|Year : 2022 | Volume
| Issue : 2 | Page : 118-124
Screening of field-caught mosquitoes for the SARS-CoV-2 virus at the southwest of Saudi Arabia
Yasser Alraey1, Gaffar S Zaman2, Sultan M Alhefdi1, Roaa A Asiri1, Sarah M Alshehri1, Sultan Z Alasmari1, Abdulrahim Hakami1, Alghamdi Ibrahim Saeed3, Ayed A Dera1
1 Department of Clinical Laboratory Sciences, Central Research Laboratory, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
2 Department of Clinical Laboratory Sciences, College of Applied Medical Sciences, King Khalid University, Abha, Saudi Arabia
3 Asir Central Hospital, Ministry of Health, Abha, Saudi Arabia
|Date of Submission||07-Nov-2022|
|Date of Decision||15-Nov-2022|
|Date of Acceptance||18-Nov-2022|
|Date of Web Publication||27-Dec-2022|
Dr. Yasser Alraey
Department of Clinical Laboratory Sciences, Central Research Laboratory, College of Applied Medical Sciences, King Khalid University, Abha
Source of Support: None, Conflict of Interest: None
Background: It was observed that the severe acute respiratory syndrome-Coronavirus (SARS-CoV-2) virus is more transmissible in places that are more clustered and where mosquitoes breed in significant numbers. Therefore, we wanted to study the potential influence of mosquitoes in the COVID-19 transmission in the field settings. Materials and Methods: This research was applied in the Asir region of Saudi Arabia. A questionnaire survey was meted out to the quarantined participants diagnosed with the COVID-19 infection. Mosquito traps were located at places with the high rate of human vector contact. A total of 464 female mosquitos were screened for the SARS-CoV-2 S gene utilizing real time reverse transcriptase–polymerase chain reaction. In addition, blood-fed mosquitoes were tested through sequencing of the cytochrome b gene region to identify the source of blood meals. Results: People having low economic status and places with high population density, such as parks and malls, resulted in the individuals “high risk of being infected with the COVID-19 infection. Most of the collected blood-fed mosquitoes exhibited a high match-sequencing with human. All tested mosquitoes had not manifested any SARS-CoV-2S gene amplification. Conclusion: Our results highlight the association of low economic status of individuals and the individuals” behavior of frequently visiting parks, with an elevated risk of contracting the COVID-19 infection. Moreover, using a field experiment for the first time in Saudi Arabia, our data supported the theory that the SARS-CoV-2 virus cannot be transmitted through mosquitoes, either by blood meal or by mechanical transmission.
Keywords: COVID-19, mosquitoes, real-time reverse transcriptase–polymerase chain reaction, severe acute respiratory syndrome-coronavirus-2, vector transmission
|How to cite this article:|
Alraey Y, Zaman GS, Alhefdi SM, Asiri RA, Alshehri SM, Alasmari SZ, Hakami A, Saeed AI, Dera AA. Screening of field-caught mosquitoes for the SARS-CoV-2 virus at the southwest of Saudi Arabia. King Khalid Univ J Health Sci 2022;7:118-24
|How to cite this URL:|
Alraey Y, Zaman GS, Alhefdi SM, Asiri RA, Alshehri SM, Alasmari SZ, Hakami A, Saeed AI, Dera AA. Screening of field-caught mosquitoes for the SARS-CoV-2 virus at the southwest of Saudi Arabia. King Khalid Univ J Health Sci [serial online] 2022 [cited 2023 Mar 22];7:118-24. Available from: https://www.kkujhs.org/text.asp?2022/7/2/118/365762
| Introduction|| |
Since the outbreak of COVID-19 cases, which is caused by the severe acute respiratory syndrome-Coronavirus-2 (SARS-CoV-2), more than 6.6 million COVID-19 cases-associated deaths has been reported worldwide (until mid-November 2022). It has been found that coronaviruses are found mainly in the mammalian and avian species. Since the onset of the pandemic, scientists from all over the world are trying to identify the natural transmission cycles and find out treatment modalities in all aspects of the disease (Pharmacophore-based virtual screening and molecular docking simulation, etc.). Travelling bans, social distancing, safety precautions in touching various kinds of materials, and quarantines are commonly the initial steps taken that are meant to be preventive against novel infectious diseases. Nonetheless, these ancient methods are more often of insubstantial benefit for immensely transmissible diseases. If these methods are imposed on people with rigorous regulation, or in a very disorganized manner, they may prove to be unproductive., It is crucial to interrupt the transmission sequences, which is the most productive pathway for a swift containment of the sharp increase in the magnitude of cases of COVID-19. Home isolation is now becoming conventional in many countries as one of the strategies to curtail the COVID-19 infection. However, the SARS-CoV-2 virus is immensely contagious and has numerous transmission pathways; no medical personnel has any immunity against it. Therefore, it is important to make sure that cluster infection does not occur in families during the isolation interval of the affected individual for no <14 days, except on the condition that outstanding infection control is effectuated. Otherwise, the asymptomatic carriers that have not been diagnosed, later on become a fundamental cause of the outbreak in the community.,
Earlier, it was believed that the replication of the SARS-CoV-2 virus occurs predominantly in the respiratory tract of the infected patient and is less effective in other variety of cells. However, the most frightening aspect is detecting the virus in the peripheral blood, especially in those cases with critical disease,,, which has further aggravated the fear pertaining the vector-borne transmission of the virus. Nevertheless, the likelihood that mosquitoes are able to transmit SARS-CoV-2 was proclaimed improbable by the World Health Organization (WHO) at the beginning of the epidemic. In several experimental surveys that showed that SARS-CoV-2 virus was incapable of reproducing inside the colony-reared Aedes mosquito cells., and intrathoracically SARS-CoV infected colony-reared Aedes Albopictus, Aedes aegypti, and Culex quinquefasciatus in the United States. Contemporary research involving COVID-19-infected patients could not detect the virus presence in their peripheral blood., Another evidence suggesting a lack of viremia is that no SARS-CoV-2 infections have occurred from organ transplants or blood transfusions.
Since a very high viremia is required for the mechanical transmission of viruses by mosquito, even when mosquitoes might be interrupted while sucking on a SARS-CoV-2 patient, contamination of the mouthparts would occur. Although the duration of virus infectivity on contaminated surfaces is unknown, it seems highly unlikely that mechanical transmission by hematophagous arthropods would occur. Studies to find out the potential role of the mosquitoes in transmissibility of the SARS-CoV-2 virus are required because some previous research studies have demonstrated that mosquitoes may become infected with the virus even when they are exposed to the magnitudes of infectious virus that are suspected to be insignificant for infection.,,
So far, literature has not adequately reported on the capability of mosquitoes in SARS-CoV-2 transmission following the intake of a blood meal from an infected SARS-CoV-2 patient. The preceding studies have scrutinized SARS-CoV-2 reproduction in only the colony-reared mosquito varieties. Further, since the COVID-19 infection is recent, no comprehensive studies comparing the susceptibility to the transmission in human beings by mosquitoes have been thoroughly researched, especially in Saudi Arabia. Therefore, our main aim was to find out the possibility of mosquitoes to transmit SARS-CoV-2 infection to humans by utilising reverse transcriptase–polymerase chain reaction (RT-PCR) of SARS-CoV-2 screening on field mosquito strains collected from the public areas in Asir region of the KSA.
| Materials and Methods|| |
In this study, we first conducted a quick questionnaire survey on the quarantined COVID-19-infected people to determine the most-visited places by them during the 14 days prior to their infection. The study was carried out with the ethical approval from the Standing Committee on Ethics of Scientific Research at King Khalid University (Approval Number-ECM#2020-1211) and Regional Committee for Research Ethics at Ministry of Health (MoH) (Approval Number-H-06-B-091).
Mosquito collection from the field
This study was conducted in Abha city at the Asir region during the summer from June to September 2020. Five popular parks in Abha city (high altitude) were chosen for this study: Alandalus, Alshalal, Alsaad, and Almatar park. Further, Mohayel and Al Darb parks in Mohayel and Al Darb cities were included in this study, as it is characterised with low sea level [Figure 1].
|Figure 1: Map showing the location of the Asir region and the sampling parks present within certain cities (Abha, Al Darb and Mhayel cities). Made with ArcGIS 10 (ESRI, Redlands CA)|
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For sample collection, the centers for disease control light trap baited with yeast-generated CO2 was applied for three consecutive nights per week from 7:00 pm to 6:00 am. The collected mosquitoes were kept in Eppendorf tubes containing absolute ethanol for RNA preservation., Groups of five mosquitoes were pooled in one Eppendorf tube based on the mosquito species, place, and the time of collection.
Mosquito screening for severe acute respiratory syndrome-coronavirus-2 S gene
Samples were then homogenized and used for DNA and RNA extraction using magna pure DNA and viral NA kit (Roche). Subsequently, the extracted samples were tested using the Real Star® SARS-CoV-2 RT-PCR Kit 1.0 (Altona diagnostic), to detect the SARS-CoV-2 (target S gene labelled with the fluorophore Cy5) and B-βCoV (target E gene labelled with the fluorophore FAM™). Based on the kit procedure, positive control contains both targets, B-βCoV and SARS-CoV-2 and negative controls (H2O) were used per testfor quality control check. Furthermore, the internal control was added during samples preparation procedure to confirm reagents integrity, which can be identified by specific probe labeled with the fluorophore JOE™.
The Real time RT-PCR was run by LightCycler 480 II (LC480II, Roche company, Basel, Switzerland) and performed according to the manufacturer's instructions. The samples with a cycle threshold value below 35 (fluorophore Cy5 channel) were considered positive amplification of the SARS-CoV-2 S gene. Negative results were detected when there isno amplification for SARS-CoV-2 (fluorophore Cy5) with positive amplification for internal control (fluorophore JOE™).
Mosquito blood meal analysis
The blood-fed mosquitoes were tested through sequencing cytochrome b (cyt-b) gene region from the host mitochondrial DNA to identify the blood meal source utilising modified universal primers Cytb For (5'-CCC CTC AGA ATG ATA TTT GTC CTC A-3') and Cytb Rev (5'-CAT CCA ACA TCT CAG CAT GAT GAA A-3') to amplify a 383 bp segment of the Cyt-b gene. The amplified genes were purified by a QIAquick Gel Extraction Kit, sent for sequencing, and matched with the GenBank database. Species sequences with the highest identity were chosen as the most likely blood meal source. Earlier modified BOOTSCAN algorithms has been utilized to screen nucleotide sequence alignments for any evidence that recombination might have occurred without identifying of the nonrecombinant sequences used as reference.
R version 1.3.1056 (R project, Statistical Software): was used for all data analysis. A binomial test was applied to calculate the significant difference in the expected proportion of visited places. Expected proportion differences with P < 0.05 in the binomial analysis were considered as being statistically significant. SPSS Statistics for Windows, Version 24.0. Armonk, NY, USA was employed in this study for data descriptive analysis.
| Results|| |
Questionnaire survey result
First, our main aim was to find out the quarantined individuals due to COVID-19 infection, in various hotels of Asir region. A preliminary data of quarantined people in Asir district was collected from MoH between the month of March and September 2020. The data showed that 4031 individuals were quarantined in hotels due to COVID-19 infection [Figure 2].
|Figure 2: The frequency of individuals whom quarantined in hotels due to COVID-19 infection in Asir region from March to September 2020, Data was collected from Saudi ministry of health|
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Among those patients, 350 (221 males and 129 females) participants infected by the COVID-19 were considered. The majority of subjects were middle-aged and 57% of them (201) were characterised as low income. The socio-demographic data are represented in [Table 1]. Loss of smell and taste with headache were the most common symptoms found in participants and the majority of these symptoms lasted from 4 to 10 days [Figure 3].
|Figure 3: Most common symptoms with period of 350 quarantined participants infected by COVID-19 in Asir region|
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|Table 1: Demographic characteristics of 350 quarantined participants infected by COVID-19 in Asir region|
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Furthermore, the majority of those participants were nonsmokers and have not complained from any chronic diseases. In addition, only few of them had contacted animals during 14 days before the appearance of the symptoms, and cat were the most contacted animal [Figure 4].
|Figure 4: Health information and animal contact of 350 quarantined participants infected by COVID-19 in Asir region|
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We inquired the respondents regarding their most-visited places within 14 days before the appearance of the symptoms. Places with high population density, such as parks and malls, likely put individuals at high risk of COVID-19 infection as represented in [Table 2], especially with parks (258 out of 350 subjects) as showing a significant difference in expected proportion (P ≤ 0.001). This underscores the possible risk factors of the COVID-19 infection present within parks that should be investigated, such as the effect of mosquitoes on transmissibility.
|Table 2: Possible risk areas that participant visited 14 days ahead of COVID-19 symptoms appearance|
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Collected mosquito species and severe acute respiratory syndrome-coronavirus-2 S gene real-time reverse transcriptase–polymerase chain reaction
A total of 464 female mosquito species namely Culex (n = 213), Anopheles (n = 176), and Aedes (n = 75) were collected from the concerned areas of Asir region. 94 tubes of five pooled mosquitos were molecularly tested for the presence of the SARS-CoV-2 virus by standard methods. The 464 female mosquitos were screened for the SARS-CoV-2 S gene utilizing qRT-PCR. In addition, blood-fed mosquitoes were tested through sequencing of the cyt-b gene region to identify the source of blood meals. Positive and negative controls worked as expected, and the positive amplification of the internal control was detected in the 94 tubes. However, all tubes showed negative amplification of the SARS-CoV-2 S gene.
Blood meal analysis
A total of 125 blood-fed mosquitos were analyzed to identify the blood sources. Based on the date and collection sites, samples were pooled into 25 tubes (with 5 mosquitoes in each tube). 7 tubes of pooled samples showed mixed blood meals that could not be identified by sequencing. Among the remaining tubes (n = 18), 16 tubes showed a high match to humans (Homo sapiens-KX697454) with identity 99% (1e-150). At the same time, only two tubes revealed a high match to cats (Felissilvestris-KP202275) with identity 94% (3e-150).
| Discussion|| |
The information collected in this survey was mostly focused on the demographic profile, economic, and social situation to identify possible risk groups for the COVID-19 infection. Although the sample size is small, it was fairly evident that individuals with low income faced a greater risk of being infected with the COVID-19 infection. In this respect, our research is similar to various other studies that have shown that economically poor classes of people were the most adversely affected groups by the COVID-19 pandemic.,,, This may be related to the personal conduct and attitude of the individuals, their social activities, places visited by them, and their socioeconomic level. These factors enhance the risks of contracting the COVID-19 infection. Therefore, further studies are required to substantiate the association between the individuals with low socioeconomic status and the risks of contracting the COVID-19 infection in terms of prevention.
Places with high population density, such as parks and shopping malls, likely put individuals at a high risk of contracting the infection, especially in parks, as showing a significant difference in expected proportion. This coincides with the observations and study conducted by Bhadra et al., Rocklov et al. and Rocklov and Sjodin,, who found a positive correlation between the COVID-19 infection and the subsequent mortality, with the density of the population. On the other hand, we noticed that most quarantined participants were not exposed to close interactions observed in places such as health centres, government offices, sports centres, hotels, schools, colleges, and the public transport. This highlights the possible risk factors of COVID-19 infection present within parks; as noticed in some other studies.,
In the Asir region of Saudi Arabia, with the approach of the favorable season for the breeding of mosquitoes, the COVID-19 pandemic triggered the apprehension of the health authorities and the general population for potential dissemination of the disease through mosquito bites. As some studies that have shown the presence of SARS-CoV-2 virus in the blood of some of the infected patients. In our research, we wanted to investigate the possible involvement of mosquitoes in the transmission of the COVID-19 infection, either by blood meal or mechanical transmission through the contamination of mosquito body parts. Among mosquito species, Culex (mainly Culex pipiens and Culiseta longiareolata) is the most frequently found in the Asir province. Others comprise of Culiseta, Anopheles, and Aedinespp.
Mosquito collection was carried out during the summer season. This time was considered owing to the summer vacation, when the prevalence of the infection and outdoor human activities were at their highest in the Asir region, as it is one of the most attractive tourist areas in Saudi Arabia during the period. Therefore, considering the aim of this study and our questionnaire survey analysis, mosquito traps were located at places where the rate of human-vector contact was high, such as parks. This was verified by mosquito blood meal sources analysis which revealed that most of the 125 blood-fed mosquitoes had fed on humans. Based on our collection, most Culex species were collected in Abha city (~2200 m above sea level), whereas most Aedes and Anopheles species were collected from Al Darb and Mhayel cities (~400 m above sea level).
The whole mosquito's body was used for RNA and DNA extraction to investigate the possible mechanical and viral blood meal transmission of SARS-CoV-2. However, the field-collected mosquitoes did not show any positive amplification of the SARS-CoV-2 S gene. This can be due to two reasons. The first reason is that field-collected mosquitoes have not come in contact with infected persons. The second reason could be that he mosquitoes cannot transmit SARS-CoV-2 virus as demonstrated in previous studies, which supports WHO's notion of the mosquitoes' inertness in the COVID-19 transmission. To exclude the first reason, it is very difficult to assure the presence of COVID-19 infected individuals at the selected parks. Therefore, we tried to collect mosquitoes at the time when the circulation of SARS-CoV-2 among the general population was high (June to September 2020). The number of recorded COVID-19 cases had reached a peak, based on the Saudi MoH records. However, although it is a known fact that the environmental determinants on the human genome may also impact scenario responsible for current wide spread epidemics, we were unable to pursue it and will make another project for it in the near future.
| Conclusion|| |
Our results highlight the association between low socioeconomic status and the risk of the COVID-19 infection. Moreover, it underscores the possible risk factor of contracting the infection in open parks, which need future investigations to be carried out in terms of COVID-19 prevention. In addition, concluding from our field experiment, our data supports the WHO's assertion that SARS-CoV-2 cannot be transmitted by mosquito, either by blood meal or mechanical transmission. To our knowledge, nobody has conducted a significant number of field studies in Saudi Arabia, comparable to this study, to investigate the mosquitoes' involvement in the transmission of SARS-CoV-2.
Ethics approval and consent to participate
The study was carried out under ethical approval from the Standing Committee on Ethics of Scientific Research at King Khalid University (Approval Number-ECM#2020-1211) and Regional Committee for Research Ethics at MoH (Approval Number-H-06-B-091).
Availability of data and material
All data generated or analyzed during this study are included in this published article.
Financial support and sponsorship
This project was funded by King Khalid University, Institute for Research and Consulting Studies Administration of Contracts grant# 24-3-S-2020.
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4]
[Table 1], [Table 2]