Smear-positive Samples Research Articles

Evaluating culture-free targeted next-generation sequencing for diagnosing drug-resistant tuberculosis: a multicentre clinical study of two end-to-end commercial workflows.

Drug-resistant tuberculosis remains a major obstacle in ending the global tuberculosis epidemic. Deployment of molecular tools for comprehensive drug resistance profiling is imperative for successful detection and characterisation of tuberculosis drug resistance. We aimed to assess the diagnostic accuracy of a new class of molecular diagnostics for drug-resistant tuberculosis. We conducted a prospective, cross-sectional, multicentre clinical evaluation of the performance of two targeted next-generation sequencing (tNGS) assays for drug-resistant tuberculosis at reference laboratories in three countries (Georgia, India, and South Africa) to assess diagnostic accuracy and index test failure rates. Eligible participants were aged 18 years or older, with molecularly confirmed pulmonary tuberculosis, and at risk for rifampicin-resistant tuberculosis. Sensitivity and specificity for both tNGS index tests (GenoScreen Deeplex Myc-TB and Oxford Nanopore Technologies [ONT] Tuberculosis Drug Resistance Test) were calculated for rifampicin, isoniazid, fluoroquinolones (moxifloxacin, levofloxacin), second line-injectables (amikacin, kanamycin, capreomycin), pyrazinamide, bedaquiline, linezolid, clofazimine, ethambutol, and streptomycin against a composite reference standard of phenotypic drug susceptibility testing and whole-genome sequencing. Between April 1, 2021, and June 30, 2022, 832 individuals were invited to participate in the study, of whom 720 were included in the final analysis (212, 376, and 132 participants in Georgia, India, and South Africa, respectively). Of 720 clinical sediment samples evaluated, 658 (91%) and 684 (95%) produced complete or partial results on the GenoScreen and ONT tNGS workflows, respectively, with 593 (96%) and 603 (98%) of 616 smear-positive samples producing tNGS sequence data. Both workflows had sensitivities and specificities of more than 95% for rifampicin and isoniazid, and high accuracy for fluoroquinolones (sensitivity approximately ≥94%) and second line-injectables (sensitivity 80%) compared with the composite reference standard. Importantly, these assays also detected mutations associated with resistance to critical new and repurposed drugs (bedaquiline, linezolid) not currently detectable by any other WHO-recommended rapid diagnostics on the market. We note that the current format of assays have low sensitivity (≤50%) for linezolid and more work on mutations associated with drug resistance is needed. This multicentre evaluation demonstrates that culture-free tNGS can provide accurate sequencing results for detection and characterisation of drug resistance from Mycobacterium tuberculosis clinical sediment samples for timely, comprehensive profiling of drug-resistant tuberculosis. Unitaid.

Evaluation of Diagnostic Methods and Rifampicin Resistance in Pulmonary Tuberculosis: A Hospital-Based Study.

Background Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis, predominantly affecting the lungs (pulmonary TB) and is a significant public health challenge in India. The study aims to analyze demographic, radiological, and clinical subgroups of pulmonary TB cases, examine the relationship between smear acid-fast bacillus (AFB examination) and cartridge-based nucleic acid amplification test (CBNAAT), evaluate CBNAAT sensitivity for Mycobacterium tuberculosis (MTB) in new and previously treated patients, and determine the proportion of rifampicin resistance. Methods This hospital-based prospective study was conducted among patients diagnosed with pulmonary TB at the Respiratory Medicine Department of a Government Hospital over 16 months (August 2019 to December 2020). The study included 150 diagnosed TB cases (new and previously treated). Data collection encompassed demographic details, clinical symptoms, comorbidities, radiological findings (chest X-ray), and microbiological results (smear AFB examination, CBNAAT). Sputum samples were subjected to Ziehl-Neelsen staining and CBNAAT for MTB detection and rifampicin resistance testing. Statistical analysis was performed using IBM SPSS Statistics version 21.0 (IBM Corp., Armonk, NY, USA). Results Of the 150 patients, 69.3% were male, and 48% were aged 21-40 years. The majority had a BMI of 18.5-24.9 kg/m² (50%) and resided in urban areas (63.3%). Common symptoms included cough (95.3%), fever (80%), and weight loss (74%). Cavitary lesions on chest X-ray were observed in 84% of patients. Smear microscopy detected MTB in 72.7% of cases, while CBNAAT detected MTB in 94% of cases. CBNAAT sensitivity for smear-positive and smear-negative samples was 93.97% and 94.12%, respectively. Rifampicin resistance was found in 3% of new cases and 6% of previously treated cases. The sensitivity of smear microscopy was 77.33%, and the sensitivity of CBNAAT was 94%. Conclusion The study underscores the high burden of pulmonary TB and the utility of CBNAAT in detecting MTB and rifampicin resistance, particularly in smear-negative samples. The findings highlight the necessity of universal drug susceptibility testing (DST) for effective TB management and the importance of addressing drug resistance to improve treatment outcomes.

Evaluation of Diagnostic Performance of Line Probe Assay on Smear-Negative Samples of Pulmonary Tuberculosis.

This study aims to compare the performance of lineprobeassay (LPA) onsmear-negative samples with that of smear-positive samples for diagnosingpulmonarytuberculosis (PTB) andfirst-line drugsensitivitytesting (FLDST). A total of 196 sputum samples including both smear-positive (112) and negative (84) samples of patients suspected of PTBwere subjected to LPA for TB detection and FL DST. TB culture followed by MPT 64 Ag was done and conventional FL DST was performed on all culture-positive isolates. Results of LPA on smear-negative were compared with smear-positive samples. The LPAconfirmed the diagnosis of PTB in 104/112 smear-positive cases but in only 36/84 smear-negative cases. The assay had 47.36%, 72.72%, and 88.88% sensitivity and 86.96%, 95.23%, and 95.65% specificity in smear-negative cases compared to 89.09%, 95.83%, and 98.07% sensitivity and 100%, 98.36%, and 98.24% specificity in smear-positive cases for detecting Mycobacterium tuberculosis (MTB), rifampicin (RMP)resistance, and isoniazid(INH) resistance, respectively. LPA performance was better on smear-positive than smear-negative sputum samples. Further larger studies are needed to justify the use of LPA on smear-negative pulmonary samples for diagnosis.

Prevalence of Smear-Positive, Rifampicin-Resistant Mycobacterium tuberculosis and Related Factors Among Residents with Cough in Northern Ethiopian Refugee Health Facilities.

To ascertain the prevalence of Mycobacterium tuberculosis (M.tb) among refugees suspected of tuberculosis (TB) and related risk factors, including smear-positive and Rifampicin-resistant M.tb. A cross-sectional study was conducted between January 2020 and May 2020 among 384 refugees in four refugee camps in Northwest Tigray, Ethiopia. Socio-demographic and clinical data were collected from refugees with a history of cough for more than two weeks prospectively. Spot-spot sputum samples were collected and transported in an ice box to the Shire Suhul Hospital Microbiology laboratory; and then examined using a Fluorescent Microscope. All smear-positive samples were further processed by GeneXpert to detect Rifampicin-resistant MTB. Data were analyzed using SPSS version 21 and a p-value <0.05 was considered statistically significant. The overall prevalence of smear-positive PTB infection was 5.5% (21/384), but No TB case was resistant to Rifampicin detected by GeneXpert MTB/RIF assay. About 70% of the smear-positive pulmonary TB identified were females. Five (23.8%) of the smear-positive pulmonary tuberculosis cases were co-infected by HIV. Sharing of drink and food materials (AOR = 4.36, 95% CI = 1.19-15.89), active TB contact (AOR 7.24, 95% CI = 1.62-32.125), BMI (AOR = 5.23, 95% CI = 1.28-21.29), opening window practice (AOR = 4.32, 95% CI = 1.02-18.30) and HIV status (AOR = 9.36, 95% CI = 1.64-53.35) were statistically significant predisposing factors. The prevalence of smear-positive pulmonary TB among northwest Tigray refugee camps was still high. The prevalence of TB/HIV co-infection was also high. Minimizing close contact with active TB cases, reducing malnutrition, rapid TB/HIV screening, and establishing a ventilation system can reduce the transmission of TB among refugees.

What is New in the Diagnosis of Childhood Tuberculosis?

The fact that almost half of the 1 million cases of childhood tuberculosis (TB)globally remain undiagnosed jeopardizes the TB elimination goal. Fortunately, there are new advances in this fieldwhich have the potential to bridge this diagnostic gap. Advances in imaging include computer assisted interpretation of chest X-rays (CXRs), point of care ultrasound (POCUS) and faster and superior computed tomography/ magnetic resonance imaging (CT/ MRI) protocols. The urine lipoarabinomannan test has proved to be a good point of care test for diagnosing TB in Human immunodeficiency virus (HIV) infected children. Stool and nasopharyngeal aspirates are emerging as acceptable alternatives for gastric lavage and induced sputum for diagnosing intrathoracic tuberculosis. Xpert MTB/RIF Ultra has improved sensitivitycompared to Xpert MTB/RIF for diagnosing both pulmonary/ extrapulmonary TB. Xpert XDR is another commercially available accurate point of care test for detecting resistance to drugs other than rifampicin in smear positive samples. Other molecular methods including new line probe assays, pyrosequencing, whole genome sequencing, and targeted next generation sequencing are extremely promising but not available commercially at present. The C-Tb skin test isan acceptable alternative to the tuberculin skin test and interferon gamma release assays for diagnosis of latent infection. There is an urgent need to incorporate some of these advances in the existingdiagnostic algorithms of childhood TB.

Mutational analysis in drug resistant Mycobacterium tuberculosis in Western Uttar Pradesh

BackgroundMulti-drug resistant tuberculosis (MDR-TB) results in treatment failure and poor clinical outcomes. This study was carried out with the aim to determine the pattern of drug resistance against Mycobacterium tuberculosis towards first line ATT (anti-tubercular treatment) in sputum smear–positive patients using Line Probe Assay (LPA). MethodsA cross sectional prospective study was carried out in a tertiary care Hospital of Meerut. A total of 898 sputum samples (on spot and early morning) collected from 449 suspected pulmonary tuberculosis patients as per RNTCP guidelines were screened by microscopy. Decontamination was done by N-acetyl-l-cysteine and sodium hydroxide. Then smear positive samples were subjected to 1st line drug susceptibility testing (DST) using LPA GenoType® MTBDRplus (HAIN Life Science) assay, a molecular method which allows rapid detection of Rifampicin (Rif) and Isoniazid (INH) resistance. ResultsThe overall burden of MDR TB in this geographical area was 7.9 %. Mono-resistance with Rif alone was around 2.8 %. However, the mono-resistance with INH (inhA gene) and INH (katG gene) was 2.8 % and 1.1 % respectively. Drug resistance of Rif was due to mutations in rpoB gene while resistances to INH were more commonly due to mutation in inhA gene followed by katG gene. TB was more commonly seen in the age group of 30–59 years (43.8 %) and predominantly in males. ConclusionTuberculosis positivity rate is high in Western Uttar Pradesh. Burden of MDR TB in Western Uttar Pradesh was similar to National data. Line probe assay can be used as a primary method to diagnose multi drug resistant TB as done in present study which can help in earlier initiation of correct therapy.

Status of quality indicators in a mycobacteriology culture laboratory, Hawassa, Sidama, Ethiopia.

We aimed to assess performance parameters in a Hawassa Tuberculosis Culture Laboratory, in the Sidama Regional Public Health Institute. A cross-sectional survey was conducted between 27 October 2020 and 31 May 2021, on 439 clinical specimens. The specimens were processed using standard procedures, and the final suspension was inoculated into a Microbial Growth Indicator Tube and Lowenstein-Jensen media slant. Ziehl-Neelsen staining and the Bioline test kit were used to identify and confirm Mycobacterium tuberculosis. The data were analyzed using the IBM Statistical Package for Social Sciences (SPSS, version 26). Out of a total of 439 specimens that were processed, the recovery rates for smear-positive specimens were 61% (25 out of 41) and 58.5% (24 out of 41) for the Mycobacterial Growth Indicator Tube, and the Lowenstein-Jensen methods, respectively. For smear-negative samples, the recovery rates were 4.5% (18 out of 398) for both methods. Only 4 (0.9%) specimens were rejected. The mean turnaround times to detect mycobacteria from smear-positive samples were 14 and 32 days for the Mycobacterial Growth Indicator Tube and Lowenstein-Jensen methods, respectively. The standard deviations were ±6.3 days and ±9.7 days, respectively. For smear-negative samples, the mean turnaround times were 17.7 and 31 days for the Mycobacterial Growth Indicator Tube and Lowenstein-Jensen methods, respectively. The standard deviations were ±9.2 days and ±9.6 days, respectively. The contamination rates for the Mycobacterial Growth Indicator Tube and Lowenstein-Jensen methods were 9.8% (43 out of 439) and 9.6% (42 out of 439), respectively. The detection rate of nontuberculosis mycobacteria was 1.4% (6 out of 439). It demands attention to improve the low recovery rate among smear-negative cultures and culture contamination rates.

A Novel Microfluidic Dielectrophoresis Technology to Enable Rapid Diagnosis of Mycobacteria tuberculosis in Clinical Samples

To achieve the global efforts to end tuberculosis, affordable diagnostics suitable for true point-of-care implementation are required to reach the missing millions. In addition, diagnostics with increased sensitivity and expanded drug susceptibility testing are needed to address drug resistance and to diagnose low-bacterial burden cases. The laboratory-on-a-chip technology described herein used dielectrophoresis to selectively isolate Mycobacterium tuberculosis from sputum samples, purifying the bacterial population ahead of molecular confirmation by multiplex real-time quantitative PCR. After optimization using a panel of 50 characterized sputum samples, the performance of the prototype was assessed against the current gold standards, screening 100 blinded sputum samples using characterized and biobanked sputum provided by Foundation for Innovative New Diagnostics. Concordance with culture diagnosis was 100% for smear-negative samples and 87% for smear-positive samples. Of the smear-positive samples, the high burden sample concordance was 100%. Samples were diagnosed on the basis of visual assessment of the dielectrophoresis array and by multiplex real-time quantitative PCR assay. The results described herein demonstrate the potential of the CAPTURE-XT technology to provide a powerful sample preparation tool that could function as a front-end platform for molecular detection. This versatile tool could equally be applied as a visual detection diagnostic, potentially associated withbacterial identification for low-cost screening or coupled with an expanded PCR assay for genotypic drug susceptibility testing.

Efficacy of TrueNat cartridge extracted DNA for detecting drug resistant tuberculosis by line probe assay

BackgroundCurrently for diagnosing Mycobacterium tuberculosis and its drug resistance, two sputum samples are required. One of them is subjected to TrueNat™ and if positive the other sample is subjected to line probe assay (LPA). This study was done to evaluate whether TrueNat extracted DNA can be directly used for performing LPA in a diagnostic laboratory setting to decrease patient turn-around time. MethodsTotal 45 smear positive sputum samples were subjected to TrueNat™ MTB detection and first and second line (FL and SL) LPA testing in parallel. DNA extracted by Trueprep® Cartridge was also tested by LPA and results were compared. Further, TrueNat extracted DNA from 20 samples was divided into 6 aliquots each, two of which were stored at 4 °C, 37 °C and 55 °C (under humidification) each. One aliquot from stored DNA at each temperature was used for FL & SL LPA on day three and the other on day eight. The blots thus obtained were compared with those of conventional LPA at day 1. ResultsFor FL-LPA, TrueNat extracted DNA gave valid results for all 45 (100%) samples but conventionally extracted DNA could give results for 44 (97.8%) samples. Likewise, for SL-LPA, valid results were obtained for 40 (88.9%) and 35 (77.8%) samples respectively using TrueNat extracted DNA and conventionally extracted DNA respectively. All samples with invalid LPA results had Ct values ≥ 28 by TrueNat PCR. LPA results were obtained for all the 20 samples using stored DNA at all temperatures and duration. ConclusionsTrueNat extracted DNA can be used for performing LPA under field conditions for selected samples.

Molecular Characterization of katG and inhA Mutations by Genotype MTBDRplus Line Probe Assay To Guide Isoniazid and Ethionamide Use for Drug-Resistant Tuberculosis.

Introduction Drug-resistant tuberculosis (TB) continues to be a global health threat in all its forms. Significant resistance has been observed against isoniazid (INH), one of the most important therapeutic options for treating TB. Molecular testing methods such as line probe assay (LPA) provide rapid diagnosis and early management. Mutations in different genes can be detected, which indicate INH and ethionamide (ETH) drug resistance. We aimed to determine the frequency of these mutations in katG and inhA genes by LPA to guide INH and ETH use for drug-resistant TB. Materials and methods Two consecutive sputum samples were collected from each patient, followed by decontamination by N‑acetyl‑L‑cysteine and sodium hydroxide method. LPA was performed on the decontaminated samples by GenoType MTBDRplus, and the strips were analyzed. Results Out of the 3,398 smear-positive samples tested by LPA, valid results were found in 3,085 (90.79%) samples. Of the 3,085 samples, INH resistance was seen in 295 samples (9.56%), of which mono INH resistance was in 204 samples, and 91 were multidrug resistant. katG S315T was the most common mutation responsible for high-level INH resistance. At the same time, inhA c15t was the most common mutation associated with low-level INH resistance and ETH cross-resistance. The average turnaround time for the processing and reporting of samples was five days. Conclusions The high burden of INH resistance is alarming and can be a major obstacle to TB elimination. Although molecular methods have reduced the reporting time leading to early management of the patients still, a large knowledge gap persists.

Role of line probe assay in detection of Mycobacterium tuberculosis in children with pulmonary tuberculosis

BackgroundIndia is the highest TB burden country in the world with almost 27 lakh cases reported in 2019. Pediatric tuberculosis in India accounts for almost 31% of global TB burden. Despite such high mortality and morbidity in children, diagnosis of pulmonary TB in children still remains very challenging. Material and methodsThis cross-sectional study was conducted in a tertiary care hospital in Delhi, India. Children between 1 and 12 months with clinical suspicion of pulmonary tuberculosis who had not previously taken ATT were included.Early morning gastric aspirate samples were collected after overnight fasting, on two days. Both days sputum sample were subjected to sputum smear microscopy and one of the two samples was subjected to line probe assay (LPA), cartridge based nucleic acid amplification (CBNAAT) and mycobacterium growth in tube (MGIT-960). Results84 children with pulmonary tuberculosis were enrolled. The most common presenting complaint was fever seen in 83 patients (98.8%). Only 17 (20.24%) were sputum smear positive by Ziehl- Neelsen (ZN) staining. LPA was positive in 47 (55.95%) samples and among these positive samples both INH and RIF resistance was detected in 2 (4.26%) samples. CBNAAT was positive in 53 patients (63%). Growth in liquid culture media (MGIT 960) was observed in 44 (52.38%) samples. Among 17 smear positive samples, LPA was detected in 14 (82.35%) samples and among 67 smear negative sample LPA was detected in 33 (49.25%) samples. LPA had 63.46% sensitivity, 100% specificity in detecting mycobacterium tuberculosis. DiscussionWHO's recommendation for using LPAs has been limited to culture isolates or smear-positive sputum specimens. New data has since been generated on the use of LPAs as newer versions of LPA have been developed over past few years.Previous studies conducted using LPA version 1.0 reported much lower detection rate of mycobacterium tuberculosis in smear negative specimens.With the availability of newer versions such as Hain GenoTypeMTBDRplusversion 2 and Nipro NTM + MDRTB detection kit 2, the diagnostic utility of LPA may be enhanced.

Molecular investigations on outbreaks of ovine theileriosis among sheep and goats in Haryana, India.

During February 2020 to October 2020, four outbreaks of theileriosis in small ruminants were recorded with overall morbidity, mortality and case fatality rates of 27.95%, 17.46% and 62.5%, respectively. The disease was characterized by high fever (up to 106°F), superficial lymphadenopathy, anaemia, anorexia, lethargy, respiratory distress and death. The presence of pleomorphic intra-erythrocytic piroplasms of Theileria species in Giemsa's stained blood smears was a common finding in all the episodes. Significant haematological alterations including high total leucocyte count and low haemoglobin and packed cell volume were characteristic. Necropsy findings of the icteric liver, enlarged spleen, pulmonary oedema and abomasal ulcerations were observed in three flocks. Smear-positive blood samples from all the episodes were screened by PCR using 18S rRNA gene-specific primer sets for T. lestoquardi, T. luwenshuni, T. uilenbergi and T. ovis. T. lestoquardi which was detected in all four flocks, while there was co-infection of T. ovis in two flocks. Phylogenetic analysis revealed that T. ovis and T. lestoquardi identified in this study had 100% and ~ 99.86% homology, respectively, with the published sequences used for comparison. This is the first confirmed report of outbreaks of malignant ovine theileriosis in the Haryana state of India which caused high morbidity, mortality and case fatality among sheep and goats. Further studies on theilerioses in small ruminants are required to understand epidemiology better.

Comparative evaluation of geneXpert MTB/RIF assay and Ziehl-Neelsen staining for the diagnosis of tuberculosis

Background: Ziehl-Neelsen (ZN) staining for the diagnosis of tuberculosis is frequently used. Since ZN smear technique has poor sensitivity, molecular tool like GeneXpert assay have recently been introduced in Nepal. This study compared GeneXpert MTB/RIF assay with ZN stain microscopy for the diagnosis of tuberculosis. Objectives: To compare and evaluate GeneXpert MTB/RIF assay and Ziehl-Neelsen staining for the diagnosis of tuberculosis. Methods: An analytical observational study was conducted in Manipal Teaching Hospital, Pokhara from February 2020 to August 2021 after ethical clearance. A total of 355 clinical specimens (Sputum, Broncho-alveolar lavage, pus, lymph node aspirate etc) were collected and processed for Zeihl-Neelson staining and GeneXpert assay as per the recommended guidelines. Results: Zeihl-Neelson staining detected acid-fast bacilli (AFB) in 17 (4.78%) specimens, while GeneXpert assay showed positivity in 37 (10.42%). All the 17 smear positive sputum samples yielded positive result by GeneXpert also. AFB were not detected in any of the 318 samples negative by GeneXpert. Fifteen (83.3%) of the 18 specimens that showed high to medium density of bacilli by GeneXpert were positive by Ziehl-Neelsen staining. Only two (10.5%) of the 19 specimens with low bacillary density by GeneXpert were positive by Ziehl-Neelsen Staining. These differences were statistically significant (p &lt;0.001). Out of 37 positive specimens, one yielded rifampicin resistant Mycobacterium tuberculosis. Conclusion: ZN smear, though, rapid, lacks sensitivity. GeneXpert, on the other hand, can be relied upon, as it detected significantly higher number of cases, demonstrated bacillary density and drug resistance.

Comparison of Ziehl-Neelsen Microscopy Technique and Genexpert in the Diagnosis of Tuberculosis in Sudanese Patients

Aim: The quality of tuberculosis diagnosis service is indispensable for early infection control and preventing unnecessary anti-tubercular therapy use. TB diagnosis in Sudan mainly relies on detecting tubercule bacilli using the Ziehl-Neelsen (ZN) staining technique. Thus, the current study aimed to compare the ZN microscopy with GeneXpert in diagnosing pulmonary tuberculosis (PTB) in TB laboratories center in Khartoum state, Sudan. Methods: A cross-sectional study was carried out at the TB laboratories diagnosis center in Khartoum state from April 2019 to January 2020. A total of 183 sputum samples were processed and examined by the Ziel-Neelsen (ZN) technique and GeneXpert assay for the detection of tubercle bacilli. Results: Out of 183 sputum samples analyzed, 80.9% were AFB smear-positive, of which 65.6% were male patients. Most patients (57.4%) were within the age group of 20 – 39 years old. All smear-positive samples were detected positive for Mycobacterium tuberculosis by GeneXpert. However, one case of smear-positive PTB was given a negative result by GeneXpert. These results demonstrate that GeneXpert is crucial for confirming TB cases to control TB infection and avoid unnecessary anti-tubercular drug use. Conclusion: ZN microscopy can result in false positivity, which may lead to the misuse of anti-tuberculosis drugs. A positive test should also be confirmed by the GeneXpert and culture techniques. Keywords: Mycobacterium tuberculosis, Tuberculosis, GeneXpert, Ziel-Neelsen stain

PERFORMANCE OF XPERT MTB/RIF ULTRA AND GENOTYPE MTBDRPLUS ASSAY IN THE DIAGNOSIS OF DRUG-RESISTANT TB IN TIRANA, ALBANIA

TYPE: Late Breaking Abstract TOPIC: Chest Infections PURPOSE: In a low resource setting, the tuberculosis (TB) culture may pose a problem for case detection and initiation of second-line treatment due to both financial restraints and technical challenges. There is a need for rapid, reliable and easily accessible tools for diagnosis of drug resistant TB. This comparative analysis was performed to assess diagnostic performance characteristics of GeneXpert MTB/RIF Ultra and MTBDRplus Assay. METHODS: A systematic random sampling procedure was used to select sputum samples from a pool of samples received at the National TB Reference Laboratory for TB diagnosis between January to December 2021. Fifty-three smear positive sputum samples were subjected to testing by GeneXpert MTB/RIF Ultra, MTBDRplus Assay, BACTEC MGIT Culture and drug susceptibility testing (DST). Sensitivity, specificity and predictive values were then determined to assess the performance characteristics of the two assays compared to DST as a reference standard. RESULTS: For diagnosis of rifampicin resistance, GeneXpert MTB/RIF Ultra had a sensitivity, specificity, positive predictive value, and negative predictive value of 66.7, 100, 100 and 98.1% respectively while MTBDRplus assay had 100, 100, 100 and 100%. For diagnosis of isoniazid resistance, MTBDRplus assay had a sensitivity, specificity, positive predictive value, and negative predictive value of 87.5, 100, 100, and 97.8% compared to DST. CONCLUSIONS: MTBDRplus assay shows a slightly better performance characteristic to GeneXpert MTB/RIF Ultra, regarding detection of rifampicin resistance. CLINICAL IMPLICATIONS: MTBDRplus assay may represent an alternative to culture with regards to detection of both rifampicin and isoniazid resistance in smear positive samples. DISCLOSURE: No significant relationships. KEYWORD: drug resistant tuberculosis

Trends in rifampicin and isoniazid resistance in patients with presumptive TB.

BACKGROUND: Early diagnosis of drug-resistant TB (DR-TB) is crucial in preventing the spread of the disease in the community. Introduction of upfront decentralised drug susceptibility testing to district-level as part of universal drug susceptibility testing (UDST) policy increased the feasibility of rapid and early testing for drug resistance closer to the patient and has resulted in reduced circumstances for transmission. The introduction of the first-line line-probe assay (FL-LPA), GenoType® MTBDRplus v2, has had an extensive impact on the management of multidrug-resistant TB (MDR-TB) in India.MATERIALS and METHODS: Sputum samples of patients with presumptive TB and DR-TB from selected districts of Tamil Nadu received through National TB Elimination Programme (NTEP) were subjected to FL-LPA as per programme guidelines. In this study, we present trends in genotypic resistance to isoniazid (INH) and rifampicin (RIF) during the 4 years (2016-2019) among these patients. Band patterns were analysed as per the updated GLI (Global Laboratory Initiative) LPA interpretation and reporting guidelines.RESULTS: A total of 26,349 samples were received during the study period. Smear-positive samples (n = 20231) were directly subjected to FL-LPA; smear-negative samples were cultured in liquid media and M. tuberculosis-positive cultures were tested using FL-LPA. A total of 18,441 were MTB-positive on FL-LPA. INH monoresistance, RIF monoresistance and MDR-TB was observed in respectively 8.7%, 1.1% and 3.3% of the samples. There was a decreasing trend in all types of resistance observed particularly after 2017 (P < 0.001). MDR-TB showed a steady decrease from 5.6% to 1.8%. S531L (19.5%) and S315T (61.1%) were the most common mutations identified in the rpoB and katG genes, respectively. The percentage of inhA-c-15t promoter mutation, indicating low-level INH resistance, showed a consistent increase (P < 0.001).CONCLUSION: The impact of the UDST policy on the NTEP may have led to this decreasing trend in RIF and INH resistance observed in the study period. The increase in low-level INH resistance mutation inhA-c-15t may be associated with ethionamide/prothionamide resistance, and this should be taken into account when designing DR-TB regimen.

The occurrence of multidrug-resistant Mycobacterium tuberculosis from patients of pulmonary tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is one of the leading causes of death in the world. The resource constraints make it difficult to diagnose and monitor the cases of MDR-TB. GeneXpert is a recognized tool used to diagnose the patients of pulmonary tuberculosis in clinical settings across the globe. The present one-year cross-sectional study was conducted to estimate the occurrence of MDR-TB in patients with pulmonary TB. A total of 1000 patients suspected of pulmonary tuberculosis were included in this study. A random convenient sampling technique was done to collect the sputum samples (twice) from the patients. Samples were processed for the detection of Mycobacterium tuberculosis using conventional detection methods like the Ziehl Nelson staining method and fluorescent microscopy. Additionally, Cepheid GeneXpert was used for molecular detection of MDR-TB in smear-positive samples of pulmonary tuberculosis by amplifying the rifampicin resistance determining region (RRDR; rpoB gene). All the tests were performed in the biosafety level III lab of District Headquarters Hospital Nankana Sahib. It was observed that 103 (10.3%) individuals were diagnosed as positive for tuberculosis among 1000 patients. Among these 103 TB positive cases, there were 11 (10.7%) patients diagnosed with rifampicin resistance gene (RR-Gene) of Mycobacterium tuberculosis. Overall findings of the study showed that MDR-TB is prevalent in pulmonary TB patients and GeneXpert is the most sensitive technique for early diagnosis of the disease, which may be very helpful in the treatment and control of this public health menace in low and middle-income countries.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Detection of Mycobacterium tuberculosis on stool specimens by PCR among patients with pulmonary tuberculosis.

Background:To detect Mycobacterium tuberculosis on stool specimens by polymerase chain reaction (PCR) among patients with pulmonary tuberculosis. Detection of M. tuberculosis complex in sputum forms the basis of diagnosis of pulmonary tuberculosis. However, some patients tend to swallow sputum and some are unable to produce sputum. Based on the survival of M. tuberculosis in the gastric fluid, swallowed organisms may be detectable in stool samples.Methods:The study was carried out on 30 cases each in four groups: sputum smear-positive and sputum smear-negative adults, pediatric patients suspected of pulmonary tuberculosis along with healthy controls. The samples were processed for direct microscopy for acid-fast bacilli (AFB) and M. tuberculosis culture. Stool PCR was done on all 120 samples.Results:AFB was demonstrated in 42 and cultured in 39 out of 240 samples. PCR-targeting IS6110 gene showed positive results in 24 (20%) out of 120 stool samples. PCR in stool showed the highest positivity in sputum smear-positive samples followed by gastric aspirates and sputum smear-negative samples.Conclusion:Stool PCR is a potentially useful diagnostic method for pulmonary tuberculosis.

Evaluation of the performance of the BD MAX MDR-TB test in the diagnosis of Mycobacterium tuberculosis complex in extrapulmonary and pulmonary samples

ABSTRACT Background The BD MAX MDR-TB is a recently marketed molecular test for detecting Mycobacterium tuberculosis complex (MTC), rifampin, and isoniazid drug resistance. Research Design and Methods This study aimed to evaluate the BD MAX MDR-TB test performance in 933 extrapulmonary and 774 pulmonary samples. Results Test MTC detecting sensitivity was 90.6%, 82.5%, and the specificity was 98.5%, 98.9%, in pulmonary and extrapulmonary samples, respectively. In smear-positive samples, sensitivity, and specificity were 100% for all samples. However, in smear-negative samples, the test’s sensitivity and specificity were 82.3%, 98.5% in pulmonary samples, and 76.7%, 98.9% in extrapulmonary samples. Test sensitivity in detecting isoniazid resistance was 71.4%, specificity 96.8%, and in detecting rifampin resistance was 100%, specificity 93.9%, respectively. Conclusions BD MAX MDR-TB is a reliable, rapid, user-friendly test for detecting MTC in extrapulmonary and pulmonary samples and its resistance toward isoniazid and rifampin. It can be used as an alternative to the Xpert system assays.