Your browser doesn't support javascript.
loading
Show: 20 | 50 | 100
Results 1 - 10 de 10
Filter
1.
Am J Respir Crit Care Med ; 194(5): 621-30, 2016 09 01.
Article in English | MEDLINE | ID: mdl-26910495

ABSTRACT

RATIONALE: The development of molecular diagnostics that detect both the presence of Mycobacterium tuberculosis in clinical samples and drug resistance-conferring mutations promises to revolutionize patient care and interrupt transmission by ensuring early diagnosis. However, these tools require the identification of genetic determinants of resistance to the full range of antituberculosis drugs. OBJECTIVES: To determine the optimal molecular approach needed, we sought to create a comprehensive catalog of resistance mutations and assess their sensitivity and specificity in diagnosing drug resistance. METHODS: We developed and validated molecular inversion probes for DNA capture and deep sequencing of 28 drug-resistance loci in M. tuberculosis. We used the probes for targeted sequencing of a geographically diverse set of 1,397 clinical M. tuberculosis isolates with known drug resistance phenotypes. We identified a minimal set of mutations to predict resistance to first- and second-line antituberculosis drugs and validated our predictions in an independent dataset. We constructed and piloted a web-based database that provides public access to the sequence data and prediction tool. MEASUREMENTS AND MAIN RESULTS: The predicted resistance to rifampicin and isoniazid exceeded 90% sensitivity and specificity but was lower for other drugs. The number of mutations needed to diagnose resistance is large, and for the 13 drugs studied it was 238 across 18 genetic loci. CONCLUSIONS: These data suggest that a comprehensive M. tuberculosis drug resistance diagnostic will need to allow for a high dimension of mutation detection. They also support the hypothesis that currently unknown genetic determinants, potentially discoverable by whole-genome sequencing, encode resistance to second-line tuberculosis drugs.


Subject(s)
Antitubercular Agents/pharmacology , Drug Resistance, Multiple, Bacterial/genetics , Molecular Diagnostic Techniques , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/genetics , Drug Resistance, Multiple, Bacterial/drug effects , Genes, Bacterial/drug effects , Genes, Bacterial/genetics , Humans , Mutation/drug effects , Mutation/genetics , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/isolation & purification , Sequence Analysis, DNA , Tuberculosis, Multidrug-Resistant/drug therapy , Tuberculosis, Multidrug-Resistant/microbiology
2.
J Antimicrob Chemother ; 71(1): 17-26, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26472768

ABSTRACT

The emergence of drug resistance continues to plague TB control, with a global increase in the prevalence of MDR-TB. This acts as a gateway to XDR-TB and thus emphasizes the urgency for drug development and optimal treatment options. Bedaquiline is the first new anti-TB drug approved by the FDA in 40 years and has been shown to be an effective treatment option for MDR Mycobacterium tuberculosis infection. Bedaquiline has also recently been included in clinical trials for new regimens with the aim of improving and shortening treatment periods. Alarmingly, efflux-mediated bedaquiline resistance, as well as efflux-mediated cross-resistance to clofazimine, has been identified in treatment failures. This mechanism of resistance results in efflux of a variety of anti-TB drugs from the bacterial cell, thereby decreasing the intracellular drug concentration. In doing so, the bacillus is able to render the antibiotic treatment ineffective. Recent studies have explored strategies to reverse the resistance phenotype conferred by efflux pump activation. It was observed that the addition of efflux pump inhibitors partially restored drug susceptibility in vitro and in vivo. This has significant clinical implications, especially in MDR-TB management where treatment options are extremely limited. This review aims to highlight the current efflux pump inhibitors effective against M. tuberculosis, the effect of efflux pump inhibitors on mycobacterial growth and the clinical promise of treatment with efflux pump inhibitors and standard anti-TB therapy.


Subject(s)
Antitubercular Agents/pharmacology , Biological Transport, Active/drug effects , Drug Resistance, Bacterial/drug effects , Membrane Transport Proteins/metabolism , Mycobacterium tuberculosis/drug effects , Antitubercular Agents/metabolism , Clofazimine/metabolism , Clofazimine/pharmacology , Diarylquinolines/metabolism , Diarylquinolines/pharmacology , Humans
3.
J Clin Microbiol ; 52(5): 1322-9, 2014 May.
Article in English | MEDLINE | ID: mdl-23784122

ABSTRACT

Alternative diagnostic methods, such as sequence-based techniques, are necessary for increasing the proportion of tuberculosis cases tested for drug resistance. Despite the abundance of data on drug resistance, isolates can display phenotypic resistance but lack any distinguishable markers. Furthermore, because resistance-conferring mutations develop under antibiotic pressure, different drug regimens could favor unique single-nucleotide polymorphisms (SNPs) in different geographical regions. A total of 407 isolates were collected from four geographical regions with a high prevalence of drug-resistant tuberculosis (India, Moldova, the Philippines, and South Africa). The "hot spot" or promoter sequences of nine genes (rpoB, gyrA, gyrB, katG, inhA promoter, ahpC promoter, eis promoter, rrs, and tlyA) associated with resistance to four types of antibiotics (rifampin, isoniazid, fluoroquinolones, and aminoglycosides) were analyzed for markers. Four genes contributed largely to resistance (rpoB, gyrA, rrs, and katG), two genes contributed moderately to resistance (the eis and inhA promoters), and three genes contributed little or no resistance (gyrB, tlyA, and the ahpC promoter) in clinical isolates. Several geographical differences were found, including a double mutation in rpoB found in 37.1% of isolates from South Africa, the C→T mutation at position -12 of the eis promoter found exclusively in 60.6% of isolates from Moldova, and the G→A mutation at position -46 of the ahpC promoter found only in India. These differences in polymorphism frequencies emphasize the uniqueness of isolates found in different geographical regions. The inclusion of several genes provided a moderate increase in sensitivity, and elimination of the examination of other genes might increase efficiency.


Subject(s)
Mycobacterium tuberculosis/genetics , Polymorphism, Single Nucleotide/genetics , Tuberculosis, Multidrug-Resistant/genetics , Antibiotics, Antitubercular/pharmacology , Bacterial Proteins/genetics , DNA, Bacterial/genetics , Genes, Bacterial/genetics , India , Microbial Sensitivity Tests/methods , Moldova , Mycobacterium tuberculosis/drug effects , Philippines , Promoter Regions, Genetic/genetics , South Africa , Tuberculosis/drug therapy , Tuberculosis/microbiology
4.
PLoS One ; 8(7): e70178, 2013.
Article in English | MEDLINE | ID: mdl-23936157

ABSTRACT

BACKGROUND: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. AIM: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Löwenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. METHOD: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. RESULTS: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. CONCLUSION: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility.


Subject(s)
Genotype , Mycobacterium tuberculosis/genetics , Tuberculosis, Pulmonary/microbiology , Coinfection , Cross-Sectional Studies , Culture Media , Humans , Mycobacterium tuberculosis/growth & development , Polymerase Chain Reaction , Sensitivity and Specificity , Sputum/microbiology , Tuberculosis, Pulmonary/diagnosis
5.
PLoS One ; 8(5): e63057, 2013.
Article in English | MEDLINE | ID: mdl-23667572

ABSTRACT

BACKGROUND: Treatment-related outcomes in patients with extensively drug-resistant tuberculosis (XDR-TB) are poor. However, data about the type, frequency and severity of presumed drug-associated adverse events (AEs) and their association with treatment-related outcomes in patients with XDR-TB are scarce. METHODS: Case records of 115 South-African XDR-TB patients were retrospectively reviewed by a trained researcher. AEs were estimated and graded according to severity [grade 0 = none; grade 1-2 = mild to moderate; and grade 3-5 = severe (drug stopped, life-threatening or death)]. FINDINGS: 161 AEs were experienced by 67/115(58%) patients: 23/67(34%) required modification of treatment, the offending drug was discontinued in 19/67(28%), reactions were life-threatening in 2/67(3.0%), and 6/67(9.0%) died. ∼50% of the patients were still on treatment at the time of data capture. Sputum culture-conversion was less likely in those with severe (grade 3-5) vs. grade 0-2 AEs [2/27(7%) vs. 24/88(27%); p = 0.02]. The type, frequency and severity of AEs was similar in HIV-infected and uninfected patients. Capreomycin, which was empirically administered in most cases, was withdrawn in 14/104(14%) patients, implicated in (14/34) 41% of the total drug withdrawals, and was associated with all 6 deaths in the severe AE group (renal failure in five patients and hypokalemia in one patient). CONCLUSION: Drug-associated AEs occur commonly with XDR-TB treatment, are often severe, frequently interrupt therapy, and negatively impact on culture conversion outcomes. These preliminary data inform on the need for standardised strategies (including pre-treatment counselling, early detection, monitoring, and follow-up) and less toxic drugs to optimally manage patients with XDR-TB.


Subject(s)
Antitubercular Agents/adverse effects , Antitubercular Agents/therapeutic use , Extensively Drug-Resistant Tuberculosis/drug therapy , Demography , Dose-Response Relationship, Drug , Extensively Drug-Resistant Tuberculosis/complications , Extensively Drug-Resistant Tuberculosis/microbiology , Extensively Drug-Resistant Tuberculosis/mortality , Female , HIV Infections/complications , Humans , Kaplan-Meier Estimate , Male , Multivariate Analysis , Mycobacterium tuberculosis , South Africa/epidemiology , Treatment Outcome
6.
Clin Infect Dis ; 56(4): 503-8, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23090928

ABSTRACT

BACKGROUND: Diagnosis of drug resistance and timely initiation of multidrug-resistant (MDR) tuberculosis therapy are essential to reduce transmission and improve patient outcomes. We sought to determine whether implementation of the rapid MTBDRplus diagnostic shortened the time from specimen collection to patient MDR tuberculosis therapy initiation. METHODS: We conducted a retrospective cohort analysis of 197 MDR tuberculosis patients treated at Brewelskloof, a rural tuberculosis hospital in Western Cape Province, South Africa, between 2007 and 2011. RESULTS: Eighty-nine patients (45%) were tested using conventional liquid culture and drug susceptibility testing (DST) on solid medium and 108 (55%) were tested using the MTBDRplus assay after positive acid-fast bacilli or culture. Median time from sample taken to therapy initiation was reduced from 80 days (interquartile range [IQR] 62-100) for conventional DST to 55 days (IQR 37.5-78) with the MTBDRplus. Although the laboratory processing time declined significantly, operational delays persisted both in the laboratory and the clinical infrastructure for getting patients started on treatment. In multivariate analysis, patients tested using the MTBDRplus test had a reduced risk of starting treatment 60 days or more after sputum collection of 0.52 (P < .0001) compared with patients tested with culture-based DST, after adjustment for smear status and site of disease. CONCLUSIONS: Use of MTBDRplus significantly reduced time to MDR tuberculosis treatment initiation. However, DST reporting to clinics was delayed by more than 1 week due, in part, to laboratory operational delays, including dependence on smear and culture positivity prior to MTBDRplus performance. In addition, once MDR tuberculosis was reported, delays in contacting patients and initiating therapy require improvements in clinical infrastructure.


Subject(s)
Antitubercular Agents/therapeutic use , Mycobacterium tuberculosis/genetics , Tuberculosis, Multidrug-Resistant/genetics , Adult , Cohort Studies , Female , Genotype , Humans , Male , Microbial Sensitivity Tests/methods , Middle Aged , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/isolation & purification , Retrospective Studies , Sensitivity and Specificity , South Africa , Time Factors , Tuberculosis, Multidrug-Resistant/diagnosis , Young Adult
7.
Evol Med Public Health ; 2013(1): 65-74, 2013 Jan.
Article in English | MEDLINE | ID: mdl-24481187

ABSTRACT

BACKGROUND AND OBJECTIVES: Multidrug resistant (MDR) bacteria are a growing threat to global health. Studies focusing on single antibiotics have shown that drug resistance is often associated with a fitness cost in the absence of drug. However, little is known about the fitness cost associated with resistance to multiple antibiotics. METHODOLOGY: We used Mycobacterium smegmatis as a model for human tuberculosis (TB) and an in vitro competitive fitness assay to explore the combined fitness effects and interaction between mutations conferring resistance to rifampicin (RIF) and ofloxacin (OFX); two of the most important first- and second-line anti-TB drugs, respectively. RESULTS: We found that 4 out of 17 M. smegmatis mutants (24%) resistant to RIF and OFX showed a statistically significantly higher or lower competitive fitness than expected when assuming a multiplicative model of fitness effects of each individual mutation. Moreover, 6 out of the 17 double drug-resistant mutants (35%) had a significantly higher fitness than at least one of the corresponding single drug-resistant mutants. The particular combinations of resistance mutations associated with no fitness deficit in M. smegmatis were the most frequent among 151 clinical isolates of MDR and extensively drug-resistant (XDR) Mycobacterium tuberculosis from South Africa. CONCLUSIONS AND IMPLICATIONS: Our results suggest that epistasis between drug resistance mutations in mycobacteria can lead to MDR strains with no fitness deficit, and that these strains are positively selected in settings with a high burden of drug-resistant TB. Taken together, our findings support a role for epistasis in the evolution and epidemiology of MDR- and XDR-TB.

8.
J Clin Microbiol ; 50(3): 995-1002, 2012 Mar.
Article in English | MEDLINE | ID: mdl-22170931

ABSTRACT

Genotyping of multidrug-resistant (MDR) Mycobacterium tuberculosis strains isolated from tuberculosis (TB) patients in four South African provinces (Western Cape, Eastern Cape, KwaZulu-Natal, and Gauteng) revealed a distinct population structure of the MDR strains in all four regions, despite the evidence of substantial human migration between these settings. In all analyzed provinces, a negative correlation between strain diversity and an increasing level of drug resistance (from MDR-TB to extensively drug-resistant TB [XDR-TB]) was observed. Strains predominating in XDR-TB in the Western and Eastern Cape and KwaZulu-Natal Provinces were strongly associated with harboring an inhA promoter mutation, potentially suggesting a role of these mutations in XDR-TB development in South Africa. Approximately 50% of XDR-TB cases detected in the Western Cape were due to strains probably originating from the Eastern Cape. This situation may illustrate how failure of efficient health care delivery in one setting can burden health clinics in other areas.


Subject(s)
Antitubercular Agents/pharmacology , Biodiversity , Drug Resistance, Multiple, Bacterial , Mycobacterium tuberculosis/classification , Mycobacterium tuberculosis/isolation & purification , Tuberculosis, Multidrug-Resistant/microbiology , Bacterial Proteins , Genetic Variation , Genotype , Humans , Molecular Epidemiology , Molecular Typing , Mutation , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , Oxidoreductases , Promoter Regions, Genetic , South Africa
10.
J Clin Microbiol ; 44(10): 3539-43, 2006 Oct.
Article in English | MEDLINE | ID: mdl-16928967

ABSTRACT

Drug resistance among children with culture-confirmed tuberculosis (TB) provides an accurate measure of transmitted drug resistance within the community. We describe the genotype diversity in children with culture-confirmed TB and investigate the relationship between genotype and drug resistance. A prospective study was conducted from March 2003 through August 2005 at Tygerberg Children's Hospital, in the Western Cape Province of South Africa. All children (<13 years of age) diagnosed with culture-confirmed TB were included. Genotype analysis and phenotypic drug susceptibility testing were performed on the first culture-positive isolate from each patient. Mutation analysis was performed on all drug-resistant isolates. Spoligotyping was successfully performed on isolates from 391/399 (98%) children diagnosed with culture-confirmed TB. Drug susceptibility testing was also performed on 391 isolates; 49 (12.5%) were resistant to isoniazid, and 20 (5.1%) of these were resistant to both isoniazid and rifampin. Beijing was the most common genotype family, identified in 130/391 (33.2%) cases, followed by LAM in 114/391 (29.2%) cases. The presence of both Beijing and Haarlem genotype families was significantly associated with drug resistance (26/49 [53.1%] versus 113/342 [33.0%]; odds ratio, 1.7; 95% confidence interval, 1.0 to 2.9). The high prevalence of Beijing and LAM in children with culture-confirmed TB reflects considerable transmission of these genotype families within the community. The overrepresentation of Beijing and Haarlem genotype families in children with drug-resistant TB demonstrates their contribution to transmitted drug resistance and their potential importance in the emergent drug-resistant TB epidemic.


Subject(s)
Antitubercular Agents/therapeutic use , Drug Resistance, Multiple, Bacterial , Tuberculosis/drug therapy , Tuberculosis/epidemiology , Child , Child, Preschool , Female , Genotype , Humans , Infant , Infant, Newborn , Male , Mycobacterium tuberculosis/drug effects , Mycobacterium tuberculosis/genetics , South Africa/epidemiology
SELECTION OF CITATIONS
SEARCH DETAIL
...