Validation of an indigenous assay for rapid molecular detection of rifampicin resistance in presumptive multidrug-resistant pulmonary tuberculosis patients.

BACKGROUND & OBJECTIVES: There is a need for an affordable, easy, high-sensitivity test usable at the peripheral health facility for diagnosis of drug-resistant (DR) tuberculosis (TB) to interrupt disease transmission. Nucleic acid amplification tests (NAATs) for early detection of DR-TB are ideal to bring testing near to the patient. TruenatTM MTB (Mycobacterium tuberculosis) and TruenatTM MTB-RIF (rifampicin) is an indigenous chip-based real-time polymerase chain reaction (PCR) based test for detection of multidrug-resistant (MDR) TB. The test involves extraction of DNA using automated, battery operated Trueprep instrument and real-time PCR performed on the Truelab analyzer. We report here multicentric validation of Truenat MTB-RIF for detection of DR-TB in suspected DR-TB patients. METHODS: Consecutive patients aged 18-65 yr, with symptoms suggestive of TB and with a history of previous treatment, reporting to the National TB Elimination Programme (NTEP) clinics under four national institutes, namely AIIMS (All India Institute of Medical Sciences, New Delhi), NITRD (National Institute of Tuberculosis and Respiratory Diseases, New Delhi), NIRT (National Institute for Research in Tuberculosis, Chennai) and ICMR-National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra, were included in the study. Two sputum samples (one spot and one morning) were collected from each patient, after obtaining informed written consent. The samples were subjected to smear, GeneXpert and MGIT 960 culture (and drug susceptibility testing to RIF) (surrogate for MDR-TB) to serve as reference tests. The samples were coded to ensure blinding and subjected to Truenat MTB-RIF. Truenat MTB-RIF Version 1.5 was used for testing 1084 samples for RIF resistance, while Version 2.0 was used to test another 1201 samples. RESULTS: Truenat MTB-RIF Version 1.5 in comparison with comprehensive laboratory reference standards yielded sensitivity and specificity of 76.2 and 94.7 per cent, respectively for the detection of RIF resistance in 1084 samples, collected across four sites. Based on the analysis of discordant samples, Version 2.0 of Truenat was developed by the manufacturer and this was further tested on additional 1201 samples, yielding a sensitivity of 87.5 per cent and specificity of 99.5 per cent. INTERPRETATION & CONCLUSIONS: Multicentric trial of TruenatTM MTB-RIF demonstrated a great potential of this point of care NAAT for detection of MDR-TB. The test would be useful in limited resource settings and inaccessible areas without need for any additional infrastructure.

Multi-centric evaluation of Truenat MTB and MTB-RIF Dx assays for diagnosis of extrapulmonary tuberculosis.

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

Cost-Effectiveness of Currently Available Diagnostic Tools for Diagnosis of Pediatric Tuberculosis Under National Tuberculosis Elimination Program.

In India, children do not get diagnosed with tuberculosis (TB) for reasons such as lack of screening modality at the health-care settings, inadequate sputum sample, and low detection rate. This study aims to assess various modalities for diagnosis of pediatric TB and their cost-effectiveness. Cost-effectiveness was found for various diagnostic modalities for TB diagnosis in children of India below 15 years of age. TrueNat MTB was the intervention being compared to GeneXpert MTB and sputum microscopy. Evidence pertinent to effectiveness and cost per test, and health benefits in terms of disability adjusted life years were researched and documented. Modeling a cohort of children through a decision tree and assimilating costs and disability-adjusted life years (DALYs) at each step gave results in the form of cost-effectiveness. Interventions were compared by calculating the cost-effectiveness ratio. The results revealed that TrueNat is more cost effective (Rs. 9450/DALY averted) compared to GeneXpert MTB/RIF (Rs. 9750/DALY averted). The incremental cost effectiveness ratio of TrueNat with respect to GeneXpert was found to be Rs. 5925 per DALY averted. Diagnosis through TrueNat point of care (POC) will avert 962 more DALYs compared to GeneXpert. As is evident from the results, TrueNat does alleviate disability caused by TB in children as more DALYs are averted. At an additional cost of Rs. 5925 to avert one DALY, which is below the gross domestic product (GDP) per capita for India (for 2021, it was $2277), TrueNat can have significant health benefits.

National TB elimination programme - What has changed.

BACKGROUND: Before the start of Coronavirus (COVID-19) pandemic, TB was the leading cause of death due to a single infectious agent, ranking well above HIV/AIDS. Almost one-fourth of the world's population is infected with M. tuberculosis. TB is curable and preventable. About 85% of people who develop TB can be successfully treated with drug regimens of 6 months. Universal health coverage (UHC) is necessary to ensure that all those with the disease can access these treatments. Research breakthroughs (e.g., newer rapid diagnostic techniques, drugs, newer vaccine) are needed to rapidly reduce the number of new cases each year (TB incidence) worldwide. OBJECTIVE: Changes in the National TB Elimination Programme since its inception. CONTENT: The Government of India launched the "National TB Programme" in 1962 as District TB Centre model involved with BCG vaccination and TB treatment to fight tuberculosis, a major public health problem. The tuberculosis control programme has come a long way since then and has undergone major changes over the past few years The Ministry of Health and Family Welfare has developed the "National Strategic Plan" for Tuberculosis Elimination (2017-25) which encapsulates the bold and innovative steps required to eliminate TB in India by 2025, five years ahead of the global targets. By 2020 it was clear that the NSP- 2017-25 will not be able to meet these objectives, so another new NSP India 2025 had been launched in 2020. India has been actively involved in TB control activities for more than 50 years now. TB still continues to be a severe health problem in India. The country is now better prepared to tackle TB than before. It now has advanced and effective interventions and technologies for diagnosis, treatment and care of TB cases.

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.

Evaluation of the Indian TrueNAT micro RT-PCR device with GeneXpert for case detection of pulmonary tuberculosis.

To evaluate the performance of TrueNAT (RT Micro PCR device) assay in comparison with GeneXpert on sputum samples from pulmonary cases of tuberculosis. 274 samples were processed to detect MTB by ZN smear examination, MGIT culture and molecular methods that included RT-PCR (ABI 7500 & TrueNAT) and GeneXpert for case detection of TB. The overall performance of the test with MGIT(Mycobacterium Growth Indicator Tube) culture as gold standard, sensitivity of smear, RT PCR/TrueNAT and Genexpert was 61.5% (CI:53.3-69.3%), 94.7% (CI:89.8-97.6%) & 96.0% (CI: 91.5-98.5%), respectively. Amongst the S+ (108) samples, RT-PCR/TrueNAT and GeneXpert showed a sensitivity of 99% (CI:94.9%-99.8%) and 100% (98.6%-100.0%), respectively. High concordance was observed between GeneXpert and TrueNAT for case detection of TB. The GeneXpert MTB/RIF test was independent on the user's skills. It has a short turn-around time and simultaneously detects RIF resistance with M. tuberculosis in less than 3h. The TrueNAT MTB has good sensitivity and specificity in case detection with hands on time of less than 3h as well as fits the requirements in resourcelimited health care settings. Larger, multi-site studies are required to obtain better estimates of the performance of TrueNAT MTB.