Comparison of MGIT 960 with Lowenstein Jensen Media for Recovery of Mycobacteria from Extrapulmonary Specimens in Southern India

Introduction: Prevalence of Extrapulmonary Tuberculosis (EPTB) due to Mycobacteriumtuberculosis and Non-Tuberculous Mycobacteria (NTM) are on the rise especially in a developing country like India. Smear Microscopy (SM) is commonly used for detection of mycobacteria. Due to the paucibacillary nature in the extrapulmonary specimens SM pose a problem in detection. Though molecular methods are increasingly used now-a-days but there are possibilities that these reactions may get inhibited due to the presence of inhibitors in the extrapulmonary specimens. Aim: To compare Mycobacterium Growth Indicator Tubes (MGIT 960) with Lowenstein Jensen (LJ) medium for the detection of mycobacteria. Materials and Methods: The current prospective study was conducted on 1879 extrapulmonary specimens collected from a tertiary care hospital during the study period from July 2018 to March 2020. Specimens were subjected to Ziehl Neelsen (ZN) staining and Auramine Phenol (AP) staining. Culture was done in both LJ media and MGIT 960 culture. Positive mycobacterial cultures were subjected to MPT64 Immunochromatographic Test (ICT). Data were analysed using the Statistical Package for Social Sciences (SPSS®) for Windows® release 21.0 (SPSS Inc., Chicago, IL, USA). Results: A total of 129 (6.9%) and 105 (5.6%) mycobacteria was isolated by MGIT 960 and by LJ culture respectively among 1879 extrapulmonary specimens. MGIT 960 identified 118 (91.5%) as Mycobacterium tuberculosis complex and 11 (8.5%) as NTM among the total mycobacteria isolated. Out of 105 mycobacteria grown by LJ culture, 95 (90.5%) and 10 (9.5%) were identified as Mycobacterium tuberculosis and NTM, respectively. The rate of contamination associated with MGIT 960 and LJ culture was 4.6% and 4.3% respectively. The Time to Detection (TTD) was found to be significantly shorter for isolation of Mycobacterium tuberculosis by MGIT 960 culture compared to LJ culture. Conclusion: In the current study, authors compared MGIT 960 with solid LJ culture for recovery of both Mycobacterium tuberculosisComplex and NTM from extrapulmonary specimens and authors found increased recovery by MGIT 960 compared to LJ culture and also shorter duration of detection for Mycobacterium tuberculosis by MGIT 960 with comparable contamination rates.

In Vitro Activity of Methylene Blue on Mycobacterium Tuberculosis Complex Isolates

Background: Methylene blue (MB) is used for bacterial staining, and as an antidote drug. We aimed to investigate the antimicrobial effects of MB against Mycobacterium tuberculosis complex clinical isolates. Methods: Seventeen stored Mycobacterium tuberculosis complex isolates were inoculated into Mycobacteria Growth Indicator Tubes (MGIT) and incubated in Automated Mycobacterial Detection System (AMDS). MGIT tubes containing MB blue at concentrations of 0.2, 2, 20, 1000 µg ml-1 and control were prepared. Antibiograms were performed using AMDS. Results: Six isolates were susceptible to MB at all concentrations and five were susceptible to only 1000 µg ml-1 MB. Three isolates were susceptible to 1000 and 20 µg ml-1 MB. Susceptibility rate was found 94% when the critical concentration was accepted 400 GU (1/100 of control). Conclusions: MB may become an alternative anti-tuberculosis agent especially in the topical form of this drug due to their well-known side effects and dosing regimens.

The local technical validation of new plasma tube with a mechanical separator

BackgroundUnsuitable samples are common problem for laboratories. The blood collection tubes need to be validated or verified prior to their being used in the routine laboratory for reducing this situation.ObjectiveWe aim to compare the technical qualifications of routinely used BD Vacutainer® Serum Separator Tubes™ II Advance Plus with BD Vacutainer® Barricor™ LH Plasma Tubes for local technical validation.Materials and methodsApparently healthy 150 voluntary subjects were enrolled in the study. Samples were collected in two separated tubes by a single phlebotomist. Twelve quality indicators were used to compare these two different types of tubes for local technical validation. Differences (%) between them were calculated with the formula proposed by EFLM. In case of any difference of less than 1% for each indicator, the evaluated tube was considered as non-inferior.ResultsIndicators, such as tubes with physical defects, that fail to create vacuum, not properly fitting into the blood collection device, under filling (10%), cracked tubes, tubes exterior surface contaminated with blood, hemolysed specimens, including fibrin strand/mass in the sample, red blood cell adhesion, poor/incomplete barrier formation were found non-inferior in Barricor™ tubes. White particulate matter (WPM) was observed in 24.6% of Barricor™. Therefore, the last indicator (tubes including gel/foreign material/WPM in sample after centrifugation) was found inferior for Barricor™.ConclusionTechnical local validation studies should be encouraged in terms of quality management. It was thought that WPM would not cause any interference in a properly filled tube. In addition to, Barricor™ was also found to be technically acceptable when evaluated through using all other indicators.

Validating a 14-Drug Microtiter Plate Containing Bedaquiline and Delamanid for Large-Scale Research Susceptibility Testing ofMycobacterium tuberculosis

ABSTRACTThe UKMYC5 plate is a 96-well microtiter plate designed by the CRyPTIC Consortium (Comprehensive Resistance Prediction for Tuberculosis: an International Consortium) to enable the measurement of MICs of 14 different antituberculosis (anti-TB) compounds for >30,000 clinicalMycobacterium tuberculosisisolates. Unlike the MYCOTB plate, on which the UKMYC5 plate is based, the UKMYC5 plate includes two new (bedaquiline and delamanid) and two repurposed (clofazimine and linezolid) compounds. UKMYC5 plates were tested by seven laboratories on four continents by use of a panel of 19 external quality assessment (EQA) strains, including H37Rv. To assess the optimal combination of reading method and incubation time, MICs were measured from each plate by two readers, using three methods (mirrored box, microscope, and Vizion digital viewing system), after 7, 10, 14, and 21 days of incubation. In addition, all EQA strains were subjected to whole-genome sequencing and phenotypically characterized by the 7H10/7H11 agar proportion method (APM) and by use of MGIT960 mycobacterial growth indicator tubes. We concluded that the UKMYC5 plate is optimally read using the Vizion system after 14 days of incubation, achieving an interreader agreement of 97.9% and intra- and interlaboratory reproducibility rates of 95.6% and 93.1%, respectively. The mirrored box had a similar reproducibility. Strains classified as resistant by APM, MGIT960, or the presence of mutations known to confer resistance consistently showed elevated MICs compared to those for strains classified as susceptible. Finally, the UKMYC5 plate records intermediate MICs for one strain for which the APM measured MICs close to the applied critical concentration, providing early evidence that the UKMYC5 plate can quantitatively measure the magnitude of resistance to anti-TB compounds that is due to specific genetic variation.

Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

ABSTRACTIntroductionDetecting laboratory cross-contamination and mixed tuberculosis infection are important goals of clinical Mycobacteriology laboratories.ObjectivesTo develop a method detecting mixtures of different M. tuberculosis lineages in laboratories performing Mycobacterial next generation sequencing (NGS).SettingPublic Health England National Mycobacteriology Laboratory Birmingham, which performs Illumina sequencing on DNA extracted from positive Mycobacterial Growth Indicator tubes.MethodsWe analysed 4,156 samples yielding M. tuberculosis from 663 MiSeq runs, obtained during development and production use of a diagnostic process using NGS. Counts of the most common (major) variant, and all other variants (non-major variants) were determined from reads mapping to positions defining M. tuberculosis lineages. Expected variation was estimated during process development.ResultsFor each sample we determined the non-major variant proportions at 55 sets of lineage defining positions. The non-major variant proportion in the two most mixed lineage defining sets (F2 metric) was compared with that in the 47 least mixed lineage defining sets (F47 metric). Three patterns were observed: (i) not mixed by either metric, (ii) high F47 metric suggesting mixtures of multiple lineages, and (iii) samples compatible with mixtures of two lineages, detected by differential F2 metric elevation relative to F47. Pattern (ii) was observed in batches, with similar patterns in the H37Rv control present in each run, and is likely to reflect cross-contamination. During production, the proportions of samples in each pattern were 97%, 2.8%, and 0.001%, respectively.ConclusionThe F2 and F47 metrics described could be used for laboratory process control in laboratories sequencing M. tuberculosis.