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.
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.


METHODS
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.


RESULTS
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.
National Tuberculosis Reference Laboratory, National Institute of Hygiene, Rabat, Morocco; Laboratory of Microbiology and Molecular Biology, Faculty of Sciences, University Mohammed V, Rabat, Morocco.
Nada
N
Bouklata
INSERMU1018, Lille, France; CNRS UMR8204, Lille, France; Center for Infection and Immunity of Lille (CIIL), Institut Pasteur de Lille, France; Université de Lille, Lille, France; Genoscreen, Lille, France.
Philip
P
Supply
National Tuberculosis Reference Laboratory, National Institute of Hygiene, Rabat, Morocco.
Sanae
S
Jaouhari
National Tuberculosis Reference Laboratory, National Institute of Hygiene, Rabat, Morocco.
Reda
R
Charof
Laboratory of Cell Immunology, Department of Immunology, National Institute of Hygiene, Rabat, Morocco.
Fouad
F
Seghrouchni
Laboratory of Microbiology and Molecular Biology, Faculty of Sciences, University Mohammed V, Rabat, Morocco.
Khalid
K
Sadki
Department of Epidemiology and Public Health, Faculty of Medicine and Pharmacy of Fes, Sidi Mohamed Ben Abdillah University, Fes, Morocco.
Youness
Y
El Achhab
Department of Epidemiology and Public Health, Faculty of Medicine and Pharmacy of Fes, Sidi Mohamed Ben Abdillah University, Fes, Morocco.
Chakib
C
Nejjari
Laboratory of Microbiology and Molecular Biology, Faculty of Sciences, University Mohammed V, Rabat, Morocco.
Abdelkarim
A
Filali-Maltouf
National Tuberculosis Reference Laboratory, National Institute of Hygiene, Rabat, Morocco.
Ouafae
O
Lahlou
School of Public Health and Management System Health, University Mohamed VI of Sciences and Health, Casablanca, Morocco.
Rajae
R
El Aouad
D000293:Adolescent; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016000:Cluster Analysis; D024881:Drug Resistance, Bacterial / Q000235:genetics; D005260:Female; D056426:Genetic Loci / Q000235:genetics*; D014644:Genetic Variation; D006801:Humans; D020071:Interspersed Repetitive Sequences / Q000235:genetics*; D008297:Male; D008875:Middle Aged; D018598:Minisatellite Repeats / Q000235:genetics*; D009018:Morocco; D058885:Multilocus Sequence Typing / Q000379:methods*; D009169:Mycobacterium tuberculosis / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D055815:Young Adult