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.

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.

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.

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.