a-tuberculosis-molecular-bacterial-load-assay-tb-mbla

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological ...

Instituto Nacional de Saúde (INS), Ministério da Saúde

Tuberculosis (TB) represents a serious public health problem in Mozambique, with an estimated incidence rate of 548 cases per 100,000 population in 2011. Information on the molecular epidemiology of Mycobacterium tuberculosis (MTB) strains circulating in Mozambique is limited. This study provides the first description of the genetic diversity of MTB strains circulating in Beira city, the second largest town in Mozambique.

Genetic diversity of Mycobacterium tuberculosis isolates obtained from patients with pulmonary tuberculosis in Beira city, Mozambique.

The World Health Organization End Tuberculosis (TB) strategy has called for the development of-and increased access to-effective tools for diagnosis and treatment of TB disease.  , the causative agent of TB, is categorized as a highly infectious agent. Consequently, diagnostic tests that involve comprehensive manipulation of specimens from presumed tuberculosis cases must be performed in a category 3 laboratory. We have evaluated the use of heat inactivation to render TB samples safe to work with while preserving RNA for downstream molecular tests. Using  bacillus Calmette-Guérin (BCG) cultures and TB-positive sputum samples, we show that boiling for 20 min at 80, 85, and 95°C inactivates all  bacilli. The efficiency of inactivation was verified by culturing heat-treated and untreated (live) fractions of BCG and TB sputum samples for 42 days. No growth was observed in the cultures of heat-treated samples. In contrast, the optical density of untreated BCG in Middlebrook 7H9 broth rose from 0.04 to 0.85, and the untreated sputum samples flagged positive at 3 days of incubation in mycobacterial growth indicator tubes. Quantification of reference genes 16S rRNA, transfer-messenger RNA (tmRNA), pre-16S rRNA, and  by reverse transcriptase quantitative PCR (RT-qPCR) showed minimal loss in estimated bacterial load. The loss was RNA species dependent, <1 log, 1.1 log, 1.3 log, and 2.4 log estimated CFU/ml for 16S rRNA, tmRNA, pre-16S rRNA, and , respectively. The RNA loss was independent of inactivation temperature. These findings show that heat inactivation could obviate the need for category 3 laboratories to perform RNA-based testing of TB samples.

Khalide Azam

Instituto Nacional de Saúde (INS), Ministério da Saúde

A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)