Molecular Typing of Mycobacterium Tuberculosis Complex by 24-Locus Based MIRU-VNTR Typing in Conjunction with Spoligotyping to Assess Genetic Diversity of Strains Circulating in Morocco

Abstract

Background: Standard 24-locus Mycobacterial Interspersed Repetitive Unit Variable Number Tandem Repeat (MIRU-VNTR) typing allows to get an improved resolution power for tracing TB transmission and predicting different strain (sub) lineages in a community.

Methodology: During 2010-2012, a total of 168 Mycobacterium tuberculosis Complex (MTBC) isolates were collected by cluster sampling from 10 different Moroccan cities, and centralized by the National Reference Laboratory of Tuberculosis over the study period. All isolates were genotyped using spoligotyping, and a subset of 75 was genotyped using 24-locus based MIRU-VNTR typing, followed by first line drug susceptibility testing. Corresponding strain lineages were predicted using MIRU-VNTRplus database.

Principal findings: Spoligotyping resulted in 137 isolates in 18 clusters (2-50 isolates per cluster: clustering rate of 81.54%) corresponding to a SIT number in the SITVIT database, while 31(18.45%) patterns were unique of which 10 were labelled as "unknown" according to the same database. The most prevalent spoligotype family was LAM; (n = 81 or 48.24% of isolates, dominated by SIT42, n = 49), followed by Haarlem (23.80%), T superfamily (15.47%), >Beijing (2.97%), > U clade (2.38%) and S clade (1.19%). Subsequent 24-Locus MIRU-VNTR typing identified 64 unique types and 11 isolates in 5 clusters (2 to 3isolates per cluster), substantially reducing clusters defined by spoligotyping only. The single cluster of three isolates corresponded to two previously treated MDR-TB cases and one new MDR-TB case known to be contact a same index case and belonging to a same family, albeit residing in 3 different administrative regions. MIRU-VNTR loci 4052, 802, 2996, 2163b, 3690, 1955, 424, 2531, 2401 and 960 were highly discriminative in our setting (HGDI >0.6).

Conclusions: 24-locus MIRU-VNTR typing can substantially improve the resolution of large clusters initially defined by spoligotyping alone and predominating in Morocco, and could therefore be used to better study tuberculosis transmission in a population-based, multi-year sample context.

Fig 1. Genetic tree based on spoligotyping and 24-locus MIRU-VNTR data of 75 M. tuberculosis isolates from10 Moroccan cities.

A dendogram was generated using the UPGMA algorithm using tools available from the MIRU-VNTRplus identification database (see text). Isolates are identified according to their corresponding spoligotype international type (SIT; boxed), according to the SITVIT database.