McAulay K, Saito K, Warrier T, Walsh KF, Mathurin LD, Royal-Mardi G, Lee MH, Ocheretina O, Pape JW, Fitzgerald DW, Nathan CF. Differentially Detectable Mycobacterium tuberculosis Cells in Sputum from Treatment-Naive Subjects in Haiti and Their Proportionate Increase after Initiation of Treatment. MBio. 2018 Nov 20;9(9). PMID: 30459198 [PubMed] PMCID: PMC6247085
Recent reports indicate that the sputum of 80% or more of treatment-naive subjects with tuberculosis recruited in England or South Africa contained more viable cells detected by limiting dilution (LD) in liquid culture than detected as CFU. Efforts to generate such differentially detectable (DD) populations have been difficult to reproduce, and the LD assay is prone to artifact. Here, we applied a stringent version of the LD assay to sputum from 33 treatment-naive, HIV-negative Haitian subjects with drug-sensitive tuberculosis (TB) and to a second sputum sample after two weeks of standard treatment with isoniazid, rifampin, pyrazinamide, and ethambutol (HRZE) for 13 of these subjects. Twenty-one percent had statistically defined levels of DD in their pretreatment sputum at an average proportional excess over CFU of 3-fold. Sixty-nine percent of those who received HRZE had statistically defined levels of DD in their sputum, and of these, the mean proportionate excess over CFU was 7.9-fold. Thus, DD is detectable in pretreatment sputum from a significant proportion of subjects in the Western Hemisphere, and certain drugs or drug regimens, while reducing CFU, may at the same time increase the proportional representation of DD among the surviving bacilli. Monitoring DD may improve our ability to predict the efficacy of efforts to shorten treatment. Measurement of the reduction in CFU in sputum of patients with TB up to 2 weeks after the initiation of treatment is the gateway test for a new TB treatment. Reports have suggested that CFU assays fail to detect the majority of viable cells in sputum samples from the majority of patients when the number of is estimated by limiting dilution (LD). In an effort to avoid potential methodologic confounders, we applied a modified version of the LD assay in a study of a geographically distinct population. We confirmed that differentially detectable (DD) is often found before treatment, albeit at lower proportionate levels than in earlier reports. Strikingly, the prevalence and proportionate representation of DD increased during standard treatment. Sublethal exposure to certain antibiotics may help generate DD cells or enrich their representation among the surviving bacteria, and this may contribute to the need for prolonged treatment with those agents in order to achieve durable cures.