Detection of pyrazinamide heteroresistance in Mycobacterium tuberculosis

AbstractAimPyrazinamide (PZA) is a first-line key agent in the effective treatment of tuberculosis (TB), including PZA susceptible multidrug-resistant tuberculosis (MDR-TB). Occasionally, TB patients might have mixed infections with both drug-sensitive and -resistant strains. This is termed heteroresistance. If 10% of the bacterial population is resistant to PZA, there is an increased risk for poor treatment outcome. The aim of this study is to evaluate the ability of the three established drug susceptibility testing (DST) techniques – BACTEC MGIT 960, Wayne’s pyrazinamidase test and Sanger sequencing of the pncA gene – to detect 10% PZA heteroresistance.MethodsMixed cultures of the fully drug susceptible Mycobacterium tuberculosis H37Rv reference strain and two laboratory-generated isogenic H37Rv mutants (with C475G and T254C pncA mutations, respectively) were made in proportions of 100%, 10%, 5% and 1% of the PZA-resistant (PZA-R) strain. Corresponding mixed cultures were also made using one drug-susceptible and one PZA-resistant MDR clinical isolate with the T62G pncA mutation, both belonging to one specific MIRU cluster. Additional mixes of 50%, 75%, 90% and 99% of the PZA-R strains were prepared for the Wayne’s test. Tests were for all methods performed in duplicates at two separate occasions.ResultsUsing the MGIT system, the in vitro-generated PZA-R strains were generally detected at a 5% proportion while the clinical PZA-R isolate only was detected at the critical 10% proportion, except for one test occasion. Sanger sequencing was unable to detect 10% PZA heteroresistance. Wayne’s test also failed to detect the critical level of 10% PZA resistance; instead it displayed misguiding results determining highly resistant samples as susceptible.ConclusionHeteroresistance is caused by present drug-resistance development and/or dual infections with one resistant and one susceptible strain. Mixed infections with resistant strains may occur in up to 20% of all TB cases in high burden areas, according to limited data. This study showed that only the phenotypic BACTEC MGIT system was capable in determining the critical proportion of 10% PZA resistance, whereas neither the Sanger nor the Wayne’s test were successful in this respect. This indicates a need for diagnostic tools with increased sensitivity to determine heteroresistance in M. tuberculosis

Dynamics of Antibiotic Resistant Mycobacterium tuberculosis during Long-Term Infection and Antibiotic Treatment

For an infecting bacterium the human body provides several potential ecological niches with both internally (e.g. host immunity) and externally (e.g. antibiotic use) imposed growth restrictions that are expected to drive adaptive evolution in the bacterium, including the development of antibiotic resistance. To determine the extent and pattern of heterogeneity generated in a bacterial population during long-term antibiotic treatment, we examined in a monoclonal Mycobacterium tuberculosis infection antibiotic resistant mutants isolated from one patient during a 9-years period. There was a progressive accumulation of resistance mutations in the infecting clone. Furthermore, apparent clonal sweeps as well as co-existence of different resistant mutants were observed during this time, demonstrating that during treatment there is a high degree of dynamics in the bacterial population. These findings have important implications for diagnostics and treatment of drug resistant tuberculosis infections

Detection of first- and second-line drug resistance in Mycobacterium tuberculosis clinical isolates by pyrosequencing

Conventional phenotypic drug susceptibility testing (DST) methods for Mycobacterium tuberculosis are laborious and very time-consuming. Early detection of drug-resistant tuberculosis (TB) is essential for prevention and control of TB transmission. We have developed a pyrosequencing method for simultaneous detection of mutations associated with resistance to rifampin, isoniazid, ethambutol, amikacin, kanamycin, capreomycin, and ofloxacin. Seven pyrosequencing assays were optimized for following loci: rpoB, katG, embB, rrs, gyrA, and the promoter regions of inhA and eis. The molecular method was evaluated on a panel of 290 clinical isolates of M. tuberculosis. In comparison to phenotypic DST, the pyrosequencing method demonstrated high specificity (100%) and sensitivity (94.6%) for detection of multidrug-resistant M. tuberculosis as well as high specificity (99.3%) and sensitivity (86.9%) for detection of extensively drug-resistant M. tuberculosis. The short turnaround time combined with multilocus sequencing of several isolates in parallel makes pyrosequencing an attractive method for drug resistance screening in M. tuberculosis.Fil: Engström, Anna. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Morcillo, Nora Susana. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Imperiale, Belén Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Provincia de Buenos Aires. Ministerio de Salud. Hospital "Dr. Antonio A. Cetrángolo"; ArgentinaFil: Hoffner, Sven E.. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; SueciaFil: Juréen, Pontus. Karolinska Huddinge Hospital. Karolinska Institutet; Suecia. Swedish Institute for Communicable Disease Control; Sueci

Direct detection of Mycobacterium tuberculosis in respiratory samples from patients in Scandinavia by polymerase chain reaction

ObjectiveTo investigate the use of DNA amplification by the polymerase chain reaction (PCR) for the detection of Mycobacterium tuberculosis directly in human respiratory specimens.MethodsThe PCR assay employed was the Amplicor M. tuberculosis Test (Roche Diagnostics, Switzerland), which uses the 16S rDNA as the target template. Nine hundred and sixty samples from 741 patients in two clinical microbiology laboratories in Norway and Sweden were processed by routine culture analysis and PCR.ResultsOf the 56 specimens containing cultivatable M. tuberculosis, 49 (87.5%) were detected by PCR. Among the 904 culture-negative specimens, 897 samples were negative also by PCR and seven (0.8%) were positive by PCR. In comparison with culture, the sensitivity, specificity, and positive and negative predictive values of PCR were 91.7%, 99.6%, 94.2% and 99.4% for laboratory 1 and 80.0%, 98.7%, 76.2% and 99.0% for laboratory 2, respectively. For both laboratories combined the values were 87.5%, 99.2%, 87.5% and 99.2%.ConclusionsThese results indicate that multiple (two or three) respiratory samples from each patient should be tested, to allow sufficient accuracy in detecting M. tuberculosis in the specimens. Still, the labor-intensive format of this test necessitates strong clinical indications and patient prioritization to provide a service feasible within the current limits of routine laboratories

Outcomes of a clinical diagnostic algorithm for management of ambulatory smear and Xpert MTB/Rif negative HIV infected patients with presumptive pulmonary TB in Uganda: a prospective study

Introduction: Diagnostic guidelines for Tuberculosis (TB) in HIV infected patients previously relied on microscopy where the value of initial antibiotic treatment for exclusion of pulmonary TB (PTB) was limited. New guidelines rely on the Xpert MTB Rif test (Xpert). However, the value of the antibiotic treatment remains unclear particularly in individuals who are smear-negative and Xpert-negative-given Xpert has only moderate sensitivity for smear-negative PTB. We assessed an algorithm involving initial treatment with antibiotics prior empiric TB treatment in HIV patients with presumptive PTB who were both smear and Xpert negative. Methods: We performed a prospective study with six month follow-up to establish patient response to a course of broad spectrum antibiotics prior empiric TB treatment between March 2012 and June 2013. We calculated the proportion of patients who responded to the antibiotic treatment and those who did not. We computed the crude and adjusted odds ratios with their 95% confidence intervals, for response to the antibiotic treatment on various patient characteristics. We report treatment outcomes for patients who received broad spectrum antibiotics only or who were initiated empiric TB treatment. Results: Our cohort comprised 162 smearnegative and Xpert-negative patients, of whom 59% (96 of 162) were female, 81% (131 of 162) were on antiretroviral therapy (ART) for a median of 8.7 months. Overall, 88% (141 of 160) responded to the antibiotic treatment, 8% (12 of 160) got empiric TB treatment and 4% (7 out of 160) were treated for other respiratory disease. The odds of improvement on antibiotics were lower in patients with advanced HIV disease than in patients with early HIV disease. Adjusted odds ratios were significant for HIV clinical stage (AOR; 0.038,) and duration on ART (AOR; 1.038,). Conclusion: The majority of HIV patients with presumptive PTB with smear-negative and Xpert negative results improved on the antibiotic treatment and did not require empiric TB treatment. Initial antibiotic treatment appeared more successful in patients with less advanced HIV disease. Findings from our study suggest it is useful to initiate HIV infected patients with presumptive PTB having smear and Xpert negative results on an initial course of antibiotic treatment prior empiric TB treatment.Pan African Medical Journal 2016; 2

Populations of latent Mycobacterium tuberculosis lack a cell wall: Isolation, visualization, and whole-genome characterization

AbstractObjective/BackgroundMycobacterium tuberculosis (MTB) causes active tuberculosis (TB) in only a small percentage of infected people. In most cases, the infection is clinically latent, where bacilli can persist in human hosts for years without causing disease. Surprisingly, the biology of such persister cells is largely unknown. This study describes the isolation, identification, and whole-genome sequencing (WGS) of latent TB bacilli after 782days (26months) of latency (the ability of MTB bacilli to lie persistent).MethodsThe in vitro double-stress model of latency (oxygen and nutrition) was designed for MTB culture. After 26months of latency, MTB cells that persisted were isolated and investigated under light and atomic force microscopy. Spoligotyping and WGS were performed to verify the identity of the strain.ResultsWe established a culture medium in which MTB bacilli arrest their growth, reduce their size (0.3–0.1μm), lose their acid fastness (85–90%) and change their shape. Spoligopatterns of latent cells were identical to original H37Rv, with differences observed at spacers two and 14. WGS revealed only a few genetic changes relative to the already published H37Rv reference genome. Among these was a large 2064-bp insertion (RvD6), which was originally detected in both H37Ra and CDC1551, but not H37Rv.ConclusionHere, we show cell-wall free cells of MTB bacilli in their latent state, and the biological adaptation of these cells was more phenotypic in nature than genomic. These cell-wall free cells represent a good model for understanding the nature of TB latency

Extensively Drug-Resistant Tuberculosis, Pakistan

Frequency of extensively drug-resistant tuberculosis in Pakistan increased from 1.5% in 2006 to 4.5% in 2009 (p<0.01). To understand the epidemiology, we genotyped selected strains by using spoligotyping, mycobacterial interspersed repetitive units–variable number of tandem repeats, and IS6110 restriction fragment length polymorphism analysis

A multilaboratory, multicountry study to determine MIC quality control ranges for phenotypic drug susceptibility testing of selected first-line antituberculosis dugs, second-line injectables, fluoroquinolones, clofazimine, and linezolid

OBJECTIVES : Our objective was to establish reference minimal inhibitory concentration (MIC) quality control (QC) ranges for drug susceptibility testing of antimycobacterials, including firstline agents, second-line injectables, fluoroquinolones and World Health Organization Category 5 drugs for multidrug-resistant tuberculosis, using a 7H9 broth microdilution MIC method. METHODS : A Tier-2 reproducibility study was conducted in eight participating laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Three lots of custom-made frozen 96-well polystyrene micro titer plates were used and pre-prepared with 2X pre-diluted drugs in 7H9 broth/oleic acid albumin dextrose catalase. The QC reference strain was Mycobacterium tuberculosis (MTB) H37Rv. MIC frequency, mode and geometric mean were calculated for each drug. QC ranges were derived, based on predefined, strict CLSI criteria. Any data lying outside CLSI criteria resulted in exclusion of the entire laboratory dataset. RESULTS : Data from one laboratory were excluded due to higher MIC values than for other laboratories. QC ranges were established for eleven drugs: isoniazid (0.03–0.12 μg/ml), rifampin (0.03–0.25 μg/ml), ethambutol (0.25–2 μg/ml), levofloxacin (0.12–1 μg/ml), moxifloxacin (0.06–0.5 μg/ml), ofloxacin (0.25–2 μg/ml), amikacin (0.25–2 μg/ml), kanamycin (0.25–2 μg/ml), capreomycin (0.5–4 μg/ml), linezolid (0.25–2 μg/ml) and clofazimine (0.03–0.25 μg/ml). QC ranges could not be established for nicotinamide (pyrazinamide surrogate), prothionamide or ethionamide, which were assay non-performers. CONCLUSIONS : Using strict CLSI criteria, QC ranges against the MTB H37Rv reference strain were established for the majority of commonly used antituberculosis drugs, with a convenient 7H9 broth microdilution MIC method suitable for use in resource-limited settings.All participating laboratories received funds for this study from Janssen Pharmaceuticals except the Reference Laboratory, Division of TB Elimination, United States Centers for Disease Control and Prevention, Atlanta, GA, USA. Support for medical writing assistance was provided by Janssen Pharmaceuticals.http://jcm.asm.org2017-06-30hb2017Medical Microbiolog

A multilaboratory, multicountry study to determine bedaquiline MIC quality control ranges for phenotypic drug susceptibility testing

The aim of this study was to establish standardized drug susceptibility testing (DST) methodologies and reference MIC quality control (QC) ranges for bedaquiline, a diarylquinoline antimycobacterial, used in the treatment of adults with multidrug-resistant tuberculosis. Two tier-2 QC reproducibility studies of bedaquiline DST were conducted in eight laboratories using Clinical Laboratory and Standards Institute (CLSI) guidelines. Agar dilution and broth microdilution methods were evaluated. Mycobacterium tuberculosis H37Rv was used as the QC reference strain. Bedaquiline MIC frequency, mode, and geometric mean were calculated. When resulting data occurred outside predefined CLSI criteria, the entire laboratory data set was excluded. For the agar dilution MIC, a 4-dilution QC range (0.015 to 0.12 μg/ml) centered around the geometric mean included 95.8% (7H10 agar dilution; 204/213 observations with one data set excluded) or 95.9% (7H11 agar dilution; 232/242) of bedaquiline MICs. For the 7H9 broth microdilution MIC, a 3-dilution QC range (0.015 to 0.06 μg/ml) centered around the mode included 98.1% (207/211, with one data set excluded) of bedaquiline MICs. Microbiological equivalence was demonstrated for bedaquiline MICs determined using 7H10 agar and 7H11 agar but not for bedaquiline MICs determined using 7H9 broth and 7H10 agar or 7H9 broth and 7H11 agar. Bedaquiline DST methodologies and MIC QC ranges against the H37Rv M. tuberculosis reference strain have been established: 0.015 to 0.12 μg/ml for the 7H10 and 7H11 agar dilution MICs and 0.015 to 0.06 μg/ml for the 7H9 broth microdilution MIC. These methodologies and QC ranges will be submitted to CLSI and EUCAST to inform future research and provide guidance for routine clinical bedaquiline DST in laboratories worldwide.All participating laboratories received funds for this study from Janssen Pharmaceuticals except the Reference Laboratory, Division of TB Elimination, United States Centers for Disease Control and Prevention, Atlanta, GA, USA. Support for medical writing assistance was provided by Janssen Pharmaceuticals.http://jcm.asm.orghj2017Medical Microbiolog