Drug Susceptibility Patterns in MDR-TB Patients:Challenges for Future Regimen Design. A Cross-Sectional Study

Globally, there is substantial concern regarding the challenges of treating complex drug resistance patterns in multidrug resistant tuberculosis cases. Utilising data from three different settings (Estonia, Latvia, Romania) we sought to contrast drug susceptibility profiles for multidrug resistant tuberculosis cases, highlight the difficulties in designing universal regimen, and inform future regimen selection. Demographic and microbiological surveillance data for multidrug resistant tuberculosis cases from 2004-13 were analysed. High levels of additional resistance to currently recommended second line drugs were seen in all settings, with extensive variability between countries. Accurate drug susceptibility testing and drug susceptibility testing data are vital to inform the development of comprehensive, flexible, multidrug resistant tuberculosis guidance

Viability testing of material derived from Mycobacterium tuberculosis prior to removal from a Containment Level-III Laboratory as part of a Laboratory Risk Assessment Program

BACKGROUND: In the field of clinical mycobacteriology, Mycobacterium tuberculosis (MTB) can be a difficult organism to manipulate due to the restrictive environment of a containment level 3 (CL3) laboratory. Tests for rapid diagnostic work involving smears and molecular methods do not require CL3 practices after the organism has been rendered non-viable. While it has been assumed that after organism deactivation these techniques can be performed outside of a CL3, no conclusive study has consistently confirmed that the organisms are noninfectious after the theoretical 'deactivation' steps. Previous studies have shown that initial steps (such as heating /chemical fixation) may not consistently kill MTB organisms. METHODS: An inclusive viability study (n = 226) was undertaken to determine at which point handling of culture extraction materials does not necessitate a CL3 environment. Four different laboratory protocols tested for viability included: standard DNA extractions for IS6110 fingerprinting, crude DNA preparations for PCR by boiling and mechanical lysis, protein extractions, and smear preparations. For each protocol, laboratory staff planted a proportion of the resulting material to Bactec 12B medium that was observed for growth for 8 weeks. RESULTS: Of the 208 isolates initially tested, 21 samples grew within the 8-week period. Sixteen (7.7%) of these yielded positive results for MTB that included samples of: deactivated culture resuspensions exposed to 80°C for 20 minutes, smear preparations and protein extractions. Test procedures were consequently modified and tested again (n = 18), resulting in 0% viability. CONCLUSIONS: This study demonstrates that it cannot be assumed that conventional practices (i.e. smear preparation) or extraction techniques render the organism non-viable. All methodologies, new and existing, should be examined by individual laboratories to validate the safe removal of material derived from MTB to the outside of a CL3 laboratory. This process is vital to establish in house biosafety-validated practices with the aim of protecting laboratory workers conducting these procedures

Commercial Nucleic-Acid Amplification Tests for Diagnosis of Pulmonary Tuberculosis in Respiratory Specimens: Meta-Analysis and Meta-Regression

BACKGROUND: Hundreds of studies have evaluated the diagnostic accuracy of nucleic-acid amplification tests (NAATs) for tuberculosis (TB). Commercial tests have been shown to give more consistent results than in-house assays. Previous meta-analyses have found high specificity but low and highly variable estimates of sensitivity. However, reasons for variability in study results have not been adequately explored. We performed a meta-analysis on the accuracy of commercial NAATs to diagnose pulmonary TB and meta-regression to identify factors that are associated with higher accuracy. METHODOLOGY/PRINCIPAL FINDINGS: We identified 2948 citations from searching the literature. We found 402 articles that met our eligibility criteria. In the final analysis, 125 separate studies from 105 articles that reported NAAT results from respiratory specimens were included. The pooled sensitivity was 0.85 (range 0.36-1.00) and the pooled specificity was 0.97 (range 0.54-1.00). However, both measures were significantly heterogeneous (p<.001). We performed subgroup and meta-regression analyses to identify sources of heterogeneity. Even after stratifying by type of commercial test, we could not account for the variability. In the meta-regression, the threshold effect was significant (p = .01) and the use of other respiratory specimens besides sputum was associated with higher accuracy. CONCLUSIONS/SIGNIFICANCE: The sensitivity and specificity estimates for commercial NAATs in respiratory specimens were highly variable, with sensitivity lower and more inconsistent than specificity. Thus, summary measures of diagnostic accuracy are not clinically meaningful. The use of different cut-off values and the use of specimens other than sputum could explain some of the observed heterogeneity. Based on these observations, commercial NAATs alone cannot be recommended to replace conventional tests for diagnosing pulmonary TB. Improvements in diagnostic accuracy, particularly sensitivity, need to be made in order for this expensive technology to be worthwhile and beneficial in low-resource countries

Rapid Detection of Mycobacterium tuberculosis in Contaminated BACTEC 12B Broth Cultures by Testing with Amplified Mycobacterium Tuberculosis Direct Test

Contamination of broth cultures of acid-fast bacilli (AFB) by bacterial species other than Mycobacterium species frequently occurs. Many of these contaminated cultures require redecontamination and reincubation before the appropriate tests can be performed for identification, significantly affecting the turnaround time for reporting culture results. In this study, the Amplified Mycobacterium Tuberculosis Direct Test (MTD; Gen-Probe) was performed to detect the Mycobacterium tuberculosis complex (MTBC) in 125 BACTEC 12B broth cultures with positive growth indices. Among these, 41 grew non-AFB bacteria only, and all 41 were negative by the MTD. The remaining 84 bottles contained contaminated cultures that grew both AFB and other bacteria or yeasts. Repeat decontamination and reincubation of these specimens required a mean time of 13 days (range, 3 to 40 days). The MTD results were positive for 10 samples, 9 of which were MTBC culture positive and 1 of which grew Myobacterium celatum, a species known to cross-react in the MTD. All cultures growing other mycobacterial species were negative by the MTD. The results of this study demonstrate that the MTD is both sensitive and specific in detecting MTBC in contaminated broth cultures and that, when used selectively, the MTD can potentially rule in or out a diagnosis of MTBC as much as 12 days earlier than using nonamplified DNA probe testing alone can

Outbreak of acupuncture-associated cutaneous Mycobacterium abscessus infections

BACKGROUND: Cutaneous atypical mycobacterial infections have been increasingly described in association with cosmetic and alternative procedures. OBJECTIVE: We report an outbreak of acupuncture-associated mycobacteriosis. Between April and December 2002, 32 patients developed cutaneous mycobacteriosis after visiting an acupuncture practice in Toronto, Canada. RESULTS: Of 23 patients whose lesions were biopsied, 6 (26.1%) had culture-confirmed infection with Mycobacterium abscessus. These isolates were genetically indistinguishable by amplified fragment length polymorphism. The median incubation period was 1 month. Of 24 patients for whom clinical information was available, 23 (95.8%) had resolution of their infection. All patients developed residual scarring or hyperpigmentation. CONCLUSION: Nontuberculous mycobacteria should be recognized as an emerging, but preventable, cause of acupuncture-associated infections