Mohammed Akrim

The Genotypic Population Structure of Mycobacterium tuberculosis Complex from Moroccan Patients Reveals a Predominance of Euro-American Lineages

Background Tuberculosis (TB) remains a major health problem in Morocco. Characterization of circulating Mycobacterium tuberculosis genotypic lineages, important to understand the dynamic of the disease, was hereby addressed for the first time at a national level. Methodology/Principal Findings Spoligotyping was performed on a panel of 592 M. tuberculosis complex strains covering a 2-year period (2004–2006). It identified 129 patterns: 105 (n = 568 strains) corresponded to a SIT number in the SITVIT2 database, while 24 patterns were labeled as orphan. A total of 523 (88.3%) strains were clustered vs. 69 or 11.7% unclustered. Classification of strains within 3 large phylogenetical groups was as follows: group 1– ancestral/TbD1+/PGG1 (EAI, Bovis, Africanum), group 2– modern/TbD1−/PGG1 group (Beijing, CAS), group 3– evolutionary recent/TbD1−/PGG2/3 (Haarlem, X, S, T, LAM; alternatively designated as the Euro-American lineage). As opposed to group 3 strains (namely LAM, Haarlem, and T) that predominated (86.5% of all isolates), 6 strains belonged to group 2 (Beijing n = 5, CAS n = 1), and 3 strains (BOV_1 n = 2, BOV_4-CAPRAE) belonged to ancestral group 1 (EAI and AFRI lineage strains were absent). 12-loci MIRU-VNTR typing of the Casablanca subgroup (n = 114 strains) identified 71 patterns: 48 MITs and 23 orphan patterns; it allowed to reduce the clustering rate from 72.8% to 29.8% and the recent transmission rate from 64% to 20.2%. Conclusion The M. tuberculosis population structure in Morocco is highly homogeneous, and is characterized by the predominance of the Euro-American lineages, namely LAM, Haarlem, and T, which belong to the “evolutionary recent” TbD1−/PGG2/3 phylogenetic group. The combination of spoligotyping and MIRUs decreased the clustering rate significantly, and should now be systematically applied in larger studies. The methods used in this study appear well suited to monitor the M. tuberculosis population structure for an enhanced TB management program in Morocco.

Seroprevalence of hepatitis B and C among barbers and their clients in the Rabat region of Morocco.

A cross-sectional seroepidemiological study was conducted in the Rabat-Salé-Zemmour-Zaër region of Morocco in 2007 among 267 barbers and 529 clients, all men with no history of hepatitis B (HBV) vaccination. The overall prevalence of HBV seropositivity was 28.1% in barbers and 25.1% in clients; 1.9% and 1.7% respectively had active HBV (HBsAg positive). Risk factors for HBV included older age, low educational level, urban living, being married, history of transfusion, lack of current heterosexual relationship and liver-associated symptoms. Observations showed that HBV seropositivity was lower in clean barbershops and those using alum as an antispetic. The rate of PCR-confirmed hepatitis C virus (HCV) was only 1.1% and 1.3% in barbers and clients respectively, and was associated with increased age, drug use, history of surgery and symptoms of liver disease. Less than 1% of barbers were aware of HBV or HCV as causative agents of liver disease or jaundice.

Contribution of spoligotyping and MIRU-VNTRs to characterize prevalent Mycobacterium tuberculosis genotypes infecting tuberculosis patients in Morocco

In the present study, Mycobacterium tuberculosis complex (MTBC) clinical isolates from culture-positive TB patients in Morocco were studied by spoligotyping and 12-loci MIRU-VNTR typing methods to characterize prevalent genotypes (n = 219 isolates from 208 patients). Spoligotyping resulted in 39 unique patterns and 167 strains in 30 clusters (2-50 strains per cluster). Comparison with international database showed that 29 of 39 unique patterns matched existing shared spoligotype international types (SITs). Nine shared types containing 10 strains were newly created (SIT 2891 to SIT 2899); this led to the description of 69 SITs with 206 strains and two orphan patterns. The most prevalent spoligotype was SIT42 (LAM; n = 50 or 24% of isolates). The repartition of strains according to major MTBC clades was as follows LAM (46.1%)> Haarlem (26%) >ill-defined T superfamily (22.6%) and S clade (0.96%). On the other hand, Beijing, CAS (Central Asian) and EAI (East-African Indian) strains were absent in this setting. Subsequent 12-Loci MIRU typing resulted in a total of 25 SIT/MIT clusters (n = 66 isolates, 2-6 isolates per cluster), with a resulting recent transmission rate of 22.3%. The MIRU-VNTR patterns corresponded to 69 MITs for 138 strains and 46 orphan patterns. The most frequent patterns were MIT43 (n = 8), MIT9 (n = 7) and MIT42 (n = 7). HGDI analysis of the 12 MIRU loci showed that loci 10, 23 and 40 were highly discriminative in our setting. The results also underlined the usefulness of spoligotyping and MIRU-VNTR to detect mixed infections among certain of our TB patients. Globally, the results obtained showed that TB is almost exclusively transmitted in Morocco through evolutionary-modern MTBC lineages belonging to principal genetic groups 2/3 strains (Haarlem, LAM, T), with a high level of biodiversity seen by MIRU typing. This study provides with a 1st global snapshot of MTBC population structure in Morocco, and validates the potential use of spoligotyping in conjunction with minisatellites for future investigations in Morocco that should in future ideally include optimized 15- or 24-loci MIRU-VNTRs.

Molecular analysis of multidrug resistantMycobacterium tuberculosis isolates from Morocco

Tuberculosis remains a global threat to public health. Considerable efforts have been made to combat this disease. However, the emergence ofMycobacterium tuberculosis (Mtb) strains resistant to the major anti-tuberculosis drugs especially multidrug resistant (MDR) strains poses a deadly threat to control programs. The present study aims to identify the most common mutations within multidrug-resistantM. tuberculosis Moroccan isolates in order to use them as molecular markers for early and rapid detection of multidrug resistant strains. For that, allM. tuberculosis isolates received during 2002–2003 in the National Reference Laboratory of Tuberculosis in Morocco were subject to drug susceptibility tests for rifampicin and isoniazid and to a PCR probe method to detect specific mutation. Sequencing was performed for all genotypic rifampicin resistant isolates and also for four genotypic iso niazid resistant asolates randomly selected. Out of 187M. tuberculosis positive cultures, 46 (24.6%) were phenotypically resistant to both rifampicin and isoniazid. Nucleotide mutations in rpoB531, rpoB526, rpoB516, katG315 and inh-15 codons associated with resistance to rifampin (RIF) and isoniazid (INH) were found respectively in 37/46 (80.4%) and 43/46 (93.5%) isolates. Genotypic multi drug resistance was then confirmed in 74% (34/46) isolates. The mutations at codon 315 ofkatG gene and at codons 531, 526 and 516 ofrpoB gene are frequently found in MDR isolates which confirm their strong implication in the development of multidrug resistant tuberculosis. We concluded that these mutations are useful as molecular markers for detection of multidrug resistant isolates but are not yet sufficient to fully predictM. tuberculosis multidrug resistance.

Rifoligotyping assay: an alternative method for rapid detection of rifampicin resistance in Mycobacterium tuberculosis isolates from Morocco

One of the greatest threats to global tuberculosis (TB) control is the growing prevalence of drug resistant strains. In the past decades, considerable efforts have been made upon the development of new molecular technologies and methodologies for detection of drug resistance in Mycobacterium tuberculosis (MTB). A sensitive, specific reverse line blot assay, called rifoligotyping (RIFO), for the detection of genotypic resistance to rifampicin (RIF), was designed and evaluated. RIFO includes oligonucleotide probes specific for wild-type and mutant sequences, allowing specific and sensitive detection of both genotypes in a single assay. The RIFO was applied on 500 MTB isolates from Morocco. The results of the RIFO showed a good sensitivity (90.9%) and high specificity (100%); the positive and negative predictive values were 100% and 96.1%, respectively. This rapid, simple, economical assay provides a practical alternative for RIF genotyping, especially in low-income countries, to improve TB control and management.

HIV-1 Subtype distribution in morocco based on national sentinel surveillance data 2004-2005

BackgroundLittle is known about HIV-1 subtype distribution in Morocco. Some data suggest an emergence of new HIV subtypes. We conducted phylogenetic analysis on a nationally representative sample of 60 HIV-1 viral specimens collected during 2004-2005 through the Morocco national HIV sentinel surveillance survey.ResultsWhile subtype B is still the most prevalent, 23.3% of samples represented non-B subtypes, the majority of which were classified as CRF02_AG (15%). Molecular clock analysis confirmed that the initial introduction of HIV-1B in Morocco probably came from Europe in the early 1980s. In contrast, the CRF02_AG strain appeared to be introduced from sub-Saharan Africa in two separate events in the 1990s.ConclusionsSubtype CRF02_AG has been emerging in Morocco since the 1990s. More information about the factors introducing HIV subtype-specific transmission will inform the prevention strategy in the region.