Dodecanoic acid & palmitic acid disarms rifampicin resistance by putatively targeting mycobacterial efflux pump Rv1218c

Background & objectives: Drug-resistant tuberculosis (TB) jeopardizes the treatment process with poor outcomes. Efflux pumps (EPs) belonging to the ABC transporter family in Mycobacterium tuberculosis confer resistance to rifampicin (RMP) besides genetic mutations thus serving as a target for a potential adjunct therapeutic inhibitory molecule. Rv1218c is one such pump that was previously reported to be active in multidrug-resistant TB clinical isolates. Methods: In this study, the inhibition potential of Rv1218c-EP was tested on 8 molecules that were shortlisted by in silico methods. These molecules were subjected to the minimum inhibitory concentration (MIC) determination, checkerboard drug combination assay, ethidium bromide-DNA binding assay, and in vitro and ex vivo cytotoxicity assay. Results: Based on the outcome of the study, two molecules dodecanoic acid (DA) and palmitic acid (PA) were found to be potential enough to decrease the MIC of RMP by 8 to 1000 folds against multidrug-resistant clinical isolates and Rv1218c expressing recombinant Mycobacterium smegmatis. Interpretation & conclusions: These molecules were also found to reduce the time taken by RMP to kill these drug-resistant Mycobacteria to 48 h, unlike control isolates that survived more than 240 h of RMP exposure. The functional concentration of both molecules was non-toxic to the epithelial and blood mononuclear cells. With further comprehensive scientific validation, PA and DA could be recommended as adjunct therapeutic molecules with first-line anti-TB drugs to treat drug-resistant TB.

Diagnostic efficacy of joint detection of Xpert MTBRIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid for smear-negative pulmonary tuberculosis / 中国热带医学

@#Abstract: Objective　To investigate the diagnostic value of joint detection of Mycobacterium tuberculosis rifampicin resistance gene (Xpert MTB/RIF), Mycobacterium tuberculosis ribonucleic acid (TB-RNA) and Mycobacterium tuberculosis deoxyribonucleic acid (TB-DNA) in bronchoalveolar lavage fluid for smear-negative pulmonary tuberculosis. Methods A total of 806 patients with suspected smear-negative pulmonary tuberculosis admitted to our hospital from May 2020 to July 2022 were selected, 506 patients diagnosed as bacterial negative pulmonary tuberculosis by clinical, X-ray and sputum samples were classified as bacterial negative pulmonary tuberculosis group, and the other 300 patients with non-tuberculous pulmonary disease were classified as non-tuberculous pulmonary disease group. XpertMTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid of all patients were detected. With clinical, X-ray and sputum specimen examination of mycobacterium tuberculosis as the gold standard, the diagnostic efficacy of alveolar lavage solution Xpert MTB/RIF, TB-RNA and TB-DNA alone and in combination was analyzed. Results The positive detection rates of Xpert MTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid of the smear-negative pulmonary tuberculosis group and the non-tuberculosis pulmonary disease group were 69.96% (354/506) and 2.67% (8/300), 61.46% (311/506) and 5.00% (15/300), and 63.64% (322/506) and 8.00% (24/300), respectively. The rates in the smear-negative pulmonary tuberculosis group were higher than those in the non-tuberculosis lung disease group, and the differences were statistically significant (χ2=342.005, 246.930, 235.687, P<0.01). Compared with the gold standard, the sensitivity, specificity, accuracy, positive predictive value and negative predictive value of Xpert MTB/RIF in the diagnosis of smear-negative pulmonary tuberculosis were 69.96%, 97.33%, 80.15%, 97.79% and 65.77%, respectively; those values of TB-RNA were 61.46%, 95.00%, 73.95%, 95.40% and 59.38%, respectively; those values of TB-DNA were 63.64%, 92.00%, 74.19%, 93.06% and 60.00%, respectively; those values of combined diagnosis with Xpert MTB/RIF, TB-RNA and TB-DNA were 61.26%, 100.00%, 75.68%, 100.00% and 60.48%, respectively; the specificity and positive predictive value of combined detection were higher than those of single detection (P<0.05). Conclusions The joint detection of Xpert MTB/RIF, TB-RNA and TB-DNA in bronchoalveolar lavage fluid can improve the diagnostic efficacy of smear-negative pulmonary tuberculosis and is worthy of clinical promotion and application.

Construction of a High-Throughput Mutation Library of Mycobacterium Rifampin Resistance Gene rpoB / 中山大学学报(医学科学版)

ObjectiveTo establish a mutation library of rifampicin resistance gene rpoB. MethodsThe ΔrpoB attB：：rpoB strain of Mycobacterium smegmatis （M. smegmatis） be constructed by homologous recombination and L5 attB phage integration site exchange. Based on the L5 attB plasmid exchange system and resistance selection medium， 48 clones are selected to verify plasmid replacement efficiency. Degenerate primers are designed every 3 bases in the rifampicin resistance determining region （RRDR）， and a full-coverage mutation library of 81 bases in RRDR region is obtained by PCR amplification. The library fragments are seamlessly cloned into the vector and transformed into Escherichia coli （E. coli）to form an E. coli mutation library. Based on the principle of plasmid exchange， the mutant plasmid library is transformed into the M. smegmatis strain ΔrpoB attB：：rpoB， and the original L5 attB site plasmid is replaced to form the M. smegmatis mutant library. The genotype of the library are determined by genome extraction， library construction and high-throughput sequencing. ResultsCompared with the wild-type rpoB gene （5 600 bp）， the amplified fragment of the rpoB knockout strain is 2 200 bp， which proved that the ΔrpoB attB：：rpoB conditional knockout strain of M. smegmatis is successfully constructed. The success rate of plasmid replacement is 100%. There were 540 kinds of single amino acid mutations in both E. coli library and M. smegmatis library， 5 301 kinds of multi-point mutations in E. coli library， and 853 kinds of multi-point mutations in M. smegmatis library. The correlation coefficient between E. coli library and M. smegmatis library is 0.84. ConclusionsWe have developed a strategy to construct a library of mutants targeting the essential mycobacterial gene rpoB， and successfully established a mutant library of rifampicin resistance gene rpoB.

Statistical analysis of 484 Mycobacterium tuberculosis genomes reveals an association between single nucleotide polymorphisms on ponA1 gene and LAM and Haarlem lineages / Análisis estadístico de 484 genomas de Mycobacterium tuberculosis revela una asociación entre los polimorfismos de un solo nucleótido en el gen ponA1 y los linajes LAM y Haarlem

Abstract Objectives: Evaluate the association between rifampicin resistance and the presence of at least one SNP in the rpoB and ponA1 genes and the spoligotype defined lineages. Material and Methods: This study analyzed two databases of 484 genomes of M. tuberculosis from strains isolated from patients in the cities of Lima and Callao, for which the odds ratio (OR) was calculated considering belonging to a certain spoligotype defined lineages as an exposure factor. Results: No statistically significant association (ρ value> 0.05) was found between the presence of at least one SNP in the rpoB gene and the lineages included in the study (LAM, Haarlem, T and Beijing). However, a statistically significant association was found between the presence of at least one SNP in the ponA1 gene and the LAM and Haarlem lineages (ρ value <0.05). An association was found between the P631S SNP in the ponA1 gene and the LAM and Haarlem lineages; and the A516T SNP, of this same gene, presented an association with the LAM lineage. Likewise, an association was found between rifampicin resistance and the LAM lineage. Conclusions: The presence of SNPs in the ponA1 gene is associated with the LAM and Haarlem lineages.

Resumen Objetivos: Evaluar la asociación entre la resistencia a rifampicina y la presencia de al menos un SNP en los genes rpoB y ponA1 y los linajes definidos por espoli gotipos. Material y Métodos: Este estudio analizó dos bases de datos de 484 genomas de M. tuberculosis de cepas aisladas de pacientes de las ciudades de Lima y Callao, para lo cual se calculó el odds ratio (OR) considerando la pertenencia a determinado linaje definido por espoligotipos como un factor de exposición. Resultados: No se encontró una asociación estadísticamente significativa (valor de ρ >0.05) entre la presencia de al menos un SNP en el gen rpoB y los linajes incluidos en el estudio (LAM, Haarlem, T y Beijing). No obstante, se halló una asociación estadísticamente significativa entre la presencia de al menos un SNP en el gen ponA1 y los linajes LAM y Haarlem (valor de ρ <0.05). Se encontró una asociación entre el SNP P631S del gen ponA1 y los linajes LAM y Haarlem; y el SNP A516T, de este mismo gen, presentó una asociación con el linaje LAM. Asimismo, se halló una asociación entre la resistencia a rifampicina y el linaje LAM. Conclusiones: La presencia de SNPs en el gen ponA1 está asociada con los linajes LAM y Haarlem.

Assessment of rifampicin resistance in re-treatment cases of pulmonary tuberculosis using gene-xpert

Objective:To assess therifampicin resistance in re-treatment cases of pulmonary tuberculosis using gene-xpert.Material and methods:This cross sectional study was conducted at Department of Pulmonology Bahawal Victoria Hospital, Bahawalpur.Total 145 patients who came for re-treatment of TB either male or female having age 20-60 years were selected for this study.Results:Mean age of the patients was 45.79 ± 11.65 years and mean duration of TB was 5.44 ± 2.9 years.Rifampicin resistance was found in 55 (38%) cases by using gene-xpert test. Rifampicin resistance is significantly associated with gender but insignificantly associated with age and duration of TB.Conclusion:In present study very high percentage of rifampicin resistance was found in retreatment cases of TB by using gen-xpert test.Significant association of gender with rifampicin resistance was noted but insignificant association of age and duration of TB with rifampicin resistance was detected.

Development of molecular markers of Mycobacterium tuberculosis rifampicin resistance gene rpoB by PARMS technology / 生物工程学报

The purpose of this study is to provide a simple and reliable genetic typing approach for molecular drug susceptibility test of Mycobacterium tuberculosis, through the developing of fluorescence molecular marker of rifampicin resistance gene rpoB. Eleven fluorescent molecular markers of the rpoB gene were established by using the sequence difference between the amino acid positions 531, 526, 516, 511 and 513 of rpoB gene of rifampicin-resistant strains and the alleles of rifampicin-sensitive strains, combined with the PARMS technique (Penta-primer amplification refractory mutation system). We used 104 clinical isolates of Mycobacterium tuberculosis to validate this marker and it was verified by sequencing as 100% correct. These samples were also tested with proportional drug sensitivity test. The coincidence rate was 94.23%. The molecular markers had high reliability for genotyping of rpoB gene. It can also detect low-concentration drug-resistant samples (511/533 unit point mutations) whose phenotypic susceptibility cannot be detected. The eleven sets of fluorescent molecular markers could cover 92%-96% of rpoB gene mutation types of rifampicin-resistant strains, and provide new idea for rapid detection of rifampin-resistant Mycobacterium tuberculosis.

Occurrence of Rifampicin Resistant and HIV Co-Infection Tuberculosis in Ikere-Ekiti, Nigeria

@# Tuberculosis is a major cause of disability and death worldwide. One of the targets of Sustainable Goal Development for 2030 is to put an end to tuberculosis epidemics. This study accessed the occurrence of tuberculosis, its co-infection with HIV and rifampicin resistance in the study area, as well as the reliability of acid fast bacilli (AFB) test in tuberculosis (TB) diagnosis. Methods: The study, which was made up of both retrospective and prospective TB investigations, was carried out at the State Specialist Hospital, Ikere–Ekiti, Nigeria, covering April 2014 to March 2017. Sputum samples from 1227 individuals with suspected cases of TB, made up of 496 (40.42%) males and 731 (58.11%) females, were analysed for TB by smear microscopy for AFB and molecular determination using GeneXpert machine. Results: A total of 141 (11.49%) individuals were diagnosed tuberculosis positive using the GeneXpert machine, while 78 (6.36%) tested positive by AFB technique. Eleven (7.8 %) of the 141 tuberculosis-positive cases were rifampicin resistant; also a tuberculosis - HIV co-infection rate of 25.53% was obtained. The risk factors associated with TB in the study were smoking, alcoholism, over-crowding and HIV co-infection. Using the GeneXpert as a standard, the AFB’s sensitivity, specificity, positive predictive value (PPV) and negative predictive value were 43.26, 98.43, 78.20 and 93.04% respectively. Conclusion: The high occurrence of rifampicin resistant tuberculosis and HIV-TB co-infection are of serious concern. The low sensitivity and PPV values, as well as its inability to detect drug resistant TB, undermine the reliability of AFB.

Value of GeneXpertMTB/RIF and Hain techniques for the diagnosis of sputum smear negative tuberculosis and for the assessment of rifampin resistance / 公共卫生与预防医学

Objective To investigate the application value of Xpert mycobacterium tuberculosis/rifampicin (GeneXpertMTB/RIF) and Hain techniques for the diagnosis of sputum smear negative tuberculosis and for the assessment of rifampin resistance. Methods A total of 254 patients with suspected pulmonary tuberculosis treated in our hospital from March 2018 to June 2019 were enrolled. GeneXpertMTB/RIF, Hain techniques, Roche culture and drug susceptibility testing were performed. Taking Roche culture results as a gold standard, the efficacy of GeneXpertMTB/RIF and Hain techniques in detecting sputum smear-negative pulmonary tuberculosis was analyzed by ROC curve. Based on the results of drug susceptibility testing, the efficacy of GeneXpertMTB/RIF and Hain techniques in detecting rifampicin resistance was analyzed. Results The sensitivity, specificity and accuracy of GeneXpertMTB/RIF in the diagnosis of sputum smear negative pulmonary tuberculosis were 82.18%, 86.93% and 83.52%, respectively. The sensitivity, specificity and accuracy of Hain technique in the diagnosis of sputum smear negative pulmonary tuberculosis were 67.33%, 77.12% and 73.23%, respectively. The sensitivity, specificity and accuracy of GeneXpertMTB/RIF in evaluating rifampicin resistance were 73.33%, 98.84% and 95.05%, respectively. The sensitivity, specificity and accuracy of Hain technique in evaluating rifampicin resistance were 86.67%, 82.56% and 83.17%, respectively. Conclusion Compared with Hain technology, GeneXpertMTB/RIF had better clinical value for diagnosis of sputum smear negative tuberculosis and for the assessment of rifampin resistance. This study demonstrates that GeneXpertMTB/RIF technique has a good application value in the rapid diagnosis and treatment of sputum smear negative pulmonary tuberculosis.

Microbiological diagnosis of tuberculous meningitis: Phenotype to genotype

Tuberculous meningitis (TBM) is a commonly encountered central nervous system infection. Characteristic clinical, imaging and cerebrospinal fluid parameters help clinicians to make a prompt presumptive diagnosis that enables them to start empirical anti-tuberculosis treatment. There are several close mimic to TBM, such as partially treated pyogenic meningitis, fungal meningitis, sarcoidosis, meningeal metastases and meningeal lymphomatosis. Microbiological confirmation instils a sense of confidence amongst treating physicians. With conventional phenotypic methods (cerebrospinal fluid microscopy and culture), in more than 50 per cent patients, microbiological confirmation is not achieved. Moreover, these methods take a long time before providing conclusive results. Negative result does not rule out Mycobacterium tuberculosis infection of the brain. Genotypic methods, such as IS 6110 polymerase chain reaction and automated Xpert M. tuberculosis/rifampicin (MTB/RIF) assay system improved the TBM diagnostics, as results are rapidly available. Xpert MTB/RIF assay, in addition, detects rifampicin resistance. Xpert MTB/RIF Ultra is advanced technology which has higher (60-70%) sensitivity and is being considered a game-changer in the diagnostics of TBM. A large number of TBM cases remain unconfirmed. The situation of TBM diagnostics will remain grim, if low-cost technologies are not widely available. Till then, physicians continue to rely on their clinical acumen to start empirical anti-tuberculosis treatment.

Diagnostic Accuracy Of Xpertmtb/Rif Assay In Detection Of Pulmonary Tuberculosis And Rifampicin Resistance In Comparison With Conventional Methods.

Introduction: Tuberculosis is one of the oldest diseases with high morbidity and mortality. India accounts for one- fourth of the global TB burden (2.7million cases). Material and methods: A total 1056 patients were included in this study. All patients were requested to give two sputum samples, spot sputum sample (at the time of visit) and early morning sputum for mycobacterial testing. All spot samples tested with smear microscopy and Xpert MTB/Rif assay. Rifampicin resistant samples compared with conventional method. Results: Out of 1056 sputum samples GeneXpert MTB Rif assay detected positive 204 (19.3%), not detected 827 (78.31%). There is significant difference founded for positivity in spot samples and early morning sputum samples (P =0.0026). Rifampicins resistant were 8 (3.9%) andshown 100% sensitivity, specificity with Conventional method. Conclusion: For diagnosing tuberculosis and detecting Rifampicin resistance GeneXpert MTB/RIF has been especially recommended.

Evaluation of cepheid GeneXpert MTB/RIF assay for Mycobacterium tuberculosis detection and rifampicin resistance in clinical specimen

Background: Timely diagnosis and treatment of tuberculosis is important to treat the disease and to reduce transmission. The WHO recommends using GeneXpert MTB in developing, high-burden countries.  A study was conducted to evaluate the performance of Xpert assay for the detection of M. tuberculosis and rifampicin resistance in clinical specimen.Methods: About 615 consecutive samples were simultaneously subjected to culture and phenotypic drug susceptibility test for M. tuberculosis and analysis by GeneXpert assay. Confirmed Mycobacterium tuberculosis in a positive culture was used as a reference standard for TB diagnosis.Results: The assay achieved a sensitivity of 96.75% (268/277) and 76.47% (26/34) for smear positive and smear negative pulmonary specimen respectively. In extrapulmonary specimen, the sensitivity was 50% (1/2) and 42.8% (3/7) for smear positive and smear negative specimen respectively. An additional 48 M. tuberculosis were detected by Xpert assay which were smear and culture negative. The Xpert assay identified 100% of the phenotypic rifampicin susceptible isolates and 74.19% of the phenotypic rifampicin resistant isolates. Discordant results were seen in 8 (2.76%) isolates. 6 of these isolates were confirmed to be rifampicin resistant by the reference lab.Conclusions: Present study indicates that Xpert MTB/RIF assay is an effective and rapid tool for the rapid diagnosis of Mycobacterium tuberculosis. The sensitivity is comparable to culture in smear positive specimen but less sensitive than culture for smear negative specimen. In cases with high index of suspicion or discordance for rifampicin results, confirmation should be done by other methods due to false negative results on Xpert assay.

Prevalence of Rifampicin Resistance Tuberculosis in Newly Diagnosed and Previously Treated Pulmonary Tuberculosis Patients Attending the Department of Pulmonary Medicine , Muzaffarnagar Medical College, Muzaffarnagar

Background: Multidrug-resistant tuberculosis (MDR-TB) prevalence is increasing throughout the world. Although, most important risk factor for development of MDR-TB is inadequate and irregular previous treatment for tuberculosis, but newly treated patients are also at risk due to either spontaneous mutations or transmission of drug-resistant strains. The conventional tuberculosis drug susceptibility tests are sensitive and specific but they are not rapid. Newer molecular methods help in rapid diagnosis. GeneXpert MTB/RIF is a rapid method to detect MTB and rifampicin resistance. Rifampicin resistance is an initial indicator of multidrug resistance. We have tried to ascertain the prevalence of Rifampicin resistance in newly diagnosed and previously treated pulmonary tuberculosis patients. Methods: This study was a prospective cross sectional study that involved all newly diagnosed and previously treated pulmonary tuberculosis patients attending the Department of Pulmonary Medicine, Muzaffarnagar Medical College, Muzaffarnagar from January 2018 to Dec 2018. In the District Hospital, Muzaffarnagar,the sputum specimens were sent for GeneXpert MTB/RIF .On the basis of the result, the patients were labelled as Rifampicin resistance. Results: A total no of 118 sputum samples were sent to District Hospital,Muzaffarnagar for GeneXpert MTB/RIF . Among these 118 patients, 102 patients were newly diagnosed pulmonary tuberculosis and 16 patients were previously treated pulmonary tuberculosis . Prevalence of Rifampicin resistance TB was found to be 0.98 % among newly diagnosed pulmonary tuberculosis, and 25 % among previously treated pulmonary tuberculosis patients. Conclusion: In conclusion, our results showed that the prevalence of Rifampicin resistance TB was high in previously treated pulmonary tuberculosis patient in comparison to newly diagnosed pulmonary tuberculosis patient. Proper administration of first line drugs for susceptible cases is most efficient method in preventing drug resistance.

A Retrospective Review of Rifampicin-Resistant Mycobacterium tuberculosis between 2015 and 2017 in Port Harcourt, Nigeria

Background:The diagnosis and treatment of drug resistant tuberculosis (TB) is a significant challenge for the control of TB in Nigeria. Aim: The study was carried out to assess the prevalence of rifampicin-resistant TB at the point of initial diagnosis among subjects suspected of TB.Methods: A retrospective review of Mycobacterium tuberculosis (MTB)and rifampicin resistance detected by GeneXpert™ analysis between 2015 and 2017 in Braithwaite Memorial Specialist Hospital was carried out. Results: A total of 6733 samples were received and analyzed in the period under review, 1252 (19%) were positive for Mycobacterium tuberculosisand 5841 (81%) were negative. The Original ResearchArticle prevalence of Mycobacterium tuberculosis was 24.56%, 20.11% and 16.86% from 2015 to 2017 respectively. There was a significant decline in the prevalence of MTB from 2015 to 2017 (2= 33.59, p = 0.0001). Rifampicin (RIF) resistance was 5.42%, 5.86% and 6.22% respectively from 2015 to 2017;but the trend was not statistically significant (2 = 0.21; p = 0.6418). Conclusion: The study showed that despite a decrease in the prevalence of tuberculosis infection there was an increase in rifampicin resistance from 5.42% to 6.22% between 2015 and 2017. There is an urgent need to improve the management of TB in the Port Harcourt metropolis to improve treatment outcomes and to prevent the proliferation of drug resistant strains

Pulmonary Mycobacterium kyorinense disease: A case report and review of literature

We report here the first case of pulmonary infection due to Mycobacterium kyorinense in a 55-year-old hypertensive woman treated for pulmonary tuberculosis earlier on two occasions. She presented with productive cough, intermittent episode of left-sided chest pain, loss of appetite, low-grade fever, and breathlessness. Sputum cultures revealed non-tuberculous mycobacteria (NTM). She remained persistently symptomatic with sputum cultures positive for acid-fast bacilli even after 6 months of treatment. Hence, a 16SrRNA gene amplification and sequencing were done that revealed M. kyorinense. Based on the guidelines of the American Thoracic Society, she was started on weight-based dosing of clarithromycin, levofloxacin, ethambutol, isoniazid and injection amikacin daily. The patient improved symptomatically and became culture-negative after 3 months of therapy with the above regimen and continued to be culture negative for 12 months of treatment. She continues to remain symptom-free without evidence of any clinical or bacteriological relapse.

Diagnostic accuracy of Xpert MTB/RIF assay in extrapulmonary tuberculosis

Introduction: The WHO endorsed Xpert Mycobacterium tuberculosis/rifampicin (MTB/RIF) assay, has been evaluated for pulmonary TB in a number of studies but very few have investigated it for extrapulmonary specimens. The present study evaluates the performance of Xpert MTB/RIF assay in the diagnosis of extrapulmonary TB (EPTB). Aim and Objectives: The aim of the study is to determine sensitivity and specificity of Xpert MTB/RIF assay for diagnosis of EPTB and RIF resistance in comparison to culture on Lowenstein–Jensen (LJ) medium and proportion method (PM), respectively. Materials and Methods: A total of 738 specimens from clinically suspected cases of EPTB were subjected to Ziehl–Neelsen staining, Xpert MTB/RIF assay and culture on LJ medium. PM was done on MTB isolates. Results: The sensitivity, specificity of Xpert MTB/RIF assay for diagnosis of EPTB were 84.91% (95% confidence interval [CI] 72.41%–93.25%) and 86.72% (95% CI 83.94%–89.17%) and for RIF resistance detection were 60.00% (95% CI 32.29%–83.66%) and 94.74% (95% CI 73.97%–99.87%), respectively. Among culture-positive cases, the sensitivity of Xpert MTB/RIF assay was 94.12% in smear positive and 80.56% in smear-negative cases. Xpert MTB/RIF showed maximum sensitivity of MTB detection from lymph node specimens (100% [95% CI 54.07%–100.00%]) and other body fluids (100% [95% CI 15.81%–100.00%]). Conclusion: The present study establishes Xpert MTB/RIF assay as a promising tool in the rapid diagnosis of EPTB and detection of RIF resistance.

An evaluation of false-positive rifampicin resistance on the Xpert MTB/RIF

BACKGROUND Mycobacterium tuberculosis (MTB) is one of the most significant causes of mortality and morbidity. Early diagnose is important especially in multiple drug resistant tuberculosis to avoid transmission. Traditional techniques requires at least one to three weeks for diagnosis of tuberculosis. Diagnostic delays with multiple drug resistant tuberculosis are associated with worse clinical outcomes and increased transmission The Xpert MTB/RIF assay is one of the new diagnostic device for the diagnosis of tuberculosis and rapid detection of rifampicin resistance. OBJECTIVE We assessed the performance of Xpert MTB/RIF assay for detecting rifampicin resistance using phenotypic drug susceptibility tests as automated BD MGIT 960. METHODS Total of 2136 specimens were included in the study. Xpert MTB/RIF testing was performed on samples, using version 4 cartridges, according to the manufacturer's recommendations. The MTBC culture and first-line phenotypic DST were performed in automated BD MGIT 960 (Becton & Dickinson, USA) according to the recommendations of the manufacturer. Agar proportion was used in the case of inconsistency for rifampicin resistance. FINDINGS Thirty-four samples (19 respiratory and 15 nonrespiratory samples) were determined as positive for M. tuberculosis complex by Xpert MTB/RIF (Cepheid GeneXpert® System, USA). Xpert MTB/RIF assay detected 4/34 (11.7%) specimens as rifampicin resistant. One of the rifampicin resistant isolates was determined susceptible in MGIT 960 automated system. This isolate was also tested with agar proportion method and found susceptible to rifampicin. MAIN CONCLUSION The Xpert MTB/RIF assay can be used as first-line assay for the detection of M. tuberculosis. However, microbiologists must be aware of the limitations of the assay.

Detection of tuberculosis drug resistance: a comparison by Mycobacterium tuberculosis MLPA assay versus Genotype®MTBDRplus

BACKGROUND To cope with the emergence of multidrug-resistant tuberculosis (MDR-TB), new molecular methods that can routinely be used to screen for a wide range of drug resistance related genetic markers in the Mycobacterium tuberculosis genome are urgently needed. OBJECTIVE To evaluate the performance of multiplex ligaton-dependent probe amplification (MLPA) against Genotype® MTBDRplus to detect resistance to isoniazid (INHr) and rifampicin (RIFr). METHOD 96 culture isolates characterised for identification, drug susceptibility testing (DST) and sequencing of rpoB, katG, and inhA genes were evaluated by the MLPA and Genotype®MTBDRplus assays. RESULTS With sequencing as a reference standard, sensitivity (SE) to detect INHr was 92.8% and 85.7%, and specificity (SP) was 100% and 97.5%, for MLPA and Genotype®MTBDRplus, respectively. In relation to RIFr, SE was 87.5% and 100%, and SP was 100% and 98.8%, respectively. Kappa value was identical between Genotype®MTBDRplus and MLPA compared with the standard DST and sequencing for detection of INHr [0.83 (0.75-0.91)] and RIFr [0.93 (0.88-0.98)]. CONCLUSION Compared to Genotype®MTBDRplus, MLPA showed similar sensitivity to detect INH and RIF resistance. The results obtained by the MLPA and Genotype®MTBDRplus assays indicate that both molecular tests can be used for the rapid detection of drug-resistant TB with high accuracy. MLPA has the added value of providing information on the circulating M. tuberculosis lineages.

Drug Resistant Mycobacterium tuberculosis in Benue, Nigeria.

Tuberculosis is an important opportunistic infection in HIV/AIDS. Benue state is the highest HIV endemic state in Nigeria and investigation of tuberculosis cases and Mycobacterial resistance patterns are needed. A retrospective study with a review of reports of Mycobacterium tuberculosis and the rifampicin resistance was detected by the Cepheid GeneXpert MTB/RIF system was carried out from July, 2012 to September, 2013 in Federal Medical Centre, Makurdi and Nigeria Airforce Hospital, Makurdi. Pulmonary tuberculosis was detected in 21.5% (n=303/1407) of the total sample. Rifampicin resistance of 13.5% (31/230) and 15.1% (11/73) of the pulmonary tuberculosis positive cases was detected in Federal Medical Centre and Nigeria Airforce Base Hospital respectively, with an average prevalence of 13.9% (42/303). Re-treatment cases comprised 81% (34/42) whereas new cases constituted 19% (8/42) of all Rifampicin resistant Mycobacterium tuberculosis positive cases (X 2 = 6.51; p<0.05). Mean age was 30 years and there was no significant statistical difference in gender. Rifampicin resistant Mycobacterium tuberculosis is prevalent in Benue state, Nigeria especially, in the young adults. Therefore, laboratory facilities for rapid diagnosis of the drug resistant M. tuberculosis should be scaled up across the country. This remains an important step to achieve maximal impact in managing drug resistance in Nigeria.

Luciferase reporter phage phAE85 for rapid detection of rifampicin resistance in clinical isolates of Mycobacterium tuberculosis

OBJECTIVE@#To evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic.@*METHODS@#One hundred and ninety primary isolates on Lowenstein-Jensen medium were tested. Middlebrook 7H9 complete medium with and without rifampicin at 2 μg/mL was inoculated with standard inoculum from suspensions of the clinical isolate. After incubation for 72 h, LRP was added. Following 4 h of further incubation, light output from both control and test was measured as relative light units. Strains exhibiting a reduction of less than 50% relative light units in the drug containing vial compared to control were classified as resistant. Results were compared with the conventional minimum inhibitory concentration method (MIC) of drug susceptibility testing.@*RESULTS@#The two methods showed high level of agreement of 97% (CI 0.94, 0.99) and P value was 0.000 1. The sensitivity and specificity of LRP assay for detection of rifampicin resistance were 91% (CI 0.75, 0.98) and 99% (CI 0.95, 1.00) respectively. Time to detection of resistance by LRP assay was 3 d in comparison with 28 d by the minimum inhibitory concentration method.@*CONCLUSIONS@#LRP assay with phAE85 is 99% specific, 91% sensitive and is highly reproducible. Thus the assay offers a simple procedure for drug sensitivity testing, within the scope of semi-automation.

Variations in the occurrence of specific rpoB mutations in rifampicin-resistant Mycobacterium tuberculosis isolates from patients of different ethnic groups in Kuwait.

Background & objectives: Frequency of resistance-conferring mutations vary among isoniazid- and ethambutol-resistant Mycobacterium tuberculosis isolates obtained from patients of various ethnic groups. This study was aimed to determine the occurrence of specific rpoB mutations in rifampicin-resistant M. tuberculosis isolates from tuberculosis patients of various ethnic groups in Kuwait. Methods: Rifampicin-resistant M. tuberculosis isolates (n=119) from South Asian (n=55), Southeast Asian (n=23), Middle Eastern (n=39) and other (n=2) patients and 107 rifampicin-susceptible isolates were tested. Mutations in rpoB were detected by DNA sequencing. Polymorphisms at katG463 and gyrA95 were detected by PCR-RFLP for genetic group assignment. Results: None of rifampicin-susceptible but 116 of 119 rifampicin-resistant isolates showed rpoB mutation(s). Mutations among isolates from South Asian patients were distributed at rpoB516 (20%), rpoB526 (24%) and rpoB531 (27%) while 78 and 51 per cent of isolates from Southeast Asian and Middle Eastern patients, respectively, contained a mutated rpoB531. All isolates with rpoB N-terminal and cluster II mutations were obtained from Middle Eastern and South Asian patients. Most isolates from South Asian (84%) and Southeast Asian (70%) patients belonged to genetic group I while nearly all remaining isolates belonged to genetic group II. Isolates from Middle Eastern patients were distributed among genetic group I (46%), genetic group II (33%) and genetic group III (21%). Interpretation & conclusions: The occurrence of specific rpoB mutations varied considerably in rifampicin-resistant M. tuberculosis isolates obtained from patients of different ethnic groups within the same country. The present data have important implications for designing region-specific rapid methods for detecting majority of rifampicin-resistant strains.