Cost analysis of rapid diagnostics for drug-resistant tuberculosis.

BACKGROUND: Growth-based drug susceptibility testing (DST) is the reference standard for diagnosing drug-resistant tuberculosis (TB), but standard time to result (TTR) is typically ≥ 3 weeks. Rapid tests can reduce that TTR to days or hours, but accuracy may be lowered. In addition to the TTR and test accuracy, the cost of a diagnostic test may affect whether it is adopted in clinical settings. We examine the cost-effectiveness of rapid diagnostics for extremely drug-resistant TB (XDR-TB) in three different high-prevalence settings. METHODS: 1128 patients with confirmed TB were enrolled at clinics in Mumbai, India; Chisinau, Moldova; and Port Elizabeth, South Africa. Patient sputum samples underwent DST for first and second line TB drugs using 2 growth-based (MGIT, MODS) and 2 molecular (Pyrosequencing [PSQ], line-probe assays [LPA]) assays. TTR was the primary measure of effectiveness. Sensitivity and specificity were also evaluated. The cost to perform each test at each site was recorded and included test-specific materials, personnel, and equipment costs. Incremental cost-effectiveness ratios were calculated in terms of $/day saved. Sensitivity analyses examine the impact of batch size, equipment, and personnel costs. RESULTS: Our prior results indicated that the LPA and PSQ returned results in a little over 1 day. Mean cost per sample without equipment or overhead was $23, $28, $33, and $41 for the MODS, MGIT, PSQ, and LPA, respectively. For diagnosing XDR-TB, MODS was the most accurate, followed by PSQ, and LPA. MODS was quicker and less costly than MGIT. PSQ and LPA were considerably faster but cost more than MODS. Batch size and personnel costs were the main drivers of cost variation. CONCLUSIONS: Multiple factors must be weighed when selecting a test for diagnosis of XDR-TB. Rapid tests can greatly improve the time required to diagnose drug-resistant TB, potentially improving treatment success, and preventing the spread of XDR-TB. Faster time to result must be weighed against the potential for reduced accuracy, and increased costs. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02170441 .

Shedding light on the performance of a pyrosequencing assay for drug-resistant tuberculosis diagnosis.

BACKGROUND: Rapid molecular diagnostics, with their ability to quickly identify genetic mutations associated with drug resistance in Mycobacterium tuberculosis clinical specimens, have great potential as tools to control multi- and extensively drug-resistant tuberculosis (M/XDR-TB). The Qiagen PyroMark Q96 ID system is a commercially available pyrosequencing (PSQ) platform that has been validated for rapid M/XDR-TB diagnosis. However, the details of the assay's diagnostic and technical performance have yet to be thoroughly investigated in diverse clinical environments. METHODS: This study evaluates the diagnostic performance of the PSQ assay for 1128 clinical specimens from patients from three areas of high TB burden. We report on the diagnostic performance of the PSQ assay between the three sites and identify variables associated with poor PSQ technical performance. RESULTS: In India, the sensitivity of the PSQ assay ranged from 89 to 98 % for the detection of phenotypic resistance to isoniazid, rifampicin, fluoroquinolones, and the injectables. In Moldova, assay sensitivity ranged from 7 to 94 %, and in South Africa, assay sensitivity ranged from 71 to 92 %. Specificity was high (94-100 %) across all sites. The addition of eis promoter sequencing information greatly improved the sensitivity of kanamycin resistance detection in Moldova (7 % to 79 %). Nearly all (89.4 %) sequencing reactions conducted on smear-positive, culture-positive specimens and most (70.8 %) reactions conducted on smear-negative, culture-positive specimens yielded valid PSQ reads. An investigation into the variables influencing sequencing failures indicated smear negativity, culture negativity, site (Moldova), and sequencing of the rpoB, gyrA, and rrs genes were highly associated with poor PSQ technical performance (adj. OR > 2.0). CONCLUSIONS: This study has important implications for the global implementation of PSQ as a molecular TB diagnostic, as it demonstrates how regional factors may impact PSQ diagnostic performance, while underscoring potential gene targets for optimization to improve overall PSQ assay technical performance. TRIAL REGISTRATION: ClinicalTrials.gov ( #NCT02170441 ). Registered 12 June 2014.

MTBDRplus and MTBDRsl Assays: Absence of Wild-Type Probe Hybridization and Implications for Detection of Drug-Resistant Tuberculosis.

Accurate identification of drug-resistantMycobacterium tuberculosisis imperative for effective treatment and subsequent reduction in disease transmission. Line probe assays rapidly detect mutations associated with resistance and wild-type sequences associated with susceptibility. Examination of molecular-level performance is necessary for improved assay result interpretation and for continued diagnostic development. Using data collected from a large, multisite diagnostic study, probe hybridization results from line probe assays, MTBDRplusand MTBDRsl, were compared to those of sequencing, and the diagnostic performance of each individual mutation and wild-type probe was assessed. Line probe assay results classified as resistant due to the absence of wild-type probe hybridization were compared to those of sequencing to determine if novel mutations were inhibiting wild-type probe hybridization. The contribution of absent wild-type probe hybridization to the detection of drug resistance was assessed via comparison to a phenotypic reference standard. In our study, mutation probes demonstrated significantly higher specificities than wild-type probes and wild-type probes demonstrated marginally higher sensitivities than mutation probes, an ideal combination for detecting the presence of resistance conferring mutations while yielding the fewest number of false-positive results. The absence of wild-type probe hybridization without mutation probe hybridization was determined to be primarily the result of failure of mutation probe hybridization and not the result of novel or rare mutations. Compared to phenotypic culture-based drug susceptibility testing, the absence of wild-type probe hybridization without mutation probe hybridization significantly contributed to the detection of phenotypic rifampin and fluoroquinolone resistance with negligible increases in false-positive results.

Performance Comparison of Three Rapid Tests for the Diagnosis of Drug-Resistant Tuberculosis.

BACKGROUND: The aim of this study was to compare the performance of several recently developed assays for the detection of multi- and extensively drug-resistant tuberculosis (M/XDR-TB) in a large, multinational field trial. METHODS: Samples from 1,128 M/XDR-TB suspects were examined by Line Probe Assay (LPA), Pyrosequencing (PSQ), and Microscopic Observation of Drug Susceptibility (MODS) and compared to the BACTEC MGIT960 reference standard to detect M/XDR-TB directly from patient sputum samples collected at TB clinics in India, Moldova, and South Africa. RESULTS: Specificity for all three assays was excellent: 97-100% for isoniazid (INH), rifampin (RIF), moxifloxacin (MOX) and ofloxacin (OFX) and 99-100% for amikacin (AMK), capreomycin (CAP) and kanamycin (KAN) resistance. Sensitivities were lower, but still very good: 94-100% for INH, RIF, MOX and OFX, and 84-90% for AMK and CAP, but only 48-62% for KAN. In terms of agreement, statistically significant differences were only found for detection of RIF (MODS outperformed PSQ) and KAN (MODS outperformed LPA and PSQ) resistance. Mean time-to-result was 1.1 days for LPA and PSQ, 14.3 days for MODS, and 24.7 days for MGIT. CONCLUSIONS: All three rapid assays evaluated provide clinicians with timely detection of resistance to the drugs tested; with molecular results available one day following laboratory receipt of samples. In particular, the very high specificity seen for detection of drug resistance means that clinicians can use the results of these rapid tests to avoid the use of toxic drugs to which the infecting organism is resistant and develop treatment regiments that have a higher likelihood of yielding a successful outcome.

Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates.

We report the discovery and confirmation of 23 novel mutations with previously undocumented role in isoniazid (INH) drug resistance, in catalase-peroxidase (katG) gene of Mycobacterium tuberculosis (Mtb) isolates. With these mutations, a synonymous mutation in fabG1 (g609a), and two canonical mutations, we were able to explain 98% of the phenotypic resistance observed in 366 clinical Mtb isolates collected from four high tuberculosis (TB)-burden countries: India, Moldova, Philippines, and South Africa. We conducted overlapping targeted and whole-genome sequencing for variant discovery in all clinical isolates with a variety of INH-resistant phenotypes. Our analysis showed that just two canonical mutations (katG 315AGC-ACC and inhA promoter-15C-T) identified 89.5% of resistance phenotypes in our collection. Inclusion of the 23 novel mutations reported here, and the previously documented point mutation in fabG1, increased the sensitivity of these mutations as markers of INH resistance to 98%. Only six (2%) of the 332 resistant isolates in our collection did not harbor one or more of these mutations. The third most prevalent substitution, at inhA promoter position -8, present in 39 resistant isolates, was of no diagnostic significance since it always co-occurred with katG 315. 79% of our isolates harboring novel mutations belong to genetic group 1 indicating a higher tendency for this group to go down an uncommon evolutionary path and evade molecular diagnostics. The results of this study contribute to our understanding of the mechanisms of INH resistance in Mtb isolates that lack the canonical mutations and could improve the sensitivity of next generation molecular diagnostics.

Evaluation of pyrosequencing for detecting extensively drug-resistant Mycobacterium tuberculosis among clinical isolates from four high-burden countries.

Reliable molecular diagnostics, which detect specific mutations associated with drug resistance, are promising technologies for the rapid identification and monitoring of drug resistance in Mycobacterium tuberculosis isolates. Pyrosequencing (PSQ) has the ability to detect mutations associated with first- and second-line anti-tuberculosis (TB) drugs, with the additional advantage of being rapidly adaptable for the identification of new mutations. The aim of this project was to evaluate the performance of PSQ in predicting phenotypic drug resistance in multidrug- and extensively drug-resistant tuberculosis (M/XDR-TB) clinical isolates from India, South Africa, Moldova, and the Philippines. A total of 187 archived isolates were run through a PSQ assay in order to identify M. tuberculosis (via the IS6110 marker), and to detect mutations associated with M/XDR-TB within small stretches of nucleotides in selected loci. The molecular targets included katG, the inhA promoter and the ahpC-oxyR intergenic region for isoniazid (INH) resistance; the rpoB core region for rifampin (RIF) resistance; gyrA for fluoroquinolone (FQ) resistance; and rrs for amikacin (AMK), capreomycin (CAP), and kanamycin (KAN) resistance. PSQ data were compared to phenotypic mycobacterial growth indicator tube (MGIT) 960 drug susceptibility testing results for performance analysis. The PSQ assay illustrated good sensitivity for the detection of resistance to INH (94%), RIF (96%), FQ (93%), AMK (84%), CAP (88%), and KAN (68%). The specificities of the assay were 96% for INH, 100% for RIF, FQ, AMK, and KAN, and 97% for CAP. PSQ is a highly efficient diagnostic tool that reveals specific nucleotide changes associated with resistance to the first- and second-line anti-TB drug medications. This methodology has the potential to be linked to mutation-specific clinical interpretation algorithms for rapid treatment decisions.

The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD): design of a multi-site, head-to-head study of three rapid tests to detect extensively drug-resistant tuberculosis.

BACKGROUND: Drug-resistant tuberculosis (DR-TB) remains a threat to global public health, owing to the complexity and delay of diagnosis and treatment. The Global Consortium for Drug-resistant Tuberculosis Diagnostics (GCDD) was formed to develop and evaluate assays designed to rapidly detect DR-TB, so that appropriate treatment might begin more quickly. This paper describes the methodology employed in a prospective cohort study for head-to-head assessment of three different rapid diagnostic tools. METHODS: Subjects at risk of DR-TB were enrolled from three countries. Data were gathered from a combination of patient interviews, chart reviews, and laboratory testing from each site's reference laboratory. The primary outcome of interest was reduction in time from specimen arrival in the laboratory to results of rapid drug susceptibility tests, as compared with current standard mycobacterial growth indicator tube (MGIT) drug susceptibility tests. RESULTS: Successful implementation of the trial in diverse multinational populations is explained, in addition to challenges encountered and recommendations for future studies with similar aims or populations. CONCLUSIONS: The GCDD study was a head-to-head study of multiple rapid diagnostic assays aimed at improving accuracy and precision of diagnostics and reducing overall time to detection of DR-TB. By conducting a large prospective study, which captured epidemiological, clinical, and biological data, we have produced a high-quality unique dataset, which will be beneficial for analyzing study aims as well as answering future DR-TB research questions. Reduction in detection time for XDR-TB would be a major public health success as it would allow for improved treatment and more successful patient outcomes. Executing successful trials is critical in assessment of these reductions in highly variable populations. TRIAL REGISTRATION: ClinicalTrials.gov NCT02170441.

Energy metabolism and drug efflux in Mycobacterium tuberculosis.

The inherent drug susceptibility of microorganisms is determined by multiple factors, including growth state, the rate of drug diffusion into and out of the cell, and the intrinsic vulnerability of drug targets with regard to the corresponding antimicrobial agent. Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), remains a significant source of global morbidity and mortality, further exacerbated by its ability to readily evolve drug resistance. It is well accepted that drug resistance in M. tuberculosis is driven by the acquisition of chromosomal mutations in genes encoding drug targets/promoter regions; however, a comprehensive description of the molecular mechanisms that fuel drug resistance in the clinical setting is currently lacking. In this context, there is a growing body of evidence suggesting that active extrusion of drugs from the cell is critical for drug tolerance. M. tuberculosis encodes representatives of a diverse range of multidrug transporters, many of which are dependent on the proton motive force (PMF) or the availability of ATP. This suggests that energy metabolism and ATP production through the PMF, which is established by the electron transport chain (ETC), are critical in determining the drug susceptibility of M. tuberculosis. In this review, we detail advances in the study of the mycobacterial ETC and highlight drugs that target various components of the ETC. We provide an overview of some of the efflux pumps present in M. tuberculosis and their association, if any, with drug transport and concomitant effects on drug resistance. The implications of inhibiting drug extrusion, through the use of efflux pump inhibitors, are also discussed.

Pyrosequencing for rapid detection of extensively drug-resistant Mycobacterium tuberculosis in clinical isolates and clinical specimens.

Treating extensively drug-resistant (XDR) tuberculosis (TB) is a serious challenge. Culture-based drug susceptibility testing (DST) may take 4 weeks or longer from specimen collection to the availability of results. We developed a pyrosequencing (PSQ) assay including eight subassays for the rapid identification of Mycobacterium tuberculosis complex (MTBC) and concurrent detection of mutations associated with resistance to drugs defining XDR TB. The entire procedure, from DNA extraction to the availability of results, was accomplished within 6 h. The assay was validated for testing clinical isolates and clinical specimens, which improves the turnaround time for molecular DST and maximizes the benefit of using molecular testing. A total of 130 clinical isolates and 129 clinical specimens were studied. The correlations between the PSQ results and the phenotypic DST results were 94.3% for isoniazid, 98.7% for rifampin, 97.6% for quinolones (ofloxacin, levofloxacin, or moxifloxacin), 99.2% for amikacin, 99.2% for capreomycin, and 96.4% for kanamycin. For testing clinical specimens, the PSQ assay yielded a 98.4% sensitivity for detecting MTBC and a 95.8% sensitivity for generating complete sequencing results from all subassays. The PSQ assay was able to rapidly and accurately detect drug resistance mutations with the sequence information provided, which allows further study of the association of drug resistance or susceptibility with each mutation and the accumulation of such knowledge for future interpretation of results. Thus, reporting of false resistance for mutations known not to confer resistance can be prevented, which is a significant benefit of the assay over existing molecular diagnostic methods endorsed by the World Health Organization.

Predicting extensively drug-resistant Mycobacterium tuberculosis phenotypes with genetic mutations.

Molecular diagnostic methods based on the detection of mutations conferring drug resistance are promising technologies for rapidly detecting multidrug-/extensively drug-resistant tuberculosis (M/XDR TB), but large studies of mutations as markers of resistance are rare. The Global Consortium for Drug-Resistant TB Diagnostics analyzed 417 Mycobacterium tuberculosis isolates from multinational sites with a high prevalence of drug resistance to determine the sensitivities and specificities of mutations associated with M/XDR TB to inform the development of rapid diagnostic methods. We collected M/XDR TB isolates from regions of high TB burden in India, Moldova, the Philippines, and South Africa. The isolates underwent standardized phenotypic drug susceptibility testing (DST) to isoniazid (INH), rifampin (RIF), moxifloxacin (MOX), ofloxacin (OFX), amikacin (AMK), kanamycin (KAN), and capreomycin (CAP) using MGIT 960 and WHO-recommended critical concentrations. Eight genes (katG, inhA, rpoB, gyrA, gyrB, rrs, eis, and tlyA) were sequenced using Sanger sequencing. Three hundred seventy isolates were INHr, 356 were RIFr, 292 were MOXr/OFXr, 230 were AMKr, 219 were CAPr, and 286 were KANr. Four single nucleotide polymorphisms (SNPs) in katG/inhA had a combined sensitivity of 96% and specificities of 97 to 100% for the detection of INHr. Eleven SNPs in rpoB had a combined sensitivity of 98% for RIFr. Eight SNPs in gyrA codons 88 to 94 had sensitivities of 90% for MOXr/OFXr. The rrs 1401/1484 SNPs had 89 to 90% sensitivity for detecting AMKr/CAPr but 71% sensitivity for KANr. Adding eis promoter SNPs increased the sensitivity to 93% for detecting AMKr and to 91% for detecting KANr. Approximately 30 SNPs in six genes predicted clinically relevant XDR-TB phenotypes with 90 to 98% sensitivity and almost 100% specificity.

Alcohol, hospital discharge, and socioeconomic risk factors for default from multidrug resistant tuberculosis treatment in rural South Africa: a retrospective cohort study.

BACKGROUND: Default from multidrug-resistant tuberculosis (MDR-TB) treatment remains a major barrier to cure and epidemic control. We sought to identify patient risk factors for default from MDR-TB treatment and high-risk time periods for default in relation to hospitalization and transition to outpatient care. METHODS: We retrospectively analyzed a cohort of 225 patients who initiated MDR-TB treatment between 2007 through 2010 at a rural TB hospital in the Western Cape Province, South Africa. RESULTS: Fifty percent of patients were cured or completed treatment, 27% defaulted, 14% died, 4% failed treatment, and 5% transferred out. Recent alcohol use was common (63% of patients). In multivariable proportional hazards regression, older age (hazard ratio [HR]= 0.97 [95% confidence interval 0.94-0.99] per year of greater age), formal housing (HR=0.38 [0.19-0.78]), and steady employment (HR=0.41 [0.19-0.90]) were associated with decreased risk of default, while recent alcohol use (HR=2.1 [1.1-4.0]), recent drug use (HR=2.0 [1.0-3.6]), and Coloured (mixed ancestry) ethnicity (HR=2.3 [1.1-5.0]) were associated with increased risk of default (P<0.05). Defaults occurred throughout the first 18 months of the two-year treatment course but were especially frequent among alcohol users after discharge from the initial four-to-five-month in-hospital phase of treatment, with the highest default rates occurring among alcohol users within two months of discharge. Default rates during the first two months after discharge were also elevated for patients who received care from mobile clinics. CONCLUSIONS: Among patients who were not cured or did not complete MDR-TB treatment, the majority defaulted from treatment. Younger, economically-unstable patients and alcohol and drug users were particularly at risk. For alcohol users as well as mobile-clinic patients, the early outpatient treatment phase is a high-risk period for default that could be targeted in efforts to increase treatment completion rates.

Characterization of W-Beijing isolates of Mycobacterium tuberculosis from the Western Cape.

The purpose of this simple study was to characterize a panel of clinical isolates of Mycobacterium tuberculosis obtained from the Western Cape region of South Africa where new clinical vaccine trials are beginning, in the low dose aerosol guinea pig infection model. Most of the strains tested grew well in the lungs and other organs of these animals, and in most cases gave rise to moderate to very severe lung damage. We further observed that the current BCG vaccine was highly protective against two randomly selected strains, giving rise to significantly prolonged survival.

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.

Genomic analysis identifies targets of convergent positive selection in drug-resistant Mycobacterium tuberculosis.

M. tuberculosis is evolving antibiotic resistance, threatening attempts at tuberculosis epidemic control. Mechanisms of resistance, including genetic changes favored by selection in resistant isolates, are incompletely understood. Using 116 newly sequenced and 7 previously sequenced M. tuberculosis whole genomes, we identified genome-wide signatures of positive selection specific to the 47 drug-resistant strains. By searching for convergent evolution--the independent fixation of mutations in the same nucleotide position or gene--we recovered 100% of a set of known resistance markers. We also found evidence of positive selection in an additional 39 genomic regions in resistant isolates. These regions encode components in cell wall biosynthesis, transcriptional regulation and DNA repair pathways. Mutations in these regions could directly confer resistance or compensate for fitness costs associated with resistance. Functional genetic analysis of mutations in one gene, ponA1, demonstrated an in vitro growth advantage in the presence of the drug rifampicin.

Epidemic spread of multidrug-resistant tuberculosis in Johannesburg, South Africa.

Numerous reports have documented isolated transmission events or clonal outbreaks of multidrug-resistant Mycobacterium tuberculosis strains, but knowledge of their epidemic spread remains limited. In this study, we evaluated drug resistance, strain diversity, and clustering rates in patients diagnosed with multidrug-resistant (MDR) tuberculosis (TB) at the National Health Laboratory Service (NHLS) Central TB Laboratory in Johannesburg, South Africa, between March 2004 and December 2007. Phenotypic drug susceptibility testing was done using the indirect proportion method, while each isolate was genotyped using a combination of spoligotyping and 12-MIRU typing (12-locus multiple interspersed repetitive unit typing). Isolates from 434 MDR-TB patients were evaluated, of which 238 (54.8%) were resistant to four first-line drugs (isoniazid, rifampin, ethambutol, and streptomycin). Spoligotyping identified 56 different strains and 28 clusters of variable size (2 to 71 cases per cluster) with a clustering rate of 87.1%. Ten clusters included 337 (77.6%) of all cases, with strains of the Beijing genotype being most prevalent (16.4%). Combined analysis of spoligotyping and 12-MIRU typing increased the discriminatory power (Hunter Gaston discriminatory index [HGDI] = 0.962) and reduced the clustering rate to 66.8%. Resolution of Beijing genotype strains was further enhanced with the 24-MIRU-VNTR (variable-number tandem repeat) typing method by identifying 15 subclusters and 19 unique strains from twelve 12-MIRU clusters. High levels of clustering among a variety of strains suggest a true epidemic spread of MDR-TB in the study setting, emphasizing the urgency of early diagnosis and effective treatment to reduce transmission within this community.

The rationale for using rifabutin in the treatment of MDR and XDR tuberculosis outbreaks.

Genetically related Mycobacterium tuberculosis strains with alterations at codon 516 in the rpoB gene were observed amongst a substantial number of patients with drug resistant tuberculosis in the Eastern Cape Province (ECP) of South Africa. Mutations at codon 516 are usually associated with lower level rifampicin (RIF) resistance, while susceptibility to rifabutin (RFB) remains intact. This study was conducted to assess the rationale for using RFB as a substitution for RIF in the treatment of MDR and XDR tuberculosis outbreaks. Minimum inhibitory concentrations (MICs) of 34 drug resistant clinical isolates of M tuberculosis were determined by MGIT 960 and correlated with rpoB mutations. RFB MICs ranged from 0.125 to 0.25 µg/ml in the 34 test isolates thereby confirming phenotypic susceptibility as per critical concentration (CC) of 0.5 µg/ml. The corresponding RIF MICs ranged between 5 and 15 µg/ml, which is well above the CC of 1.0 µg/ml. Molecular-based drug susceptibility testing provides important pharmacogenetic insight by demonstrating a direct correlation between defined rpoB mutation and the level of RFB susceptibility. We suggest that isolates with marginally reduced susceptibility as compared to the epidemiological cut-off for wild-type strains (0.064 µg/ml), but lower than the current CC (≤0.5 µg/ml), are categorised as intermediate. Two breakpoints (0.064 µg/ml and 0.5 µg/ml) are recommended to distinguish between susceptible, intermediate and RFB resistant strains. This concept may assist clinicians and policy makers to make objective therapeutic decisions, especially in situations where therapeutic options are limited. The use of RFB in the ECP may improve therapeutic success and consequently minimise the risk of ongoing transmission of drug resistant M. tuberculosis strains.

embB306 mutations as molecular indicators to predict ethambutol susceptibility in Mycobacterium tuberculosis.

BACKGROUND: Discordant results in conventional susceptibility testing of ethambutol against Mycobacterium tuberculosis may lead to underreporting of drug resistance. METHODS: A 240-bp region of the embB gene in 111 clinical isolates of M. tuberculosis was sequenced and examined for mutations linked to ethambutol resistance. The phenotypic susceptibility levels of the isolates were quantified by the BACTEC™ MGIT 960™ TB System and correlated with the genotypic test results. These data were analyzed to find information that could be used to clarify discordant ethambutol susceptibility test results. RESULTS: Mutations M306I (n = 56), M306V (n = 18) and M306L (n = 3) in M. tuberculosis showed decreased susceptibility to ethambutol. The minimum inhibitory concentrations (MICs) in 73% (56/77) of embB306 mutants were at or just above the critical concentration (MICs, 5.0 to ≤12.5 µg/ml) of ethambutol reflecting borderline (or intermediate) resistance. Eight ethambutol-resistant isolates lacked embB mutations, probably due to mutational alterations elsewhere in the genome. CONCLUSION: Our findings suggest that clinical isolates containing embB306 mutations with MICs overlapping the critical concentration are associated with discordant ethambutol susceptibility test results. The clinical significance of borderline resistance in combination treatment of tuberculosis remains to be determined before alternative ethambutol breakpoints are considered.

Discordant drug susceptibility for mycobacterium tuberculosis within families.

Children with presumed tuberculosis who are in contact with a multidrug-resistant source case should be treated according to the drug susceptibility of the source case's isolate. However, it is important to obtain a microbiologic diagnosis as it is possible for the child to have a different susceptibility profile to the source case. We present 2 such cases.

Impact of drug resistance on clinical outcome in children with tuberculous meningitis.

BACKGROUND: Tuberculous meningitis (TBM) is associated with delayed diagnosis and poor outcome in children. This study investigated the impact of drug resistance on clinical outcome in children with TBM. METHODS: All children (0-13 years) were included if admitted to Tygerberg Children's Hospital, Cape Town, South Africa, from January 2003 to April 2009 with a diagnosis of either confirmed TBM, or probable TBM with mycobacterial isolation from a site other than cerebrospinal fluid. Mycobacterial samples underwent drug susceptibility testing to rifampin and isoniazid. Children were treated with isoniazid, rifampin, pyrazinamide and ethionamide according to local guidelines. RESULTS: One hundred twenty-three children were included; 13% (16 of 123) had any form of drug resistance, and 4% (5 of 123) had multidrug-resistant tuberculosis. Time from start of symptoms to appropriate treatment was longer in children with any drug resistance (median: 31 days versus 9 days; P=0.001). In multivariable analysis, young age (P=0.013) and multidrug-resistant tuberculosis (adjusted odds ratio: 12.4 [95% confidence interval: 1.17-132.3]; P=0.037) remained risk factors for unfavorable outcome, and multidrug-resistant tuberculosis remained a risk for death (adjusted odds ratio: 63.9 [95% confidence interval: 4.84-843.2]; P=0.002). We did not detect any difference in outcome between those with isolates resistant to only isoniazid and those with fully susceptible strains (adjusted odds ratio: 0.22 [confidence interval: 0.03-1.87]; P=0.17). CONCLUSION: Multidrug-resistant TBM in children has poor clinical outcome and is associated with death. We did not find any difference in the outcomes between children with isoniazid monoresistant TBM and those with drug-susceptible TBM. One explanation could be the local treatment regimen. Further investigation of this regimen is indicated.