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Abstract Background A supervised land cover classification was developed from very high resolution IKONOS satellite data and extensive ground truth sampling of a ca. 10 sq km malaria-endemic lowland in western Kenya. The classification was then applied to an investigation of distribution of larval Anopheles habitats. The hypothesis was that the distribution and abundance of aquatic habitats of larvae of various species of mosquitoes in the genus Anopheles is associated with identifiable landscape features. Results and discussion The classification resulted in 7 distinguishable land cover types, each with a distinguishable vegetation pattern, was highly accurate (89%, Kappa statistic = 0.86), and had a low rate of omission and commission errors. A total of 1,198 habitats and 19,776 Anopheles larvae of 9 species were quantified in samples from a rainy season, and 184 habitats and 582 larvae from a dry season. Anopheles gambiae s.l. was the dominant species complex (51% of total) and A. arabiensis the dominant species. Agricultural land covers (mature maize fields, newly cultivated fields, and pastured grasslands) were positively associated with presence of larval habitats, and were located relatively close to stream channels; whilst nonagricultural land covers (short shrubs, medium shrubs, tall shrubs, and bare soil around residences) were negatively associated with presence of larval habitats and were more distant from stream channels. Number of larval habitats declined exponentially with distance from streams. IKONOS imagery was not useful in direct detection of larval habitats because they were small and turbid (resembling bare soil), but was useful in localization of them through statistical associations with specific land covers. Conclusion A supervised classification of land cover types in rural, lowland, western Kenya revealed a largely human-modified and fragmented landscape consisting of agricultural and domestic land uses. Within it, larval habitats of Anopheles vectors of human malaria were associated with certain land cover types, of largely agricultural origin, and close to streams. Knowledge of these associations can inform malaria control to gather information on potential larval habitats more efficiently than by field survey and can do so over large areas.

Interleukin (IL)-1beta is a cytokine released as part of the innate immune response to Plasmodium falciparum. Because the role played by IL-1beta polymorphic variability in conditioning the immunopathogenesis of severe malarial anemia (SMA) remains undefined, relationships between IL-1beta promoter variants (-31C/T and -511A/G), SMA (hemoglobin [Hb] level <6.0 g/dL), and circulating IL-1beta levels were investigated in children with parasitemia (n= 566) from western Kenya. The IL-1beta promoter haplotype -31C/-511A (CA) was associated with increased risk of SMA (Hb level <6.0 g/dL; odds ratio [OR], 1.98 [95% confidence interval {CI}, 1.55-2.27]; P < .05) and reduced circulating IL-1beta levels (p <.05). The TA (-31T/-511A) haplotype was nonsignificantly associated with protection against SMA (OR, 0.52 [95% CI, 0.18-1.16]; p =.11) and elevated IL-1beta production ( p<.05). Compared with the non-SMA group, children with SMA had significantly lower IL-1beta levels and nonsignificant elevations in both IL-1 receptor antagonist (IL-1Ra) and the ratio of IL-1Ra to IL-1beta. The results presented demonstrate that variation in IL-1beta promoter conditions susceptibility to SMA and functional changes in circulating IL-1beta levels. [ABSTRACT FROM AUTHOR]

A randomized, placebo-controlled treatment trial was conducted among 546 anemic (hemoglobin concentration, 7-11 g/dL) children aged 2-36 months in an area with intense malaria transmission in western Kenya. All children used bednets and received a single dose of sulfadoxine-pyrimethamine (SP) on enrollment, followed by either intermittent preventive treatment (IPT) with SP at 4 and 8 weeks and daily iron for 12 weeks, daily iron and IPT with SP placebo, IPT and daily iron placebo, or daily iron placebo and IPT with SP placebo (double placebo). The mean hemoglobin concentration at 12 weeks, compared with that for the double-placebo group, was 1.14 g/dL (95% confidence interval [CI], 0.82-1.47 g/dL) greater for the IPT+iron group, 0.79 g/dL (95% CI, 0.46-1.10 g/dL) greater for the iron group, and 0.17 g/dL (95% CI, -0.15-0.49 g/dL) greater for the IPT group. IPT reduced the incidence of malaria parasitemia and clinic visits, but iron did not. The combination of IPT and iron supplementation was most effective in the treatment of mild anemia. Although IPT prevented malaria, the hematological benefit it added to that of a single dose of SP and bednet use was modest. [ABSTRACT FROM AUTHOR]

We detected a cluster of dengue virus infections in children in Kenya during July 2014-June 2015. Most cases were serotype 1, but we detected all 4 serotypes, including co-infections with 2 serotypes. Our findings implicate dengue as a cause of febrile illness in this population and highlight a need for robust arbovirus surveillance.

Background: Use of insecticide treated bednets (ITNs) was a breakthrough in the fight against malaria. However, ITNs are only effective when properly used. Recent reports indicate low compliance in ITN usage and changes in biting times of malaria vectors with early and late biting cases recorded when people are not sleeping under their nets. Hence, there is a need to develop methods to supplement or replace the use of ITNs for malaria vector control. A field trial was conducted to investigate the effect and longevity of prototypes of long lasting impregnated UV protected eave nets, curtains and door hangers (fully screened houses), compared to houses with bednets only, in traditional East African houses., Materials and Methods: A randomised controlled trial was carried out in the Ahero rice irrigation scheme in Nyando district, Western Kenya. Eighty houses with open eaves were randomly selected. Forty of these houses were fully screened (FSH+LLINs) with long lasting insecticide-treated nets/curtains used to screen the eaves, windows and doors. The FSH materials were produced with anti-UV additives. The other 40 houses served as controls. Long lasting insecticide-treated bednets (LLINs) were suspended over all sleeping areas in the control and intervention houses. Indoor resting Anopheles mosquitoes were collected using pyrethrum spray catches (PSC) during both dry and wet seasons. Indoor population densities of anophelines were compared between intervention (FSH+LLINs) and control (LLINs) houses. Loss of insecticide (deltamethrin) was compared after 12 and 24 months for both the FSH materials and LLINs through bioassays and chemical analyses., Results: In the FSH+LLINs houses densities of indoor resting Anopheles funestus and An. arabiensis were reduced by 82% (RR=0.18, 95% CI 0.09-0.36, P<0.0001) and 70% (RR=0.30, 95% CI 0.15-0.58, P<0.0001), respectively. No significant difference was recorded for indoor resting Culex spp. (RR=0.95, 95% CI 0.48-1.86, P=0.8). The population of indoor resting bloodfed An. arabiensis and An. funestus was reduced by 72% (RR=0.22, 95% CI 0.09-0.51, P<0.0001) and 84% (RR=0.16, 95% CI 0.07-0.33, P<0.0001) in the FSH+LLINs houses and LLIN houses, respectively. Insecticide loss in eave nets did not depend on the side of the house where the nets were placed. The eave nets showed little loss of bio efficacy over the 12-24 months period., Conclusions: The study revealed that the use of insecticide-treated nets on the eaves and windows combined with door hangers largely impeded entrance of anopheline mosquitoes into houses and can be used to compliment LLINs for household protection. The eave nets were suspended from wood structures near the eave and remained in place when walls were re-plastered. The nets are therefore not depending on daily compliance behaviour and provide protection for the entire household., Competing Interests: The authors declare that they have no competing interests. The tested products are not commercialised and the Netprotect WHO recommendation was withdrawn in 2012 and this net is no longer commercially available., (Copyright © 2016: Odhiambo et al.)

Introduction: The voltage gated sodium channel mutation Vgsc-1014S (kdr-east) was first reported in Kenya in 2000 and has since been observed to occur at high frequencies in the local Anopheles gambiae s.s., Population: The mutation Vgsc-1014F has never been reported from An. gambiae Complex complex mosquitoes in Kenya., Findings: Molecularly confirmed An. gambiae s.s. (hereafter An. gambiae) and An. arabiensis collected from 4 different parts of western Kenya were genotyped for kdr from 2011 to 2013. Vgsc-1014F was observed to have emerged, apparently, simultaneously in both An. gambiae and An. arabiensis in 2012. A portion of the samples were submitted for sequencing in order to confirm the Vgsc-1014F genotyping results. The resulting sequence data were deposited in GenBank (Accession numbers: KR867642-KR867651, KT758295-KT758303). A single Vgsc-1014F haplotype was observed suggesting, a common origin in both species., Conclusion: This is the first report of Vgsc-1014F in Kenya. Based on our samples, the mutation is present in low frequencies in both An. gambiae and An. arabiensis. It is important that we start monitoring relative frequencies of the two kdr genes so that we can determine their relative importance in an area of high insecticide treated net ownership.

Plasmodium falciparum parasites that are resistant to artemisinins have been detected in Southeast Asia. Resistance is associated with several polymorphisms in the parasite's K13-propeller gene. The molecular epidemiology of these artemisinin resistance genotypes in African parasite populations is unknown. We developed an assay to quantify rare polymorphisms in parasite populations that uses a pooled deep-sequencing approach to score allele frequencies, validated it by evaluating mixtures of laboratory parasite strains, and then used it to screen P. falciparum parasites from >1100 African infections collected since 2002 from 14 sites across sub-Saharan Africa. We found no mutations in African parasite populations that are associated with artemisinin resistance in Southeast Asian parasites. However, we observed 15 coding mutations, including 12 novel mutations, and limited allele sharing between parasite populations, consistent with a large reservoir of naturally occurring K13-propeller variation. Although polymorphisms associated with artemisinin resistance in P. falciparum in Southeast Asia are not prevalent in sub-Saharan Africa, numerous K13-propeller coding polymorphisms circulate in Africa. Although their distributions do not support a widespread selective sweep for an artemisinin-resistant phenotype, the impact of these mutations on artemisinin susceptibility is unknown and will require further characterization. Rapid, scalable molecular surveillance offers a useful adjunct in tracking and containing artemisinin resistance., (© The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.)

Cytophilic immunoglobulin (IgG) subclass responses (IgG1 and IgG3) to Plasmodium falciparum antigens have been associated with protection from malaria, yet the relative importance of transmission intensity and age in generation of subclass responses to pre-erythrocytic and blood-stage antigens have not been clearly defined. We analyzed IgG subclass responses to the pre-erythrocytic antigens CSP, LSA-1, and TRAP and the blood-stage antigens AMA-1, EBA-175, and MSP-1 in asymptomatic residents age 2 years or older in stable (n=116) and unstable (n=96) transmission areas in Western Kenya. In the area of stable malaria transmission, a high prevalence of cytophilic (IgG1 and IgG3) antibodies to each antigen was seen in all age groups. Prevalence and levels of cytophilic antibodies to pre-erythrocytic and blood-stage P. falciparum antigens increased with age in the unstable transmission area, yet IgG1 and IgG3 responses to most antigens for all ages in the unstable transmission area were less prevalent and lower in magnitude than even the youngest age group from the stable transmission area. The dominance of cytophilic responses over non-cytophilic (IgG2 and IgG4) was more pronounced in the stable transmission area, and the ratio of IgG3 over IgG1 generally increased with age. In the unstable transmission area, the ratio of cytophilic to non-cytophilic antibodies did not increase with age, and tended to be IgG3-biased for pre-erythrocytic antigens yet IgG1-biased for blood-stage antigens. The differences between areas could not be attributed to active parasitemia status, as there were minimal differences in antibody responses between those positive and negative for Plasmodium infection by microscopy in the stable transmission area. Individuals in areas of unstable transmission have low cytophilic to non-cytophilic IgG subclass ratios and low IgG3:IgG1 ratios to P. falciparum antigens. These imbalances could contribute to the persistent risk of clinical malaria in these areas and serve as population-level, age-specific biomarkers of transmission., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background: Monitoring local malaria transmission intensity is essential for planning evidence-based control strategies and evaluating their impact over time. Anti-malarial antibodies provide information on cumulative exposure and have proven useful, in areas where transmission has dropped to low sustained levels, for retrospectively reconstructing the timing and magnitude of transmission reduction. It is unclear whether serological markers are also informative in high transmission settings, where interventions may reduce transmission, but to a level where considerable exposure continues., Methods: This study was conducted through ongoing KEMRI and CDC collaboration. Asembo, in Western Kenya, is an area where intense malaria transmission was drastically reduced during a 1997-1999 community-randomized, controlled insecticide-treated net (ITN) trial. Two approaches were taken to reconstruct malaria transmission history during the period from 1994 to 2009. First, point measurements were calculated for seroprevalence, mean antibody titre, and seroconversion rate (SCR) against three Plasmodium falciparum antigens (AMA-1, MSP-119, and CSP) at five time points for comparison against traditional malaria indices (parasite prevalence and entomological inoculation rate). Second, within individual post-ITN years, age-stratified seroprevalence data were analysed retrospectively for an abrupt drop in SCR by fitting alternative reversible catalytic conversion models that allowed for change in SCR., Results: Generally, point measurements of seroprevalence, antibody titres and SCR produced consistent patterns indicating that a gradual but substantial drop in malaria transmission (46-70%) occurred from 1994 to 2007, followed by a marginal increase beginning in 2008 or 2009. In particular, proportionate changes in seroprevalence and SCR point estimates (relative to 1994 baseline values) for AMA-1 and CSP, but not MSP-119, correlated closely with trends in parasite prevalence throughout the entire 15-year study period. However, retrospective analyses using datasets from 2007, 2008 and 2009 failed to detect any abrupt drop in transmission coinciding with the timing of the 1997-1999 ITN trial., Conclusions: In this highly endemic area, serological markers were useful for generating accurate point estimates of malaria transmission intensity, but not for retrospective analysis of historical changes. Further investigation, including exploration of different malaria antigens and/or alternative models of population seroconversion, may yield serological tools that are more informative in high transmission settings.

Background: Achieving optimal iron status in children in malaria-endemic areas may increase the risk of malaria. Malaria itself may contribute to iron deficiency, but the impact of an interruption in malaria transmission on the prevalence of iron deficiency is unknown., Objectives: We aimed to determine whether 1) iron status improved in children living in 2 Kenyan villages with a documented cessation in malaria transmission and 2) changes in iron status correlated with changes in hemoglobin., Design: We measured iron [hemoglobin, ferritin, soluble transferrin receptor (sTfR)] and inflammatory [C-reactive protein (CRP)] markers in paired plasma samples from 190 children aged 4-59 mo at the beginning (May 2007) and end (July 2008) of a documented 12-mo period of interruption in malaria transmission in 2 highland areas in Kenya with unstable malaria transmission and ongoing malaria surveillance., Results: Between May 2007 and July 2008, mean (±SD) hemoglobin increased from 10.8 ± 1.6 to 11.6 ± 1.6 g/dL. Median (25th, 75th percentile) ferritin increased from 17.0 (9.7, 25.6) to 22.6 (13.4, 34.7) μg/L (P < 0.001), whereas median sTfR decreased from 32.4 (26.3, 43.2) to 27.7 (22.1, 36.0) nmol/L (P < 0.001). Median CRP was low (<1 mg/L in both years) and did not change significantly. Iron deficiency prevalence (ferritin <12 μg/L, or <30 μg/L if CRP ≥10 mg/L) decreased from 35.9% (95% CI: 28.9%, 43.0%) to 24.9% (18.5%, 31.2%) (P = 0.005). The prevalence of iron deficiency with anemia (hemoglobin <11.0 g/dL) declined from 27.2% (20.7%, 33.8%) to 12.2% (7.4%, 17.1%) (P < 0.001). Improvement in iron status correlated with an increase in hemoglobin and was greater than explained by physiologic changes expected with age., Conclusions: In this area of unstable malaria transmission, the prevalence of iron deficiency in children decreased significantly after the interruption of malaria transmission and was correlated with an increase in hemoglobin. These findings suggest that malaria elimination strategies themselves may be an effective way to address iron deficiency in malaria-endemic areas., (© 2014 American Society for Nutrition.)

Background: It has been speculated that widespread and sustained use of insecticide treated bed nets (ITNs) for over 10 years in Asembo, western Kenya, may have selected for changes in the location (indoor versus outdoor) and time (from late night to earlier in the evening) of biting of the predominant species of human malaria vectors (Anopheles funestus, Anopheles gambiae sensu stricto, and Anopheles arabiensis)., Methods: Mosquitoes were collected by human landing catches over a six week period in June and July, 2011, indoors and outdoors from 17 h to 07 h, in 75 villages in Asembo, western Kenya. Collections were separated by hour of the night, and mosquitoes were identified to species and tested for sporozoite infection with Plasmodium falciparum. A subset was dissected to determine parity. Human behavior (time going to bed and rising, time spent indoors and outdoors) was quantified by cross-sectional survey. Data from past studies of a similar design and in nearby settings, but conducted before the ITN scale up commenced in the early 2000s, were compared with those from the present study., Results: Of 1,960 Anopheles mosquitoes collected in 2011, 1,267 (64.6%) were morphologically identified as An. funestus, 663 (33.8%) as An. gambiae sensu lato (An. gambiae s.s. and An. arabiensis combined), and 30 (1.5%) as other anophelines. Of the 663 An. gambiae s.l. collected, 385 were successfully tested by PCR among which 235 (61.0%) were identified as An. gambiae s.s. while 150 (39.0%) were identified as An. arabiensis. Compared with data collected before the scale-up of ITNs, daily entomological inoculation rates (EIRs) were consistently lower for An. gambiae s.l. (indoor EIR = 0.432 in 1985-1988, 0.458 in 1989-1990, 0.023 in 2011), and An. arabiensis specifically (indoor EIR = 0.532 in 1989-1990, 0.039 in 2009, 0.006 in 2011) but not An. funestus (indoor EIR = 0.029 in 1985-1988, 0.147 in 1989-1990, 0.010 in 2009 and 0.103 in 2011). Sporozoite rates were lowest in 2009 but rose again in 2011. Compared with data collected before the scale-up of ITNs, An. arabiensis and An. funestus were more likely to bite outdoors and/or early in the evening (p < 0.001 for all comparisons). However, when estimates of human exposure that would occur indoors (πi) or while asleep (πs) in the absence of an ITN were generated based on human behavioral patterns, the changes were modest with >90% of exposure of non-ITN users to mosquito bites occurring while people were indoors in all years. The proportion of bites occurring among non-ITN users while they were asleep was ≥90% for all species except for An. arabiensis. For this species, 97% of bites occurred while people were asleep in 1989-1990 while in 2009 and 2011, 80% and 84% of bites occurred while people were asleep for those not using ITNs. Assuming ITNs prevent a theoretical maximum of 93.7% of bites, it was estimated that 64-77% of bites would have occurred among persons using nets while they were asleep in 1989-1990, while 20-52% of bites would have occurred among persons using nets while they were asleep in 2009 and 2011., Conclusions: This study found no evidence to support the contention that populations of Anopheles vectors of malaria in Asembo, western Kenya, are exhibiting departures from the well-known pattern of late night, indoor biting characteristic of these typically highly anthropophilic species. While outdoor, early evening transmission likely does occur in western Kenya, the majority of transmission still occurs indoors, late at night. Therefore, malaria control interventions such as ITNs that aim to reduce indoor biting by mosquitoes should continue to be prioritized.

Background: Intestinal schistosomiasis caused by Schistosoma mansoni and urinary schistosomiasis caused by Schistosoma haematobium are widely distributed parasites in several localities of the Lake Victoria basin of Kenya, the former being more prevalent. In Kenya, transmission of the intestinal form of bilharzia (S. mansoni) tends to be closely confined to narrow zones along the shores of large bodies of water such as Lake Victoria where it is endemic and the intermediate host is found. The prevalence of S. mansoni along the Kenyan Lake Victoria basin ranges between 40% and 80%., Objective: To assess the community's knowledge and perceptions of schistosomiasis prevalence, transmission and control in relation to aquatic habitats in the Lake Victoria basin of Kenya., Design: Community-based cross-sectional study., Setting: The Kenyan Lake Victoria basin., Subjects: Two hundred and forty three individuals (both women and men residing in the beaches and surrounding areas) were interviewed about their knowledge and perceptions regarding schistosomiasis., Results: The community regarded schistosomiasis as a naturalistic disease not caused by supernatural forces but by an agent of contamination in water. Knowledge on schistosomiasis transmission and control was low, with 42% of the respondents having no idea on how schistosomiasis is contracted, while 22% and 18% of the respondents mentioned contact with contaminated water and drinking / eating dirty water / food, respectively. Most respondents were familiar with the snails' habitats, but had poor knowledge on aquatic plants harbouring snails, as 57% of the respondents did not know about aquatic plants being associated with schistosomiasis snails. Only 3% of the respondents associated snails with schistosomiasis transmission. Sixty percent (60%) of the respondents mentioned use of tablets and injections as means of treating schistosomiasis, while 38% had no idea how it is treated and 2% mentioned use of local herbs and services of medicine men., Conclusion: Majority of Kenyan Lake Victoria basin inhabitants had little awareness about schistosomiasis despite high prevalence of the disease in the region. There is need to adapt prevention and control strategies to the people's livelihoods. There is also need to target the less advantaged members of the community such as women, uneducated and subsistence farmers for intense health education strategies aimed at increasing participation in the control of schistosomiasis. Study to elicit divergence between biomedical and local understandings of schistosomiasis/bilharzia is suggested.

Background: Predictive models of malaria vector larval habitat locations may provide a basis for understanding the spatial determinants of malaria transmission., Methods: We used four landscape variables (topographic wetness index [TWI], soil type, land use-land cover, and distance to stream) and accumulated precipitation to model larval habitat locations in a region of western Kenya through two methods: logistic regression and random forest. Additionally, we used two separate data sets to account for variation in habitat locations across space and over time., Results: Larval habitats were more likely to be present in locations with a lower slope to contributing area ratio (i.e. TWI), closer to streams, with agricultural land use relative to nonagricultural land use, and in friable clay/sandy clay loam soil and firm, silty clay/clay soil relative to friable clay soil. The probability of larval habitat presence increased with increasing accumulated precipitation. The random forest models were more accurate than the logistic regression models, especially when accumulated precipitation was included to account for seasonal differences in precipitation. The most accurate models for the two data sets had area under the curve (AUC) values of 0.864 and 0.871, respectively. TWI, distance to the nearest stream, and precipitation had the greatest mean decrease in Gini impurity criteria in these models., Conclusions: This study demonstrates the usefulness of random forest models for larval malaria vector habitat modeling. TWI and distance to the nearest stream were the two most important landscape variables in these models. Including accumulated precipitation in our models improved the accuracy of larval habitat location predictions by accounting for seasonal variation in the precipitation. Finally, the sampling strategy employed here for model parameterization could serve as a framework for creating predictive larval habitat models to assist in larval control efforts.

Historically, the malaria vectors in western Kenya have been Anopheles funestus, Anopheles gambiae s.s., and Anopheles arabiensis. Of these species, An. funestus populations declined the most after the introduction of insecticide-treated bed nets (ITNs) in the 1990s in Asembo, and collections of An. funestus in the region remained low until at least 2008. Contrary to findings during the early years of ITN use in Asembo, the majority of the Anopheles collected here in 2010 and 2011 were An. funestus. Female An. funestus had characteristically high Plasmodium falciparum sporozoite rates and showed nearly 100% anthropophily. Female An. funestus were found more often indoors than outdoors and had relatively low mortality rates during insecticide bioassays. Together, these results are of serious concern for public health in the region, indicating that An. funestus may once again be contributing significantly to the transmission of malaria in this region despite the widespread use of ITNs/long-lasting insecticidal nets (LLINs).

A DNA-DNA hybridization method, reverse dot blot analysis (RDBA), was used to identify Anopheles gambiae s.s. and Anopheles arabiensis (Diptera: Culicidae) hosts. Of 299 blood-fed and semi-gravid An. gambiae s.l. collected from Kisian, Kenya, 244 individuals were identifiable to species; of these, 69.5% were An. arabiensis and 29.5% were An. gambiae s.s. Host identifications with RDBA were comparable with those of conventional polymerase chain reaction (PCR) followed by direct sequencing of amplicons of the vertebrate mitochondrial cytochrome b gene. Of the 174 amplicon-producing samples used to compare these two methods, 147 were identifiable by direct sequencing and 139 of these were identifiable by RDBA. Anopheles arabiensis bloodmeals were mostly (94.6%) bovine in origin, whereas An. gambiae s.s. fed upon humans more than 91.8% of the time. Tests by RDBA detected that two of 112 An. arabiensis contained blood from more than one host species, whereas PCR and direct sequencing did not. Recent use of insecticide-treated bednets in Kisian is likely to have caused the shift in the dominant vector species from An. gambiae s.s. to An. arabiensis. Reverse dot blot analysis provides an opportunity to study changes in host-feeding by members of the An. gambiae complex in response to the broadening distribution of vector control measures targeting host-selection behaviours., (© 2013 The Royal Entomological Society.)

In holoendemic Plasmodium falciparum transmission regions, malarial anemia is a leading cause of childhood morbidity and mortality. Identifying biomarkers of malaria disease severity is important for identifying at-risk groups and for improved understanding of the molecular pathways that influence clinical outcomes. We have previously shown that decreased cyclooxygenase (COX)-2-derived prostaglandin E2 (PGE2) levels are associated with enhanced clinical severity in cerebral malaria, malarial anemia, and malaria during pregnancy. Since children with malaria often have increased incidence of additional infections, such as bacteremia and HIV-1, we extend our previous findings by investigating COX-2 and PGE2 in children with falciparum malaria and co-infection with either bacteremia or HIV-1. Plasma bicyclo-PGE2/creatinine levels and peripheral blood COX-2 transcripts were significantly reduced in co-infected children relative to those with malaria mono-infection. Furthermore, suppression of circulating bicyclo-PGE2 was significantly associated with reduced hemoglobin levels in both mono- and co-infected children with malaria, suggesting that bicyclo-PGE2 may represent both a marker and mediator of malaria pathogenesis., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Field and laboratory investigations revealed phenotypic, target site and metabolic resistance to permethrin in an Anopheles gambiae s.s. (Diptera: Culicidae) population in Bungoma District, a region in western Kenya in which malaria is endemic and rates of ownership of insecticide-treated bednets are high. The sensitivity of individual An. gambiae s.l. females as indicated in assays using World Health Organization (WHO) test kits demonstrated reduced mortality in response to permethrin, deltamethrin and bendiocarb. Estimated time to knock-down of 50% (KDT50 ) of the test population in Centers for Disease Control (CDC) bottle bioassays was significantly lengthened for the three insecticides compared with that in a susceptible control strain. Anopheles arabiensis from all three sites showed higher mortality to all three insecticides in the WHO susceptibility assays compared with the CDC bottle assays, in which they showed less sensitivity and longer KDT50 than the reference strain for permethrin and deltamethrin. Microplate assays revealed elevated activity of β-esterases and oxidases, but not glutathione-S-transferase, in An. gambiae s.s. survivors exposed to permethrin in bottle bioassays compared with knocked down and unexposed individuals. No An. arabiensis showed elevated enzyme activity. The 1014S kdr allele was fixed in the Bungoma An. gambiae s.s. population and absent from An. arabiensis, whereas the 1014F kdr allele was absent from all samples of both species. Insecticide resistance could compromise vector control in Bungoma and could spread to other areas as coverage with longlasting insecticide-treated bednets increases., (© 2012 The Authors. Medical and Veterinary Entomology © 2012 The Royal Entomological Society.)

Background: Operational vector sampling methods lack standardization, making quantitative comparisons of malaria transmission across different settings difficult. Human landing catch (HLC) is considered the research gold standard for measuring human-mosquito contact, but is unsuitable for large-scale sampling. This study assessed mosquito catch rates of CDC light trap (CDC-LT), Ifakara tent trap (ITT), window exit trap (WET), pot resting trap (PRT), and box resting trap (BRT) relative to HLC in western Kenya to 1) identify appropriate methods for operational sampling in this region, and 2) contribute to a larger, overarching project comparing standardized evaluations of vector trapping methods across multiple countries., Methods: Mosquitoes were collected from June to July 2009 in four districts: Rarieda, Kisumu West, Nyando, and Rachuonyo. In each district, all trapping methods were rotated 10 times through three houses in a 3 × 3 Latin Square design. Anophelines were identified by morphology and females classified as fed or non-fed. Anopheles gambiae s.l. were further identified as Anopheles gambiae s.s. or Anopheles arabiensis by PCR. Relative catch rates were estimated by negative binomial regression., Results: When data were pooled across all four districts, catch rates (relative to HLC indoor) for An. gambiae s.l (95.6% An. arabiensis, 4.4% An. gambiae s.s) were high for HLC outdoor (RR = 1.01), CDC-LT (RR = 1.18), and ITT (RR = 1.39); moderate for WET (RR = 0.52) and PRT outdoor (RR = 0.32); and low for all remaining types of resting traps (PRT indoor, BRT indoor, and BRT outdoor; RR < 0.08 for all). For Anopheles funestus, relative catch rates were high for ITT (RR = 1.21); moderate for HLC outdoor (RR = 0.47), CDC-LT (RR = 0.69), and WET (RR = 0.49); and low for all resting traps (RR < 0.02 for all). At finer geographic scales, however, efficacy of each trap type varied from district to district., Conclusions: ITT, CDC-LT, and WET appear to be effective methods for large-scale vector sampling in western Kenya. Ultimately, choice of collection method for operational surveillance should be driven by trap efficacy and scalability, rather than fine-scale precision with respect to HLC. When compared with recent, similar trap evaluations in Tanzania and Zambia, these data suggest that traps which actively lure host-seeking females will be most useful for surveillance in the face of declining vector densities.

In holoendemic Plasmodium falciparum transmission areas such as western Kenya, severe malarial anemia [SMA, hemoglobin (Hb) < 6.0 g/dL, with any density parasitemia] is the most common clinical manifestation of severe malaria resulting in high rates of pediatric morbidity and mortality in these regions. Previous studies associated interleukin (IL)-13 with pathogenesis of different infectious diseases, including P. falciparum malaria. However, the functional roles of polymorphic variants within the IL-13 promoter in conditioning susceptibility to SMA remain largely unexplored. As such, the association between the IL-13 variants -7402 T/G (rs7719175) and -4729G/A (rs3091307) and susceptibility to SMA was determined in children (n = 387) presenting with clinical symptoms of falciparum malaria and resident in a holoendemic transmission region in western Kenya. Our results indicated no difference in the proportions of individual genotypes among children presenting with non-SMA (n = 222) versus SMA (n = 165). Similarly, there was no associations between the individual genotypes (-7402 T/G and -4729G/A) and SMA. Additional analyses, however, revealed that proportions of individuals with -7402 T/-4729A (TA) haplotype was significantly higher in children presenting with SMA than non-SMA group (P = 0.043). A further multivariate logistic regression analyses, controlling for confounding factors, demonstrated that carriage of the TA haplotype was associated with increased susceptibility to SMA (OR; 1.564, 95% CI; 1.023-2.389, P = 0.039). In addition, circulating levels of IL-13 were comparable between the clinical groups as well as across genotypes and haplotypes. Collectively, findings presented here suggest that haplotypes within the IL-13 promoter at -7402 T/G and -4729G/A may modulate SMA pathogenesis, but do not affect circulating IL-13 levels.

We studied the effect on malaria incidence, mosquito abundance, net efficacy, net use rate, chemical analysis, and holes of a long lasting insecticide treated bed net (Netprotect) in western Kenya, 2007-2010. Nets were hung in 150 households 6 months before they were hung in a second, 2 km away. Indoor resting densities were monitored by pyrethrum spray catch and malaria cases by passive detection using clinical manifestations and rapid diagnostic test. The probability of finding An. arabiensis in the control area was 2.6 times higher than that in intervention area during the first 6 months. Human blood feeding index of Anopheles funestus declined 17%. After bed nets were hung in the second area, malaria incidence declined 25% down to the level in the first area. Incidence remained at this low level for 2 years. 90% of collected nets were efficacious after 3-year use. Deltamethrin dosage declined from 1.9 to 0.5 g/kg over 3 years. Attrition rate after 3 years was 21%. WHO hole index changed from 333 to 114 to 381 over the three years. This index summarizes the numbers of holes in size categories and multiplies with the mean hole area per category. It is very sensitive to the impact of big holes in a few nets.

In holoendemic Plasmodium falciparum transmission areas, severe malaria primarily occurs in children aged <48 months and manifests as severe malarial anemia [SMA; hemoglobin (Hb) < 6.0 g/dL]. Induction of high levels of prostaglandin-E(2) (PGE(2)) through inducible cyclooxygenase-2 (COX-2) is an important host-defense mechanism against invading pathogens. We have previously shown that COX-2-derived PGE(2) levels are reduced in children residing in hyperendemic transmission regions with cerebral malaria and in those with mixed sequelae of anemia and hyperparasitemia. Our in vitro studies further demonstrated that reduced PGE(2) was due to downregulation of COX-2 gene products following phagocytosis of malarial pigment (hemozoin, PfHz). However, as COX-2-PGE(2) pathways and the impact of naturally acquired PfHz on erythropoietic responses have not been determined in children with SMA, plasma and urinary bicyclo-PGE(2)/creatinine and leukocytic COX-2 transcripts were determined in parasitized children (<36 months) stratified into SMA (n = 36) and non-SMA (Hb ≥ 6.0 g/dL; n = 38). Children with SMA had significantly reduced plasma (P = 0.001) and urinary (P < 0.001) bicyclo-PGE(2)/creatinine and COX-2 transcripts (P = 0.007). There was a significant positive association between Hb and both plasma (r = 0.363, P = 0.002) and urinary (r = 0.500, P = 0.001)] bicyclo-PGE(2)/creatinine. Furthermore, decreased systemic bicyclo-PGE(2)/creatinine was associated with inefficient erythropoiesis (i.e., reticulocyte production index; RPI < 2.0, P = 0.026). Additional analyses demonstrated that plasma (P = 0.031) and urinary (P = 0.070) bicyclo-PGE(2)/creatinine and COX-2 transcripts (P = 0.026) progressively declined with increasing concentrations of naturally acquired PfHz by monocytes. Results presented here support a model in which reduced COX-2-derived PGE(2), driven in part by naturally acquired PfHz by monocytes, promotes decreased erythropoietic responses in children with SMA., (Copyright © 2012 Wiley Periodicals, Inc.)

The KEMRI/Centers for Disease Control and Prevention (CDC) Health and Demographic Surveillance System (HDSS) is located in Rarieda, Siaya and Gem Districts (Siaya County), lying northeast of Lake Victoria in Nyanza Province, western Kenya. The KEMRI/CDC HDSS, with approximately 220 000 inhabitants, has been the foundation for a variety of studies, including evaluations of insecticide-treated bed nets, burden of diarrhoeal disease and tuberculosis, malaria parasitaemia and anaemia, treatment strategies and immunological correlates of malaria infection, and numerous HIV, tuberculosis, malaria and diarrhoeal disease treatment and vaccine efficacy and effectiveness trials for more than a decade. Current studies include operations research to measure the uptake and effectiveness of the programmatic implementation of integrated malaria control strategies, HIV services, newly introduced vaccines and clinical trials. The HDSS provides general demographic and health information (such as population age structure and density, fertility rates, birth and death rates, in- and out-migrations, patterns of health care access and utilization and the local economics of health care) as well as disease- or intervention-specific information. The HDSS also collects verbal autopsy information on all deaths. Studies take advantage of the sampling frame inherent in the HDSS, whether at individual, household/compound or neighbourhood level.

Development of protective immunity against Plasmodium falciparum is partially mediated through binding of malaria-specific IgG to Fc gamma (γ) receptors. Variations in human FcγRIIA-H/R-131 and FcγRIIIB-NA1/NA2 affect differential binding of IgG sub-classes. Since variability in FcγR may play an important role in severe malarial anemia (SMA) pathogenesis by mediating phagocytosis of red blood cells and triggering cytokine production, the relationship between FcγRIIA-H/R131 and FcγRIIIB-NA1/NA2 haplotypes and susceptibility to SMA (Hb < 6.0 g/dL) was investigated in Kenyan children (n = 528) with acute malaria residing in a holoendemic P. falciparum transmission region. In addition, the association between carriage of the haplotypes and repeated episodes of SMA and all-cause mortality were investigated over a 3-year follow-up period. Since variability in FcγR can alter interferon (IFN)-γ production, a mediator of innate and adaptive immune responses, functional associations between the haplotypes and IFN-γ were also explored. During acute malaria, children with SMA had elevated peripheral IFN-γ levels (P = 0.006). Although multivariate logistic regression analyses (controlling for covariates) revealed no associations between the FcγR haplotypes and susceptibility to SMA during acute infection, the FcγRIIA-131H/FcγRIIIB-NA1 haplotype was associated with decreased peripheral IFN-γ (P = 0.046). Longitudinal analyses showed that carriage of the FcγRIIA-131H/FcγRIIIB-NA1 haplotype was associated with reduced risk of SMA (RR 0.65, 95% CI 0.46-0.90; P = 0.012) and all-cause mortality (P = 0.002). In contrast, carriers of the FcγRIIA-131H/FcγRIIIB-NA2 haplotype had increased susceptibility to SMA (RR 1.47, 95% CI 1.06-2.04; P = 0.020). Results here demonstrate that variation in the FcγR gene alters susceptibility to repeated episodes of SMA and mortality, as well as functional changes in IFN-γ production.

Severe malarial anemia (SMA) is a leading cause of morbidity and mortality in children residing in regions where Plasmodium falciparum transmission is holoendemic. Although largely unexplored in children with SMA, interleukin-18 (IL-18) is important for regulating innate and acquired immunity in inflammatory and infectious diseases. As such, we selected two functional single-nucleotide polymorphisms (SNPs) in the IL-18 promoter (-137G→C [rs187238] and -607C→A [rs1946518]) whose haplotypes encompass significant genetic variation due to the presence of strong linkage disequilibrium among these variants. The relationship between the genotypes/haplotypes, SMA (hemoglobin [Hb], <5.0 g/dl], and longitudinal clinical outcomes were then investigated in Kenyan children (n = 719). Multivariate logistic regression analyses controlling for age, gender, sickle cell trait, glucose-6-phosphate dehydrogenase (G6PD) deficiency, HIV-1, and bacteremia revealed that carriage of the -607AA genotype was associated with protection against SMA (odds ratio [OR] = 0.440 [95% confidence interval {CI} = 0.21 to 0.90], P = 0.031) in children with acute infection. In contrast, carriers of the -137G/-607C (GC) haplotype had increased susceptibility to SMA (OR = 2.050 [95% CI = 1.04 to 4.05], P = 0.039). Measurement of IL-18 gene expression in peripheral blood leukocytes demonstrated that elevated IL-18 transcripts were associated with reduced hemoglobin concentrations (ρ = -0.293, P = 0.010) and that carriers of the "susceptible" GC haplotype had elevated IL-18 transcripts (P = 0.026). Longitudinal investigation of clinical outcomes over a 3-year follow-up period revealed that carriers of the rare CC haplotype (∼1% frequency) had 5.76 times higher mortality than noncarriers (P = 0.001). Results presented here demonstrate that IL-18 promoter haplotypes that condition elevated IL-18 gene products during acute infection are associated with increased risk of SMA. Furthermore, carriage of the rare CC haplotype significantly increases the risk of childhood mortality.

Areas where Plasmodium falciparum transmission is holoendemic are characterized by high rates of pediatric severe malarial anemia (SMA) and associated mortality. Although the etiology of SMA is complex and multifactorial, perturbations in inflammatory mediator production play an important role in the pathogenic process. As such, the current study focused on identification of inflammatory biomarkers in children with malarial anemia. Febrile children (3 to 30 months of age) presenting at Siaya District Hospital in western Kenya underwent a complete clinical and hematological evaluation. Children with falciparum malaria and no additional identifiable anemia-promoting coinfections were stratified into three groups: uncomplicated malaria (hemoglobin [Hb] levels of ≥11.0 g/dl; n = 31), non-SMA (Hb levels of 6.0 to 10.9 g/dl; n = 37), and SMA (Hb levels of <6.0 g/dl; n = 80). A Luminex hu25-plex array was used to determine potential biomarkers (i.e., interleukin 1β [IL-1β], IL-1 receptor antagonist [IL-1Ra], IL-2, IL-2R, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12p70, IL-13, IL-15, IL-17, tumor necrosis factor alpha [TNF-α], alpha interferon [IFN-α], IFN-γ, granulocyte-macrophage colony-stimulating factor [GM-CSF], macrophage inflammatory protein 1 alpha [MIP-1α], MIP-1β, IFN-inducible protein of 10 kDa [IP-10], monokine induced by IFN-γ [MIG], eotaxin, RANTES, and monocyte chemoattractant protein 1 [MCP-1]) in samples obtained prior to any treatment interventions. To determine the strongest biomarkers of anemia, a parsimonious set of predictor variables for Hb was generated by least-angle regression (LAR) analysis, controlling for the confounding effects of age, gender, glucose-6-phosphate dehydrogenase (G6PD) deficiency, and sickle cell trait, followed by multiple linear regression analyses. IL-12p70 and IFN-γ emerged as positive predictors of Hb, while IL-2R, IL-13, and eotaxin were negatively associated with Hb. The results presented here demonstrate that the IL-12p70/IFN-γ pathway represents a set of biomarkers that predicts elevated Hb levels in children with falciparum malaria, while activation of the IL-13/eotaxin pathway favors more profound anemia.

Background: Plasmodium falciparum malaria remains a leading cause of morbidity and mortality among African children. Innate immunity provides the first line of defence against P. falciparum infections, particularly in young children that lack naturally-acquired malarial immunity, such as the population examined here. Consistent with the fact that elevated interleukin (IL)-12 is an important component of the innate immune response that provides protective immunity against malaria, we have previously shown that suppression of IL-12 in African children is associated with the development of severe malarial anaemia (SMA). Since the role of IL12B variants in conditioning susceptibility to SMA remains largely unexplored, the association between a single nucleotide polymorphism (1188A→C, rs3212227), SMA (Hb<6.0 g/dL), circulating IL-12p40/p70 levels, and longitudinal clinical outcomes in Kenyan children (n = 756) residing in a holoendemic falciparum malaria transmission area were investigated., Results: Multivariate logistic regression analysis in children with acute malaria (n = 544) demonstrated that carriers of the C allele had increased susceptibility to SMA (CC: OR, 1.674; 95% CI, 1.006-2.673; P = 0.047, and AC: OR, 1.410; 95% CI, 0.953-2.087; P = 0.086) relative to wild type (AA). Although children with SMA had lower IL-12p40/p70 levels than the non-SMA group (P = 0.037), levels did not differ significantly according to genotype. Longitudinal analyses in the entire cohort (n = 756) failed to show any significant relationships between rs3212227 genotypes and either susceptibility to SMA or all-cause mortality throughout the three year follow-up., Conclusion: The rs3212227 is a marker of susceptibility to SMA in children with acute disease, but does not appear to mediate functional changes in IL-12 production or longitudinal outcomes during the acquisition of naturally-acquired malarial immunity.

Since the etiologies and clinical outcomes of bacteremia in children with Plasmodium falciparum infections, particularly in areas of holoendemic malaria transmission, are largely unexplored, blood cultures and comprehensive clinical, laboratory, hematological, and nutritional parameters for malaria-infected children (aged 1 to 36 months, n = 585 patients) were investigated at a rural hospital in western Kenya. After the exclusion of contaminant microorganisms, the prevalence of bacteremia was 11.7% in the cohort (n = 506), with nontyphoidal Salmonella spp. being the most common isolates (42.4%). Bacteremia was found to occur in a significantly higher proportion of females than males and was associated with elevated blood glucose concentrations and lowered malaria parasite and hemoglobin (Hb) levels compared to those in abacteremic participants. In addition, the incidences of respiratory distress and severe malarial anemia (SMA; Hb level of <6.0 g/dl) were nonsignificantly greater in children with bacteremia. Mortality was 8.5-fold higher in children with bacteremia. Multivariate logistic regression analyses revealed that bacteremia was significantly associated with reduced incidences of high-density parasitemia (HDP; ≥ 10,000/μl) and increased incidences of malnutrition (i.e., underweight; weight-for-age Z score of <-2 using the NCHS system). Since previous studies showed that bacteremia caused by Gram-negative organisms is associated with enhanced anemia and mortality, multivariate logistic regression was also performed separately for randomly age- and gender-matched children with bacteremia caused by Gram-negative organisms (n = 37) and for children found to be abacteremic (n = 74). These results revealed that the presence of bacteremia caused by Gram-negative organisms was significantly associated with reduced HDP, enhanced susceptibility to respiratory distress, SMA (Hb level of <6.0 g/dl), and being underweight (Z score, <-2). Data presented here from a region of holoendemic P. falciparum transmission demonstrate that although bacteremia is associated with reduced malaria parasitemia, a number of unfavorable clinical outcomes, including malnutrition, respiratory distress, anemia, and mortality, are elevated in children with bacteremia, particularly in cases of Gram-negative origin.

Background: Malaria vector control in Africa depends upon effective insecticides in bed nets and indoor residual sprays. This study investigated the extent of insecticide resistance in Anopheles gambiae s.l., Anopheles gambiae s.s. and Anopheles arabiensis in western Kenya where ownership of insecticide-treated bed nets has risen steadily from the late 1990s to 2010. Temporal and spatial variation in the frequency of a knock down resistance (kdr) allele in A. gambiae s.s. was quantified, as was variation in phenotypic resistance among geographic populations of A. gambiae s.l., Methods: To investigate temporal variation in kdr frequency, individual specimens of A. gambiae s.s. from two sentinel sites were genotyped using RT-PCR from 1996-2010. Spatial variation in kdr frequency, species composition, and resistance status were investigated in additional populations of A. gambiae s.l. sampled in western Kenya in 2009 and 2010. Specimens were genotyped for kdr as above and identified to species via conventional PCR. Field-collected larvae were reared to adulthood and tested for insecticide resistance using WHO bioassays., Results: Anopheles gambiae s.s. showed a dramatic increase in kdr frequency from 1996 - 2010, coincident with the scale up of insecticide-treated nets. By 2009-2010, the kdr L1014S allele was nearly fixed in the A. gambiae s.s. population, but was absent in A. arabiensis. Near Lake Victoria, A. arabiensis was dominant in samples, while at sites north of the lake A. gambiae s.s was more common but declined relative to A. arabiensis from 2009 to 2010. Bioassays demonstrated that A. gambiae s.s. had moderate phenotypic levels of resistance to DDT, permethrin and deltamethrin while A. arabiensis was susceptible to all insecticides tested., Conclusions: The kdr L1014S allele has approached fixation in A. gambiae s.s. populations of western Kenya, and these same populations exhibit varying degrees of phenotypic resistance to DDT and pyrethroid insecticides. The near absence of A. gambiae s.s. from populations along the lakeshore and the apparent decline in other populations suggest that insecticide-treated nets remain effective against this mosquito despite the increase in kdr allele frequency. The persistence of A. arabiensis, despite little or no detectable insecticide resistance, is likely due to behavioural traits such as outdoor feeding and/or feeding on non-human hosts by which this species avoids interaction with insecticide-treated nets.

Background: Identification of high-risk malaria foci can help enhance surveillance or control activities in regions where they are most needed. Associations between malaria risk and land-use/land-cover are well-recognized, but these environmental characteristics are closely interrelated with the land's topography (e.g., hills, valleys, elevation), which also influences malaria risk strongly. Parsing the individual contributions of land-cover/land-use variables to malaria risk requires examining these associations in the context of their topographic landscape. This study examined whether environmental factors like land-cover, land-use, and urban density improved malaria risk prediction based solely on the topographically-determined context, as measured by the topographic wetness index., Methods: The topographic wetness index, an estimate of predicted water accumulation in a defined area, was generated from a digital terrain model of the landscape surrounding households in two neighbouring western Kenyan highland communities. Variables determined to best encompass the variance in this topographic wetness surface were calculated at a household level. Land-cover/land-use information was extracted from a high-resolution satellite image using an object-based classification method. Topographic and land-cover variables were used individually and in combination to predict household-level malaria in the communities through an iterative split-sample model fitting and testing procedure. Models with only topographic variables were compared to those with additional predictive factors related to land-cover/land-use to investigate whether these environmental factors improved prediction of malaria based on the shape of the land alone., Results: Variables related to topographic wetness proved most useful in predicting the households of individuals contracting malaria in this region of rugged terrain. Other variables related to human modification of the environment also demonstrated clear associations with household malaria. However, these land-cover/land-use variables failed to produce unambiguous improvements in statistical predictive models controlling for important topographic factors, with none improving prediction of household-level malaria more than 75% of the time., Conclusions: Topographic wetness values in this region of highly varied terrain more accurately predicted houses at greater risk of malaria than did consideration of land-cover/land-use characteristics. As such, those planning control or local elimination strategies in similar highland regions may use topographic and geographic characteristics to effectively identify high-receptivity regions that may require enhanced vigilance.

Background: Insecticide-treated nets protect users from mosquito bites, thereby preventing transmissions of mosquito borne pathogens. Repeated washing of nets removes insecticide on the netting rendering them ineffective within a short period. Long-lasting insecticide-treated nets (LLINs) offer longer time protection against such bites because they are more wash resistant, and are preferred to conventionally treated nets. However, there is limited information on the effect of repeated washing of LLINs on the feeding success and survival of wild malaria vectors., Methods: The current study evaluated the effect of repeated washing of four brands of LLINs on the feeding success and survival rates of Anopheles gambiae sl reared from wild strains. In this study, two- to five-day old F1s, reared from gravid mosquitoes collected from an area with a high coverage of LLINs were offered blood meals through protective barriers of the above LLINs. Mosquitoes were exposed for a period of 10 minutes each time. Nets were tested unwashed and subsequently after every 5th through wash 15. After exposure mosquitoes were sorted out according to their feeding status. They were then held under normal laboratory conditions for 24 hours and mortality was scored in both fed and unfed., Results: It was observed that mosquitoes did not feed through a barrier of unwashed LLINs. However, the feeding success and survival rates increased with successive number of washes and were also net brand dependant. After 15 washes, 49% of vectors succeeded to feed through a protective barrier of PermaNet 2.0 and 50% of the fed died after 24 hrs while after the same number of washes 60% of vectors succeeded to feed through Olyset brand of LLINs and all of them survived. In general, more mosquitoes survived after feeding through Olyset compared to the other four brands that were evaluated. When efficacy of individual LLINs was compared by a t-test analysis to a conventionally treated net, the results were not significantly different statistically for Olyset (p = 0.239) and NetProtect (TNT) (p = 0.135). However, the results were highly significant when comparison was made with PermaNet and Interceptor (BASF); p values 0.015 and 0.025 respectively., Conclusion: The result of this study shows that repeated washing of LLINs at short time intervals using local washing methods may render them infective within a short time in preventing local vectors from feeding.

Household-level water treatment products provide safe drinking water to at-risk populations, but relatively few people use them regularly; little is known about factors that influence uptake of this proven health intervention. We assessed uptake of these water treatments in Nyanza Province, Kenya, November 2003-February 2005. We interviewed users and non-user controls of a new household water treatment product regarding drinking water and socioeconomic factors. We calculated regional use-prevalence of these products based on 10 randomly selected villages in the Asembo region of Nyanza Province, Kenya. Thirty-eight percent of respondents reported ever using household-level treatment products. Initial use of a household-level product was associated with having turbid water as a source (adjusted odds ratio [AOR] = 16.6, p = 0.007), but consistent usage was more common for a less costly and more accessible product that did not address turbidity. A combination of social marketing, retail marketing, and donor subsidies may be necessary to extend the health benefits of household-level water treatment to populations most at risk.

Background: The current study was undertaken to determine the optimal wash-drying regimen and the effects of different washing procedures on the efficacy, and durability of four brands of newly introduced long-lasting insecticide-treated nets (LLINs) under tropical conditions., Methods: In the current study, the following four LLINs were tested: Olyset, PermaNet 2.0, BASF and TNT. Nets were divided into three sets; one set was washed by hand rubbing and air-dried either hanging or spread on the ground in direct sunlight or hanging or spread on the ground under the shade. A second set was washed using the WHO protocol (machine) and the third set was washed by beating the nets on rocks. The biological activities of the nets were assessed by a three-minute bioassay cone test and the residual insecticide contents were determined using high performance liquid chromatography (HPLC) procedure., Results: Nets that were dried hanging under the shade retained more insecticide, 62.5% and recorded higher mortality compared to nets which were dried lying on the ground in direct sunlight 58.8%, nets dried under the shade spread on the ground 56.3%, and 57.8% for nets dried hanging in direct sunlight. It was also observed that nets washed by the standard WHO protocol, retained more insecticide and were more effective in killing mosquitoes compared to nets washed by local methods of hand rubbing and beating on rocks. There were significant differences between drying regimens (p < 0.0001) and between washing procedures (p < 0.001) respectively. However, the effect of net type was statistically insignificant. The statistical differences on individual nets were also compared, for PermaNet and TNT there were no significant differences observed between the four drying regimens (p = 0.7944 and 0.4703) respectively). For BASF and Olyset, the differences were significant (p < 0.001 and p > 0.0001)., Conclusion: The results of this study suggest that washing and drying regimen influence the insecticidal activity of LLINs. The standard WHOPES washing protocol underestimates the amount of insecticide washed from LLINs compared to the abrasive washing procedures that are used in the field. This suggests that there is need to educate net users to adopt a more gentle washing procedure while handling LLINs. The education should accompany net distribution campaigns.

The epidemiology of malaria in urban environments is poorly characterized, yet increasingly problematic. We conducted an unmatched case-control study of risk factors for malarial anemia with high parasitemia in urban Kisumu, Kenya, from June 2002 through February 2003. Cases (n = 80) were hospital patients with a hemoglobin level < or = 8 g/dL and a Plasmodium parasite density > or = 10,000/microL. Controls (n = 826) were healthy respondents to a concurrent citywide knowledge, attitude, and practice survey. Children who reported spending at least one night per month in a rural area were especially at risk (35% of cases; odds ratio = 9.3, 95% confidence interval [CI] = 4.4-19.7, P < 0.0001), and use of mosquito coils, bed net ownership, and house construction were non-significant, potentially indicating that malaria exposure during rural travel comprises an important element of risk. Control of severe malaria in an urban setting may be complicated by Plasmodium infections acquired elsewhere. Epidemiologic studies of urban malaria in low transmission settings should take travel history into account.

Background: High coverage of insecticide-treated bed nets in Asembo and low coverage in Seme, two adjacent communities in western Nyanza Province, Kenya; followed by expanded coverage of bed nets in Seme, as the Kenya national malaria programme rolled out; provided a natural experiment for quantification of changes in relative abundance of two primary malaria vectors in this holoendemic region. Both belong to the Anopheles gambiae sensu lato (s.l.) species complex, namely A. gambiae sensu stricto (s.s.) and Anopheles arabiensis. Historically, the former species was proportionately dominant in indoor resting collections of females., Methods: Data of the relative abundance of adult A. gambiae s.s. and A. arabiensis sampled from inside houses were obtained from the literature from 1970 to 2002 for sites west of Kisumu, Kenya, to the region of Asembo ca. 50 km from the city. A sampling transect was established from Asembo (where bed net use was high due to presence of a managed bed net distribution programme) eastward to Seme, where no bed net programme was in place. Adults of A. gambiae s.l. were sampled from inside houses along the transect from 2003 to 2009, as were larvae from nearby aquatic habitats, providing data over a nearly 40 year period of the relative abundance of the two species. Relative proportions of A. gambiae s.s. and A. arabiensis were determined for each stage by identifying species by the polymerase chain reaction method. Household bed net ownership was measured with surveys during mosquito collections. Data of blood host choice, parity rate, and infection rate for Plasmodium falciparum in A. gambiae s.s. and A. arabiensis were obtained for a sample from Asembo and Seme from 2005., Results: Anopheles gambiae s.s. adult females from indoor collections predominated from 1970 to 1998 (ca. 85%). Beginning in 1999, A. gambiae s.s decreased proportionately relative to A. arabiensis, then precipitously declined to rarity coincident with increased bed net ownership as national bed net distribution programmes commenced in 2004 and 2006. By 2009, A. gambiae s.s. comprised proportionately ca. 1% of indoor collections and A. arabiensis 99%. In Seme compared to Asembo in 2003, proportionately more larvae were A. gambiae s.s., larval density was higher, and more larval habitats were occupied. As bed net use rose in Seme, the proportion of A. gambiae larvae declined as well. These trends continued to 2009. Parity and malaria infection rates were lower in both species in Asembo (high bed net use) compared to Seme (low bed net use), but host choice did not vary within species in both communities (predominantly cattle for A. arabiensis, humans for A. gambiae s.s.)., Conclusions: A marked decline of the A. gambiae s.s. population occurred as household ownership of bed nets rose in a region of western Kenya over a 10 year period. The increased bed net coverage likely caused a mass effect on the composition of the A. gambiae s.l. species complex, resulting in the observed proportionate increase in A. arabiensis compared to its closely related sibling species, A. gambiae s.s. These observations are important in evaluating the process of regional malaria elimination, which requires sustained vector control as a primary intervention.

Plasmodium falciparum malaria is a leading global cause of infectious disease burden. In areas in which P. falciparum transmission is holoendemic, such as western Kenya, severe malarial anemia (SMA) results in high rates of pediatric morbidity and mortality. Although the pathophysiological basis of SMA is multifactorial, we recently discovered that suppression of unexplored hematopoietic growth factors that promote erythroid and myeloid colony development, such as stem cell growth factor (SCGF) (C-type lectin domain family member 11A [CLEC11A]), was associated with enhanced development of SMA and reduced erythropoietic responses. To extend these investigations, the relationships between a novel SCGF promoter variant (-539C/T, rs7246355), SMA (hemoglobin [Hb] < 6.0 g/dl), and reduced erythropoietic responses (reticulocyte production index [RPI], <2.0) were investigated with Kenyan children (n = 486) with falciparum malaria from western Kenya. Circulating SCGF was positively correlated with hemoglobin levels (r = 0.251; P = 0.022) and the reticulocyte production index (RPI) (r = 0.268; P = 0.025). Children with SMA also had lower SCGF levels than those in the non-SMA group (P = 0.005). Multivariate logistic regression analyses controlling for covariates demonstrated that individuals with the homologous T allele were protected against SMA (odds ratio, 0.57; 95% confidence interval [95% CI] 0.34 to 0.94; P = 0.027) relative to CC (wild-type) carriers. Carriers of the TT genotype also had higher SCGF levels in circulation (P = 0.018) and in peripheral blood mononuclear cell culture supernatants (P = 0.041), as well as an elevated RPI (P = 0.005) relative to individuals with the CC genotype. The results presented here demonstrate that homozygous T at -539 in the SCGF promoter is associated with elevated SCGF production, enhanced erythropoiesis, and protection against the development of SMA in children with falciparum malaria.

Highland areas where malaria transmission is unstable are targets for malaria elimination because transmission decreases to low levels during the dry season. In highland areas of Kipsamoite and Kapsisiywa, Kenya (population approximately 7,400 persons), annual household indoor residual spraying with a synthetic pyrethroid was performed starting in 2005, and artemether/lumefantrine was implemented as first-line malaria treatment in October 2006. During April 2007-March 2008, no microscopy-confirmed cases of malaria occurred at the sites. In 4 assessments of asymptomatic persons during May 2007-April 2008, a total of <0.3% of persons were positive for asexual Plasmodium falciparum by microscopy or PCR at any time, and none were positive by PCR at the last 2 sample collections. Our findings show that in such areas, interruption and eventual elimination of malaria transmission may be achievable with widespread annual indoor residual spraying of households and artemisinin combination therapy.

In areas of holoendemic Plasmodium falciparum transmission, severe malarial anemia (SMA) is a leading cause of pediatric morbidity and mortality. Although many soluble mediators regulate erythropoiesis, it is unclear how these factors contribute to development of SMA. Investigation of novel genes dysregulated in response to malarial pigment (hemozoin [PfHz]) revealed that stem cell growth factor (SCGF; also called C-type lectin domain family member 11A [CLEC11A]), a hematopoietic growth factor important for development of erythroid and myeloid progenitors, was one of the most differentially expressed genes. Additional experiments with cultured peripheral blood mononuclear cells (PBMCs) demonstrated that PfHz decreased SCGF/CLEC11A transcriptional expression in a time-dependent manner. Circulating SCGF levels were then determined for Kenyan children (n = 90; aged 3 to 36 months) presenting at a rural hospital with various severities of malarial anemia. SCGF levels in circulation (P = 0.001) and in cultured PBMCs (P = 0.004) were suppressed in children with SMA. Circulating SCGF also correlated positively with hemoglobin levels (r = 0.241; P = 0.022) and the reticulocyte production index (RPI) (r = 0.280; P = 0.029). In addition, SCGF was decreased in children with reduced erythropoiesis (RPI of <2) (P < 0.001) and in children with elevated levels of naturally acquired monocytic PfHz (P = 0.019). Thus, phagocytosis of PfHz promotes a decrease in SCGF gene products, which may contribute to reduced erythropoiesis in children with SMA.

Regulated upon activation, normal T-cell expressed, and secreted (RANTES, CCL-5) is an important immunoregulatory mediator that is suppressed in children with malarial anemia (MA). Although pro-inflammatory (e.g., TNF-alpha, IL-1beta and IFN-gamma) and anti-inflammatory (e.g., IL-4, IL-10 and IL-13) cytokines regulate RANTES production, their effect on RANTES in children with MA has not been determined. Since intraleukocytic malarial pigment, hemozoin (Hz), causes dysregulation in chemokine and cytokine production, the impact of naturally acquired Hz (pfHz) on RANTES and RANTES-regulatory cytokines (TNF-alpha, IFN-gamma, IL-1beta, IL-4, IL-10, and IL-13) was examined. Circulating RANTES levels progressively declined with increasing levels of pigment-containing monocytes (PCM) (P=0.035). Additional experiments in cultured peripheral blood mononuclear cells (PBMC) showed that monocytic acquisition of pfHz (in vivo) was associated with suppression of RANTES under baseline (P=0.001) and stimulated conditions (P=0.072). Although high PCM levels were associated with decreased circulating IFN-gamma (P=0.003) and IL-10 (P=0.010), multivariate modeling revealed that only PCM (P=0.048, beta=-0.171) and IL-10 (P<0.0001, beta=-0.476) were independently associated with RANTES production. Subsequent in vitro experiments revealed that blockade of endogenous IL-10 significantly increased RANTES production (P=0.028) in PBMC from children with naturally acquired Hz. Results here demonstrate that monocytic acquisition of Hz suppresses RANTES production in children with MA through an IL-10-dependent mechanism.

Plasmodium falciparum malaria is one of the leading global causes of morbidity and mortality with African children bearing the highest disease burden. Among the various severe disease sequelae common to falciparum malaria, severe malarial anemia (SMA) in pediatric populations accounts for the greatest degree of mortality. Although the patho-physiological basis of SMA remains unclear, dysregulation in inflammatory mediators, such as interleukin (IL)-10, appear to play an important role in determining disease outcomes. Since polymorphic variability in innate immune response genes conditions susceptibility to malaria, the relationship between common IL-10 promoter variants (-1,082A/G, -819T/C, and -592A/C), SMA (Hb < 6.0 g/dL), and circulating inflammatory mediator levels (i.e., IL-10, TNF-alpha, IL-6 and IL-12) were investigated in parasitemic Kenyan children (n = 375) in a holoendemic P. falciparum transmission area. Multivariate logistic regression analyses demonstrated that the -1,082G/-819C/-592C (GCC) haplotype was associated with protection against SMA (OR; 0.68, 95% CI, 0.43-1.05; P = 0.044) and increased IL-10 production (P = 0.029). Although none of the other haplotypes were significantly associated with susceptibility to SMA, individuals with the -1,082A/-819T/-592A (ATA) haplotype had an increased risk of SMA and reduced circulating IL-10 levels (P = 0.042). Additional results revealed that the IL-10:TNF-alpha ratio was higher in the GCC group (P = 0.024) and lower in individuals with the ATA haplotype (P = 0.034), while the IL-10:IL-12 ratio was higher in ATA haplotype (P = 0.006). Results presented here demonstrate that common IL-10 promoter haplotypes condition susceptibility to SMA and functional changes in circulating IL-10, TNF-alpha, and IL-12 levels in children with falciparum malaria.

In areas where levels of transmission of Plasmodium falciparum are high and stable, the age-related acquisition of high-level immunoglobulin G (IgG) antibodies to preerythrocytic circumsporozoite protein (CSP) and liver-stage antigen 1 (LSA-1) has been associated with protection from clinical malaria. In contrast, age-related protection from malaria develops slowly or not at all in residents of epidemic-prone areas with unstable low levels of malaria transmission. We hypothesized that this suboptimal clinical and parasitological immunity may in part be due to reduced antibodies to CSP or LSA-1 and/or vaccine candidate blood-stage antigens. Frequencies and levels of IgG antibodies to CSP, LSA-1, thrombospondin-related adhesive protein (TRAP), apical membrane antigen 1 (AMA-1), erythrocyte binding antigen 175 (EBA-175), and merozoite surface protein 1 (MSP-1) were compared in 243 Kenyans living in a highland area of unstable transmission and 210 residents of a nearby lowland area of stable transmission. Levels of antibodies to CSP, LSA-1, TRAP, and AMA-1 in the oldest age group (>40 years) in the unstable transmission area were lower than or similar to those of children 2 to 6 years old in the stable transmission area. Only 3.3% of individuals in the unstable transmission area had high levels of IgG (>2 arbitrary units) to both CSP and LSA-1, compared to 43.3% of individuals in the stable transmission area. In contrast, antibody levels to and frequencies of MSP-1 and EBA-175 were similar in adults in areas of stable and unstable malaria transmission. Suboptimal immunity to malaria in areas of unstable malaria transmission may relate in part to infrequent high-level antibodies to preerythrocytic antigens and AMA-1.

In an area with unstable malaria transmission, detection of Plasmodium falciparum infection in 379 symptomatic individuals was assessed by microscopy and three polymerase chain reaction (PCR) methodologies. P. falciparum infection was detected in 25% of patients by microscopy, 37% by nested PCR, 41% by merozoite surface protein-2 (MSP-2) PCR, and 45% by a ligase detection reaction-fluorescent microsphere assay (LDR-FMA). Of the 64 individuals who were LDR-FMA positive, microscopy negative and did not receive treatment, 8 (12.5%) had persistent symptoms and returned for treatment. Malaria attributable fraction (MAF) in symptomatic individuals was 14.6% by microscopy (95% confidence interval [CI] = 6.6-21.8%) and 28.2% by nested PCR (95% CI = 17.9-37.2%). In this highland area, P. falciparum infection in symptomatic individuals is detected more frequently by PCR than microscopy, and most frequently by LDR-FMA. P. falciparum infection appears to resolve without treatment in most LDR-FMA-positive, microscopy-negative individuals, but is persistent in a subset of these individuals and requires treatment.

Background: Transmission of Plasmodium falciparum generally decreases with increasing elevation, in part because lower temperature slows the development of both parasites and mosquitoes. However, other aspects of the terrain, such as the shape of the land, may affect habitat suitability for Anopheles breeding and thus risk of malaria transmission. Understanding these local topographic effects may permit prediction of regions at high risk of malaria within the highlands at small spatial scales., Methods: Hydrologic modelling techniques were adapted to predict the flow of water across the landscape surrounding households in two communities in the western Kenyan highlands. These surface analyses were used to generate indices describing predicted water accumulation in regions surrounding the study area. Households with and without malaria were compared for their proximity to regions of high and low predicted wetness. Predicted wetness and elevation variables were entered into bivariate and multivariate regression models to examine whether significant associations with malaria were observable at small spatial scales., Results: On average, malaria case households (n = 423) were located 280 m closer to regions with very high wetness indices than non-malaria "control" households (n = 895) (t = 10.35, p < 0.0001). Distance to high wetness indices remained an independent predictor of risk after controlling for household elevation in multivariate regression (OR = 0.93 [95% confidence interval = 0.89-0.96] for a 100 m increase in distance). For every 10 m increase in household elevation, there was a 12% decrease in the odds of the house having a malaria case (OR = 0.88 [0.85-0.90]). However, after controlling for distance to regions of high predicted wetness and the community in which the house was located, this reduction in malaria risk was not statistically significant (OR = 0.98 [0.94-1.03])., Conclusion: Proximity to terrain with high predicted water accumulation was significantly and consistently associated with increased household-level malaria incidence, even at small spatial scales with little variation in elevation variables. These results suggest that high wetness indices are not merely proxies for valley bottoms, and hydrologic flow models may prove valuable for predicting areas of high malaria risk in highland regions. Application in areas where malaria surveillance is limited could identify households at higher risk and help focus interventions.

Background: Urban malaria is likely to become increasingly important as a consequence of the growing proportion of Africans living in cities. A novel sampling strategy was developed for urban areas to generate a sample simultaneously representative of population and inhabited environments. Such a strategy should facilitate analysis of important epidemiological relationships in this ecological context., Methods: Census maps and summary data for Kisumu, Kenya, were used to create a pseudo-sampling frame using the geographic coordinates of census-sampled structures. For every enumeration area (EA) designated as urban by the census (n = 535), a sample of structures equal to one-tenth the number of households was selected. In EAs designated as rural (n = 32), a geographically random sample totalling one-tenth the number of households was selected from a grid of points at 100 m intervals. The selected samples were cross-referenced to a geographic information system, and coordinates transferred to handheld global positioning units. Interviewers found the closest eligible household to the sampling point and interviewed the caregiver of a child aged < 10 years. The demographics of the selected sample were compared with results from the Kenya Demographic and Health Survey to assess sample validity. Results were also compared among urban and rural EAs., Results: 4,336 interviews were completed in 473 of the 567 study area EAs from June 2002 through February 2003. EAs without completed interviews were randomly distributed, and non-response was approximately 2%. Mean distance from the assigned sampling point to the completed interview was 74.6 m, and was significantly less in urban than rural EAs, even when controlling for number of households. The selected sample had significantly more children and females of childbearing age than the general population, and fewer older individuals., Conclusion: This method selected a sample that was simultaneously population-representative and inclusive of important environmental variation. The use of a pseudo-sampling frame and pre-programmed handheld GPS units is more efficient and may yield a more complete sample than traditional methods, and is less expensive than complete population enumeration.

Severe malarial anemia (SMA) is a leading cause of mortality among children in sub-Saharan Africa. Although the novel cytokine, interleukin (IL)-23, promotes anemia in chronic inflammatory diseases, the role of IL-23 in SMA remains undefined. Since IL-23 and IL-12 share the IL-12p40 subunit and IL-12Rbeta1 receptor, and are down-regulated by IL-10, relationships among these cytokines were explored in Kenyan children with varying severities of malarial anemia. Children with malarial anemia had increased circulating IL-23 and IL-10 and decreased IL-12 relative to healthy controls. Enhanced anemia severity and elevated parasitemia were associated with increased IL-10 relative to IL-23 and IL-12. Further exploration of the relationships among the cytokines using an in vitro model in which peripheral blood mononuclear cells were treated with synthetic hemozoin (sHz, malarial pigment) revealed that IL-12p35 and IL-23p19 transcripts had a sustained induction over 72 h, while IL-12p40 and IL-10 message peaked at 24 h, and rapidly declined thereafter. Taken together, results here show that IL-23 is elevated in children with malarial anemia, and that IL-10 and IL-12 appear to have important regulatory effects on IL-23 production during childhood malaria.

The status of resistance was investigated in Anopheles gambiae sensu lato and An. funestus (Diptera: Culicidae) mosquitoes from western Kenya to four classes of insecticides approved by World Health Organization for indoor residual spraying. The prevalence of the knockdown-resistance (kdr) mutation associated with resistance to pyrethroids and DDT was determined in An. gambiae s.l.. Standard World Health Organization diagnostic bioassay kits for DDT (an organochlorine), fenitrothion (an organophosphate), bendiocarb (a carbamate), and the pyrethoirds, lambdacyhalothrin and permethrin, were used. Knockdown every 10 min and mortality 24 h after exposure were noted. Controls not treated with insecticides and with the susceptible An. gambiae KISUMU strain were included in the bioassays. The presence of the kdr gene was determined using a standard diagnostic polymerase chain reaction assay. Over 98% mortality was observed for tests with all insecticides for both An. gambiae s.l. and An. funestus. Knockdown rates were not significantly different between An. gambiae s.l. and the KISUMU strain control. 50% and 95% knockdown times were either slightly lower than those for the KISUMU strain or higher by factors of less than 1.6. The mean frequency of the East African kdr mutation was 24.7% in An. gambiae sensu strictu. Based on conventional criteria where susceptibility is defined by mortality rates >98% 24 h after exposure, no evidence for resistance was found, implying that vector control measures employing any of the insecticides tested would be unhampered by resistance. The observed frequencies of the kdr mutation do not appear to compromise the effectiveness of the insecticides. The need for continuous monitoring of the status of insecticide resistance and of the impact of any observed resistance on the efficacy of vector control programs employing insecticides is apparent.

Objectives: (1) To determine whether mortality rates were raised in sick children in the 30 days after visiting first-level health facilities in an area under demographic surveillance in western Kenya, (2) to identify the types of illnesses associated with increased mortality and (3) to estimate the effectiveness of appropriate treatment., Methods: All sick children (2-59 months of age) who attended one of the seven participating first-level health facilities from May to August 2003 were identified. A standardized mortality ratio was computed to compare their mortality rate in the 30 days after a sick visit with that of the community under active demographic and health surveillance. A multivariate Cox Proportional Hazards model was used to identify illnesses associated with death and to estimate the protective effectiveness of appropriate treatment for potentially life-threatening diseases., Results: A total of 1383 eligible children made 1697 sick visits; 33 (2.4%) died within 30 days. Compared with children 2-59 months in the general population, sick children had a 5.3 times greater mortality rate [95% confidence interval (CI) 3.8-7.5]. In a multivariate survival analysis, significant risk factors for mortality included age <24 months [Hazard Ratio (HR) 4.4, 95% CI 1.5-12.6], malnutrition (HR 15.5, 95% CI 6.1-39.8), severe pneumonia (HR 12.9, 95% CI 3.0-56.4) and anaemia (HR 3.3, 95% CI 1.5-7.2). Appropriate treatment for a child's most severe illness reduced mortality by 78% (95% CI 57-89%)., Conclusion: We estimate that improvements in diagnosis and appropriate treatment at first-level health facilities for children 2-59 months could reduce overall under-5 mortality in the area by 12-14%.

Anopheles arabiensis, an important vector of malaria in sub-Saharan Africa shows great versatility in utilizing a broad range of larval habitats. Most studies on the population genetic structure of this species have focused on adult mosquitoes collected mainly indoors and have found populations within relatively short distances to be panmictic. Various authors suggested that the incipient speciation between the chromosomal forms of Anopheles gambiae s.s. in West Africa involved adaptation to different habitats of larvae. To investigate if similar process occurs in An. arabiensis, we analyzed samples from four different larval habitats alongside a sample of adult mosquitoes collected indoors at eight microsatellite loci. We tested the hypothesis that An. arabiensis is subdivided into populations that are adapted to exploit different larval habitats. Overall, the level of genetic differentiation among the larval subpopulations was low with the mean pairwise Fst estimate based on information from all the loci being 0.0232 (P=0.1691) although differentiation was only significant for comparisons between certain larval samples and the adult samples. In addition, there was strong evidence against subdivision among larvae when multilocus simultaneous assignment tests were used. These results do not provide sufficient support for the hypothesis that An. arabiensis is subdivided into subpopulations that specialize to exploit different larval habitats.

Background: Identification of Anopheles gambiae s.s. and Anopheles arabiensis from field-collected Anopheles gambiae s.l. is often necessary in basic and applied research, and in operational control programmes. The currently accepted method involves use of standard polymerase chain reaction amplification of ribosomal DNA (rDNA) from the 3' 28S to 5' intergenic spacer region of the genome, and visual confirmation of amplicons of predicted size on agarose gels, after electrophoresis. This report describes development and evaluation of an automated, quantitative PCR method based upon TaqMan single nucleotide polymorphism (SNP) genotyping., Methods: Standard PCR, and TaqMan SNP genotyping with newly designed primers and fluorophore-labeled probes hybridizing to sequences of complementary rDNA specific for either An. gambiae s.s. or An. arabiensis, were conducted in three experiments involving field-collected An. gambiae s.l. from western Kenya, and defined laboratory strains. DNA extraction was from a single leg, sonicated for five minutes in buffer in wells of 96-well PCR plates., Results: TaqMan SNP genotyping showed a reaction success rate, sensitivity, and species specificity comparable to that of standard PCR. In an extensive field study, only 29 of 3,041 (0.95%) were determined to be hybrids by TaqMan (i.e., having rDNA sequences from both species), however, all but one were An. arabiensis by standard PCR, suggesting an acceptably low (ca. 1%) error rate for TaqMan genotyping in mistakenly identifying species hybrids., Conclusion: TaqMan SNP genotyping proved to be a sensitive and rapid method for identification of An. gambiae s.l. and An. arabiensis, with a high success rate, specific results, and congruence with the standard PCR method.