Vector composition, abundance, biting patterns and malaria transmission intensity in Madang, Papua New Guinea: assessment after 7 years of an LLIN-based malaria control programme

Background A malaria control programme based on distribution of long-lasting insecticidal bed nets (LLINs) and artemisinin combination therapy began in Papua New Guinea in 2009. After implementation of the programme, substantial reductions in vector abundance and malaria transmission intensity occurred. The research reported here investigated whether these reductions remained after seven years of sustained effort. Methods All-night (18:00 to 06:00) mosquito collections were conducted using human landing catches and barrier screen methods in four villages of Madang Province between September 2016 and March 2017. Anopheles species identification and sporozoite infection with Plasmodium vivax and Plasmodium falciparum were determined with molecular methods. Vector composition was expressed as the relative proportion of different species in villages, and vector abundance was quantified as the number of mosquitoes per barrier screen-night and per person-night. Transmission intensity was quantified as the number of sporozoite-infective vector bites per person-night. Results Five Anopheles species were present, but vector composition varied greatly among villages. Anopheles koliensis, a strongly anthropophilic species was the most prevalent in Bulal, Matukar and Wasab villages, constituting 63.7–73.8% of all Anopheles, but in Megiar Anopheles farauti was the most prevalent species (97.6%). Vector abundance varied among villages (ranging from 2.8 to 72.3 Anopheles per screen-night and 2.2–31.1 Anopheles per person-night), and spatially within villages. Malaria transmission intensity varied among the villages, with values ranging from 0.03 to 0.5 infective Anopheles bites per person-night. Most (54.1–75.1%) of the Anopheles bites occurred outdoors, with a substantial proportion (25.5–50.8%) occurring before 22:00. Conclusion The estimates of vector abundance and transmission intensity in the current study were comparable to or higher than estimates in the same villages in 2010–2012, indicating impeded programme effectiveness. Outdoor and early biting behaviours of vectors are some of the likely explanatory factors. Heterogeneity in vector composition, abundance and distribution among and within villages challenge malaria control programmes and must be considered when planning them.

Estimating Malaria Attributable Fractions With Changing Transmission Intensity: Bayesian Latent Class Vs Logistic Models

Background Asymptomatic carriage of malaria parasites is common in high transmission intensity areas and confounds clinical case definitions for research studies. This is important for investigations that aim to identify immune correlates of protection from clinical malaria. The proportion of fevers attributable to malaria parasites is widely used to define different thresholds of parasite density associated with febrile episodes. We investigated whether varying intensity of malaria transmission had a significant impact on parasite density thresholds. We used the same dataset to explore an alternative statistical approach using the probability of developing fevers as a choice over threshold cut-offs as the former has been reported to increase predictive power. Methods Data from children monitored longitudinally between 2005 and 2017 from Junju and Chonyi in Kilifi, Kenya were analysed. We compare the performance of Bayesian-latent class and logistic power models in estimating malaria attributable fractions and probabilities of having fever given a parasite density with changing malaria transmission intensity. Zero-inflated beta regressions were used to assess the impact of using probabilities to evaluate anti-merozoite antibodies as correlates of protection compared with multilevel binary regression.ResultsMalaria transmission intensity declined from over 49% to 5% between 2006 and 2017 respectively. During this period, malaria attributable fraction varied between 27%-59% using logistic regression compared to 10%-36% using the Bayesian latent class approach. Both models estimated similar patterns of fevers attributable to malaria with changing transmission intensities. The former performed well in estimating the probabilities of having fever, while the latter was efficient in determining the parasite density threshold. However, compared to the logistic power model, the Bayesian algorithm yielded lower estimates for both attributable fractions and probabilities of fever. In modelling the association of merozoite antibodies and clinical malaria, both approaches resulted in comparable estimates, but the utilization of probabilities had a better statistical fit. ConclusionsMalaria attributable fractions varied with an overall decline in the malaria transmission intensity in this setting but did not significantly impact the outcomes of analyses aimed at identifying immune correlates of protection. These data confirm the statistical advantage of using probabilities over binary data.

Malaria prevalence and long-lasting insecticidal net use in rural western Uganda: results of a cross-sectional survey conducted in an area of highly variable malaria transmission intensity

Background Long-lasting insecticidal nets (LLINs) remain a cornerstone of malaria control, but strategies to sustain universal coverage and high rates of use are not well-defined. A more complete understanding of context-specific factors, including transmission intensity and access to health facilities, may inform sub-district distribution approaches and tailored messaging campaigns. Methods A cross-sectional survey of 2190 households was conducted in a single sub-county of western Uganda that experiences highly variable malaria transmission intensity. The survey was carried out approximately 3 years after the most recent mass distribution campaign. At each household, study staff documented reported LLIN use and source among children 2 to 10 years of age and performed a malaria rapid diagnostic test. Elevation and distance to the nearest health facility was estimated for each household. Associations between parasite prevalence and LLIN use were estimated from log binomial regression models with elevation and distance to clinic being the primary variables of interest. Results Overall, 6.8% (148 of 2170) of children age 2–10 years of age had a positive RDT result, yielding a weighted estimate of 5.8% (95% confidence interval [CI] 5.4–6.2%). There was substantial variability in the positivity rates among villages, with the highest elevation villages having lower prevalence than lowest-elevation villages (p < .001). Only 64.7% (95% CI 64.0–65.5%) of children were reported to have slept under a LLIN the previous night. Compared to those living < 1 km from a health centre, households at ≥ 2 km were less likely to report the child sleeping under a LLIN (RR 0.86, 95% CI 0.83–0.89, p < .001). Households located farther from a health centre received a higher proportion of LLINs from government distributions compared to households living closer to health centres. Conclusions LLIN use and sourcing was correlated with household elevation and estimated distance to the nearest health facility. The findings suggest that current facility-based distribution strategies are limited in their reach. More frequent mass distribution campaigns and complementary approaches are likely required to maintain universal LLIN coverage and high rates of use among children in rural Uganda.

Molecular characterization of Plasmodium falciparum PHISTb proteins as potential targets of naturally-acquired immunity against malaria

Background: Plasmodium falciparum causes the deadliest form of malaria in humans. Upon infection, the host’s infected red blood cells (iRBCs) are remodelled by exported parasite proteins to provide a niche for parasite development and maturation. Methods: Here we analysed the role of three PHISTb proteins Pf3D7_0532400, Pf3D7_1401600, and Pf3D7_1102500 by expressing recombinant proteins and evaluated antibody responses against these proteins using immune sera from malaria-exposed individuals from Kenya and The Gambia in Africa. Results: Children and adults from malaria-endemic regions recognized the three PHISTb proteins. Responses against PHISTb proteins varied with malaria transmission intensity in three different geographical sites in Kenya (Siaya and Takaungu) and The Gambia (Sukuta). Antibody responses against PHISTb antigens Pf3D7_1102500 and Pf3D7_1401600 were higher in Sukuta, a low transmission region in Gambia, compared to Siaya, a high transmission region in western Kenya, unlike Pf3D7_0532400. Anti-PHIST responses indicate negative correlation between antibody levels and malaria transmission intensity for Pf3D7_1102500 and Pf3D7_1401600. We report a correlation in antibody responses between schizont and gametocyte extract, but this is not statistically significant (cor=0.102, p=0.2851, CI=95%) and, Pf3D7_0532400 (cor=0.11, p=0.249, CI=95%) and Pf3D7_1401600 (cor=0.02, p=0.7968, CI=95%). We report a negative correlation in antibody responses between schizont and Pf3D7_1102500 (cor=-0.008, p=0.9348, CI=95%). There is a correlation between gametocyte extract and Pf3D7_1401600 (cor=-0.0402, p=0.6735, CI=95%), Pf3D7_1102500 (cor=0.0758, p=0.4271, CI=95%) and Pf3D7_0532400 (cor=0.155, p=0.1028, CI=95%). Acquisition of anti-PHIST antibodies correlates with exposure to malaria for Pf3D7_0532400 (p=0.009) but not Pf3D7_1102500 and Pf3D7_1401600 (p=0.507 and p=0.15, respectively, CI=95%). Children aged below 2 years had the lowest antibody levels which do not correlate with age differences. Conclusions: Collectively, these findings provide evidence of natural immunity against PHISTb antigens that varies with level of malaria exposure and underscore their potential as possible serological markers to P. falciparum infection aimed at contributing to malaria control through vaccine development.

Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

Background Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. Methods High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results The prevalence of KIR3DS1, KIR2DL5, KIR2DS5, and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6 vs 13.2%: p = 0.006, 57.2 vs 66.4%: p = 0.005, 33.2 vs 46.6%: p < 0.001, and 19.7 vs 26.7%: p = 0.014, respectively) or Jinja (7.6 vs 18.1%: p < 0.001, 57.2 vs 63.8%: p = 0.048, 33.2 vs 43.5%: p = 0.002, and 19.7 vs 30.4%: p < 0.001, respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p = 0.043, with no significant difference between Kanungu and Tororo (26.7%), p = 0.296. Conclusions The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput, multiplex, real-time HLA-C genotyping PCR method developed will be useful in disease-association studies involving large cohorts.

Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. The aim of this study was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches.Methods: High throughput multiplex quantitative real-time PCR method was used to genotype KIR genetic variants and copy number variation and a high-throughput real-time PCR method was developed to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes may partly explain differences in transmission intensity of malaria since these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method developed will be useful in disease association studies involving large cohorts.

Estimating the extrinsic incubation period of malaria using a mechanistic model of sporogony

During sporogony, malaria-causing parasites infect a mosquito, reproduce and migrate to the mosquito salivary glands where they can be transmitted the next time blood-feeding occurs. The time required for sporogony, or extrinsic incubation period (EIP), is a crucial determinant of malaria transmission intensity. The EIP is typically estimated as the time for a given percentile of infected mosquitoes to have salivary gland sporozoites (the infectious parasite life stage). Many mechanisms, however, affect the observed sporozoite prevalence including the human-to-mosquito transmission probability and possibly differences in mosquito mortality according to infection status. To account for these various mechanisms, we present a mechanistic mathematical model (“mSOS”), which explicitly models key processes at the parasite, mosquito and observational scales. Fitting this model to experimental data, we find greater variation in EIP than previously thought: we estimated the range between two percentiles of the distribution, EIP10–EIP90 (at 27°C), as 4.5 days, compared to 0.9 days using existing methods. This pattern holds over the range of study temperatures included in the dataset. Increasing temperature from 21°C to 34°C decreased the EIP50 from 16.1 to 8.8 days and the human-to-mosquito transmission probability from 84% to 42%. Our work highlights the importance of mechanistic modelling of sporogony to (1) improve estimates of malaria transmission under different environmental conditions or disease control programs and (2) evaluate novel interventions that target the mosquito life stages of the parasite.Author summaryAnopheles mosquitoes become infected with malaria-causing parasites when blood feeding on an infectious human host. The parasites then process through a number of life stages, which begin in the mosquito gut and end in the salivary glands, where the newly formed infectious parasites can be transmitted to another host the next time a mosquito blood-feeds. The large variability in parasite numbers and development times that exists between mosquitoes, environments and parasites, mean that understanding parasite population dynamics from individual mosquito dissections is difficult. Here, we introduce a mathematical model of the mosquito life stages of parasites that mimics key characteristics of the biology. We show that the model’s parameters can be chosen so that its predictions correspond with experimental observations. In doing so, we estimate key system characteristics that are crucial determinants of malaria transmission intensity. Our work is a step towards a realistic model of within-mosquito parasite dynamics, which is increasingly important given that many recently proposed disease interventions specifically target mosquito life stages of the parasite.

Diversity of KIR genes and their HLA-C ligands in Ugandan populations with historically varied malaria transmission intensity

Background: Malaria is one of the most serious infectious diseases in the world. The malaria burden is greatly affected by human immunity, and immune responses vary between populations. Genetic diversity in KIR and HLA-C genes, which are important in immunity to infectious diseases, is likely to play a role in this heterogeneity. Several studies have shown that KIR and HLA-C genes influence the immune response to viral infections, but few studies have examined the role of KIR and HLA-C in malaria infection, and these have used low-resolution genotyping. Our aim was to determine whether genetic variation in KIR and their HLA-C ligands differ in Ugandan populations with historically varied malaria transmission intensity using more comprehensive genotyping approaches. Methods: We used high throughput multiplex quantitative real-time PCR method to genotype KIR genetic variants and copy number variation and developed a high-throughput real-time PCR method to genotype HLA-C1 and C2 allotypes for 1,344 participants, aged 6 months to 10 years, enrolled from Ugandan populations with historically high (Tororo District), medium (Jinja District) and low (Kanungu District) malaria transmission intensity. Results: The prevalence of KIR3DS1, KIR2DL5, KIR2DS5 and KIR2DS1 genes was significantly lower in populations from Kanungu compared to Tororo (7.6% vs. 13.2%: p=0.006, 57.2% vs. 66.4%: p=0.005, 33.2% vs. 46.6%: p<0.001 and 19.7% vs. 26.7%: p=0.014 respectively) or Jinja (7.6% vs.18.1%: p<0.001, 57.2% vs. 63.8%: p=0.048, 33.2% vs. 43.5%: p=0.002 and 19.7% vs. 30.4%: p<0.001 respectively). The prevalence of homozygous HLA-C2 was significantly higher in populations from Kanungu (31.6%) compared to Jinja (21.4%), p=0.043, with no significant difference between Kanungu and Tororo (26.7%), p=0.296. Conclusions: The KIR3DS1 , KIR2DL5, KIR2DS5 and KIR2DS1 genes are potentially beneficial in malaria as these genes have been positively selected for in places with historically high malaria transmission intensity. The high-throughput multiplex real-time HLA-C genotyping PCR method we have developed will be useful in disease association studies involving large cohorts.

Molecular characterization of Plasmodium falciparum PHISTb proteins as potential targets of naturally-acquired immunity against malaria

Background: Plasmodium falciparum causes the deadliest form of malaria in humans. Upon infection, the host’s infected red blood cells (iRBCs) are remodelled by exported parasite proteins in order to provide a niche for parasite development and maturation. Methods: Here we analysed the role of three PHISTb proteins Pf3D7_0532400, Pf3D7_1401600, and Pf3D7_1102500 by expressing recombinant proteins and evaluated antibody responses against these proteins using immune sera from malaria-exposed individuals from Kenya and The Gambia in Africa. Results: Our findings show that children and adults from malaria-endemic regions recognized the three PHISTb proteins. Responses against the PHISTb proteins varied with malaria transmission intensity in three different geographical sites in Kenya (Siaya and Takaungu) and The Gambia (Sukuta). Antibody responses against PHISTb antigens Pf3D7_1102500 and Pf3D7_1401600 were higher in Sukuta, a low transmission region in the Gambia, as compared to Siaya, a high transmission region in western Kenya, unlike Pf3D7_0532400. Anti-PHIST responses show a negative correlation between antibody levels and malaria transmission intensity for two PHIST antigens, Pf3D7_1102500 and Pf3D7_1401600. However, we report a correlation in antibody responses between schizont extract and Pf3D7_0532400 (p=0.00582). Acquisition of anti-PHIST antibodies was correlated with exposure to malaria for PHISTb protein Pf3D7_0532400 (p=0.009) but not the other PHIST antigens Pf3D7_1102500 and Pf3D7_1401600 (p=0.507 and p=0.15, respectively, CI=95%). Children aged below 2 years had the lowest antibody levels, but the responses do not correlate with age differences. Conclusions: Collectively, these findings provide evidence of natural immunity against PHISTb antigens that varies with level of malaria exposure and underscore potential for these parasite antigens as possible serological markers to P. falciparum infection aimed at contributing to malaria control through vaccine development.