Malaria transmission in two rural communities in the forest zone of Ghana

Abstract

Malaria transmission was assessed in two rural communities, Kona and Afamanaso in Sekyere South district, Ashanti Region, in the forest zone of Ghana to provide baseline data for ongoing clinical studies and the evaluation of the effect of interventions. Altogether, 3,479 Anopheles gambiae and 1,157 Anopheles funestus were caught by human landing catches. Sporozoite rates determined by either microscopy of salivary glands or enzyme-linked immunosorbent assay (ELISA) for Plasmodium falciparum in the two villages were 6.6% vs. 8.9% for the main vector A. gambiae and 3.2% vs. 6.3% for A. funestus. ELISA tests of dissected specimens compared to microscopy of salivary glands were 1.3 and 2.0 times more positive for A. gambiae and A. funestus, respectively. Plasmodium infections of 122 microscopically positive salivary glands of A. gambiae were identified by real-time PCR as 95 (77.9%) P. falciparum, 7 (5.7%) Plasmodium malariae, 7 (5.7%) Plasmodium ovale and 1 (0.8%) mixed infection of P. falciparum and P. malariae. Transmission in the area was found to be intense and perennial with some seasonal variations during the study period from Dec. 2003 to Aug. 2005. Although the two villages were only 10 km apart from each other, Annual Biting Rates (ABRs) and Annual Entomological Inoculation Rates (AEIRs) were much higher at Afamanaso (11,643 vs. 866) than at Kona (5,329 vs. 490). Most of the transmission (91.4%) occurred during bedtime hours from 21 to 6 h. It is important to note that there was still a substantial transmission before 21 h with AEIRs of 57.3 at Afamanso and 38.7 at Kona. The distribution of impregnated bednets alone, therefore, may not be sufficiently effective.

Full text

ORIGINAL PAPER
Malaria transmission in two rural communities in the forest
zone of Ghana
Ayimbire Abonuusum &Kofi Owusu-Daako &
Egbert Tannich &Jürgen May &Rolf Garms &
Thomas Kruppa
Received: 17 September 2010 / Accepted: 26 November 2010 /Published online: 14 December 2010
#Springer-Verlag 2010
Abstract Malaria transmission was assessed in two rural
communities, Kona and Afamanaso in Sekyere South district,
Ashanti Region, in the forest zone of Ghana to provide
baseline data for ongoing clinical studies and the evaluation of
the effect of interventions. Altogether, 3,479 Anopheles
gambiae and 1,157 Anopheles funestus were caught by
human landing catches. Sporozoite rates determined by
either microscopy of salivary glands or enzyme-linked
immunosorbent assay (ELISA) for Plasmodium falciparum
in the two villages were 6.6% vs. 8.9% for the main vector
A. gambiae and 3.2% vs. 6.3% for A. funestus. ELISA tests
of dissected specimens compared to microscopy of salivary
glands were 1.3 and 2.0 times more positive for A. gambiae
and A. funestus,respectively.Plasmodium infections of 122
microscopically positive salivary glands of A. gambiae were
identified by real-time PCR as 95 (77.9%) P. falciparum,7
(5.7%) Plasmodium malariae, 7 (5.7%) Plasmodium ovale
and 1 (0.8%) mixed infection of P. falciparum and P.
malariae. Transmission in the area was found to be intense
and perennial with some seasonal variations during the study
period from Dec. 2003 to Aug. 2005. Although the two
villages were only 10 km apart from each other, Annual
Biting Rates (ABRs) and Annual Entomological Inoculation
Rates (AEIRs) were much higher at Afamanaso (11,643 vs.
866) than at Kona (5,329 vs. 490). Most of the transmission
(91.4%) occurred during bedtime hours from 21 to 6 h. It is
important to note that there was still a substantial transmis-
sion before 21 h with AEIRs of 57.3 at Afamanso and 38.7
at Kona. The distribution of impregnated bednets alone,
therefore, may not be sufficiently effective.
Introduction
Malaria is the leading cause of mortality among children
under 5 years old and pregnant women in Ghana and
accounts for 40–60% outpatient attendance and for more
income and workdays lost than any other disease (Asante
et al. 2004). In the forest zone of the Ashanti Region,
parasitaemia peaks at a prevalence of 93% in 11-year-old
children and declines to a plateau of 20% in adults, as
reported by Browne et al. (2000). Intensities of transmis-
sion, as defined by Entomological Inoculation Rates
(EIRs), have so far been determined in the northern
savanna areas (Appawu et al. 2004), in the coastal forest
and coastal savannah (Appawu et al. 2001) and the forest-
savanna transitional zone (Owusu-Agyei et al. 2009), but
not in the main forest region of Ghana.
The present study aimed to describe malaria transmis-
sion in two communities in the forest zone by analysing
monthly (MBR) and annual biting rates (ABRs) and
EIRs by the two vectors Anopheles gambiae and
Anopheles funestus. A clinical study conducted in parallel
had shown that in this homogeneous forest environment
malaria incidences of 3 to 15-month-old babies were
highly heterogeneous between villages (Kreuels et al.
2008).
A. Abonuusum
Kumasi Centre for Collaborative Research in Tropical Medicine,
Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana
K. Owusu-Daako
Kwame Nkrumah University of Science and Technology,
Kumasi, Ghana
E. Tannich :J. May :R. Garms (*):T. Kruppa
Bernhard Nocht Institute for Tropical Medicine,
Bernhard-Nocht-Str. 74,
20359 Hamburg, Germany
e-mail: garms@bni-hamburg.de
Parasitol Res (2011) 108:1465–1471
DOI 10.1007/s00436-010-2195-1

Materials and methods
Study sites
Studies were carried out in Afamanaso and Kona, two towns in
the Afigya Sekyere District (714 km
2
, population 131,658,
Ghana Statistical Service, 2000) in the north-eastern part of
the Ashanti Region in the forest zone of Ghana. The district
capital is Agona, located 27 km north of Kumasi (Fig. 1a).
The area is characterised by semi-deciduous forest and
farmland. The river Ofin with its tributaries meanders through
the district including a forest reserve, providing pools of water
and flooded marshy areas in the rainy season (Fig. 1b).
Afamanaso (6
°
56′N, 1
°
30′W), a rural village in the
plain, 290 m above sea level and 2.5 km off the main road,
is a typical farming village with a population of 2,508
(Ghana Statistical Service 2000). Most of the houses are
made of mud with thatched roofs or covered with
corrugated iron. Kona (6
°
52′N, 1
°
30′W) is a fast-
developing town, 305–320 m above sea level, situated on a
small mountain crest on the Kumasi–Ejura Road, with a
population of 5,853, engaged in crafts and trading. Most of
Fig. 1 Maps of Ghana (a) with
vegetation zones: 1Sudan
savannah, 2interior wooded
savanna, 3semi-deciduous
forest, 4rainforest, 5coastal
savannah, 6strand and
mangrove; and bthe study area
in Afigya Sekyere District with
its capital Agona and the two
towns Afamanaso and Kona
Fig. 2 Monthly biting rates (MBR) of A. gambiae and A. funestus in
Afamanaso from Mar 2004 to Aug 2005 and monthly rainfall (mm)
measured in Kona (Mar 2004 to Aug 2005)
Fig. 3 Monthly biting rates (MBR) of A. gambiae and A. funestus in
Kona from Mar 2004 to Aug 2005 and monthly rainfall (mm)
measured in Kona (Mar 2004 to Aug 2005)
1466 Parasitol Res (2011) 108:1465–1471

the houses are made of cement bricks. The peasant farmers
of both villages mainly cultivate cocoa, plantain, maize,
palm oil and fruit. Some farmers rear livestock, such as
poultry, goats and sheep, but not cattle.
Three seasons can be distinguished: a minor rainy
season from Mar to Jun, a major rainy season from July
toOct,andadryseasonfrom Nov to Feb (Meteorolog-
ical Service, Kumasi). Monthly rainfall was measured in
Kona from Mar 2004 to Aug 2005 (Figs. 2,3). Total
rainfall for 1 year (Sept 2004 to Aug 2005) was 2,124 mm,
with a minimum of 25 mm in Jan 2005 and a maximum of
480 mm in May 2005. The average monthly rainfall was
177 mm. It was exceptional that the minor rainy season in
2005 had more rainfall than the previous major rainy
season of 2004.
Mosquito collection
Human landing catches (HLC) were performed twice a month
at three sites in each town beginning Dec 2003 in Kona and
Mar 2004 in Afamanaso until the end of Aug 2005. One
mosquito collector caught from 1800 h to midnight and a
second one from midnight to 0600 h. Collectors rotated from
site to site to compensate for possible differences in individual
attraction to mosquitoes. The mosquitoes collected were kept
in cool boxes and transported to the laboratory for further
processing the next morning.
Identification and processing of mosquitoes
Mosquitoes were sorted into Anopheles species and Culicinae.
The former were further identified using the keys of Gillies
and Coetzee (1987); culicines were counted and discarded.
From A. gambiae s.l., legs and wings were removed before
dissection and retained for species identification by polymer-
ase chain reaction (PCR), following the protocol of Scott et al.
(1993). Anopheles females were dissected under a stereomi-
croscope, their midgut and ovaries were removed, and the
latter were examined under a compound microscope to
determine parity by inspection of the ovarian tracheoles
(Detinova 1962). Salivary glands were examined for spor-
ozoites, using a compound microscope. Sporozoite positive
salivary glands were stored at −80°C for later determination
of Plasmodium species by real-time PCR (Mangold et al.
2005). Head and thorax of all Anopheles females were
examined for the presence of circumsporozoite (CS) P.
falciparum antigen, using the enzyme-linked immunosorbent
assay (ELISA) (Wirtz 1987). Head and thorax of nulliparous
females were used as negative control. A mosquito was
considered infective if it was found positive by salivary gland
dissection and/or ELISA.
Statistics
Statistica for Windows, 1993, StatSoft Inc., Tulsa, OK,
USA, was used for the statistical analysis of the results.
Differences between percentages and chi
2
values were
analysed with the Quick Probability Calculator of this
programme.
Results
Vector collection
Altogether, 4,636 Anopheles mosquitoes were collected
during 217 full-night HLCs in the two villages, 63.6%
(2,948) in Afamanaso and 36.4% (1,688) in Kona. Morpho-
Table 1 Mean annual biting rates (ABR), bites per person per night
(b/p/n), annual entomological inoculation rates (AEIR), entomological
inoculation rates per person per night (EIR/p/n) and sporozoite rates of
A. gambiae and A. funestus in Afamanaso and Kona for the study
period XII.2003 to VIII.2005
Afamanaso
A. gambiae A. funestus Total
ABR 7,451 4,192 11,643
b/p/n 20.4 11.5 31.9
Annual EIR 638 228 866
EIR/p/n 1.7 0.6 2.3
Sporozoite rate % 8.6 5.4 7.4
Kona
ABR 4,896 433 5,329
b/p/n 13.4 1.2 14.6
Annual EIR 457 33 490
EIR/p/n 1.3 0.1 1.4
Sporozoite rate % 9.3 7.5 9.2
Glands Removed Not removed Significance
No. examined No. +ve (%) No. examined No. +ve (%) Pvalue
A. gambiae 2,302 163 (7.1) 1,175 105 (8.9) 0.052
A. funestus 554 19 (3.4) 602 38 (6.3) 0.024
Table 2 Comparison of results
of ELISA tests of A. gambiae
and A. funestus from which
salivary glands had been
removed for microscopy with
those with salivary glands
Parasitol Res (2011) 108:1465–1471 1467

logically, 75% (3,479) were identified as A. gambiae s.l. and
25% (1,157) as A. funestus. One hundred thirty-five A.
gambiae were classified by PCR as A. gambiae s.s.; DNA of
three specimens did not amplify.
Parous rates
Parous rates of the Anopheles females were high through-
out. Mean parous rates of A. gambiae and A. funestus were
85.8% vs. 85.2% at Afamanaso and 84.0% vs. 82.2% at
Kona. Parous rates of A. gambiae were significantly higher
(P=0.0029) in the dry season (90%) than in the major rainy
season (83%). Parous rates of A. funestus of the dry and the
major rainy season did not vary significantly at 85.3% vs.
84.3%, respectively (P=0.71).
Annual and seasonal biting activities
Anopheles biting activities were perennial but varied season-
ally. When MBRs of months with more or less than 100 mm
rainfall were compared, means of MBRs were always lower in
months with less rain. Differences, however, were only
significant for A. gambiae in Kona (P= 0.024, Mann–
Whitney Utest). A. funestus contributed 36% of the bites
in Afamanaso but only played a minor role of 8.9% of bites
in Kona (Figs. 2and 3, Table 1). A person passing a night at
Afamanaso received an average 31.9 bites per night (b/p/n),
more than twice as much as a person in Kona at 14.6 b/p/n.
Infection rates
Altogether, 4,634 Anopheles females were tested for the
presence of P. falciparum CS protein. Salivary glands were
removed from 2,858 mosquitoes and were examined
microscopically for the presence of sporozoites; 6.6% of
2,280 A. gambiae and 3.2% of 570 A. funestus turned out to
be positive. Of the non-dissected mosquitoes (N= 1174),
8.9% of A. gambiae and 6.3% A. funestus were positive in
ELISA, indicating that the ELISA was 1.3 times more
sensitive for A. gambiae and 2.0 times more sensitive for
A. funestus. Differences were significant (P=0.00084 vs. P=
0.023). When ELISA results for mosquitoes with and
without salivary glands were compared, differences were
only significant for A. funestus (Table 2). When microscop-
ically negative mosquitoes were retested with ELISA, 3.1%
of 2,154 A. gambiae and 1.9% of 536 A. funestus became
positive. For the calculation of infection rates and EIRs,
mosquitoes, either positive in microscopy and or ELISA,
were used (A. gambiae 9.1%, A. funestus 5.8%). Infection
rates of A. funestus were always lower than those of A.
gambiae (Table 1).
Entomological inoculation rates (EIR)
Annual entomological inoculation rate (AEIR) was 866 in
Afamanaso and 490 in Kona. The contribution of A.
Fig. 4 Hourly biting activities in % of all Anopheles gambiae (N=
3479) and A. funestus (N=1157) caught at Kona and Afamanaso
Table 3 Total numbers, numbers (%) of infected A. gambiae and A. funestus caught before (1800–2100 h), during bedtime (2100-0400 h) and in
early morning hours (0400–0600 h) in Afamanaso and Kona, and percentages of all females and all infected females caught during these time
intervals
Time interval hours A. gambiae A. funestus A. gam. +A. fun. % of all caught % of +ves caught
No. No.(%)+ ve No. No. (%)+ ve No. No.(%) +ve
Afamanaso
1800–2100 139 15 (10.8) 54 5 (9.3) 194 20 (10.3) 6.6 8.7
2100–0400 1,678 153 (9.1) 868 44 (5.1) 2,546 197 (7.7) 86.4 85.7
0400–0600 138 10 (7.2) 71 3 (4.2) 209 13 (6.2) 7.1 5.7
Kona
1800–2100 104 12 (11.5) 29 1 (3.5) 133 13 (9.8) 7.9 8.4
2100–0400 1,311 117 (8.9) 130 14 (10.8) 1,441 131 (9.1) 85.4 84.5
0400–0600 109 11 (10.1) 5 0 (0) 114 11 (9.6) 6.8 7.1
1468 Parasitol Res (2011) 108:1465–1471

funestus reached 35.7% in Afamanao but only 7.2% in
Kona (Table 1).
Hourly biting activities and risk of transmission
Biting activities of both A. gambiae and A. funestus started
as early as 1800 h, peaked between 2300 and 0200 h, and
persisted until 0600 h in the morning (Fig. 4). There were
no significant differences between the two species (chi
2
test
of homogeneity by Brandt–Snedecor, Sachs 1999,P=0.75).
The percentage of infected A. gambiae and A. funestus
caught before, during bedtime and in the early morning
hours were assessed to calculate the risk to be bitten by
mosquitoes or to pick up an infection during different time
intervals (Table 3). In both communities, 85% of all bites
and infected bites occurred during bedtime hours between
2100 h in the evening and 0400 h in the morning, and about
91% between 2100 and 0600 h in the morning. Infection
rates of both species did not change significantly (chi
2
=
1.91, P=0.38 for A. gambiae, chi
2
=0.71, P=0.38 for A.
funestus) during the three time intervals (Table 3). It is
important to note that high transmission rates occurred
before bedtime from 1800 to 2100 h with an EIR of 57.3 at
Afamanaso and 38.7 at Kona.
Identification of Plasmodium species
Plasmodium infections in 121 of 139 microscopically
positive salivary glands (110 A. gambiae,11A. funestus,
18 did not amplify) could be identified by real-time PCR
(Table 4); the distribution of Plasmodium species was
87.6% P. falciparum, 5.8% P. malariae and 5.8% P. ovale.
As expected, no P. vivax infection was detected. One A.
gambiae contained a mixed infection of P. falciparum with
P. malariae (Table 4).
The kdr gene in Anopheles gambiae in the study area
The knock down resistance (kdr) gene was highly prevalent in
the A. gambiae populations of the two study villages and
only absent in 3 of the 109 successfully amplified specimens
(Table 5).
Discussion
Altogether, 3,479 A. gambiae and 1,157 A. funestus,
which had been caught by HLCs in the two study villages
Kona and Afamanaso, were examined. Samples of A.
gambiae from both villages were identified as A. gambiae
sensu stricto, which agrees with the results of Tuno et al.
(2010), who furthermore recorded a high human blood
ratio and strong endophilic behaviour of this species. The
genotype of A. gambiae was not determined. It can be
assumed that it was the S form which predominates in the
forest region of Ghana and is positively associated with
malaria (De Souza et al. 2010). All 52 specimens collected
at Kumasi were identified as S form and carried the kdr
mutation. (Yawson et al. 2004). This was in accordance
with the high kdr (89.9% homozygous) detected in our
material from Kona and Afamanaso.
The sporozoite rates for A. gambiae were always higher
than those of A. funestus which corroborates with the
findings of Owusu-Agyei et al. (2009) from the forest
transitional zone in Brong Ahafo, north of Kumasi. This is
in contrast to the coastal forest, the coastal savannah and
Table 4 Plasmodium species from infected salivary glands in A. gambiae and A. funestus
P. falciparum
No. (%)
P. malariae
No. (%)
P. ovale
No. (%)
P. falciparum
& P. malariae
No. (%)
Not amplified
No. (%)
Total
A. gam. 95 (77.9) 7 (5.7) 7 (5.7) 1 (0.8) 12 (9.8) 122
A. fun. 11 (64.7) 0 0 0 6 (35.3) 17
Total 106 (76.3) 7 (5.0) 7 (5.0) 1 (0.7) 18 (13.0) 139
Table 5 Presence of the kdr gene in Anopheles gambiae in the study area
Town No.
amplified
Homozygous
resistant (RR)
Heterozygous
resistant (RS)
Homozygous
susceptible (SS)
Not
amplified
Afamanaso 61 53 (86.9%) 6 (9.8%) 2 (3.3%) 5
Kona 48 45 (93.8%) 2 (4.2%) 1 (2.1%) 13
Totals 109 98 (89.9%) 8 (7.3%) 3 (2.8%) 18
Parasitol Res (2011) 108:1465–1471 1469

also to the northern savannah of Ghana where infection
rates of A. funestus were higher than those of A. gambiae
(Appawu et al. 2001,2003; Okoye et al. 2005).
The malaria transmission in both study villages was
perennial and intensive with annual EIRs of 866 for
Afamanaso and 490 for Kona. This was comparable with
the transmission in the Sudan savannah (418) of northern
Ghana (Appawu et al. 2004) and higher than that measured
in the forest savannah transitional zone around Kintampo
(269) and the coastal forest at Dodowa (21.9) (Owusu-
Agyei et al. 2009; Appawu et al. 2001).
Biting rates and transmission varied in both villages with
rainfall. A. gambiae was the main vector contributing 81%
of the transmission. A. funestus was the secondary vector.
However, the contribution of A. funestus differed in the two
villages, 36% in Afamanaso but only 7.7% in Kona.
Similarly A. funestus was found to be the secondary vector
also in Dodowa (Appawu et al. 2001), the area around
Kintampo (Owusu-Agyei et al. 2009) and in the Kassena
Nankana District of the northern savannah (Appawu et al.
2004). Differences in the amount of transmission in Kona and
Afamanaso were in parallel with the number of malaria
episodes per year of small children of 2.2 and 1.1,
respectively (Kreuels et al. 2008). The larger population in
Kona (5,853, Afamanaso 2,508) might lead to a dilution of
man vector contact and a main reason for the difference in
biting rates and EIRs determined in both villages. Differ-
ences of transmission and species composition of the
vector populations are further influenced by the distinct
topographies of the two study sites. Afamanaso is a village
in the plain surrounded by the Ofin river and swampy
areas, while Kona is located on a ridge with only small
streams in the valley.
The hourly biting activities were similar for both vectors
with peaks from 23.00-02.00 h as described by Gillies and
de Meillon (1968). It was important to note that essential
biting and transmission occurred before bedtime from
1800–2100 h in both villages with AEIRs of 57.3 in
Afamanaso and 38.7 in Kona. These AEIRs were even
higher than the AEIR of 22 measured for whole nights at
Dodowa in the coastal forest (Appawu et al. 2004), but
malaria prevalences in the human population of 42.2% in
April and 51.3% in August (Afari et al. 1995) were
comparable with 50.7% determined in Ashanti Region
(Browne et al. 2000). It is to be feared that the use of
impregnated long-lasting bednets may not be effective in
preventing transmission in areas with such high early-
evening transmission rates: i.e. before bedtime. Children are
only sufficiently protected by bednets in areas with no
transmission in the early evening hours, e.g. Dodowa of the
coastal forest (Appawu et al. 2001). It is another problem
that in Afamanaso 15.9% of women and 37% of men sleep
outside (Tuno et al. 2010).
Acknowledgements The study was partially funded by the Bundes-
ministerium für Bildung und Forschung (grant 01KA0202). We are
indebted to the vector collectors and the people in Kona and
Afamanaso for continuous cooperation. We appreciate the support of
the staff of Kumasi Centre for Collaborative Research in Tropical
Medicine (KCCR). In particular we acknowledge the assistance of Dr.
Christof Berberich, head of KCCR laboratories. Visits of Rolf Garms
in Ghana were made possible by support of the German Senior
Experten Service in 2003, 2005 and 2006. Ayimbire Abonuusum
thanks the Bernhard Nocht Institute for Tropical Medicine for a 1-year
scholarship and research stay in Hamburg, Germany and the Kwame
Nkrumah University of Science and Technology, Kumasi, Ghana for a
PhD grant. We thank Dr. Jean Pierre Lin for proofreading of the final
version of the manuscript.
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