Line Probe Assay Research Articles

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.

Effect of tellurite on growth of extensively drug resistant (XDR) Mycobacterium tuberculosis and action on mycobacterial drug efflux pump

In the present study genotypic resistance of bacterial strain to first- and second-line anti-tuberculosis drugs was determined by Line probe assay. Toxic effect of tellurite on the growth of Mycobacterium tuberculosis (MTB) was determined by growing cells in different concentrations of tellurite (1 to 5 mM). Morphological effects of tellurite and uptake of metal in bacterial cells were confirmed by Scanning electron microscope (SEM) and energy dispersive X-ray (EDX) analysis. Change in fold expression of the efflux pump gene was measured by RT-PCR. Mycobacterial strain was characterized as XDR-TB based on the genotypic resistance to rifampicin and isoniazid, along with resistance to fluoroquinolones and second line injectable. XDR-TB showed black colonies in tellurite presence and growth was delayed (2–3 weeks) when compared with the control. The reduced cell size, metal accumulation and the characteristic tellurite peaks appeared in metal-treated cells. MTB showed MIC value of 1 mM and had high susceptibility for higher concentrations (2–5 mM). However, no significant metal inhibitory effect on the mmpL7 efflux system was determined. Tellurite shows significant growth reduction potential against XDR-TB strain. However, the exact mechanism of action needs to be elucidated with further research.

Detection of katG and inhA Genes Mutation in Rifampicin-Resistant Mycobacterium Tuberculosis Using Line Probe Assay (LPA) In Kebbi, Nigeria

Elimination of tuberculosis remains a public health menace, due to multidrug resistant strains of Mycobacterium tuberculosis, that are resistant to the first-line anti-tubercular drugs. Consequently, detection of resistant strains depends on early diagnosis. Intervention of molecular techniques is essential to reduce mortality and morbidity rates. This study aimed to detect katG and inhA gene mutations among rifampicin resistant strain of Mycobacterium tuberculosis in Kebbi State. A cross-sectional study was conducted which covered all the five major health zones in Kebbi State. Two hundred and forty (240) acid fast bacilli confirmed sputum samples were recruited and subjected to Gene Xpert testing. Positive sputum samples were later subjected to Line Probe Assay technique. 14 sputum samples were resistant to rifampicin out of the 240 confirmed acid fast bacilli sputum samples while 226 were sensitive to rifampicin. Mutations were found at the frequency of 100% for katG gene (katG/S315T) in all the 14 rifampicin resistant samples (rpoB/S531L) at the different health zones in kebbi state using the line probe assay. The research has also detected inhA gene mutation at a frequency of 100% in all rifampicin resistant samples showing that mutation is associated with multi resistance to isoniazid and rifampicin. This study confirms that resistance due to katG and inhA mutation is a better surrogate of multidrug-resistant tuberculosis among TB patients in Kebbi State compared to the single-marker analysis. Hence, early molecular detection of rifampicin resistant strains will suffice in determining the management of multidrug resistant tuberculosis in Kebbi State.

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

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). 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. 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. 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.

Head-to-head comparison of genotyping of human papillomavirus by real-time multiplex PCR assay using type-specific primers and SPF10-PCR-based line probe assay.

The SPF10-polymerase chain reaction(PCR)-based line probe assay (LiPA-25) with high analytical sensitivity and specificity for human papillomavirus(HPV) genotyping in clinical samples has been widely used in vaccine and epidemiologic studies. A real-time multiplex PCR assay using type-specific primers (Hybribio-23) with low workload and cost has been developed recently. The study aimed to compare the performance of LiPA-25 and Hybribio-23 in selected 1731 cervical swab and 117 tissue samples, with a focus on 20 common HPV types (14 high-risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68/73; 6 low-risk: 6, 11, 42, 43, 44, and 53). The level of agreement of two assays was determined using Cohen's Kappa (κ) statistics. A total of 1296 (74.9%) swab samples were identified as HPV-positive by Hybribio-23 or LiPA-25, of which 814 (62.8%) samples exhibited concordant, 358 (27.6%) showed additional or fewer types (compatible), and 124 (9.6%) were discordant. In addition, the two assays showed a perfect agreement for 20 HPV-combined detection (κ = 0.838) and 17 individual HPV types (all κ > 0.800), a good agreement for HPV31 (κ = 0.792) and 43 (κ = 0.696), and a moderate agreement for HPV42 (κ = 0.504). Hybribio-23 was significantly more sensitive for HPV58, 59, 68/73, 42, 43, and 44, and less sensitive for HPV35 and 66 than LiPA-25 (McNemar's test: all p < 0.05). For 117 HPV-positive tissue specimens, the identification of genotypes was 85.2% identical, 12.2% compatible, and only 2.6% discordant. The agreement for HPV31 (κ = 0.786), 68/73 (κ = 0.742), and HPV53 (κ = 0.742) was good, while for other types (all κ > 0.853) and 20 HPV-combined detection (κ = 0.936) was perfect (all p > 0.05). In conclusion, Hybribio-23 and LiPA-25 are comparable. Hybribio-23 could be used for the detection and genotyping of HPV in cervical samples for epidemiological and vaccine studies worldwide.

Fluoroquinolone resistance mutations among Mycobacterium tuberculosis and their interconnection with treatment outcome.

Fluoroquinolone (FQ) antibiotics are among the most potent second-line antitubercular drugs these days. The aim of the study was to analyze the frequency and pattern of genetic mutation in preextensive (pre-XDR) and extensively drug-resistant Mycobacterium tuberculosis using second-line line probe assay (LPA) and to compare drug-resistant mutations with different treatment outcomes. Sputum, lymph node aspirate, and cold accesses from patients with rifampicin-resistant Tuberculosis (TB) were subjected to first-line and second-line LPA (Genotype MTBDRsl by Hain Life Science, Germany) to assess additional drug resistance to fluoroquinolones (levofloxacin and moxifloxacin). Final treatment outcomes as per the National TB Elimination Program were assessed and compared with the mutation profile. One hundred and fifty subjects were observed to have mutations associated with resistance to FQs and constituted the final study population. The most frequent mutation observed among GyrA drug resistance mutation was D94G (Gyr A MUT3C, 44/150, 66%) corresponding to high-level resistance to levofloxacin and moxifloxacin. The same mutation was associated with poor treatment outcome as died or treatment failure (odds ratio 2.50, relative risk 1.67, P = 0.043). The most common hetero-resistance mutation pattern observed in GyrA gene was wild type plus Asp94Gly mutation in 24.6% of isolates. GyrA MUT3C hybridization corresponding to single-point mutation of aspartic acid to glycine at codon 94 constitutes the most common mutation in GyrA gene locus in M. tuberculosis with significant association with treatment outcome as died compared to those with treatment outcome as cured.

Evaluation of the Novaplex II HPV28 Detection Assay for HPV Typing in Formalin-Fixed, Paraffin-Embedded Tissues

Prophylactic human papillomavirus (HPV) vaccines are recommended for prevention of HPV-associated cancers. Type-specific detection of HPV in formalin-fixed, paraffin-embedded (FFPE) tissues retrieved from diagnostic pathology laboratories is important in monitoring the impact of HPV vaccines. However, few typing assays have been validated for testing FFPE samples. Results of the Novaplex II HPV28 Detection (Novaplex) assay were compared with those from the reference assay (Linear Array with reflex Line Probe Assay) on 708 FFPE samples from cervical lesions. Novaplex showed high type-specific concordance with the reference method for HPV16/18, 9 types targeted by the Gardasil 9 vaccine, 14 high-risk types, and 21 types covered by comparison assays. The rate of inadequate samples was low in both approaches (reference, 3.4%; Novaplex, 1.7%). The proportion of discrepant types was less than 3.5% and positive concordance was greater than 75.0%. Furthermore, the type-specific positive agreement (92.0% to 98.0%), negative agreement (96.0% to 99.0%), and accuracy (97.0% to 99.0%) was high. Cohen's κ ranged from 0.86 to 0.89, indicating excellent agreement between Novaplex and reference assays. The results show that Novaplex is a suitable method for detection of HPV in FFPE tissues.

Second-line drug resistance markers as proxy indicators of time to sputum culture conversion among second-line drug resistant tuberculosis patients tested in Uganda: A cross-sectional study

Background Increased tuberculosis disease burden arises as a result of low treatment success rates stemming from the emergence of second-line drug resistance. We aimed at determining the usefulness of second-line drug (SLD) resistance markers as proxy indicators of time to sputum culture conversion; a renowned predictor of Tuberculosis treatment outcome, among SLD-resistant tuberculosis (TB) patients tested at the Uganda National TB Reference Laboratory (NTRL). Methods A cross-sectional study was conducted on 72 bacteriologically confirmed SLD resistant TB patients with datasets including culture conversion time and second line probe assay mutation profiles between 01/06/2017 and 31/12/2019. The data were then imported into STATA v15 for descriptive statistical analysis, Univariate cox proportional hazard model analysis and Kaplan-Meier survival curves at a 5% level of significance; p-value ≤0.05. Results Results indicate the median time was achieved at 3 (0–12) months across the studied patients. The rrs G1484T mutation associated with conferring drug resistance to injectable agents was observed to have the shortest median conversion time of 1.5 months, longest by the gryB E540D at 5 months. A single mutation in the gryA gene locus showed higher converted proportions 70.8% (58.9–81.0) than those that had two 8.3% (3.1–17.3) or three 2.7% (0.3–10.0) mutations. Conclusions The studied second-line drug resistance markers had no statistically significant association with the time to sputum culture conversion, although increased drug resistance levels reduced the converted proportions and stressed the need to utilize molecular diagnostics data and other crucial variables to better comprehend proxy indicators of SLD resistant tuberculosis management.

Head-to-head comparison of genotyping of human papillomavirus by GP5+/6+-PCR-based reverse dot blot hybridization assay and SPF10-PCR-based line probe assay.

The SPF10-PCR-based line probe assay (LiPA-25) for human papillomavirus (HPV) genotyping with high analytical sensitivity and specificity was widely used in HPV vaccine clinical trials and epidemiologic studies. In the study, we aimed to compare a novel GP5+/6+-PCR-based reverse dot blot hybridization assay (Yaneng-23) with LiPA-25. The performance of two assays was evaluated in 1735 cervical swab and 117 tissue samples, with a focus on 19 common HPV types (14 high-risk: 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, and 68/73; 5 low-risk: 6, 11, 42, 43, and 53). A total of 1197 (69.0%) swab samples were identified as HPV-positive by two assays. Of these, 878 (73.4%) samples displayed absolute agreement (concordant), 255 (21.3%) showed additional or fewer types (compatible), and the remaining 64 (5.3%) samples were discordant. Additionally, the two assays showed an excellent strength of agreement for 19 HPV-combined detection (κ = 0.886) and 17 individual HPV types (all κ > 0.800), and displayed a good agreement for HPV39 (κ = 0.780) and 42 (κ = 0.699). Yaneng-23 was more sensitive than LiPA-25 for HPV58, 59, 68/73, 42, 43 and 53 (McNemar's test: all p < 0.05), while LiPA-25 was more sensitive for HPV31, 39, 52, and 66 than Yaneng-23 (all p < 0.05). In 113 HPV-positive tissue specimens, the identification of genotypes was 82.3% identical and 17.7% compatible. The agreement between the tests for HPV45 (κ = 0.796) and 51 (κ = 0.742) was good, and for other types (all κ > 0.843) and 19 HPV-combined detection (κ = 0.929) was perfect (all p > 0.05). In conclusion, Yaneng-23 and LiPA-25 are comparable. Yaneng-23 could be used for the detection and genotyping of HPV in cervical samples for epidemiological and vaccine studies worldwide.

Evaluation of Two Different Preparation Protocols for MALDI-TOF MS Nontuberculous Mycobacteria Identification from Liquid and Solid Media.

Nontuberculous mycobacteria (NTM) identification is essential for establishing the relevance of the isolate and for appropriate antimicrobial therapy. Traditionally, NTM identification is performed by using Line Probe Assays (LPA), a costly and time-consuming technique requiring trained personnel. MALDI-TOF MS is a promising tool for NTM identification, and its use is rapidly growing. We evaluated the newly introduced MBT Mycobacteria kit (MBT) and the MycoEx preparation protocol (Bruker Daltonics, Germany) for NTM MALDI-TOF MS identification using LPA results as a reference. Fifty NTM grown on 7H11 agar and MGIT broth were analyzed with both protocols using the Bruker Microflex® LT MALDI-TOF MS (Bruker Daltonics) instrument. MBT and MycoEx provided identification results in 97.0% and 95.0% of the cases, respectively. With both protocols, 100% of the provided results agreed with LPA with no registered mismatch. MBT achieved an elevated number of highly probable identifications (88.0% vs. 83.0%) and a higher reproducibility rate of correct results (86.6% vs. 75.8%) in comparison to MycoEx. This study provides results about MBT performance for liquid and solid media, underlining the strengths and weakness under different conditions. Our results suggest that MALDI-TOF MS could provide a great advantage for timely and cost-saving NTM identification with potential implications for patient outcome.

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.

Comparison of xpert MTB/RIF assay, line probe assay, and culture in diagnosis of pulmonary tuberculosis on bronchoscopic specimen.

In patients unable to expectorate good quality sputum or with minimal to none sputum production, bronchoscopic specimens may be collected. The objective of the study is to determine the use of Xpert MTB/RIF assay and line probe assay (LPA) in the diagnosis of pulmonary TB (PTB) using specimens collected by bronchoscopy in a tertiary care center. Bronchoscopy specimens received in the TB laboratory were processed by microscopy, Xpert MTB/RIF assay, LPA, and mycobacteria growth indicator tube (MGIT) culture. Results of MGIT culture are considered gold standard. Of the 173 specimens tested, MTB was detected in 48 (27.74%) samples by any of the above methods. Positivity in bronchoalveolar lavage was 31.4% (44/140) and in bronchial wash was 12.1% (4/33). Detection by microscopy, Xpert assay, and culture was 20 (11.56%), 45 (26.01%), and 38 (21.96%), respectively. Culture detected MTB in three additional specimens compared to Xpert assay. Xpert assay detected MTB in 45 (26%) specimens which include 10 specimens which were negative by culture. LPA detected MTB in 18 (90%) out of 20 smear-positive specimens. RIF resistance was detected in 20 (41.7%) specimens by Xpert and/or MGIT culture drug susceptibility testing (DST). Isoniazid (INH) resistance was detected in 19 specimens by LPA and MGIT culture DST. Bronchoscopy can provide alternative respiratory specimens for diagnosing PTB in patients with difficulty to expectorate sputum. The utility of Xpert MTB/RIF as a rapid, sensitive, and specific test should always be supplemented with culture in difficult-to-obtain and precious respiratory specimens. LPA plays an important role in rapid detection of INH monoresistance.

Evaluation of the GeneXpert Mycobacterium tuberculosis/Rifampicin Assay for Early Detection of Extrapulmonary Tuberculosis and Rifampicin Resistance in Aligarh Region of Northern India

Purpose: Early diagnosis of extrapulmonary tuberculosis (TB) with simultaneous detection of rifampicin (RIF) resistance (RR) directly from the sample is a revolution brought by GeneXpert Mycobacterium TB (MTB/RIF) assay. Line probe assay (LPA) is not recommended for extrapulmonary/nonsputum specimens; GeneXpert MTB/RIF provides a diagnostic option for these. Hence, the following study aimed to evaluate the performance of GeneXpert MTB/RIF assay in nonsputum samples. Materials and Methods: A total number of 642 extrapulmonary and nonsputum respiratory samples were subjected to Ziehl–Neelsen (ZN) staining technique, fluorescent microscopy (FM), and GeneXpert MTB/RIF Assay. All samples were cultured on a solid Lowenstein–Jensen (LJ) medium and culture-positive samples were subjected to GenoType MTBDRplus LPA and drug susceptibility testing by 1% proportion method on a solid LJ medium. Results: GeneXpert MTB/RIF assay detected MTB in 25.9% (166/642) of the samples. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of ZN staining technique and FM were 21.52%, 99.35%, 91.89%, and 78.77% and 27.22%, 99.78%, 97.73%, and 80.07%, respectively. Sensitivity, specificity, PPV, and NPV of GeneXpert MTB/RIF in the detection of MTB were 93.75%, 96.53%, 90.36%, and 97.80%, and RIF RR was 92.0%, 97.74%, 88.46%, and 98.48%, respectively. LPA performed on positive culture growth showed 100% sensitivity and specificity in the detection of RIF and isoniazid resistance. Conclusion: GeneXpert MTB/RIF showed high sensitivity and specificity in detecting MTB and simultaneous RIF RR among the nonsputum samples which had a good concordance to LPA. It provides a judicious option for early detection of multidrug-resistant TB in extrapulmonary and nonsputum respiratory samples.

Drug resistance patterns and treatment outcomes in DR-TB patients at a tertiary care centre in Mumbai

BackgroundDrug-resistant tuberculosis (DR-TB) is a major health problem and threatens Tuberculosis (TB) control and outcomes globally. India holds one-fourth of global DR-TB burden.1 Aims1- To study drug resistance patterns and outcomes in DR-TB patients under National Tuberculosis Elimination Programme (NTEP) at a tertiary care-centre. 2- To correlate outcome of DR-TB with drug resistance patterns. MethodsIt is a retrospective study of 302 Drug Resistant Tuberculosis patients from Jan 2020 to May 2022. Common mutations of drug resistance, pyrazinamide resistance in DR-TB patients, correlation of High dose Moxifloxacin sensitivity by Line Probe Assay (LPA) and drug sensitivity test (DST), outcome of DR-TB patients with drug resistance patterns and correlation of outcome of DR-TB patients with their initial body-weight were studied. ResultsKat G was the most common mutation in Isoniazid (96%) resistance for MDR TB as well as Isoniazid Mono-resistance TB (p = 0.001). 91% cases with MDR-TB were resistant to pyrazinamide. 51.2% cases had low dose Fluroquinolone resistance. 18.8% cases had low and high dose Fluroquinolone resistance. 8.5% cases had resistance to injectables. 21.7% of cases who were resistant to High dose Moxifloxacin on second line LPA were found to be sensitive on DST. Outcomes were not dependent on the LPA resistance patterns. Body-weight greater than 45 Kg at the time of initiation of treatment was associated with better outcomes (p = 0.007). ConclusionDR-TB patients are resistant to pyrazinamide in nearly all cases; hence pyrazinamide is not suitable for initial replacement sequence. Second line resistance doesn't impact outcome in DR-TB patients.

Heteroresistance to rifampicin & isoniazid in clinical samples of patients with presumptive drug-resistant tuberculosis in Central India.

A combination of resistant and susceptible Mycobacterium tuberculosis (MTB) isolated from clinical specimens is referred to as heteroresistance. Heteroresistance leads to difficulties in drug resistance testing and may adversely affect treatment outcomes. The present study estimated the proportion of heteroresistance among MTB in clinical samples of presumptive drug-resistant tuberculosis (TB) patients in Central India. A retrospective analysis of data generated from line probe assay (LPA) at a tertiary care hospital in Central India between January 2013 and December 2018 was carried out. A heteroresistant MTB in a sample was indicated by the presence of both wild-type and mutant-type patterns on an LPA strip. Data analysis was carried out on interpretable 11,788 LPA results. Heteroresistance in MTB was detected in 637 (5.4%) samples. Of these, heteroresistance in MTB was detected in 413 (64.8%), 163 (25.5%) and 61 (9.5%) samples with respect to rpoB, katG and inhA genes, respectively. Heteroresistance is considered a preliminary step in the development of drug resistance. Delayed or suboptimal anti-tubercular therapy in patients with heteroresistance of MTB may elicit full clinical resistance and negatively impact the National TB Elimination Programme. Further studies are, however, needed to determine the impact of heteroresistance on treatment outcomes in individual patients.

Molecular characterization of genetic mutations with fitness loss in pulmonary tuberculosis patients associated with HIV co-infection in Northwest Amhara, Ethiopia.

Molecular approaches to identifying resistance-conferring mutations suggest a revolution in the field of tuberculosis. The aim of the study was to determine the association between resistance-conferring mutations with fitness loss in Mycobacterium tuberculosis clinical isolates and HIV co-infection in the Amhara region of Ethiopia. A laboratory-based cross-sectional study was conducted between September 2022 and June 2023. A line probe assay was performed on 146 culture-positive clinical isolates. Logistic regression analysis was used to measure the strength of the association between the drug-resistance-conferring mutations with fitness loss in M. tuberculosis isolates and tuberculosis/HIV co-infection. A p-value ⩽ 0.05 was considered statistically significant. A total of 11 distinct mutations at four genetic loci among 19 resistant isolates were detected. The frequency of rifampicin, isoniazid, and fluoroquinolones resistance-conferring mutations was identified in 12 (8.2%), 17 (11.6%), and 2 (1.4%) of the isolates, respectively. The most prominent specific mutations were S450L (5/9, 55.6%), S315T (11/11, 100%), C-15T (4/4, 100%), and D94G (1/1, 100%). Double mutations were observed in 10 (52.6%) multidrug-resistant tuberculosis isolates; the most common were detected in both the rpoB and katG genes (8/10, 80.0%). The HIV-co-infected tuberculosis patients carried a higher proportion of low fitness of non-rpoB S450L variants than those tuberculosis patients without HIV (80.0% vs 14.3%) and showed a significant association (cOR = 0.042, 95% CI: 0.002-0.877, p = 0.041), but not with the low fitness of non-katG S315T variants (cOR = 3.00, 95% CI: 0.348-25.870, p = 0.318). This study provides valuable information on the genetic variants with fitness loss associated with HIV co-infection, but requires further whole-genome-based mutation analysis.

Clinical utility of target-based next-generation sequencing for drug-resistant TB.

BACKGROUND: In high TB burden countries, access to drug susceptibility testing is a major bottleneck. Targeted next-generation sequencing (tNGS) is a promising technology for rapid resistance detection. This study assessed the role of tNGS for the diagnosis of drug-resistant TB (DR-TB).METHODS: A total of 161 samples from bacteriologically confirmed TB cases were subjected to tNGS using the Deeplex® Myc-TB kit and sequenced using the MiSeq platform. These samples were also processed for conventional phenotypic DST (pDST) using 13 drugs on Mycobacteria Growth Indicator Tube and line-probe assays (MTBDRplus and MTBDRsl).RESULTS: There were 146 DR-TB and 15 drug-susceptible TB (DS-TB) samples. About 70% of patients with DR-TB had no previous TB treatment history. Overall, 88.2% had rifampicin-resistant/multidrug-resistant TB (RR/MDR-TB), 58.5% pre-extensively drug-resistant TB (pre-XDR-TB) and 9.2% had XDR-TB as defined by the WHO (2020). Around 8% (n = 13) of samples were non-culturable; however, identified 8 were resistant to first and second-line drugs using tNGS. Resistance frequency was similar across methods, with discordance in drugs less reliable using pDST or with limited mutational representation within databases. Sensitivities were aligned with literature reports for most drugs. We observed 10% heteroresistance, while 75% of strains were of Lineages 2 and 3.CONCLUSIONS: Programme data supported tNGS in the diagnosis of DR-TB for early treatment using individualised regimens.

Performance Evaluation of MolecuTech REBA Myco-ID Using HybREAD480 for Identification of Nontuberculous Mycobacteria.

Rapid and accurate identification of nontuberculous mycobacteria (NTM) species is essential for the diagnosis and treatment of NTM disease. MolecuTech REBA Myco-ID (YD Diagnostics, Yongin, Korea) is a line probe assay for identification of NTM species and can be performed using HybREAD480, an instrument for automating the post-PCR steps. In this study, we assessed the performance of MolecuTech REBA Myco-ID using HybREAD480. Seventy-four reference strains, including 65 Mycobacterium strains and nine non-Mycobacterium strains within the order Mycobacteriales, were used to determine the analytical specificity of MolecuTech REBA Myco-ID. The clinical performance of this assay was evaluated with 192 clinical Mycobacterium strains, and the assay results were compared to those of multigene sequencing-based typing. The accuracy of MolecuTech REBA Myco-ID for the 74 reference strains and 192 clinical strains was 77.0% (57/74; 95% confidence interval [CI], 65.8 - 86.0%) and 94.3% (181/192; 95% CI, 90.0 - 97.1%), respectively. Although some rarely isolated NTM species are misidentified, the most commonly isolated NTM species, including M. avium complex, M. abscessus subsp. abscessus, M. abscessus subsp. massiliense, and M. fortuitum com-plex, were all correctly identified. Of note, all M. lentiflavum strains tested (reference strain, n = 1; clinical strain, n = 10) were misidentified as M. gordonae. MolecuTech REBA Myco-ID using HybREAD480 was accurate for identifying commonly isolated NTM species and for discriminating between M. abscessus subsp. abscessus and M. abscessus subsp. massiliense. However, the main limitations of this assay, including misidentification of some rarely isolated NTM species and cross-reactivity between M. lentiflavum and M. gordonae, should be considered.

Risk factors of multidrug resistant tuberculosis among patients with tuberculosis at selected multidrug resistance treatment initiative centres in southern Ethiopia: a case-control study

ObjectiveTo identify the risk factors for multidrug resistant tuberculosis (MDR-TB) among patients with TB at selected MDR-TB treatment initiative centres, southern Ethiopia, 2021.DesignAn unmatched case-control study was employed.SettingMultidrug resistance treatment...

Anti-Tuberculosis Therapy in The Drug Resistant Tuberculosis Patients of Kashmir Valley

Drug-resistant tuberculosis (DR-TB) strains are a significant threat to global TB control. Due to a lack of resources and inadequate healthcare infrastructure, it is difficult to identify and monitor drug resistance situations in low and middle-income nations. The study aimed to evaluate the anti-tuberculosis therapy regimen in drug-resistant tuberculosis patients of the mountainous valley of Kashmir. The current study was carried out at Government Medical College, Department of Pulmonary Medicine, Chest Diseases Hospital (CDH), Srinagar, Kashmir. A total of 1100 suspected tuberculosis subjects were included in the study, and out of 1100 TB subjects, 195 were documented medication resistance cases. Of the total 1100 Line Probe Assay (LPA) tests done, 69 (6.2%) such samples in the Tuberculosis band were absent. Of the real samples tested by LPA, Tuberculosis band (TUB) was present in 1031 samples (93.7%), of which 24 (2.3%) were multidrugresistant (MDR), 121 (11.7%) were rifampicin mono-resistant, 42 (4.0%) were isoniazid mono-resistant, 8 (0.77%) were extensively drugresistant (XDR), and 836 (81%) samples were pan-sensitive. Smoking remained an independent risk factor for poor treatment outcomes in the multivariate analysis (p-value = 0.014, OR = 4.356, 95% CI [1.425�12.45]). This study found a low incidence of Drug-Resistant Tuberculosis (DR-TB). Most of the cases were resistant to first-line anti-tuberculosis drugs. Special attention should be paid to high-risk DR-TB patients to improve treatment results even further. Further studies are recommended with a large sample size.