there are large among - site variations in the abundance and temporal dynamics of malaria vector populations indicating that the risk of parasite transmission differs among sites . even in one topographic area , mosquito vectors and malaria infections
may not be distributed homogeneously , and some households within the same area have a higher malaria incidence than others [ 24 ] .
many factors may be responsible for this spatial heterogeneity of malaria vectors and transmission intensity such as land use and land cover changes , topography , house building materials , and design and the level of household protection measures against mosquitoes [ 510 ] . in most cases , it is difficult to identify the factor that contributes most to these variations . in many african highlands ,
malaria resurgence has been attributed largely to the rise in drug - resistant parasites , although other factors are also likely to be important , such as poor health systems , land use such as deforestation and swamp reclamation [ 6 , 13 , 14 ] , population growth and migration , and climate variability [ 16 , 17 ] . in western kenya , malaria is predominantly a rural disease , and the main malaria vectors are anopheles gambiae sensu stricto , an .
anopheles gambiae generally increases in density after the start of the long rains , while an .
funestus density is seen to vary in direct proportion to the proximity of permanent breeding grounds rather than rainfall . in the adult stage , these anopheline species share many of the same habitats . in the usambara mountains , tanzania , and in western kenya , balls
reported that altitude plays an important role in determining malaria infection due to its effect on temperature .
temperature decreases with increasing altitude , and at lower altitudes , the high temperature levels accelerate the sporogonic cycle of malaria parasites in the presence of vectors and the breeding habitats .
land use such as deforestation and swamp reclamation by eliminating shade modifies the local climate and microclimate , and in the presence of stagnant water , new habitats for malaria vectors are formed [ 6 , 14 ] .
consequently , the new habitats provide new breeding grounds leading to increased vector densities and subsequently an increase in malaria transmission . over the past four decades
, deforestation and swamp cultivation have widely occurred in western kenya , and these are now thought to be a major contributing factor to the abundance of breeding habits and the survival of malaria vectors . the ever - increasing human population and the need for food security place large pressure on land and threaten the survival of undisturbed natural forests and swamps .
the current study was undertaken to investigate the dynamics of malaria transmission in three different sites in western kenya .
the hypothesis being tested is that malaria risk is high in transformed swamp sites of nyalenda and lunyerere and not fort ternan .
the study was carried out in western kenya in two highland villages , lunyerere and fort ternan , and the lowland periurban nyalenda , a suburb of kisumu city .
fort ternan ( 0  12  s and 35  20  e ) is a rural village in kericho county located on the slopes of nandi hills lying between 1480 and 1650  m. the area is hilly with sharp , v - shaped valleys with high rainfall favouring agriculture .
farming in fort ternan is done on large scale with the main crops being sugarcane , maize , and to some extent coffee .
lunyerere ( 0  06  n and 34  43  e ) village is located in vihiga county , on the eastern side of the kakamega forest , about 5  km north of the equator , with an altitude ranging from 1460 to 1550  m. the area is characterized by broad u - shaped valleys that are prone to flooding offering excellent mosquito breeding habitats .
majority of the valley bottoms in this area were previously forested covered with natural swamps that were fed by water through underground seepage .
however , in recent times , the land has been cleared to farmland , where the community members practice small - scale food crop farming .
nyalenda ( 0  06  s and 34  46  e , 1100  m ) is a periurban area located on the outskirts of kisumu city .
kisumu is situated on the northeastern tip of winam gulf , an inlet of lake victoria .
nyalenda is fairly flat area fed by natural springs that produce abundant water used for irrigation on small - scale gardens .
the area was previously a swamp but due to an increase in population in urban kisumu , farming for food crops has been encouraged as a way of ensuring food security for the expanding population .
more information of the study areas including the larval species and abundance can be found in imbahale et al . .
arabiensis to be the most abundant in fort ternan and nyalenda , 71% and 93% , respectively , whereas an .
ten houses were randomly selected in lunyerere and nyalenda for adult mosquito sampling , while in fort ternan , 20 houses were randomly selected .
most of the sentinel houses consisted of mud walls and thatched roofs , while a few had iron sheet roofs and cemented walls . in each site , adult mosquitoes were collected monthly from the sentinel houses by centres for disease control ( cdc ) battery - operated light traps ( model 512 ; john w. hock company , gainesville , fl , usa ) and pyrethrum spray catches ( pscs ) .
pyrethrum spray catch began in march 2006 , while cdc light trap collections began later in july 2006 . on each sampling occasion , the cdc light trap catches preceded the psc catches by 24  h throughout the study .
light traps were installed in the sentinel houses near the foot end of the bed , next to an untreated bed net   and operated from 18.00 pm to 06.00 hours in each house .
one day after the cdc light trap collections , pscs were made between 08  :  00 and 11  :  00 am using simple flit guns to spray inside closed rooms with 2% pyrethrum extract synergised with piperonyl butoxide in kerosene .
ten minutes were allowed before closed rooms were reentered , and the mosquitoes were collected from the sheets that had been laid out in the rooms .
female anopheles mosquitoes were identified morphologically according to gillies and coetzee , stored , and dried on silica gel at room temperature pending further analysis .
although culicine mosquitoes do not transmit malaria , mosquitoes of this genus are mainly nuisance biters and were also recorded during the sampling .
mosquito sampling took place in the same sentinel houses throughout the study . in any event such as abandoning of the houses by occupants
gambiae complex were identified to the species level using the polymerase chain reaction ( pcr ) method ; for this purpose , dna of adult female an .
gambiae were extracted from one wing or leg . the head and thorax of each female an .
funestus were tested singly for plasmodium falciparum sporozoites using the standard enzyme - linked immunosorbent assay as described by beier et al .   at the walter reed army institute laboratory based at kisian , kisumu , kenya .  
a house - to - house population survey was done in each study location to identify households with children aged between 2 and 10 years .
these children were then enrolled to form a study cohort of 100 children per study site . in fort
ternan , children were enrolled from houses located in the valley and up the valley .
consent was sought from the parents / guardians before the child was enrolled in the study . each child
was then given a unique code that was used to track the same child throughout the study from june 2006 to april 2008 .
during the surveys , the children were followed to their respective schools , while those not in schools were followed in their respective homes .
blood samples were collected monthly by the standard finger - prick method ; thick and thin smears were prepared on labeled slides .
core body temperature of the children was measured with a braun thermoscan ( frankfurt , germany ) ear probe thermometer , and each child was tested with a fresh sterile ear plug . the thin and thick blood smears were air dried .
thereafter , the thin and thick smears were fixed in methanol and stained in 4% giemsa for 30 minutes .
an experienced technician examined the slides under 1,000 magnification by using oil immersion to identify and count the parasite species .
random checks were carried out on the slide counts ( to include at least 10% of all slides ) by independent microscopists to ensure quality control .
parasite density was scored against 200 leukocytes when the slide was positive ; otherwise , the whole slide was carefully scanned before being declared negative .
any child that was clinically ill at the survey date was taken to the nearest public health facility for treatment free of charge .
a child was considered clinically ill if he / she had fever ( a core body temperature  37.5c ) and malaria parasites identified from the blood smear .
malaria parasite incidence studies in fort ternan commenced in june 2006 while in lunyerere and nyalenda in january 2007 .  
automatic weather stations were installed , one at the fort ternan health centre , the other one at lyanaginga health centre about 30  km from lunyerere , and another at the kenya medical research institute ( kemri ) , centre for global research , kisian , about 17  km from nyalenda ( but at the same altitude as nyalenda ) .
the weather stations measured temperature and humidity at 2  m above ground ( ventilated probe ; vaisala , finland ) and precipitation ( rain gauge , eijkelkamp , the netherlands ) throughout the study period .
the weather variables were recorded on a 21x microdatalogger ( campbell scientific inc . , uk ) at an interval of 15 minutes from march 2006 to april 2008 .
detailed description of how the weather station works has been provided in paaijmans et al . .
for lunyerere and fort ternan , all variables were measured as expected . in kisian , however , the weather station experienced technical problems for several months ; hence , humidity data are not available for nyalenda . as a proxy
, we have used the average relative humidity data from the kenya airports authority based at kisumu airport , midway between nyalenda and kisian .
institutional ethical clearance was given by the kenya medical research institute ( kemri ) and wageningen university and research centre ( wur ) , the netherlands , protocol approval numbers 1121 and 512 .
in addition , consent was obtained from the parents / guardians , community elders , and house owners .  
only female mosquitoes were included in data analysis as they are responsible for disease transmission .
sporozoite rate calculations were based on the total anopheles female catch from cdc light traps and psc collections . the mean
sporozoite rate was estimated , and the annual entomological inoculation rate ( eir ) was calculated by multiplying the sporozoite rate by the mean biting rate / night multiplied by 365 days .
due to political instability in the country between december 2007 and march 2008 , we were unable to work in our study sites for the months of january , february , and march 2008 ; hence , data for adult mosquito sampling and malaria incidence for this period are not available in some sites .   for entomological survey data , the proportion of mosquitoes caught were compared using chi - square test , while for the other datasets , we perform regression analysis to adjust for the confounding effect of covariates .
we fit a negative binomial generalized additive model ( gam ) with log link to study the association between risk of malaria parasite and the site while adjusting for body temperature and month , with site entering the model parametrically and month , temperature , and their interaction as nonparametrically smoothed functions .
month was defined as a fraction of time given by year + ( month 1)/12 . to study the association between the risk of having malaria parasite and weather variables
, we fit poisson gam ( with log link ) with site , mean monthly relative humidity , monthly rainfall , and temperature as covariates , with the latter two entering the model as smoothed functions and total monthly slides read as the offset .
the gam was used to allow the inclusion of nonparametric smooth functions to model the potential nonlinear dependence of malaria parasites on weather variables and other covariates .
the assumption of this model is that 
 ( 1)log[e(y)]=0+1x1+2x2++spk(xpk )  + +sp(xp ) , 
 where y , is the count of parasites , e(y ) is the expected value of this count , xi  ( i = 1,2 ,  , p ) are the covariates , and spk  sp and the smoothing functions .
negative binomial was assumed for the parametric part in the former case because the observed data were highly aggregated ; they had a variance - mean ratio much greater than 1 , thus violating poisson assumptions .
the contribution of variables in the regression models was assessed with the use of likelihood ratio ( lr ) and wald tests , with all tests performed at 5% level .
of 13640 adult mosquitoes caught , 422 ( 122 males and 300 females ) were anophelines and 13218 ( 3407 males and 9811 females ) culicines .  
of the 300 female anophelines , 21 , 117 , and 162 were collected from fort ternan , lunyerere , and nyalenda , respectively . among the 300 ,
the results indicated that overall 85 ( 31% : 95% ci 2637% ) of the 273 mosquitoes were an .
s.s , but the proportion was significantly greater in lunyerere ( 52% ) than nyalenda ( 19% ) and fort ternan ( 5% ; p < 0.001 ) . for an .
arabiensis , the overall was 76 ( 28% : 95% ci 2333 ) , but the proportion was significantly higher in nyalenda ( 50% ) than in fort ternan ( 5% ) and lunyerere ( 3% ; p < 0.001 ) . for an .
funestus , the total was 17 ( 6% : 95% ci 39% ) , detected in two sites only fort ternan ( 68% ) and lunyerere ( 2% ; p < 0.001 ) .
culicine species collected include culex spp . , mansonia spp . , and coquillettidia spp . in terms of the methods
used , the results indicated that overall , when all the female anophelines per house in all the study sites were combined , the cdc light trap ( 52.3% ) caught more mosquitoes compared to psc ( 47.7% ) , although not statistically significant ( p = 0.689 ) .
the mean number of mosquitoes per house per night collected with the psc was 0.26  0.039 , while that of the cdc light trap was 0.15  0.027 . in fort
ternan , none of the adult anophelines tested by elisa were found to be infected by plasmodium falciparum sporozoites .
however , in lunyerere , mosquitoes infected with p. falciparum were recorded on four occasions in june , september and november in 2007 and march 2008 . in nyalenda ,
sporozoite - positive mosquitoes were recorded on two occasions in march 2006 and january 2007 .
the mean biting rate was 0.001579 , 0.015909 , and 0.015238 per person per night in fort ternan , nyalenda , and lunyerere , respectively . in fort
ternan , none of the adult anopheles tested by elisa were found to be infected by plasmodium falciparum sporozoites .
conversely , in lunyerere and nyalenda , the p. falciparum sporozoite rates were 11.1% and 1.2% , respectively .
the entomological inoculation rates per annum were 0.00 , 6.91 , and 61.79 infective bites / person / year for fort ternan , nyalenda , and lunyerere , respectively .
a total of 1735 from fort ternan , 1847 from lunyerere , and 1196 from nyalenda blood slides were read , of which 60 ( 3.9% ) , 106 ( 6.6% ) , and 46 ( 4.2% ) slides , respectively , were positive for p. falciparum , the only malaria parasite identified from the study population .
the proportion of children with malaria parasites was 4.4% over the whole sampling period with significant differences among the study sites ( p = 0.002 ) .
the negative binomial gam results indicated a significant interaction between temperature and month ( p < 0.0001 ) . site , month , and body temperature were all associated with the risk of having malaria parasite ( p < 0.0001 ) .
compared to nyalenda , the risk of having malaria parasites was significantly lower in fort ternan ( rr = 0.48 , 95% ci : 0.470.49 ) but significantly higher in lunyerere ( rr = 3.82 , 95% ci : 3.454.24 ) , after adjusting for body temperature and month .
overall , the mean monthly rainfall , relative humidity , and temperature were 105.92  10.82 , 67.16  1.04 , and 20.99  0.26 , respectively .
a summary of the means ( together with their associated standard errors ) of the weather variables for each site separately and all sites combined is presented in table 1 , while the monthly rainfall and humidity in figure 2 .
table 1 results show a significantly higher air temperature in nyalenda than the other two sites .
the multivariable poisson gam results indicated that rainfall was significantly associated with the occurrence of p. falciparum malaria parasites ( p = 0.0146 ) , but not relative humidity ( p = 0.2875 ) and inconclusive for air temperature ( lr test p = 0.0678 ) after adjusting for site .
results of this study show a heterogeneous distribution of vectors and the risk of being infected with malaria within sites only a few kilometres apart .
lunyerere and fort ternan , both highland villages at similar altitudes , exhibit markedly different mosquito vector densities and risk of malaria infection . among the vectors collected ,
rainfall , body temperature , site , and month were all associated with the occurrence of plasmodium parasites among the children cohort .
the differences in malaria risk among the sites can be explained by vector species of local importance , availability of breeding habitats , topography , farming activities , terrain characteristic , preferred host , and environmental conditions among others .  

, the most efficient malaria vector , was abundant in lunyerere , and due to its anthropophilic behavior , high sporozoite rates were recorded in this site compared to nyalenda and fort ternan . in the same location , the proportion ( 12.5% ) of an .
arabiensis recorded during the study was high in the kakamega area when compared to previous studies carried out in nearby villages that did not record the presence of this species [ 1 , 5 ] .
arabiensis in aquatic habitats   and indoors during adult collections indicates that changes in environmental conditions must have occurred to favour its breeding in lunyerere . a substantial number of an .
funestus was collected from fort ternan , but none was found to be infected with parasites .
one possibility is that the species collected might have been a nonvector sibling of the an .
we did not carry out pcr analysis to identify the specific sibling species within the an .
gambiae larvae were sampled in man - made habitats   at the same period in fort ternan , but few adult vectors were collected from indoors .
previous work by koenraadt and others   in the same village found few adult anophelines in indoor collections , although anopheline larvae were found in nearby watering sites for cattle and in tyre tracks .
the low numbers of vector species caught resting indoors can not explain the presence of malaria parasites in the children cohort .
these findings are consistent with the results of ototo and others , who detected no sporozoites in fort ternan after eight months sampling , implying that after taking a blood meal the vectors either rest outdoors or perhaps most of the biting takes place outdoors .
the results obtained from lunyerere and fort ternan are similar to the findings of atieli and others   who found flat - bottomed valleys to have higher larval and adult densities compared to narrow valleys .
gambiae s.s [ 33 , 34 ] , and it has been found to dominate irrigated areas such as rice fields   among other habitats , which may explain its abundance in nyalenda .
anopheles arabiensis exhibits exophilic and exophagic behavior , and because adult mosquito collections were done indoors throughout this study , we may have missed p. falciparum sporozoite - positive mosquitoes of this species in all sites .
recent studies suggest existence of outdoor biting malaria vectors that may contribute to considerable transmission , which went hitherto unnoticed [ 36 , 37 ]
. it would therefore be beneficial for future studies to consider incorporating both indoor and outdoor mosquito catches .
anopheles coustani , a potential vector species for malaria transmission , was present in all sites .
the risk of being infected with malaria was lower in fort ternan but higher in lunyerere when compared to nyalenda .
these findings can partly be explained by the annual eirs recorded which show distinct differences among the study sites with the rural village of lunyerere having a higher eir ( 61.79 ) in contrast to fort ternan where the eir was zero throughout the study period .
the periurban nyalenda recorded an eir ( 6.91 ) eleven times lower than that of lunyerere .
for lunyerere , small - scale food crop production in a transformed swamp area with underground water seepage ensures vector breeding throughout the year which is not the case with fort ternan , an area under large - scale farming .
nyalenda being relatively flat , transformed swamp area supplied with water throughout the year with a significantly higher average air temperature compared to the other two , one would expect a higher risk to infection here .
the presence of breeding habitats throughout the year with favourable temperatures leads to accelerated sporogonic cycle of p. falciparum parasites hence posing a higher risk of infection but that was not the case when compared to lunyerere .
nyalenda being a periurban site , the population was found to be equipped with more knowledge on mosquito and malaria control than the counterparts in the two villages .
consequently , more households used protective measures such as insecticide - treated bednets   lowering the risk of being infected .
finally , microscopy was adopted as the standard method used to examine malaria parasites in a cohort of children throughout the study period .
although microscopy still remains the standard diagnostic method for malaria parasites , a number of studies have shown that it may fail to detect low parasitemia levels that are common in asymptomatic individuals when compared to pcr [ 40 , 41 ] .
asymptomatic individuals are able to sustain malaria transmission , and thus , the failure of microscopy to identify such individuals could mean that our result represents an underestimate of the real situation . nevertheless , historical records of malaria parasite prevalence in the highlands of western kenya reported higher rates than what was found in the current study .
the reduction in malaria cases is attributed to the adoption of the roll back malaria initiative   by the kenyan government since the year 2006 , which scaled up the use of insecticide - treated bednets [ itns ] in the areas studies . however , in spite of these control measures , low levels of transmission continue given the recorded p. falciparum parasite , calling for integrated approaches that are complementary to the use of itns . in conclusion , these results show that the risk of being infected with malaria in western kenya is heterogeneously distributed , both temporally and spatially depending on the topography , farming activities such as swamp reclamation , biology of vector species concerned , availability of mosquito breeding habitats , and behavioural characteristics of the population at risk .
the presence of malaria parasite - infected children in all the study sites provides evidence of local malaria transmission , although in fort ternan the mosquito density was too low to explain the presence of malaria parasites in the cohort of children .
future studies need to consider both indoors and outdoor resting vectors to get a more insight on how malaria is transmitted in fort ternan .
 the main objective was to investigate malaria transmission dynamics in three different sites , two highland villages ( fort ternan and lunyerere ) and a lowland peri - urban area ( nyalenda ) of kisumu city . 
 adult mosquitoes were collected using psc and cdc light trap while malaria parasite incidence data was collected from a cohort of children on monthly basis . 
 rainfall , humidity and temperature data were collected by automated weather stations . 
 negative binomial and poisson generalized additive models were used to examine the risk of being infected , as well as the association with the weather variables . 
 anopheles gambiae s.s . was most abundant in lunyerere , an . 
 arabiensis in nyalenda and an . 
 funestus in fort ternan . 
 the cdc light traps caught a higher proportion of mosquitoes ( 52.3% ) than psc ( 47.7% ) , although not significantly different ( p = 0.689 ) . 
 the eir 's were 0 , 61.79 and 6.91 bites / person / year for fort ternan , lunyerere and nyalenda . 
 site , month and core body temperature were all associated with the risk of having malaria parasites ( p < 0.0001 ) . 
 rainfall was found to be significantly associated with the occurrence of p. falciparum malaria parasites , but not relative humidity and air temperature . 
 the presence of malaria parasite - infected children in all the study sites provides evidence of local malaria transmission . 
1. Introduction
2. Materials and Methods
3. Results 
4. Discussion 