Rapid Detection Of Rifampicin Resistance Research Articles

RpoB Gene Mutation Characteristics of Rifampicin-resistant Tuberculosis in Anqing, China

Background: Rifampicin resistant tuberculosis is a serious problem faced by tuberculosis control in China, and rapid detection of rifampicin resistance is urgently needed. Objectives: This study aimed to describe the molecular characteristics and frequency of RNA polymerase β subunit (rpoB) gene mutations in rifampicin-resistant tuberculosis (RR-TB) in the Anqing area. Methods: The rpoB gene fragment was amplified by polymerase chain reaction (PCR), and all isolates were sequenced for mutations in the rpoB gene. The mutations were obtained by comparing the sequencing results with the MUBII database. In addition, logistic regression was used to analyze the relationship between rpoB mutations and rifampicin (RIF) resistance. Results: There were 152 males and 42 females in this study, and the mean age was 56.60 ± 17.91 years. Mutations in the rpoB gene were a risk factor for rifampicin resistance (β = 5.271, P < 0.001 OR = 195.192). Among the 19 RR-TB strains, 16 (84.21%) had mutations in the ropB gene, and three (1.71%) of 175 rifampicin-sensitive strains were mutated. The mutation sites of five strains (31.58%) were at the codon 526 and five strains (31.58%) at the codon 531. However, there were two strains at the codon 513 and two strains at the codon 533 (15.79%), and two strains (10.53%) were double mutations. Conclusions: The mutation characteristics of the rpoB gene in the Anqing area are complex, and rpoB mutation detection can be used as an indicator to screen drug resistance of RIF.

Genexpert rapid detection of rifampicin resistance among patients with pulmonary tuberculosis

The routine conventional phenotypic bacteriological tests available for pulmonary tuberculosis (PTB) diagnosis, such as microscopy by Ziehl Neelsen staining, and Lowenstein Jensen method of solid culture, requires a long time for revealing positivity, resulting in a delayed diagnosis. After Mycobacterium tuberculosis (MTB) genome was discovered, nucleic acid amplification techniques were developed with more rapid detection and identification of rifampicin resistance in respiratory and extra-pulmonary specimens. GeneXpert is a DNA-polymerase chain reaction technique based on NAA, which allows an accurate genotypic method for a quick diagnosis of PTB diagnosis. The aim of the study was to assess retrospectively the yield of genotypic assay GeneXpert in revealing multidrug resistant TB compare to phenotypic confirmation by solid culture. 512 patients with positive and negative smears of suspected PTB were investigated by conventional phenotypic assays. PTB diagnosis assessed by positive genotypic assay and confirmed by phenotypic conventional solid culture method was 78.12%. The yield of GeneXpert in revealing rifampicin resistance was high (n=79/512; 15.43%) with 12.5% additional resistance against isoniazid revealed by positive solid cultures and drug susceptibility test. GeneXpert assay is a very useful method for rapid detection of MTB and drug resistance, which facilitates timely diagnosis and appropriate management of pulmonary tuberculosis.

GeneXpert: A new tool for the rapid detection of rifampicin resistance in mycobacterium tuberculosis

Background: present study was planned to evaluate the diagnostic accuracy of GeneXpert assay for the detection of rifampicin resistance in mycobacterium tuberculosis using conventional drug susceptibility testing as gold standard. Methods and materials: This cross-sectional study was conducted at Jinnah Hospital, Lahore,/Allama Iqbal Medical College, Lahore, Pakistan, from January 2012 to December 2014, and comprised clinically and radiologically diagnosed tuberculosis suspected cases. Pulmonary and extra-pulmonary specimens were collected from strong tuberculosis suspects. All specimens were processed for Ziehl Neelsen staining, Lowenstein-Jensen culture and GeneXpert assay. All mycobacterium tuberculosis positive cases on Lowenstein-Jensen culture were further processed for drug susceptibility testing. Results: Of the 2,200 cases, 840(49.46%) were positive for mycobacterium tuberculosis on GeneXpert assay. Of them, 134(15.6%) cases showed rifampicin resistance on GeneXpert assay. The sensitivity, specificity, positive predictive value and negative predictive value of GeneXpert assay for rifampicin resistance were 127(98.3%), 704(99.1%), 127(94.7%) and 704(99.4%), respectively, by comparing the results with drug susceptibility testing. Conclusion: GeneXpert assay was an extremely helpful diagnostic tool for the detection of rifampicin resistance in tuberculosis suspects with fairly high sensitivity and specificity along with short turnout time

High-resolution melt curve analysis for rapid detection of rifampicin resistance in Mycobacterium tuberculosis: a single-centre study in Iran.

This study aimed to compare the diagnostic accuracy of high-resolution melting (HRM) analysis in comparison with Xpert MTB/RIF as well as conventional drug susceptibility testing (DST) for the detection of rifampicin (RIF) resistance in Mycobacterium tuberculosis in Iran. A comparative cross-sectional study was carried out from April 2017 to September 2018. A total of 80 culture-positive clinical samples selected during the study period were analysed for detection of RIF-resistant TB by conventional DST, Xpert MTB/RIF, and sequencing. Sensitivity and specificity of the HRM calculated according to DST was our reference standard test in this study. The overall sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of HRM assay were found to be 100%, 89.33%, 38.46%, and 100% respectively. The analysis demonstrated that the diagnostic accuracy of HRM tests is insufficient to replace Xpert MTB/RIF and conventional DST. HRM tests have the advantage of time to result and may be used in combination with culture. Further work to improve molecular tests would benefit from standardized reference standards and the methodology.

Molecular Epidemiology of <i>Mycobacterium tuberculosis</i> among Pulmonary Tuberculosis Patients in Ilorin, Nigeria

Background: Tuberculosis (TB) is an infectious disease and it is a public health concern due to the endemic nature of the disease. Nigeria’s health systems still rely heavily on out-dated tuberculosis diagnostic tests; including sputum smear microscopy and chest radiography. This necessitated the urgent need to explore a more reliable, effective method that can rapidly enhance simultaneous detection of Mycobacterium tuberculosis (MTB) and rifampicin (RIF) resistance. Aims: We evaluated the prevalence of MTB and rifampicin resistance and the influence of socio-demographic factors. Methods: A longitudinal, descriptive study that employs the Xpert MTB/RIF automated molecular method for rapid detection of rifampicin resistance. Secondary data were collected from the medical record from March 2017 to December 2018. Primary data were collected by direct clinical observations and analysis with gene expert machine from January 2019-March 2019. Results: Study populations were 1893 subjects pooled as secondary data at the Gene Expert laboratory, with a mean age of 44.92 years and were categorized based on sex, 1125 (59.4%) males and 768 (40.6%) females respectively. The MTB detected were 117 (15.7%) for males while 76 (9.9%) were positive for females. 948 (84.3%) were negative for males and 692 (90.1%) negative for females. Also, the categories for age were grouped into three but the older age groups (29 - 54) and (55 - 80) were more vulnerable to MTB having a p = 0.506 which is statistically non-significant; only 14 (5.5%) have RIF while 239 (94.5%) do not have RIF resistance strains. Logistic regression analysis of MTB detected showed a significant association (p 0.05) for age. The prevalence rate for RIF Resistance is 5.53%, there is a statistical difference p = 0.001 between RIF Resistance and MTB in Ilorin. Conclusion: The utilization of validated molecular technique for MTB strain identification has shown stronger epidemiological impetus for an improved care management outcome specifically in resources limited areas.

Cepheid GeneXpert Mycobacterium tuberculosis/Rifampicin assay for the rapid detection of Mycobacterium tuberculosis and identification of Rifampicin resistance

CONTEXT: Rapid diagnosis and detection of Rifampicin (RIF) resistance are essential for effective disease management of tuberculosis (TB). Cartridge-based nucleic acid amplification test also known as GeneXpert Mycobacterium tuberculosis (MTB)/RIF assay is a novel integrated diagnostic system for the diagnosis of TB and rapid detection of RIF resistance in clinical specimens. AIM: In the present study, we determine the performance of the MTB/RIF assay for the rapid diagnosis of TB and detection of RIF resistance in pulmonary specimens obtained from presumptive multidrug-resistant (MDR) TB cases. SUBJECTS AND METHODS: This is a cross-sectional observational study conducted in culture and drug-susceptibility testing laboratory between January 2014 and December 2014. A total of 1994 sputum samples were obtained from presumptive MDR-TB cases of Dharwad and Belgaum districts of Karnataka. All samples were tested on GeneXpert for MTB/RIF detection. RESULTS: A total of 1994 presumptive pulmonary TB clinical samples were received of which 840 (42.1%) were MTB complex (MTBC) positive and 1154 (57.9%) were negative. The MTB/RIF assay also detected 127 (6.4%) RIF-resistant specimen and 713 (35.6%) RIF-susceptible specimens. CONCLUSIONS: The MTB/RIF test is a simple and rapid method, and staff with adequate training can perform this test with minimal laboratory setup. It helps to avoid the injudicious use of the anti-TB drug and offers high potential for the diagnosis of TB and RIF resistance due to its capacity for direct detection of MTBC, its rapidity and its simplicity.

Flash Diagnostic Tool for MDR TB in HIV Era

Tuberculosis (TB) is one of the commonest opportunistic infection and the leading cause of death in HIV patients in developing countries. HIV infection is a well recognised risk factor for both activation of initial infection and reactivation of latent infection. This study was done to find out the co-prevalence and the trend of HIV infection among tuberculosis patients and to determine the prevalence of MDR Tuberculosis in HIV positive patients using Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, United States).The sputum samples are received from patients attending ICTC clinic at Osmania General Hospital, Hyderabad. Sputum specimen was processed for TB detection by Cartridge Based Nucleic Acid Amplification testing (CB-NAAT) using Xpert MTB/RiF assay technology. A total of 125 HIV patients were in the study. Out of these patients 20 MTB cases were detected. Of these 20 cases 18(90%) cases were sensitive to Rifampicin (RIF) and 2(10%) cases were showing resistance to Rifampicin (RIF) drug. Co-existence of HIV and tuberculosis is high and there is high Prevalence of MDR tuberculosis in HIV patients. The Gene Xpert MTB/RIF assay is a novel integrated diagnostic device that performs sample processing and real-time PCR analysis in a single hands-free step for the diagnosis of tuberculosis and rapid detection of RIF resistance in clinical specimens.

Assessing the utility of Xpert(®) MTB/RIF as a screening tool for patients admitted to medical wards in South Africa.

Many hospital inpatients in South Africa have undiagnosed active and drug-resistant tuberculosis (TB). Early detection of TB is essential to inform immediate infection control actions to minimize transmission risk. We assessed the utility of Xpert® MTB/RIF (GeneXpert) as a screening tool for medical admissions at a large public hospital in South Africa. Consecutive adult patients admitted to medical wards between March-June 2013 were enrolled; sputum specimens were collected and tested by GeneXpert, smear microscopy, and culture. Chest X-rays (CXRs) were conducted as standard care for all patients admitted. We evaluated the proportion of patients identified with TB disease through each diagnostic method. Among enrolled patients whose medical charts were available for review post-discharge, 61 (27%) were diagnosed with TB; 34 (56% of diagnosed TB cases) were GeneXpert positive. When patients in whom TB was identified by other means were excluded, GeneXpert yielded only four additional TB cases. However, GeneXpert identified rifampicin-resistant TB in one patient, who was initially diagnosed based on CXR. The utility of GeneXpert for TB screening was limited in an institution where CXR is conducted routinely and which serves a population in which TB and TB/HIV co-infection are highly prevalent, but it allowed for rapid detection of rifampicin resistance.

Rapid detection of rifampicin resistance in Mycobacterium tuberculosis by high-resolution melting curve analysis.

High-resolution melting curve analysis (HRMA) can be used to screen for mutations in genes without the need for specific probes, with low turnaround time and high cost-effectiveness. To detect the sensitivity and specificity of a line-probe assay (LPA) and HRMA in comparison with BACTEC™ MGIT™ 960 for the detection of rifampicin (RMP) resistance. A total of 219 Mycobacterium tuberculosis isolates tested by MGIT 960 for RMP susceptibility were tested with HRMA and LPA. Discordant samples were processed for sequencing of the RMP resistance-determining region (RRDR) of the rpoB gene. HRMA identified 93 of 103 (90.3%) isolates that were resistant and 113/116 (97.4%) that were susceptible on MGIT 960, with a sensitivity and specificity of respectively 90.3% and 97.4%. HRMA identified 117/119 (98.3%) LPA-susceptible and 94/100 (94%) resistant isolates, with 98.3% specificity and 94% sensitivity. Two isolates that were susceptible on LPA but resistant on HRMA showed silent mutations at 539 and 541 codons on sequencing, while 6 isolates that were susceptible on HRMA but resistant on LPA showed D516V (n = 4) and H526C/D (n = 2) mutations. Four isolates (3.9%) that were resistant on MGIT were susceptible on all three genotypic methods, which could be due to mutations outside the RRDR or efflux pumps. HRMA shows good potential as a rapid screening tool for the detection of drug resistance.

Evaluation of GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculosis and Rapid Detection of Rifampicin Resistance in Clinical Specimens

Evaluation of GeneXpert MTB/RIF Assay for the Diagnosis of Tuberculosis and Rapid Detection of Rifampicin Resistance in Clinical Specimens

Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco

One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management.

Detection of Mutations in rpob Gene of Clinically Isolated M. Tuberculosis by DNA Sequencing

Aim: A population-based study was performed to detect the mutations in 81-bp rifampicin resistance determining region (RRDR) of rpoB gene in clinically isolated M. tuberculosis strains. Methods: Polymerase chain reaction (PCR) mediated direct DNA sequencing was used for rapid detection of rifampicin resistance of M. tuberculosis. Results: Among the 150 isolates, 115 were rifampicin sensitive and exhibited a wild-type pattern on PCR mediated direct sequencing, and remaining 35 isolates were found to be resistant. The codons most frequently involved in mutation were codon 531(40%), 526(23%), and 516(15%).Total twenty four kinds of mutation, which 18 point mutation, 4 insertion & 2 deletion were observed in 81-bp RRDR region of rpoB gene. Conclusion: The sequencing analysis for genotypic evaluation of rifampicin resistance is a highly sensitive assay and provides a practical alternative to in vitro testing in M. tuberculosis. Information on the profile of rpoB mutations in M. tuberculosis provides an improved diagnosis of rifampicin resistance by increasing the efficacy of gene sequencing based test.

Utility of the REBA MTB-rifa® assay for rapid detection of rifampicin resistant Mycobacterium Tuberculosis

BackgroundDrug-resistant tuberculosis (TB), including resistance to both rifampicin (RIF) and isoniazid (INH) referred to as multidrug-resistant tuberculosis (MDR-TB), has become an increasing global threat in recent years. Effective management of patients infected with MDR-TB strains requires identifying such patients by performing conventional drug-susceptibility testing (DST) on bacteria isolated from sputum, a process that can take up to 2 months. This delay in diagnosis can result in worsening and continued transmission of MDR-TB. Molecular methods that rely upon nucleic acid amplification of specific alleles known to be associated with resistance to specific drugs have been helpful in shortening the time to detect drug resistant TB.MethodsWe investigated the utility of the REBA MTB-Rifa®, a commercially available line probe assay (LPA) for detecting rifampicin (RIF) resistance in the RIF resistance-determining region (RRDR) of the rpoB gene. Altogether, 492 Mycobacterium tuberculosis (M. tuberculosis) clinical isolates and additional 228 smear- and culture-positive sputum samples with confirmed M. tuberculosis were collected from subjects with suspected MDR-TB in South Korea. The results were compared with conventional phenotypic DST and sequencing of the rpoB gene.ResultsA total of 215 of the 492 isolates were resistant to RIF by conventional DST, and of which 92.1% (198/215) were MDR-TB strains. The REBA MTB-Rifa® assay identified RIF resistance in 98.1% (211/215) of these isolates but failed to identify resistance in four phenotypically RIF resistant isolates. These four isolates lacked mutations in the RRDR but three were confirmed to be MDR-TB strains by sequencing. The sensitivity and specificity of this test for clinical isolates was thus 98.1% (211/215) and 100% (277/277), respectively. When applied directly to 228 smear positive sputum samples, the sensitivity and the specificity of REBA MTB-Rifa® assay was 100% (96/96, 132/132), respectively.ConclusionsThese findings support the use of the REBA MTB-Rifa® assay for rapid detection of RIF resistance on clinical isolates and smear positive sputum samples. The results also suggest that RIF resistance is a good surrogate marker of MDR-TB in South Korea and the need to add more probes to other LPAs which can cover newly identified mutations relevant to RIF resistance.

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

ObjectiveTo evaluate luciferase reporter phage (LRP) phAE85 in rapid detection of rifampicin resistance in a region where TB is endemic. MethodsOne 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. ResultsThe 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. ConclusionsLRP 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.

Selected culture and drug-susceptibility testing methods for drug-resistant Mycobacterium tuberculosis screening in resource-constrained settings

Evaluation of: Adikaram CP, Perera J, Wijesundera SS. The manual Mycobacteria Growth Indicator Tube and the Nitrate Reductase Assay for the rapid detection of rifampicin resistance of M. tuberculosis in low resource settings. BMC Infect. Dis. 12, 326 (2012).The control of drug-resistant tuberculosis (TB) requires an accurate and a rapid diagnostic for the detection of critical patterns of drug resistance. Rifampicin resistance is a good predictor of the presence of multidrug-resistant TB. Traditionally, in vitro susceptibility methods for Mycobacterium tuberculosis are time consuming and laborious. More rapid methods are available but are very expensive for routine use in low-resource settings. According to the article under evaluation, the Manual Mycobacteria Growth Indicator Tube and Nitrate Reductase Assay, in liquid medium, agreed well with the agar proportion method for the rapid detection of rifampicin resistance in low-resource settings. The results of both susceptibility tests will be available in less than 2 weeks and the cost per test is low. Major efforts are needed to improve the diagnostic and treatment success rate among patients with drug-resistant TB. Since 2007, the WHO has promoted new diagnostic tests such as Xpert Mycobacterium tuberculosis/rifampicin; point-of-care tests are nowadays under development.

Pyrosequencing for the rapid detection of rifampicin resistance in <I>Mycobacterium tuberculosis</I>: a meta-analysis [Review article

Multidrug-resistant tuberculosis is a major threat to the control of tuberculosis and to public health. Whereas most conventional methods of drug susceptibility testing (DST) are precise but time consuming, pyrosequencing is a rapid, high-throughput technique. To conduct a meta-analysis to evaluate the overall accuracy of pyrosequencing for the detection of rifampicin (RMP) resistance. We searched PubMed, Web of Science, Elsevier and BIOSIS databases according to a written protocol and explicit study selection criteria. A summary receiver operating characteristic curve (SROC) and Cochrane (Q*) index were calculated to perform this meta-analysis using Meta-Disc software. Twelve studies involving 594 specimens with RMP resistance and 793 RMP-susceptible specimens met the inclusion criteria. Of these, 11 were based on Mycobacterium tuberculosis clinical isolates. The overall sensitivity and specificity were estimated at respectively 0.94 (95%CI 0.92-0.96) and 0.98 (95%CI 0.97-0.99). The area under the SROC curve was 0.99 and the Cochrane (Q*) index was 0.96. For clinical specimens, the overall sensitivity and specificity estimates were respectively 0.89 (range 0.52-1.00) and 0.99 (range 0.95-1.00). This meta-analysis shows that pyrosequencing is a highly sensitive and specific tool for the detection of RMP resistance in M. tuberculosis. The pyrosequencing assay is conducted in a high-throughput format, with a turnaround time of <2 h, making it substantially faster than conventional DST methods. We propose that pyrosequencing applied directly to clinical specimens instead of M. tuberculosis isolates could be of greater clinical value.

The manual mycobacteria growth indicator tube and the nitrate reductase assay for the rapid detection of rifampicin resistance of M. Tuberculosisin low resource settings

BackgroundTuberculosis (TB) is a disease of poverty that contributes significantly to ill-health in developing countries. Drug resistant TB is a major challenge to disease control. Early diagnosis and rapid determination of drug sensitivity is of paramount importance in eradication of TB. Although automated liquid culture based methods are available for rapid detection of drug resistance, the high cost of these tests prevent them from being used routinely in low resource settings. This study compares two phenotypic methods, the manual Mycobacteria Growth Indicator Tube (MGIT) and the Nitrate Reductase Assay (NRA) in liquid medium, with the agar proportion method (APM), the gold standard for susceptibility testing of Mycobacterium tuberculosis.MethodologyFourteen day old M. tuberculosis strains (n=373) grown on solid media were used for drug susceptibility testing by APM, NRA and the manual MGIT method. Rifampicin free and rifampicin incorporated (final concentration, 1 μg/ml) media were inoculated with the recommended concentrations of mycobacterial suspensions and incubated at 37°C in 5% CO2. In the APM, the proportion of colonies in the drug containing medium was determined. In the NRA, the colour change in the medium was compared with a standard colour series after day 6 and day 12 of incubation. Growth in the MGIT was detected using the manual MGIT reader from day 2 onwards. The 2 methods were compared with the gold standard, APM to determine sensitivity and specificity and agreement between the methods was calculated using kappa statistics.ResultsThirty one (31) rifampicin resistant isolates were identified. When compared with the APM, the sensitivity of detection of rifampicin resistance was 85% for the NRA and 93% for the manual MGIT and the specificity was 99% and 100% respectively. Both assays, NRA (κ=0.86) and manual MGIT method (κ= 0.94) were in excellent agreement with the APM. The mean turnaround time for manual MGIT method and NRA were 08 days and 10 days respectively.ConclusionThe NRA in liquid medium and manual MGIT are useful alternatives to APM for drug susceptibility testing of M. tuberculosis in low resource settings.

Molecular characterization of rpoB gene encoding the RNA polymerase β subunit in rifampin-resistant Mycobacterium tuberculosis strains from south India

Polymerase chain reaction (PCR) mediated direct DNA sequencing was evaluated for rapid detection of Rifampicin resistance (RMP r ) of Mycobacterium tuberculosis. After amplification of the rpoB gene, the product was sequenced using ABI 310 Genetic Analyzer and the rifampicin resistance in M. tuberculosis were assessed using the sequence pattern. Among the 100 isolates, 83 isolates were rifampicin sensitive, exhibited a wild-type pattern on PCR mediated direct sequencing, and remaining 17 isolates were resistant. All the RMP r isolates were multidrug resistant, that is, resistant to at least Rifampicin and isoniazid. The mutation rate at the most effected codon 526 in the 81 bp rifampin resistance-determining region (RRDR) of rpoB gene was determined to be 35.29% in isolates from South India. In addition, the mutation rate at the most affected codon 451 was in the out side of the 81bp RRDR region of rpoB gene. A synonym mutation was detected (Thr→Thr) at codon 526 in one isolate, which was inside the active site (codons 507-533) of RMP binding in the rpoB gene region. The sequencing analysis for genotypic evaluation of Rifampicin resistance in highly sensitive assay provides a practical alternative to in vitro testing in M. tuberculosis . Key words: Rifampin resistance, Mycobacterium tuberculosis , rpoB gene, sequencing, mutation.

Diagnostic test of rifampicin resistance in mycobacterium tuberculosis: a meta‐analysis

To evaluate available evidence on the diagnostic value of radioactivity-free cultivation and detection technologies for rapid detection of rifampicin resistance in Mycobacterium tuberculosis. A fully recursive literature search was conducted in PubMed, EMBASE, Biosis, Web of Science (all 1990-2010), CBMWeb (1978-2010), and Google Scholar. QUADAS items were used to evaluate the quality of included studies. Sensitivity, specificity, Summary receiver-operating curve SEN, SPE, SROC, and related techniques were used to assess the diagnostic value of radioactivity-free Mycobacterium tuberculosis cultivation and detection technologies. Six studies were included in the final analysis. The MB/BacT, BACTEC MGIT 960, and Manual MGIT systems were highly sensitive and specific for detecting rifampicin-resistant TB. The summary SEN and summary SPE of the MB/BacT and BACTEC MGIT 960 systems were 100%, 99%, 100%, and 96%, respectively. The SROC of the BACTEC MGIT 960 system was 0.9943. We recommended that the BACTEC 460 system be replaced by MB/BacT or BACTEC MGIT 960 as the final diagnostic test for rifampicin resistance in Mycobacterium tuberculosis. More studies are needed on the diagnostic value of other radioactivity-free cultivation and detection technologies to reliably determine their sensitivity and specificity for this bacterium.

Evaluation of a rapid screening test for rifampicin resistance in re-treatment tuberculosis patients in the Eastern Cape.

Patients with multidrug-resistant (MDR) tuberculosis (TB) are at high risk of treatment failure. It is anticipated that early identification of MDR-TB and appropriate treatment will improve patient outcome and disease control. We evaluated the rapid detection of rifampicin resistance in previously treated TB patients, directly from acidfast bacilli (AFB)-positive sputum using a phage-based test, FASTPlaque-Response (Biotec Laboratories Ltd, Ipswich, UK). The ability of rifampicin resistance to predict MDR-TB was also determined. A prospective study was done comparing performance of the rapid phage test with conventional culture and drug susceptibility testing (DST) in AFB-positive TB patients. Five primary health clinics and one TB referral centre in the Port Elizabeth Metropolitan area, Eastern Cape. Sensitivity, specificity and overall accuracy of the phage test were determined compared with gold standard culture and DST. Discrepant results were resolved by molecular detection of mutations conferring rifampicin resistance. The proportion of rifampicin-resistant strains that were MDR was also determined. Previously treated patients were at a high risk of MDRTB (35.7%). Sensitivity, specificity and overall accuracy of FASTPlaque-Response for rifampicin resistance determination were 95.4% (95% confidence interval (CI): 91.0 - 99.8%), 97.2% (95% CI: 94.5 - 99.9%) and 96.5% (95% CI: 94.1 - 98.9%) respectively compared with conventional DST (unresolved), calculated for specimens that had both FASTPlaque-Response and conventional DST results available. FASTPlaque-Response results were available in 2 days instead of 28 - 85 days with conventional DST. However, only 70.6% of FASTPlaque-Response results were interpretable compared with 86.3% of conventional DST results. The majority (95.5%) of rifampicinresistant strains were MDR-TB. Rapid detection of rifampicin resistance using FASTPlaque-Response could contribute to improved management of patients at risk of MDR-TB, such as previously treated patients. However, improvement in control of specimen-related contamination is needed to ensure that a higher proportion of FASTPlaque-Response results are interpretable. Where indicated, early modification of therapy could improve patient prognosis and reduce disease transmission.