR) using rpoB, mpb64 and IS6110 gene on a variety of EPTB samples and to compare the performance of different gene targets. All qPCR positive samples were subjected to high resolution melt-curve analysis (HRM analysis) for rpoB and katG gene to evaluate its potential for MDR screening among different sample types. MethodsReal-time PCR using rpoB, mpb64 and IS6110 genes was carried out on 200 cases of study group and 100 cases of non-TB control group. The study group consisted of 100 culture-confirmed and 100 clinically suspected cases of EPTB. Phenotypic drug susceptibility testing (DST) for culture isolates was performed by the 1% indirect agar proportion method. DNA extracted from all qPCR positive samples was subjected to rpoB and katG HRM analysis for screening of MDR. Sequencing was used to confirm the results of HRM analysis and the results were also compared with phenotypic DST in all culture positive cases. ResultsThe sensitivity of qPCR using rpoB, mpb64 and IS6110 was 86.5%, 86.5% and 76.5%, respectively. All isolates from the control group were negative by all the three targets, giving a specificity of 100%. HRM analysis detected MDR in 22/200 (11%) isolates. 3/200 (1.5%) had mono-rifampicin resistance while 8/200 (4%) had mono-isoniazid resistance. HRM analysis identified an additional 4 MDR cases directly from the samples which were negative by culture. On sequencing, mutations were observed at codon 531 (60%); 533 (16%); 516 (12%) and 526 (12%) of the rpoB gene and at codon 315 (100%) of the katG gene. There was 100% concordance in the results of phenotypic DST, HRM analysis and sequencing. ConclusionThe HRM analysis can play a promising role in the reliable and rapid screening of EPTB samples for detection of MDR.

Assessment of Sputum Quality and Its Importance in the Rapid Diagnosis of Pulmonary Tuberculosis

Background: Globally, Tuberculosis (TB) still remains a major public health problem. In India, Pulmonary Tuberculosis (PTB) is the most common form of the disease; however, Extrapulmonary Tuberculosis (EPTB) comprises 10 to 20% of all TB cases. The diagnosis of EPTB cases is difficult because of paucibacillary nature and consequently is associated with low sensitivity of Zhiel- Neelson (ZN) smear and culture on Lowenstein-Jensen (LJ) media as gold standard. The present study comparatively evaluates the utility of real time quantitative PCR over nested PCR and other conventional techniques for the detection of M. tuberculosis Complex (Mtc) in clinical isolates of PTB and EPTB samples at a tertiary care centre, Shri Ram Murti Smarak Institute of Medical Sciences (SRMSIMS), Bareilly. Methods and Findings: In total, 205 both pulmonary (24) and extrapulmonary (181) specimens were processed for ZN smear, culture on LJ media, nested PCR and real time quantitative PCR using IS6110 as a gene target. Out of 205 samples, none of the sample was found to be smearpositive and only 28 (14%) samples were found to be culture positive. The nested PCR and real time quantitative PCR positivity was observed 100% in culture positive specimens. However in majority of culture negative specimens the sensitivity was 100% in both nested PCR and real time quantitative PCR assays for EPTB and PTB specimens. The specificity was better in case of real time quatitative PCR as compared to nested PCR, 45.8% and 20% in case of nested PCR for nonrespiratory and respiratory specimens respectively. The specificity was increased upto 86.1 and 100% with real time quantitative PCR for EPTB and PTB cases respectively. Conclusions: The combined analysis of nested PCR, real time quantitative PCR and other lab investigations can be very useful in the rapid diagnosis of M. tuberculosis in paucibacillary extrapulmonary tuberculosis samples in Indian scenario.

TBSURE: An Improved Multiplex Real-Time PCR for TB Detection and Differentiation from Nontuberculous Mycobacterial Diseases in Adult Indian Population

Background: Tuberculosis (TB), an infectious disease caused by bacterium Mycobacterium tuberculosis, has crossed the borders of developing nations and has now emerged as disease of global emergency due to an alarming increase in cases of co-infection with human immunodeficiency virus (HIV). Conventional diagnostics used for TB diagnosis like microscopy and culture although reliable are time consuming. Rapid diagnosis of the disease is required for early commencement of treatment of TB patients. The diagnosis of extra pulmonary tuberculosis (EPTB) is more troublesome due to limited accessibility of infection sites and lack of accurate differentiation from other granulomatous diseases. Currently, WHO advises use of Xpert MTB/RIF assay for EPTB diagnosis, but this assay cannot be used in resource constrained settings prevalent in most of parts of India. Aim: We aimed to develop a multiplex real-time PCR assay which not only detects Mycobacterium tuberculosis complex (MTBC) but also differentiates them from nontuberculous mycobacteria (NTM). Methods: For development and validation of TBSURE, we included 3, 709 EPTB samples from adult Indian patients suspected for TB. TBSURE is a real time PCR diagnostic which uses three Taqman probes based on IS6110, MPB64 and 16S rRNA genes. The sensitivity and specificity of TBSURE was compared with the goldstandard culture test and XpertMTB/RIF assay for TB detection. Results and Conclusion: TBSURE gave a sensitivity of 92% and specificity of 91% as compared to Xpert MTB/RIF assay, when samples from TB suspected individuals were subjected to both the tests. As compared to AFB culture, TBSURE gave a sensitivity of 93.1% and specificity of 96.5%. Thus, TBSURE is an in-house efficient and affordable diagnostic test for EPTB diagnosis.

Role of line probe assay in detection of extra-pulmonary tuberculosis/multidrug-resistant tuberculosis: The experience from Kerala state, India

Background: There are limited data on use of line probe assay (LPA) for detection of TB and Rifampicin resistance among extra-pulmonary tuberculosis (EPTB) patients under specific TB control programme settings. Our aim was to compare the positivity of LPA with Lowenstein–Jensen (LJ) and Bactec MGIT and to test its utility in faster testing and in patients on treatment. Methodology: The data of all 387 EPTB samples processed in two years by the state-level accredited laboratory in 2013–2014 were selected for the study. The laboratory used standard N-acetyl-L-cysteine – sodium hydroxide processing method for all samples except for cerebrospinal fluid and urine samples before conducting Ziehl-Neelsen smear microscopy, LPA and culture on both LJ and MGIT. Those samples with a negative LPA result if culture positive was subjected to a second LPA as per laboratory protocol. Doubtful Rifampicin resistant (RR) results were cleared by phenotypic testing by MGIT. Anti-TB treatment (ATT) duration was correlated with positivity of these tests later. Results and Discussion: LPA done on 321 processed samples out of 387 EPTB samples identified eight RR cases among 136 positive results within two to four days. Performing LPA on culture positive isolates identified an additional seven RR cases, thus reducing turn-around-time by two to eight weeks for the susceptibility results. It also demonstrated far better positivity in smear negative (15.2%) and in smear positive samples (74.3%) compared to culture methods as the test demonstrated clearly, better positivity among patients on ATT for more than two months. Conclusion: LPA in association with culture is an excellent combination with very good results in rapid identification of EPTB along with detection of Rifampicin resistance. LPA gives far superior positivity compared to MGIT and solid cultures among patients under ATT.