Undetected Multidrug-Resistant Tuberculosis Amplified by First-line Therapy in Mixed Infection

Abstract

Infections with >1 Mycobacterium tuberculosis strain(s) are underrecognized. We show, in vitro and in vivo, how first-line treatment conferred a competitive growth advantage to amplify a multidrug-resistant M. tuberculosis strain in a patient with mixed infection. Diagnostic techniques that identify mixed tubercle bacilli populations are needed to curb the spread of multidrug resistance.A s the number of multidrug-resistant tuberculosis (TB) cases continues to rise, so does the amplification of multidrug-resistant Mycobacterium tuberculosis strains during treatment (1,2). This amplification is generally assumed to result from in vivo evolution of drug resistance caused by poor therapy compliance or, in high-incidence settings, from exogenous reinfection with a multidrug-resistant strain. We report a case in which emergence of multidrug resistance did not result from in vivo acquisition of drug resistance by a drugsensitive strain or from exogenous reinfection with an already resistant strain. By integrating epidemiologic, microbiological, and molecular strain typing data with in vitro competitive growth experiments, we provide evidence for an initial mixed infection with a drug-sensitive strain and an undetected drug-resistant strain that outgrew the sensitive strain under the selection pressure of first-line chemotherapy.M. tuberculosis strains in sputum from TB-infected patients or in samples from the disease site are generally identified by strain typing a single broth culture or colony grown on solid medium. However, this method does not enable identification of mixed infections, and any treatment regimen would be determined on the basis of the drug sensitivity of the strain with the fastest growth rate in the in vitro culture. Use of suboptimal drug combinations could lead to selection of a slower growing, drug-resistant strain already present in the host and thus to treatment failure.Studies of artificially mixed M. tuberculosis strains before and after culture showed that culturing can reduce the clonal complexity of the strains and that, in most samples (6/10), only 1 strain will be identified in mixed infections after culture (3). This suggests that mixed infections and clonal complexity are underrepresented in culture-based diagnoses of TB. In support of this suggestion, the results of molecular-based methods that use strain-specific PCR showed that 2.1%-19.0% of patients with active TB in moderate to high incidence countries were simultaneously infected with >2 strains (1,2,4-10).Possible co-infection of patients with drug-sensitive and drug-resistant M. tuberculosis strains has been described (1,2), and modeling of the effect of such co-infection on the long-term dynamics of tuberculous infection has led to the hypothesis that persons with this type of infection may retain small populations of drug-resistant bacteria that can flourish after the host receives treatment (11). van Rie et al. showed the amplification of a drug-resistant strain after treatment and postulated selection of drug-resistant st...

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

“…Furthermore, the advent of high resolution tests for genetic variation has revealed that individuals may simultaneously harbor infections with more than one distinct strain of M. tuberculosis (Warren et al, 1999;Sola et al, 2003;Kremer et al, 1999;van Embden et al, 1993;Imaeda, 1985). This phenomenon, which we will refer to as "mixed infection", has been linked with poor treatment outcome when the co-infecting strains differ with respect to drug susceptibility (van Rie et al, 2005;Hingley-Wilson et al, 2013) and is predicted to influence the impact of population-level interventions against tuberculosis (Cohen et al, 2008;Rodrigues et al, 2007;Colijn et al, 2009;Sergeev et al, 2011;Mills et al, 2013).…”

Rifampin Heteroresistance in Mycobacterium tuberculosis Cultures as Detected by Phenotypic and Genotypic Drug Susceptibility Test Methods

“…The prevalence of heteroresistance is unknown and is presumably dependent on the local resistance epidemiology. Findings of heteroresistance are accidental, and simple methods for the detection are needed (6).…”

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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).…”

“…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 Wilson et al, 2013).As an alternative to culture and phenotypic sensitivity testing, the WHO has recently recommended a new, rapid, automated, real time amplification based TB diagnostic test, the Xpert MTB/RIF assay (WHO, 2011). This system allows simultaneous detection of M. tuberculosis and rifampicin resistance mutations in a closed system, suitable for use in a simple laboratory setting, while providing a result in less than two hours directly from sputum samples (Helb et al, 2010).…”