Molecular diagnostic tests for isoniazid-resistant tuberculosis: a scoping review.

Smear-Negative Pulmonary Tuberculosis in Industrialized Countries

Whole genome sequencing (WGS) is an increasingly useful tool for tuberculosis (TB) diagnosis and disease management. In this study, we evaluated the utility of user-friendly WGS tools in reporting resistance profiles and identifying lineages of clinical TB isolates from South Korea. Forty clinical samples from TB patients showing discrepancies between their rapid molecular and conventional drug susceptibility tests were used in this study. Among these clinical isolates, 37 strains were successfully evaluated via WGS software, using the GenTB, TB Profiler, PhyResSE, CASTB, and Mykrobe. More accurate and faster susceptibility results could be obtained with isoniazid than with rifampin. Using the phenotypic test as the gold standard, the isoniazid concordance rate between phenotypic drug susceptibility test (DST) and WGS (GenTB: 45.9%, TB profiler: 40.5%, PhyResSE: 40.5%, CASTB: 48.6%, and Mykrobe: 43.2%) was much higher than between phenotypic DST and rapid molecular genotypic DST (18.9%) among the 37 strains. In contrast, the rifampin concordance rate between phenotypic DST and WGS and that between phenotypic DST and rapid molecular genotypic DST was similar (81.1-89.2%). We also found novel mutations associated with INH in katG and ahpC gene region, not covered by the line probe assay. In addition, lineage analysis identified 81.1% of these samples as L2 East Asian lineage strains, and 18.9% as L4 Euro-American lineage strains. WGS may play a pivotal role in TB diagnosis and the detection of drug resistance, genetic diversity, and transmission dynamics in the near future because of its accuracy, speed, and extensibility.

For the diagnosis of pulmonary tuberculosis phenotypic and genotypic tests were analysed. On 269 pulmonary specimens comprising of Sputum (139), Bronchoalveolar lavage (B. A. L.) (90), Pleural fluid (21) and Endotracheal secretions (E. T.) (19) ZeihlNeelson (ZN) stain, Automated TB culture, Line Probe Assay (LPA), Nucleic Acid Amplification Test (NAAT) (TrueNat) and Real Time PCR (RTPCR) (Hi media) were performed. A total of 123 (45.72%) specimens were positive either by single or multiple tests together. Of these highest positivity was seen in 72 (58.54%) sputum specimens followed by 35 (28.46%) B. A. L., 9 (7.32%) pleural fluids and 7 (5.69%) E. T. secretions. Highest positivity amongst the tests was seen with 121 (98.37%) NAAT test, 119 (96.74%) Culture and 116 (94.31%) RTPCR while it was low with 83 (67.48%) ZN stain and 81 (65.85%) LPA test. All five tests were positive in 77 (62.60%) and Culture, NAAT and RTPCR were simultaneously positive 36 (29.27%). Molecular tests of NAAT and RTPCR are most favored tests for diagnosis of TB which can be substantiated by Culture if required.

Background: The treatment of non-tuberculous mycobacterial (NTM) infections is challenging because of the difficulty in obtaining phenotypic (pDST) and/or molecular (mDST) drug susceptibility testing and the need of a multi-drug regimen. Objectives: The objective was to describe the in vitro susceptibility patterns of various NTM species through an analysis of susceptibility results obtained on isolates collected between 2018 and 2023. Methods: Species identification and mutations in rrs or rrl genes (mDST) were identified by a line probe assay, while the pDST was performed by broth microdilution and interpreted according to CLSI criteria. Results: We analysed 337 isolates of NTM belonging to 15 species/subspecies. The Mycobacterium avium complex (MAC) was the most common (62%); other species identified included M. gordonae (11%), M. kansasii (5%), the M. abscessus complex (8%), M. chelonae (6%), and M. fortuitum (2%). The results of pDST (claritromycin and amikacin) and mDST (rrl and rrs genes) on 66 NTM strains showed that while wild-type rrl and rrs occurred in 86.3% and 94% strains, respectively, the pDST showed 88% sensitivity for clarithromycin and 57.5% for amikacin. The main incongruity was observed for macrolides. Conclusions: Most NTM are likely to be susceptible to macrolides and aminoglycosides. The molecular identification of resistant genotypes is accurate and strongly recommended for optimal patient management.

Effect of Nucleic Acid Amplification for Mycobacterium tuberculosis on Clinical Decision Making in Suspected Extrapulmonary Tuberculosis

Molecular Diagnosis of Tuberculosis and Drug Resistance

Profiling Genomic Alterations Of Diffuse Large B-Cell Lymphoma (DLBCL) At Diagnosis, Relapse, and Transformation, Using a Novel Clinical Diagnostic Targeted Sequencing Platform

Determination of the predictive factors for diagnostic positivity of nucleic acid amplification tests for diagnosing pulmonary tuberculosis

Accuracy of line probe assays for the diagnosis of pulmonary and multidrug-resistant tuberculosis: a systematic review and meta-analysis.

BackgroundWe assessed the coverage of molecular drug susceptibility testing (mDST) among patients with pulmonary multidrug/rifampicin-resistant tuberculosis (MDR/RR-TB) in South Korea and identified factors influencing the lack of mDST implementation.MethodsThis retrospective study included patients with pulmonary MDR/RR-TB who initiated tuberculosis (TB) treatment between January 2015 and September 2021. Data were obtained from the K-TB-N cohort, an integrated national TB database linking three datasets. We assessed mDST coverage, temporal trends and factors associated with the lack of mDST implementation. mDST was defined as the use of the Xpert MTB/RIF assay or line probe assay (LPA) for isoniazid and rifampicin (first-line LPA).ResultsIn total, 4637 patients were included in the analysis. Of the 4637 patients, 1342 (28.9%) did not undergo mDST; whereas, 3295 (71.1%) underwent mDST. Over the study period, a statistically significant annual increase in mDST coverage was observed, escalating from 49.1% in 2015 to 96.9% in 2021 (p<0.001). Throughout the study, the coverage of the Xpert MTB/RIF assay remained lower than that of LPA (22.1% vs 64.2%, p<0.001). Multivariable logistic regression analysis identified several factors independently associated with a decreased likelihood of mDST being conducted, including TB treatment initiation in secondary general hospitals, small hospitals or primary clinics, as well as in non-public-private mix (PPM) participating institutions. In addition, transfers between PPM-participating and non-participating institutions during the treatment period and sputum acid-fast bacilli smear-negative status were significantly associated with lower mDST uptake.ConclusionAlthough the increasing mDST coverage is a positive development, further efforts are needed to achieve nationwide and universal implementation, particularly for the Xpert MTB/RIF assay, in South Korea.

ABSTRACTNew non-sputum biomarker tests for active tuberculosis (TB) diagnostics are of the highest priority for global TB control. We performed in-depth proteomic analysis using the 4,000-plex SOMAscan assay on 1,470 serum samples from seven countries where TB is endemic. All samples were from patients with symptoms and signs suggestive of active pulmonary TB that were systematically confirmed or ruled out for TB by culture and clinical follow-up. HIV coinfection was present in 34% of samples, and 25% were sputum smear negative. Serum protein biomarkers were identified by stability selection using L1-regularized logistic regression and by Kolmogorov-Smirnov (KS) statistics. A naive Bayes classifier using six host response markers (HR6 model), including SYWC, kallistatin, complement C9, gelsolin, testican-2, and aldolase C, performed well in a training set (area under the sensitivity-specificity curve [AUC] of 0.94) and in a blinded verification set (AUC of 0.92) to distinguish TB and non-TB samples. Differential expression was also highly significant (P < 10−20) for previously described TB markers, such as IP-10, LBP, FCG3B, and TSP4, and for many novel proteins not previously associated with TB. Proteins with the largest median fold changes were SAA (serum amyloid protein A), NPS-PLA2 (secreted phospholipase A2), and CA6 (carbonic anhydrase 6). Target product profiles (TPPs) for a non-sputum biomarker test to diagnose active TB for treatment initiation (TPP#1) and for a community-based triage or referral test (TPP#2) have been published by the WHO. With 90% sensitivity and 80% specificity, the HR6 model fell short of TPP#1 but reached TPP#2 performance criteria. In conclusion, we identified and validated a six-marker signature for active TB that warrants diagnostic development on a patient-near platform.

Background: Many studies have compared the efficacy of Gene Xpert (cartridge-based nucleic acid amplification test [CBNAAT]) and line probe assay (LPA) techniques; however, a systematic and meticulous comparison of these two techniques is lacking. Objective: To study the accuracy of GeneXpert Mycobacterium tuberculosis (MTB)/rifampicin (RIF) assay and LPA in the detection of MTB complex in smear-positive retreatment cases of pulmonary tuberculosis (TB). Materials and Methods: This diagnostic evaluation study was conducted on 300 retreatment cases of pulmonary TB. The patients who were receiving retreatment were identified by sputum for acid fast Bacilli by Ziehl–Neelsen staining method and were instructed to provide sputum samples for CBNAAT and LPA. These sputum samples were then processed for liquid culture as part of the routine diagnostic procedure. Results: Majority of the patients (52.3%) were of 31–45 years of age. Males (56%) were more than females (44%). Most of the cases were relapse (88%). Ninety-three percent were reactive on sputum culture. Seventy percent had RIF resistance on CBNAAT. Isoniazid and RIF resistance was seen in 69.7% on LPA. 23.3% were sensitive on LPA and 7% had isoniazid resistance. The sensitivity, specificity, positive predictive value, and negative predictive for CBNAAT as compared with LPA were 75%, 100%, 100%, and 22.2%, respectively. Conclusion: Both LPA and CBNAAT are equally effective in detecting MTB in the given sputum sample. However, LPA is preferred over can be very helpful as it can point out the Isoniazid (INH) and Rifampicin (RIF) resistant nature of the bacterium.

BackgroundThe dual challenge of low diagnostic sensitivity of microscopy test and technical challenge of performing a TB culture test poses a problem for case detection and initiation of Tuberculosis (TB) second-line treatment. There is thus need for a rapid, reliable and easily accessible assay. This comparative analysis was performed to assess diagnostic performance characteristics of GeneXpert MTB/RIF and Line Probe Assay (LPA).MethodsThree hundred twenty nine sputum samples of patients across the 47 counties in Kenya suspected to have drug resistant TB were picked and subjected to GeneXpert, LPA and Culture MGIT at the National TB Reference Laboratory. Sensitivity, specificity and predictive values were then determined to assess the performance characteristics of the various assays.ResultsAgainst culture MGIT as the gold standard for TB diagnosis, GeneXpert had a sensitivity, specificity, positive predictive value, and negative predictive value of 78.5, 64.9, 59.4 and 82.2% respectively while LPA had 98.4, 66.0, 65.4 and 98.4%. For diagnosis of rifampicin mono-resistance GeneXpert had a moderate agreement (Kappa 0.59, P < 0.01) (sensitivity 62.50%, specificity 96.50%) while LPA that had almost perfect agreement (Kappa = 0.89, p < 0.01) with a (sensitivity 90.0% and specificity 99.1%).ConclusionLPA has a better performance characteristic to GeneXpert and an alternative to culture with regards to detection of RIF’s mono-resistance.

With the rapid development of new advanced molecular detection methods, identification of new genetic mutations conferring pathogen resistance to an ever-growing variety of antimicrobial substances will generate massive genomic datasets for public health and clinical laboratories. Keeping up with specialized standard coding for these immense datasets will be extremely challenging. This challenge prompted our effort to create a common molecular resistance Logical Observation Identifiers Names and Codes (LOINC) panel that can be used to report any identified antimicrobial resistance pattern. To develop and utilize a common molecular resistance LOINC panel for molecular drug susceptibility testing (DST) data exchange in the U.S. National Tuberculosis Surveillance System using California Department of Public Health (CDPH) and New York State Department of Health as pilot sites. We developed an interface and mapped incoming molecular DST data to the common molecular resistance LOINC panel using Health Level Seven (HL7) v2.5.1 Electronic Laboratory Reporting (ELR) message specifications through the Orion Health™ Rhapsody Integration Engine v6.3.1. Both pilot sites were able to process and upload/import the standardized HL7 v2.5.1 ELR messages into their respective systems; albeit CDPH identified areas for system improvements and has focused efforts to streamline the message importation process. Specifically, CDPH is enhancing their system to better capture parent-child elements and ensure that the data collected can be accessed seamlessly by the U.S. Centers for Disease Control and Prevention. The common molecular resistance LOINC panel is designed to be generalizable across other resistance genes and ideally also applicable to other disease domains. The study demonstrates that it is possible to exchange molecular DST data across the continuum of disparate healthcare information systems in integrated public health environments using the common molecular resistance LOINC panel.

Aims: To evaluate genotypic Line Probe Assay (LPA) for rapid detection of Multidrug Resistant Tuberculosis (MDRTB) directly from sputum samples in comparison with Drug Susceptibility Testing (DST) on phenotypic MBBacT liquid media. Study Design: Data analysis from 86 Mycobacterium tuberculosis (MTB) strains was done using SPSS version 17. Place and Duration of Study: Department of Microbiology, JSS Medical College, Mysore, Karnataka, between January 2011 to January 2012. Methodology: MDRTB rate detected by LPA assay from 92 samples by noting the mutations in hot spot region of rpoB gene, katG and inhA regulatory region and compared with DST on MBBacT liquid media. Research Article Annual Research & Review in Biology, 4(1): 246-257, 2014 247 Results: Out of 86 MTB isolates, resistant rates for Rifampicin (RIF) and Isoniazid (INH) were 41.8%, 39.53% by LPA and 45.34%, 55.81% by MBBacT. LPA assay showed sensitivity and specificity as 92.35%, 100% for RIF resistance detection and 70.83%, 100% for INH resistance detection, 94.74%, 100% for MDRTB detection compared to conventional DST results. Conclusion: This study showed that LPA has high detection rate for RIF resistance. However to improve the detection of INH resistance in MTB strains additional probes are to be included in LPA. LPA has good sensitivity and specificity for MDRTB detection with turnaround time of less than 48 hours.