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.

BACKGROUND: Little 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. RESULTS: While 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. CONCLUSIONS: Subtype 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.

Using geostatistics to better understand the epidemiology of animal rabies in Morocco: what is the contribution of the predictive value?

This study aims to characterize the spatial distribution of animal rabies in Morocco in order to provide appropriate control approaches. Descriptive analyses of the epidemiological data show that the number of reported canine rabies cases greatly underestimates the true incidence of the disease. Underreporting subsequently affects the coherence of its spatial distribution. To perform accurate geographic distribution mapping of the disease based on interpolation methods, a data set was created using data between 2000 and 2018 to compare the derived disease cases with known true values in order to identify disease clusters. The subsequent interpolation was conducted using Ordinary Kriging regression methods and the semi variogram to focus on short distances and reduce uncertainty. The estimated clusters of rabies were evaluated using a cross validation step which revealed predicted cases close to the true values. To improve the precision of analysis, the authors displayed georeferenced dog and human rabies cases reported during the last three years, demonstrating reliable results that correspond to the estimated cluster areas similar to the true disease incidence on the field. This work highlights a strong correlation between infrastructure projects (i.e. railways, roads, facilities) and rabies epizootics for several specific locations. This study is the first attempt to use geostatistics to build upon the understanding of animal rabies in Morocco and shed light on the most appropriate strategies to sustainably reduce and mitigate the risk of rabies. There has been little literature on the use of kriging methods in animal health research. Thus, this study also aimed to explore a novel method in the veterinary sciences to establish kriging as a valid and coherent analysis tool to identify the extent to which the geostatistic area can objectively support understanding on animal rabies and saw it as being highly instrumental in coping with gaps in the data.

Outbreak Investigation of a Multipathogen Foodborne Disease in a Training Institute in Rabat, Morocco: Case-Control Study.

BACKGROUND: On June 18, 2017, the public health service was alerted about 43 students in the training institute in Rabat who were admitted to the emergency room for acute gastroenteritis following the uptake of a meal a day before. OBJECTIVE: This study aimed to investigate the foodborne disease outbreak by confirming the outbreak, identifying the source of contamination, and recommending control measures. METHODS: We conducted a case-control study. Cases and controls were selected in a ratio of 1:1. We defined a case as any member of the training institute who attended the Ramadan buffet in the institute's restaurant and who had presented, in the weekend of June 16 to 20, 2017, symptoms of diarrhea or vomiting with at least one of the following signs: abdominal pain, fever, headache, nausea, and dizziness. A control was defined as anyone who attended the Ramadan buffet in the institute's restaurant but had not presented any symptoms from June 16 to 20, 2017. We conducted a bivariate and multivariable analysis. Stools of ill students were collected, and a food specimen was collected for bacterial testing. RESULTS: A total of 50 cases and 50 controls were selected. Among the cases, males were predominant (43/50, 86%); the median age was 21 years. A total of 47 cases sought medical care. There were no hospitalizations and no deaths. The episode was short with an estimated average incubation period of 9 hours. The epidemic curve oriented toward a common source of contamination. Among food items, briwates were strongly associated with the illness with an odd ratio of 14.23 (95% CI 5.04-40.04; P<.001). Laboratory testing of briwates showed presence of Escherichia coli O157 and Staphylococcus aureus. CONCLUSIONS: This foodborne disease outbreak was likely caused by briwates that was contaminated with S aureus and E coli. We recommended strengthening hygiene measures. Food handling techniques should be taught as part of continuous professional development for food handlers.

Evaluation of the Yield of Histopathology in the Diagnosis of Lymph Node Tuberculosis in Morocco, 2017: Cross-Sectional Study.

BACKGROUND: The frequency of occurrence of extrapulmonary tuberculosis (EPTB) has been increasing globally over the last two decades. In Morocco, EPTB cases account for 46% of the patients reported with a new episode of tuberculosis (TB). Lymph node TB (LNTB) is the most common form of EPTB. In line with the guidelines of the National TB Program, the diagnosis is mainly based on clinical evidence, including histopathology. OBJECTIVE: This study aimed to evaluate the yield of histopathology testing in the diagnosis of LNTB. METHODS: This cross-sectional, prospective study was conducted among patients with cervical lymph node who were enrolled in the study from November 2016 to May 2017 in three regions of Morocco. We compared the outcomes of histopathological testing with those of bacteriology. Sensitivity (Se), specificity (Sp), positive predictive value (PPV), and negative predictive value (NPV) of histopathology testing were calculated. Culture and Xpert tests were used as the gold standard Laboratoty Testing. RESULTS: A total of 262 patients were enrolled in this study. The Se, Sp, PPV, and NPV of histopathology testing were 95.6% (129/135), 64.6% (82/127), 74.1% (129/174), and 93.2% (82/88), respectively, in the presence of granuloma with or without caseous necrosis and were 84.4% (114/135), 74.8% (95/127), 78.1% (114/146), and 81.9% (95/116), respectively, in the presence of granuloma with caseous necrosis. The granuloma with caseous necrosis was associated with increased PPV and Sp of histopathology testing (P<.05). CONCLUSIONS: The presence of the granuloma with caseous necrosis in the histopathological examination had significantly improved the yield of histopathology testing for the diagnosis of LNTB. The findings recommend to maintain histopathology testing in establishing the LNTB diagnosis and to explore other techniques to improve it.

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.

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.

Evaluation of conventional molecular diagnosis of Mycobacterium tuberculosis in clinical specimens from Morocco.

INTRODUCTION: Tuberculosis is a major public health threat, annually affecting new individuals worldwide, especially those in developing countries. Rapid detection of the agent and effective treatment are two important factors in controlling this disease. METHODOLOGY: The present study aimed to evaluate polymerase chain reaction (PCR) as a rapid and direct molecular method for the diagnosis of Mycobacterium tuberculosis (MTB) in 70 clinical specimens (62 sputum samples, six cerebrospinal fluids, and two biopsies) using heat shock protein (hsp65) as the gene target. Automated sequencing of the same gene was used for the identification of MTB to the species level. RESULTS: The sensitivity of PCR was 81.13%, with specificity of 88.24%; the positive and negative predictive values were 95.56% and 60%, respectively. CONCLUSION: Based on these results, the hsp65 gene sequence can be used to differentiate the members of MTB complex from non-tuberculosis mycobacteria (NTM).

Comparison of a DNA Based PCR Approach with Conventional Methods for the Detection of Mycobacterium tuberculosis in Morocco.

BACKGROUND: Worldwide, tuberculosis (TB) is a major public health problem and the rapid diagnosis and appropriate chemotherapy become the first priority and a serious challenge to improve TB treatment. In the objective of early TB diagnosis and rapid detection of Mycobacterium tuberculosis (MTB) in the clinical specimens, the utility of the Polymerase Chain Reaction (PCR) using the Insertion Sequence 6110 "IS6110" as target was compared to conventional methods. METHODS: Out of 305 patients with different clinical manifestations: suspected, new, drug relapse, drug failure and chronic cases were enrolled in this study and tested by mycobacteriological and PCR techniques for the investigation about the tubercle bacilli. RESULTS: The results of the in house "IS6110" PCR showed a good sensitivity (92.4%) and high specificity (98.0%), the positive and negative predictive values were 96.4 % and 95.3 % respectively. CONCLUSION: This study showed clearly that the PCR testing using the "IS6110" in the routine analysis is a potential tool for the rapid TB diagnosis, especially for critical cases and would be of great interest to help the clinician in the misdiagnosed critical cases by the traditional radiology.

Analysis of isoniazid, streptomycin and ethambutol resistance in Mycobacterium tuberculosis isolates from Morocco.

BACKGROUND: Drug-resistant tuberculosis is a major problem worldwide. Based on the knowledge of specific mutations occurring in Mycobacterium tuberculosis genome, drug resistance can be detected earlier. The aim of this study was to determine the prevalence of the most common mutations associated with resistance to Isoniazid (INH), Streptomycin (SM) and Ethambutol (EMB) in Mycobacterium tuberculosis isolates from Morocco in order to select target mutations to develop tests for rapid detection of drug-resistant Mycobacterium tuberculosis Moroccan isolates. METHODOLOGY: A total of 199 M. tuberculosis isolates collected from the National Tuberculosis Reference Laboratory in Morocco were subject to katG, inhA, rrs, rpsL and emb mutation analysis by PCR probe-based assay. The genotypic results were then compared to drug susceptibility testing results for the corresponding drugs. RESULTS: Among 66 phenotypically INH resistant isolates, 80.3% (53/66) were found to be genotypically INH resistant from which 77.3% (51/66) and 3% (2/66) had respective mutations in katG315 and inhp-15 codons. Of the 58 phenotypically SM resistant isolates, genotypic SM resistance was confirmed in 17.2% (10/58) cases. Nucleotide mutations at codons 43 and 88 of rpsL gene and at codon 512 of rrs gene were found respectively in 12.1% (7/58); 1.7% (1/58) and 3.4% (2/58) of the phenotypically SM resistant Mycobacterium tuberculosis isolates. Finally, mutations at codon 306 of embB gene were identified in 42.3% (11/26) of Mycobacterium tuberculosis isolates phenotypically EMB resistant. CONCLUSION: This study showed that a large proportion of Mycobacterium tuberculosis resistant isolates from Morocco carry a large number of mutations in different codons (especially katG315, embB306 and rpsL43) of the corresponding genes associated with drug resistance. Thus, molecular analysis based on the identification of such mutations is useful but not fully sufficient to predict all drug resistance cases. Based on these results, rapid drug resistance genotyping can be used as an adjunct to the traditional culture based methods in reference laboratories.