Drug Susceptibility Testing Methods Research Articles

Unveiling the complexity of rifampicin drug susceptibility testing in Mycobacterium tuberculosis: comparative analysis with next-generation sequencing.

Introduction. The discordance between phenotypic and molecular methods of rifampicin (RIF) drug susceptibility testing (DST) in Mycobacterium tuberculosis poses a significant challenge, potentially resulting in misdiagnosis and inappropriate treatment.Hypothesis/gap statement. A comparison of RIF phenotypic and molecular methods for DST, including whole genome sequencing (WGS), may provide a better understanding of resistance mechanisms.Aim. This study aims to compare RIF DST in M. tuberculosis using two phenotypic and molecular methods including the GeneXpert RIF Assay (GX) and WGS for better understanding.Methodology. The study evaluated two phenotypic liquid medium methods [Lowenstein-Jensen (LJ) and Mycobacterium Growth Indicator Tube (MGIT)], one targeted molecular method (GX), and one WGS method. Moreover, mutational frequency in ponA1 and ponA2 was also screened in the current and previous RIF resistance M. tuberculosis genomic isolates to find their compensatory role.Results. A total of 25 RIF-resistant isolates, including nine from treatment failures and relapse cases with both discordant and concordant DST results on LJ, MGIT and GX, were subjected to WGS. The phenotypic DST results indicated that 11 isolates (44%) were susceptible on LJ and MGIT but resistant on GX. These isolates exhibited multiple mutations in rpoB, including Thr444>Ala, Leu430>Pro, Leu430>Arg, Asp435>Gly, His445>Asn and Asn438>Lys. Conversely, four isolates that were susceptible on GX and MGIT but resistant on LJ were wild type for rpoB in WGS. However, these isolates possessed several novel mutations in the PonA1 gene, including a 10 nt insertion and two nonsynonymous mutations (Ala394>Ser, Pro631>Ser), as well as one nonsynonymous mutation (Pro780>Arg) in PonA2. The discordance rate of RIF DST is higher on MGIT than on LJ and GX when compared to WGS. These discordances in the Delhi/CAS lineages were primarily associated with failure and relapse cases.Conclusion. The WGS of RIF resistance is relatively expensive, but it may be considered for isolates with discordant DST results on MGIT, LJ and GX to ensure accurate diagnosis and appropriate treatment options.

Prospective evaluation of thin-layer agar colour test in routine diagnosis of multidrug-resistant TB.

<sec><title>BACKGROUND</title>This study evaluated the diagnostic performance of the thin-layer agar MDR/XDR-TB Colour Test (CT), a Mycobacterium tuberculosis complex (MTBC) detection and direct drug susceptibility testing (DST) method with routine sputum, bronchoalveolar lavage and pleural fluid specimen.</sec><sec><title>METHODS</title>In a prospective study, the time and rate of MTBC detection were compared between CT, Löwenstein-Jensen, and MGIT media. Times until DST result, sensitivities, and specificities were evaluated between CT and MGIT 960 indirect DST.</sec><sec><title>RESULTS</title>The cultivation of 177 pulmonary specimens resulted in 83 MTBC-positive cultures. The sensitivity of CT for MTBC detection was 81.3% with a median time of 20 days compared to 13 days and 93.5% for MGIT. The sensitivity of CT for DST results was 100% for isoniazid and levofloxacin and 94.7% for rifampicin. The specificities for isoniazid and rifampicin DST were 97.3% and 98.0% for levofloxacin. The median time until a DST result was significantly shorter with CT than the BACTEC MGIT 960 system, 20 and 27 days, respectively, independent of the specimen type used.</sec><sec><title>CONCLUSIONS</title>The CT is a highly accurate and fast initial diagnostic test for high-incidence settings and could also be used as a first culture and direct DST in peripheral settings.</sec>.

Proficiency of phenotypic drug susceptibility testing for Mycobacterium tuberculosis in China, 2008-2021.

To analyze the results of proficiency testing for anti-tuberculosis drug susceptibility testing (DST) in China. Number of laboratory participating the proficiency testing performed DST, and the sensitivity, specificity, reproducibility, and accordance rate were calculated from data of 13 rounds proficiency testing results for DST from 2008 to 2021. A total of 30 and 20 strains of Mycobacterium tuberculosis with known susceptibility results were sent to each laboratory in 2008 to 2019, 2020 and 2021, respectively. The number of participating laboratories ranged from 30 in 2009 to 546 in 2021. L-J DST was the predominant method. The specificity presented relatively higher than sensitivity. Improvement of specificity were observed for all drugs through the years, while sensitivity did not show improvement for amikacin and capreomycin. Accordance rate of pyrazinamide and kanamycin and reproducibility of capreomycin and pyrazinamide were not significantly improved through the years. Most of the participating laboratories significantly improved the quality of their DST through the consecutive rounds of proficiency testing except for second-line injectable drugs and pyrazinamide. The results highlight the importance of developing novel and/or improving existing methods for phenotypic DST for certain drugs.

Evaluation of a real-time PCR assay performance to detect Mycobacterium tuberculosis, rifampicin, and isoniazid resistance in sputum specimens: a multicenter study in two major cities of Indonesia.

Tuberculosis (TB) is one of the major global health issues due to its high mortality rate, especially in low- and middle-income countries. One of the key success points of the TB eradication program is early TB diagnosis, which requires rapid and accurate diagnostic testing. This study aimed to evaluate the performance of a newly developed RT-PCR kit (Indigen MTB/DR-TB RT-PCR) in a routine TB clinical setting. A multi-fluorescence RT-PCR assay was designed and developed to detect regions within IS6110, rpoB, katG, and inhA of the Mycobacterium tuberculosis (MTB) genes. Sputum specimens were obtained from suspected TB patients who visited TB healthcare facilities in two major cities of Indonesia from September 2022 to May 2023. Specimens were assessed using Indigen MTB/DR-TB RT-PCR, acid-fast bacillus (AFB) smear microscopy, MTB culture, and drug susceptibility testing (DST) methods. Fisher's exact test (χ2) was used to analyze the Indigen performance relative to culture methods. The performance of Indigen MTB/DR-TB RT-PCR to detect MTB was assessed using 610 sputum specimens obtained from suspected patients. The overall sensitivity and specificity were 94.12% (95% CI: 90.86-96.48%) and 98.32% (95% CI: 96.20-99.46%), respectively. When the analysis was performed on AFB smear-negative TB subjects (386 subjects), a lower sensitivity level was found at 78.57% (95% CI: 68.26-86.78%), while the specificity level remained similar at 98.34% (95% CI: 96.18-99.46%). The overall performance of Indigen MTB/DR-TB RT-PCR to detect MTB showed substantial agreement with the MTB culture method (kappa value 0.93). In comparison to DST, the sensitivity and specificity levels of Indigen to detect RIF resistance or INH resistance were 78.2% (95% CI: 61.8-90.2%) and 82.8% (95% CI: 64.2-94.2%), respectively, while the specificity level for both groups was at 100% (95% CI, 87.7-100%). Indigen MTB/DR-TB RT-PCR demonstrated reliable performance for TB molecular diagnostic testing and can be implemented in routine TB diagnostic settings.

Identification of Mycobacterium tuberculosis Resistance to Common Antibiotics: An Overview of Current Methods and Techniques.

Multidrug-resistant tuberculosis (MDR-TB) is an essential cause of tuberculosis treatment failure and death of tuberculosis patients. The rapid and reliable profiling of Mycobacterium tuberculosis (MTB) drug resistance in the early stage is a critical research area for public health. Then, most traditional approaches for detecting MTB are time-consuming and costly, leading to the inappropriate therapeutic schedule resting on the ambiguous information of MTB drug resistance, increasing patient economic burden, morbidity, and mortality. Therefore, novel diagnosis methods are frequently required to meet the emerging challenges of MTB drug resistance distinguish. Considering the difficulty in treating MDR-TB, it is urgently required for the development of rapid and accurate methods in the identification of drug resistance profiles of MTB in clinical diagnosis. This review discussed recent advances in MTB drug resistance detection, focusing on developing emerging approaches and their applications in tangled clinical situations. In particular, a brief overview of antibiotic resistance to MTB was present, referred to as intrinsic bacterial resistance, consisting of cell wall barriers and efflux pumping action and acquired resistance caused by genetic mutations. Then, different drug susceptibility test (DST) methods were described, including phenotype DST,genotype DST and novel DST methods. The phenotype DST includes nitrate reductase assay, RocheTM solid ratio method, and liquid culture method and genotype DST includes fluorescent PCR, GeneXpert, PCR reverse dot hybridization, ddPCR, next-generation sequencing and gene chips. Then, novel DST methods were described, including metabolism testing, cell-free DNA probe, CRISPR assay, and spectral analysis technique. The limitations, challenges, and perspectives of different techniques for drug resistance are also discussed. These methods significantly improve the detection sensitivity and accuracy of multidrug-resistant tuberculosis (MRT) and can effectively curb the incidence of drug-resistant tuberculosis and accelerate the process of tuberculosis eradication.

Evaluation of Antibiotic Penicillin G Activities Based on Electrochemical Measurement of a Tetrazolium Salt.

This study focused on the electrochemical properties of tetrazolium salts to develop a simple method for evaluating viable bacterial counts, which are indicators of drug susceptibility. Considering that the oxidized form of tetrazolium, which has excellent cell membrane permeability, changes to the insoluble reduced form formazan inside the cell, the number of viable cells was estimated based on the reduction current of the tetrazolium remaining in the bacterial suspension. Dissolved oxygen is an important component of bacterial activity. However, it interferes with the electrochemical response of tetrazolium. We estimated the number of viable bacteria in the suspension based on potential-selective current responses that were not affected by dissolved oxygen. Based on solubility, cell membrane permeability, and characteristic electrochemical properties of the tetrazolium salt 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium, we developed a method for rapidly measuring viable bacteria within one-fifth of the time required by conventional colorimetric methods for drug susceptibility testing.

Rapid detection of drug-resistant Mycobacterium tuberculosis by Modified MODS assay suitable for resource-poor settings.

Cross contamination and biosafety are concerns with the microscopic observation drug susceptibility assay. To address these issues, we modified the MODS technique in the current study. Two hundred and seventy-five samples were processed on LJ media and drug susceptibility was performed by the Indirect agar proportion method. A modified MODS test was done in tissue culture bottles. GenoType MTBDRplus assay was performed to detect the resistance and mutational pattern associated with the resistances. Sensitivity, specificity, positive predictive value, and negative predictive value for the detection of tuberculosis by modified MODS were 97.44%, 80.00%, 97.44%, and 80.00% respectively. The perfect agreement was seen between modified MODS and the Indirect agar proportion method for drug susceptibility testing of isoniazid (kappa = 0.923) and rifampicin (kappa = 1). The contamination rate, cost and TAT for modified MODS were less as compared to the solid media. In the case of MDR-TB isolates S531L (66.66%) was the most prevalent mutation in the rpoB gene followed by S315T2 mutation (58.33%) and T8C (41.66%) in katG and inhA gene respectively. In hetero-resistant strains, C-15T mutation (37.50%) was the most common followed by A-16G (12.50%) in the inhA gene. In INH mono-resistant strains only two mutations were observed i.e., S-315T1(50%) and C-15T (50%) in the katG and inhA genes respectively. Modified MODS proved to be cost-effective and user-friendly, with minimal risk to the handler and no cross-contamination between samples were observed. Hence, it can be used in low-income countries for early detection of tuberculosis and its resistance.

Diagnostic Performance and Comparative Analysis of Conventional Drug Susceptibility Testing, Line Probe Assay, and GeneXpert in Extrapulmonary Tuberculosis Cases: A Cross-sectional Observational Study

Introduction: Tuberculosis (TB), a known threat to mankind for ages, causes a significant burden on healthcare worldwide. The alarming rise of Extrapulmonary Tuberculosis (EPTB) has led healthcare professionals to opt for molecular diagnostics. Despite the availability of rapid diagnostics, conventional culture Drug Susceptibility Testing (DST) is still considered the gold standard in diagnosing TB. Aim: To assess the diagnostic accuracy of culture DST which is considered the gold standard. Materials and Methods: This cross-sectional observational study included 150 suspected EPTB patients and was conducted over a period of one year (September 2018 to August 2019) at Lokmanya Tilak Municipal Medical College and Sion Hospital (LTMMC), a tertiary care hospital in central Mumbai, Maharashtra, India. The hospital is located adjacent to Dharavi, the largest slum area with a high population density, which contributes to the majority of patients attending clinics. Patients attending the pulmonary medicine Outpatient Department (OPD) with extrapulmonary manifestations and suspected cases of EPTB were enrolled in the study. Samples, excluding sputum, were sent to the Department of Microbiology, LTMMC, Sion Hospital for GeneXpert analysis. The samples were further evaluated by microscopy, GeneXpert, and culture DST to detect the presence of Mycobacterium tuberculosis (M. tuberculosis) and drug susceptibility, respectively. Patients positive for Pulmonary Tuberculosis (PTB) were excluded from the study. Categorical variables were described using percentages. A Chi-square test was applied, and a p-value of &lt;0.05 was considered significant. All statistical analyses were performed using SPSS statistical software (IBM SPSS version 26.0, Armonk, N.Y.). Results: Out of the 150 extrapulmonary samples, 23 (15.33%) samples were culture positive for M. tuberculosis and were subjected to DST using the 1% proportion method and GeneXpert assay. The MDR isolates were tested using the firstline Line Probe Assay (LPA). Eleven (47.8%) showed resistance to first-line antitubercular drugs. Among the 12 new cases, only 2 (16.7%) showed resistance compared to 9 (81.8%) in previously treated cases. A discordance of 8.7% was observed between DST with GeneXpert and LPA. Additionally, a discordance of 8.7% was observed between DST and LPA for rifampicin resistance and 4.3% for isoniazid resistance. Conclusion: The paucibacillary nature of extrapulmonary samples contributes to the challenging diagnosis of EPTB cases, leading to increased drug resistance. Highlighting the importance of the conventional solid culture DST method, this study strongly recommends the use of conventional DST accompanied by LPA for extrapulmonary cases.

Prevalence of antibiotic resistance in Clostridium difficile: A meta-analysis

In recent years, the resistance of Clostridium difficile to various common mainstream antibiotics cannot be ignored, the incidence of CDI in many areas is also increasing year by year, posing a great threat to public safety. Literature collection was carried out on 6 open databases, and high-quality literature data conforming to classification criteria were screened and extracted. The WPR and WPF of each antibacterial drug were calculated using the random effects model. Subgroup analysis was used to investigate the influence of regional and experimental methods. Results: A total of 40 studies were included. The WPR of vancomycin was 0.0372, 95%CI: [0.0148; 0.0671], I: 93.7%, WPF was 0.2195, I: 94.0%. The WPR of fidaxomicin was 0.0051, 95%CI: [0.0000; 0.0205], I: 82.7%, WPF was 0.1757, I: 93.6%. The WPR of metronidazole was 0.0318, 95%CI: [0.0056; 0.0727], I: 91.6%. Among the three antibiotics, the highest resistance of C. difficile to metronidazole were mainly distributed in the Middle East and Africa. The conclusion seems to indicate that C. difficile is most sensitive to Fidaxomicin, and the clinical cure rate is better than vancomycin. However, the clinical cure failure rate increased significantly from 2007 to 2019, while the drug resistance rate did not change significantly, suggesting that other factors besides drug resistance may affect the therapeutic effect of fidaxomicin on CDI. There was no significant difference in the results of AGAR dilution and e test, the main methods for drug susceptibility testing.

Discordance between genotypic and phenotypic methods for the detection of rifampicin and isoniazid resistant Mycobacterium tuberculosis and the correlation with patient treatment outcomes

BackgroundAccurate drug susceptibility testing (DST) of Mycobacterium tuberculosis (MTB) is essential for proper patient management. We investigated discordance between genotypic (Xpert MTB/RIF and MTBDRplus) and phenotypic (MGIT 960) methods for the detection of rifampicin (RIF) and isoniazid (INH) resistant MTB and its correlation with patient treatment outcomes in Jimma, Southwest Oromia, Ethiopia. MethodsA retrospective study was conducted on 57 stored MTB isolates with known Xpert RIF resistance status (45 RIF resistant and 12 RIF susceptible) at Jimma University Mycobacteriology Research Center from November 2, 2021, to December 28, 2022. We did MTBDRplus and phenotypic DST (using the Mycobacterial Growth Indicator Tube (MGIT) system). The Xpert and MTBDRplus results were compared using phenotypic DST as a reference standard method. The treatment outcome was determined as per national guideline. The discordance between the genotypic and phenotypic DST was calculated using GraphPad software. ResultsAmong the 57 MTB isolates, six (10.5 %) had discordant results between the two DST methods. Xpert yielded five discordant results for RIF when compared with phenotypic DST (kappa coefficient (κ) = 0.76, 95 % confidence interval 0.56–0.96). The MTBDRplus compared with phenotypic DST gave three discordant results for RIF (κ = 0.86, 95 % confidence interval 0.71–1.00) and three for INH (κ = 0.86, 95 % confidence interval 0.70–1.00). Compared with Xpert, MTBDRplus yielded lower discordance with phenotypic DST for RIF. Out of six patients with discordant results, three had unfavorable outcomes while the other three were cured. Of the three patients with unfavorable outcomes, only one patient has received an inappropriate treatment regimen. There was no correlation between unfavorable outcomes and incorrect treatment regimens due to discordant results (Χ2 = 0.404; P = 0.525). ConclusionsDiscordance between genotypic and phenotypic DST for RIF or INH occurred in 10.5 % of isolates. Only one patient with discordant results has received an inappropriate treatment regimen, resulting in an unfavorable outcome. The impact of parallel use of rapid molecular assay with phenotypic DST on patient treatment outcomes requires further study.

Genetic Aspects of Mycobacterium Tuberculosis Resistance to New Anti-Tuberculosis Drugs

We analyzed 57 publications devoted to study of genetic mechanisms of formation of drug resistance of Mycobacterium tuberculosis to bedaquiline, delamanid, pretomanid, linezolid, and clofazimine. Identification of genetic factors of MTB drug resistance make the basis to search for new targets in development of anti-tuberculosis drugs and methods for drug susceptibility testing.

Genetic Characterization Conferred Co-Resistance to Isoniazid and Ethionamide in Mycobacterium tuberculosis Isolates from Southern Xinjiang, China.

Ethionamide (ETH), a structural analogue of isoniazid (INH), is used for treating multidrug-resistant tuberculosis (MDR-TB). Due to the common target InhA, INH and ETH showed cross-resistance in M. tuberculosis. This study aimed to explore the INH and ETH resistant profiles and genetic mutations conferring independent INH- or ETH-resistance and INH-ETH cross-resistance in M. tuberculosis circulating in south of Xinjiang, China. From Sep 2017 to Dec 2018, 312 isolates were included using drug susceptibility testing (DST), spoligotyping, and whole genome sequencing (WGS) to analyze the resistance characteristics for INH and/or ETH. Among the 312 isolates, 185 (58.3%) and 127 (40.7%) belonged to the Beijing family and non-Beijing family, respectively; 90 (28.9%) were INH-resistant (INHR) with mutation rates of 74.4% in katG, 13.3% in inhA and its promoter, 11.1% in ahpC and its upstream region, 2.2% in ndh, 0.0% in mshA, whilst 34 (10.9%) were ETH-resistant (ETHR) with mutation rates of 38.2% in ethA, 26.2% in inhA and its promoter, and 5.9% in ndh, 0.0% in ethR or mshA; and 25 (8.0%) were INH-ETH co-resistant (INHRETHR) with mutation rates of 40.0% in inhA and its promoter, and 8% in ndh. katG mutants tended to display high-level resistant to INH; and more inhA and its promoter mutants showed low-level of INH and ETH resistance. The optimal gene combinations by WGS for the prediction of INHR, ETHR, and INHRETHR were, respectively, katG+inhA and its promoter (sensitivity: 81.11%, specificity: 90.54%), ethA+inhA and its promoter+ndh (sensitivity: 61.76%, specificity: 76.62%), and inhA and its promoter+ndh (sensitivity: 48.00%, specificity: 97.65%). This study revealed the high diversity of genetic mutations conferring INH and/or ETH resistance among M. tuberculosis isolates, which would facilitate the study on INHR and/or ETHR mechanisms and provide clues for choosing ETH for MDR treatment and molecular DST methods in south of Xinjiang, China.

Comparison of in vitro fosfomycin susceptibility testing methods with agar dilution for carbapenem resistant Klebsiella pneumoniae and Escherichiacoli.

The emergence of Extensively drug resistant (XDR) pathogens like Carbapenem Resistant Klebsiella pneumoniae (CR Kpn) and Carbapenem Resistant Escherichia coli (CR Eco) has limited therapeutic options for treating them. Fosfomycin a broad-spectrum antibiotic, has emerged as a potential treatment option in combination with other agents. It is therefore important that accurate drug susceptibility testing (DST) results of fosfomycin should be available to all clinical microbiology laboratories. Agar dilution which is the recommended method for fosfomycin DST is not convenient to adopt in a routine set-up. This study aimed to determine the susceptibility pattern of CR Kpn and CR Eco to fosfomycin and to evaluate the discrepancies of the available manual MIC based alternative methods. Agar dilution (AD), broth microdilution (BMD), E-test and Ezy MIC test were performed on 235 CR-Kpn and Eco isolates respectively. Of 177 CR Kpn, 31.63% (n​=​56/177) of the isolates were susceptible by AD. Categorical Agreement (CA) by BMD, E-test and Ezy MIC were lower than the acceptable limit while Very Major Errors (VMEs) and Major Errors (MEs) were beyond the acceptable limits. In the case of CR Eco, 96.55% (n​=​56/58) were susceptible by AD. CA of 100% (n​=​58/58) was shown by both BMD and Ezy MIC while 86.20% (n​=​50/58) was shown by E-test, with no VME observed for CR Eco. ME was only observed for E-test method. The alternative methods were in poor agreement with AD method for CR Kpn and for CR Eco, BMD and Ezy MIC have shown reliable results.

Crystal violet decolorization assay: a simplified colorimetric test for the rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates

The increased prevalence of multi-drug resistant M. tuberculosis is quite possibly the direst and most difficult task for the early diagnosis and treatment. A rapid, reliable, and inexpensive diagnostic method is the need of the hour. The current study on crystal violet decolorization assay explores the possibility to develop a rapid and simple detection method to detect multi-drug-resistant tuberculosis isolates by comparing the results with the traditional liquid culture drug susceptibility testing method based on their sensitivity, specificity, positive predictive value, and negative predictive value. 70 isolates were used for the study and were detected as multi-drug resistant, mono drug-resistant, and sensitive by using crystal violet decolourization assay and further compared with the results of DST and using H37Rv as the standard control strain. The sensitivity, specificity, positive predictive value, and negative predictive value of crystal violet decolorization assay (Rifampicin: 100%, 94.60%, 100% and 82.40%; isoniazid: 100%, 94.10%, 100%, 86.40%) are calculated and the percentage were compared with the conventional liquid culture drug susceptibility testing for M. tuberculosis using rifampicin and isoniazid. Crystal violet decolourization assay is rapid, reproducible, and doesn't require any highly experienced personal or sophisticated laboratory instruments for interpretation. This assay is found to be nearly as reliable as conventional liquid culture drug susceptibility testing and may thus be of great help in phenotypic confirmation of multi-drug resistant tuberculosis by providing results more rapidly.

Evaluation of two colorimetric microplate microtiter tests for susceptibility testing of clinical isolates of Mycobacterium tuberculosis to first-line antituberculosis drugs.

Drug-resistance tuberculosis (TB) is one of the most important global public health problems. Accurate and rapid drug-susceptibility testing is critical for the effective treatment of TB patients. Various colorimetric methods are used for anti-TB drug-susceptibility testing (DST) and minimum inhibitory concentration (MIC) determination. This study was conducted to evaluate the resazurin microtiter assay (REMA) and malachite green decolorization assay (MGDA). A total of 65 Mycobacterium tuberculosis strains isolated from patients with suspected TB using REMA and malachite green microtiter assay methods were tested against streptomycin (SM), isoniazid (INH), rifampicin (RIF), and ethambutol (ETB). The Mycobacterial Growth Indicator Tube 960 DST method was accepted as the gold standard in the evaluation of test results. The sensitivity of REMA and MGDA tests was found to be 87.5% and 62.5% for INH, respectively. RIF and SM sensitivity for both tests was 100%. While ETB sensitivity was 81.8 for the REMA test, this rate was 60% for the MGDA test. Specificity of both tests varied between 92.5% and 98.2% according to the drug types. REMA and MGDA are a simple, rapid, and low cost. They can be used as an alternative test for drug-susceptibility testing and MIC determination. Extensive studies and standardization are needed for the routine use of both tests.

Pre-extensively drug-resistant tuberculosis among pulmonary multidrug-resistant tuberculosis patients in Eastern Nigeria.

Pre-extensively drug-resistant tuberculosis (Pre-XDR-TB), an emerging form of drug-resistant tuberculosis, is challenging efforts at tuberculosis control, leading to treatment failure among multidrug-resistant tuberculosis (MDR-TB) patients and progression to extensively drug-resistant tuberculosis (XDR-TB). We determined the rate of Pre-XDR-TB among multidrug-resistant patients in Southeast, Nigeria. A prospective laboratory-based study was carried out at the South East Zonal Tuberculosis Reference Laboratory from January 2021 to December 2021. Second-line drug (SLD) resistance was performed on 225 sputum samples of multidrug-resistant patients prior to treatment initiation using GenoType MTBDRsl genotypic drug susceptibility testing (DST) method. The rate of Pre-XDR-TB among 225 MDR-TB cases was 3.1%. Fluoroquinolone-resistant Pre-XDR-TB was observed (100%) in previously treated tuberculosis cases. Only one (0.4%) case showed resistance to both fluoroquinolone (FQ) and one second-line injectable drug (XDR-TB). The extensively drug-resistant case observed was a de-novo resistance. Exactly 0.9% of the multidrug-resistant cases showed resistance to second-line injectables. The prevalence of Pre-XDR-TB among MDR-TB cases was high. There is need for rapid detection of Pre-XDR-TB among MDR-TB cases before treatment initiation.

Whole-genome sequencing of presumptive MDR-TB isolates from a tertiary healthcare setting in Mumbai

Whole-genome sequencing (WGS) of Mycobacterium tuberculosis (MTB), proven to be a better alternative when compared with the combined sensitivity and specificity of all other modalities for diagnosis of tuberculosis (TB), aids epidemiological surveillance investigations by combining the current research with diagnostics.This study was conducted to identify and resolve operational challenges in performing WGS-based drug resistance testing (DRT) for MTB in a TB culture and drug susceptibility testing (DST) laboratory. Three critical, non-redundant steps for WGS-based DRT were tested: viz. DNA extraction, high-throughput paired-end next-generation sequencing (NGS), and genomic analysis pipeline for automated reporting of WGS-based DRT. DNA was extracted from 100 liquid culture isolates on a mycobacterial growth indicator tube (MGIT) using DNEASY Ultraclean Microbial Kit (Qiagen, USA) as per the manufacturer's instructions. Illumina paired-end sequencing was performed. All analysis steps were automated using custom python scripts, requiring no intervention. Variant calling was performed as per the World Health Organization (WHO) technical guide. The number of cultures resistant to rifampicin, isoniazid, pyrazinamide, ethambutol, and streptomycin was 89, 88, 35, 67, and 73, respectively. Resistance to amikacin, kanamycin, and capreomycin was found in 15, 17, and 15 cultures, respectively. Seventy cultures were resistant to fluoroquinolones, four were resistant to ethionamide, and 12 were resistant to linezolid. Six cultures were resistant to only one of the 18 drugs tested. Seventy-five cultures were resistant to more than three anti-TB drugs. One culture was resistant to 13 of the 18 anti-TB drugs tested for this study. The maximum number of variants were observed in the rpoB gene (n=93, 93%), wherein the Ser450Leu was the predominant mutation (n=68, 73%). Ser315Thr was the most common variant (n=86, 97%) that encoded resistance to isoniazid. The Lys43Arg variant encodes resistance to streptomycin and was the third most predominant variant (n=65, 89%). In addition to the high levels of resistance observed in the dataset, we also observed a high proportion of Beijing strains (n=63, 63%). Compared with results from routine diagnostics based on the 'Guidelines on Programmatic Management of Drug-Resistant TB (PMDT) in India', none of the samples had DST available for all 18 drugs. This represents a gap in PMDT guidelines. The WGS-DRT must be considered as the primary DST method after a sample is flagged rifampicin-resistant by cartridge-based nucleic acid amplification testing (CBNAAT).With several research studies currently underway globally to identify novel variants associated with drug resistance and classifiy their minimum inhibitory coefficients, WGS-DRT presents a scalable technology that updates analytical pipelines, relegating the need for changing microbiological protocols.

Novel rrs mutations in second-line injectable drug-resistant clinical isolates of Mycobacterium tuberculosis from the Punjab province of Pakistan

IntroductionPhenotypic drug susceptibility testing is the most common approach to assess drug-resistant isolates; however, molecular methods of drug susceptibility testing are fast, accurate hence, offer less time for transmission during the diagnosis period. As data on the molecular methods regarding injectable drug resistance in the Punjab province of Pakistan is limited, therefore in this study, we aimed to analyze the mutations in the rrs gene behind second-line injectable drug resistance. Material and methodsMycobacterium tuberculosis isolates were collected from the sputum of 5362 TB suspects. The strains confirmed for resistant to injectable drugs through drug susceptibility testing were further proceeded. The 1537bp rrs gene was amplified with the help of three sets of primers with overlapping regions and DNA sequencing was performed. Obtained sequences were aligned with reference sequence to find mutations. RFLP-PCR method was also optimized for rapid detection of a common (143bp and 205bp) rrs gene mutation. ResultsAmong 172 rifampicin resistance isolates, 163(95%) were resistant to both rifampicin and isoniazid, and 9 (5%) were resistant to only rifampicin. Among the resistant samples, 12 (6.9%) samples were resistant to all three injectable drugs. Sixty out of 172 (34.9%) samples showed resistance to at least one drug and 10 (5.8%) samples were resistant to two drugs among the 3 s-line drugs. Sequencing analysis showed novel mutations in different samples at positions 443InsC, 19DelT, 29G>A, 48C>T, 50G>C, 265InsT, 423T>G, 476InsA, 446A>G, 563DelA, 695G>A, 805DelA, 900G>A, and 1510A>G, while some already reported mutations at position 1401A>G, 1402A>G, and 1484G>T were also observed. MIC of novel rrs gene mutations in KAN, CAP, and AMK resistant isolates were found between 2.5 mg/L-3.05 mg/L, 2.08 mg/L-3.0 mg/L, and 2.1 mg/L-2.7 mg/L respectively. ConclusionNovel mutations in the rrs gene reported in this study may confer second-line injectable drugs resistance in Mtb. This molecular insight into second-line injectable drug resistance is useful for better management of resistance Mtb in high burden countries.

Implementation and continued validation of the malaria Plasmodium falciparum lactate dehydrogenase-based colorimetric assay for use in antiplasmodial drug screening

Antimalarial drug discovery has been facilitated by the development of various in vitro drug susceptibility testing methods suitable for medium-throughput or high-throughput campaigns. Among many, the Plasmodium falciparum lactate dehydrogenase (PfLDH) assay has acceptable demand on equipment, labour, technical skills and affordability and offers a good opportunity for scientists in low- and middle-income countries to participate in the global effort of discovering future antimalarial drugs. Hence, to enable our search for novel antimalarial drugs, we implemented and examined assay conditions and validated the PfLDH-based method in our laboratory using a reference set of standard antimalarial drugs with known activity against Plasmodium falciparum strains. The PfLDH assay revealed acceptable linearity profiles of R2 = 0.97 and 0.92 for Pf3D7 and PfDd2, respectively, achieved at 2% parasitaemia and 1% haematocrit. The detection and quantitation limits (DL and QL) of the PfLDH-based assay were 0.09% and 0.4% parasitemia, respectively. The assay showed an acceptable average Z-factor between 0.76 and 0.79 and was considerably robust. The average interassay reproducibility via percent coefficient of variation (%CV) was 5.47 between independent experiments. Overall, the PfLDH-based method produced a reliable and reproducible drug screening profile for in vitro assays in our setting. There were no significant interassay variability or hazards of other screening assays.

Detection of Mycobacterium tuberculosis Rifampicin Resistance Conferred by Borderline rpoB Mutations: Xpert MTB/RIF is Superior to Phenotypic Drug Susceptibility Testing

ObjectiveTo compare the ability of detection of borderline rifampicin resistance in Mycobacterium tuberculosis between molecular assay and phenotypic drug susceptibility tests.MethodsFifty-seven isolates with His445Leu, Asp435Tyr, Leu452Pro, Leu430Pro, His445Asn, Ile491Phe, and His445Ser mutations in rpoB gene identified by whole-genome sequencing conferring borderline rifampicin resistance were included. Molecular-based Xpert MTB/RIF, phenotypic Löwenstein–Jensen (L-J) medium-based drug susceptibility test (DST) with a critical concentration of 40.0μg/mL and minimal inhibitory concentration (MIC) assay were performed to detect borderline rifampicin resistance.ResultsWhen using Xpert MTB/RIF, 48/57 (84.2%) isolates exhibited resistance to rifampicin. 25/57 (43.9%) and 33/57 (57.9%) isolates showed rifampicin resistance by L-J medium-based DST with 4 and 6 weeks of incubation, respectively. 30/57 (52.6%) and 40/57 (70.2%) strains were resistant to rifampicin by MIC method at cutoff values of 1.0 and 0.5μg/mL, respectively. The detection rate of rifampicin resistance of Xpert MTB/RIF was significantly higher than that of phenotypic methods (p < 0.001). Of the 57 isolates with borderline rpoB mutations, 5 (8.8%) had MICs of 0.25 or 0.12μg/mL, 22 (38.6%) had MICs of 0.5μg/mL or 1.0μg/mL, and 30 (52.6%) other isolates showed MICs ≥2.0μg/mL.ConclusionMolecular-based Xpert MTB/RIF showed superior ability to detect borderline rifampicin resistance over phenotypic DST methods. Extending the incubation time of L-J DST or lowering the cutoff value of the MIC method can improve borderline rifampicin resistance detection.