A Tuberculosis Molecular Bacterial Load Assay (TB-MBLA)

Abstract

Tuberculosis is caused by Mycobacterium tuberculosis (Mtb), a pathogen classified by the United Nations (UN) as a dangerous category B biological substance. For the sake of the workers' safety, handling of all samples presumed to carry Mtb must be conducted in a containment level (CL) 3 laboratory. The TB molecular bacterial load assay (TB-MBLA) test is a reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) test that quantifies Mtb bacillary load using primers and dual-labelled probes for 16S rRNA. We describe the use of heat inactivation to render TB samples noninfectious while preserving RNA for the TB-MBLA. A 1 mL aliquot of the sputum sample in tightly closed 15 mL centrifuge tubes is boiled for 20 min at either 80 °C, 85 °C, or 95 °C to inactivate Mtb bacilli. Cultivation of the heat inactivated and control (live) samples for 42 days confirmed the death of TB. The inactivated sample is then spiked with 100 µL of the extraction control and RNA is extracted following the standard RNA isolation procedure. No growth was observed in the cultures of heat treated samples. The isolated RNA is subjected to real-time RT-qPCR, which amplifies a specific target in the Mtb 16S rRNA gene, yielding results in the form of quantification cycles (Cq). A standard curve is used to translate Cq into bacterial load, or estimated colony forming units per mL (eCFU/mL). There is an inverse relationship between Cq and the bacterial load of a sample. The limitation is that heat inactivation lyses some cells, exposing the RNA to RNases that cause a loss of <1 log 10 eCFU/mL (i.e., <10 CFU/mL). Further studies will determine the proportion of very low burden patients that cause false negative results due to heat inactivation.

Molecular bacterial load assay versus culture for monitoring treatment response in adults with tuberculosis

“…TB-MBLA process in a protocol has a three-step consisting of (1) extraction of total RNA, (2) enzymatic genomic DNA removal, and (3) RT-qPCR where cycle threshold is transformed to bacterial load. 24 , 28 When mycobacterial cells are killed by anti-TB drugs, there is a decrease in rRNA amount and thus easily estimates the number of viable cells in a patient’s sputum sample. A decline in rRNA has been defined as a surrogate biomarker of microbial viability and bactericidal activity for anti-TB regimen, due to a cellular abundance of 16S rRNA and half-life being shorter than that of DNA.…”

“…measuring and quantifying bacterial load in the sputum of a patient. TB-MBLA process in a protocol has a three-step consisting of (1) extraction of total RNA, (2) enzymatic genomic DNA removal, and (3) RT-qPCR where cycle threshold is transformed to bacterial load 24,28. When mycobacterial cells are killed by anti-TB drugs, there is a decrease in rRNA amount and thus easily estimates the number of viable cells in a patient's sputum sample.…”

Bacterial Load Comparison of the Three Main Lineages of Mycobacterium tuberculosis Complex in West Africa

“…The TB molecular bacterial load assay (TB-MBLA) is a reverse transcriptase real-time quantitative PCR (RT-qPCR) method that detects and quantifies viable MTBC in sputum by determining the amount of MTBC 16S ribosomal RNA (16S rRNA). It is a twostep process consisting of a) extraction of total RNA and b) RT-qPCR (Sabiiti et al, 2017(Sabiiti et al, , 2020b.…”

Perspective for Precision Medicine for Tuberculosis

“…Mycobacterial culture is the most relevant measure of treatment response, but mycobacterial growth is slow and, as treatment progresses, the time to a positive result increases (90). Here, rapid molecular tests detecting only viable bacteria such as the molecular bacterial load assay (MBLA) may be promising for the future treatment monitoring of tuberculosis patients (91).…”