Detection Of Multidrug-resistant Mycobacterium Tuberculosis Research Articles

The occurrence of multidrug-resistant Mycobacterium tuberculosis from patients of pulmonary tuberculosis.

Multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is one of the leading causes of death in the world. The resource constraints make it difficult to diagnose and monitor the cases of MDR-TB. GeneXpert is a recognized tool used to diagnose the patients of pulmonary tuberculosis in clinical settings across the globe. The present one-year cross-sectional study was conducted to estimate the occurrence of MDR-TB in patients with pulmonary TB. A total of 1000 patients suspected of pulmonary tuberculosis were included in this study. A random convenient sampling technique was done to collect the sputum samples (twice) from the patients. Samples were processed for the detection of Mycobacterium tuberculosis using conventional detection methods like the Ziehl Nelson staining method and fluorescent microscopy. Additionally, Cepheid GeneXpert was used for molecular detection of MDR-TB in smear-positive samples of pulmonary tuberculosis by amplifying the rifampicin resistance determining region (RRDR; rpoB gene). All the tests were performed in the biosafety level III lab of District Headquarters Hospital Nankana Sahib. It was observed that 103 (10.3%) individuals were diagnosed as positive for tuberculosis among 1000 patients. Among these 103 TB positive cases, there were 11 (10.7%) patients diagnosed with rifampicin resistance gene (RR-Gene) of Mycobacterium tuberculosis. Overall findings of the study showed that MDR-TB is prevalent in pulmonary TB patients and GeneXpert is the most sensitive technique for early diagnosis of the disease, which may be very helpful in the treatment and control of this public health menace in low and middle-income countries.

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen "Z-N" stained smear and culture on Lowenstein-Jenson media "LJ"). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

ASSESSMENT OF THE MICROSCOPIC– OBSERVATION- DRUG - SUSCEPTIBLITY ASSAY (MODS) AND GENEXPERT ASSAY FOR DIAGNOSIS OF PULMONARY TUBERCULOSIS

Background: Tuberculosis (TB) is considered the most common and highly contagious disease worldwide. Early and accurate diagnosis of TB with detection of multidrug-resistant Mycobacterium tuberculosis (M.TB) is of primary importance for both patient management and infection control. Objectives: To evaluate the performance of Microscopic-Observation Drug-Susceptibility (MODS) assay and GeneXpert MTB/RIF( GeneXpert ) assay in diagnosis of TB and detection of MDR-TB directly from sputum specimens in comparison to the convential diagnostic methods (direct Ziehl-Neelsen stained smear and culture on Lowenstein-Jenson media LJ). Patients and Methods: After approval of institutional ethical committee a total of 120 suspected pulmonary TB patients admitted to chest hospitals and dispensaries in Egypt (Giza, Cairo, Alexandria, Dakahlia, Suez, Beheira, Monufia, Qena). Their age was more than 16 years; all of them did not receive any anti tubercular drugs. Sputum samples were collected from each patient and subjected to direct ZN smear, culture and sensitivity of M.TB on LJ and Middlebrook 7H9 medium for MODS assay. Finally Genexpert M.TB/RIF assay was done directly for all sputum samples. Results: Infection rate among males was higher than female (81.7% & 18.7%). Also, it was observed that 56.7% of sputum samples gave positive direct Z-N smear, while 64.2% were positive for both LJ culture &MODS assay. On the other hand, by Genexpert, 68% of cases were positive. The sensitivity and specificity of the various diagnostic methods were studied in relation to LJ culture (gold standard). It was found that MODS assay was rapid that results were obtained in a median of 8 days ranged from (7– 21 days), while median time of LJ was 21dayes ranged from (21– 60). MODS had 100% sensitivity and specificity. Direct smear was less sensitive (88.3%), and GeneXpert was less specific (88.4%), but more rapid results were obtained in 2 hours. Finally, detection of MDR-TB using various methods for 77 isolated M.TB strains ranged from 16.9-19.5% to isoniazid (INH) and 9.8%-18.2% to rifampicin (RMP) by various diagnostic methods with statistically insignificant difference between them. Conclusion: MODS was an optimal alternative rapid diagnostic technique for TB and detection of M.TB and MDR-TB in resource limited settings, results were obtained in a median of 8 days. On the other hand, Gene Xpert was a novel integrated diagnostic PCR analysis in a single hand free step for rapid diagnosis of TB and detection of RMP resistance in clinical specimens in 2 hours but it was expensive.

Detection of mutations associated with resistance to rifampicin and isoniazid in <italic>Mycobacterium tuberculosis</italic> by polymerase chain reaction-ligase detection reaction

Mycobacterium tuberculosis (MTB) infection remains a serious infectious disease worldwide, causing 8.8 million new infections and 1.45 million deaths in 2010 [1]. The emergence of drug-resistant strains of MTB poses a significant threat to the control of the disease globally. Multidrugresistant MTB (MDR-TB), defined as being resistant to at least rifampicin (RMP) and isoniazid (INH), and extensively drug-resistant TB (XDR-TB), defined as being additionally resistant to any fluoroquinolone and one injectable secondline drug, are associated with higher treatment failure rates. China is the second largest nation in terms of the number of the MTB infection, and 9.3% of the cases are MDR-TB [2]. MDR-TB arises from spontaneous chromosomal mutations at low frequency, but clinical MDR-TB largely occurs as a result of man-made selection during disease treatment of these genetic alterations through erratic drug supply, suboptimal physician prescription, and poor patient adherence [3]. S315T/M mutation in katG and mutations in the promoter region of inhA operon are the main mechanisms of INH resistance [4,5]. Mutations at positions 516, 526, and 531 in rpoB are the most frequent mutations in RMP-resistant MTB strains [6]. Since the transmission of the selected MDR-TB strains causes a large number of MDR-TB cases [7,8], the need for rapid, reliable, and cost-effective drug susceptibility testing (DST) is urgent. Polymerase chain reaction (PCR) followed by ligase detection reaction (LDR) method has been widely used to detect various single nucleotide polymorphisms and drug-resistant mutations with high sensitivity and low cost [9–12]. In the present study, we established a PCR-LDR assay for the simultaneous detection of the mutations related to MDR-TB, and evaluated its performance using isolates and clinical samples. Here, a total of 120 MTB isolates obtained from pulmonary TB patients at The Third Hospital of Changzhou, during July, 2011 and March, 2012 were collected and subjected to DST for RMP and INH in the BACTEC MGIT 960 instrument with medium, and DST supplement (Becton Dickinson and Company, Sparks, USA) as recommended by the manufacturer. All the 120 MTB isolates were detected by phenotypic DST, PCR-LDR, and DNA sequencing. The specific primers and probes (Supplementary Tables S1 and S2) for both wild type and mutant type targeting codon 315 of the katG gene, codon 15 of the inhA promoter region, codons 516, 526, and 531 of rpoB. All the primers and probes were synthesized by Invitrogen (Shanghai, China). Genome DNA of MTB was extracted by a commercial kit (Fosun Diagnostics, Shanghai, China) according to manufacturer’s instruction. All isolates were detected by both the PCR-LDR assay and sequencing. The multiplex PCR-LDR method was the same as described in previous studies [11,12]. The amplifications and LDR were performed in a PE 9600 thermal cycler (Life Technology, Foster City, USA). The LDR products were electrophoresed on an ABI 377 DNA Sequencer (Life Technology) for 30 min. Results were analyzed using the GeneMapper software (Life Technology). Meanwhile, the PCR products of rpoB and inhA promoter region were purified and sequenced by Life Technology. The results of phenotypic DST, PCR-LDR, and DNA sequencing were summarized in Table 1. Phenotypic DST of MTB isolates indicated that 26 (21.7%) at low level (1 mg/ ml) and 11 (9.2%) at high level (10 mg/ml) resistance to INH, while 17 (14.2%) at low-level and 9 (7.5%) at highlevel resistance to RMP. Among them, 13 (10.8%) were resistant to both INH and RMP at low level, or MDR. PCR-LDR assays for the detection of MDR-TB were carried out on 120 isolates. The S315M in katG was detected in 16 (13.3%) of 120 isolates. No S315T mutation in katG and G15T mutation in the promoter region of inhAwas detected. The D516V/G, H526Y/D, S531/L/W mutations in rpoB were detected in 1 (0.8%), 3 (2.5%), and 8 (6.7%) of 120 isolates, respectively. The typical results of PCR-LDR were shown in Fig. 1 and Supplementary Fig. S1. Acta Biochim Biophys Sin 2014, 46: 78–81 |a The Author 2013. Published by ABBS Editorial Office in association with Oxford University Press on behalf of the Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences. DOI: 10.1093/abbs/gmt119. Advance Access Publication 11 November 2013

Microscopic observation drug susceptibility assay in the diagnosis of multidrug-resistant tuberculosis

Early detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) is of primary importance for both patient management and infection control. Optimal methods for identifying MDR-TB in a timely and affordable manner in resource-limited settings are not yet available. To evaluate the performance of a low-technology but rapid drug susceptibility testing method, the microscopic observation drug susceptibility assay (MODS), in the concurrent detection of M. tuberculosis and its susceptibility to isoniazid (INH) and rifampin (RMP) directly from sputum specimens. A total of 115 smear-positive TB patients admitted to Abbasia Chest Hospital, Cairo, Egypt, were simultaneously tested using MODS and the BACTEC MGIT 960 mycobacterial detection system for the detection of M. tuberculosis and the identification of MDR-TB samples. MODS detected 112 (97.4%) samples and BACTEC MGIT detected 115 (100%). Of the 115 isolates tested for susceptibility to INH, RMP and MDR-TB, complete agreement between MODS and MGIT results was found among respectively 92.9%, 95.5% and 97.3% of samples. The sensitivity, specificity, and positive and negative predictive values of MODS in the detection of MDR-TB were respectively 95.3%, 98.6%, 97.6% and 97.1%. MODS results were obtained in a median of 8 days (range 5-21). MODS is an optimal alternative method for timely and affordable identification of MDR-TB in resource-limited settings.

Nitrate reductase assay using sodium nitrate for rapid detection of multidrug resistant tuberculosis.

We validated the nitrate reductase assay (NRA) for the detection of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) using sodium nitrate (NaNO3) in replacement of potassium nitrate (KNO3) as nitrate source. NaNO3 is cheaper than KNO3 and has no restriction on use which facilitates the implementation of NRA to detect MDR-TB.

Evaluation of nitrate reduction assay, resazurin microtiter assay and microscopic observation drug susceptibility assay for first line antitubercular drug susceptibility testing of clinical isolates of M. tuberculosis

Background Drug resistant tuberculosis (TB) is a growing concern worldwide. Early detection of multidrug-resistant Mycobacterium tuberculosis is of primary importance for both patient management and infection control. Optimal method for identifying drug-resistant M. tuberculosis in a timely and affordable way in resource-limited settings is not yet available. Aim This study evaluated; nitrate reductase assay (NRA), resazurin microtiter assay (REMA) and microscopic observation drug susceptibility assay (MODS) against the conventional 1% proportion method (PM) for the detection of resistance to first line antitubercular drugs, in M. tuberculosis clinical isolates. Methods A total of one hundred and five clinical isolates of M. tuberculosis; 50 pan sensitive and 55 pan resistant were tested with NRA, REMA and MODS. The 1% proportion method on Lowenstein–Jensen medium was used as reference test. Results Of all three methods which were tested NRA was found to be most sensitive and specific. Sensitivity for rifampicin resistance detection was 100%, 94.55% and 92.73% by NRA, REMA and MODS respectively. NRA and REMA were found to be 100% specific, while the MODS was 98% specific for detection of rifampicin resistance. Test results with all these methods were obtained within 8–14 days. Conclusion Rapid non-conventional and inexpensive methods may serve as a replacement for 1% proportion method in resource limited settings.

Utility of nitrate reductase assay for detection of multidrug-resistant Mycobacterium tuberculosis in a low resource setting.

Introduction. The performance of a drug susceptibility test may change when moving from the research stage to implementation on a population level in actual public health practice. Objective. The performance of a rapid drug susceptibility test was described for detecting multidrug-resistant Mycobacterium tuberculosis when implemented in the routine workflow of a low-resource reference laboratory. Materials and methods. A prospective study was done comparing the performance of the nitrate reductase assay with the conventional proportion method for rifampicin and isoniazid on 364 isolates were obtained from multidrug-resistant tuberculosis risk patients referred from diffrent Colombian laboratories. Results. When compared with the proportion method, the nitrate reductase assay sensitivity was 86.8% and 84.9% for rifampicin and isoniazid, respectively, whereas nitrate reductase assay specificity was 100% for isoniazid and rifampicin. Nitrate reductase assay sensitivity was significantly higher when the age of isolate was less than 70 days. A sensitivity of 94.4% dropped to 78.1% for rifampicin resistance for fresh and old isolates, respectively (Fisher exact test, p=0.05). For isoniazid resistance using fresh and old isolates, 94.7% vs.74.3% sensitivities, were achieved (chi square test, p=0.03). The proportion of nitrate reductase assay ambiguous results was significantly higher in multidrug-resistant than in non-multidrug-resistant isolates (17.6% vs. 4.0%, chi square test, p<0.005). Conclusions. The nitrate reductase assay demonstrated provided reliable results for antibiotic resistance. However, using old cultures leds to a higher proportion of false sensitive results; furthermore, the nitrate reductase assay capability to detect multidrug-resistant tuberculosis decreased due to a higher proportion of non-interpretable results.

A rapid detection of multidrug-resistant Mycobacterium tuberculosis by a nitrate reductase assay on blood agar

The susceptibility of 49 Mycobacterium tuberculosis clinical isolates to isoniazid (INH) and rifampisin (RIF) (28 multi-drug resistant-tuberculosis samples) was determined by a nitrate reductase assay (NRA) on blood agar. Agreement between the NRA and other testing methods was found to be 93.8% for both INH and RIF. The sensitivity, specificity, positive predictive value and negative predictive value for INH were 92.8%, 94.2%, 86.6% and 97%, respectively. The sensitivity, specificity, positive predictive value and negative predictive value for RIF were 90.4%, 96.4%, 95% and 93.1%. In conclusion, we show here that blood agar can be used effectively for the NRA test.

Fast track method for the identification of multi–drug resistant tuberculosis on direct clinical specimen using combined drug media

Objective To combine isoniazid (INH) and rifampicin (RIF) in a single media to detect and evaluate multi-drug resistant Mycobacterium tuberculosis (MDR-TB) strains using clinical specimens by direct and indirect drug susceptibility testing (DST). Methods Drug susceptibility testing for INH and RIF using individual and combined drug media was performed by minimum inhibitory concentration (MIC) method on direct clinical specimens. Results The combined drug media showed complete concordance with individual drug media in the detection of MDR-TB by direct DST method and 89% efficiency with indirect DST method. Susceptibility results were available by 3 weeks after the receipt of clinical specimen using direct DST on combined drug media. Conclusions Combined drug media can be used as a fast track method in large scale studies warranting detection of MDR-TB.

Combined drug medium with isoniazid and rifampicin for identification of multi-drug resistant Mycobacterium tuberculosis

A low-cost method of detecting multi-drug resistant Mycobacterium tuberculosis (MDR-TB) with the possibility of quick adoption in a resource limited setting is urgently required. We conducted a study combining isoniazid and rifampicin in a single LJ medium, to detect MDR-TB strains. Combined and individual drug media showed 100% concordance for the detection of MDR-TB and susceptible strains by proportion method. Considering the results, combined isoniazid and rifampicin containing medium could be considered for use in settings where the sole detection of MDR-TB strains is justified.

Rapid detection of multidrug-resistant Mycobacterium tuberculosis in Cotonou (Benin) using two low-cost colorimetric methods: resazurin and nitrate reductase assays

We have evaluated two simple, rapid and low-cost colorimetric methods for the detection of multidrug-resistant Mycobacterium tuberculosis. A total of 151 M. tuberculosis strains were tested for resistance to rifampicin (RMP) and isoniazid by resazurin microplate assay (REMA) and nitrate reductase assay (NRA) in comparison with the conventional proportion method (PM) on Löwenstein-Jensen medium. A complete agreement was found between NRA and PM, while one false RMP-susceptible result was found by REMA. REMA and NRA tests are rapid and inexpensive, and could be good alternatives to the conventional PM in low-resource countries.

Distribution of rpoB mutations among multidrug-resistant Mycobacterium tuberculosis (MDRTB) strains from Thailand and development of a rapid method for mutation detection

Since rifampicin resistance is a surrogate marker for multidrug-resistant Mycobacterium tuberculosis (MDRTB), the present study aimed to investigate rpoB mutations conferring rifampicin resistance in M. tuberculosis strains from Thailand, and to develop a rapid, inexpensive and simple PCR-based method for rapid detection of MDRTB. Overall, 267 M. tuberculosis isolates, including 143 MDRTB isolates, were investigated. Isolates of the Beijing strain predominated among the MDRTB isolates (79.1%), but accounted for only 45.5% of the susceptible isolates. Mutations in the rpoB gene were found most commonly at codons 531, 526 and 516 (58%, 25.2% and 9.1%, respectively). A multiplex allele-specific PCR was developed and tested with 216 clinical isolates. In comparison with the proportion method, the method showed 94.2% sensitivity and 100% specificity, and had a 100% positive predictive value and a 95% negative predictive value, which suggested that this method could be useful for screening for MDRTB, particularly in resource-limited countries.

Rapid and low-cost colorimetric method using 2,3,5-triphenyltetrazolium chloride for detection of multidrug-resistant Mycobacterium tuberculosis

A rapid and inexpensive method for the detection of drug resistance in Mycobacterium tuberculosis is essential for the effective control of tuberculosis. The aim of this study was to evaluate a colorimetric method using 2,3,5-triphenyltetrazolium chloride (TTC) for antibiotic susceptibility testing of M. tuberculosis isolates. Eleven multidrug-resistant (MDR) isolates of M. tuberculosis and 12 isolates which were susceptible to rifampicin (RIF) and isoniazid (INH) were used. The test was performed with a critical concentration of 0.2 microg ml(-1) for INH and 2.0 microg ml(-1) for RIF in 7H9GC broth with 0.625 microg TTC ml(-1). Each isolate was inoculated under these conditions and inspected daily for colour changes; the results were obtained after a mean of 4.9 days. The sensitivity and specificity of this method were 100 % and 92 %, respectively, for both antibiotics. Considering the speed, technical ease and cost-effectiveness of this method, the TTC assay is a good alternative method for drug susceptibility testing of M. tuberculosis isolates.