| Abstrakt: |
Background: Mycobacterium tuberculosis (M.tb) is a biosafety level 3 pathogen that kills over 1 million people annually. Detection of M.tb by culture or molecular tests requires level III laboratory infrastructure which is expensive to establish in resource poor countries Objective: To assess the effectiveness of heat and Guanidine thiocyanate (GTC) for inactivation of M.tb prior to molecular tests application in healthcare laboratories. Method: We used M.tb, H37Rv reference strain and pooled pulmonary TB (PTB) sputum. A 0.5 McFarland standard, approximate to 1.50x107 CFU/mL of M.tb, H37Rv was serially diluted to 10 CFU/mL in Middlebrook 7H9. For sputum, five sputum samples were pooled and homogenised for 30 minutes. GeneXpert MTB/RIF Ultra was performed to check the initial bacterial load and serially diluted the sputum to obtain six 10-fold dilutions. For each dilution of M.tb, H37Rv or sputum, three replicates of 1 mL were boiled at 80°C for 20 minutes, mixed with 4 mL of GTC for 15 minutes and 2 mL was decontaminated by NALC-NaOH for MGIT liquid culture (controls) following BD MGIT protocol. RNA was extracted following the TB-MBLA protocol. Results: No M.tb growth observed in MGIT culture for GTC, and heat treated H37Rv cultures and sputum (p<0.001) compared to controls. All untreated H37Rv cultures were MGIT positive except the last diluted culture. For sputum, all dilutions were MGIT positive except the last two dilutions. The average bacterial load (BL±SD) of untreated H37Rv neat culture was 6.36±0.33log10 compared to 4.22±0.1log10 for heat (p=0.36) and 5.57±0.03log10 for GTC treated (p=0.71), respectively. In contrast to H37Rv cultures, GTC treated sputum had average BL of 5.35±3.14log10 compared to 3.48±3.14log10 for heat-treated sputum (p=0.57). Conclusion: Heat or GTC inactivate M.tb preserving adequate RNA for molecular tests. These methods can be applied for inactivation of TB samples prior to molecular tests in healthcare settings without level III laboratory infrastructure. [ABSTRACT FROM AUTHOR] |
