PLOS NEGLECTED TROPICAL DISEASES
RESEARCH ARTICLE
Entomological surveillance on Aedes aegypti
during covid 19 period in Cape Coast, Ghana:
Risk of arboviral outbreaks, multiple
insecticide resistance and distribution of
F1534C, V410L and V1016I kdr mutations
Joana Ayettey1, Aikins Ablorde2, Godwin K. Amlalo3, Ben A. Mensah1, Andreas
a1111111111 A. Kudom 1ID *
a1111111111
1 Department of Conservation Biology and Entomology, University of Cape Coast, Cape Coast–Ghana,
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2 Center for International Health, Ludwig Maximilian University of Munich, Germany, 3 Department of
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Parasitology, Noguchi Memorial Institute for Medical Research, University of Ghana, Accra Ghana
a1111111111
* akudom@ucc.edu.gh
Abstract
OPEN ACCESS
Citation: Ayettey J, Ablorde A, Amlalo GK, Mensah
BA, Kudom AA (2023) Entomological surveillance Background
on Aedes aegypti during covid 19 period in Cape
Coast, Ghana: Risk of arboviral outbreaks, multiple The study assessed the risk of transmission of Aedes-borne arboviruses in a community at
insecticide resistance and distribution of F1534C, Cape Coast during the Covid-19 restriction period in 2020 based on entomological indices.
V410L and V1016I kdr mutations. PLoS Negl Trop
The spatial distribution of insecticide resistance was also assessed in Ae. aegypti population
Dis 17(5): e0011397. https://doi.org/10.1371/
journal.pntd.0011397 from Cape Coast.
Editor: Mariangela Bonizzoni, Universita degli Studi
di Pavia, ITALY Methods
Received: September 8, 2022 Three larval indices were calculated from a household larval survey in 100 randomly
selected houses. WHO susceptibility bioassay was performed on female adult Ae. aegypti
Accepted: May 22, 2023
that were reared from the larvae collected from household containers and other receptacles
Published: May 31, 2023
located outside houses against four insecticides. The mosquitoes were also screened for
Peer Review History: PLOS recognizes the F1534C, V1016I, and V410L kdr mutations.
benefits of transparency in the peer review
process; therefore, we enable the publication of
all of the content of peer review and author Results
responses alongside final, published articles. The
The estimated larval indices in the study community were House index– 34%, Container
editorial history of this article is available here:
https://doi.org/10.1371/journal.pntd.0011397 index– 22.35%, and Breteau index– 2.02. The mosquito population was resistant to Delta-
© methrin (0.05%), DDT (4%), Fenitrothion (1%), and Bendiocarb (0.1%). A triple kdr muta-Copyright: 2023 Ayettey et al. This is an open
access article distributed under the terms of the tion, F1534C, V410L and V1016I were detected in the mosquito population.
Creative Commons Attribution License, which
permits unrestricted use, distribution, and Conclusion
reproduction in any medium, provided the original
author and source are credited. The study found the risk of an outbreak of Aedes-borne diseases lower in the covid-19 lock-
Data Availability Statement: The authors confirm down period than before the pandemic period. The low risk was related to frequent clean-up
that all data underlying the findings are fully exercises in the community during the Covid-19 restriction period. Multiple insecticide
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PLOS NEGLECTED TROPICAL DISEASES Risk of Aedes arboviral outbreaks in Covid 19 period in Cape Coast, Ghana
available without restriction. All relevant data are resistance couple with three kdr mutations detected in the study population could affect the
within the paper and its Supporting Information effectiveness of control measures, especially in emergency situations. The study supports
files.
sanitation improvement as a tool to control Ae. aegypti and could complement insecticide-
Funding: The author(s) received no specific based tools in controlling this vector.
funding for this work.
Competing interests: The authors have declared
that no competing interests exist
Author summary
The study assessed the risk of transmission of Aedes-borne arboviruses in a community in
Cape Coast during the Covid-19 restriction period in 2020. Three larval indices were cal-
culated from the household larval survey in 100 randomly selected houses in the commu-
nity. Insecticide susceptibility of the female adult Ae. aegypti to four insecticides was
performed. The results showed that the risk of a potential outbreak of Aedes-borne dis-
eases in the Covid 19 restriction period was much lower than in 2017 when a similar
assessment was done. The low risk was related to frequent clean-up exercises in the com-
munity during the Covid-19 restriction period. The mosquito population was resistant to
deltamthrin, DDT, bendiocarb and fenitrothion insecticides. However, resistance level
was higher in the mosquitoes collected in houses than the mosquitoes collected outside
houses. Insecticide use in houses may have contributed to the different level of resistance
observed. The study detected F1534C, V1016I and V410L kdr mutations in Ae. aegypti
population. The combination of these mutations is known to generate high levels of pyre-
throid resistance in the mosquito. The study agrees with sanitation improvement as a tool
to control Ae. aegypti and could complement insecticide-based tools in controlling this
vector.
Introduction
Aedes aegypti is a medically important mosquito due to its involvement in the transmission of
several arboviruses to humans. In the last decade, there have been several epidemics of Ae.
aegypti-borne diseases in many countries. In 2016, two linked urban yellow fever outbreaks
were reported in Luanda (Angola) and Kinshasa (the Democratic Republic of the Congo) in
Central Africa [1]. In the same year, large dengue outbreaks in the Region of the Americas
reported more than 2.38 million cases [1]. In 2015–2016, the Zika virus disease outbreak was
also reported in some countries in South America. Sadly, as the world battles with the Covid-
19 pandemic, there is also a surge of Aedes-borne arboviral diseases in some countries. For
instance, between January and June 2020, about 1.6 million dengue cases, 37,000 chikungunya
cases, and 7000 Zika cases have been reported in the region of the Americas [2]. The combined
impact of both Covid-19 and outbreaks of Aedes-borne arboviral diseases could have poten-
tially devastating consequences, particularly in low-resource countries. This calls for advanced
preparation by endemic countries against any Aedes-borne disease outbreak.
In urban areas, Ae. aegypti normally breeds in water storage containers as well as other
man-made receptacles that can hold water [3,4]. There is a serious concern that the use of
these storage receptacles could increase in this pandemic period. Because it is anticipated that
the demand for water in households would increase due to some Covid-19 mitigation mea-
sures such as regular washing of hands and other hygienic practices that require a constant
flow of water. In fact, for this reason, the Ghana government declared a ‘free water for all’ ini-
tiative on 5 April 2020 where water was supplied for free in the country as a relief measure
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PLOS NEGLECTED TROPICAL DISEASES Risk of Aedes arboviral outbreaks in Covid 19 period in Cape Coast, Ghana
during the Covid-19 restriction period [5]. An increase in water demand, particularly in
households with limited access to pipe water, may lead to increased use of water storage con-
tainers. The danger is that if the containers are not managed well, they could serve as a breed-
ing place for Ae. aegypti and other container-breeding mosquitoes. A similar situation was
reported during a drought period in Australia in 2008 [6]. Household water storage practices
in response to drought resulted in new habitats for domestic container-breeding mosquitoes
[6]. Such conditions could affect the epidemiology of mosquito-borne diseases and their
control.
In Ghana, about 49% of households use pipe-borne water as the main source of water for
general use. However, only 10.6% of households have piped-borne water inside their premises
(GLSS7). Before the Covid-19 outbreak, more than 80% of households in Ghana stored water
in plastic containers/buckets (GLSS7). The high density of Ae. aegypti population in Ghana
has been attributed to water storage practices and poor sanitation conditions [4,7,8]. With the
anticipated increase in water storage practices in the Covid-19 period, the question is, would
the risk of a potential outbreak of Aedes-borne arboviruses increase as compared to the period
before the pandemic?
Emergency control measures presently depend on insecticide-based strategies, especially,
pyrethroid insecticides. Unfortunately, the emergence and development of insecticide resis-
tance among mosquito vectors could affect the effectiveness of insecticide-based strategies. For
example, evidence of reduced effectiveness of space spray for control against pyrethroid-resis-
tant Ae. aegypti has been reported in the island of Martinique [9]. This calls for continuous
monitoring of insecticide resistance status of vector populations and development of resistance
management strategies. Management of insecticide resistance requires understanding of the
major resistance mechanisms in the vector population as well as patterns of cross resistance
among various classes of insecticides. Two major physiological mechanisms involved in resis-
tance among vector mosquitoes are 1) alteration of target sites, and 2) increased metabolic
detoxification. However, knockdown resistance (kdr) mutation in the voltage-gated sodium
channel, which is responsible for conferring cross resistance between pyrethroids, and DDT
are the most studied mechanism in Ae. aegypti [10–12]. About ten major kdr mutations have
been detected in Ae. aegypti [13]. The widely distributed kdr mutation in Ae. aegypti popula-
tion in Africa is F1534C mutation, which together with V1016I were first detected in Africa
from Ae. aegypti population from Ghana [14]. The mutations have since been found in differ-
ent African countries [15–17]. As part of the entomological surveillance on Aedes aegypti dur-
ing covid 19 period in Cape Coast, we investigated the spatial distribution of insecticide
resistance in the study area. The question is, in case of an outbreak, is the current insecticide-
based mosquito control strategy completely reliable for emergency control? Answers to these
questions may help prepare in advance against any potential disease outbreaks from Ae.
aegypti in the country.
Materials and methods
Study area
The study was conducted in Cape Coast (5˚060N 1˚150W; 5.1˚N 1.25˚W), a coastal city of
approximately 122 km2 of land, which is about 165 km west of Accra, the capital of Ghana.
Cape Coast has a population of about 189,925 to the 2021 Census (Ghana Statistical Service).
The major rainy season is between May and July with mean monthly relative humidity ranging
from 85 to 99%. The total monthly rainfall and mean daily maximum temperature for 2017
and 2022 have been summarized in Fig 1. The city has several coastal thickets (remnant for-
ests) containing several species of primates [18].
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Fig 1. Total monthly rainfall and mean monthly maximum daily temperature recorded for Cape Coast in 2017 and 2020.
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Covid 19 situation in Ghana
In Ghana, there have been 168,580 confirmed cases of Covid-19 with 1,459 deaths as of August
2022 [19]. The first case of Covid 19 was reported in March 2020 and the country saw a sharp
rise in infections in many parts of the country. Initially, the government of Ghana banned all
public gatherings, including conferences, workshops, funerals, festivals, political rallies and
church activities. Basic and high schools, as well as universities, were closed to reduce the
spread of the virus. The government also imposed 21-day partial lockdown restrictions on
Accra, Kumasi, and Kasoa, after they emerged as hotspots for the spread of the disease. This
period is what this study described as Covid 19 restriction period. This study was conducted
from July to September 2020 in Duakor, a community in Cape Coast. During this period,
schools were still closed. However, the final year students of both the universities and the high
schools were back to school to write their final examinations. In that period, piped water was
supplied for free in the country by the government of Ghana as a relief measure against the
impact of Covid 19. The risk of transmission from the present study was compared with a sim-
ilar study conducted in 2017 in the same area. The total rainfall during the present study
period, July to September 2020 and the previous study April to July 2017 were 12.8 mm and
125.6 mm respectively.
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Household larval survey
Duakor is a suburb of Cape Coast with a poor structural layout. Most households depend on
rainwater, communal taps, and wells. Containers are of great importance in this area for the
storage of water. Following the procedures outlined by WHO [20], a survey frame of houses in
Duakor was obtained and a simple random sampling method was employed to choose 100
houses from the community for the study. Pre-adult mosquito stages were collected from the
selected households during the entomological survey. The number of receptacles in and
around each house was recorded and the water containers were searched for immature mos-
quito forms, which were collected for identification in the laboratory. The water containers
found were recorded as 1) those used for water storage (category A) and 2) discarded contain-
ers (category B). From the house data, House Index (HI), Container Index (CI), and Breteau
Index (BI) were calculated [20]. The House Index (HI) was expressed as the percentage of
houses infested with Aedes larvae and Container index (CI) as the percentage of containers
infested with the larvae while Breteau index (BI) was expressed as the number of positive con-
tainers per 100 inspected houses.
Insecticide resistance bioassay
The level of insecticide resistance of Ae. aegypti to Deltamethrin (0.05%), DDT (4%), Feni-
trothion (0.1%) and Bendiocarb (0.1%) were determined following WHO standard protocols
using the WHO impregnated papers and test kits [21]. The first emergence (F0) of Ae. aegypti
collected as larvae from household water storage containers (HWSC) located inside houses as
well as abandoned car tires (ACT) and abandoned plastic containers found outside the resi-
dential houses were used for the susceptibility bioassay. About twenty-five, unfed 3-to-5-day-
old females were exposed to each insecticide with four replications for each of the three vector
populations. The laboratory conditions for the bioassay were 29 ± 1˚C and relative humidity
of 75 ± 10%. After the exposure, mosquitoes were transferred to holding tubes, fed with 10%
sugar solution and knockdown read after one hour. Total death or mortality was read after 24
hours. For control, mosquitoes were exposed to paper without any insecticides. The mortalities
caused by the insecticides were expressed in percentages.
Screening for kdr mutations
DNA of hundred adult female mosquitoes from the three populations (HWSC, ACT, APC)
was extracted using the QuantaBio Extracta kit according to the manufacturer’s direction.
Knock-down resistance (kdr) mutations were screened using qPCR melting curve analysis.
The Saavedra-Rodriguez et al. [22] (V410L) and Estep et al. [23] (V1016 and F1534C) proto-
cols were optimized and modified for this work. For V410L detection, a total of 20μL reaction
contained 0.05μM of V410fw and L410fw primers and 0.1μM of primer 410rev, 9.5μL of 2x
Sybr Hi-Rox Mix (Bioline), 1μL of genomic DNA and DNase-free water. V1016I detection was
done in a 20μL reaction consisting of 8.2μL of 2x Sybr Hi-Rox Mix (Bioline), 0.15μM of
Val1016f primer, 0.2μM each of lle1016f and lle1016r primers, 2μL DNA template and DNase-
free water. The cycling condition for this procedure is 95˚C for 3minutes, 40x (95˚C: 10sec,
60˚C:10sec,72˚C: 30sec) 95˚C with the melting condition of 65˚-95˚ inc 0.2˚C per 10sec. Each
20μL reaction for V1016I consisted of 8μL of 2x Sybr Hi-Rox Mix (Bioline), 0.15μM of
Val1016f primer, 0.2μM each of lle1016f and lle1016r primers, 2μL DNA template and DNase-
free water, with the same cycling conditions as the V410L. F1534C was screened in a reaction
volume of 20μL containing 8μL of 2x Sybr Hi-Rox Mix (Bioline), 0.3μM of Cys1534+ primer,
0.3μM each of Phe1534+ and 1534- primers, 2μL DNA and DNase-free water. The cycling
conditions for F1534C included 3min at 95˚C, 37 cycles of (10 sec at 95˚C, 10 sec at 57˚C, 30
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sec at 72˚C) and 95˚C for 10 seconds. A melting curve analysis was performed at the end of the
PCR cycle at 65˚C—95˚C. The mutant gene (resistant) and wild type (susceptible) respectively
produced melting curve peaks at 83˚C and 86˚C for V410L, 80˚C and 86˚C for V1016I, and
86˚C and 82˚C for F1534C. A chi-square test for deviation from Hardy-Weinberg equilibrium
of the genotype frequency distribution was performed using Gene-Cal.
Results
Household container survey
In the houses surveyed, 850 water-holding containers were found in 100 houses. About 22% of
the containers in 32 houses were positive for mosquito larvae (Fig 2). A total of 2829 larvae
and 481 pupae were collected from the positive containers (Table 1). Water storage containers
in the houses (Category A containers) harboured 84% and 96% of total larvae and pupae that
were collected respectively. Pupal productivity was highest for buckets (57.4%) whereas pupal
productivity for cement tanks and barrel/drums were 29.9% and 12.8% respectively (Fig 3).
Aedes larvae constituted 66.8% of the total larvae with 27.4% being Culex while 4.3% and
1.45% were Lutzia and Anopheles larvae respectively. All the 481 pupae collected were Aedes
mosquitoes. The estimated larval indices in the study community were House index– 34%,
Container index– 22.35%, and Breteau index– 2.02.
Fig 2. Spatial distribution of households that were positive or negative for mosquito larval-infested items. The shapefiles were created with ArcGIS from
GPS coordinates collected in the field, and the base map was sourced from OpenStreetMap (https://www.openstreetmap.org).
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Table 1. Number of containers found in households with infestations of mosquito larvae and the total number recorded.
Items Category Specific Items Number of Items (% Total larvae Percentage of larvae: Total pupae
infested) collected Aedes Culex Lutzia Anopheles collected
Category A: household water storage bucket 306 (2.9%) 65 100 - - - 265
containers 5–20 L plastic 18 (61%) 569 37.4 59.8 2.8 - 0
containers
Concrete tank 39 (51%) 1088 57.7 33.6 4.9 3.8 138
barrel/drum 54 (19%) 656 95.9 0 4.1 0 59
bowls/jars 24 (0%) 0 - - - - 0
Total 441 (11%) 2378 64.6 29.7 4.0 1.7 462
Category B: discarded items tires 13 (39%) 241 88.8 - 11.2 0 15
coconut shell 313 (0%) - - - - - -
bowls/jars 15 (33%) 100 30.0 70.0 - - 0
bottles 170 (76%) 110 100 - - - 4
Total 511 (27%) 451 78.5 15.5 6.0 0 19
https://doi.org/10.1371/journal.pntd.0011397.t001
Fig 3. Different containers infested with Aedes aegypti larvae in Duakor community. Cement tank (A), drum (b) of
categeory A items and coconut shells (C) and bottles (D) of category B items.
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Insecticide resistance in the Aedes aegypti population collected from
household water storage containers (inside houses) and abandoned
automobile tires and plastic containers (outside residential houses)
In total, Ae. aegypti from the study community were resistant to the four insecticides (Fig 4).
However, resistance differed in the Ae. aegypti collected from the different breeding habitats
(Fig 5). Besides deltamethrin, Ae. aegypti collected from the household water containers
(HWSC) were more resistant to DDT (P = .002), Fenitrothion 1% (P = .005), and Bendiocarb
0.1% (P = 0.002) than those collected from the abandoned automobile tires (ACT) or plastic
containers (APT) outside residential houses. It was more pronounced in Bendiocarb and DDT
insecticides (Fig 5). A mean %mortality of 32 ± 20 and 24 ± 14.9 of Bendiocarb and DDT were
recorded for the vector population collected from household water storage containers, which
were significantly lower than 94 ± 2.8 and 74.7 ± 9.2 for the population collected from automo-
bile tires and 88 ± 45.7 and 60 ± 12.7 for those collected from abandoned plastic containers
against the same insecticides respectively. Similarly, the vector population collected from out-
side the residential houses were almost susceptible (ACT: 97.3 ± 4.6; APC: 98 ± 2.3) to Feni-
trothion whereas the insecticide caused a mean %mortality of 74 ± 12.4 to the vector
population collected from HWSC.
Among the 100 mosquitoes, 98, 97 and 96 were successfully genotyped for the F1534C,
V410L and V1016I kdr mutations, respectively (Tables 2 and S1). The homozygote mutant at
codons 1016 and 410 was absent whereas the wild type of F1534C was rarely (2%) observed.
F1534C mutation was widely distributed in the study population with the heterozygote F /
C1534 genotype mutant dominating in the population. Higher allelic frequency of F1534C
mutation was observed in the Ae aegypti population collected from HWSC compared to those
collected outside residential houses (ACT and APC) (Table 2) (X2 = 33.93, df = 2, p< .0001).
Fig 4. Susceptibility of female adult Aedes aegypti from Cape Coast to Deltamethrin (0.05%), DDT (4%),
Fenitrothion (0.1%) and Bendiocarb (0.1%) using WHO test kits.
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Fig 5. Susceptibility of female adult Aedes aegypti collected from household water storage containers (HWSC)
(inside houses) and those collected from abandoned car tires (ACT) and abandoned plastic containers (APC)
(outside residential houses) to Deltamethrin (0.05%), DDT (4%), Fenitrothion (0.1%) and Bendiocarb (0.1%)
using WHO test kits.
https://doi.org/10.1371/journal.pntd.0011397.g005
However, V1016I kdr mutation was absent in the population from the HWSC whereas low
allele frequency was observed in the populations collected outside the residential houses (APC
and ACT). Nine genotypes across the three kdr mutations were identified from the 93 Ae.
aegypti population from Cape Coast (Fig 6). The genotype FC/VL/VV was the most frequently
observed (37%, n = 93). Individuals that were homozygous for the three kdr mutations were
absent. Overall, the genotype frequency distribution of V1016I was consistent with Hardy-
Weinberg equilibrium (X2 = 0.233, df = 1, p = .63). However, the genotype frequency distribu-
tion of F1534C and V410L were not in Hardy-Weinberg equilibrium (X2 = 7.6, df = 1, p =
.006; X2 = 28.3, df = 1, p< .0001).
Table 2. V1016I, V410L and F1534C genotype numbers and the allelic frequency of the I, L and C mutations of Ae. aegypti collected from different breeding
habitats.
Source of mosquito V1016I V410L F1534C Kdr allele frequencies
V/V V/I I/I V/V V/L L/L F/F F/C C/C I L C
Household water storage containers 23 0 0 10 12 0 0 2 22 0 0.27 0.96
Abandoned car tires outside houses 43 7 0 9 41 0 2 32 16 0.07 0.41 0.64
Plastic containers outside houses 21 2 0 10 15 0 0 16 8 0.04 0.30 0.67
Total 87 9 0 29 68 0 2 50 46 0.05 0.35 0.72
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Fig 6. Frequency of the combined V1016I, V410L and F1534C genotypes of Aedes aegypti from Cape Coast.
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Discussion
The study assessed the risk of transmission of Aedes-borne arboviruses in the Duakor commu-
nity in Cape Coast during the Covid-19 lockdown period in 2020 based on three larval indices:
container index, house index and Breteau index. The results were compared with a previous
study conducted in 2017 in the community before the Covid-19 pandemic. The assumption
was that Covid-19 mitigation strategies could increase the demand for water, which in turn,
might influence significant changes in householder behavior towards water storage practices.
A change in behavior that would lead to increased use of water storage containers may pro-
mote the breeding of Ae. aegypti and other container-breeding mosquitoes. This may lead to
an outbreak of mosquito-borne diseases. Among the three indices determined in the study,
only the container index marginally exceeded the WHO threshold. Both Breteau and house
indices were below the WHO threshold. Contrary to the assumption made, the result on the
larval indices suggested a lower risk of transmission of Aedes-borne diseases in the Covid-19
restriction period compared to the 2017 survey period.
In the previous study in Cape Coast in 2017, discarded and abandoned containers/recepta-
cles (category B) were important breeding grounds in all the four communities and accounted
for 60% of the infested containers. Water storage containers were less important in the three
communities that had better access to pipe water. However, water storage containers together
with discarded items (category B items) were equally important in Duakor community (the
study area of the present study), which had the least access to piped-borne water. Duakor
accounted for 77% of infested household water storage containers among the four study com-
munities [4]. In this study, fewer category B items were observed to have been infested with
mosquito larvae, and this may have contributed to the lower larval indices compared to the
study before the pandemic [4]. The category B containers contributed only 4% to the total
pupae collected in the study. During the Covid-19 restriction period, there were frequent
clean-up exercises in both public and residential areas [24] leading to the removal of discarded
items from the compounds. This may have improved the sanitation condition in the study
community resulting in fewer mosquito-infested category B items. The total precipitation dur-
ing the present survey period (July-September) was extremely lower than the survey period
(April-July) in the previous study (Fig 1). This could also have contributed to the differences in
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the infested category B items recorded between the two studies. However, we think precipita-
tion might not have contributed much on the infestation of category B items in the present
study. For precipitation to influence the level of infestation of category B items, discarded con-
tainers must first be available. Unlike the previous study, discarded items were extremely low
in this study due to the clean-up exercise and other factors mentioned earlier. It is worth men-
tioning that the bottles and coconut shells, which formed 95% of category B items in the pres-
ent study were technically not discarded or abandoned items (Fig 3C and 3D). The bottles and
the coconut shells were packed because of restricted movement during that period. They were
waiting to be transported to their destinations.
Furthermore, the Government of Ghana’s free water initiative greatly improved water
accessibility in Ghana during the Covid-19 restriction period [25] and this initiative may have
minimized water stress in the community. The limitation of this study was that the larval sur-
vey was conducted only in one community. A larval survey from different communities with
different levels of accessibility to pipe-borne water may have provided a better picture of the
situation than one community. However, Covid-19 restrictions made it very difficult to
include other communities during the study period. Another limitation of the study was that
the infectious status of the mosquitoes collected were not determined. Although, entomologi-
cal surveillance tools such as the use of larval or pupal indices to assess the risk of outbreak of
Aedes borne diseases remain important, especially in low endemic areas like Ghana, informa-
tion on the infectious status of the mosquitoes could have given a better picture of the situa-
tion. A future study that would determine the infectious status of the vector together with the
entomological indices may give a better risk assessment of the situation in study area.
Notwithstanding, health education on mosquito prevention in the community is still
needed. All the houses that were surveyed in the study community had water storage contain-
ers that were either partially covered or not covered at all. The practice of storing water has
been reported to be a reason for the increase in mosquito productivity [26,27]. It was therefore
not surprising that the majority (86%) of the mosquitoes collected in the study came from
water storage containers. Health education such as the promotion of the use of lids to cover
long-term water storage containers can drastically reduce the presence of Aedes larvae in the
community [28]. The promotion of this simple technique could have been incorporated into
the Covid-19 public health campaigns. A recent study in Cape Coast found that intensive pub-
lic health education during the pandemic has greatly improved health knowledge among the
population [29]. However, the improved knowledge was related to chronic diseases, nutrition,
hygiene, and risky health behaviors. Improving the knowledge of anti-mosquito strategies in
the community may also help further lower the population density of Aedes mosquitoes and
ultimately the risk of an outbreak of Aedes-borne diseases.
The most productive containers are identified by determining the relative contribution that
a particular container type makes to the overall production of Aedes pupae [20]. The most pro-
ductive containers from this study were concrete tanks and medium to large plastic containers.
However, buckets were the most productive contributing to more than 50% of the pupae col-
lected in this study. This is consistent with the findings from other countries where concrete
washbasins, drums and buckets are the most productive household containers for Aedes mos-
quitoes [30–32]. Mosquito larvae feed primarily on aquatic microorganisms that colonize
detritus in breeding habitats and the chemicals produced by the microorganisms also influence
the oviposition behaviors of adult female mosquitoes [33]. Under normal conditions, it may
take some time for microorganisms to colonize and build up their population in water-filled
containers. It is therefore not surprising that this study and many other findings have shown
containers (e.g., cement tanks, barrels) that are used to store water for a long period to be
more productive than containers (e.g., bowls, jars) that are mostly used to store water for short
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PLOS NEGLECTED TROPICAL DISEASES Risk of Aedes arboviral outbreaks in Covid 19 period in Cape Coast, Ghana
period [27,30,32]. Targeting these containers for vector control may greatly reduce the popula-
tion of Aedes in the community.
Although Ae. aegypti constituted the major mosquito larvae collected, Culex, Anopheles and
Lutzia mosquitoes were also found in the household containers. These species are known to
co-exist in breeding habitats in Ghana. Lutzia is a well-known mosquito predator whilst
Aedes, Culex, Anopheles are also known to exhibit interspecies predatory activities [34,35].
Indeed, the fourth instar Lutzia larva can consume up to 24 fourth instar Aedes larvae per day
[36]. In a mixture of different mosquito larvae, Appawu et al [34] reported that Lutzia larvae
exhibited a significant preference for Ae. aegypti larvae compared to An. gambiae s. I. and Cx
quinquefasciatus. Interaction of these mosquito larvae in the containers may have a significant
influence on the resulting adult populations of Ae. aegypti. Further study is needed to elucidate
the impact of such complex interaction among mosquito larvae in household containers on
Aedes’ productivity.
Chemical control remains an important part of most control measures against mosquito
vectors. In this study, Ae. aegypti population from Cape Coast was highly resistant to deltame-
thrin (pyrethroid), DDT (organochlorine) and bendiocarb (carbamate). But exhibited moder-
ate resistance to fenithrothion (organophosphate). Resistance of the mosquitoes to the
different classes of insecticides is worrying and could affect the efficacy of insecticide based
control tools [9]. The level of resistance to deltamethrin and DDT is consistent with the results
from other parts of the country [4,14,37]. However, the high level of resistance to carbamate
(bendiocarb) was unexpected. Unlike, pyrethroid insecticides, carbamate insecticides are not
normally employed in household vector control tools. Furthermore, urban agricultural with its
associated insecticide use are not important activitiy in Cape Coast Metropolis. Thus, the
source of resistance particularly to carbamates is not clearly known. In fact, the source of resis-
tance in Ae. aegypti population from Ghana and many African countries remains less obvious
[12]. Nonetheless, domestic use of insecticides could be a very important source, particularly
for pyrethroid insecticides [38–40]. The result from the insecticide bioassay showed that the
mosquitoes collected from containers located inside houses were more resistant to the insecti-
cides than the mosquitoes collected from containers located outside houses (Fig 4). This differ-
ential resistance could be explained by the heavy use of insecticides in houses [38–40]. Most
houses depend on daily use of insecticide-based tools such as insecticide treated nets and mos-
quito coils for protection against mosquito bites. This could put Ae. aegypti that lives and ovi-
posit in containers located in houses under higher insecticide selection pressure than the
mosquitoes that breed in containers outside houses. Understanding the spatial distribution of
insecticide resistance among the mosquito population may help improve resistance manage-
ment strategies.
In this study, F1534C, V1016I, and V410L kdr mutations were detected from the Ae. aegypti
population from Cape Coast. Single or multiple of these kdr mutations have been associated
with resistance to different pyrethroid insecticides and DDT [38,41,42]. F1534C mutation was
previously detected in Cape Coast and other parts of Ghana and remains the most widespread
mutation in Ae. aegypti population in Africa [43]. We report for the first time the detection of
V410L and V1016I mutations in Ae. aegypti population outside Accra, Ghana. In the first
report of the detection of V1016I mutation in Africa about six years ago [14], a single Ae.
aegypti mosquito from Accra (Ghana) was found with the mutation. Thus, it is alarming to
observe the spread of the mutation together with the V410L in Cape Coast within this short
time. The combination of F1534C and V1016I have been shown to generate high levels of
resistance to pyrethroid [22,41]. In fact, Vera-Maloof et al. [41] suggested that high pyrethroid
resistance in Ae. aegypti requires the sequential evolution of F1534C and V1016I mutations. It
is for this reason that the high frequency of the F1534C mutation recorded in this study in
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PLOS NEGLECTED TROPICAL DISEASES Risk of Aedes arboviral outbreaks in Covid 19 period in Cape Coast, Ghana
addition to the V1016I mutation is worrying. However, the contribution of V410L to the pyre-
throid resistance remains unclear. This mutation was recently detected in a population from
Accra [44] as well as Ghana’s neighboring countries of Cote d’Ivoire [15] and Burkina Faso
[38]. However, all the three studies did not find the V410L contribution to pyrethroid resis-
tance in their respective countries. Nevertheless, V410L alone or in combination with the
F1534C mutation has been shown to reduce the sensitivity of mosquito sodium channels
expressed in Xenopus oocytes to pyrethroids [45]. A recent study found high frequencies of the
V410L kdr mutation to be associated with pyrethroid resistance and its combination with
F1534C and V1016I was also found to influence the survival of Ae. aegypti after exposure to
pyrethroid insecticide in a field cage tests [46].
Similar to the results from the bioassay, higher frequency of the resistant allele of F1534C
mutation was found in the mosquito population collected inside houses than those collected
outside the houses (Table 2). This supports the earlier suggestion that household use of insecti-
cide may be contributing to the selection of resistance in the vector population. However, the
three kdr mutations detected in this study cannot fully explain the multiple insecticide resis-
tance found in the mosquito populations from Cape Coast. The resistance to bendiocarb and
fenitrothion in the vector population indicates the existence of other important mechanisms.
Previous study in Cape Coast detected metabolic resistance through elevated activity of mixed-
function oxidase, esterase and glutathione-S-transferase from biochemical assays in Ae. aegypti
population [4]. Similar result has also been reported in Ae. aegypti population from Accra [44].
This mechanism could explain some of the resistant phenotype found in this study. Further
study is needed to elucidate all the important resistance mechanisms and the potential source
of resistance for Ae. aegypti in the study area.
Conclusion
The study found the risk of an outbreak of Aedes-borne diseases lower in the covid-19 lock-
down period than before the pandemic period. Although the study sample is very limited,
valuable lessons could be drawn from it concerning the control of Ae. aegypti. In the previous
study in the community, a high number of discarded items were infested with mosquito larvae
and this contributed to the high larval indices. However, improved sanitation conditions
through the clean-up exercise during the restriction period in the community may have caused
lower larval indices than what was observed in 2017 before the pandemic. Multiple insecticide
resistance couple with three kdr mutations among the Ae. aegypti population in Cape Coast
could affect the effectiveness of control measures, especially in emergency situations. The
study supports sanitation improvement as a tool to control Ae. aegypti [27,47] and could com-
plement insecticide-based tools in controlling this vector.
Supporting information
S1 Table. The genotype frequencies of V1016I, V410L and F1534C kdr mutations in Aedes
aegypti population from Cape Coast.
(XLSX)
Acknowledgments
We are very grateful to Mr Francis Abortri who facilitated community entry and helped in
data collection during the larval survey. JA also obtained CIH One Health Scholarship. We
also thank the Department of Conservation Biology and Entomology, at the University of
Cape Coast for their support.
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PLOS NEGLECTED TROPICAL DISEASES Risk of Aedes arboviral outbreaks in Covid 19 period in Cape Coast, Ghana
Author Contributions
Conceptualization: Andreas A. Kudom.
Data curation: Joana Ayettey, Andreas A. Kudom.
Formal analysis: Andreas A. Kudom.
Investigation: Joana Ayettey, Andreas A. Kudom.
Methodology: Joana Ayettey, Aikins Ablorde, Godwin K. Amlalo, Ben A. Mensah, Andreas A.
Kudom.
Project administration: Andreas A. Kudom.
Resources: Andreas A. Kudom.
Supervision: Ben A. Mensah, Andreas A. Kudom.
Validation: Andreas A. Kudom.
Writing – original draft: Joana Ayettey, Ben A. Mensah, Andreas A. Kudom.
Writing – review & editing: Ben A. Mensah, Andreas A. Kudom.
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