Human NCR3 gene variants rs2736191 and rs11575837 alter longitudinal risk for development of pediatric malaria episodes and severe malarial anemia.

BACKGROUND: Plasmodium falciparum malaria is a leading cause of pediatric morbidity and mortality in holoendemic transmission areas. Severe malarial anemia [SMA, hemoglobin (Hb) < 5.0 g/dL in children] is the most common clinical manifestation of severe malaria in such regions. Although innate immune response genes are known to influence the development of SMA, the role of natural killer (NK) cells in malaria pathogenesis remains largely undefined. As such, we examined the impact of genetic variation in the gene encoding a primary NK cell receptor, natural cytotoxicity-triggering receptor 3 (NCR3), on the occurrence of malaria and SMA episodes over time. METHODS: Susceptibility to malaria, SMA, and all-cause mortality was determined in carriers of NCR3 genetic variants (i.e., rs2736191:C > G and rs11575837:C > T) and their haplotypes. The prospective observational study was conducted over a 36 mos. follow-up period in a cohort of children (n = 1,515, aged 1.9-40 mos.) residing in a holoendemic P. falciparum transmission region, Siaya, Kenya. RESULTS: Poisson regression modeling, controlling for anemia-promoting covariates, revealed a significantly increased risk of malaria in carriers of the homozygous mutant allele genotype (TT) for rs11575837 after multiple test correction [Incidence rate ratio (IRR) = 1.540, 95% CI = 1.114-2.129, P = 0.009]. Increased risk of SMA was observed for rs2736191 in children who inherited the CG genotype (IRR = 1.269, 95% CI = 1.009-1.597, P = 0.041) and in the additive model (presence of 1 or 2 copies) (IRR = 1.198, 95% CI = 1.030-1.393, P = 0.019), but was not significant after multiple test correction. Modeling of the haplotypes revealed that the CC haplotype had a significant additive effect for protection against SMA (i.e., reduced risk for development of SMA) after multiple test correction (IRR = 0.823, 95% CI = 0.711-0.952, P = 0.009). Although increased susceptibility to SMA was present in carriers of the GC haplotype (IRR = 1.276, 95% CI = 1.030-1.581, P = 0.026) with an additive effect (IRR = 1.182, 95% CI = 1.018-1.372, P = 0.029), the results did not remain significant after multiple test correction. None of the NCR3 genotypes or haplotypes were associated with all-cause mortality. CONCLUSIONS: Variation in NCR3 alters susceptibility to malaria and SMA during the acquisition of naturally-acquired malarial immunity. These results highlight the importance of NK cells in the innate immune response to malaria.

Clinical malaria incidence and health seeking pattern in geographically heterogeneous landscape of western Kenya.

BACKGROUND: Malaria remains a public health problem in Kenya despite sustained interventions deployed by the government. One of the major impediments to effective malaria control is a lack of accurate diagnosis and effective treatment. This study was conducted to assess clinical malaria incidence and treatment seeking profiles of febrile cases in western Kenya. METHODS: Active case detection of malaria was carried out in three eco-epidemiologically distinct zones topologically characterized as lakeshore, hillside, and highland plateau in Kisumu County, western Kenya, from March 2020 to March 2021. Community Health Volunteers (CHVs) conducted biweekly visits to residents in their households to interview and examine for febrile illness. A febrile case was defined as an individual having fever (axillary temperature ≥ 37.5 °C) during examination or complaints of fever and other nonspecific malaria related symptoms 1-2 days before examination. Prior to the biweekly malaria testing by the CHVs, the participants' treatment seeking methods were based on their behaviors in response to febrile illness. In suspected malaria cases, finger-prick blood samples were taken and tested for malaria parasites with ultra-sensitive Alere® malaria rapid diagnostic tests (RDT) and subjected to real-time polymerase chain reaction (RT-PCR) for quality control examination. RESULTS: Of the total 5838 residents interviewed, 2205 residents had high temperature or reported febrile illness in the previous two days before the visit. Clinical malaria incidence (cases/1000people/month) was highest in the lakeshore zone (24.3), followed by the hillside (18.7) and the highland plateau zone (10.3). Clinical malaria incidence showed significant difference across gender (χ2 = 7.57; df = 2, p = 0.0227) and age group (χ2 = 58.34; df = 4, p < 0.0001). Treatment seeking patterns of malaria febrile cases showed significant difference with doing nothing (48.7%) and purchasing antimalarials from drug shops (38.1%) being the most common health-seeking pattern among the 2205 febrile residents (χ2 = 21.875; df = 4, p < 0.0001). Caregivers of 802 school-aged children aged 5-14 years with fever primarily sought treatment from drug shops (28.9%) and public hospitals (14.0%), with significant lower proportions of children receiving treatment from traditional medication (2.9%) and private hospital (4.4%) (p < 0.0001). There was no significant difference in care givers' treatment seeking patterns for feverish children under the age of five (p = 0.086). Residents with clinical malaria cases in the lakeshore and hillside zones sought treatment primarily from public hospitals (61.9%, 60/97) traditional medication (51.1%, 23/45) respectively (p < 0.0001). However, there was no significant difference in the treatment seeking patterns of highland plateau residents with clinical malaria (p = 0.431).The main factors associated with the decision to seek treatment were the travel distance to the health facility, the severity of the disease, confidence in the treatment, and affordability. CONCLUSION: Clinical malaria incidence remains highest in the Lakeshore (24.3cases/1000 people/month) despite high LLINs coverage (90%). The travel distance to the health facility, severity of disease and affordability were mainly associated with 80% of residents either self-medicating or doing nothing to alleviate their illness. The findings of this study suggest that the Ministry of Health should strengthen community case management of malaria by providing supportive supervision of community health volunteers to advocate for community awareness, early diagnosis, and treatment of malaria.

Influence of landscape heterogeneity on entomological and parasitological indices of malaria in Kisumu, Western Kenya.

BACKGROUND: Identification and characterization of larval habitats, documentation of Anopheles spp. composition and abundance, and Plasmodium spp. infection burden are critical components of integrated vector management. The present study aimed to investigate the effect of landscape heterogeneity on entomological and parasitological indices of malaria in western Kenya. METHODS: A cross-sectional entomological and parasitological survey was conducted along an altitudinal transect in three eco-epidemiological zones: lakeshore along the lakeside, hillside, and highland plateau during the wet and dry seasons in 2020 in Kisumu County, Kenya. Larval habitats for Anopheles mosquitoes were identified and characterized. Adult mosquitoes were sampled using pyrethrum spray catches (PSC). Finger prick blood samples were taken from residents and examined for malaria parasites by real-time PCR (RT-PCR). RESULTS: Increased risk of Plasmodium falciparum infection was associated with residency in the lakeshore zone, school-age children, rainy season, and no ITNs (χ2 = 41.201, df = 9, P < 0.0001). Similarly, lakeshore zone and the rainy season significantly increased Anopheles spp. abundance. However, house structures such as wall type and whether the eave spaces were closed or open, as well as the use of ITNs, did not affect Anopheles spp. densities in the homes (χ2 = 38.695, df = 7, P < 0.0001). Anopheles funestus (41.8%) and An. arabiensis (29.1%) were the most abundant vectors in all zones. Sporozoite prevalence was 5.6% and 3.2% in the two species respectively. The lakeshore zone had the highest sporozoite prevalence (4.4%, 7/160) and inoculation rates (135.2 infective bites/person/year). High larval densities were significantly associated with lakeshore zone and hillside zones, animal hoof prints and tire truck larval habitats, wetland and pasture land, and the wet season. The larval habitat types differed significantly across the landscape zones and seasonality (χ2 = 1453.044, df = 298, P < 0.0001). CONCLUSION: The empirical evidence on the impact of landscape heterogeneity and seasonality on vector densities, parasite transmission, and Plasmodium infections in humans emphasizes the importance of tailoring specific adaptive environmental management interventions to specific landscape attributes to have a significant impact on transmission reduction.

Risk associations of submicroscopic malaria infection in lakeshore, plateau and highland areas of Kisumu County in western Kenya.

BACKGROUND: Persons with submicroscopic malaria infection are a major reservoir of gametocytes that sustain malaria transmission in sub-Saharan Africa. Despite recent decreases in the national malaria burden in Kenya due to vector control interventions, malaria transmission continues to be high in western regions of the country bordering Lake Victoria. The objective of this study was to advance knowledge of the topographical, demographic and behavioral risk factors associated with submicroscopic malaria infection in the Lake Victoria basin in Kisumu County. METHODS: Cross-sectional community surveys for malaria infection were undertaken in three eco-epidemiologically distinct zones in Nyakach sub-County, Kisumu. Adjacent regions were topologically characterized as lakeshore, hillside and highland plateau. Surveys were conducted during the 2019 and 2020 wet and dry seasons. Finger prick blood smears and dry blood spots (DBS) on filter paper were collected from 1,777 healthy volunteers for microscopic inspection and real time-PCR (RT-PCR) diagnosis of Plasmodium infection. Persons who were PCR positive but blood smear negative were considered to harbor submicroscopic infections. Topographical, demographic and behavioral risk factors were correlated with community prevalence of submicroscopic infections. RESULTS: Out of a total of 1,777 blood samples collected, 14.2% (253/1,777) were diagnosed as submicroscopic infections. Blood smear microscopy and RT-PCR, respectively, detected 3.7% (66/1,777) and 18% (319/1,777) infections. Blood smears results were exclusively positive for P. falciparum, whereas RT-PCR also detected P. malariae and P. ovale mono- and co-infections. Submicroscopic infection prevalence was associated with topographical variation (χ2 = 39.344, df = 2, p<0.0001). The highest prevalence was observed in the lakeshore zone (20.6%, n = 622) followed by the hillside (13.6%, n = 595) and highland plateau zones (7.9%, n = 560). Infection prevalence varied significantly according to season (χ2 = 17.374, df = 3, p<0.0001). The highest prevalence was observed in residents of the lakeshore zone in the 2019 dry season (29.9%, n = 167) and 2020 and 2019 rainy seasons (21.5%, n = 144 and 18.1%, n = 155, respectively). In both the rainy and dry seasons the likelihood of submicroscopic infection was higher in the lakeshore (AOR: 2.71, 95% CI = 1.85-3.95; p<0.0001) and hillside (AOR: 1.74, 95% CI = 1.17-2.61, p = 0.007) than in the highland plateau zones. Residence in the lakeshore zone (p<0.0001), male sex (p = 0.025), school age (p = 0.002), and living in mud houses (p = 0.044) increased the risk of submicroscopic malaria infection. Bed net use (p = 0.112) and occupation (p = 0.116) were not associated with submicroscopic infection prevalence. CONCLUSION: Topographic features of the local landscape and seasonality are major correlates of submicroscopic malaria infection in the Lake Victoria area of western Kenya. Diagnostic tests more sensitive than blood smear microscopy will allow for monitoring and targeting geographic sites where additional vector interventions are needed to reduce malaria transmission.

A cross-sectional observational study investigating the association between sedges (swamp grasses, Cyperaceae) and the prevalence of immature malaria vectors in aquatic habitats along the shore of Lake Victoria, western Kenya.

Background: Strategies that involve manipulations of the odour-orientation of gravid malaria vectors could lead to novel attract-and-kill interventions. Recent work has highlighted the potential involvement of graminoid plants in luring vectors to oviposition sites. This study aimed to analyse the association between water-indicating graminoid plants (Cyperaceae, sedges), other abiotic and biotic factors and the presence and abundance of early instar Anopheles larvae in aquatic habitats as a proxy indicator for oviposition. Methods: A cross-sectional survey of 110 aquatic habitats along the shores of Lake Victoria was done during the rainy season. Habitats were sampled for mosquito larvae using the sweep-net method and habitat characteristics recorded. Results: Anopheles arabiensis was the dominant species identified from aquatic habitats. Larvae of the secondary malaria vectors such as Anopheles coustani, An. rufipes and An. maculipalpis were found only in habitats covered with graminoids, whereas An. arabiensis, An. ziemanni and An. pharoensis were found in both habitats with and without graminoid plants. The hypothesis that sedges might be positively associated with the presence and abundance of early instar Anopheles larvae could not be confirmed. The dominant graminoid plants in the habitats were Panicum repens, Cynodon dactylon in the Poaceae family and Cyperus rotundus in the Cyperaceae family. All of these habitats supported abundant immature vector populations. The presence of early instar larvae was significantly and positively associated with swamp habitat types (OR=22, 95% CI=6-86, P<0.001) and abundance of late Anopheles larvae (OR=359, CI=33-3941, P<0.001), whilst the association was negative with tadpole presence (OR=0.1, CI=0.0.01-0.5, P=0.008). Conclusions: Early instar malaria vectors were abundant in habitats densely vegetated with graminoid plants in the study area but specific preference for any of the graminoids could not be detected. In search for oviposition cues, it might be useful to screen for chemical volatiles released from all dominant plant species.

Antibiotic Resistance of Escherichia coli from Humans and Black Rhinoceroses in Kenya.

Upsurge of antibiotic resistance in wildlife poses unprecedented threat to wildlife conservation. Surveillance of antibiotic resistance at the human-wildlife interface is therefore needed. We evaluated differences in antibiotic resistance of Escherichia coli isolates from human and the endangered black rhinoceros in Lambwe Valley, Kenya. We used standard microbiological techniques to carry out susceptibility assays using eight antibiotics of clinical and veterinary importance. Standard PCR method was used to characterize antibiotic resistance genes. There was no difference in resistance between E. coli isolates from human and those from rhinoceros (U = 25, p = 0.462). However, higher resistance in isolates from humans was noted for cotrimoxazole (p = 0.000, OR = 0.101), ceftriaxone (p = 0.005, OR = 0.113) and amoxicillin/clavulanic acid (p = 0.017, OR = 0.258), whereas isolates from rhinoceros showed higher gentamicin resistance (p = 0.001, OR = 10.154). Multi-drug resistance phenotype was 69.0% in humans and 43.3% in rhinoceros. Isolates from both species contained blaTEM, tetA, tetB, dfrA1 and sul1 genes. Resistance profiles in the two species suggest potential for cross-transfer of resistance genes or exposure to comparable selective pressure and call for a multi-sectorial action plan on surveillance of antibiotic resistance at the human-wildlife interface. Genome-wide studies are needed to explicate the direction of transfer of genes that confer antibiotic resistance at the human-wildlife interface.

Hormonal correlates of natal dispersal and rank attainment in wild male baboons.

In many mammals, maturational milestones such as dispersal and the attainment of adult dominance rank mark stages in the onset of reproductive activity and depend on a coordinated set of hormonal and socio-behavioral changes. Studies that focus on the link between hormones and maturational milestones are uncommon in wild mammals because of the challenges of obtaining adequate sample sizes of maturing animals and of tracking the movements of dispersing animals. We examined two maturational milestones in wild male baboons-adult dominance rank attainment and natal dispersal-and measured their association with variation in glucocorticoids (fGC) and fecal testosterone (fT). We found that rank attainment is associated with an increase in fGC levels but not fT levels: males that have achieved any adult rank have higher fGC than males that have not yet attained an adult rank. This indicates that once males have attained an adult rank they experience greater energetic and/or psychosocial demands than they did prior to attaining this milestone, most likely because of the resulting participation in both agonistic and sexual behaviors that accompany rank attainment. In contrast, natal dispersal does not produce sustained increases in either fGC or fT levels, suggesting that individuals are either well adapted to face the challenges associated with dispersal or that the effects of dispersal on hormone levels are ephemeral for male baboons.

Puberty and dispersal in a wild primate population.

This article is part of a Special Issue "Puberty and Adolescence". The onset of reproduction is preceded by a host of organismal adjustments and transformations, involving morphological, physiological, and behavioral changes. In highly social mammals, including humans and most nonhuman primates, the timing and nature of maturational processes are affected by the animal's social milieu as well as its ecology. Here, we review a diverse set of findings on how maturation unfolds in wild baboons in the Amboseli basin of southern Kenya, and we place these findings in the context of other reports of maturational processes in primates and other mammals. First, we describe the series of events and processes that signal maturation in female and male baboons. Sex differences in age at both sexual maturity and first reproduction documented for this species are consistent with expectations of life history theory; males mature later than females and exhibit an adolescent growth spurt that is absent or minimal in females. Second, we summarize what we know about sources of variance in the timing of maturational processes including natal dispersal. In Amboseli, individuals in a food-enhanced group mature earlier than their wild-feeding counterparts, and offspring of high-ranking females mature earlier than offspring of low-ranking females. We also report on how genetic admixture, which occurs in Amboseli between two closely related baboon taxa, affects individual maturation schedules.

Life at the top: rank and stress in wild male baboons.

In social hierarchies, dominant individuals experience reproductive and health benefits, but the costs of social dominance remain a topic of debate. Prevailing hypotheses predict that higher-ranking males experience higher testosterone and glucocorticoid (stress hormone) levels than lower-ranking males when hierarchies are unstable but not otherwise. In this long-term study of rank-related stress in a natural population of savannah baboons (Papio cynocephalus), high-ranking males had higher testosterone and lower glucocorticoid levels than other males, regardless of hierarchy stability. The singular exception was for the highest-ranking (alpha) males, who exhibited both high testosterone and high glucocorticoid levels. In particular, alpha males exhibited much higher stress hormone levels than second-ranking (beta) males, suggesting that being at the very top may be more costly than previously thought.

Endocrinology of year-round reproduction in a highly seasonal habitat: environmental variability in testosterone and glucocorticoids in baboon males.

In conditions characterized by energetic constraints, such as in periods of low food availability, some trade-offs between reproduction and self-maintenance may be necessary; even year-round breeders may then be forced to exhibit some reproductive seasonality. Prior research has largely focused on female reproduction and physiology, and few studies have evaluated the impact of environmental factors on males. Here we assessed the effects of season and ambient temperatures on fecal glucocorticoid (fGC) and testosterone (fT) levels in male baboons in Amboseli, Kenya. The Amboseli basin is a highly challenging, semiarid tropical habitat that is characterized by strongly seasonal patterns of rainfall and by high ambient temperatures. We previously reported that female baboons were impacted by these challenging environmental conditions. We ask here whether male baboons in the same environment and groups as females exhibit similar physiological effects. We found that after accounting for male age and individual variability, males exhibited higher fGC levels and lower fT levels during the dry season than during the wet season. Furthermore, fT but not fGC levels were lower in months of high average daily maximum temperatures, suggesting a direct impact of heat on testes. Our results demonstrate that male baboons, like females, experience ecological stress that alters their reproductive physiology. The impact of the environment on male reproduction deserves more attention both in its own right and because alteration in male physiology may contribute to the reduction in female fertility observed inchallenging environments.

Life history context of reproductive aging in a wild primate model.

The pace of reproductive aging has been of considerable interest, especially in regard to the long postreproductive period in modern women. Here we use data for both sexes from a 37-year longitudinal study of a wild baboon population to place reproductive aging within a life history context for this species, a primate relative of humans that evolved in the same savannah habitat as humans did. We examine the patterns and pace of reproductive aging, including birth rates and reproductive hormones for both sexes, and compare reproductive aging to age-related changes in several other traits. Reproductive senescence occurs later in baboon females than males. Delayed senescence in females relative to males is also found in several other traits, such as dominance status and body condition, but not in molar wear or glucocorticoid profiles. Survival, health, and well-being are the product of risk factors in morphological, physiological, and behavioral traits that differ in rate of senescence and in dependence on social or ecological conditions; some will be very sensitive to differences in circumstances and others less so.

A genome-wide screen for normally methylated human CpG islands that can identify novel imprinted genes.

DNA methylation is a covalent modification of the nucleotide cytosine that is stably inherited at the dinucleotide CpG by somatic cells, and 70% of CpG dinucleotides in the genome are methylated. The exception to this pattern of methylation are CpG islands, CpG-rich sequences that are protected from methylation, and generally are thought to be methylated only on the inactive X-chromosome and in tumors, as well as differentially methylated regions (DMRs) in the vicinity of imprinted genes. To identify chromosomal regions that might harbor imprinted genes, we devised a strategy for isolating a library of normally methylated CpG islands. Most of the methylated CpG islands represented high copy number dispersed repeats. However, 62 unique clones in the library were characterized, all of which were methylated and GC-rich, with a GC content >50%. Of these, 43 clones also showed a CpG(obs)/CpG(exp) >0.6, of which 30 were studied in detail. These unique methylated CpG islands mapped to 23 chromosomal regions, and 12 were differentially methylated regions in uniparental tissues of germline origin, i.e., hydatidiform moles (paternal origin) and complete ovarian teratomas (maternal origin), even though many apparently were methylated in somatic tissues. We term these sequences gDMRs, for germline differentially methylated regions. At least two gDMRs mapped near imprinted genes, HYMA1 and a novel homolog of Elongin A and Elongin A2, which we term Elongin A3. Surprisingly, 18 of the methylated CpG islands were methylated in germline tissues of both parental origins, representing a previously uncharacterized class of normally methylated CpG islands in the genome, and which we term similarly methylated regions (SMRs). These SMRs, in contrast to the gDMRs, were significantly associated with telomeric band locations (P =.0008), suggesting a potential role for SMRs in chromosome organization. At least 10 of the methylated CpG islands were on average 85% conserved between mouse and human. These sequences will provide a valuable resource in the search for novel imprinted genes, for defining the molecular substrates of the normal methylome, and for identifying novel targets for mammalian chromatin formation.