Magnitude and sources of bias in the detection of mixed strain M. tuberculosis infection

Abstract

High resolution tests for genetic variation reveal that individuals may simultaneously host more than one distinct strain of Mycobacterium tuberculosis. Previous studies find that this phenomenon, which we will refer to as "mixed infection", may affect the outcomes of treatment for infected individuals and may influence the impact of population-level interventions against tuberculosis. In areas where the incidence of TB is high, mixed infections have been found in nearly 20% of patients; these studies may underestimate the actual prevalence of mixed infection given that tests may not be sufficiently sensitive for detecting minority strains. Specific reasons for failing to detect mixed infections would include low initial numbers of minority strain cells in sputum, stochastic growth in culture and the physical division of initial samples into parts (typically only one of which is genotyped). In this paper, we develop a mathematical framework that models the study designs aimed to detect mixed infections. Using both a deterministic and a stochastic approach, we obtain posterior estimates of the prevalence of mixed infection. We find that the posterior estimate of the prevalence of mixed infection may be substantially higher than the fraction of cases in which it is detected. We characterize this bias in terms of the sensitivity of the genotyping method and the relative growth rates and initial population sizes of the different strains collected in sputum.

Bacterial Genotyping of Central Nervous System Tuberculosis in South Africa: Heterogenic Mycobacterium tuberculosis Infection and Predominance of Lineage 4

“…Genetic heterogeneity in lung tissue, in which multiple lineages can be found in a single individual at the same time, is in fact not new. Numerous reports, including a South African cohort in the same region as the current study, report 10 to 20% of sputum cultures containing more than one M. tuberculosis lineage (15)(16)(17)(18)20). However, this is likely an underestimation of the actual amount of genetic heterogenic infections due to the difficulty of isolating these strains and differences in genotyping methods (17,18,27).…”

“…The presence of genomic heterogeneity so far has not been described in cerebrospinal fluid (CSF) of patients with TBM. Since detection is restricted to our limited ability to culture bacteria from CSF and is dependent on the sensitivity of the genotyping method used (17)(18)(19), the actual number of patients with genomic heterogeneity in the CNS is most likely underestimated.…”

M. tuberculosis microvariation is common and is associated with transmission: analysis of three years prospective universal sequencing in England

“…Our study has a number of limitations. Firstly, we used culture to amplify M. tuberculosis isolates, which may reduce diversity, 25 and so may underestimate the importance of microvariation in TB infection. The introduction of culture independent sequencing methods 26 may address this.…”

Identifying mixed Mycobacterium tuberculosis infection and laboratory cross-contamination during Mycobacterial sequencing programs

“…A second class of mixture, which we found was rarely detected in this setting, is compatible with co-infection with two organisms of differing lineage within the patient. This kind of mixture is clinically relevant (7, 11), and may be under-detected using the laboratory process we describe here, since culture based amplification can reduce diversity in the sample inoculated (10). Its frequency may rise if direct-from-sample sequencing is employed, or if samples from high-endemicity areas are studied, but here we identified only one probable case of such mixtures, and five other possible cases.…”

“…Reported rates of mixed infection vary markedly as reviewed (3, 5), with between 10 and 30% reported in areas of current (5–7) or historical (8, 9) high prevalence. Much lower rates are reported in low incidence countries (3), although systematic under-detection is likely to occur due to both to the limited representation of bacteria in single sputum samples of pulmonary disease, and to the decrease in diversity occurring during differential strain growth in broth culture (10).…”