Acetic Acid Enables Molecular Enumeration of Mycobacterium tuberculosis from Sputum and Eliminates the Need for a Biosafety Level 3 Laboratory.

BACKGROUND: Improved monitoring of Mycobacterium tuberculosis response to treatment is urgently required. We previously developed the molecular bacterial load assay (MBLA), but it is challenging to integrate into the clinical diagnostic laboratory due to a labor-intensive protocol required at biosafety level 3 (BSL-3). A modified assay was needed. METHODS: The rapid enumeration and diagnostic for tuberculosis (READ-TB) assay was developed. Acetic acid was tested and compared to 4 M guanidine thiocyanate to be simultaneously bactericidal and preserve mycobacterial RNA. The extraction was based on silica column technology and incorporated low-cost reagents: 3 M sodium acetate and ethanol for the RNA extraction to replace phenol-chloroform. READ-TB was fully validated and compared directly to the MBLA using sputa collected from individuals with tuberculosis. RESULTS: Acetic acid was bactericidal to M. tuberculosis with no significant loss in 16S rRNA or an unprotected mRNA fragment when sputum was stored in acetic acid at 25°C for 2 weeks or -20°C for 1 year. This novel use of acetic acid allows processing of sputum for READ-TB at biosafety level 2 (BSL-2) on sample receipt. READ-TB is semiautomated and rapid. READ-TB correlated with the MBLA when 85 human sputum samples were directly compared (R2 = 0.74). CONCLUSIONS: READ-TB is an improved version of the MBLA and is available to be adopted by clinical microbiology laboratories as a tool for tuberculosis treatment monitoring. READ-TB will have a particular impact in low- and middle-income countries (LMICs) for laboratories with no BSL-3 laboratory and for clinical trials testing new combinations of anti-tuberculosis drugs.

Multiple drug-resistant Mycobacterium tuberculosis: evidence for changing fitness following passage through human hosts.

Recent studies have shown a difference in the genotype of resistant bacteria following passage in animals compared to those passaged in vitro. This suggests that organisms rapidly adapt to their environment of growth. We sought to investigate whether this phenomenon occurred in human infection and whether changes could be detected in the fitness (growth velocity) of isolates transmitted between human hosts. Isogenic strains of Mycobacterium tuberculosis were obtained from a well-documented hospital outbreak. The subjects included those who were HIV seropositive and immunocompromised. The relative fitness of each sample was measured using growth competition in vitro. The results confirmed that our method of measuring fitness was not influenced by the storage conditions of the isolates, and demonstrated that the fitness of genetically similar isolates obtained from different patients in the outbreak differed significantly, as reflected in the growth velocity of the strains. This study provides the first evidence that multiple drug resistant M. tuberculosis strains adapt to the environment of their human host.