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.

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.

Background Clinical courses and treatment outcomes are largely unknown in patients with adult-onset immunodeficiency associated with anti-interferon-gamma autoantibodies due to the fact that it was recently recognized and anti-IFN-γ auto-Abs detection is not widely available. Methods and findings Non-HIV-infected adult patients with detectable anti-IFN-γ auto-Abs diagnosed and followed at Siriraj Hospital, Bangkok, Thailand during January 2013 to November 2016 were prospectively studied. At each follow-up visit, patients were classified as stable or active disease according to symptoms and signs, and all proven OIs were recorded. Laboratory parameters, including erythrocyte sedimentation rate, C-reactive protein, and anti-IFN-γ auto-Abs level, were compared between active and stable disease episodes. We identified 80 patients with this clinical syndrome and followed them up during study period. Seventy-nine patients developed overall 194 proven opportunistic infections. Mycobacterium abscessus (34.5%) and Salmonella spp. (23.2%) were the two most common pathogens identified among these patients. Sixty-three patients were followed for a median of 2.7 years (range 0.6–4.8 years). Eleven (17.5%) patients achieved the drug-free remission period for at least 9 months. Four patients died. Anti-IFN-γ auto-Abs concentration was significantly lower at baseline and decreased over time in the drug-free remission group compared to another group ( p = 0.001). C-reactive protein, erythrocyte sedimentation rate and white cell count were found to be useful biomarkers for determining disease activity during follow-up. Conclusions Reinfection or relapse of OIs is common despite long-term antimicrobial treatment in patients with anti-IFN-γ auto-Abs. Treatment to modify anti-IFN-γ auto-Abs production may improve long-term outcomes in this patient population.

Mycobacterium haemophilum is a nontuberculous mycobacterium that can infect immunocompromised patients. Because of special conditions required for its culture, this bacterium is rarely reported and there are scarce data for long-term outcomes. We conducted a retrospective study at Siriraj Hospital, Bangkok, Thailand, during January 2012–September 2017. We studied 21 patients for which HIV infection was the most common concurrent condition. The most common organ involvement was skin and soft tissue (60%). Combination therapy with macrolides and fluoroquinolones resulted in a 60% cure rate for cutaneous infection; adding rifampin as a third drug for more severe cases resulted in modest (66%) cure rate. Efficacy of medical therapy in cutaneous, musculoskeletal, and ocular diseases was 80%, 50%, and 50%, respectively. All patients with central nervous system involvement showed treatment failures. Infections with M. haemophilum in HIV-infected patients were more likely to have central nervous system involvement and tended to have disseminated infections and less favorable outcomes.

Recently a newly identified clinical syndrome of disseminated non-tuberculous mycobacterial diseases (with or without other opportunistic infections in adult patients who were previously healthy, has been recognized in association with an acquired autoantibody to interferon-gamma. This syndrome is emerging as an important cause of morbidity and mortality, especially among people of Asian descent. Trigger for the production of this autoantibody remains unknown, but genetic factors are strongly suspected to be involved. We compared HLA genotyping between 32 patients with this clinical syndrome, and 38 controls. We found that this clinical syndrome was associated with very limited allele polymorphism, with HLA-DRB1 and DQB1 alleles, especially HLA-DRB1*15:01, DRB1*16:02, DQB1*05:01 and DQB1*05:02. Odds ratio of DRB1*15:01, DRB1*16:02, DQB1*05:01 and DQB1*05:02 were 7.03 (95% CI, 2.18–22.69, P<0.0001, 9.06 (95% CI, 2.79–29.46, P<0.0001), 6.68 (95% CI, 2.29–19.52, P = 0.0004), and 6.64 (95% CI, 2.30–19.20, P = 0.0004), respectively. Further investigation is warranted to provide better understanding on pathogenesis of this association.

Genotypic methods for drug susceptibility testing of Mycobacterium tuberculosis are desirable to speed the diagnosis and proper therapy of tuberculosis (TB). However, the numbers of genes and polymorphisms implicated in resistance have proliferated, challenging diagnostic design. We developed a microfluidic TaqMan array card (TAC) that utilizes both sequence-specific probes and high-resolution melt analysis (HRM), providing two layers of detection of mutations. Twenty-seven primer pairs and 40 probes were designed to interrogate 3,200 base pairs of critical regions of the inhA, katG, rpoB, embB, rpsL, rrs, eis, gyrA, gyrB, and pncA genes. The method was evaluated on 230 clinical M. tuberculosis isolates from around the world, and it yielded 96.1% accuracy (2,431/2,530) in comparison to that of Sanger sequencing and 87% accuracy in comparison to that of the slow culture-based susceptibility testing. This TAC-HRM method integrates assays for 10 genes to yield fast, comprehensive, and accurate drug susceptibility results for the 9 major antibiotics used to treat TB and could be deployed to improve treatment outcomes.

Human lymphatic filariasis is caused by filarial worms such as Brugia malayi for which the major reservoir is domestic cats. However, domestic cats or dogs also carry nonhuman filaria such as Brugia pahangi and Dirofilaria immitis. We have developed a single-tube, real-time PCR with a high-resolution melting (HRM) analysis assay for detection and identification of B. malayi, B. pahangi, and D. immitis in blood samples. The designated primer pair in the PCR can amplify a 114-bp region of mitochondrial 12S rRNA genes of these filarial worms. Subsequently, the HRM assay showed a specific melting temperature for each species. The assay showed the highest sensitivity and specificity in comparison with DNA sequences after assessment with 34 cat and 14 dog blood samples. This assay could be helpful for epidemiological studies of reservoirs and vectors.