Use of multiplex allele-specific polymerase chain reaction (MAS-PCR) to detect multidrug-resistant tuberculosis in Panama

The frequency of individual genetic mutations conferring drug resistance (DR) to Mycobacterium tuberculosis has not been studied previously in Central America, the place of origin of many immigrants to the United States. The current gold standard for detecting multidrug-resistant tuberculosis (MDR-TB) is phenotypic drug susceptibility testing (DST), which is resource-intensive and slow, leading to increased MDR-TB transmission in the community. We evaluated multiplex allele-specific polymerase chain reaction (MAS-PCR) as a rapid molecular tool to detect MDR-TB in Panama. Based on DST, 67 MDR-TB and 31 drug-sensitive clinical isolates were identified and cultured from an archived collection. Primers were designed to target five mutation hotspots that confer resistance to the first-line drugs isoniazid and rifampin, and MAS-PCR was performed. Whole-genome sequencing confirmed DR mutations identified by MAS-PCR, and provided frequencies of genetic mutations. DNA sequencing revealed 70.1% of MDR strains to have point mutations at codon 315 of the katG gene, 19.4% within mabA-inhA promoter, and 98.5% at three hotspots within rpoB. MAS-PCR detected each of these mutations, yielding 82.8% sensitivity and 100% specificity for isoniazid resistance, and 98.4% sensitivity and 100% specificity for rifampin resistance relative to DST. The frequency of individual DR mutations among MDR strains in Panama parallels that of other TB-endemic countries. The performance of MAS-PCR suggests that it may be a relatively inexpensive and technically feasible method for rapid detection of MDR-TB in developing countries.     

Mutations linked with antimicrobial resistance in Mycobacterium tuberculosis isolated from tuberculosis patients in the Samara region

A total of 234 M. tuberculosis isolates were used to demonstrate the leading role of mutations in, respectively, codon 531 of gene rpoB (90.0%) and codon 315 of gene katG (92.9%), in the development of resistance to rifampicin and isoniazid by the methods of reverse hybridization with oligonucleotide probes and the sequencing of gene stretches. The levels of primary resistance of M. tuberculosis to rifampicin, isoniazid and multiresistance, according to the molecular-genetic analysis, were 41.0%, 57.7% and 37.2% respectively. The coincidence of the results of the bacteriological and molecular-genetic analyses of the antimicrobial resistance of the isolates was 90.4% and 95.3% for isoniazid and rifampicin respectively. The prevalence of individual types of mutations, linked with antimicrobial resistance, in the presence of a considerable spread of strains of the family Beijing in the region may be indicative of the limited number of M. tuberculosis clones circulating in the region.     

A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe array

AIMS: To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. METHODS AND RESULTS: A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC-6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88.5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR-ahpC intergenic region (86.5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88.5% and 100% for rifampicin resistance, and 86.5% and 100% for isoniazid resistance, respectively. CONCLUSION: A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. SIGNIFICANCE AND IMPACT OF THE STUDY: This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated.     

The use of macroarrays for the identification of MDR Mycobacterium tuberculosis

The emergence of Mycobacterium tuberculosis (Mtb), resistant to both isoniazid (INH) and rifampicin (RIF) (MDR-TB), is an increasing threat to tuberculosis control programs. Susceptibility testing of Mtb complex isolates by phenotypic methods requires a minimum of 14 days from a primary specimen. This can be reduced significantly if molecular analysis is used. Low density oligonucleotide arrays (macroarrays) have been used successfully for the detection of RIF resistance in Mtb. We describe the use of macroarray technology to identify Mtb complex isolates resistant to INH and/or RIF. The macroarray MDR-Mtb screen has been designed to detect mutations in the RIF resistance determining region (RRDR) of Mtb rpoB and loci in katG and mabA-inhA associated with INH resistance. A panel of Mtb isolates containing 38 different RRDR genotypes, 4 different genotypes within codon 315 of katG and 2 genotypes at mabA-inhA was used to validate the macroarray. The wild type (WT) genotype was correctly identified at all three loci. Of the 37 mutant rpoB genotypes, 36 were correctly detected; the single mutant not detected contained a 9 base insertion. All mutations within katG and mabA-inhA were correctly identified. We conclude that this low cost, rapid system can usefully detect the mutations associated with the vast majority of MDR-Mtb.     

Rapid Molecular Detection of Multidrug-Resistant Tuberculosis by PCR-Nucleic Acid Lateral Flow Immunoassay

Several existing molecular tests for multidrug-resistant tuberculosis (MDR-TB) are limited by complexity and cost, hindering their widespread application. The objective of this proof of concept study was to develop a simple Nucleic Acid Lateral Flow (NALF) immunoassay as a potential diagnostic alternative, to complement conventional PCR, for the rapid molecular detection of MDR-TB. The NALF device was designed using antibodies for the indirect detection of labeled PCR amplification products. Multiplex PCR was optimized to permit the simultaneous detection of the drug resistant determining mutations in the 81-bp hot spot region of the rpoB gene (rifampicin resistance), while semi-nested PCR was optimized for the S315T mutation detection in the katG gene (isoniazid resistance). The amplification process additionally targeted a conserved region of the genes as Mycobacterium tuberculosis (Mtb) DNA control. The optimized conditions were validated with the H37Rv wild-type (WT) Mtb isolate and Mtb isolates with known mutations (MT) within the rpoB and katG genes. Results indicate the correct identification of WT (drug susceptible) and MT (drug resistant) Mtb isolates, with the least limit of detection (LOD) being 10⁴ genomic copies per PCR reaction. NALF is a simple, rapid and low-cost device suitable for low resource settings where conventional PCR is already employed on a regular basis. Moreover, the use of antibody-based NALF to target primer-labels, without the requirement for DNA hybridization, renders the device generic, which could easily be adapted for the molecular diagnosis of other infectious and non-infectious diseases requiring nucleic acid detection.     

Epidemic levels of drug resistant tuberculosis (MDR and XDR-TB) in a high HIV prevalence setting in Khayelitsha, South Africa

Background

Although multidrug-resistant tuberculosis (MDR-TB) is emerging as a significant threat to tuberculosis control in high HIV prevalence countries such as South Africa, limited data is available on the burden of drug resistant tuberculosis and any association with HIV in such settings. We conducted a community-based representative survey to assess the MDR-TB burden in Khayelitsha, an urban township in South Africa with high HIV and TB prevalence.

Methodology/Principal Findings

A cross-sectional survey was conducted among adult clinic attendees suspected for pulmonary tuberculosis in two large primary care clinics, together constituting 50% of the tuberculosis burden in Khayelitsha. Drug susceptibility testing (DST) for isoniazid and rifampicin was conducted using a line probe assay on positive sputum cultures, and with culture-based DST for first and second-line drugs. Between May and November 2008, culture positive pulmonary tuberculosis was diagnosed in 271 new and 264 previously treated tuberculosis suspects (sample enriched with previously treated cases). Among those with known HIV status, 55% and 71% were HIV infected respectively. MDR-TB was diagnosed in 3.3% and 7.7% of new and previously treated cases. These figures equate to an estimated case notification rate for MDR-TB of 51/100,000/year, with new cases constituting 55% of the estimated MDR-TB burden. HIV infection was not significantly associated with rifampicin resistance in multivariate analyses.

Conclusions/Significance

There is an extremely high burden of MDR-TB in this setting, most likely representing ongoing transmission. These data highlight the need to diagnose drug resistance among all TB cases, and for innovative models of case detection and treatment for MDR-TB, in order to interrupt transmission and control this emerging epidemic.     

Will tuberculosis become resistant to all antibiotics?

The discovery of high prevalences of antibiotic resistance in some pathogens, in some parts of the world, has provoked fears of a widespread loss of drug efficacy. Here, we use a mathematical model to investigate the evolution of resistance to four major anti-tuberculosis drugs (isoniazid, rifampicin, ethambutol and streptomycin) in 47 sites around the world. The model provides a new method of estimating the relative risk of treatment failure for patients carrying drug-resistant strains and the proportion of patients who develop resistance after failing treatment. Using estimates of these two quantities together with other published data, we reconstructed the epidemic spread of isoniazid resistance over the past 50 years. The predicted median prevalence of resistance among new cases today was 7.0% (range 0.9-64.3%), close to the 6.3% (range 0-28.1%) observed. Predicted and observed prevalences of resistance to isoniazid plus rifampicin (multidrug-resistant or MDR-TB) after 30 years of combined drug use were also similar, 0.9% (0.1-5.9%) and 1.0% (range 0-14.1%), respectively. With current data, and under prevailing treatment practices, it appears that MDR-TB will remain a localized problem, rather than becoming a global obstacle to tuberculosis control. To substantiate this result, further measurements are needed of the relative fitness of drug-resistant strains.     

Comparison of drug resistance genotypes between Beijing and non-Beijing family strains of Mycobacterium tuberculosis in Korea

The frequency of resistance genotypes among Beijing and non-Beijing strains was compared using a reverse blot hybridization assay to detect mutations within genes associated with rifampicin (rpoB) and isoniazid (katG, inhA, and ahpC) resistance. Of the 743 Mycobacterium tuberculosis isolates, 569 (77%) belonged to Beijing family. The proportion of Beijing strains was significantly higher among MDR-TB isolates than among drug-susceptible strains (82% vs. 72%, p<0.01). Genotype analysis of the rpoB gene revealed significantly lower rates of the Ser531Leu mutation rate among Beijing vs. non-Beijing MDR-TB strains (41% vs. 66%, p<0.005). While the mutation for Ser315Thr in the katG gene was more common among Beijing vs. non-Beijing family strains (65% vs. 50%, p<0.01), the mutation rate of promoter region of the inhA gene was lower among Beijing strains compared with non-Beijing strains (14% vs. 25%, p<0.05). Reverse hybridization successfully detected over 80% of isoniazid-resistant strains and over 92% of rifampicin-resistant strains among Korean isolates. Significant differences in mutation rates in the rpoB, katG, and inhA genes between Beijing strains and non-Beijing strains could explain discrepancies in mutation rates of genotypes in different countries. Reverse hybridization was useful for rapid detection of isoniazid and rifampicin resistant strains.     

A population-based study of first and second-line drug-resistant tuberculosis in a high-burden area of the Mexico/United States border

The resistance of 139 Mycobacterium tuberculosis (MTB) isolates from the city of Monterrey, Northeast Mexico, to first and second-line anti-TB drugs was analysed. A total of 73 isolates were susceptible and 66 were resistant to anti-TB drugs. Monoresistance to streptomycin, isoniazid (INH) and ethambutol was observed in 29 cases. Resistance to INH was found in 52 cases and in 29 cases INH resistance was combined with resistance to two or three drugs. A total of 24 isolates were multidrug-resistant (MDR) resistant to at least INH and rifampicin and 11 MDR cases were resistant to five drugs. The proportion of MDR-TB among new TB cases in our target population was 0.72% (1/139 cases). The proportion of MDR-TB among previously treated cases was 25.18% (35/139 cases). The 13 polyresistant and 24 MDR isolates were assayed against the following seven second-line drugs: amikacin (AMK), kanamycin (KAN), capreomycin (CAP), clofazimine (CLF), ethionamide (ETH), ofloxacin (OFL) and cycloserine (CLS). Resistance to CLF, OFL or CLS was not observed. Resistance was detected to ETH (10.80%) and to AMK (2.70%), KAN (2.70%) and CAP (2.70%). One isolate of MDR with primary resistance was also resistant to three second-line drugs. Monterrey has a high prevalence of MDR-TB among previously treated cases and extensively drug-resistant-MTB strains may soon appear.     

Molecular characterization of mutation associated with rifampicin and isoniazid resistance in Mycobacterium tuberculosis isolates

Nucleotide changes in catalase peroxidase (Kat G) gene and gene encoding the beta subunit of RNA polymerase (rpo B), responsible for isoniazid and rifampicin drug resistance were determined in the clinical isolates of Mycobacterium tuberculosis by PCR-RFLP, Line probe assay and DNA sequencing. PCR-RFLP test was performed by HapII cleavage of an amplified fragment of Kat G gene to detect the transversion 315AGC-->ACC(Ser-->Thr) which is associated with INH drug resistance. The Line probe assay kit was evaluated to detect the mutation in 81bp RMP resistance determining region of rpo B gene associated with RMP drug resistance. These results were validated by DNA sequencing and drug susceptibility test. Kat G S 315 T mutation was found in 74.19% strains of M. tuberculosis from Delhi. This mutation was not found in any of the susceptible strains tested. The line probe assay kit and DNA sequencing identified 18 isolates as RMP resistant with specific mutation, while one of the RMP resistant strain was identified as RMP susceptible, with a concordance of 94.73% with the phenotypic drug susceptibility result. Majority (8 of 19, 42.1%) of resistant isolates involved base changes at codon 531 of rpo B gene. Both PCR-RFLP and Line probe assay test can be used in many of the clinical microbiology laboratories for early detection of isoniazid and rifampicin drug resistance in clinical isolates of M. tuberculosis.     

High Isoniazid Resistance Rates in Rifampicin Susceptible Mycobacterium tuberculosis Pulmonary Isolates from Pakistan

Background

Rapid new diagnostic methods (including Xpert MTB/RIF assay) use rifampicin resistance as a surrogate marker for multidrug resistant tuberculosis. Patients infected with rifampicin susceptible strains are prescribed first line anti-tuberculosis therapy. The roll out of such methods raises a concern that strains with resistance to other first line anti-tuberculosis drugs including isoniazid will be missed and inappropriate treatment given. To evaluate implications of using such methods review of resistance data from high burden settings such as ours is essential.

Objective

To determine resistance to first line anti-tuberculosis drugs amongst rifampicin susceptible pulmonary Mycobacterium tuberculosis (MTB) isolates from Pakistan.

Materials and Methods

Data of pulmonary Mycobacterium tuberculosis strains isolated in Aga Khan University Hospital (AKUH) laboratory (2009–2011) was retrospectively analyzed. Antimicrobial susceptibility profile of rifampicin susceptible isolates was evaluated for resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin.

Results

Pulmonary specimens submitted to AKUH from 2009 to 2011 yielded 7738 strains of Mycobacterium tuberculosis. These included 54% (n 4183) rifampicin susceptible and 46% (n: 3555) rifampicin resistant strains. Analysis of rifampicin susceptible strains showed resistance to at least one of the first line drugs in 27% (n:1133) of isolates. Overall isoniazid resistance was 15.5% (n: 649), with an isoniazid mono-resistance rate of 4% (n: 174). Combined resistance to isoniazid, pyrazinamide, and ethambutol was noted in 1% (n: 40), while resistance to isoniazid, pyrazinamide, ethambutol, and streptomycin was observed in 1.7% (n: 70) of strains.

Conclusions

Our data suggests that techniques (including Xpert MTB/RIF assay) relying on rifampicin susceptibility as an indicator for initiating first line therapy will not detect patients infected with MTB strains resistant to other first line drugs (including isoniazid). The roll out of these techniques must therefore be accompanied by strict monitoring ensuring early resistance detection to increase chances of improved patient outcomes.     

Impact of reduced vancomycin susceptibility on the therapeutic outcome of MRSA bloodstream infections

Background

Resistance to anti-tuberculosis drugs is a serious public health problem. Multi-drug resistant tuberculosis (MDR-TB), defined as resistance to at least rifampicin and isoniazid, has been reported in all regions of the world. Current phenotypic methods of assessing drug susceptibility of M. tuberculosis are slow. Rapid molecular methods to detect resistance to rifampicin have been developed but they are not affordable in some high prevalence countries such as those in sub Saharan Africa. A simple multi-well plate assay using mycobacteriophage D29 has been developed to test M. tuberculosis isolates for resistance to rifampicin. The purpose of this study was to investigate the performance of this technology in Kampala, Uganda.

Methods

In a blinded study 149 M. tuberculosis isolates were tested for resistance to rifampicin by the phage assay and results compared to those from routine phenotypic testing in BACTEC 460. Three concentrations of drug were used 2, 4 and 10 μg/ml. Isolates found resistant by either assay were subjected to sequence analysis of a 81 bp fragment of the rpoB gene to identify mutations predictive of resistance. Four isolates with discrepant phage and BACTEC results were tested in a second phenotypic assay to determine minimal inhibitory concentrations.

Results

Initial analysis suggested a sensitivity and specificity of 100% and 96.5% respectively for the phage assay used at 4 and 10 μg/ml when compared to the BACTEC 460. However, further analysis revealed 4 false negative results from the BACTEC 460 and the phage assay proved the more sensitive and specific of the two tests. Of the 39 isolates found resistant by the phage assay 38 (97.4%) were found to have mutations predictive of resistance in the 81 bp region of the rpoB gene. When used at 2 μg/ml false resistant results were observed from the phage assay. The cost of reagents for testing each isolate was estimated to be 1.3US$ when testing a batch of 20 isolates on a single 96 well plate. Results were obtained in 48 hours.

Conclusion

The phage assay can be used for screening of isolates for resistance to rifampicin, with high sensitivity and specificity in Uganda. The test may be useful in poorly resourced laboratories as a rapid screen to differentiate between rifampicin susceptible and potential MDR-TB cases.     

Management of extensively drug-resistant tuberculosis in Peru: cure is possible

Aim

To describe the incidence of extensive drug-resistant tuberculosis (XDR-TB) reported in the Peruvian National multidrug-resistant tuberculosis (MDR-TB) registry over a period of more than ten years and present the treatment outcomes for a cohort of these patients.

Methods

From the Peruvian MDR-TB registry we extracted all entries that were approved for second-line anti-TB treatment between January 1997 and June of 2007 and that had Drug Susceptibility Test (DST) results indicating resistance to both rifampicin and isoniazid (i.e. MDR-TB) in addition to results for at least one fluoroquinolone and one second-line injectable (amikacin, capreomycin and kanamycin).

Results

Of 1,989 confirmed MDR-TB cases with second-line DSTs, 119(6.0%) XDR-TB cases were detected between January 1997 and June of 2007. Lima and its metropolitan area account for 91% of cases, a distribution statistically similar to that of MDR-TB. A total of 43 XDR-TB cases were included in the cohort analysis, 37 of them received ITR. Of these, 17(46%) were cured, 8(22%) died and 11(30%) either failed or defaulted treatment. Of the 14 XDR-TB patients diagnosed as such before ITR treatment initiation, 10 (71%) were cured and the median conversion time was 2 months.

Conclusion

In the Peruvian context, with long experience in treating MDR-TB and low HIV burden, although the overall cure rate was poor, a large proportion of XDR-TB patients can be cured if DST to second-line drugs is performed early and treatment is delivered according to the WHO Guidelines.     

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.     

Characterization of mutations causing rifampicin and isoniazid resistance of Mycobacterium tuberculosis in Syria

In order to characterize mutations causing rifampicin and isoniazid resistance of M. tuberculosis in Syria, 69 rifampicin resistant (Rif(r)) and 72 isoniazid resistant (Inh(r)) isolates were screened for point mutations in hot spots of the rpoB, katG and inhA genes by DNA sequencing and real time PCR. Of 69 Rif(r) isolates, 62 (90%) had mutations in the rifampin resistance determining region (RRDR) of the rpoB gene, with codons 531 (61%), 526 (13%), and 516 (8.7%) being the most commonly mutated. We found two new mutations (Asp516Thr and Ser531Gly) described for the first time in the rpoB-RRDR in association with rifampicin resistance. Only one mutation (Ile572Phe) was found outside the rpoB-RRDR. Of 72 Inh(r) strains, 30 (41.6%) had a mutation in katGcodon315 (with Ser315Thr being the predominant alteration), and 23 (32%) harbored the inhA(-15C-->T) mutation. While the general pattern of rpoB-RRDR and katG mutations reflected those found worldwide, the prevalence of the inhA(-15C-->T mutation was above the value found in most other countries, emphasizing the great importance of testing the inhA(-15C-->T) mutation for prediction of isoniazid resistance in Syria. Sensitivity of a rapid test using real time PCR and 3'-Minor groove binder (MGB) probes in detecting Rif(r) and Inh(r) isolates was 90% and 69.4%, respectively. This demonstrates that a small set of MGB-probes can be used in real time PCR in order to detect most mutations causing resistance to rifampicin and isoniazid.     

Extensively drug-resistant tuberculosis: current challenges and threats

Extensively drug-resistant tuberculosis (XDR-TB) is defined as tuberculosis caused by a Mycobacterium tuberculosis strain that is resistant to at least rifampicin and isoniazid among the first-line antitubercular drugs (multidrug-resistant tuberculosis; MDR-TB) in addition to resistance to any fluroquinolones and at least one of three injectable second-line drugs, namely amikacin, kanamycin and/or capreomycin. Recent studies have described XDR-TB strains from all continents. Worldwide prevalence of XDR-TB is estimated to be c. 6.6% in all the studied countries among multidrug-resistant M. tuberculosis strains. The emergence of XDR-TB strains is a reflection of poor tuberculosis management, and controlling its emergence constitutes an urgent global health reality and a challenge to tuberculosis control activities in all parts of the world, especially in developing countries and those lacking resources and as well as in countries with increasing prevalence of HIV/AIDS.     

Contribution of efflux to the emergence of isoniazid and multidrug resistance in Mycobacterium tuberculosis

Multidrug resistant (MDR) tuberculosis is caused by Mycobacterium tuberculosis resistant to isoniazid and rifampicin, the two most effective drugs used in tuberculosis therapy. Here, we investigated the mechanism by which resistance towards isoniazid develops and how overexpression of efflux pumps favors accumulation of mutations in isoniazid targets, thus establishing a MDR phenotype. The study was based on the in vitro induction of an isoniazid resistant phenotype by prolonged serial exposure of M. tuberculosis strains to the critical concentration of isoniazid employed for determination of drug susceptibility testing in clinical isolates. Results show that susceptible and rifampicin monoresistant strains exposed to this concentration become resistant to isoniazid after three weeks; and that resistance observed for the majority of these strains could be reduced by means of efflux pumps inhibitors. RT-qPCR assessment of efflux pump genes expression showed overexpression of all tested genes. Enhanced real-time efflux of ethidium bromide, a common efflux pump substrate, was also observed, showing a clear relation between overexpression of the genes and increased efflux pump function. Further exposure to isoniazid resulted in the selection and stabilization of spontaneous mutations and deletions in the katG gene along with sustained increased efflux activity. Together, results demonstrate the relevance of efflux pumps as one of the factors of isoniazid resistance in M. tuberculosis. These results support the hypothesis that activity of efflux pumps allows the maintenance of an isoniazid resistant population in a sub-optimally treated patient from which isoniazid genetically resistant mutants emerge. Therefore, the use of inhibitors of efflux should be considered in the development of new therapeutic strategies for preventing the emergence of MDR-TB during treatment.     

Temperature-mediated heteroduplex analysis for the detection of drug-resistant gene mutations in clinical isolates of Mycobacterium tuberculosis by denaturing HPLC, SURVEYOR nuclease

Denaturing high-performance liquid chromatography (DHPLC) is a relatively new technique, which utilizes heteroduplex formation between wild-type and mutated DNA strands to identify point mutations. Heteroduplex molecules are separated from homoduplex molecules by ion-pair, reverse-phase liquid chromatography on a special column matrix with partial heat denaturation of the DNA strands. In order to investigate the application of this method for point mutation detection in drug-resistant genes of Mycobacterium tuberculosis, katG, rpoB, embB, gyrA, pncA and rpsL genes, which are responsible for isoniazid, rifampicin, ethambutol, fluoroquinolone, pyrazinamide and streptomycin resistance, respectively, were detected by temperature-mediated DHPLC in 10 multidrug-resistant and 10 drug-susceptible clinical isolates. The DHPLC data were compared with those from a conventional MIC test. The results show that DHPLC is cost-effective with high capacity and accuracy, and is potentially useful for genotypic screening for mutations associated with anti-tuberculosis drug resistance.     

Programmatically Selected Multidrug-Resistant Strains Drive the Emergence of Extensively Drug-Resistant Tuberculosis in South Africa

Background

South Africa shows one of the highest global burdens of multidrug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (TB). Since 2002, MDR-TB in South Africa has been treated by a standardized combination therapy, which until 2010 included ofloxacin, kanamycin, ethionamide, ethambutol and pyrazinamide. Since 2010, ethambutol has been replaced by cycloserine or terizidone. The effect of standardized treatment on the acquisition of XDR-TB is not currently known.

Methods

We genetically characterized a random sample of 4,667 patient isolates of drug-sensitive, MDR and XDR-TB cases collected from three South African provinces, namely, the Western Cape, Eastern Cape and KwaZulu-Natal. Drug resistance patterns of a subset of isolates were analyzed for the presence of commonly observed resistance mutations.

Results

Our analyses revealed a strong association between distinct strain genotypes and the emergence of XDR-TB in three neighbouring provinces of South Africa. Strains predominant in XDR-TB increased in proportion by more than 20-fold from drug-sensitive to XDR-TB and accounted for up to 95% of the XDR-TB cases. A high degree of clustering for drug resistance mutation patterns was detected. For example, the largest cluster of XDR-TB associated strains in the Eastern Cape, affecting more than 40% of all MDR patients in this province, harboured identical mutations concurrently conferring resistance to isoniazid, rifampicin, pyrazinamide, ethambutol, streptomycin, ethionamide, kanamycin, amikacin and capreomycin.

Conclusions

XDR-TB associated genotypes in South Africa probably were programmatically selected as a result of the standard treatment regimen being ineffective in preventing their transmission. Our findings call for an immediate adaptation of standard treatment regimens for M/XDR-TB in South Africa.     

广泛耐药结核分枝杆菌耐药机制及其疾病诊断的研究进展

自20世纪90年代以来,全球结核病疫情回升,结核分枝杆菌耐药是其中的一个重要原因.广泛耐药结核病是指在耐多药结核病(即同时对异烟肼和利福平耐药的结核分枝杆菌引起的结核病)的基础上,还对氟喹诺酮类药物和至少3种二线静脉用抗结核药物(卷曲霉素、卡那霉素、阿米卡星)中的1种耐药的结核分枝杆菌引起的结核病.我国是结核病高流行国...