A persistent 8-year infection by a Beijing Mycobacterium tuberculosis strain 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 opportunitiesand challenged some of the advantages associated with Beijing strains.
Extensive application of genotyping strategies to characterize Mycobacterium tuberculosis has enabled us to identify strains that are advantageous because of their virulence, ability to acquire resistance, or efficiency in causing severe outbreaks.Once such strains are identified, their fingerprints can be used as a reference to closely monitor their emergence in populations where they remain undetected (1). However, this approach requires universal population-based systematic fingerprinting programs.Alternatively, strain-specific PCRs can be used to track the emergence of theoretically advantageous strains (2, 3). Wholegenome sequencing (WGS) has simplified the identification of genotypic peculiarities (based on specific single nucleotide polymorphisms [SNPs]) from which we can design strain-specific PCRs (4, 5).We developed an allele-specific oligonucleotide (ASO)-multiplex PCR method based on WGS data to track the presence of a susceptible Beijing strain in Madrid, Spain. This strain was responsible for a severe outbreak on Gran Canaria Island (GCI) in the 1990s, and it was eventually shown to be the cause of around one-quarter of the total number of cases on the island (6). The GCI strain was still found in 2007 and 2008 as shown after the application of a specific PCR (7). In Madrid, we identified a nonadherent case with persistently active infectious tuberculosis (8 years) caused by the Gran Canaria strain (8), which obliged us to fast track potential secondary cases.(The results of this study were partially presented at the 2015 ESM Meeting, Riga, Latvia, 28 June to 1 July 2015.)The persistent case was diagnosed in our institution in 2006. The mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) pattern of this isolate coincided with the pattern described for the GCI strain (7). Using fingerprinting data from 2006 to 2008, we only detected 3 patients, all of whom were linked to the GCI outbreak. Given that the population-based genotyping program running in our area was discontinued at the end of 2008, we were unable to identify potential secondary cases that occurred from 2009 to 2014, when previously exposed cases were more likely to develop the disease. To update the 2009 to 2014 gap, we developed an ASO-PCR method to retrospectively fast track the GCI strain by directly analyzing stored isolates.WGS of 4 isolates from our case was performed as indicated elsewhere (4). We followed standard library preparation protocols. Us...