Mixed-Strain Mycobacterium tuberculosis Infections among Patients Dying in a Hospital in KwaZulu-Natal, South Africa

Abstract

We performed spoligotyping and 24-locus mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing on M. tuberculosis culture-positive biopsy specimens collected from adults dying in a hospital in KwaZulu-Natal. Of 56 culture-positive samples genotyped, we detected mixed strains in five (9%) and clonal heterogeneity in an additional four (7%).The application of molecular approaches for detecting variation among Mycobacterium tuberculosis isolates has generated new appreciation for the diversity present within this relatively genetically conserved bacterial species (4, 17). Genotyping methods, such as insertion sequence typing (IS6110), spacer oligonucleotide typing (spoligotyping), and mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing, have been used to identify transmission chains (1,10,18,26), to classify strains into families and lineages (2,5,9,18,27,29), to identify episodes of exogenous reinfection (3,20,25,31), and, most recently, to detect the presence of within-host genetic heterogeneity (11,19,24,33).Genetic heterogeneity of M. tuberculosis within a host may arise by one of two mechanisms: (i) within-host diversification following a single infection event or (ii) reinfection resulting in a mixed infection with more than one strain (12, 16). The clinical consequences of within-host diversity are most obvious when manifesting as subpopulations of bacteria with resistance to tuberculosis (TB) antibiotics, either reflecting acquired drug resistance (mechanism 1) or transmitted drug resistance (mechanism 2). Furthermore, because individuals can be simultaneously infected by strains with different phenotypic characteristics (e.g., growth rates, drug resistance), the withinhost competition between strains may influence the clinical outcomes for coinfected patients (30) and may affect the population dynamics of the pathogen in the community (6,8).While within-host M. tuberculosis genetic diversity has been documented in many settings, systematic efforts to measure the prevalence of these complex infections have rarely been attempted. In this study, we report the results of a genotyping analysis on isolates collected from young adults dying in a hospital in KwaZulu-Natal, South Africa.We conducted limited autopsies on adults aged 20 to 45 years who died after admission to Edendale Hospital in KwaZulu-Natal, South Africa, between October 2008 and August 2009. The incidence of tuberculosis in KwaZulu-Natal is 1,094 cases/100,000 persons per year, and the HIV prevalence among women in antenatal clinical settings is 39% (14, 21). Our primary aim was to investigate the burden of tuberculosis as a contributing cause of death in this highly vulnerable community. Of the 240 decedents enrolled in the study, 94% were HIV seropositive. Fifty-eight percent of those on treatment for tuberculosis at the time of death were still infected with viable M. tuberculosis, while 42% of those not receiving treatment for TB also had positive M. tuberculosis cul...

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

“…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).…”

“…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). Those which follow population-based designs to determine the proportion of clonally complex TB cases are performed mainly in epidemiological contexts with a markedly high incidence of TB (8,10,29,30,35) and/or where the possibility of overexposure is more likely (28).…”

Mixed Infections and Rifampin Heteroresistance among Mycobacterium tuberculosis Clinical Isolates

“…Currently, mycobacterial interspersed repetitive unit-variable-number tandem repeat (MIRU-VNTR)-based methods have been the most widely used to detect mixed infections (5). Mixed infections are defined by the presence of strains with different MIRU-VNTR patterns at two or more loci in the same sputum, lymph, or other sample while having clonal heterogeneity with a different MIRU-VNTR pattern at a single locus (7)(8)(9).…”

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“…In addition, it represents an open ended one size fits all approach that could allow the reunification of TB microbiology with other sputum microbiology, particularly as metagenomics has already been shown to work on other respiratory tract pathogens, including bacteria and viruses (Lysholm et al, 2012;Fischer et al, 2014). It also aids in the detection of mixed infections (Chan et al, 242 243 244 245 246 247 248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268 269 270 271 272 273 274 275 276 277 278 279 280 281 282 283 2013; Koser et al, 2013), which are clinically important, but hard to recognise (Shamputa et al, 2004;Warren et al, 2004;Cohen et al, 2011;Wang et al, 2011;Hingley Wilson et al, 2013).…”

“…Furthermore, as isolation of mycobacteria in pure culture and sensitivity testing remain onerous, in resource poor settings these steps are omitted and, even in well resourced laboratories, typically only one or a few single colony subcultures are followed up from each sample. This leads to under recognition of mixed infections, where more than one strain from the M. tuberculosis complex is present or where TB co occurs with infection by other mycobacteria (Shamputa et al, 2004;Warren et al, 2004;Cohen et al, 2011;Wang et al, 2011). This can lead to difficulties in treatment when strains or species susceptible to conventional anti tuberculous treatment co exist with resistant strains or species within the same patient (Hingley .…”