Multidrug-resistant Tuberculosis Detection, Latvia

To improve multidrug-resistant tuberculosis (MDR-TB) detection, we successfully introduced the rpoB gene mutation line probe assay into the national laboratory in Latvia, a country with epidemic MDR-TB. The assay detected rifampin resistance with 91% sensitivity and 96% specificity within 1 to 5 days (vs. 12–47 days for BACTEC)

Geographic Differences in Time to Culture Conversion in Liquid Media: Tuberculosis Trials Consortium Study 28. Culture Conversion Is Delayed in Africa

Tuberculosis Trials Consortium Study 28, was a double blind, randomized, placebo-controlled, phase 2 clinical trial examining smear positive pulmonary Mycobacterium tuberculosis. Over the course of intensive phase therapy, patients from African sites had substantially delayed and lower rates of culture conversion to negative in liquid media compared to non-African patients. We explored potential explanations of this finding.In TBTC Study 28, protocol-correct patients (n = 328) provided spot sputum specimens for M. tuberculosis culture in liquid media, at baseline and weeks 2, 4, 6 and 8 of study therapy. We compared sputum culture conversion for African and non-African patients stratified by four baseline measures of disease severity: AFB smear quantification, extent of disease on chest radiograph, cavity size and the number of days to detection of M. tuberculosis in liquid media using the Kaplan-Meier product-limit method. We evaluated specimen processing and culture procedures used at 29 study laboratories serving 27 sites.African TB patients had more extensive disease at enrollment than non-African patients. However, African patients with the least disease by the 4 measures of disease severity had conversion rates on liquid media that were substantially lower than conversion rates in non-African patients with the greatest extent of disease. HIV infection, smoking and diabetes did not explain delayed conversion in Africa. Some inter-site variation in laboratory processing and culture procedures within accepted practice for clinical diagnostic laboratories was found.Compared with patients from non-African sites, African patients being treated for TB had delayed sputum culture conversion and lower sputum conversion rates in liquid media that were not explained by baseline severity of disease, HIV status, age, smoking, diabetes or race. Further investigation is warranted into whether modest variation in laboratory processes substantially influences the efficacy outcomes of phase 2 TB treatment trials or if other factors (e.g., nutrition, host response) are involved.ClinicalTrials.gov NCT00144417

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

Mycobacterium tuberculosis Transmission from Human to Canine

A 71-year-old woman from Tennessee, USA with a 3-week history of a productive, nonbloody cough was evaluated. Chest radiograph showed infiltrates and atelectasis in the upper lobe of the right lung. A tuberculosis (TB) skin test resulted in a 14-mm area of induration. Sputum stained positive for acid-fast bacilli (AFB) and was positive for Mycobacterium tuberculosis by DNA probe and culture. Treatment was initiated with isoniazid, rifampicin, and pyrazinamide. After 14 days of daily, directly observed therapy, the patient complained of nausea, vomiting and diarrhoea. Treatment adjustments were made, and therapy was completed 11 months later with complete recovery. Six months after the patient\u27s TB diagnosis, she took her three and a half-year-old male Yorkshire Terrier to a veterinary clinic with cough, weight loss, and vomiting of several months\u27 duration. Initial sputum sample was negative on AFB staining. Eight days after discharge from a referral veterinary teaching hospital with a presumptive diagnosis of TB, the dog was euthanized due to urethral obstruction. Liver and tracheobronchial lymph node specimens collected at necropsy were positive for M. tuberculosis complex by polymerase chain reaction. The M. tuberculosis isolates from the dog and its owner had an indistinguishable 10-band pattern by IS6110-based restriction fragment length polymorphism genotyping

Evaluation of Xpert MTB/RIF Versus AFB Smear and Culture to Identify Pulmonary Tuberculosis in Patients With Suspected Tuberculosis From Low and Higher Prevalence Settings

Background. The Xpert MTB/RIF (Xpert) assay is a rapid nucleic acid amplification test widely used in settings of high tuberculosis prevalence to detect tuberculosis as well as rpoB mutations associated with rifampin resistance. Data are needed on the diagnostic performance of Xpert in lower-prevalence settings to inform appropriate use for both tuberculosis detection and the need for respiratory isolation. Methods. Xpert was compared to 2 sputum samples, each evaluated with acid-fast bacilli (AFB) smear and mycobacterial culture using liquid and solid culture media, from participants with suspected pulmonary tuberculosis from the United States, Brazil, and South Africa. Results. Of 992 participants enrolled with evaluable results, 22% had culture-confirmed tuberculosis. In 638 (64%) US participants, 1 Xpert result demonstrated sensitivity of 85.2% (96.7% in participants with AFB smear-positive [AFB+] sputum, 59.3% with AFB smear-negative [AFB–] sputum), specificity of 99.2%, negative predictive value (NPV) of 97.6%, and positive predictive value of 94.9%. Results did not differ between higher- and low-prevalence settings. A second Xpert assay increased overall sensitivity to 91.1% (100% if AFB+, 71.4% if AFB–), with specificity of 98.9%. In US participants, a single negative Xpert result predicted the absence of AFB+/culture-positive tuberculosis with an NPV of 99.7%; NPV of 2 Xpert assays was 100%, suggesting a role in removing patients from airborne infection isolation. Xpert detected tuberculosis DNA and mutations associated with rifampin resistance in 5 of 7 participants with rifampin-resistant, culture-positive tuberculosis. Specificity for rifampin resistance was 99.5% and NPV was 98.9%. Conclusions. In the United States, Xpert testing performed comparably to 2 higher-tuberculosis-prevalence settings. These data support the use of Xpert in the initial evaluation of tuberculosis suspects and in algorithms assessing need for respiratory isolation

Worldwide Emergence of Extensively Drug-resistant Tuberculosis

Mycobacterium tuberculosis strains are becoming resistant to not only the most powerful first-line drugs but also many second-line drugs

Evaluation of a U.S. Public Health Laboratory Service for the Molecular Detection of Drug Resistant Tuberculosis

Crucial to interrupting the spread of tuberculosis (TB) is prompt implementation of effective treatment regimens. We evaluated satisfaction, comfort with interpretation, and use of molecular results from a public health service provided by the Centers for Disease Control and Prevention (CDC) for the molecular detection of drug resistant Mycobacterium tuberculosis complex (MTBC). An electronic survey instrument was used to collect information anonymously from U.S. Public Health Laboratories (PHL) that submitted at least one isolate of MTBC to CDC from September 2009 through February 2011. Over 97% of those responding expressed satisfaction with the turnaround time for receiving results. Twenty-six PHL (74%) reported molecular results to healthcare providers in less than two business days. When comparing the molecular results from CDC with their own phenotypic drug susceptibility testing, 50% of PHL observed discordance. No respondents found the molecular results difficult to interpret and 82% were comfortably discussing them with TB program officials and healthcare providers. Survey results indicate PHL were satisfied with CDC’s ability to rapidly provide interpretable molecular results for isolates of MTBC submitted for determination of drug resistance. To develop educational materials and strategies for service improvement, reasons for discordant results and areas of confusion need to be identified

ethA, inhA, and katG Loci of Ethionamide-Resistant Clinical Mycobacterium tuberculosis Isolates

Ethionamide (ETH) is a structural analog of the antituberculosis drug isoniazid (INH). Both of these drugs target InhA, an enzyme involved in mycolic acid biosynthesis. INH requires catalase-peroxidase (KatG) activation, and mutations in katG are a major INH resistance mechanism. Recently an enzyme (EthA) capable of activating ETH has been identified. We sequenced the entire ethA structural gene of 41 ETH-resistant Mycobacterium tuberculosis isolates. We also sequenced two regions of inhA and all or part of katG. The MICs of ETH and INH were determined in order to associate the mutations identified with a resistance phenotype. Fifteen isolates were found to possess ethA mutations, for all of which the ETH MICs were ≥50 μg/ml. The ethA mutations were all different, previously unreported, and distributed throughout the gene. In eight of the isolates, a missense mutation in the inhA structural gene occurred. The ETH MICs for seven of the InhA mutants were ≥100 μg/ml, and these isolates were also resistant to ≥8 μg of INH per ml. Only a single point mutation in the inhA promoter was identified in 14 isolates. A katG mutation occurred in 15 isolates, for which the INH MICs for all but 1 were ≥32 μg/ml. As expected, we found no association between katG mutation and the level of ETH resistance. Mutations within the ethA and inhA structural genes were associated with relatively high levels of ETH resistance. Approximately 76% of isolates resistant to ≥50 μg of ETH per ml had such mutations