Cross-transmission of MDR-TB in Kuwait. Molecular characterization of multidrug-resistant Mycobacterium tuberculosis (MDR-TB) isolates identifies local transmission of infection in Kuwait, a country with a low incidence of TB and MDR-TB

Background: Increasing incidence of multidrug-resistant Mycobacterium tuberculosis infections are hampering global TB control efforts. Kuwait is a low tuberculosis incidence country and ~1% of M. tuberculosis strains are resistant to rifampicin and isoniazid (MDR-TB). This study detected mutations in seven genes predicting resistance to rifampicin, isoniazid, ethambutol, pyrazinamide and streptomycin in MDR-TB strains. Sequence data were combined with spoligotypes for detecting local transmission of MDR-TB in Kuwait.Methods: Ninety-three MDR-TB strains isolated from 12 Kuwaiti and 81 expatriate patients and 50 pansusceptible strains were used. Phenotypic drug susceptibility was determined by MGIT 460TB/960 system. Mutations conferring resistance to rifampicin, isoniazid, ethambutol, streptomycin and pyrazinamide were detected by Genotype MTBDRplus assay and/or PCR-sequencing of three rpoB regions, katG codon 315 (katG315) + inhA regulatory region, three embB regions, rpsL + rrs-500-900 regions and pncA. Phylogenetic tree was constructed from concatenated sequences by MEGA7 software. Spoligotyping kit was used, spoligotypes were identified by SITVIT2 and phylogenetic tree was constructed by using MIRU-VNTRplus software. Additional PCR-sequencing of gidB and rpsA was performed for cluster isolates.Results: Pansusceptible isolates contained wild-type sequences. Mutations in rpoB and katG + inhA were detected in 93/93 and 91/93 MDR-TB strains, respectively. Mutations were also detected in ethambutol-resistant, streptomycin-resistant and pyrazinamide-resistant MDR-TB isolates in embB, rpsL + rrs and pncA, respectively. Phylogenetic analysis of concatenated sequences showed unique patterns for 51 isolates while 42 isolates grouped in 16 clusters. Spoligotyping identified 35 patterns with 18 isolates exhibiting unique patterns and 75 isolates grouped in 17 patterns. Beijing genotype was most common (32/93) and 11 isolates showed nine orphan patterns. Interestingly, 18 isolates clustered by both methods and were isolated from TB patients typically within a span of <2 years. Eight of nine clusters were confirmed completely or nearly completely by additional gidB and rpsA sequence data.Conclusions: Our study provides first insight into molecular epidemiology of MDR-TB in Kuwait and identified seven potential clusters of local transmission of MDR-TB involving 2-6 subjects which had escaped detection by routine surveillance studies. Prospective detection of resistance-conferring mutations can identify possible cases of local transmission of MDR-TB in low MDR-TB settings.