Drug resistance in Mycobacterium tuberculosis presents an enormous public health threat. It is typically defined as >1% of drug resistant colonies using the agar proportion method. Detecting small numbers of drug resistant Tb in a population, also known as heteroresistance, is challenging with current methodologies. Here we have utilized digital PCR to detect heteroresistance within M. tuberculosis populations with excellent accuracy versus the agar proportion method. We designed dual TaqMan-MGB probes to detect wild-type and mutant sequences of katG (315), rpoB (531), gyrA (94,95) and rrs (1401), genes that associate with resistance to isoniazid, rifampin, fluoroquinolone, and aminoglycoside respectively. We generated heteroresistant mixtures of susceptible and extensively drug resistant Tb, followed by DNA extraction and digital PCR. Digital PCR yielded a close approximation to agar proportion's percentages of resistant colonies, and yielded 100% concordance with agar proportion's susceptible/resistant results. Indeed, the digital PCR method was able to identify mutant sequence in mixtures containing as little as 1000∶1 susceptible:resistant Tb. By contrast, real-time PCR or PCR followed by Sanger sequencing were less sensitive and had little resolution to detect heteroresistance, requiring fully 1∶1 or 10∶1 susceptible:resistant ratios in order to detect resistance. Our assay can also work in sputum so long as sufficient quantities of Tb are present (>1000 cfu/ml). This work demonstrates the utility of digital PCR to detect and quantify heteroresistance in drug resistant Tb, which may be useful to inform treatment decisions faster than agar proportion.

bGiven the increases in drug-resistant tuberculosis, laboratory capacities for drug susceptibility testing are being scaled up worldwide. A laboratory must decide among several endorsed methodologies. We evaluated 87 Mycobacterium tuberculosis isolates for concordance of susceptibility results across six methods: the L-J proportion method, MGIT 960 SIRE AST, Gene/Xpert MTB/ RIF, GenoType MTBDRplus line probe assay, MycoTB MIC plate, and a laboratory-developed mycobacteriophage quantitative PCR (qPCR)-based method. Most (80%) isolates were multidrug resistant. Of the culture-based methods, the mycobacteriophage qPCR method was fastest, the L-J proportion method was the slowest, and the MGIT method required the most repeat testing (P < 0.05). For isoniazid (INH), 82% of isolates were susceptible by all methods or resistant by all methods, whereas for rifampin (RIF), ethambutol (EMB), and streptomycin (STR), such complete concordance was observed in 77%, 50%, and 51% of isolates, respectively (P < 0. G iven the increasing rates of multidrug-resistant tuberculosis (MDR-TB) isolates worldwide and the emergence of extensively drug-resistant TB, the development of rapid and accurate methods for drug susceptibility testing (DST) of Mycobacterium tuberculosis isolates is a global priority. The culture-based proportion method that employs Löwenstein-Jensen medium is a World Health Organization (WHO)-recommended method that has been in wide use for over 50 years (1, 2). Such solid medium-based DST methods are slow, requiring readings at 4 to 6 weeks, which delays the detection of drug resistance and risks inappropriate treatment and spread of drug-resistant strains. This deficiency has led to the development of newer DST methodologies, including liquid culture systems and molecular line probe assays, which have also received recommendations from WHO (3, 4).As a consequence, laboratories have seen an accumulation of methods from which they must choose, and a given specimen or isolate may be tested across a variety of methods. A natural consequence is that discrepancies between methods may be encountered. Such discrepancies may be of little consequence in certain scenarios, such as streptomycin (STR) resistance in settings that use primarily isoniazid (INH), rifampin (RIF), pyrazinamide (PZA), and ethambutol (EMB), but they may have critical implications for treating MDR-TB in areas where the arsenal of drugs is limited in number and potency. Discordance is becoming a vexing aspect for TB clinicians and will likely increase in frequency as new methodologies are adopted, yet its extent has received little attention. Most diagnostic evaluations examine one new method against one gold standard reference method, not several methods against each other. Additionally, most diagnostic evaluations are performed on predominantly drug-susceptible isolates, often highly susceptible clinical isolates or reference strains, and thus discrepancies between methods would be expected to be rare.For this reason, we prospectively examined 87 mos...

Background. d-cycloserine is used to treat multidrug-resistant tuberculosis. Its efficacy, contribution in combination therapy, and best clinical dose are unclear, also data on the d-cycloserine minimum inhibitory concentration (MIC) distributions is scant. Methods. We performed a systematic search to identify pharmacokinetic and pharmacodynamic studies performed with d-cycloserine. We then performed a combined exposure-effect and dose fractionation study of d-cycloserine in the hollow fiber system model of tuberculosis (HFS-TB). In parallel, we identified d-cycloserine MICs in 415 clinical Mycobacterium tuberculosis (Mtb) isolates from patients. We utilized these results, including intracavitary concentrations, to identify the clinical dose that would be able to achieve or exceed target exposures in 10 000 patients using Monte Carlo experiments (MCEs). Results. There were no published d-cycloserine pharmacokinetics/pharmacodynamics studies identified. Therefore, we performed new HFS-TB experiments. Cyloserine killed 6.3 log 10 colony-forming units (CFU)/mL extracellular bacilli over 28 days. Efficacy was driven by the percentage of time concentration persisted above MIC (%T MIC), with 1.0 log 10 CFU/mL kill achieved by %T MIC = 30% (target exposure). The tentative epidemiological cutoff value with the Sensititre MYCOTB assay was 64 mg/L. In MCEs, 750 mg twice daily achieved target exposure in lung cavities of 92% of patients whereas 500 mg twice daily achieved target exposure in 85% of patients with meningitis. The proposed MCE-derived clinical susceptibility breakpoint at the proposed doses was 64 mg/L. Conclusions. Cycloserine is cidal against Mtb. The susceptibility breakpoint is 64 mg/L. However, the doses likely to achieve the cidality in patients are high, and could be neurotoxic.

Amplicon-based Next Generation Sequencing (NGS) is an emerging method for Mycobacterium tuberculosis drug susceptibility testing (DST) but has not been well described. We examined 158 clinical multidrug-resistant M. tuberculosis isolates via NGS of 11 resistance-associated gene regions covering 3519 nucleotides. Across these gene regions, complete resistance or heteroresistance (defined as 1%-99% mutation) was present in at least one isolate in 6.3% of loci. The number of isolates with heteroresistance was highest for gyrA codon 94, rpoB codons 526 and 531, and embB codons 306, 372 and 406 (range 11–26% of isolates exhibited heteroresistance). 57% of MDR strains had heteroresistance of one or more recognized resistance-associated mutation. Heteroresistant loci generally exhibited high or low degrees of mutation (>90% or <10%). The deep sensitivity of NGS for detecting low level pncA heteroresistance appeared to improve genotypic-phenotypic PZA susceptibility correlations over that of Sanger. NGS demonstrates that heteroresistance in TB in the regions of key genes is common and will need to be bioinformatically managed. The clinical significance of such heteroresistance is unclear, and further study of pncA should be pursued.

c MIC testing for Mycobacterium tuberculosis is now commercially available. Drug susceptibility testing by the MycoTB MIC plate has not been directly compared to that by the Bactec MGIT 960. We describe a case of extensively drug-resistant tuberculosis (XDR-TB) in Tanzania where initial MIC testing may have prevented acquired resistance. From testing on archived isolates, the accuracy with the MycoTB plate was >90% for important first-and second-line drugs compared to that with the MGIT 960, and clinically useful quantitative interpretation was also provided.A mong other factors, the total numbers of drugs and drug class to which a Mycobacterium tuberculosis isolate is resistant predict the treatment outcomes (1, 2). Despite the advantage of rapid susceptibility testing results, the molecular mechanisms of drug resistance remain incomplete for many drugs used in the treatment of multidrug-resistant tuberculosis (MDR-TB) (3). Phenotypic methods rely on mycobacterial cultures, but the testing is ultimately qualitative, reporting a threshold of growth in the presence of a single "critical" drug concentration. In contrast to the quantitative results used in the testing of other rapidly growing pathogens, the critical concentrations for M. tuberculosis are based in part on the epidemiological breakpoints, which for some drugs are very near the MIC (4). The World Health Organization (WHO) endorsed phenotypic methods for drug susceptibility testing (DST), including the agar proportion method (APM) on solid medium and an automated system in liquid medium, the Bactec MGIT 960 (BD, Franklin Lakes, NJ, USA).To overcome the prior limitations with MIC testing, a commercially available microtiter plate of lyophilized antituberculosis drugs (Sensititre MycoTB; Trek Diagnostics, Cleveland, OH, USA) was introduced (5). Prior comparisons of the MycoTB plate with the APM on Middlebrook agar found excellent agreement for most drugs (Ͼ94%) (5-7). To our knowledge, the MycoTB plate has not been compared to the Bactec MGIT 960.The Kilimanjaro Clinical Research Institute in Moshi, Tanzania, supports a biosafety level 3 facility for M. tuberculosis research and receives study specimens from the national MDR-TB hospital, the Kibong'oto Infectious Diseases Hospital (KIDH). We hence compared DST results from the MycoTB plate and the Bactec MGIT 960 using archived specimens and now describe an illustrative clinical case from the KIDH.Isolates were originally obtained from patients with known or suspected MDR-TB who had provided written informed consent for a protocol approved by the institutional review boards of the Tanzania National Institute for Medical Research and the University of Virginia. The M. tuberculosis complex was identified by Gen-Probe (San Diego, CA). Molecular testing results for rpoB or inhA and katG mutations by the Xpert MTB/RIF (Cepheid, Sunnyvale, CA, USA) and the GenoType MTBDRplus (Hain Lifescience, Nehren, Germany), respectively, were recorded if the tests were performed for clinical purposes. In previous studies, ...