Application of Replicate Organism Detection and Counting Method (RODAC) in Measuring Mycobacterium Tuberculosis Contamination in High Burden Laboratories

Background: Technicians working in high burden tuberculosis (TB) laboratories pose a higher risk of being infected by Mycobacterium tuberculosis from clinical samples. Contamination control is mandatory to detect the release of bacteria into the working environment and to minimize the risk of exposure to the workers. The contamination measurement is rarely performed due to the lack of standard methodology. This study optimized and applied a unique culture-based method named Replicate Organism Detection and Counting (RODAC) plates to assess the presence of M. tuberculosis contaminant in the laboratory with high burden of clinical samples. Methods: RODAC was applied on twenty working surfaces in the Mycobacteriology Laboratory of Universitas Padjadjaran. The results of RODAC were compared with DNA-based detection from the same working surfaces using in-house IS6110 real-time PCR (IS6110-qPCR). The detection limit of the RODAC plate was 19.6 CFU mL-1.Results: From all working surfaces tested, two distinct colonies were found on RODAC plate stamped on the Ziehl-Neelsen staining basin. Those colonies were identified as M. tuberculosis and non-tuberculous mycobacteria (NTM), as confirmed by the MPT64 antigen test and the presence of acid-fast bacilli. IS6110-qPCR detected the presence of M. tuberculosis DNA in ten sampling points, including the ZN staining basin, incubators, and microscopy areas. IS6110-qPCR detected more working surface contamination versus RODAC. However, it was noted that RODAC, which was a culture-based method, detected live bacteria, while PCR could not distinguish between live and dead bacteria.Conclusion: The application of the RODAC plate is more suitable for monitoring the contamination of live bacteria in the working environment and to inform a proper corrective action

Application of Replicate Organism Detection and Counting Method (RODAC) in Measuring Mycobacterium Tuberculosis Contamination in High Burden Laboratories

Background: Technicians working in high burden tuberculosis (TB) laboratories pose a higher risk of being infected by Mycobacterium tuberculosis from clinical samples. Contamination control is mandatory to detect the release of bacteria into the working environment and to minimize the risk of exposure to the workers. The contamination measurement is rarely performed due to the lack of standard methodology. This study optimized and applied a unique culture-based method named Replicate Organism Detection and Counting (RODAC) plates to assess the presence of M. tuberculosis contaminant in the laboratory with high burden of clinical samples. Methods: RODAC was applied on twenty working surfaces in the Mycobacteriology Laboratory of Universitas Padjadjaran. The results of RODAC were compared with DNA-based detection from the same working surfaces using in-house IS6110 real-time PCR (IS6110-qPCR). The detection limit of the RODAC plate was 19.6 CFU mL-1. Results: From all working surfaces tested, two distinct colonies were found on RODAC plate stamped on the Ziehl-Neelsen staining basin. Those colonies were identified as M. tuberculosis and non-tuberculous mycobacteria (NTM), as confirmed by the MPT64 antigen test and the presence of acid-fast bacilli. IS6110-qPCR detected the presence of M. tuberculosis DNA in ten sampling points, including the ZN staining basin, incubators, and microscopy areas. IS6110-qPCR detected more working surface contamination versus RODAC. However, it was noted that RODAC, which was a culture-based method, detected live bacteria, while PCR could not distinguish between live and dead bacteria. Conclusion: The application of the RODAC plate is more suitable for monitoring the contamination of live bacteria in the working environment and to inform a proper corrective action

Accuracy of LED-FM and ZN method in direct and decontaminated smears.

<p>LED-FM = Light emitting diode-fluorescence microscopy, ZN = Ziehl-Neelsen, CI = confidence interval.</p

Accuracy of LED-FM and ZN in two study groups.

<p>LED-FM = Light emitting diode-fluorescence microscopy, ZN = Ziehl-Neelsen, PPV = positive predictive value, NPV = negative predictive value, CI = confidence interval.</p

ROC curve for LED-FM.

<p>The optimal threshold for sensitivity and specificity of LED-FM was assessed using ROC analysis. Maximum area under the curve (AUC 0.85, 95% CI 0.78–0.92) was obtained at threshold of ≥2 AFB/length (sensitivity 75,5%, specificity 90,0%,). ROC = receiver operating characteristic, LED-FM =  Light emitting diode-fluorescence microscopy, AFB = Acid Fast Bacilli, CI = confidence interval.</p

Implementation of LED fluorescence microscopy for diagnosis of pulmonary and HIV-associated tuberculosis in a hospital setting in Indonesia

Contains fulltext : 118612.pdf (publisher's version ) (Open Access)BACKGROUND: Fluorescence microscopy (FM) has not been implemented widely in TB endemic settings and little evaluation has been done in HIV-infected patients. We evaluated diagnostic performance, time and costs of FM with light-emitting diodes technology (LED-FM), compared with conventional (Zieh-Neelsen) microscopy in a hospital in Indonesia which acts as referral centre for HIV-infected patients. METHOD: We included pulmonary tuberculosis suspects from the outpatient and HIV clinic. Direct and concentrated sputum smears were examined using LED-FM and ZN microscopy by two technicians who were blinded for the HIV-status and the result of the comparative test. Mean reading time per slide was recorded and cost of each slide was calculated. Mycobacteria culture served as the reference standard. RESULTS: Among 404 tuberculosis suspects from the outpatient clinic and 256 from the HIV clinic, mycobacteria culture was positive in 12.6% and 27%, respectively. The optimal sensitivity of LED-FM was achieved by using a threshold of >/=2 AFB/length. LED-FM had a higher sensitivity (75.5% vs. 54.9%, P<0.01) but lower specificity (90.0% vs 96.6%, P<0.01) compared to ZN microscopy. HIV was associated with a lower sensitivity but similar specificity. The average reading time using LED-FM was significantly shorter (2.23+/-0.78 vs 5.82+/-1.60 minutes, P<0.01), while costs per slide were similar. CONCLUSION: High sensitivity of LED-FM combined with shorter reading time of sputum smear slides make this method a potential alternative to ZN microscopy. Additional data on specificity are needed for effective implementation of this technique in high burden TB laboratories

Cerebral tryptophan metabolism and outcome of tuberculous meningitis : An observational cohort study

Background: Immunopathology contributes to the high mortality of tuberculous meningitis, but the biological pathways involved are mostly unknown. We aimed to compare cerebrospinal fluid (CSF) and serum metabolomes of patients with tuberculous meningitis with that of controls without tuberculous meningitis, and assess the link between metabolite concentrations and mortality. Methods: In this observational cohort study at the Hasan Sadikin Hospital (Bandung, Indonesia) we measured 425 metabolites using liquid chromatography-mass spectrometry in CSF and serum from 33 HIV-negative Indonesian patients with confirmed or probable tuberculous meningitis and 22 control participants with complete clinical data between March 12, 2009, and Oct 27, 2013. Associations of metabolite concentrations with survival were validated in a second cohort of 101 patients from the same centre. Genome-wide single nucleotide polymorphism typing was used to identify tryptophan quantitative trait loci, which were used for survival analysis in a third cohort of 285 patients. Findings: Concentrations of 250 (70%) of 351 metabolites detected in CSF were higher in patients with tuberculous meningitis than in controls, especially in those who died during follow-up. Only five (1%) of the 390 metobolites detected in serum differed between patients with tuberculous meningitis and controls. CSF tryptophan concentrations showed a pattern different from most other CSF metabolites; concentrations were lower in patients who survived compared with patients who died (9-times) and to controls (31-times). The association of low CSF tryptophan with patient survival was confirmed in the validation cohort (hazard ratio 0·73; 95% CI 0·64-0·83; p<0·0001; per each halving). 11 genetic loci predictive for CSF tryptophan concentrations in tuberculous meningitis were identified (p<0·00001). These quantitative trait loci predicted survival in a third cohort of 285 HIV-negative patients in a prognostic index including age and sex, also after correction for possible confounders (p=0·0083). Interpretation: Cerebral tryptophan metabolism, which is known to affect Mycobacterium tuberculosis growth and CNS inflammation, is important for the outcome of tuberculous meningitis. CSF tryptophan concentrations in tuberculous meningitis are under strong genetic influence, probably contributing to the variable outcomes of tuberculous meningitis. Interventions targeting tryptophan metabolism could improve outcomes of tuberculous meningitis. Funding: Royal Dutch Academy of Arts and Sciences; Netherlands Foundation for Scientific Research; Radboud University; National Academy of Sciences; Ministry of Research, Technology, and Higher Education, Indonesia; European Research Council; and PEER-Health