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ABSTRACTClonal complexity is increasingly accepted in Mycobacterium tuberculosisinfection, including mixed infections by =2 strains, which usually occur in settings with a high burden of tuberculosis and/or a high risk of overexposure to infected patients. Mixed infections can hamper diagnostic procedures; obtaining an accurate antibiogram is difficult when the susceptibility patterns of the strains differ. Here, we show how mixed infections can also prove challenging for other diagnostic procedures, even outside settings where mixed infections are traditionally expected. We show how an unnoticed mixed infection in an HIV-positive patient diagnosed in Madrid, Spain, with differences in the representativeness of the coinfecting strains in different sputum samples, markedly complicated the resolution of a laboratory cross-contamination false positivity alert.

ABSTRACTCurrent migratory movements require new strategies for rapidly tracking the transmission of high-risk imported Mycobacterium tuberculosisstrains. Whole-genome sequencing (WGS) enables us to identify single-nucleotide polymorphisms (SNPs) and therefore design PCRs to track specific relevant strains. However, fast implementation of these strategies in the hospital setting is difficult because professionals working in diagnostics, molecular epidemiology, and genomics are generally at separate institutions. In this study, we describe the urgent implementation of a system that integrates genomics and molecular tools in a genuine high-risk epidemiological alert involving 2 independent importations of extensively drug resistant (XDR) and pre-XDR Beijing M. tuberculosisstrains from Russia into Spain. Both cases involved commercial sex workers with long-standing tuberculosis (TB). The system was based on strain-specific PCRs tailored from WGS data that were transferred to the local node that was managing the epidemiological alert. The optimized tests were available for prospective implementation in the local node 33 working days after receiving the primary cultures of the XDR strains and were applied to all 42 new incident cases. An interpretable result was obtained in each case (directly from sputum for 27 stain-positive cases) and corresponded to the amplification profiles for strains other than the targeted pre-XDR and XDR strains, which made it possible to prospectively rule out transmission of these high-risk strains at diagnosis.

ABSTRACTThe strains involved in tuberculosis outbreaks are considered highly virulent and transmissible. We analyzed the case of a patient in Madrid, Spain, who was persistently infected over an 8-year period by the same Beijing Mycobacterium tuberculosisstrain. The strain was responsible for a severe outbreak on Gran Canaria Island. The case provides us with a unique opportunity to challenge our assumptions about M. tuberculosisBeijing strains. No clinical/radiological findings consistent with a virulent strain were documented, and the in vitrogrowth rate of the strain in macrophages was only moderate. No secondary cases stemming from this prolonged active case were detected in the host population. The strain did not acquire resistance mutations, despite constant treatment interruptions, and it remained extremely stable, as demonstrated by the lack of single-nucleotide-polymorphism (SNP)-based differences between the sequential isolates. Our data suggest that the general assumption about M. tuberculosisBeijing strains having advantageous properties (in terms of virulence, transmissibility, and the tendency to acquire mutations and resistance) is not always accurate.

ABSTRACTA persistent 8-year infection by a Beijing Mycobacterium tuberculosisstrain from a previous outbreak after importation from West Africa obliged us to investigate secondary cases. We developed a multiplex PCR method based on whole-genome sequencing to target strain-specific single nucleotide polymorphisms (SNPs). In 1 week, we analyzed 868 isolates stored over 6 years. Only 2 cases (immigrants from Guinea Conakry) harbored the strain, which ruled out transmission—despite opportunities—and challenged some of the advantages associated with Beijing strains.