Trends of Mycobacterium tuberculosis and rifampicin resistance at the Ho Teaching Hospital in Ghana.

BACKGROUND: Tuberculosis remains a major public health threat worldwide, causing significant morbidity and mortality, particularly in low- and middle-income countries. In recent years, efforts to combat tuberculosis have focused on strengthening healthcare systems and increasing access to diagnostics and treatment services. There is scarcity of data on the prevalence of Mycobacterium tuberculosis and rifampicin-resistant tuberculosis in the Volta region of Ghana. Therefore, the aim of this study was to determine the trends of Mycobacterium tuberculosis and rifampicin resistance in a major teaching hospital in Ghana spanning a six-year period. METHODOLOGY: A retrospective cross-sectional hospital study was conducted at Ho Teaching Hospital, Ho, Ghana. Study data included archived results on tuberculosis testing using GeneXpert from 2016-2021. Archived data on tuberculosis testing were collected and entered using Microsoft Excel 2019. IBM SPSS (v26) was used for a statistical analysis of the prevalence of tuberculosis. P-value <0.05 was considered statistically significant. RESULTS: The study included 5128 presumptive tuberculosis cases from 2016 to 2021, of which 552 were positive, revealing an overall prevalence of 10.76%. Males exhibited a significantly higher prevalence of tuberculosis (14.20%) compared to females (7.48%), with a male-to-female ratio of 2:1. The burden of tuberculosis varied significantly between age groups, with those aged 30-45 years and 46-60 years facing twice the risk compared to those under 15 years (p<0.001). Rainy seasons correlated with heightened tuberculosis occurrences (12.12%) compared to dry seasons (8.84%) (p = 0.008). Rifampicin-resistant tuberculosis was prevalent at 3.45%, slightly higher in women, particularly in the 45-59 age group (5.97%). In particular, tuberculosis prevalence exhibited fluctuations, peaking in 2016 (17.1%) and 2020 (11.5%), with a trough in 2019 (4.6%). CONCLUSION: The overall prevalence of laboratory confirmed tuberculosis was 10.76%, and resistance to rifampicin, 3.45%, indicating high infection and possible treatment failure. Considering its infectious nature, this calls for concerted efforts to curb the spread of the infection.

Rapid, antibiotic incubation-free determination of tuberculosis drug resistance using machine learning and Raman spectroscopy.

Tuberculosis (TB) is the world's deadliest infectious disease, with over 1.5 million deaths and 10 million new cases reported anually. The causative organism Mycobacterium tuberculosis (Mtb) can take nearly 40 d to culture, a required step to determine the pathogen's antibiotic susceptibility. Both rapid identification and rapid antibiotic susceptibility testing of Mtb are essential for effective patient treatment and combating antimicrobial resistance. Here, we demonstrate a rapid, culture-free, and antibiotic incubation-free drug susceptibility test for TB using Raman spectroscopy and machine learning. We collect few-to-single-cell Raman spectra from over 25,000 cells of the Mtb complex strain Bacillus Calmette-Guérin (BCG) resistant to one of the four mainstay anti-TB drugs, isoniazid, rifampicin, moxifloxacin, and amikacin, as well as a pan-susceptible wildtype strain. By training a neural network on this data, we classify the antibiotic resistance profile of each strain, both on dried samples and on patient sputum samples. On dried samples, we achieve >98% resistant versus susceptible classification accuracy across all five BCG strains. In patient sputum samples, we achieve ~79% average classification accuracy. We develop a feature recognition algorithm in order to verify that our machine learning model is using biologically relevant spectral features to assess the resistance profiles of our mycobacterial strains. Finally, we demonstrate how this approach can be deployed in resource-limited settings by developing a low-cost, portable Raman microscope that costs <$5,000. We show how this instrument and our machine learning model enable combined microscopy and spectroscopy for accurate few-to-single-cell drug susceptibility testing of BCG.

Regional differences of Mycobacterium tuberculosis complex infection and multidrug resistance epidemic in Luoyang.

BACKGROUND: Tuberculosis (TB) remains a global public health event of great concern, however epidemic data on TB covering entire areas during the special period of the COVID-19 epidemic have rarely been reported. We compared the dissemination and multidrug-resistance patterns of Mycobacterium tuberculosis complex (MTBC) in the main urban area of Luoyang City, China (including six municipal jurisdictions) and nine county and township areas under its jurisdiction, aimed to establish the epidemiology of TB in this region and to provide reference for precision anti-TB in places with similar settings. METHODS: From 2020 to 2022, sputum samples were collected from 18,504 patients with confirmed, suspected and unexcluded TB in 10 designated TB medical institutions. Insertion sequence 6110 was amplified by PCR (rpoB gene detection if necessary) to confirm the presence of MTBC. PCR-positive specimens were analyzed by multicolor melting curve analysis to detect multidrug resistance. RESULTS: Among the 18,504 specimens, 2675 (14.5%) were MTBC positive. The positive rate was higher in the main urban area than in the county and township areas (29.8% vs. 10.9%, p < 0.001). Male, re-treated and smear-positive groups were high-burden carriers of MTBC. Individuals aged > 60 years were the largest group infected with MTBC in the main urban area, compared with individuals aged < 61 years in the county and township areas. The detection of multidrug-resistant TB (MDR-TB) was higher in the main urban area than in the county and township areas (13.9% vs. 7.8%, p < 0.001). In all areas, MDR-TB groups were dominated by males, patients with a history of TB treatment, and patients aged < 61 years. Stratified analysis of MDR-TB epidemiology showed that MDR4 (INH þ RIF þ EMB þ SM) was predominant in the main urban area, while MDR3 (INH þ RIF þ SM) was predominant in the county and township areas. MDR-TB detection rate and epidemiology differed among the county and township areas. CONCLUSIONS: For local TB control, it is necessary to plan more appropriate and accurate prevention and control strategies according to the regional distribution of MTBC infection.

Whole Genome Sequencing of Mycobacterium tuberculosis under routine conditions in a high-burden area of multidrug-resistant tuberculosis in Peru.

Whole Genome Sequencing (WGS) is a promising tool in the global fight against tuberculosis (TB). The aim of this study was to evaluate the use of WGS in routine conditions for detection of drug resistance markers and transmission clusters in a multidrug-resistant TB hot-spot area in Peru. For this, 140 drug-resistant Mycobacterium tuberculosis strains from Lima and Callao were prospectively selected and processed through routine (GenoType MTBDRsl and BACTEC MGIT) and WGS workflows, simultaneously. Resistance was determined in accordance with the World Health Organization mutation catalogue. Agreements between WGS and BACTEC results were calculated for rifampicin, isoniazid, pyrazinamide, moxifloxacin, levofloxacin, amikacin and capreomycin. Transmission clusters were determined using different cut-off values of Single Nucleotide Polymorphism differences. 100% (140/140) of strains had valid WGS results for 13 anti-TB drugs. However, the availability of final, definitive phenotypic BACTEC MGIT results varied by drug with 10-17% of invalid results for the seven compared drugs. The median time to obtain results of WGS for the complete set of drugs was 11.5 days, compared to 28.6-52.6 days for the routine workflow. Overall categorical agreement by WGS and BACTEC MGIT for the compared drugs was 96.5%. Kappa index was good (0.65≤k≤1.00), except for moxifloxacin, but the sensitivity and specificity values were high for all cases. 97.9% (137/140) of strains were characterized with only one sublineage (134 belonging to "lineage 4" and 3 to "lineage 2"), and 2.1% (3/140) were mixed strains presenting two different sublineages. Clustering rates of 3.6% (5/140), 17.9% (25/140) and 22.1% (31/140) were obtained for 5, 10 and 12 SNP cut-off values, respectively. In conclusion, routine WGS has a high diagnostic accuracy to detect resistance against key current anti-TB drugs, allowing results to be obtained through a single analysis and helping to cut quickly the chain of transmission of drug-resistant TB in Peru.

Prevalence and genetic basis of Mycobacterium tuberculosis resistance to pretomanid in China.

BACKGROUND: Pretomanid is a key component of new regimens for the treatment of drug-resistant tuberculosis (TB) which are being rolled out globally. However, there is limited information on the prevalence of pre-existing resistance to the drug. METHODS: To investigate pretomanid resistance rates in China and its underlying genetic basis, as well as to generate additional minimum inhibitory concentration (MIC) data for epidemiological cutoff (ECOFF)/breakpoint setting, we performed MIC determinations in the Mycobacterial Growth Indicator Tube™ (MGIT) system, followed by WGS analysis, on 475 Mycobacterium tuberculosis (MTB) isolated from Chinese TB patients between 2013 and 2020. RESULTS: We observed a pretomanid MIC distribution with a 99% ECOFF equal to 0.5 mg/L. Of the 15 isolates with MIC values > 0.5 mg/L, one (MIC = 1 mg/L) was identified as MTB lineage 1 (L1), a genotype previously reported to be intrinsically less susceptible to pretomanid, two were borderline resistant (MIC = 2-4 mg/L) and the remaining 12 isolates were highly resistant (MIC ≥ 16 mg/L) to the drug. Five resistant isolates did not harbor mutations in the known pretomanid resistant genes. CONCLUSIONS: Our results further support a breakpoint of 0.5 mg/L for a non-L1 MTB population, which is characteristic of China. Further, our data point to an unexpected high (14/475, 3%) pre-existing pretomanid resistance rate in the country, as well as to the existence of yet-to-be-discovered pretomanid resistance genes.

The Role of Efflux Pumps transporter in Multi-drug Resistant Tuberculosis: Mycobacterial memberane protein(MmpL5).

BACKGROUND: The overexpression of efflux pumps (Eps) was reported to contribute to multidrug resistant tuberculosis (MDR-TB). Increases in Eps that expel structurally unrelated drugs contribute to reduced susceptibility by decreasing the intracellular concentration of antibiotics. In the present study, an association of mycobacterial membrane protein (MmpS5-MmpL5) Ep and its gene regulator (Rv0678) was investigated in MDR-tuberculosis isolates. METHODS: MTB strains were isolated from patients at two different intervals, i.e., once when they had persistent symptoms despite 3-15 ≥ months of treatment and once when they had started new combination therapy ≥2-3 months. Sputum specimens were subjected to Xpert MTB/rifampicin test and then further susceptibility testing using proportional method and multiplex polymerase chain reaction (PCR) were performed on them. The isolates were characterized using both 16S-23S RNA and hsp65 genes spacer (PCR-restriction fragment length polymorphism). Whole-genome sequencing (WGS) was investigated on two isolates from culture-positive specimen per patient. The protein structure was simulated using the SWISS-MODEL. The input format used for this web server was FASTA (amino acid sequence). Protein structure was also analysis using Ramachandran plot. RESULTS: WGS documented deletion, insertion, and substitution in transmembrane transport protein MmpL5 (Rv0676) of Eps. Majority of the studied isolates (n = 12; 92.3%) showed a unique deletion mutation at three positions: (a) from amino acid number 771 (isoleucine) to 776 (valine), (b) from amino acid number 785 (valine) to 793 (histidine), and (c) from amino acid number 798 (leucine) to 806 (glycine)." One isolate (7.6%) had no deletion mutation. In all isolates (n = 13; 100%), a large insertion mutation consisting of 94 amino acid was observed "from amino acid number 846 (isoleucine) to amino acid number 939 (leucine)". Thirty-eight substitutions in Rv0676 were detected, of which 92.3% were identical in the studied isolates. WGS of mycobacterial membrane proteins (MmpS5; Rv0677) and its gene regulator (Rv0678) documented no deletion, insertion, and substitution. No differences were observed between MmpS5-MmpL5 and its gene regulator in isolates that were collected at different intervals. CONCLUSIONS: Significant genetic mutation like insertion, deletion, and substitution within transmembrane transport protein MmpL5 (Rv0676) can change the functional balance of Eps and cause a reduction in drug susceptibility. This is the first report documenting a unique amino acid mutation (insertion and deletion ≥4-94) in Rv0676 among drug-resistant MTB. We suggest the changes in Mmpl5 (Rv0676) might occurred due to in-vivo sub-therapeutic drug stress within the host cell. Changes in MmpL5 are stable and detected through subsequent culture-positive specimens.

Diagnostic Performance of STANDARD™ M10 Multidrug-resistant Tuberculosis Assay for Detection of Mycobacterium tuberculosis and Rifampicin and Isoniazid Resistance in Zimbabwe.

BACKGROUND: Although Zimbabwe has transitioned out of the 30 high-burden countries, it still remained in the 30 high multidrug-resistant (MDR)/rifampicin-resistant tuberculosis (TB) burden. Rapid detection of rifampicin (RIF) and isoniazid (INH) is essential for the diagnosis of MDR-TB. The World Health Organization has recommended the use of molecular WHO-recommended rapid diagnostic (mWRD) for TB and DR-TB. STANDARD™ M10 MDR-TB assay is a new molecular rapid diagnostic assay developed by SD Biosensor for the detection of Mycobacterium tuberculosis (MTB) and RIF and INF resistance. This study aims to determine the diagnostic accuracy of STANDARD™ M10 MDR-TB assay. METHODS: The study was conducted on 214 samples with different MTB and RIF and INH resistance status. The STANDARD™ M10 MDR-TB assay was performed according to the manufacturer's instructions. Xpert MTB/RIF Ultra, MGIT culture, and phenotypic drug susceptibility testing are used as comparative methods. RESULTS: The sensitivity and specificity of STANDARD™ M10 MDR-TB assay for the detection of MTB are 99% and 97.9%, respectively. The sensitivity and specificity of the assay for detection of MDR-TB were 97.8% and 100%, respectively. CONCLUSION: The STANDARD™ M10 MDR-TB assay demonstrated high diagnostic accuracy in the detection of MTB and RIF and INH resistance. This molecular assay can also be used as an alternative to other mWRD assays.

Rapid Detection of M. tuberculosis and Its Resistance to Rifampicin and Isoniazid with the mfloDx™ MDR-TB test.

BACKGROUND: Rapid detection of tuberculosis (TB) and its resistance are essential for the prompt initiation of correct drug therapy and for stopping the spread of drug-resistant TB. There is an urgent need for increased use of rapid diagnostic tests to control the threat of increased TB and multidrug-resistant TB (MDR-TB). METHODS: EMPE Diagnostics has developed a multiplex molecular diagnostic platform called mfloDx™ by combining nucleotide-specific padlock probe-dependent rolling circle amplification with sensitive lateral flow biosensors, providing visual signals, similar to a COVID-19 test. The first test kit of this platform, mfloDx™ MDR-TB can identify Mycobacterium tuberculosis (MTB) complex and its clinically significant mutations in the rpoB and katG genes and in the inhA promotor contributing resistance to rifampicin (RIF) and isoniazid (INH), causing MDR-TB. RESULTS: We have evaluated the performance of the mfloDx™ MDR-TB test on 210 sputum samples (110 from suspected TB cases and 100 from TB-negative controls) received from a tertiary care center in India. The clinical sensitivity for detecting MTB compared to acid-fast microscopy and mycobacteria growth indicator tube (MGIT) cultures was 86.4% and 84.9%, respectively. All the 100 control samples were negative indicating excellent specificity. In smear-positive sputum samples, the mfloDx™ MDR-TB test showed a sensitivity of 92.5% and 86.4% against MGIT culture and Xpert MTB/RIF, respectively. The clinical sensitivity for the detection of RIF and INH resistance in comparison with MGIT drug susceptibility testing was 100% and 84.6%, respectively, while the clinical specificity was 100%. CONCLUSION: From the above evaluation, we find mfloDx™ MDR-TB to be a rapid and efficient test to detect TB and its multidrug resistance in 3 h at a low cost making it suitable for resource-limited laboratories.

Genotypic and phenotypic drug resistance patterns of Mycobacterium tuberculosis isolated from presumptive pulmonary tuberculosis patients in Ethiopia: A multicenter study.

BACKGROUND: The emergence of drug-resistant tuberculosis (DR-TB) has been a major obstacle to global tuberculosis control programs, especially in developing countries, including Ethiopia. This study investigated drug resistance patterns and associated mutations of Mycobacterium tuberculosis Complex (MTBC) isolates from the Amhara, Gambella, and Benishangul-Gumuz regions of Ethiopia. METHODS: A cross-sectional study was conducted using 128 MTBC isolates obtained from patients with presumptive tuberculosis (TB). Phenotypic (BACTEC MGIT 960) and genotypic (MTBDRplus and MTBDRsl assays) methods were used for drug susceptibility testing. Data were entered into Epi-info and analyzed using SPSS version 25. Frequencies and proportions were determined to describe drug resistance levels and associated mutations. RESULTS: Of the 127 isolates recovered, 100 (78.7%) were susceptible to four first-line anti-TB drugs. Any drug resistance, polydrug resistance, and multi-drug resistance (MDR) were detected in 21.3% (27), 15.7% (20), and 15% (19) of the isolates, respectively, by phenotypic and/or genotypic methods. Mono-resistance was observed for Isoniazid (INH) (2, 1.6%) and Streptomycin (STR) (2, 1.6%). There were two genotypically discordant RIF-resistant cases and one INH-resistant case. One case of pre-extensively drug-resistant TB (pre-XDR-TB) and one case of extensively drug-resistant TB (XDR-TB) were identified. The most frequent gene mutations associated with INH and rifampicin (RIF) resistance were observed in the katG MUT1 (S315T1) (20, 76.9%) and rpoB (S531L) (10, 52.6%) genes, respectively. Two MDR-TB isolates were resistant to second-line drugs; one had a mutation in the gyrA MUT1 gene, and the other had missing gyrA WT1, gyrA WT3, and rrs WT1 genes without any mutation. CONCLUSIONS: The detection of a significant proportion of DR-TB cases in this study suggests that DR-TB is a major public health problem in Ethiopia. Thus, we recommend the early detection and treatment of DR-TB and universal full first-line drug-susceptibility testing in routine system.

Large-scale statistical analysis of Mycobacterium tuberculosis genome sequences identifies compensatory mutations associated with multi-drug resistance.

Tuberculosis (TB), caused by Mycobacterium tuberculosis, has a significant impact on global health worldwide. The development of multi-drug resistant strains that are resistant to the first-line drugs isoniazid and rifampicin threatens public health security. Rifampicin and isoniazid resistance are largely underpinned by mutations in rpoB and katG respectively and are associated with fitness costs. Compensatory mutations are considered to alleviate these fitness costs and have been observed in rpoC/rpoA (rifampicin) and oxyR'-ahpC (isoniazid). We developed a framework (CompMut-TB) to detect compensatory mutations from whole genome sequences from a large dataset comprised of 18,396 M. tuberculosis samples. We performed association analysis (Fisher's exact tests) to identify pairs of mutations that are associated with drug-resistance, followed by mediation analysis to identify complementary or full mediators of drug-resistance. The analyses revealed several potential mutations in rpoC (N = 47), rpoA (N = 4), and oxyR'-ahpC (N = 7) that were considered either 'highly likely' or 'likely' to confer compensatory effects on drug-resistance, including mutations that have previously been reported and validated. Overall, we have developed the CompMut-TB framework which can assist with identifying compensatory mutations which is important for more precise genome-based profiling of drug-resistant TB strains and to further understanding of the evolutionary mechanisms that underpin drug-resistance.

Impact of line probe assay-based molecular testing on individualized treatment in patients with rifampicin-resistant tuberculosis: data from the prospective INNOVA4TB cohort study in Ukraine.

BACKGROUND: Ukraine remains a high World Health Organization priority country for drug-resistant tuberculosis (TB). Rifampicin-resistant TB (RR-TB) has a more protracted, more complicated, and more expensive treatment. In 2021, Ukraine reported 4025 RR-TB cases - 5.4 times more (751) than all 30 European Union/ European Economic Area countries together. OBJECTIVES: The objective of the study was to determine the diagnostic accuracy of line probe assay (LPA), AID Autoimmun Diagnostika GmbH, for detecting resistance to anti-TB drugs and its clinical application for selecting treatment regimens. DESIGN: A prospective observational cohort study. METHODS: From May 2019 to June 2020, we consecutively enrolled patients with active TB hospitalized at the Regional Phthisiopulmonology Center (Vinnytsia, Ukraine), aged between 18 and 82 years. The LPA was performed in the Genetic Research Laboratory at National Pirogov Memorial Medical University, Vinnytsia, Ukraine. RESULTS: A total of 84 clinical specimens and 97 culture isolates from 126 TB patients were tested during the study. Accuracy (95% confidence interval) of LPA for clinical samples in comparison with phenotypic drug susceptibility test (DST) was 80.1 (68.5-89.0) for isoniazid (H), 74.7 (62.4-84.6) for rifampicin (R), 74.4 (62.5-84.1) for ethambutol, 71.4 (41.9-91.6) for streptomycin, 84.6 (62.4-96.5) for prothionamide/ethionamide, and 84.6 (73.6-92.3) for levofloxacin (Lfx), respectively. We found a significantly higher sensitivity of LPA for H, R, and Lfx for the culture isolates compared to clinical specimens (p < 0.05). LPA detected different mutations in 6 out of 17 (35.5%) patients susceptible to R by Xpert. A shorter treatment regimen with an injectable agent demonstrated a low suitability rate of 5% (8/156) in a cohort of RR-TB patients from Ukraine. CONCLUSION: Initial LPA testing accurately identifies resistance to anti-TB drugs and facilitates the selection of an appropriate treatment regimen, minimizing exposure to empirical therapy.

Study about the impact of rapid resistance detection on the treatment of patients with tuberculosis in Ukraine written by healthcare and biomedical professionals to better understand how we can improve the results of treatment and to prevent spreading of resistant bacteriaWhy was the study done? Ukraine has over 4000 patients with tuberculosis (TB) resistant to at least one drug (rifampicin) - five times that of all 30 European Union/European Economic Area countries combined. Unfortunately, only about 60% of such patients have been successfully treated in 2019. At that time, the majority of people suffering from tuberculosis in Ukraine, after checking resistance to rifampicin, initially received standard combinations of the first-line or second-line anti-TB medicines before the result of traditionally used tests (usually few weeks later) became available to individualize the treatment. Alternatively, the sputum could be transported to some overloaded reference laboratories located hundreds of km away from the treatment places.What did the researchers do? The INNOVA4TB team implemented rapid diagnostics of drug resistance in routine practice, guiding key antibiotics use in TB patients. A total of 181 samples from 126 individuals were tested during 2019-2020.What did the researchers find? This new diagnostic technology accurately detected resistance to 9 anti-TB drugs in sputum samples. It could be helpful to select appropriate TB treatment regimens, reducing time for decision from 1 month up to 2 days. Recommended at the study time 9-month shorter standardized treatment regimen with injectable agent was suitable only for 5% of patients for whom it was indicated in Vinnytsia region of Ukraine.What do the findings mean? The study has demonstrated successful implementation of the new molecular diagnostic technology from scratch in a country with restricted resources and limited TB laboratory capacity. This test can facilitate optimal distribution of available wards among patients with different profiles of resistance and correct choice between treatment options.

Risk factors of adult isoniazid-resistant and rifampicin-susceptible tuberculosis in Nanjing, 2019-2021.

INTRODUCTION: This study aimed to analyze the risk factors associated with isoniazid-resistant and rifampicin-susceptible tuberculosis (Hr-TB) in adults. METHOD: The clinical data of 1,844 adult inpatients diagnosed with culture-positive pulmonary tuberculosis (PTB) in Nanjing Second Hospital from January 2019 and December 2021 were collected. All culture positive strain from the patient specimens underwent drug susceptibility testing (DST). Among them, 166 patients with Hr-TB were categorized as the Hr-TB group, while the remaining 1,678 patients were classified as having drug-susceptible tuberculosis (DS-TB). Hierarchical logistic regression was employed for multivariate analysis to identify variables associated with Hr-TB. RESULTS: Multivariate logistic regression analysis revealed that individuals with diabetes mellitus (DM) (OR 1.472, 95% CI 1.037-2.088, p = 0.030) and a history of previous tuberculosis treatment (OR 2.913, 95% CI 1.971-4.306, p = 0.000) were at higher risk of developing adult Hr-TB, with this risk being more pronounced in male patients. Within the cohort, 1,640 patients were newly treated, and among them, DM (OR 1.662, 95% CI 1.123-2.461, p = 0.011) was identified as risk factors for Hr-TB. CONCLUSIONS: Diabetes mellitus is a risk factor for Hr-TB in adults, and the contribution of diabetes as a risk factor was more pronounced in the newly treatment or male subgroup. And previous TB treatment history is also a risk factor for Hr-TB in adults.

Discordant results between Xpert MTB/RIF assay and Bactec MGIT 960 culture system regarding the detection of rifampin-resistant Mycobacterium tuberculosis isolates in Wenzhou, China.

This study aimed to assess the possible causes of discordant results between Xpert MTB/RIF (Xpert) and Bactec MGIT 960 Culture System (MGIT960) regarding rifampicin (RIF) susceptibility in Mycobacterium tuberculosis. Patients with previous RIF-resistant tuberculosis who were admitted to Wenzhou Central Hospital from January 2020 to December 2022 were enrolled. The isolates obtained from these patients were subjected to RIF susceptibility tests using Xpert and MGIT960, and the minimum inhibitory concentration (MIC) of RIF was determined by the MYCOTB MIC plate test. Additionally, molecular docking and molecular dynamics (MD) simulations were performed to evaluate the binding efficacy of rpoB and RIF based on rpoB mutations detected in the isolates with discordant RIF susceptibility results. A total of 28 isolates with discordant RIF susceptibility test results were detected, 15 of them were RIF susceptible with MICs ≤ 0.5 µg/mL. Twelve out of 15 isolates contained borderline RIF resistance-associated mutations [L430P (n = 6), H445N (n = 6)], 1 isolate had D435Y and Q429H double mutation, and the remaining 2 isolates had a silent (Q432Q) mutation. Compared with the affinity of RIF toward the wild type (WT) (-45.83 kcal/mol) by MD, its affinity toward L452P (-55.52 kcal/mol), D435Y (-47.39 kcal/mol), L430P (approximately -69.72 kcal/mol), H445N (-49.53 kcal/mol), and Q429H (-55.67 kcal/mol) increased. Borderline RIF resistance-associated mutations were the main cause for the discordant RIF susceptibility results between Xpert and MGIT960, and the mechanisms of the resistance need further investigated.IMPORTANCEThis study is aimed at assessing discordant results between Xpert MTB/RIF (Xpert) assay and Bactec MGIT 960 Culture System (MGIT960) regarding the detection of rifampicin (RIF)-resistant Mycobacterium tuberculosis isolates in Wenzhou, China. The discordant results of RIF between these two assays were mainly caused by borderline RIF resistance-associated mutations, subsequently by silent mutations of rpoB. Borderline RIF resistance- associated mutations detected in our study were demonstrated to not be affected by the affinity of rpoB and RIF by molecular dynamics, and the mechanism of resistance was needed to be clarified. For the discordant results of RIF by Xpert and MGIT960 that occurred, rpoB DNA sequencing was recommended to investigate its association with resistance to RIF.

Sputum culture reversion in longer treatments with bedaquiline, delamanid, and repurposed drugs for drug-resistant tuberculosis.

Sputum culture reversion after conversion is an indicator of tuberculosis (TB) treatment failure. We analyze data from the endTB multi-country prospective observational cohort (NCT03259269) to estimate the frequency (primary endpoint) among individuals receiving a longer (18-to-20 month) regimen for multidrug- or rifampicin-resistant (MDR/RR) TB who experienced culture conversion. We also conduct Cox proportional hazard regression analyses to identify factors associated with reversion, including comorbidities, previous treatment, cavitary disease at conversion, low body mass index (BMI) at conversion, time to conversion, and number of likely-effective drugs. Of 1,286 patients, 54 (4.2%) experienced reversion, a median of 173 days (97-306) after conversion. Cavitary disease, BMI < 18.5, hepatitis C, prior treatment with second-line drugs, and longer time to initial culture conversion were positively associated with reversion. Reversion was uncommon. Those with cavitary disease, low BMI, hepatitis C, prior treatment with second-line drugs, and in whom culture conversion is delayed may benefit from close monitoring following conversion.

Determinants of sputum culture conversion time in multidrug-resistant tuberculosis patients in ALERT comprehensive specialized hospital, Addis Ababa, Ethiopia: A retrospective cohort study.

INTRODUCTION: The treatment response of multi-drug resistance tuberculosis (MDR-Tuberculosis) patients is mainly dictated by the sputum culture conversion. An earlier culture conversion is a remarkable indicator of the improvement in the treatment response. In this study, we aimed to determine the time to culture conversion and its associated factors among MDR-Tuberculosis patients in All Africa Leprosy, Tuberculosis and Rehabilitation Training Center (ALERT) Hospital, Addis Ababa, Ethiopia. METHODS: A retrospective cohort study was conducted on 120 MDR-Tuberculosis patients attending ALERT Hospital from 2018-2022. Kaplan-Meier methods were used to determine the time to initial sputum culture conversion. All relevant laboratory, socio-demographic characteristics, and other clinical data were collected by chart abstraction using a structure data extraction form. The log-rank test was used to determine the survival rate. To identify the predictors of culture conversion, bivariate and multivariate Cox proportional hazard regression analysis was used. The hazard ratio (HR) with a 95% confidence interval was used to estimate the effect of each variable on the initial culture conversion. A test with a P value of < 0.05 was considered statistically significant. RESULTS: From the total of 120 study participants, 89.2% (107/120) have shown a successful culture conversion. The median age of the participants was 30 years (IQR = 12). The study participants were followed for 408.6 person-months (34.05 person-years). The median time to initial sputum culture conversion was 80 days. The median time to initial sputum culture conversion among HIV-positive and HIV-negative participants was 61 days (IQR = 58-63.5) and 88 days (IQR = 75-91), respectively. HIV-negative and patients with previous treatment history were shown to be the predictor for a prolonged time to initial sputum culture conversion, (aHR = 0.24 (95% CI: 0.1-0.4), P value <0.001) and (aHR = 0.47 (95% CI: 0.31-0.71), P value <0.001) respectively. CONCLUSION: The median time to sputum culture conversion for HIV positive was found to be 61 days in our study. Notably, patients with a history of previous anti-tuberculosis treatment, HIV-negative status, and higher bacillary load at baseline exhibited delayed culture conversion. These findings underscore the importance of considering such patient characteristics in the management of MDR-TB cases, as tailored interventions and close monitoring may lead to more favorable treatment outcomes. By identifying individuals with these risk factors early in the treatment process, healthcare providers can implement targeted strategies to optimize patient care and improve overall treatment success rates in MDR-TB management programs.

Delineating the evolutionary pathway to multidrug-resistant outbreaks of a Mycobacterium tuberculosis L4.1.2.1/Haarlem sublineage.

OBJECTIVES: We sought to capture the evolutionary itinerary of the Mycobacterium tuberculosis L4.1.2.1/Haarlem sublineage in northern Tunisia, where it caused a major multidrug-resistant (MDR) tuberculosis outbreak in a context strictly negative for HIV infection. METHODS: We combined whole genome sequencing and Bayesian approaches using a representative collection of drug-susceptible and drug-resistant L4.1.2.1/Haarlem clinical strains (n = 121) recovered from the outbreak region over 16 years. RESULTS: In the absence of drug resistance, the L4.1.2.1/Haarlem sublineage showed a propensity for rapid transmission as witnessed by the high clustering (44.6%) and recent transmission rates (25%), as well as the reduced mean distance between genome pairs. The entire pool of L4.1.2.1/Haarlem MDR strains was found to be linked to either the aforementioned major outbreak (68 individuals, 2001-2016) or to a minor, newly uncovered outbreak (six cases, 2001-2011). Strikingly, the two outbreaks descended independently from a common ancestor that can be dated back to 1886. CONCLUSIONS: Our data point to the intrinsic propensity for rapid transmission of the M. tuberculosis L4.1.2.1/Haarlem sublineage in northern Tunisia, linking the overall MDR tuberculosis epidemic to a single ancestor. These findings bring out the important role of the bacillus' genetic background in the emergence of successful MDR M. tuberculosis clones.

Drug-resistant Mycobacterium tuberculosis among Nepalese patients at a tuberculosis referral center.

BACKGROUND: Multidrug-resistant tuberculosis (MDR-TB), characterized by isoniazid and rifampicin resistance, is caused by chromosomal mutations that restrict treatment options and complicate tuberculosis management. This study sought to investigate the prevalence of pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) tuberculosis, as well as mutation pattern, in Nepalese patients with MDR/rifampicin-resistant (RR)-TB strains. METHODS: A cross-sectional study was conducted on MDR/RR-TB patients at the German Nepal Tuberculosis Project from June 2017 to June 2018. The MTBDRsl line probe assay identified pre-XDR-TB and XDR-TB. Pre-XDR-TB included MDR/RR-TB with resistance to any fluoroquinolone (FLQ), while XDR-TB included MDR/RR-TB with resistance to any FLQ and at least one additional group A drug. Mutation status was determined by comparing bands on reaction zones [gyrA and gyrB for FLQ resistance, rrs for SILD resistance, and eis for low-level kanamycin resistance, according to the GenoType MTBDRsl VER 2.0, Hain Lifescience GmbH, Nehren, Germany definition of pre-XDR and XDR] to the evaluation sheet. SPSS version 17.0 was used for data analysis. RESULTS: Out of a total of 171 patients with MDR/RR-TB, 160 had (93.57%) had MTBC, of whom 57 (35.63%) had pre-XDR-TB and 10 (6.25%) had XDR-TB. Among the pre-XDR-TB strains, 56 (98.25%) were FLQ resistant, while 1 (1.75%) was SLID resistant. The most frequent mutations were found at codons MUT3C (57.14%, 32/56) and MUT1 (23.21%, 13/56) of the gyrA gene. One patient had SLID resistant genotype at the MUT1 codon of the rrs gene (100%, 1/1). XDR-TB mutation bands were mostly detected on MUT1 (30%, 3/10) of the gyrA and rrs, MUT3C (30%, 3/10) of the gyrA, and MUT1 (30%, 3/10) of the rrs. CONCLUSIONS: Pre-XDR-TB had a significantly higher likelihood than XDR-TB, with different specific mutation bands present in gyrA and rrs genes.

Prevalence, drug resistance, and genotypic diversity of the RDRio subfamily of Mycobacterium tuberculosis in Ecuador: a retrospective analysis for years 2012-2016.

Introduction: A major sublineage within the Mycobacterium tuberculosis (MTB) LAM family characterized by a new in-frame fusion gene Rv3346c/55c was discovered in Rio de Janeiro (Brazil) in 2007, called RDRio, associated to drug resistance. The few studies about prevalence of MTB RDRio strains in Latin America reported values ranging from 3% in Chile to 69.8% in Venezuela, although no information is available for countries like Ecuador. Methods: A total of 814 MTB isolates from years 2012 to 2016 were screened by multiplex PCR for RDRio identification, followed by 24-loci MIRU-VNTR and spoligotyping. Results: A total number of 17 MTB RDRio strains were identified, representing an overall prevalence of 2.09% among MTB strains in Ecuador. While 10.9% of the MTB isolates included in the study were multidrug resistance (MDR), 29.4% (5/17) of the RDRio strains were MDR. Discussion: This is the first report of the prevalence of MTB RDRio in Ecuador, where a strong association with MDR was found, but also a very low prevalence compared to other countries in Latin America. It is important to improve molecular epidemiology tools as a part of MTB surveillance programs in Latin America to track the transmission of potentially dangerous MTB stains associated to MDR TB like MTB RDRio.

Population structure and spatial distribution of Mycobacterium tuberculosis in Ethiopia.

Ethiopia is one of the countries with a high tuberculosis (TB) burden, yet little is known about the spatial distribution of Mycobacterium tuberculosis (Mtb) lineages. This study identifies the spoligotyping of 1735 archived Mtb isolates from the National Drug Resistance Survey, collected between November 2011 and June 2013, to investigate Mtb population structure and spatial distribution. Spoligotype International Types (SITs) and lineages were retrieved from online databases. The distribution of lineages was evaluated using Fisher's exact test and logistic regression models. The Global Moran's Index and Getis-Ord Gi statistic were utilized to identify hotspot areas. Our results showed that spoligotypes could be interpreted and led to 4 lineages and 283 spoligotype patterns in 91% of the isolates, including 4% of those with multidrug/rifampicin resistance (MDR/RR) TB. The identified Mtb lineages were lineage 1 (1.8%), lineage 3 (25.9%), lineage 4 (70.6%) and lineage 7 (1.6%). The proportion of lineages 3 and 4 varied by regions, with lineage 3 being significantly greater than lineage 4 in reports from Gambella (AOR = 4.37, P < 0.001) and Tigray (AOR = 3.44, P = 0.001) and lineage 4 being significantly higher in Southern Nations Nationalities and Peoples Region (AOR = 1.97, P = 0.026) than lineage 3. Hotspots for lineage 1 were located in eastern Ethiopia, while a lineage 7 hotspot was identified in northern and western Ethiopia. The five prevalent spoligotypes, which were SIT149, SIT53, SIT25, SIT37 and SIT26 account for 42.8% of all isolates under investigation, while SIT149, SIT53 and SIT21 account for 52-57.8% of drug-resistant TB cases. TB and drug resistant TB are mainly caused by lineages 3 and 4, and significant proportions of the prevalent spoligotypes also influence drug-resistant TB and the total TB burden. Regional variations in lineages may result from both local and cross-border spread.

Evaluation of AAICare®-TB sequence analysis tool for accurate diagnosis of drug-resistant tuberculosis: A comparative study with TB-Profiler and Mykrobe.

A rapid and comprehensive drug susceptibility test is essential for eliminating drug resistant tuberculosis. Next generation sequencing (NGS) based susceptibility testing is being explored as a potential substitute for the conventional phenotypic and genotypic testing methods. However, the adoption of NGS based genotypic susceptibility testing depends on the availability of simple, accurate and efficient analysis tools. This preliminary study aimed to evaluate the performance of a Mycobacterium tuberculosis (Mtb) genome analysis pipeline, AAICare®-TB, for susceptibility prediction, in comparison to two widely used gDST prediction tools, TB-Profiler and Mykrobe. This study was performed in a National Reference Laboratory in India on presumptive drug-resistant tuberculosis (DR-TB) isolates. Whole genome sequences of the 120 cultured isolates were obtained through Illumina sequencing on a MiSeq platform. Raw sequences were simultaneously analysed using the three tools. Susceptibility prediction reports thus generated, were compared to estimate the total concordance and discordance. WHO mutation catalogue (1st edition, 2021) was used as the reference standard for categorizing the mutations. In this study, AAICare®-TB was able to predict drug resistance status for First Line (Streptomycin, Isoniazid, Rifampicin, Ethambutol and Pyrazinamide) and Second Line drugs (Fluoroquinolones, Second Line Injectables and Ethionamide) in 93 samples along with lineage and hetero-resistance as per the WHO guidelines.