The Interleukin-6 gene variants may protect against SARS-CoV-2 infection and the severity of COVID-19: a case-control study in a Moroccan population.

The symptoms of SARS-CoV-2 infection vary widely, ranging from asymptomatic cases to severe forms marked by acute respiratory distress syndrome, multi-organ damage, and fatalities. Studies indicate a correlation between specific genes and susceptibility to SARS-CoV-2 infection and disease severity, particularly involving variants in genes linked to inflammation and immune responses. The objective of this study is to investigate the association between rs1800795 (- 174 G > C) and rs1800797 (- 597 A > G) variants in the interleukin-6 (IL-6) promoter region and susceptibility to SARS-CoV-2 infection. Additionally, we aim to explore their correlation with COVID-19 severity in a Moroccan population. In this case-control study, we enrolled 270 unvaccinated COVID-19 patients, consisting of 132 with severe COVID-19 and 138 with asymptomatic-moderate COVID-19. Additionally, we included 339 SARS-CoV-2-negative group. Genotyping of rs1800795 and rs1800797 polymorphisms of the IL-6 gene was performed using predesigned TaqMan SNP genotyping. The median age of SARS-CoV-2-negative controls was 50 years, while severe COVID-19 cases exhibited a median age of 61 years. Additionally, individuals with asymptomatic to moderate COVID-19 had a median age of 36 years. We observed a significant age difference between severe and mild COVID-19 patients (p < 0.0001), and an association was noted between gender and the severity of COVID-19 (p = 0.011). The allele and genotype frequencies of the IL-6 - 597G > A and - 174G > C variants did not show significant associations with susceptibility to SARS-CoV-2 infection (p > 0.05). However, further analysis revealed that the linkage disequilibrium between rs1800797 and rs1800795 indicated that individuals with the GC* haplotype (OR = 0.04, 95% CI 0.01-0.30, p = 0.001) and AG* haplotype (OR = 0.11, 95% CI 0.03-0.46, p = 0.002) were significantly associated with protection against SARS-CoV-2 infection. Moreover, in the overdominant model, the IL-6 - 174 G/C genotype was found to be protective against the development of severe disease compared to those with the G/G-C/C genotypes (p = 0.03; OR = 0.41, 95% CI 0.18-0.96). However, correlations between complete blood count markers, hematological markers, D-dimer, C-reactive protein, and ferritin levels according to - 597 A > G and - 174G > C genotypes showed no significant differences (all p > 0.05). Our findings provide valuable insights into the pathogenesis of COVID-19, suggesting that genetic variations at the IL-6 gene may contribute to the susceptibility to severe SARS-CoV-2 infection within the Moroccan population.

Seroepidemiological assessment of SARS-CoV-2 vaccine responsiveness and associated factors in the vaccinated community of the Casablanca-Settat Region, Morocco.

Assessing the prevalence of SARS-CoV-2 IgG positivity through population-based serological surveys is crucial for monitoring COVID-19 vaccination efforts. In this study, we evaluated SARS-CoV-2 IgG positivity within a provincial cohort to understand the magnitude of the humoral response against the SARS-CoV-2 vaccine and to inform evidence-based public health decisions. A community-based cross-sectional seroprevalence study was conducted, involving 10,669 participants who received various vaccines (two doses for BBIBP-CorV/Sinopharm, Covishield vaccine, and Pfizer/BioNTech, and one dose for Johnson & Johnson's Janssen COVID-19 vaccine). The study spanned 16 provinces in the Casablanca-Settat region from February to June 2022, during which comprehensive demographic and comorbidity data were collected. We screened samples for the presence of IgG antibodies using the SARS-CoV-2 IgG II Quant assay, which quantifies antibodies against the receptor-binding domain (RBD) of the spike (S) protein, measured on the Abbott Architect i2000SR. The overall crude seroprevalence was 96% (95% CI: 95.6-96.3%), and after adjustment for assay performance, it was estimated as 96.2% (95% CI: 95.7-96.6). The adjusted overall seroprevalences according to vaccine brands showed no significant difference (96% for BBIBP-CorV/Sinopharm, 97% for ChAdOx1 nCoV-19/Oxford/AstraZeneca, 98.5% for BNT162b2/Pfizer-BioNTech, and 98% for Janssen) (p = 0.099). Participants of older age, female sex, those with a history of previous COVID-19 infection, and those with certain chronic diseases were more likely to be seropositive among ChAdOx1 nCoV-19/Oxford/AstraZeneca and BBIBP-CorV/Sinopharm vaccinee groups. Median RBD antibody concentrations were 2355 AU/mL, 3714 AU/mL, 5838 AU/mL, and 2495 AU/mL, respectively, after two doses of BBIBP-CorV/Sinopharm, ChAdOx1 nCoV-19/Oxford/AstraZeneca, BNT162b2/Pfizer-BioNTech, and after one dose of Janssen (p < 0.0001). Furthermore, we observed that participants vaccinated with ChAdOx1 nCoV-19/Oxford/AstraZeneca and BBIBP-CorV/Sinopharm with comorbid chronic diseases exhibited a more pronounced response to vaccination compared to those without comorbidities. In contrast, no significant differences were observed among Pfizer-vaccinated participants (p > 0.05). In conclusion, our serosurvey findings indicate that all four investigated vaccines provide a robust humoral immune response in the majority of participants (more than 96% of participants had antibodies against SARS-CoV-2). The BNT162b2 vaccine was found to be effective in eliciting a strong humoral response compared to the other three vaccines. However, challenges still remain in examining the dynamics and durability of immunoprotection in the Moroccan context.

Human activities and densities shape insecticide resistance distribution and dynamics in the virus-vector Culex pipiens mosquitoes from Morocco.

BACKGROUND: Mosquitoes of the Culex pipiens complex are widely distributed vectors for several arboviruses affecting humans. Consequently, their populations have long been controlled using insecticides, in response to which different resistance mechanisms have been selected. Moreover, their ecological preferences and broad adaptability allow C. pipiens mosquitoes to breed in highly polluted water bodies where they are exposed to many residuals from anthropogenic activities. It has been observed for several mosquito species that anthropization (in particular urbanization and agricultural lands) can lead to increased exposure to insecticides and thus to increased resistance. The main objective of the present study was to investigate whether and how urbanization and/or agricultural lands had a similar impact on C. pipiens resistance to insecticides in Morocco. METHODS: Breeding sites were sampled along several transects in four regions around major Moroccan cities, following gradients of decreasing anthropization. The imprint of anthropogenic activities was evaluated around each site as the percentage of areas classified in three categories: urban, agricultural and natural. We then assessed the frequencies of four known resistance alleles in these samples and followed their dynamics in five urban breeding sites over 4 years. RESULTS: The distribution of resistance alleles revealed a strong impact of anthropization, in both agricultural and urbanized lands, although different between resistance mutations and between Moroccan regions; we did not find any clear trend in the dynamics of these resistance alleles during the survey. CONCLUSIONS: Our study provides further evidence for the role of anthropic activities in the selection and maintenance of mutations selected for resistance to insecticides in mosquitoes. The consequences are worrying as this could decrease vector control capacities and thus result in epizootic and epidemic outbreaks. Consequently, concerted and integrated disease control strategies must be designed that include better management regarding the consequences of our activities.

Kinetics of specific anti-SARS-CoV-2 IgM, IgA, and IgG responses during the first 12 months after SARS-CoV-2 infection: A prospective longitudinal study.

Coronavirus 2019 (COVID-19) is a global health threat. The kinetics of antibodies against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) need to be assessed, as the long-term duration of these immunoglobulins remains largely controversial. The aim of this study was to assess the longitudinal dynamics of anti-SARS-CoV-2 antibodies against the nucleocapsid (N) protein and the receptor-binding domain (RBD) of the spike protein up to one year in a cohort of 190 COVID-19 patients. Between March and September 2021, we enrolled patients from two regional hospitals in Casablanca, Morocco. Blood samples were collected and analyzed for antibody levels. We used the commercial Euroimmun ELISA for the determination of anti-N IgM, the Abbott Architect™ SARS-CoV-2 IgG test for the detection of anti-RBD IgG, and an in-house kit for the assay of anti-N IgG and anti-N IgA. IgM and IgA antibodies were assessed 2-5, 9-12, 17-20 and 32-37 days after symptom onset. IgG antibodies were also assessed 60, 90, 120 and 360 days after symptom onset. One-third of patients developed IgM (32%), while two-thirds developed IgA (61%). One month of symptom onset, most patients developed IgG, with 97% and 93% positivity for anti-RBD IgG and anti-N IgG, respectively. The anti-RBD IgG positivity rate remained high up to one year of follow-up. However, the anti-N IgG positivity rate decreased over time, with only 41% of patients testing positive after one year's follow-up. IgG levels were significantly higher in older people (over 50 years) than in other study participants. We also found that patients who had received two doses of ChAdOx1 nCoV-19 vaccine prior to infection had a lower IgM response than unvaccinated patients. This difference was statistically significant two weeks after the onset of symptoms. We present the first study in Africa to measure the kinetics of antibody response (IgA, IgM and IgG) to SARS-CoV-2 over one year. Most participants remained seropositive for anti-RBD IgG after one year but showed a significant decline in antibody titers.

The peroxisome proliferator-activated receptor γ coactivator-1 alpha rs8192678 (Gly482Ser) variant and hepatitis B virus clearance.

BACKGROUND: Chronic hepatitis B virus (CHB) infection is still incurable a major public health problem. It is yet unclear how host genetic factors influence the development of HBV infection. The peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) has been shown to regulate hepatitis B virus (HBV). Several reports found that PPARGC1A variants are involved in a number of distinct liver diseases. Here we investigate whether the PPARGC1A rs8192678 (Gly482Ser) variant is involved in the spontaneous clearance of acute HBV infection and if it participates in chronic disease progression in Moroccan patients. METHODS: Our study included 292 chronic hepatitis B (CHB) patients and 181 individuals who spontaneously cleared-HBV infection. We genotyped the rs8192678 SNP using a TaqMan allelic discrimination assay and then explored its association with spontaneous HBV clearance and CHB progression. RESULTS: Our data showed that individuals carrying CT and TT genotypes were more likely to achieve spontaneous clearance (OR = 0.48, 95% CI (0.32-0.73), p = 0.00047; OR = 0.28, 95% CI (0.15-0.53), p = 0.00005, respectively). Subjects carrying the mutant allele T were more likely to achieve spontaneous clearance (OR = 0.51, 95% CI (0.38-0.67), P = 2.68E-06). However, when we investigated the impact of rs8192678 on the progression of liver diseases, we neither observe any influence (p > 0.05) nor found any significant association between ALT, AST, HBV viral loads, and the PPARGC1A rs8192678 genotypes in patients with CHB (p > 0.05). CONCLUSION: Our result suggests that PPARGC1A rs8192678 may modulate acute HBV infection, and could therefore represent a potential predictive marker in the Moroccan population.

Identification of novel T-cell epitopes on monkeypox virus and development of multi-epitopes vaccine using immunoinformatics approaches.

Monkeypox virus (MPV) is closely related to the smallpox virus, and previous data from Africa suggest that the smallpox vaccine (VARV) is at least 85% effective in preventing MPV. No multi-epitope vaccine has yet been developed to prevent MPV infection. In this work, we used in silico structural biology and advanced immunoinformatic strategies to design a multi-epitope subunit vaccine against MPV infection. The designed vaccine sequence is adjuvanted with CpG-ODN and includes HTL/CTL epitopes for similar proteins between vaccinia virus (VACV) that induced T-cell production in vaccinated volunteers and the first draft sequence of the MPV genome associated with the suspected outbreak in several countries, May 2022. In addition, the specific binding of the modified vaccine and the immune Toll-like receptor 9 (TLR9) was estimated by molecular interaction studies. Strong interaction in the binding groove as well as good docking scores confirmed the stringency of the modified vaccine. The stability of the interaction was confirmed by a classical molecular dynamics simulation and normal mode analysis. Then, the immune simulation also indicated the ability of this vaccine to induce an effective immune response against MPV. Codon optimization and in silico cloning of the vaccine into the pET-28a (+) vector also showed its expression potential in the E. coli K12 system. The promising data obtained from the various in silico studies indicate that this vaccine is effective against MPV. However, additional in vitro and in vivo studies are still needed to confirm its efficacy.Communicated by Ramaswamy H. Sarma.

Mix-and-match COVID-19 vaccines trigger high antibody response after the third dose vaccine in Moroccan health care workers.

Recent studies have shown that in individuals who have received two doses of COVID-19 vaccine, the level of IgG antibodies decreased over time. In addition, the resurgence of the epidemic due to variants has led the authorities in several countries, including Morocco, to extend the third dose to the entire adult population. In this study, we included 43 healthcare workers (HCWs) who were vaccinated with three doses. They were vaccinated with ChAdOx1 nCoV-19 for the first two doses and with BNT 162b2 or BBIBP-CorV vaccine for the third dose. Humoral response was assessed on the day of injection of the third dose of vaccine and one month after the third dose by measuring anti-receptor-binding domain (RBD) IgG levels. Seven months after the second dose, the median titer of anti-RBD IgG was higher in the group with a history of SARS-CoV-2 infection than in the group with no history of infection (1038 AU/mL vs. 76.05 AU/mL, respectively, p = 0.003). One month after the third dose, a significant increase in median level of anti-RBD in both groups was observed: from 76.05 AU/mL to 6127 AU/mL in the group with no history of infection and from 1038 AU/mL to 14,412 AU/mL in the group with history of infection. Notably, the BNT 162b2 vaccine elicits a high titer of anti-RBD antibody compared to the BBIBP-CorV vaccine. Median antibody titers were 21,991 AU/mL and 3640 AU/mL for BNT 162b2 and BBIBP-CorV vaccines, respectively (p = 0.0002). 23% of HCWs were infected with SARS-CoV-2 within the first two months after the third dose injection. However, all these patients developed mild symptoms and tested negative by RT-qPCR between 10 and 15 days after the onset of symptoms. Our findings support that the third dose of COVID-19 vaccine significantly improves the humoral response and protects against the severe disease.

Development and comparative evaluation of SARS-CoV-2 S-RBD and N based ELISA tests in various African endemic settings.

Management of the COVID-19 pandemic relies on molecular diagnostic methods supported by serological tools. Herein, we developed S-RBD- and N- based ELISA assays useful for infection rate surveillance as well as the follow-up of acquired protective immunity against SARS-CoV-2. ELISA assays were optimized using COVID-19 Tunisian patients' sera and prepandemic controls. Assays were further validated in 3 African countries with variable endemic settings. The receiver operating curve was used to evaluate the assay performances. The N- and S-RBD-based ELISA assays performances, in Tunisia, were very high (AUC: 0.966 and 0.98, respectively, p < 0.0001). Cross-validation analysis showed similar performances in different settings. Cross-reactivity, with malaria infection, against viral antigens, was noticed. In head-to-head comparisons with different commercial assays, the developed assays showed high agreement. This study demonstrates, the added value of the developed serological assays in low-income countries, particularly in ethnically diverse populations with variable exposure to local endemic infectious diseases.

Kinetics of SARS-CoV-2 IgM and IgG Antibodies 3 Months after COVID-19 Onset in Moroccan Patients.

Coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) poses serious global public health problems. Characterization of the immune response, particularly antibodies to SARS-CoV-2, is important for establishing vaccine strategies. The purpose of this study was to evaluate longitudinally the kinetics of anti-SARS-CoV-2 antibodies against spike protein (S1) for up to 3 months in a cohort of 169 COVID-19 patients. We enrolled COVID-19 patients at two regional hospitals in Casablanca, Morocco, between March and September 2021. Blood samples were collected and N-specific IgM and S-specific IgG levels were measured by a commercial Euroimmun ELISA. IgM antibodies were assessed 2-5 (D00), 9-12 (D07), 17-20 (D15), and 32-37 (D30) days after symptom onset; IgG antibodies were assessed at these time points plus 60 (D60) and 90 (D90) days after symptom onset. We found that at 3 months after symptom onset, 79% of patients had detectable SARS-CoV-2-specific IgG antibodies, whereas their IgM seropositivity was 19% by 1 month after symptom onset. The IgM level decreased to 0.34 (interquartile range [IQR] 0.19-0.92) at 1 month after symptom onset, whereas the IgG level peaked at D30 (3.10; IQR 1.83-5.64) and remained almost stable at D90 (2.95; IQR 1.52-5.19). IgG levels were significantly higher in patients older than 50 years than in those younger than 50 at all follow-up time points (P < 0.05). Statistical analysis showed no significant difference in median anti-S1 antibody levels among infected patients based on gender or comorbidities. This study provides information on the longevity of anti-SARS-CoV-2 IgM and IgG antibodies in COVID-19 patients.

No association between habitat, autogeny and genetics in Moroccan Culex pipiens populations.

BACKGROUND: Mosquitoes of the Culex pipiens complex are found across the globe and are the focus of many research studies. Among the temperate species C. pipiens sensu stricto (s.s.), two forms are usually described: molestus and pipiens. These two forms are indistinguishable in terms of morphology but show behavioral and physiological differences that may have consequences for their associated epidemiology. The two forms are well defined in the northern part of the species distribution, where autogeny is strictly associated with the molestus form. However, whether the two remain distinct and show the characteristic differences in behavior is less clear in North Africa, at the southern edge of their range. METHODS: The association between autogeny, as determined by ovarian dissection, and molecular forms, based on the CQ11 microsatellite marker, was studied in six Moroccan populations of C. pipiens. RESULTS: An overall low prevalence of autogeny was found at three of the Moroccan regions studied, although it reached 17.5% in the Agadir population. The prevalence of form-specific CQ11 alleles was quite similar across all populations, with the molestus allele being rarer (approx. 15%), except in the Agadir population where it reached 43.3%. We found significant deficits in heterozygotes at the diagnostic CQ11 locus in three populations, but the three other populations showed no significant departure from panmixia, which is in line with the results of a retrospective analysis of the published data. More importantly, we found no association between the autogeny status and CQ11 genotypes, despite the many females analyzed. CONCLUSIONS: There was limited evidence for two discrete forms in Morocco, where individuals carrying pipiens and molestus alleles breed and mate in the same sites and are equally likely to be capable of autogeny. These observations are discussed in the epidemiological context of Morocco, where C. pipiens is the main vector of several arboviruses.

Amplification and sequencing of entire tick mitochondrial genomes for a phylogenomic analysis.

The mitochondrial genome (mitogenome) has proven to be important for the taxonomy, systematics, and population genetics of ticks. However, current methods to generate mitogenomes can be cost-prohibitive at scale. To address this issue, we developed a cost-effective approach to amplify and sequence the whole mitogenome of individual tick specimens. Using two different primer sites, this approach generated two full-length mitogenome amplicons that were sequenced using the Oxford Nanopore Technologies' Mk1B sequencer. We used this approach to generate 85 individual tick mitogenomes from samples comprised of the three tick families, 11 genera, and 57 species. Twenty-six of these species did not have a complete mitogenome available on GenBank prior to this work. We benchmarked the accuracy of this approach using a subset of samples that had been previously sequenced by low-coverage Illumina genome skimming. We found our assemblies were comparable or exceeded the Illumina method, achieving a median sequence concordance of 99.98%. We further analyzed our mitogenome dataset in a mitophylogenomic analysis in the context of all three tick families. We were able to sequence 72 samples in one run and achieved a cost/sample of ~ $10 USD. This cost-effective strategy is applicable for sample identification, taxonomy, systematics, and population genetics for not only ticks but likely other metazoans; thus, making mitogenome sequencing equitable for the wider scientific community.

Assessment of expertise in morphological identification of mosquito species (Diptera, Culicidae) using photomicrographs.

Accurate identification of insect species is an indispensable and challenging requirement for every entomologist, particularly if the species is involved in disease outbreaks. The European MediLabSecure project designed an identification (ID) exercise available to any willing participant with the aim of assessing and improving knowledge in mosquito taxonomy. The exercise was based on high-definition photomicrographs of mosquitoes (26 adult females and 12 larvae) collected from the western Palaearctic. Sixty-five responses from Europe, North Africa and the Middle East were usable. The study demonstrated that the responders were better at identifying females (82% correct responses) than larvae (63%). When the responders reported that they were sure of the accuracy of their ID, the success rate of ID increased (92% for females and 88% for larvae). The top three tools used for ID were MosKeyTool (72% of responders), the ID key following Becker et al. [2010. Mosquitoes and their control, 2nd edn. Berlin: Springer] (38%), and the CD-ROM of Schaffner et al. [2001. Les moustiques d'Europe: logiciel d'identification et d'enseignement - The mosquitoes of Europe: an identification and training programme. Montpellier: IRD; EID] (32%), while other tools were used by less than 10% of responders. Responders reporting the identification of mosquitoes using the MosKeyTool were significantly better (80% correct responses) than non-MosKeyTool users (69%). Most responders (63%) used more than one ID tool. The feedback from responders in this study was positive, with the exercise being perceived as halfway between educational training and a fun quiz. It raised the importance of further expanding training in mosquito ID for better preparedness of mosquito surveillance and control programmes.

Title: Évaluation de l'expertise en identification morphologique des espèces de moustiques (Diptera, Culicidae) à l'aide de photomicrographies. Abstract: L'identification précise des espèces d'insectes est une exigence indispensable et difficile pour tout entomologiste, en particulier si l'espèce est impliquée dans des épidémies. Le projet européen MediLabSecure a conçu un exercice d'identification (ID) accessible à tout participant volontaire dans le but d'évaluer et d'améliorer les connaissances en taxonomie des moustiques. L'exercice était basé sur des photomicrographies haute définition de moustiques (26 femelles adultes et 12 larves) prélevées dans le Paléarctique occidental. Soixante-cinq réponses d'Europe, d'Afrique du Nord et du Moyen-Orient ont été utilisables. L'étude a démontré que les répondants étaient meilleurs pour identifier les femelles (82 % de réponses correctes) que les larves (63 %). Lorsque les répondants ont déclaré être sûrs de l'exactitude de leur ID, le taux de réussite de l'identification était meilleur (92 % pour les femelles et 88 % pour les larves). Les trois principaux outils utilisés pour les ID étaient MosKeyTool (72 % des répondants), la clé d'identification du livre de Becker et al. (38%) et le CD-ROM de Schaffner et al. (32 %), tandis que d'autres outils étaient utilisés par moins de 10 % des répondants. Les répondants déclarant identifier des moustiques à l'aide de MosKeyTool étaient significativement meilleurs (80 % de réponses correctes) que les non-utilisateurs de MosKeyTool (69 %). La plupart des répondants (63 %) ont utilisé plus d'un outil d'identification. Les commentaires des répondants de cette étude ont été positifs, l'exercice étant perçu comme à mi-chemin entre une formation pédagogique et un quiz amusant. Il a souligné l'importance d'étendre la formation complémentaire à l'identification des moustiques pour une meilleure préparation des programmes de surveillance et de contrôle des moustiques.

Anti-SARS-CoV-2 Antibody Responses 5 Months Post Complete Vaccination of Moroccan Healthcare Workers.

Data about the duration of antibodies after vaccination show that the protection against SARS-CoV-2 infection begins to decline over time. This study aims to determine anti-SARS-CoV-2 anti-S IgG levels in healthcare workers five months after the second vaccination dose. We collected samples from 82 participants who were fully vaccinated with ChAdOx1 nCoV-19 or BBIBP-CorV. We assessed anti-SARS-CoV-2 IgG antibodies using a Euroimmun ELISA and an Abbott Architect ™ SARS-CoV-2 IgG test. Of the 82 participants, 65.85% were seropositive for IgG using ELISA, and 86.59% were positive for IgG according to the Abbott Architect ™ test. Individuals vaccinated with the ChAdOx1 nCoV-19 vaccine had a median anti-S1 antibody level of 1.810 AU/mL [interquartile range (IQR), 1.080-3.7340] and 171.7 AU/mL [79.9-684.6] according to the Euroimmun ELISA and Abbott Architect test, respectively. These tests indicated that people vaccinated with BBIBP-CorV had a median anti-S1 antibody level of 1.840 AU/mL [0.810-2.960] and 126.7 AU/mL [54.9-474.3], respectively. Statistical analysis showed no significant difference between the positivity rates of the vaccinated individuals, either for gender or for age. In addition, we found no significant difference between the two vaccines. Our study provides information on the longevity of the anti-SARS-CoV-2 IgG antibodies in people at least five months after vaccination.

Serological evidence of West Nile virus infection in human populations and domestic birds in the Northwest of Morocco.

West Nile virus (WNV) was recently detected in Culex pipiens mosquitoes in Morocco. The aim of this study was to evaluate the seroprevalence of WNV in humans and in domestic birds in two regions of Morocco by the detection of IgG antibodies. Blood samples were obtained from 91 human patients and 92 domestic birds from September to December 2019. All study samples were tested using competitive enzyme-linked immunosorbent assay (cELISA) and WNV neutralization tests (VNT) were performed on positive sera. Of all samples, 4 (4.39 %) humans and 4 (4.34 %) birds were found to be seropositive for flaviviruses by the cELISA test. The VNT revealed that three of the four human samples detected positive by cELISA contained neutralizing antibodies against WNV. Two bird samples were confirmed positive by VNT. These results show a significant seroprevalence of anti-WNV antibodies and therefore suggest the active circulation and exposure of human and bird populations in the northwest of Morocco.

Distribution of insecticide resistance and molecular mechanisms involved in the West Nile vector Culex pipiens in Morocco.

BACKGROUND: Mosquitoes of the Culex pipiens complex are the vectors of several arboviruses and are thus subjected to insecticide control worldwide. However, overuse of insecticides selects for resistance. While assessing the resistance status of the vectors is required for effective and sustainable disease control, resistance has so far only been sparsely studied in Morocco. In this study, we establish a first countrywide assessment of the levels of resistance to various insecticides and the potential responsible mechanisms involved. Cx. pipiens larvae were collected from natural populations of five regions of Morocco, and their taxonomic status was determined (molecular forms). The level of their susceptibility to insecticides was assessed by single-diagnostic-dose bioassays. Molecular identification of known resistance alleles was investigated to determine the frequency of target-site mutations. RESULTS: This study confirms that Moroccan populations are an interbreeding mix of pipiens and molestus forms, with large gene flow for the resistance alleles. We also found that Cx. pipiens mosquitoes are resistant to all insecticide families, all over Morocco: resistance is high for insecticides used in mosquito control, but also present for other pesticides. Resistance alleles are similarly more frequent for mosquito control insecticides. However, their distribution is heterogeneous in the five regions, with significant genetic differentiation between populations, revealing the crucial role of local insecticide treatment practices. CONCLUSION: This study provides reference countrywide data that highlight the need for further research to refine the distribution of resistance in Morocco and to understand the role of agriculture/urban residuals in its spread. © 2020 Society of Chemical Industry.

Evidence of circulation of West Nile virus in Culex pipiens mosquitoes and horses in Morocco.

West Nile virus (WNV) is one of the most widely distributed mosquito-borne viruses in the world. In North Africa, it causes human cases of meningoencephalitis with fatalities in Algeria and in Tunisia, whereas only horses were affected in Morocco. The aims of this study were to detect WNV in mosquitoes and to determine seroprevalence of WNV in Moroccan horses by the detection of IgG antibodies. A total of 1455 mosquitoes belonging to four different species were grouped by collection site, date, and sex with 10 specimens per pool and tested for 38 arboviruses using a high-throughput chip based on the BioMark Dynamic array system. Out of 146 mosquito pools tested, one pool was positive for WNV. This positive pool was confirmed by real time RT-PCR. The serosurvey showed that 33.7% (31/92) of horses were positive for competitive enzyme-linked immunosorbent assay (cELISA) test. The flavivirus-sphere microsphere immnoassay (MIA) test, targeting three flaviviruses (WNV, Usutu virus (USUV) and Tick borne encephalitic virus (TBEV)) showed that 23 sera out of 31 were positive for WNV, two for USUV, two for USUV or WNV, and four for an undetermined flavivirus. Virus neutralization tests with USUV and WNV showed that 28 of 31 sera were positive for WNV and all sera were negative for USUV. This study reports, for the first time, the detection of WNV from Culex pipiens mosquitoes in Morocco and its circulation among horses. This highlights that the detection of arboviruses in mosquitoes could serve as an early warning signal of a viral activity to prevent future outbreaks in animals and humans.

Towards harmonisation of entomological surveillance in the Mediterranean area.

BACKGROUND: The Mediterranean Basin is historically a hotspot for trade, transport, and migration. As a result, countries surrounding the Mediterranean Sea share common public health threats. Among them are vector-borne diseases, and in particular, mosquito-borne viral diseases are prime candidates as (re)emerging diseases and are likely to spread across the area. Improving preparedness and response capacities to these threats at the regional level is therefore a major issue. The implementation of entomological surveillance is, in particular, of utmost importance. Guidance in designing entomological surveillance systems is critical, and these systems may pursue different specific objectives depending on the disease. The purpose of the proposed review is to draw up guidelines for designing effective and sustainable entomological surveillance systems in order to improve preparedness and response. However, we make it clear that there is no universal surveillance system, so the thinking behind harmonisation is to define evidence-based standards in order to promote best practises, identify the most appropriate surveillance activities, and optimise the use of resources. Such guidance is aimed at policymakers and diverse stakeholders and is intended to be used as a framework for the implementation of entomological surveillance programmes. It will also be useful to collaborate and share information with health professionals involved in other areas of disease surveillance. Medical entomologists and vector control professionals will be able to refer to this report to advocate for tailored entomological surveillance strategies. The main threats targeted in this review are the vectors of dengue virus, chikungunya virus, Zika virus, West Nile virus, and Rift Valley fever virus. The vectors of all these arboviruses are mosquitoes. METHODS: Current knowledge on vector surveillance in the Mediterranean area is reviewed. The analysis was carried out by a collaboration of the medical entomology experts in the region, all of whom belong to the MediLabSecure network, which is currently funded by the European Union and represents an international effort encompassing 19 countries in the Mediterranean and Black Sea region. FINDINGS: Robust surveillance systems are required to address the globalisation of emerging arboviruses. The prevention and management of mosquito-borne viral diseases must be addressed in the prism of a One Health strategy that includes entomological surveillance as an integral part of the policy. Entomological surveillance systems should be designed according to the entomological and epidemiological context and must have well-defined objectives in order to effect a tailored and graduated response. We therefore rely on different scenarios according to different entomological and epidemiological contexts and set out detailed objectives of surveillance. The development of multidisciplinary networks involving both academics and public authorities will provide resources to address these health challenges by promoting good practises in surveillance (identification of surveillance aims, design of surveillance systems, data collection, dissemination of surveillance results, evaluation of surveillance activities) and through the sharing of effective knowledge and information. These networks will also contribute to capacity building and stronger collaborations between sectors at both the local and regional levels. Finally, concrete guidance is offered on the vector of the main arbovirus based on the current situation in the area.

Epidemiological features of a recent zoonotic cutaneous leishmaniasis outbreak in Zagora province, southern Morocco.

BACKGROUND: Leishmania major is an endemic vector-borne disease in Morocco that causes zoonotic cutaneous leishmaniasis (ZCL), especially in arid pre-Saharan regions where its unique vector and reservoir are Phlebotomus papatasi and Meriones shawi, respectively, and may cause epidemics. In late 2017, the Zagora province, an endemic focus for ZCL in southern Morocco, had CL outbreak. The main objective of our investigation was to analyze the epidemiological features of this latest ZCL outbreak. METHODOLOGY/PRINCIPAL FINDINGS: We analyzed epidemiological features of this latest ZCL outbreak. The Regional Delegation of Health, Zagora, recorded 4,402 CL patients between October 2017 and end of March 2018. Our findings showed that 24 municipalities were affected and majority (55.1%) of infected cases belonged to the Tinzouline rural municipality. Majority of patients were females (57.2%). While all age group patients were affected, those aged <10 years were the most affected (42.1%). During this outbreak over 5 days in December 2017, we conducted a survey in Tinzouline and recruited and sampled 114 CL patients to confirm CL diagnosis by parasitological (direct examination and culture) and molecular (ITS1-PCR) methods and identify the etiological agent of infection using ITS1-PCR-RFLP and sequencing. We completed a detailed questionnaire including clinical and epidemiological data for each patient and found 72.8% of patients presenting multiple lesions (≥2), with an average number of lesions of 5.16 ± 0.5. Lesions were more prevalent in the upper limbs, with the most common type being the ulcerocrusted lesion (60.5%). We detected no associations between lesion type and patients' sex or age. CONCLUSIONS/SIGNIFICANCE: Among 114 clinically diagnosed CL patients, we confirmed 90.35% and identified L. major as the species responsible for this outbreak. Self-medication using various products caused superinfection and inflammation of lesions and complicated the diagnosis and treatment. Thus, ZCL remains a major public health problem in the Zagora province, and commitment of all stakeholders is urgently required to implement a sustainable regional control program.

G119S ace-1 mutation conferring insecticide resistance detected in the Culex pipiens complex in Morocco.

BACKGROUND: Arboviruses are controlled through insecticide control of their mosquito vector. However, inconsiderate use of insecticides often results in the selection of resistance in treated populations, so that monitoring is required to optimize their usage. Here, Culex pipiens (West Nile and Rift Valley Fever virus vector) specimens were collected from four Moroccan cities. Levels of susceptibility to the organophosphate (OP) insecticide malathion were assessed using World Health Organization (WHO)-recommended bioassays. Individual mosquitoes were tested for the presence of the G119S mutation in the ace-1 gene, the main OP-target resistance mutation. RESULTS: Bioassays showed that mosquitoes from Mohammedia were significantly more resistant to malathion than those from Marrakech. Analyzing the ace-1 genotypes in dead and surviving individuals suggested that other resistance mechanisms may be present in Mohammedia. The ace-1 resistance allele frequencies were relatively moderate (< 0.4). Their analyses in three Moroccan cities (Tangier, Casablanca and Marrakech) however showed disparities between two coexisting Cx. pipiens forms and revealed that the G119S mutation tends to be more frequent in urban than in rural collection sites. CONCLUSION: These findings provide a reference assessment of OP resistance in Morocco and should help the health authorities to develop informed and sustainable vector control programs. © 2018 Society of Chemical Industry.

Identification of mosquitoes (Diptera: Culicidae): an external quality assessment of medical entomology laboratories in the MediLabSecure Network.

BACKGROUND: Identification of vectors is of prime importance in the field of medical entomology for both operational and research purposes. An external quality assessment of mosquito identification capacities was carried out within the MediLabSecure Network, which is composed of laboratories located in 19 countries close to the European Union around the Mediterranean and Black seas. METHODS: A set of blind samples consisting of 7 or 8 adult mosquitoes and 4 larvae was given to each participant laboratory. In all, 138 adult mosquitoes and 76 larvae of different species were distributed for genus and species identification. RESULTS: All identifications were exclusively morphology based. Overall, 81% of identifications were correct at the genus level, 64% at the species level. The results were highly varied among the 19 participating laboratories. The levels of correct identifications were: 100% (three laboratories), 90-95% (four laboratories), 50-75% (six laboratories) and < 50% (six laboratories). CONCLUSIONS: This evaluation showed the need to maintain efforts in capacity building and quality control in the field of medical entomology and, more specifically, in the morphological identification of the Culicidae.