Evaluation of molecular diagnostic test for detection of adult pulmonary tuberculosis: A generic protocol.

BACKGROUND OBJECTIVES: Tuberculosis (TB) continues to be the second most-leading cause of death due to a single infectious agent as of 2022 after COVID-19. Many affordable new molecular diagnostic tools are being developed for early and more accurate diagnosis, especially for low-resource settings in low- and middle-income countries. In this context, there is a need to develop a standardized protocol for validation of new diagnostic tools. Here, we describe a generic protocol for multi-centric clinical evaluation of molecular diagnostic tests for adult pulmonary TB. METHODS: This protocol describes a cross-sectional study in TB reference laboratories in India. Adults (>18 yr) visitng the chest clinics or outpatient departments with symptoms of TB need to be enrolled consecutively till the required sample size of 150 culture positives and 470 culture negatives are met. Mycobacterium tuberculosis (Mtb) culture (mycobacteria growth indicator tube liquid culture) to be used under this protocol as the gold standard and Xpert MTB/RIF molecular test will be used as the comparator. The sputum samples will be tested by smear microscopy, Mtb culture, Xpert MTB/RIF and index molecular test as per the proposed algorithm. The specificity sensitivity, and positive/ negative predictive values are to be calculated for the index test with reference to the gold standard. DISCUSSION: TB diagnosis poses many challenges as it differs with type of disease, age group, clinical settings and type of diagnostic tests/kits used. Globally, different protocols are used by several investigators. This protocol provides standard methods for the validation of molecular tests for diagnosis of adult pulmonary TB, which can be adopted by investigators.

Assessing the utility of Truenat in extrapulmonary tuberculosis diagnosis - A NRL's experience.

Background: Diagnosis of extra pulmonary TB (EPTB) remains a big challenge. While data on utility of Xpert testing in EPTB diagnosis is enormous, there is limited data on Truenat MTB testing. Aim: In this study we aimed to evaluate the usefulness of Truenat in EPTB diagnosis. Materials and methods: The study included patients suspected and/or treated for EPTB located from Chennai district during the year 2021-2022. All processed EPTB samples were subjected to smear microscopy, culture and Truenat MTB testing. Results: Of the 195 samples tested, 38 (19.4%) samples were positive for EPTB by any one of the diagnostic methods (smear, culture, microscopy). Out of these 38, 16 (42.1 %) were positive for MTB by Truenat and negative by Culture, 12 (31.5%) were positive by culture but negative by Truenat and 8 (21%) were positive by both Truenat and/or smear and culture. The sensitivity and specificity of the test was calculated with the composite reference standard (Culture (exclusion of colonies as positives), clinical conditions, and smear) and was found to be 60% and 100% respectively. Conclusion: Truenat MTB test is a cost-effective rapid molecular test that can be used only for the diagnosis of presumptive EPTB and not on follow-up samples.

Recurrence of pulmonary tuberculosis in India: Findings from the 2019-2021 nationwide community-based TB prevalence survey.

Recurrent Tuberculosis patients contribute to a significant proportion of TB burden in India. A nationwide survey was conducted during 2019-2021 across India among adults to estimate the prevalence of TB. A total of 322480 individuals were screened and 1402 were having TB. Of this, 381 (27.1%) had recurrent TB. The crude prevalence (95% CI) of recurrent TB was 118 (107-131) per 100,000 population. The median duration between episodes of TB was 24 months. The proportion of drug resistant TB was 11.3% and 3.6% in the recurrent group and new TB patients respectively. Higher prevalence of recurrent TB was observed in elderly, males, malnourished, known diabetics, smokers, and alcohol users. (p<0.001). To prevent TB recurrence, all treated tuberculosis patients must be followed at least for 24 months, with screening for Chest X-ray, liquid culture every 6 months, smoking cessation, alcohol cessation, nutritional interventions and good diabetic management.

Comprehensive assessment of invalid and indeterminate results in Truenat MTB-RIF testing across sites under the national TB elimination program of India.

Introduction: Truenat MTB-RIF assay (Truenat), a nucleic acid amplification test (NAAT), is a real-time polymerase chain reaction (RT-PCR) chip-based assay that can detect Mycobacterium tuberculosis (Mtb) and rifampicin (RIF) drug resistance using portable, battery-operated devices. The National TB Elimination Program (NTEP) in India introduced this novel tool at the district and subdistrict level in 2020. This study aimed to assess the level and causes of inconclusive results (invalid results, errors, and indeterminate results) in MTB and RIF testing at NTEP sites and the root causes of these in the programmatic setting. Methods: Truenat testing data from 1,690 functional Truenat sites under the NTEP from April to June 2021 were analyzed to assess the rates of errors, invalid MTB results, and indeterminate RIF results. Following this analysis, 12 Truenat sites were selected based on site performance in Truenat testing, diversity of climatic conditions, and geographical terrain. These sites were visited to assess the root causes of their high and low rates of inconclusive results using a structured checklist. Results: A total of 327,649 Truenat tests performed for MTB and RIF testing were analyzed. The rate of invalid MTB results was 5.2% [95% confidence interval (CI): 5.11-5.26; n = 16,998] and the rate of errors was 2.5% (95% CI: 2.46-2.57; n = 8,240) in Truenat MTB chip testing. For Mtb-positive samples tested using the Truenat RIF chip for detection of RIF resistance (n = 40,926), the rate of indeterminate results was 15.3% (95% CI: 14.97-15.67; n = 6,267) and the rate of errors was 1.6% (95% CI: 1.53-1.78; n = 675). There was a 40.1% retesting gap for Mtb testing and a 78.2% gap for inconclusive RR results. Among the inconclusive results retested, 27.9% (95% CI: 27.23-28.66; n = 4,222) were Mtb-positive, and 9.2% (95% CI: 7.84-10.76; n = 139) were detected as RR. Conclusion: The main causes affecting Truenat testing performance include suboptimal adherence to standard operating procedures (SOPs), inadequate training, improper storage of testing kits, inadequate sputum quality, lack of quality control, and delays in the rectification of machine issues. Root cause analysis identified that strengthening of training, external quality control, and supervision could improve the rate of inconclusive results. Ensuring hands-on training of technicians for Truenat testing and retesting of samples with inconclusive results are major recommendations while planning for Truenat scale-up. The recommendations from the study were consolidated into technical guidance documents and videos and disseminated to laboratory staff working at the tiered network of TB laboratories under the NTEP in order to improve Truenat MTB-RIF testing performance.

Molecular Characterisation of M. kansasii Isolates by Whole-Genome Sequencing.

M. kansasii is the most common non-tuberculous mycobacteria, known to be causing pulmonary and extrapulmonary diseases in humans. Based on molecular methods, M. kansasii has been previously classified into seven different subtypes. Now, based on whole-genome sequence analysis, a new species designation was proposed, in which M. kansasii species was designated subtype 1 and is of pathogenic significance in both immunocompetent and immunocompromised patients. The aim of the study is to examine the distribution of subtypes, based on whole-genome sequence analysis, and identify the genetic determinants of drug resistance for the isolates. Whole-genome sequencing was performed using 12 isolates for which phenotypic DST results were available. A phylogenetic tree was constructed by alignment of each of the 12 isolates and the additional strains, as well as the M. kansasii reference strain, using the MAFFT algorithm. Based on this analysis, all 12 isolates were classified as subtype I. Drug-resistant mutations were identified by analysing the isolates with known drug-resistant loci of MTB and NTM. Although we had mutations in the drug-resistant genes, the significance of those mutations could not be explored due to the minimal availability of data available to compare. Further large-scale studies targeting the phenotypic and genotypic drug-resistance pattern, along with whole-genome analysis, will facilitate a better understanding of the resistance mechanisms involved in M. kansasii.

Genotype MTBDRsl version 2 and phenotypic drug resistance detection of Mycobacterium tuberculosis for fluoroquinolones and aminoglycosides.

Background: Information on genotypic with comparison of phenotypic drug sensitivity test of anti-tuberculosis (TB) has been reported in several studies, which have variable results. The present study aimed to assess the Genotype MTBDRsl version 2.0/Line probe assay (LPA) for the detection of fluoroquinolones (FQ) and aminoglycosides (AMGs) resistance mutations among drug-resistant Mycobacterium TB (MTB) strains and also to compare the patterns of genotypic mutations of gyrA/B, rrs, and eis with mycobacteria growth indicator tube (MGIT 960). Methods: A total of 1416 samples were subjected to Genotype MTBDRsl version 2.0 assay. One hundred and twenty sputum smear positive MTB isolates and 37 sputum smear negative MTB isolates confirmed multiple drug resistance resistant to FQ and AMG by the Genotype MTBDRsl version 2.0 were subjected to phenotypic drug susceptibility testing (DST) were analyzed. Results: The association sensitivity, specificity, positive predictive value, negative predictive value, and accuracy for the resistance detection between MGIT (DST) and the Genotype MTBDRsl version 2.0 assay was significant (P < 0.01) of moxifloxacin (MFX) concentration. Sensitivity and specificity value for kanamycin (KAN) resistance was 76% and 89%; 47% and 94% for capreomycin (CAP); and 60% and 76% for low-level KAN, respectively. Conclusion: Our results indicate that MFX (0.25and 1 µg/mL), KAN (2.5 µg/mL), and CAP (2.5 µg/mL) significantly (P < 0.01) and support the World Health Organization guidance to test FQ and AMG by genotypic test.

Resistance Profiles to Second-Line Anti-Tuberculosis Drugs and Their Treatment Outcomes: A Three-Year Retrospective Analysis from South India.

Background: Patients with first-line drug resistance (DR) to rifampicin (RIF) or isoniazid (INH) as a first-line (FL) line probe assay (LPA) were subjected to genotypic DST using second-line (SL) LPA to identify SL-DR (including pre-XDR) under the National TB Elimination Program (NTEP), India. SL-DR patients were initiated on different DR-TB treatment regimens and monitored for their outcomes. The objective of this retrospective analysis was to understand the mutation profile and treatment outcomes of SL-DR patients. Materials and Methods: A retrospective analysis of mutation profile, treatment regimen, and treatment outcome was performed for SL-DR patients who were tested at ICMR-NIRT, Supra-National Reference Laboratory, Chennai between the years 2018 and 2020. All information, including patient demographics and treatment outcomes, was extracted from the NTEP Ni-kshay database. Results: Between 2018 and 2020, 217 patients out of 2557 samples tested were identified with SL-DR by SL-LPA. Among them, 158/217 were FQ-resistant, 34/217 were SLID-resistant, and 25/217 were resistant to both. D94G (Mut3C) of gyrA and a1401g of rrs were the most predominant mutations in the FQ and SLID resistance types, respectively. Favorable (cured and treatment complete) and unfavorable outcomes (died, lost to follow up, treatment failed, and treatment regimen changed) were recorded in a total of 82/217 and 68/217 patients in the NTEP Ni-kshay database. Conclusions: As per the testing algorithm, SL- LPA is used for genotypic DST following identification of first-line resistance, for early detection of SL-DR in India. The fluoroquinolone resistance pattern seen in this study population corelates with the global trend. Early detection of fluoroquinolone resistance and monitoring of treatment outcome can help achieve better patient management.

Multi-drug loaded eugenol-based nanoemulsions for enhanced anti-mycobacterial activity.

Tuberculosis is one of the oldest bacterial infections known to mankind caused by Mycobacterium tuberculosis. The aim of this research is to optimize and formulate a multi-drug loaded eugenol based nanoemulsion system and to evaluate its ability as an antimycobacterial agent and its potential to be a low cost and effective drug delivery system. All the three eugenol based drug loaded nano-emulsion systems were optimized using response surface methodology (RSM)-central composite design (CCD) and were found stable at a ratio of 1 : 5 (oil : surfactant) when ultrasonicated for 8 minutes. The minimum inhibitory concentration (MIC) values against strains of Mycobacterium tuberculosis highly proved that these essential oil-based nano-emulsions showed more promising results and an even improved anti-mycobacterium activity on the addition of a combination of drugs. The absorbance of 1st line anti-tubercular drugs from release kinetics studies showed a controlled and sustained release in body fluids. Thus, we can conclude that this is a much more efficient and desirable method in treating infections caused by Mycobacterium tuberculosis and even its MDR/XDR strains. All these nano-emulsion systems were stable for more than 3 months.

Standardization of a stool concentration method for Mycobacterium tuberculosis detection in the pediatric population.

Background: The inability of young children to expectorate sputum and paucibacillary status of Mycobacterium tuberculosis (MTB) increases its diagnostic complexity. In this study, we aimed to standardize a stool concentration method for the detection of MTB and its drug resistance by line probe assay (LPA). Methods: The stool from 10 healthy children spiked with H37Rv in five different dilutions (1:1, 1:10, 1:100, 1:1000, and 1:10,000), and stool from 10 confirmed TB and 54 clinically diagnosed TB children were subjected to an in-house stool concentration protocol. All the processed filtrates were subjected to smear microscopy, solid culture, Xpert ultra testing, and LPA. Results: Of 10 control samples, growth was seen in four samples (neat 1:1). In smear microscopy, bacilli could be seen in eight samples (1:1 and 1:10). Xpert ultra testing could detect MTB in eight samples in all dilutions with different loads. LPA could detect MTB in all samples and dilutions. In microbiologically confirmed children, seven out of 10 stool samples tested were positive. Out of 54 children with clinically diagnosed TB, 4 (7.4%) could be confirmed by microbiological diagnosis. Conclusion: The protocol standardized in this study proves to be better working in the molecular detection of MTB.

Amine-Functionalized Silane-Assisted Preparation of AgNP-Deposited α-Ni(OH)2 Composite Materials and Their Application in Hg2+ Ion Sensing.

A facile synthetic methodology for the deposition of different concentrations of Ag nanoparticles (AgNPs) on α-Ni(OH)2 sheets (α-Ni1(OH)2-Ag0.5, α-Ni1(OH)2-Ag1, α-Ni1(OH)2-Ag2, and α-Ni1(OH)2-Ag3) is reported using N-[3-(trimethoxysilyl)propyl]diethylenetriamine (TPDT) silane. The TPDT aminosilane facilitates the formation of α-Ni(OH)2 sheets and reduces the Ag+ precursor to AgNPs, leading to the deposition of AgNPs on α-Ni(OH)2 sheets. UV-vis absorption spectroscopy, transmission microscopy analyses, X-ray photoelectron spectroscopy, X-ray diffraction, and attenuated total reflectance-Fourier transform infrared spectroscopy techniques were used to characterize the prepared α-Ni1(OH)2-Ag0.5-3 composite materials. High-angle annular dark-field scanning transmission electron microscopy-energy-dispersive X-ray spectroscopy mapping images and scanning electron microscopy-energy-dispersive X-ray spectroscopy mapping images were recorded to understand the α-Ni1(OH)2-Ag composite sheet materials. The optical sensing property of α-Ni1(OH)2-Ag0.5-3 composite materials toward toxic Hg2+ ions were investigated using a UV-vis absorption spectroscopy technique. The α-Ni1(OH)2-Ag2 composite material showed selective sensing behavior.

Comparison of two mycobacterial strains in performance of the whole blood mycobacterial growth inhibition assay in Indian children.

A major challenge in tuberculosis is identifying correlates of a protective immune response. The Mycobacterial Growth Inhibition Assay (MGIA) is a functional assay providing an integrated measure of the host immune response to mycobacteria. However, its feasibility is limited by reliance on biosafety level 3 facilities, and its performance has not been widely evaluated in TB-endemic settings. Here, we compared two mycobacterial strains (M. tuberculosis H37Rv versus attenuated M. bovis BCG) in the performance of whole-blood MGIA in 30 TB-exposed children (median age 2 years) in Chennai, India. The time-to-positivity in both assays was similar (5.7 days vs 6 days) and the mycobacterial growth of M. tuberculosis H37Rv and M. bovis BCG were correlated (r = 0.64, p<0.0001). In Bland-Altman analysis, the bias was -0.54 days (95% limit of agreement -2.08, 0.99). Collectively, our results indicate that M. tuberculosis H37Rv can be substituted with the less virulent M. bovis BCG strain to improve feasibility of the MGIA assay, particularly in low-income settings.

Tuberculosis caused by Mycobacterium orygis in wild ungulates in Chennai, South India.

We report the isolation of Mycobacterium orygis, a member of Mycobacterium tuberculosis complex (MTBC), from two black bucks (Antelope cervicapra) and one spotted deer (Axis axis) from the Guindy National Park forest range in Chennai, India. Lung tissue and lymph node samples collected during post-mortem examination were processed using NaOH method and cultured in solid and liquid media. DNA extracted from the cultured isolates was used to amplify the mpt64 gene by specific primers and the band visualized at 240 bps confirmed the isolates as a member of MTBC. Further examination of these isolates by spoligotyping and whole-genome sequencing confirmed the isolates as M. orygis and the phylogenetic tree revealed their well-clustered position with other M. orygis isolates around the globe. The deletion of RD7-RD10, RDOryx_1, RDOryx_4, RD12Oryx, RD301 and RD315 further substantiated these isolates as M. orygis. The exact source of infection in animals was untraceable and the pairwise comparison of the genomes based on single-nucleotide polymorphisms difference did not detect any events of transmission within the affected animals. Nevertheless, it would be wise to take into account the environment where there exists a high chance of transmission due to the increased human-animal interaction. Since it is well known that the pathogen is capable of causing infection in both human and animal hosts, systematic surveillance and screening of spotted deer, black buck as well as humans in the vicinity is essential for successful implementation of the One Health approach.

Comparison of performance indicators of MGIT primary culture to assess the impact of implementation of ISO 15189:2012 standards.

Performance indicators are key component and plays a major role for monitoring and continuous quality improvement of the test results. The NABL certificate of accreditation is issued in accordance with the standard ISO 15189:2012 requirements. As part of the accreditation process, the laboratory has acquired knowledge and implemented the quality system procedures. Present study analyzed the impact of the accreditation process on the "performance indicators" of MGIT primary culture and found that performance indicators have been improved significantly after implementation of NABL for almost all indicators which clearly indicate the importance of accreditation and implementation of quality procedures for reliability of valid test results.

Characterization of probes associated with rifampicin resistance in M.tuberculosis detected by GenXpert from a national reference laboratory at Chennai.

With increasing use of Xpert MTB/RIF a point of care molecular test for simultaneous detection of TB and resistance to rifampicin, a growing number of rifampicin resistant cases are being detected and notified. Insights into the variation and frequencies in the probe mutations obtained through Xpert testing in the RRTB case will form the baseline information for further investigation on drug resistance. In this study we did a retrospective analysis of the GeneXpert data obtained from patient samples received at a National reference laboratory in Chennai between the years 2014 and 2020 to look at the probe distribution, the variation in the mutation and explore its significance. Probe E mutation was most commonly identified followed by Probe D, Probe A, Probe B and Probe C. Coexistence of multiple probe mutations in low bacillary load samples could be related to prolonged amplification cycle leading to delayed hybridization of probes. In such instances reporting false RR in xpert testing is possible. The probe mutations of RR should be monitored in depth with inclusion of codon specific targets for management of drug sensitive TB. In addition, heteroresistance needs to be further tested by alternative genotypic methods to avoid false resistance.

Whole-Genome Sequencing to Identify Missed Rifampicin and Isoniazid Resistance Among Tuberculosis Isolates-Chennai, India, 2013-2016.

India has a high burden of drug-resistant tuberculosis (DR TB) and many cases go undetected by current drug susceptibility tests (DSTs). This study was conducted to identify rifampicin (RIF) and isoniazid (INH) resistance associated genetic mutations undetected by current clinical diagnostics amongst persons with DR TB in Chennai, India. Retrospectively stored 166 DR TB isolates during 2013-2016 were retrieved and cultured in Löwenstein-Jensen medium. Whole genome sequencing (WGS) and MGIT DST for RIF and INH were performed. Discordant genotypic and phenotypic sensitivity results were repeated for confirmation and the discrepant results considered final. Further, drug resistance-conferring mutations identified through WGS were analyzed for their presence as targets in current WHO-recommended molecular diagnostics. WGS detected additional mutations for rifampicin and isoniazid resistance than WHO-endorsed line probe assays. For RIF, WGS was able to identify an additional 10% (15/146) of rpoB mutant isolates associated with borderline rifampicin resistance compared to MGIT DST. WGS could detect additional DR TB cases than commercially available and WHO-endorsed molecular DST tests. WGS results reiterate the importance of the recent WHO revised critical concentrations of current MGIT DST to detect low-level resistance to rifampicin. WGS may help inform effective treatment selection for persons at risk of, or diagnosed with, DR TB.

Determining pulmonary function and the associated risk factors among stone quarry workers in a suburban area of Chennai, Tamil Nadu, India.

BACKGROUND: Stone quarrying activities generate dust and fine particulate matters of silica and heavy metals. The prolonged exposure to suspended particulates leads to fatal respiratory complications. Occupational pulmonary complications are poorly characterized among quarry workers in Tamil Nadu. OBJECTIVES: This study aims to assess pulmonary function and determine the associated risk factors among quarry workers. MATERIALS AND METHODS: A comparative, cross-sectional study was conducted from April 2014 to January 2015 among workers at seven stone quarries in Chennai and residents within a 5 Km radius. Pulmonary function tests (PFT) and sputum analysis were done. RESULTS: Overall, 670 participants were enrolled in the study, with a median age of 37 years. Comparatively, the mean PFT measures were significantly lower in quarry workers with a higher proportion of airflow obstruction and tuberculosis infection. CONCLUSION: The risk of airflow obstruction among quarry workers increased with smoking and longer duration of work years in quarry.

Autophagy Induction as a Host-Directed Therapeutic Strategy against Mycobacterium tuberculosis Infection.

Tuberculosis (TB), a bacterialinfectious disease caused by Mycobacterium tuberculosis (M.tb), which causes significant mortality in humans worldwide. Current treatment regimen involve the administration of multiple antibiotics over the course of several months that contributes to patient non-compliance leading to relapse and the development of drug-resistant M.tb (MDR and XDR) strains. Together, these facts highlight the need for the development of shorter TB treatment regimens. Host-directed therapy (HDT) is a new and emerging concept that aims to augment host immune response using drugs/compounds with or without adjunct antibiotics against M.tb infection. Autophagy is a natural catabolic mechanism of the cell that involves delivering the cytosolic constituents to the lysosomes for degradation and recycling the components; thereby maintaining the cellular and energy homoeostasis of a cell. However, over the past decade, an improved understanding of the role of autophagy in immunity has led to autophagy activation by using drugs or agents. This autophagy manipulation may represent a promising host-directed therapeutic strategy for human TB. However, current clinical knowledge on implementing autophagy activation by drugs or agents, as a stand-alone HDT or as an adjunct with antibiotics to treat human TB is insufficient. In recent years, many reports on high-throughput drug screening and measurement of autophagic flux by fluorescence, high-content microscopy, flow cytometry, microplate reader and immunoblotting have been published for the discovery of drugs that modulate autophagy. In this review, we discuss the commonly used chemical screening approaches in mammalian cells for the discovery of autophagy activating drugs against M.tbinfection. We also summarize the various autophagy-activating agents, both pre-clinical candidates and compounds approved for advanced clinical investigation during mycobacterial infection. Finally, we discuss the opportunities and challenges in using autophagy activation as HDT strategy to improve TB outcome and shorten treatment regimen.

Whole genome sequencing based differentiation between re-infection and relapse in Indian patients with tuberculosis recurrence, with and without HIV co-infection.

INTRODUCTION: Differentiation between relapse and reinfection in cases with tuberculosis (TB) recurrence has important implications for public health, especially in patients with human immunodeficiency virus (HIV) co-infection. We compared Mycobacterial Interspersed Repeat Unit (MIRU) typing and spoligotyping with whole genome sequencing (WGS) to differentiate between relapse and reinfection in patients (HIV-positive and HIV-negative) with TB recurrence. We also assessed the value of WGS to track acquired drug resistance in those with relapse after successful treatment. METHOD: Forty-one paired M. tuberculosis isolates collected from 20 HIV-positive and 21 HIV-negative patients were subjected to WGS in addition to spoligotyping and MIRU typing. Phylogenetic and Single Nucleotide Substitution (SNP) clustering analyses were performed to determine whether recurrences were due to relapse or re-infection. RESULTS: Comparison of M. tuberculosis genomes indicated that 95% of TB recurrences in the HIV-negative cohort were due to relapse, while the majority of TB recurrences (75%) in the HIV-positive cohort was due to reinfection (P = 0.0001). New drug resistance mutations were acquired in 5/24 cases (20.8%) that experienced relapse. CONCLUSIONS: WGS provided increased resolution, but differentiation between relapse and reinfection was broadly consistent with MIRU and spoligotyping. The high contribution of reinfection among HIV infected patients experiencing TB recurrence warrants further study to explore risk factors for TB exposure.