Rifampicin-resistant Mycobacterium Tuberculosis Research Articles

A Study on Rifampicin Resistant Mycobacterium Tuberculosis in a Tertiary Care Hospital Setting in Mysuru, India

A Study on Rifampicin Resistant Mycobacterium Tuberculosis in a Tertiary Care Hospital Setting in Mysuru, India

Evaluation of genetic correlation with fluoroquinolones resistance in rifampicin-resistant Mycobacterium tuberculosis isolates

ObjectiveTo detect levofloxacin (LFX) and moxifloxacin (MFX) resistance among rifampicin-resistant tuberculosis (RR-TB) isolates, and predict the resistance level based on specific mutations in gyrA and gyrB genes. MethodsA total of 686 RR-TB isolates were collected from Chinese Drug Resistance Surveillance Program from 2013 to 2020. The minimum inhibitory concentrations (MICs) of 12 anti-TB drugs were acquired using the broth microdilution method, followed by whole genome sequencing (WGS) analysis. ResultsAmong the 686 RR isolates, the most prevalent resistance was to isoniazid (80.5 %) and ethambutol (28.4 %), followed by LFX (26.1 %) and MFX (21.9 %). The resistance rate of LFX (26.1%–99.4 %) was higher than that of MFX (21.9%–83.3 %) across various drug resistance patterns. Of the 180 fluoroquinolones (FQs) resistant isolates, 168 (93.3 %) had mutations in quinolone-resistant determining regions (QRDRs) with 21 mutation types, and Asp94Gly (32.7 %, 55/168) was the predominant mutation. Isolates with mutations in Asp94Asn and Asp94Gly were associated with high levels of resistance to LFX and MFX. Using broth microdilution method as gold standard, the sensitivities of WGS for LFX and MFX were 93.3 % and 98.0 %, and the specificities were 98.6 % and 95.0 %, respectively. ConclusionThe resistance rate of LFX was higher than that of MFX among various drug resistance patterns in RR-TB isolates. The gyrA Asp94Gly was the predominant mutation type underlying FQs resistance. However, no significant difference was observed between mutation patterns in gyrA gene and resistance level of FQs.

Prevalence of Smear-Positive, Rifampicin-Resistant Mycobacterium tuberculosis and Related Factors Among Residents with Cough in Northern Ethiopian Refugee Health Facilities.

To ascertain the prevalence of Mycobacterium tuberculosis (M.tb) among refugees suspected of tuberculosis (TB) and related risk factors, including smear-positive and Rifampicin-resistant M.tb. A cross-sectional study was conducted between January 2020 and May 2020 among 384 refugees in four refugee camps in Northwest Tigray, Ethiopia. Socio-demographic and clinical data were collected from refugees with a history of cough for more than two weeks prospectively. Spot-spot sputum samples were collected and transported in an ice box to the Shire Suhul Hospital Microbiology laboratory; and then examined using a Fluorescent Microscope. All smear-positive samples were further processed by GeneXpert to detect Rifampicin-resistant MTB. Data were analyzed using SPSS version 21 and a p-value <0.05 was considered statistically significant. The overall prevalence of smear-positive PTB infection was 5.5% (21/384), but No TB case was resistant to Rifampicin detected by GeneXpert MTB/RIF assay. About 70% of the smear-positive pulmonary TB identified were females. Five (23.8%) of the smear-positive pulmonary tuberculosis cases were co-infected by HIV. Sharing of drink and food materials (AOR = 4.36, 95% CI = 1.19-15.89), active TB contact (AOR 7.24, 95% CI = 1.62-32.125), BMI (AOR = 5.23, 95% CI = 1.28-21.29), opening window practice (AOR = 4.32, 95% CI = 1.02-18.30) and HIV status (AOR = 9.36, 95% CI = 1.64-53.35) were statistically significant predisposing factors. The prevalence of smear-positive pulmonary TB among northwest Tigray refugee camps was still high. The prevalence of TB/HIV co-infection was also high. Minimizing close contact with active TB cases, reducing malnutrition, rapid TB/HIV screening, and establishing a ventilation system can reduce the transmission of TB among refugees.

A cell-free strategy for host-specific profiling of intracellular antibiotic sensitivity and resistance

Antimicrobial resistance (AMR) is a pandemic spread across multiple infectious disease-causing microbes. To provide a host-specific tool to study antibiotic susceptibility and resistance, here we develop Klebsiella pneumoniae cell-free gene expression (CFE) systems from laboratory and clinical isolates. Using proteomics, we identify relative differences and unique proteins for these new CFE systems in comparison to an Escherichia coli MG1655 CFE model. Then we profile antimicrobial susceptibility in parallel with whole cells to quantify CFE antibiotic potency. Finally, we apply this native CFE tool to study AMR variants at a proof-of-concept level. Definably we show that RpoB H526L confers a 58-fold increase in CFE resistance to rifampicin—a genotype observed in rifampicin-resistant Mycobacterium tuberculosis clinical isolates. Overall, we provide a cell-free synthetic biology strategy for the profiling of antibiotic sensitivity and resistance from K. pneumoniae. While initial extract processing requires Biosafety Level 2, the CFE system is non-living, suitable for long-term storage and study in a Biosafety Level 1 lab. We anticipate the K. pneumoniae CFE bioassay is advantageous for host-specific antimicrobial testing, the characterisation of intracellular AMR variants and potentially structure-activity relationship studies.

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.

Pulmonary tuberculosis and multidrug-resistant Mycobacterium tuberculosis in northwestern Ethiopia: a hospital-based cross-sectional study among presumptive pulmonary tuberculosis patients.

Border areas are important sites for disseminating Mycobacterium tuberculosis among individuals living in such areas. This study examined patients with suspected pulmonary tuberculosis (PTB) visiting the Abrihajira and Metema hospitals in northwest Ethiopia to investigate the prevalence of rifampicin-resistant Mycobacterium tuberculosis (RR-MTB), multidrug-resistant Mycobacterium tuberculosis (MDR-MTB), and risk factors related to Mycobacterium tuberculosis infection. A hospital-based cross-sectional study was conducted from February to August 2021 among 314 PTB presumptive patients. Xpert MTB/RIF and line probe assays (LPA) were used to process sputum samples. Data were imported into the Epi-Data 3.1 program and exported to Statistical Package for the Social Sciences (SPSS) version 20.0 (SPSS, Chicago, IL, United States) to conduct the analysis. A logistic regression analysis was used to investigate the relationship between the dependent and independent variables. A value of p of <0.05 denoted statistical significance. Of the total (314) PTB presumptive patients who participated in this study, 178 (56.69%) were men, and 165 (52.5%) were from 25 to 50 years of age with a median age of 35.00 (inter-quartile: 25-45 years). Among all patients, 12.7% had PTB by Gene Xpert and 7/314 (2.23%) were resistant to rifampicin. Among patients enrolled, 4/314 (1.27%) had MDR-MTB (resistant to RIF and INH) by LPA. Regarding the risk factors assessed, primary level of education, sputum production, night sweating, respiratory disorder, contact history of TB, history of MDR-MTB infection, history of alcohol use, and cigarette smoking showed statistical significance with the prevalence of PTB (p ≤ 0.05). This study observed a high prevalence of PTB, RR-MTB, and MDR-MTB compared with many other previous studies conducted in Ethiopia. Among the assessed risk factors that could be associated with the prevalence of PTB, eight were statistically significant. This prevalence, resistance, and statistically significant variables are the evidence to which more emphasis should be given to the country's border areas.

Study on the resistance of rifampicin-resistant Mycobacterium tuberculosis to anti-tuberculosis drugs in group A

Objective: To summarize the resistance of rifampicin-resistant Mycobacterium tuberculosis to anti-tuberculosis drugs in group A. Methods: In the retrospective study, a total of 1 226 clinical isolates from suspected multidrug-resistant pulmonary tuberculosis patients in Beijing TB control system from 2016 to 2021 were identified as Mycobacterium tuberculosis (MTB) strains by MPB64 antigen detection test. Rifampicin-resistant tuberculosis (RR-TB) strains were screened by the phenotypic drug susceptibility using the proportion method. The drug susceptibilities of Levofloxacin(LFX), Moxifloxacin(MFX), Bedaquiline(BDQ) and Linezolid(LZD)were detected by the phenotypic drug susceptibility with microplate method. The drug resistance rate, drug resistance level and minimum inhibitory concentration (MIC) distribution of four anti-tuberculosis drugs in group A were analyzed. We calculated the demographic distribution of RR-TB, multidrug-resistant tuberculosis(MDR-TB), pre-extensively drug resistant tuberculosis (pre-XDR-TB), extensively drug resistant tuberculosis (XDR-TB) patients and the cross resistance of LFX and MFX, then summarized the drug-resistance spectrum of BDQ-resistant and LZD-resistant strains and the treatment outcome of RR-TB patients. Measurement data were expressed as rate or composition ratio,χ2 test was used between and within groups, and P<0.05 was considered statistically significant. Results: Among the 1 226 suspected multidrug-resistant pulmonary tuberculosis patients, the detection rates of RR/MDR/pre-XDR/XDR-TB patients were 20.8%(255/1 226), 15.2%(186/1 226), 5.7%(70/1 226), 0.5%(6/1 226), respectively. There were statistically significant differences in the distribution of patients with the four types of drug resistance in terms of age and treatment history (χ2=14.95, P=0.020;χ2=15.91, P=0.001). The drug resistance rates of LFX, MFX, BDQ and LZD in RR-TB patients were 27.5% (70/255), 27.5% (70/255), 0.4% (1/255) and 2.4% (6/255), respectively. The MICs of LFX, MFX and LZD-susceptible MTB were mainly at 0.25 mg/L, and the MIC of BDQ-susceptible MTB was mainly concentrated at 0.03 mg/L. 25.1% (64/255) of the RR MTB were resistant to both LFX and MFX, and 6 strains were resistant to LFX or MFX, showing incomplete two-way cross resistance. One BDQ-resistant strain and six LZD-resistant strains were detected. The treatment success rate of RR-TB patients was 74.4% (151/203), and there were statistically significant differences in treatment outcomes between resistant and sensitive patients on the LFX-containing treatment regimen (Fisher's exact test, P=0.012). Conclusions: The prevalence of fluoroquinolones (LFX and MFX) resistance in rifampicin-resistant MTB is very serious. LFX and MFX show incomplete bidirectional cross-resistance. BDQ and LZD have the most promising future in the treatment of MDR-TB. Improve drug-resistance testing will help to further improve the success rate of treatment.

Emergence of bedaquiline-resistant tuberculosis and of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis strains with rpoB Ile491Phe mutation not detected by Xpert MTB/RIF in Mozambique: a retrospective observational study

In 2021, an estimated 4800 people developed rifampicin-resistant tuberculosis in Mozambique, 75% of which went undiagnosed. Detailed molecular data on rifampicin-resistant and multidrug-resistant (MDR) tuberculosis are not available. Here, we aimed at gaining precise data on the determinants of rifampicin-resistant and MDR tuberculosis in Mozambique. In this retrospective observational study, we performed whole-genome sequencing of 704 rifampicin-resistant Mycobacterium tuberculosis complex (Mtbc) strains submitted to the National Tuberculosis Reference Laboratory (NTRL) in Maputo, Mozambique, between 2015 and 2021. Phylogenetic strain classification, genomic resistance prediction, and cluster analysis were performed. Between Jan 1, 2015, and July 31, 2021, 2606 Mtbc isolates with an isoniazid or rifampicin resistance were identified in the NTRL biobank, of which, 1483 (56·9%) were from men, 1114 (42·7%) from women, and nine (0·4%) were unknown. Genome-based drug-resistant prediction classified 704 Mtbc strains as rifampicin resistant. 628 (89%) of the 704 Mtbc strains were classified MDR; of those, 146 (23%) were pre-extensively drug resistant (pre-XDR; additional fluoroquinolone resistance), and 24 (4%) extensively drug resistant (XDR; combined fluoroquinolone and bedaquiline resistance). Overall, 61 (9%) of 704 strains revealed resistance to bedaquiline: five (7%) of 76 rifampicin resistant plus bedaquiline resistant, 32 (7%) of 458 MDR plus bedaquiline resistant, and 24 (100%) of 24 XDR. Prevalence of bedaquiline resistance increased from 3% in 2016 to 14% in 2021. The cluster rate (12 single-nucleotide polymorphism threshold) was 42% for rifampicin-resistant strains, 78% for MDR strains, 94% for pre-XDR strains, and 96% for XDR Mtbc strains. 31 (4%) of 704 Mtbc strains, belonging to a diagnostic escape outbreak strain previously described in Eswatini (group_56), had an rpoB Ile491Phe mutation which is not detected by Xpert MTB/RIF (no other rpoB mutation). Of these, 23 (74%) showed additional resistance to bedaquiline, 13 (42%) had bedaquiline and fluoroquinolone resistance, and two (6%) were bedaquiline, fluoroquinolone, and delamanid resistant. Pre-XDR resistance is highly prevalent among MDR Mtbc strains in Mozambique and so is bedaquiline resistance; and the frequency of bedaquiline resistance quadrupled over time and was found even in Mtbc strains without fluoroquinolone resistance. Importantly, strains with Ile491Phe mutation were frequent, accounting for 31% (n=10) of MDR plus bedaquiline-resistant strains and 54% (n=13) of XDR Mtbc strains. Given the current diagnostic algorithms and treatment regimens, both the emergence of rifampicin resistance due to Ile491Phe and bedaquiline resistance might jeopardise MDR tuberculosis prevention and care unless sequencing-based technology is rolled out. The potential cross border spread of diagnostic escape strains needs further investigation. The German Ministry of Health through the Seq_MDRTB-Net project, the Deutsche Forschungsgemeinschaft under Germany's Excellence Strategy Precision Medicine in Inflammation and the Research Training Group 2501 TransEvo, the Leibniz Science Campus Evolutionary Medicine of the Lung, and the German Ministry of Education and Research via the German Center for Infection Research.

Bedaquiline Resistance and Molecular Characterization of Rifampicin-Resistant Mycobacterium Tuberculosis Isolates in Zhejiang, China

Bedaquiline Resistance and Molecular Characterization of Rifampicin-Resistant Mycobacterium Tuberculosis Isolates in Zhejiang, China

Impact of socio-demographic factors on prevalence of rifampicin resistance among patients attending some hospitals in Yobe state of Nigeria

Rifampicin resistance in Mycobacterium tuberculosis is a public health concern in many countries including Nigeria. There is an increase of multidrug resistant tuberculosis which results to morbidity and mortality among presumptive tuberculosis patients. Data regarding Rifampicin resistant tuberculosis in the study area are lacking. A one year prospective, cross-sectional, laboratory based study was carried out among patients attending Direct Observed Treatment Short course in Damaturu, Gashua and potiskum specialist hospitals, Yobe State, Nigeria; from January, 2020 to December, 2020. Sputum samples were collected from consented / assented participants and analyzed using MTB/RIF assay (Cepheid, GeneXpert USA). The data were analyzed using Person Chi-Square and p≤0.05 considered the presence of significant relationship and results were presented in tables and charts. Out of 400 studied participants, males had a prevalence of 2.5% (10/400) while females had 0.8% (3/400). Age group 30-39 years had the highest prevalence of 1.5%. The results show significant relationship between age, gender, and marital status, in the study area. This study confirmed the presence of Rifampicin resistant tuberculosis in the study area. Therefore, there’s need to have a public health awareness and strengthen the laboratory capacity for diagnosis and make the services available and accessible to the patients who need them.

Isoniazid Resistance Level and Associated Resistance Conferring-mutations in Rifampicin Resistant Mycobacterium tuberculosis

Background: Multidrug resistant tuberculosis (MDR-TB) is a global public health problem causing treatment failure. Rifampicin (RIF) resistance has been used as a surrogate marker for MDR-TB diagnosis, but level of isoniazid (INH) resistance and associated resistance conferring mutations for INH in rifampicin-resistant TB cases are little known. Objective: The objective of this study was to determine level of isoniazid resistance and associated resistance conferring mutations in rifampicin resistant Mycobacterium tuberculosis (MTB) in sputum samples. Methods: A total 53 RIF resistant MTB isolates in sputum, detected by Xpert-MTB RIF assay were enrolled in the study. Culture positive samples were tested by BACTEC MGIT 960 system and level of isoniazid resistance was determined, defined as minimum inhibitory concentration of INH of &gt;0.4 µg/mL and 0.1-0.4 µg/mL as high level and low level INH resistance respectively. Distribution of mutation in katG (codon 315) and inhA promoter (-5, -8, -15 and -16) genes by Real-time PCR among the different degrees of INH resistance was investigated. Results: Among the growth positive isolates, 68.8% of the resistant isolates had high level INH resistance, where katG was found to be the prominent mutation, with or without combined with inhA mutation. Positive predictive value (PPV) of katG mutation was 84.6% in detecting a high level of INH resistance. Low level resistance was present in 31.3% isolates, conferring mutation in inhA and katG in equal percentage (40%), but no detectable mutations were found in 20% low level INH resistant MTB isolates. The PPV of inhA mutation was 33.3% in detection low level resistance. Conclusion: Most of the INH resistant isolates conferred high level resistance and were associated with katG mutation. Evaluation of level INH resistance before using high dose INH will help to avoid dose dependent toxicity and to determine an appropriate treatment regimen. Bangladesh Medical Res Counc Bull 2023; 49(2): 120-125

Detection of rpoB gene mutations in clinical isolates of Rifampicin-resistant Mycobacterium tuberculosis in Makassar City, South Sulawesi, Indonesia

Detection of rpoB gene mutations in clinical isolates of Rifampicin-resistant Mycobacterium tuberculosis in Makassar City, South Sulawesi, Indonesia

Analysis of Discordance between Genotypic and Phenotypic Assays for Rifampicin-Resistant Mycobacterium tuberculosis Isolated from Healthcare Facilities in Mthatha.

The study sought to determine the rate of discordant results between genotypic and phenotypic tests for the diagnosis of drug-resistant tuberculosis (DR-TB). Sputum samples and cultured isolates from suspected DR-TB patients were, respectively, analyzed for Mycobacterium tuberculosis by Xpert® MTB/RIF (Cepheid, Sunnyvale, CA, USA) and line probe assays (LPA) (Hain, Nehren, Germany). Discrepant rifampicin (RMP)-resistant results were confirmed using BACTEC MGIT960 (BD, New York, NY, USA). Of the 224 RMP-resistant results obtained by Xpert MTB/RIF, 5.4% were susceptible to RMP by LPA. MGIT960 showed a 75% agreement with LPA. The discrepancy was attributed to either heteroresistance or DNA contamination during LPA testing in 58.3% of cases. In 25% of the samples showing agreement in RMP resistance between Xpert MTB/RIF and MGIT960, the discrepancy was attributed to laboratory errors causing false RMP susceptible results with LPA. In 16.7% of the cases, the discrepancy was attributed to false RMP susceptible results with Xpert MTB/RIF. Out of the 224 isolates, susceptibility to isoniazid (INH) by LPA was performed in 73.7% RMP-resistant isolates, of which, 80.6% were resistant. All RMP-resistant isolates by Xpert MTB/RIF were confirmed in 98.5% by LPA if TB isolates were resistant to INH, but were only confirmed in 81.3% if TB isolates were susceptible to INH (p < 0.001). In conclusion, laboratory errors should be considered when investigating discordant results.

Genetic Determinants of Rifampicin Resistance of Mycobacterium tuberculosis not Included in the Russian Molecular Genetic Test Systems

Genetic Determinants of Rifampicin Resistance of Mycobacterium tuberculosis not Included in the Russian Molecular Genetic Test Systems

Effect of compensatory evolution in the emergence and transmission of rifampicin-resistant Mycobacterium tuberculosis in Cape Town, South Africa: a genomic epidemiology study

Experimental data show that drug-resistance-conferring mutations are often associated with a decrease in the replicative fitness of bacteria in vitro, and that this fitness cost can be mitigated by compensatory mutations; however, the role of compensatory evolution in clinical settings is less clear. We assessed whether compensatory evolution was associated with increased transmission of rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa. We did a genomic epidemiological study by analysing available M tuberculosis isolates and their associated clinical data from individuals routinely diagnosed with rifampicin-resistant tuberculosis in primary care and hospitals in Khayelitsha, Cape Town, South Africa. Isolates were collected as part of a previous study. All individuals diagnosed with rifampicin-resistant tuberculosis and with linked biobanked specimens were included in this study. We applied whole-genome sequencing, Bayesian reconstruction of transmission trees, and phylogenetic multivariable regression analysis to identify individual and bacterial factors associated with the transmission of rifampicin-resistant M tuberculosis strains. Between Jan 1, 2008, and Dec 31, 2017, 2161 individuals were diagnosed with multidrug-resistant or rifampicin-resistant tuberculosis in Khayelitsha, Cape Town, South Africa. Whole-genome sequences were available for 1168 (54%) unique individual M tuberculosis isolates. Compensatory evolution was associated with smear-positive pulmonary disease (adjusted odds ratio 1·49, 95% CI 1·08-2·06) and a higher number of drug-resistance-conferring mutations (incidence rate ratio 1·38, 95% CI 1·28-1·48). Compensatory evolution was also associated with increased transmission of rifampicin-resistant disease between individuals (adjusted odds ratio 1·55; 95% CI 1·13-2·12), independent of other patient and bacterial factors. Our findings suggest that compensatory evolution enhances the in vivo fitness of drug-resistant M tuberculosis genotypes, both within and between patients, and that the in vitro replicative fitness of rifampicin-resistant M tuberculosis measured in the laboratory correlates with the bacterial fitness measured in clinical settings. These results emphasise the importance of enhancing surveillance and monitoring efforts to prevent the emergence of highly transmissible clones capable of rapidly accumulating new drug resistance mutations. This concern becomes especially crucial at present, because treatment regimens incorporating novel drugs are being implemented. Funding for this study was provided by a Swiss and South Africa joint research award (grant numbers 310030_188888, CRSII5_177163, and IZLSZ3_170834), the European Research Council (grant number 883582), and a Wellcome Trust fellowship (to HC; reference number 099818/Z/12/Z). ZS-D was funded through a PhD scholarship from the South African National Research Foundation and RMW was funded through the South African Medical Research Council.

Application of the real-time PCR Taqman allelic discrimination assay for the detection of Isoniazid and/or Rifampicin resistant Mycobacterium Tuberculosis from clinical samples

Background: Drug-resistant Tuberculosis (DR-TB) is challenging public health problem in countries with high tuberculosis ​​prevalence and limited resources. Developing and applying the most appropriate and effective methods for diagnosing DR-TB from clinical samples is necessary, allowing a more rapid detection method for large-scale screening. Methods: Applying real-time PCR Taqman allelic discrimination with a PCR Taqman probes panel to identifying the DR-TB associated mutations in rpoB and katG of Mycobacterium Tuberculosis from isolates and clinical samples. Results: Comparing results of the real-time PCR allelic and DNA sequencing results, the sensitivity and specificity for Isoniazid resistance detection by analysing katG were found 95%(75.1 - 99.8) and 100%, Rifampicin resistance determining region (RRDR) of rpoB were found 95.5(77.16 - 99.88) and 100%, respectively. The real-time PCR TaqMan allelic discrimination also showed the sensitivities 100% for both katG and rpoB, and the specificities were 93.55% (78.58 - 99.21) for the rpoB and 93.94% (79.77 - 99.26) for the katG from clinical samples. Conclusions: This study showed that the real-time PCR taqman allelic discrimination assay is useful for detection of TB and DR-TB because of an accurate and rapid diagnosis in the early stages. Key words: rug-resistant, Tuberculosis, clinical samples, real-time PCR taqman allelic discrimination assay, Mycobacterium tuberculosis.

Quinolone-3-amidoalkanol: A New Class of Potent and Broad-Spectrum Antimicrobial Agent.

Herein, we describe 39 novel quinolone compounds bearing a hydrophilic amine chain and varied substituted benzyloxy units. These compounds demonstrate broad-spectrum activities against acid-fast bacterium, Gram-positive and -negative bacteria, fungi, and leishmania parasite. Compound 30 maintained antitubercular activity against moxifloxacin-, isoniazid-, and rifampicin-resistant Mycobacterium tuberculosis, while 37 exhibited low micromolar activities (<1 μg/mL) against World Health Organization (WHO) critical pathogens: Cryptococcus neoformans, Acinetobacter baumannii, and Pseudomonas aeruginosa. Compounds in this study are metabolically robust, demonstrating % remnant of >98% after 30 min in the presence of human, rat, and mouse liver microsomes. Several compounds thus reported here are promising leads for the treatment of diseases caused by infectious agents.

Multiplex LNA probe-based RAP assay for rapid and highly sensitive detection of rifampicin-resistant Mycobacterium tuberculosis.

The World Health Organization (WHO) Global tuberculosis Report 2021 stated that rifampicin-resistant tuberculosis (RR-TB) remains a major public health threat. However, the in-practice diagnostic techniques for RR-TB have a variety of limitations including longer time, lack of sensitivity, and undetectable low proportion of heterogeneous drug resistance. Here we developed a multiplex LNA probe-based RAP method (MLP-RAP) for more sensitive detection of multiple point mutations of the RR-TB and its heteroresistance. A total of 126 clinical isolates and 78 sputum samples collected from the National Tuberculosis Reference Laboratory, China CDC, were tested by MLP-RAP assay. In parallel, qPCR and Sanger sequencing of nested PCR product assay were also performed for comparison. The sensitivity of the MLP-RAP assay could reach 5 copies/μl using recombinant plasmids, which is 20 times more sensitive than qPCR (100 copies/μl). In addition, the detection ability of rifampicin heteroresistance was 5%. The MLP-RAP assay had low requirements (boiling method) for nucleic acid extraction and the reaction could be completed within 1 h when placed in a fluorescent qPCR instrument. The result of the clinical evaluation showed that the MLP-RAP method could cover codons 516, 526, 531, and 533 with good specificity. 41 out of 78 boiled sputum samples were detected positive by MLP-RAP assay, which was further confirmed by Sanger sequencing of nested PCR product assay, on the contrary, qPCR was able to detect 32 samples only. Compared with Sanger sequencing of nested PCR product assay, both the specificity and sensitivity of the MLP-RAP assay were 100%. MLP-RAP assay can detect RR-TB infection with high sensitivity and specificity, indicating that this assay has the prospect of being applied for rapid and sensitive RR-TB detection in general laboratories where fluorescent qPCR instrument is available.

Molecular Epidemiology of Rifampicin-Resistant Mycobacterium tuberculosis by GeneXpert MTB/RIF Assay in Renal Transplant Recipients: A Single-Center Experience.

There are scarce data on the incidence and resistance pattern of rifampicin-resistant Mycobacterium tuberculosis among kidney transplant recipients. This is a retrospective, single- center study of kidney transplantrecipients suspected of M. tuberculosis infection. The GeneXpert assay we used detected mutations in the rpoB gene that confer rifampicin resistance using 5 overlapping probes (A, B, C, D, and E). The probes can detect mutations in the codons 507 to 511 (probe A), 511 to 518 (probe B), 518 to 523 (probe C), 523 to 529 (probe D), and 529 to 533 (probe E).We also detailed the treatment protocol and outcomes of kidney transplantrecipients infected with rifampicin-resistant M. tuberculosis. In total, 2700 samples were processed during the period from October 2018 to February 2022 with successful results in 2640 samples (97.04%). One hundred and ninety (7.19%) samples were positive for M.tuberculosis, and rifampicin resistance was detected in 12 (0.45%) cases (11 pulmonary, 1 genitourinary). The most common rpoB mutation was located in the region of probe E (75.0%), followed by probe A (16.6%) and in 1 combination probe DE (8.33%). The rpoB mutations were not observed in probe B and probe C. Six patients received bedaquiline-based treatmentfor a short course of 11 months, whereas the other 6 patients required a long course of 18 to 20 months. Three patients died, 2 were lost to follow-up, and 7 were cured. During treatment, 4 patients experienced acute rejection, and 1 graft loss was reported. We report for the first time the incidence and pattern of rifampicin resistance among kidney transplant recipients with tuberculosis infection. Further investigations are required for exploring the molecular and clinical phenotypes.

Rifampicin resistant Mycobacterium tuberculosis and associated factors among presumptive pulmonary tuberculosis patients in Mogadishu, Somalia.

Multi-drug resistant Mycobacterium tuberculosis is a growing public health problem in developing countries including Somalia. Although, the prevalence of multi-drug resistant tuberculosis among new and retreated cases is high, data on GeneXpert- Mycobacterium tuberculosis/rifampicin-resistant assay, which is a surrogate marker for multidrug resistance, is not well explored in Mogadishu. To determine the prevalence of rifampicin-resistant Mycobacterium tuberculosis and its associated factors among presumptive pulmonary tuberculosis patients visiting tuberculosis centers in Mogadishu, Somalia. A multicenter cross-sectional study was conducted in three tuberculosis treatment centers from March 12 to April 30, 2021. Laboratory professionals collected sputum sample consecutively from presumptive pulmonary tuberculosis participants and performed a GeneXpert assay to determine the rifampicin resistance. Socio-demographic and clinical data were collected using structured questionnaire. Logistic regression analyses were performed to assess factors associated with rifampicin resistance using an adjusted odds ratio at a 95% confidence interval. Statistical significance was considered at a p-value of less than 0.05. A total of 370 presumptive tuberculosis suspects were included; of whom 58.4% were females and the mean age of the participants was 44.3 ± 14 years. Mycobacterium tuberculosis was detected in 63 (17%) (95% confidence interval = 13.2-20.8) suspects. Of these the prevalence of rifampicin-resistant Mycobacterium tuberculosis was 35% (95% confidence interval = 30.2-39.8). Anti-tuberculosis treatment history (adjusted odds ratio = 4.1; 95% confidence interval = 1.91-6.75), monthly income less than $100 USD (adjusted odds ratio = 2.2; 95% confidence interval = 1.77-5.98) and being diagnosed with Asthma (adjusted odds ratio = 2.63; 95% confidence interval = 1.3-7.3) were significantly associated with rifampicin-resistant tuberculosis. A considerable proportion of rifampicin-resistant tuberculosis is reported in these study settings. The strong association between multidrug resistance tuberculosis and patients' retreatment history of tuberculosis, low income, and co-morbidity with asthma highlights the need for more efforts in tuberculosis treatment and monitoring programs to limit the emergence of multi-drug resistant strain in the study areas.