Study area
The study was conducted in Engari-Kakindo (0° 1′ 57.44′ S, 30° 40′ 33.74′E; Altitude = 1255 m) and Kigorogoro (0° 2′ 9.85′ S, 30° 48′ 28.54′E; Altitude = 1271 m) villages in Kazo subcounty located in Kiruhura district of southwestern Uganda (Figure 1). The area has high and perennial malaria transmission, with two peaks following the rainy seasons.
Malaria prevalence decrease from 43% in 2004 to 23% in 2010 reported in children less than five years of age. There was however high heterogeneity with nearly 10% of the area having prevalence greater than 50% [3].
The district lies within the great East African geographical rift valley. The estimated population of 290,400 (2011) and 300,800 (2012) inhabitants is mainly rural [19]. Their main occupations are agriculture, cattle rearing and cropping. The climate is equatorial with a long bi-modal rainy season from mid-August to December and from March to May. Rainfalls average 1,200 mm per annum and the temperatures 25-27°C. The households were mainly grass thatched, “igloos”, semi-permanent and permanent houses. Each household has its own water facility that serves both animal and human consumption in addition to communal valley dams and troughs. The larval habitats in the two sites were different. Engari was seen to have short shrub like vegetation with usually open water pools, foot and hoof prints. Kigorogoro however exhibited papyrus type of vegetation, swamps and large water pools usually with an emergent vegetation or shaded environment. This could explain the Anopheles species variation within the two sites. A survey carried out in Nyabushozi County, Kiruhura district, with similar environmental dynamics to Kazo found Anopheles species variations in two different villages. Anopheles gambiae was dominant species in Mugore village and An. funestus dominated in Kakyeera village [13]. The main malaria prevention methods used in these areas include use of treated and non-insecticide treated bed nets. The bed net usage in children under 5 years old was 44.5% in 2010 [3]. No indoor house spraying (IRS) with pyrethroids has ever been conducted in Engari and Kigorogoro.
Entomological methods
Selection of the houses and seasons
Two rounds of mosquito captures were done in Engari in April 2011 during the rainy season and in February 2012 during dry season and one round in Kigorogoro during the dry season. Monthly rainfall data obtained from the Mbarara meteorological station showed 132.1 mm of rainfall within 16 rain days during April 2011 and 23.3 mm of rainfall within 5 rain days during February 2011. The maximum and minimum temperatures reported from the same source were 26.5°C and 16.0°C during April 2011 and 30.8°C and 15.7°C during February 2011 respectively (unpublished data). The February 2011 meteorology data was used to predict the February 2012 weather.
Twenty houses in each village were selected following specific criteria which includes; older house constructions, outside commercial centres, close to lowland and without kitchen inside the main houses to avoid the effect of smoke on mosquito collection. Mosquito collections were performed during a preliminary study in Engari during the rainy season (April 2011).
Mosquito collection and identification
Human landing catches (HLC) were carried out in three houses per village during six days in the rainy season in Engari and ten days during the dry season in Engari and Kigorogoro. Indoor HLC was performed between 6 p.m.- 6 a.m. and outdoor collection between 6 p.m. to midnight.
Morning indoor resting catches (MIRC) was performed in six houses in Engari and Kigorogoro, different from the ones of HLC, for ten days during dry season and six days during the rainy season in Engari. The collections were done using a hand held aspirator between 6-8:30 a.m. In addition Pyrethrum spray catches (PSC) were carried out in 20 houses in each village on the last day of mosquito collections between 8:30 a.m. and midday during the two seasons.
Female adult Anopheles mosquitoes used for WHO resistance testing were reared from larvae, as described in Service et al [14].
Morphological keys were used to identify malaria vectors collected [15,20]. All mosquitoes belonging to the Anopheles family were kept in individual tubes containing silica gel for further laboratory testing. Only females heads and thoraxes from HLC, MIRC and PSC were processed for detection of circumsporozoite protein (CSP) of P. falciparum sporozoites using an enzyme-linked immunosorbent assay (ELISA) technique [16]. According to Echodu et al., 14.2% (n = 1544) of An. gambiae s.l. analysed using PCR were identified as An. gambiae S.S. and no An. arabiensis was found. In addition, 20% (n = 270) of An. funestus group analysed were found to be A. funestus, An. leesoni, and An. parensis using PCR in Nyabushozi County, Kiruhura district [13]. These species variations observed in a similar setting to this current study gives a comparison picture of what would have been expected in Kazo subcounty, Kiruhura district.
Entomological indicators
Human biting rates (HBRs) for Anopheles were calculated as number of bites per human per night obtained from HLC. The calculation of outdoor HBR considered that an average villager enters the house by 10 p.m. latest. Sporozoite rates (SRs) were the proportions of female Anopheles found positive to CSP antigens. Daily entomological inoculation rates (EIRs i.e. number of infected bites per human per night) were obtained using the average number of Anopheles CSP positive collected by HLC per nights of collection for each village and season. Monthly EIRs were extrapolated for April 2011 and February 2012. The indoor resting density (IRD) at the house level for each Anopheles species was calculated as the number of Anopheles females captured at each round of MIRC divided by the total number of houses inspected, in each village and season.
Insecticide bioassays
The insecticide resistance status of the malaria vectors was assessed using mosquitoes collected by larval collection and adults from HLC and/or MIRC when low numbers of larvae were found.
Insecticide susceptibility assays were performed using two to three days old adult female A. gambiae s.l and A. funestus fed on 5% glucose and adults of unknown ages from HLC and MIRC. Five replicates of 20 adult female Anopheles were exposed to each of the four different insecticide-impregnated papers (DDT 4%, bendiocarb 0.1%, deltamethrin 0.05% and pirimiphos methyl 1.0%) in standard WHO test tubes. One batch of 20 adult female Anopheles was subjected to untreated papers to act as controls. All tests were undertaken at 25°C ± 2°C and 70–80% relative humidity during one hour.
Cumulative knockdown (KD) counts were recorded every 5-10 minutes for the respective exposure periods. After exposure, mosquitoes were supplied with 5% glucose solution, and mortality was recorded at 24 hours post-exposure. KT50 was the time after which 50% of the Anopheles tested were knocked down and KD60 as the proportion of Anopheles knocked down after 60 minutes [17,18].
Data analysis
Data were double entered using Epidata V.3.1 (Epidata, Odense, Denmark). Statistical analysis was performed using Stata 12 (College Station, Texas). Descriptive statistics were presented for each village and season of capture. Fisher exact test was used to compare proportions.
Ethical considerations
The study obtained ethical clearance from the Mbarara University, Faculty of Medicine research committee (FREC), Mbarara institutional review committee and Uganda national council of science and technology (UNCST). Informed written consent from the heads of the households and from the collectors were obtained. In addition malaria prophylaxis (doxycycline) was provided to the collectors performing HLC. Blood smear slides and rapid diagnostic tests each week from the collectors for malaria diagnosis. If the blood slides were found positive, the participant was treated with artemether-lumefantrine (Coartem®) according to the Ugandan national guidelines. A follow-up blood slide smear check was performed on a weekly basis until day 42 when the RDT turned out negative to ensure that all parasites were fully cleared; if not, the worker was referred to the hospital to receive the second line treatment.