Malaria Journal
BioMed Central
Open Access
Research
A pre-intervention study of malaria vector abundance in Rio Muni,
Equatorial Guinea: Their role in malaria transmission and the
incidence of insecticide resistance alleles
Frances C Ridl*1, Chris Bass2, Miguel Torrez3, Dayanandan Govender1,
Varsha Ramdeen1, Lee Yellot3, Amado Edjang Edu4, Christopher Schwabe5,
Peter Mohloai6, Rajendra Maharaj1 and Immo Kleinschmidt7
Address: 1Malaria Research Lead Programme, Medical Research Council, 491 Ridge Road, Durban, South Africa, 2Department of Biological
Chemistry, Rothamsted Research, Harpenden, AL5 2JQ, UK, 3Equatorial Guinea Malaria Control Initiative, Apdo # 606, Bata, Equatorial Guinea,
4C/O.U.A., Zona Sanitaria s/n, Bata-Litoral, Equatorial Guinea, 5Medical Care Development International, 8401 Colesville Rd, Silver Spring,
Maryland, 20910, USA, 6One World Development Group International, Punta Europa, Carretera Aeropuerto, Malabo, Bioco Norte, Equatorial
Guinea and 7London School of Hygiene and Tropical Medicine, Keppel St, London, WC1E 7HT, UK
Email: Frances C Ridl* - fridl@mrc.ac.za; Chris Bass - chris.bass@bbsrc.ac.uk; Miguel Torrez - mtorrez@gmail.com;
Dayanandan Govender - dgovender@mrc.ac.za; Varsha Ramdeen - vramdeen@mrc.ac.za; Lee Yellot - lyellot@mcd.org;
Amado Edjang Edu - edjang_1@hotmail.com; Christopher Schwabe - cschwabe@mcd.org; Peter Mohloai - mohloaip@hotmail.com;
Rajendra Maharaj - rmaharaj@mrc.ac.za; Immo Kleinschmidt - Immo.Kleinschmidt@lshtm.ac.uk
* Corresponding author
Published: 29 September 2008
Malaria Journal 2008, 7:194
doi:10.1186/1475-2875-7-194
Received: 24 June 2008
Accepted: 29 September 2008
This article is available from: http://www.malariajournal.com/content/7/1/194
© 2008 Ridl et al; licensee BioMed Central Ltd.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract
Background: Following the success of the malaria control intervention on the island of Bioko,
malaria control by the use of indoor residual spraying (IRS) and long-lasting insecticide-treated nets
(LLITN) was extended to Rio Muni, on the mainland part of Equatorial Guinea. This manuscript
reports on the malaria vectors present and the incidence of insecticide resistant alleles prior to the
onset of the programme.
Methods: Anopheles mosquitoes were captured daily using window traps at 30 sentinel sites in Rio
Muni, from December 2006 to July 2007. The mosquitoes were identified to species and their
sporozoite rates, knockdown resistance (kdr) and acetylcholinesterase (AChE) sensitivity
measured, to define the role of vector species in malaria transmission and their potential
susceptibility to insecticides.
Results: A total of 6,162 Anopheles mosquitoes were collected of which 4,808 were
morphologically identified as Anopheles gambiae s.l., 120 Anopheles funestus, 1,069 Anopheles
moucheti, and 165 Anopheles nili s.l.. Both M and S molecular forms of Anopheles gambiae s.s. and
Anopheles melas were identified. Anopheles ovengensis and Anopheles carnevalei were the only two
members of the An. nili group to be identified. Using the species-specific sporozoite rates and the
average number of mosquitoes per night, the number of infective mosquitoes per trap per 100
nights for each species complex was calculated as a measure of transmission risk. Both kdr-w and
kdr-e alleles were present in the S-form of An. gambiae s.s. (59% and 19% respectively) and at much
lower frequencies in the M-form (9.7% and 1.8% respectively). The kdr-w and kdr-e alleles cooccurred in 103 S-form and 1 M-form specimens. No insensitive AChE was detected.
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Conclusion: Anopheles gambiae s.s, a member of the Anopheles gambiae complex was shown to be
the major vector in Rio Muni with the other three groups playing a relatively minor role in
transmission. The demonstration of a high frequency of kdr alleles in mosquito populations before
the onset of a malaria control programme shows that continuous entomological surveillance
including resistance monitoring will be of critical importance to ensure the chosen insecticide
remains effective.
Background
Malaria is a major endemic disease in Rio Muni, the mainland part of Equatorial Guinea situated at 1.512°N
10.267° on the west coast of Central Africa. Estimates
from a Plasmodium falciparum prevalence survey conducted in 2007 among children between two and 15 years
of age showed site-specific parasitaemias to vary from
54% to 89% with an average of 72% (unpublished data I.
Kleinschmidt and L. Benavente). Following the success of
the Bioko Island Malaria Control Project (BIMCP) [1,2]
malaria control has been extended to Rio Muni under the
Equatorial Guinea Malaria Control Initiative (EGMCI) by
a staged roll-out of indoor residual house spraying (IRS)
in Litoral and Kie-Ntem provinces and long-lasting insecticide-treated net (LLITN) distribution in the other two
provinces (Cento Sur and Wele Nzas). Extensive information and education campaigns are being conducted and
all areas will benefit from the introduction of free artemisinin-based combination therapy starting in July 2008.
This initiative, to substantially reduce malaria on the
mainland using IRS and LLITNs is being funded by the
Global Fund to fight Aids, Tuberculosis and Malaria
(GFATM) and Marathon Oil Company and is run in partnership with the government of Equatorial Guinea, Medical Care Development International (MCDI), One World
Development Group International (OWDGI), Medical
Research Council of South Africa (MRC), Harvard and
Yale Universities and the London School of Hygiene and
Tropical Medicine.
The tropical all year round humid climate and the many
rivers and streams, both fast and slow flowing, provide
ideal breeding conditions for different malaria vectors.
Earlier studies have shown Anopheles gambiae sensu lato
(s.l.) and Anopheles funestus to be the major vectors of
malaria on the mainland of Equatorial Guinea [3-5]. This
paper reports on the composition, density, infectivity,
knockdown resistance (kdr) and insensitive acetylcholinesterase (iAChE) status of malaria vector species exiting
houses through window traps before the start of the intervention.
Materials and methods
Entomological monitoring
In November 2006, window traps were installed at six
houses at each of 30 sentinel sites selected in each of the
four provinces (Figure 1); eleven sites in the coastal province, Litoral, which includes Bata, the principle city on the
mainland, five sites in Centro Sur which comprises the
centre of the mainland, six sites in Kie Ntem in the northeast and eight sites in Welas Nzas in the south-east. The
extensive distribution of the sentinel sites throughout the
country facilitated localized monitoring of malaria vectors. These data are useful for evaluating the effectiveness
of IRS and LLITNs and for long term planning regarding
choice of appropriate insecticide.
Mosquito collections from window traps were described
previously by Sharp et al [2]. Briefly, the contents of window traps were emptied daily by the home owner into
pre-labelled specimen jars containing isopropanol. Night
control sheets specifying the nights worked were documented and both jars and sheets were collected and
replaced at four week intervals. Mosquitoes were collected
during an eight month period, from December 2006 to
July 2007, before the start of the first spray round to establish a baseline for comparison between pre- and postintervention periods.
Identification of vector species, molecular forms, kdr and
AChE mutations
Mosquitoes were separated into Culicinae and Anophelinae
and counted. Anophelines were morphologically identified into An. gambiae s.l., An. funestus, Anopheles moucheti
and Anopheles nili s.l., using the keys described by Gillies
and De Meillon [6], Gillies and Coetzee [7] and Hervy et
al [8] and, subsequently, stored in isopropanol.
DNA was extracted from the head and thorax [9] of a subsample of mosquitoes to determine family member species using Polymerase Chain Reaction (PCR). The protocols of Scott et al [10], Koekemoer et al [11], Kengne et al
[12,13] were used to identify family members of An. gambiae s.l., An. funestus, An. nili s.l. and An. moucheti respectively and the molecular forms of An.gambiae sensu stricto
(s.s.) were determined according to the method described
by Fanello et al [14]. The presence of the west-African LeuPhe and east-African Leu-Ser kdr mutations was determined in 505 An. gambiae s.s individuals using the TaqMan PCR protocol described by Bass et al [15]. A new
TaqMan assay devised by Bass et al [16] was used to determine Plasmodium falciparum sporozoite rates. Insensitive
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Figure
Map
of the
1 sentinel sites in the four provinces of Rio Muni, Equatorial Guinea
Map of the sentinel sites in the four provinces of Rio Muni, Equatorial Guinea.
AChE was determined using the PCR method described
by Weil et al [17].
tor Cycle Sequencing kit followed by analysis on a 310
Automated DNA Sequencer (PE Applied Biosystems).
Transmission risk
The number of infective mosquitoes per trap per 100
nights for each species complex was calculated as a measure of transmission risk.
Statistical analysis
The genotypic frequencies at the kdr locus were compared
to Hardy-Weinberg expectations using the exact test procedures implemented in GenePOP (ver.3.4) software
[18].
Sequence analysis of kdr
The results of kdr genotyping using the TaqMan assay were
verified by sequencing the relevant region of the sodium
channel gene in 50 of the 505 samples analysed. This was
carried out using two primers (MosF1, 5'-GATAATGTGGATAGATTCCCCG-3 and MosR1, 5'-CGTTGGTGCAGACAAGGATG-3') flanking the mutation site. PCR
reactions (25 μl) contained 1 μl of genomic DNA, 12.5 μl
of 2× PCR master mix (Promega) and 100 ng of each
primer. PCR products were ethanol precipitated and direct
sequenced using an internal primer (MosSeq1 5'-CCATGATCTGCCAAGATGGA-3') and the ABI BigDye Termina-
Results
Mosquito collections and molecular identification
A total of 6,162 Anopheles mosquitoes were collected of
which 4,808 (78%) were morphologically identified as
An. gambiae s.l., 120 (2%) An. funestus, 1,069 (17%) An.
moucheti and 165 (3%) An. nili s.l. (Table 1). The four
identified family groups were found sympatrically in all
four provinces. Large variations in mosquito numbers and
mosquito species composition existed between sentinel
sites and also between different months.
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Table 1: Total number of anophelines caught per province
December 2006–July 2007
Province
An. gambiae s.l.
An. funestus
An. moucheti
An. nili s.l.
Centro Sur
Litoral
Wele Nzas
Kie-Ntem
225
3262
580
741
3
76
29
12
382
36
153
498
26
113
23
3
Total
4808
120
1069
165
Anopheles gambiae s.s. and Anopheles melas were the only
two members of the An. gambiae complex to be identified
(n = 930). An. gambiae s.s was identified from 29 of the 30
sentinel sites and accounted for 776 of the An. gambiae s.l.
identifications. An. melas was predominantly identified
from the two coastal port cities of Cogo and Mbini where
they made up 88% (n = 112) and 79% (n = 67) respectively of the total An. gambiae s.l. identified at these two
sites. Two specimens were also collected from Yengue, a
town in the north-west of Rio Muni, bordering on Cameroon. The S-molecular form of An. gambiae s.s. was found
in all four provinces and the M-form, only in Litoral,
where it accounted for 44% of identifications (n = 350).
The two forms were found sympatrically and no hybrids
were identified.
Anopheles moucheti was the second most abundant vector
accounting for 17% of the total and was identified from
22 sites. It was the only member of the An. moucheti group
to be identified. An. funestus was collected from fourteen
sentinel sites and was the only member of the An. funestus
group to be identified and accounted for 2% of the total
number of Anopheline mosquitoes caught.
Anopheles nili s.l. accounted for 3% of the total Anopheles
population and was found at seven sentinel sites:
Ayamiken, Ayene, Machinda, Ngong, Niefang, Nkue and
Yengue. Of the 151 An. nili s.l. tested, 98 and 53 were
identified as Anopheles ovengensis and Anopheles carnevalei
respectively. Anopheles carnevalei was only identified from
Yengue where it accounted for 50% of the total An. nili s.l.
identified.
0.4 respectively. Using the species complex sporozoite
prevalence (3.5%, 1.6% 2.6% and 4.1% respectively) the
number of infective mosquitoes per trap per 100 nights
for each species was 0.5, 0.06, 0.01 and 0.02 respectively.
Kdr allele frequencies in M and S molecular forms of An.
gambiae s.s
393 S and 113 M molecular forms of An. gambiae s.s. were
analysed for the presence of kdr-w and kdr-e alleles using a
recently described TaqMan assay (Table 2). The results
using the new assay were compared with sequencing in a
subset of 50 of the specimens analysed and the two methods were found to be in complete agreement. Kdr-e and
kdr-w resistance alleles were present in S forms with a
higher frequency of the kdr-w allele (59%) than the kdr-e
allele (19%). Both alleles also occurred in the M-forms
but at much lower frequencies of 9.7% for kdr-w and 1.8%
for kdr-e. Both the kdr-w and kdr-e alleles were present in S
form samples in all four provinces with frequencies of the
kdr-w allele of 51% in Litoral, 47% in Centro Sur, 64% in
Wele Nzas and 73% in Kie Ntem and frequencies of the
kdr-e allele of 32% in Litoral, 24% in Centro Sur, 8% in
Wele Nzas and 14% in Kie Ntem. The kdr-w and kdr-e alleles were found to co-occur in a single M form specimen
and in 103 S form specimens (Table 2). Sample numbers
were sufficient to compare kdr gene frequencies with
Hardy Weinberg expectations in populations collected
from a number of sites. These included Bata City in Litoral, Bisun in Centro Sur, Mongomeyen in Wele Nzas and
Ebebiyin in Kie-Ntem. Genotypic frequencies of both M
and S form populations in Bata City showed significant
deviations from Hardy-Weinberg expectations with a heterozygote deficit (P < 0.001). The same was true of the S
form population in Bisun in this instance due to a heterozygote excess (P < 0.05). The genotypic frequencies of the
S form populations at the other two localities were not significantly different from Hardy Weinberg expectations (P
= 1 for the Mongomeyen population and P = 0.39 for the
Ebebiyin population).
AChE resistance
All 200 mosquitoes tested for insensitive AChE were
found to be susceptible.
Discussion
Plasmodium falciparum sporozoite and transmission
rates
Sporozoite rates were 4.1% (n = 49) for An. funestus, 4.1%
(n = 74) for An. ovengensis, 3.3% (n = 603) for An. gambiae
s.s. and 1.6% (n = 126) for An. moucheti. The sporozoite
rate for An. melas was 4.4% (n = 137). Anopheles carnevalei
was not shown to be involved in transmission although
numbers tested were low (n = 52). The estimated number
of An. gambiae s.l., An. moucheti, An. nili s.l. and An. funestus per window trap per 100 nights was 15.5, 3.4, 0.5 and
Anopheles gambiae s.l. was shown to be the main vector
within this geographical region with the other three species playing a relatively minor role due to their low densities. A sporozoite rate of 4.4% for An. melas indicates its
involvement in malaria transmission in the two sentinel
sites from which it was identified.
Previous studies in Equatorial Guinea have shown An.
gambiae s.l. and An. funestus to be the main vectors of
malaria [3-5]. Elsewhere in West and Central Africa An.
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Table 2: Kdr genotype frequencies in An. gambiae s.s. in Rio Muni, 2006–2007
Province
Litoral
District
Locality
Bata
Yengue
Bata City
Ayamiken
Machinda
Ngolo
Etofili-Lubi
Centro
Ukomba
Ncolombong
Cogo
Mbini
Cogo
Centro Sur
Niefang
Evinayong
Wele Nzas
Akurenam
Anisok
Mongomo
Nsork
Kie-Ntem
Micomiseng
Ebebiyin
Niefang
Bisun
Bicurga
Evinayong
Akurenam
Ayene
Anisok
Mongomeyen
Mongomo
Asok
Nsork
Aconibe
Nkue
Ngong
Ebebiyin
n
molecular form
25
33
5
1
9
29
1
9
2
60
12
11
19
6
5
1
1
13
48
5
17
5
23
3
49
4
1
11
6
17
26
49
gambiae s.l., An. funestus, An. moucheti and An. nili s.l. have
been shown to be effective vectors with EIR rates ranging
from 1–1000 infective bites per year recorded [19]. In this
study, all four identified groups from Rio Muni have been
shown to be involved in transmission of malaria with An.
gambiae s.s. being the major vector. Although An. funestus
was found to have the highest sporozoite rate, the number
caught was very low hence it was not shown to be a major
vector.
Anopheles nili has recently been described as a complex
consisting of four member species based on morphological criteria: An. nili, Anopheles somalicus, An. carnevalei and
An. ovengensis [12].Anopheles carnevalei is relatively rare in
occurrence and has so far only been reported from the
equatorial forests of Ivory Coast and Cameroon [20] and
from a village in Equatorial Guinea, Yengue [21,22].
Anopheles ovengensis has been reported from southern
Cameroon [23]. The results of this study further provide
proof of the distribution of An. ovengensis to extend
throughout the northern part of Equatorial Guinea as was
M
S
S
S
M
S
M
S
S
M
S
M
S
M
S
M
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S/S
S/Rw
S/Re
20
3
1
0
6
1
1
0
0
51
4
8
1
4
1
1
0
0
3
2
2
1
2
0
4
0
0
1
0
2
0
1
4
3
3
1
0
0
0
0
0
9
1
2
0
1
4
0
0
3
20
2
3
0
11
0
19
0
1
2
1
9
6
0
1
5
1
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2
5
0
1
0
1
0
2
0
0
0
1
2
1
0
kdr genotypes
Rw/Rw
0
10
0
0
1
11
0
6
0
0
1
1
2
0
1
0
1
2
6
1
7
1
4
2
19
4
0
7
3
2
19
29
Re/Re
Re/Rw
0
2
0
0
1
6
0
1
0
0
1
0
2
0
0
0
0
1
2
0
0
0
0
0
0
0
0
0
0
0
0
1
0
10
0
0
1
11
0
2
2
0
5
0
14
0
0
0
0
5
12
0
4
3
5
1
5
0
0
1
1
2
0
18
suggested by Awono-Ambene [23] and confirms the presence of An. carnevalei in Yengue, a village in the north-west
of the mainland where it was sympatric with An. ovengensis. These collections were all made from window traps
thus indicating some degree of endophilic behaviour
although previous studies suggest predominately
exophilic habits [23].
Resistance of mosquitoes to insecticides usually arises
through one of two mechanisms, or a combination of the
two; metabolic resistance due to increased production of
detoxifying enzymes and target site resistance due to
mutations in the sodium channel, acetylcholinesterase or
GABA receptor [24]. Kdr is a target site resistance of the
sodium channel and is one of the mechanisms conferring
resistance to pyrethroid and DDT insecticides. Two mutations have been described, a leucine-phenylalanine substitution originally found in west-African An. gambiae s.l.
[25] and a leucine-serine substitution found in east-African An. gambiae s.l. [26]. However, recent studies in Cameroon and Gabon have shown that these mutations are
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not unique to these geographical regions and that there is
considerable overlap with both being present in the same
populations [27,28]. Both the resistance alleles were identified in the populations examined in this study. The kdrw and kdr-e alleles were present at low frequencies in M
forms (9.7% and 1.8%) and in much higher frequencies
in S forms with the frequency of the kdr-w allele 59% and
the frequency of the kdr-e allele 19%. The observed gene
frequencies are in close concordance with those reported
recently in the neighbouring country of Cameroon where
kdr-w and kdr-e alleles were present in M form populations at frequencies of 6.3% for kdr-w and 1.1% for kdr-e
and in S form populations at frequencies of 52.7 and
13.9% [29]. This correlation in observed gene frequencies
could indicate that the kdr alleles have migrated from
Cameroon to Equatorial Guinea or vice versa. It would be
interesting in future to sequence the sodium channel gene
regions flanking the kdr locus, in particular intron I
upstream of the mutation site, as this will provide evidence as to whether the kdr mutations have arisen once
and spread between the two countries or represent independent mutation events. It may also reveal the extent of
migration between populations in Equatorial Guinea and
Cameroon. Interestingly the gene frequencies we
observed on the mainland differ significantly from those
seen on the island part of Equatorial Guinea, Bioko,
where prior to the onset of the spray programme, 50% of
the M-forms carried the kdr-w allele in either the
homozygous or heterozygous form while it was completely absent in the S-form [30,2]. However, previous
studies have suggested that An. gambiae populations on
Bioko are to a large extent isolated from mainland populations [29].
As reported previously in the neighbouring countries of
Cameroon and Gabon [28,29] we observed a large
number of Re/Rw genotypes in the localities sampled in
this study (26% of S form mosquitoes carried this genotype). Indeed this was the predominant genotype seen in
S form mosquitoes after the Rw/Rw genotype (34%). A
recent study has shown that this genotype confers a significant degree of resistance to DDT, although the level of
resistance is not significantly greater than that conferred
by Rw/Rw [29]. Significantly we also recorded this genotype in a single M form specimen. This result was confirmed by sequencing and to our knowledge represents
the first report of this genotype in M form mosquitoes.
Further screening for kdr in M form populations in Equatorial Guinea will reveal the extent of this genotype in the
M form but this initial study indicates it may be currently
found at an extremely low frequency.
In the Bata City area of Litoral both M and S populations
showed significant deviations from Hardy-Weinberg
expectations (P < 0.001) and this was due to a heterozy-
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gote deficit. As kdr is a recessive trait [25,26] and only
homozygous genotypes express the resistance phenotype,
studies need to be implemented to determine the origin of
the insecticide selection pressure as is observed from the
high frequency of homozygous resistant individuals in
Bata City before IRS. Clearly the presence of both kdr-e
and kdr-w alleles at high frequencies in these populations
may have implications for the effectiveness of the current
vector control programme which is based on pyrethroid
insecticides.
AChE is the target site of organophosphates and carbamate insecticides and insensitive AChE in mosquitoes
coincides with high insecticide resistance to these insecticide classes. No insensitive AChE was detected in this
baseline study indicating continued efficacy of these
insecticide classes. Coleman et al [31] reviewed published
insecticide resistance data in Africa and found eight sites
with reported carbamate resistance and 13 sights with
organophosphate resistance. They attributed this to the
limited application of carbamates and organophophates
in large-scale vector control and the lack of resistance
monitoring.
This study provides contemporary information on the distribution of malaria species and their role in malaria
transmission in Rio Muni, Equatorial Guinea. It also provides useful information on measures of insecticide resistance for the vector control programme. Pyrethroids have
been selected as the insecticide of choice for the first spray
round due to its low toxicity in humans, its longer residual
effect and for cost efficacy and procurement implications.
WHO susceptibility tests in 2000 showed resistance to
DDT but susceptibility to both deltamethrin and permethrin [5,32]. In bioassays conducted in 2007 with
alphacypermethrin (Fendona), there was 95.5% mortality
in the exposed group by the end of the observation period
(24 hrs) (personnel communication M. Torrez 2007). In
West Africa, large scale agricultural pyrethroid use has
resulted in very high insecticide resistance [3]. However in
Equatorial Guinea pyrethroids have not been widely used
as an agricultural insecticide. Therefore this studies findings of kdr mutations at such high frequency in mosquito
populations in Equatorial Guinea (particularly in S form
populations) is unexpected. Nevertheless, the presence of
kdr alleles at the observed frequencies could impact on the
choice of insecticide for future spray rounds and will
require ongoing monitoring and evaluation to ensure the
chosen insecticide remains effective, a process the EGMCI
has put in place. Carbamates remain a viable alternative in
the absence of insensitive AChE and have been successfully used in a number of spray programmes including on
the island of Bioko and in Mozambique [2,33]. Further
biochemical testing is planned to determine whether or
not other resistant mechanisms are present in the mos-
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quito populations and to assess if these develop as a result
of selection pressure exerted by the IRS and ITN vector
control operations.
6.
Competing interests
7.
The authors declare that they have no competing interests.
Authors' contributions
FCR co-designed the study, carried out laboratory analyses
of mosquitoes, participated in data analysis and interpretation and was involved in the drafting of the manuscript.
CB carried out kdr laboratory analyses, analysed and interpreted the kdr results and assisted in the drafting of the
manuscript. MT carried out the susceptibility assays and
contributed to the drafting of the manuscript. DG managed the database, assisted with the analysis of results and
contributed to the manuscript. VR assisted with laboratory analyses and helped draft the manuscript. LY was
responsible for the IRS programme, monitoring of window traps and helped draft the manuscript. AE was
responsible for the window trap collections and preparation of mosquitoes for analysis and helped draft the manuscript. CS was responsible for the overall management of
the control programme and assisted in drafting the manuscript. PM assisted with mosquito collections and helped
draft the manuscript. RM helped draft the manuscript and
critical evaluation thereof. IK co-designed and coordinated the study and was involved in the drafting of the
manuscript and critical evaluation thereof. All authors
read and approved the manuscript.
Acknowledgements
The authors would like to thank Drs Adel Chaouch, Brian Linder and Susan
Rynard from Marathon Oil Company for their encouragement and constructive participation in the EGMCI; Dr Gloria Nseng, Director of the
National Malaria Control Programme in Equatorial Guinea for her oversight and direction of the malaria control efforts in the country and Mr
Simon Abaga, Ministry of Health and Social Welfare Entomologist, for his
support of field-based activities; Jaime Kuklinski, Ruben Biebeda and Blas
Abeso from One World Development Group International (OWDGI) for
the collection of mosquitoes and management of vector monitoring component of the fieldwork in the EGMCI; Natashia Morris for the preparation
of the map; Dr Mike Coleman for his interest and support in this project
and Inbarani Naidoo for critically reading the manuscript.
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