Molecular Detection of Mixed Infections of Mycobacterium tuberculosis Strains in Sputum Samples from Patients in Karonga District, Malawi

Abstract

The occurrence of mixed infections of Mycobacterium tuberculosis is no longer disputed. However, their frequency, and the impact they may have on our understanding of tuberculosis (TB) pathogenesis and epidemiology, remains undetermined. Most previous studies of frequency applied genotyping techniques to cultured M. tuberculosis isolates and found mixed infections to be rare. PCR-based techniques may be more sensitive for detecting multiple M. tuberculosis strains and can be applied to sputum. To date, one study in South Africa has used a PCR approach and suggested that mixed infection could be common. We investigated mixed infections in northern Malawi using two lineage-specific PCR assays targeting the Latin AmericanMediterranean (LAM) and Beijing lineages. Compared with spoligotyping, the specificity and sensitivity of both assays was 100%. From 160 culture-positive sputa, mixed LAM and non-LAM strains were detected in 4 sputa belonging to 2 (2.8%) patients. Both patients were HIV positive, with no history of TB. Cultured isolates from both patients showed only LAM by PCR and spoligotyping. In a set of 377 cultured isolates, 4 were mixed LAM and non-LAM. Only one showed evidence of more than one M. tuberculosis strain using IS6110-based restriction fragment length polymorphism (IS6110-RFLP) and spoligotyping analyses. Corresponding sputa for the 4 isolates were unavailable. Mixed Beijing and non-Beijing strains were not detected in this study. Mixed infections appear to be rare in our setting and are unlikely to affect findings based on DNA fingerprinting data. Molecular methods, which avoid the selective nature of culture and target distinct strains, are well suited to detection of mixed infections.It used to be thought that tuberculosis in the human host was caused by infection with a single Mycobacterium tuberculosis strain (25). This belief was first challenged when phage typing identified the presence of more than one M. tuberculosis strain in isolates obtained from a single patient (2,17). With the development of DNA fingerprinting (28) and other PCR-based genotyping methods (15, 27) to differentiate M. tuberculosis strains, the presence of multiple strains within a single patient has been demonstrated during routine genotyping studies (5,19,20,24,33). The results from these and further studies designed to specifically examine mixed infections (1,10,20,22,23,29,32) have shown that they occur in different geographical settings among both HIV-negative and HIV-positive tuberculosis (TB) patients.While the occurrence of mixed infections of M. tuberculosis is no longer questioned, the rate at which they occur is unknown. Yet this is of fundamental importance, as much of our understanding of tuberculosis pathogenesis and epidemiology has assumed that mixed infection is rare. The frequency is likely to differ according to the level of M. tuberculosis transmission in different settings. If mixed infections are common, it would necessitate reinterpretation of studies on recent infection, the importance of reinfe...

Systematic Survey of Clonal Complexity in Tuberculosis at a Populational Level and Detailed Characterization of the Isolates Involved

“…Our data demonstrate that clonal complexity in the infection by M. tuberculosis is not anecdotal, especially in cases with simultaneous infection at independent sites. The real figures for clonal complexity in this study could be even higher if we had included more than a single sputum specimen in our screening design (13,18,19,28).…”

“…The number of studies on clonal complexity in M. tuberculosis infection has increased in recent years (8,10,18). However, some of these studies examine anecdotal cases (2) and others analyze this phenomenon only in specific M. tuberculosis lineages (35) or specific phenotypes (34).…”

Standard Genotyping Overestimates Transmission of Mycobacterium tuberculosis among Immigrants in a Low-Incidence Country

“…Infections with multiple strains could potentially also influence the identification of molecular clusters, as individual strains in an infection with multiple strains cannot be resolved by MIRU-VNTR typing. The prevalence and relevance of such multiple infections need to be studied further (40)(41)(42). A potential limitation of our study is the definition of transmission clusters by WGS.…”

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

“…Recently, 12% of mixed M. tuberculosis infections were identified by whole-genome sequencing (28); however, this method is limited by the cost, complexity of data analysis, and the short sequencing reads used to account for repeat regions (5). Mixed Beijing and non-Beijing strains caused the most pathogen-pathogen compatibility (10,14,29,30); however, the mixed non-Beijing strains were also reported to be primary in some regions (12,31). Besides, although Beijing strains were commonly present in mixed infections, their spoligotypes usually were masked by the spoligotypes of non-Beijing strains (14).…”