Geographic expansion of the introduced Aedes albopictus and other native Aedes species in the Democratic Republic of the Congo.

BACKGROUND: Aedes albopictus has been reported in several Central African countries, including the Democratic Republic of the Congo (DRC). The establishment of this mosquito species poses a serious threat as a vector of various infectious diseases. Although Ae. albopictus has been reported in the western region of the DRC, information about its distribution is still scarce in the country. The aim of this study was to investigate the current nationwide distribution of the invasive Ae. albopictus, as well as other native Aedes mosquitoes, in the DRC and to identify suitable areas for its future expansion. METHODS: Two entomological surveys were conducted in 2017-2019 and 2022. Based on the occurrence sites of Ae. albopictus, important environmental variables were identified. Then, geographical areas suitable for Ae. albopictus establishment were determined using the maximum entropy model. The distribution and abundance of Ae. albopictus were also compared with those of the major native Aedes species. RESULTS: Aedes albopictus was found in the western, northern, central, and eastern regions of the DRC, but it was not found in the southeastern region. The maximum entropy model predicted that most parts of the DRC are suitable for the establishment of this mosquito. The unsuitable areas encompassed the eastern highlands, known for their low temperatures, and the southeastern highlands, which experience both low temperatures and a long dry season. The native Aedes species found were Aedes aegypti, Aedes simpsoni, Aedes africanus, and Aedes vittatus. Aedes albopictus dominated in the western and northern regions, while Ae. aegypti was more prevalent in other regions. CONCLUSIONS: Aedes albopictus has been well established in the western and northern regions of the DRC. This mosquito is expanding its distribution while replacing the native Aedes species. Most of the country is suitable for the establishment of this mosquito species, except the highlands of the eastern and the southeastern regions.

Utilizing a novel high-resolution malaria dataset for climate-informed predictions with a deep learning transformer model.

Climatic factors influence malaria transmission via the effect on the Anopheles vector and Plasmodium parasite. Modelling and understanding the complex effects that climate has on malaria incidence can enable important early warning capabilities. Deep learning applications across fields are proving valuable, however the field of epidemiological forecasting is still in its infancy with a lack of applied deep learning studies for malaria in southern Africa which leverage quality datasets. Using a novel high resolution malaria incidence dataset containing 23 years of daily data from 1998 to 2021, a statistical model and XGBOOST machine learning model were compared to a deep learning Transformer model by assessing the accuracy of their numerical predictions. A novel loss function, used to account for the variable nature of the data yielded performance around + 20% compared to the standard MSE loss. When numerical predictions were converted to alert thresholds to mimic use in a real-world setting, the Transformer's performance of 80% according to AUROC was 20-40% higher than the statistical and XGBOOST models and it had the highest overall accuracy of 98%. The Transformer performed consistently with increased accuracy as more climate variables were used, indicating further potential for this prediction framework to predict malaria incidence at a daily level using climate data for southern Africa.

Exploring the Association between Ambient Temperature and Daily Hospital Admissions for Diarrhea in Mopani District, Limpopo Province, South Africa.

Diarrhea contributes significantly to global morbidity and mortality. There is evidence that diarrhea prevalence is associated with ambient temperature. This study aimed to determine if there was an association between ambient temperature and diarrhea at a rural site in South Africa. Daily diarrheal hospital admissions (2007 to 2016) at two large district hospitals in Mopani district, Limpopo province were compared to average daily temperature and apparent temperature (Tapp, 'real-feel' temperature that combined temperature, relative humidity, and wind speed). Linear regression and threshold regression, age-stratified to participants ≤5 years and >5 years old, considered changes in daily admissions by unit °C increase in Tapp. Daily ranges in ambient temperature and Tapp were 2-42 °C and -5-34 °C, respectively. For every 1 °C increase in average daily temperature, there was a 6% increase in hospital admissions for diarrhea for individuals of all ages (95% CI: 0.04-0.08; p < 0.001) and a 4% increase in admissions for individuals older than 5 years (95% CI: 0.02-0.05; p < 0.001). A positive linear relationship between average daily Tapp and all daily diarrheal admissions for children ≤5 years old was not statistically significant (95% CI: -0.00-0.03; p = 0.107). Diarrhea is common in children ≤5 years old, however, is more likely triggered by factors other than temperature/Tapp, while it is likely associated with increased temperature in individuals >5 years old. We are limited by lack of data on confounders and effect modifiers, thus, our findings are exploratory. To fully quantify how temperature affects hospital admission counts for diarrhea, future studies should include socio-economic-demographic factors as well as WASH-related data such as personal hygiene practices and access to clean water.

Evaluation of the protective efficacy of Olyset®Plus ceiling net on reducing malaria prevalence in children in Lake Victoria Basin, Kenya: study protocol for a cluster-randomized controlled trial.

BACKGROUND: In the Lake Victoria Basin of western Kenya, malaria remains highly endemic despite high coverage of interventions such as insecticide-impregnated long-lasting insecticidal nets (LLIN). The malaria-protective effect of LLINs is hampered by insecticide resistance in Anopheles vectors and its repurposing by the community. Ceiling nets and LLIN with synergist piperonyl butoxide (PBO-LLIN) are novel tools that can overcome the problems of behavioral variation of net use and metabolic resistance to insecticide, respectively. The two have been shown to reduce malaria prevalence when used independently. Integration of these two tools (i.e., ceiling nets made with PBO-LLIN or Olyset®Plus ceiling nets) appears promising in further reducing the malaria burden. METHODS: A cluster-randomized controlled trial is designed to assess the effect of Olyset®Plus ceiling nets on reducing malaria prevalence in children on Mfangano Island in Homa Bay County, where malaria transmission is moderate. Olyset®Plus ceiling nets will be installed in 1315 residential structures. Malaria parasitological, entomological, and serological indicators will be measured for 12 months to compare the effectiveness of this new intervention against conventional LLIN in the control arm. DISCUSSION: Wider adoption of Olyset®Plus ceiling nets to complement existing interventions may benefit other malaria-endemic counties and be incorporated as part of Kenya's national malaria elimination strategy. TRIAL REGISTRATION: UMIN Clinical Trials Registry UMIN000045079. Registered on 4 August 2021.

Discovery of super-insecticide-resistant dengue mosquitoes in Asia: Threats of concomitant knockdown resistance mutations.

Aedes aegypti (Linnaeus, 1762) is the main mosquito vector for dengue and other arboviral infectious diseases. Control of this important vector highly relies on the use of insecticides, especially pyrethroids. The high frequency (>78%) of the L982W substitution was detected at the target site of the pyrethroid insecticide, the voltage-gated sodium channel (Vgsc) of A. aegypti collected from Vietnam and Cambodia. Alleles having concomitant mutations L982W + F1534C and V1016G + F1534C were also confirmed in both countries, and their frequency was high (>90%) in Phnom Penh, Cambodia. Strains having these alleles exhibited substantially higher levels of pyrethroid resistance than any other field population ever reported. The L982W substitution has never been detected in any country of the Indochina Peninsula except Vietnam and Cambodia, but it may be spreading to other areas of Asia, which can cause an unprecedentedly serious threat to the control of dengue fever as well as other Aedes-borne infectious diseases.

Novel Antiviral Efficacy of Hedyotis diffusa and Artemisia capillaris Extracts against Dengue Virus, Japanese Encephalitis Virus, and Zika Virus Infection and Immunoregulatory Cytokine Signatures.

Currently, there are no specific therapeutics for flavivirus infections, including dengue virus (DENV) and Zika virus (ZIKV). In this study, we evaluated extracts from the plants Hedyotis diffusa (HD) and Artemisia capillaris (AC) to determine the antiviral activity against DENV, ZIKV, and Japanese encephalitis virus (JEV). HD and AC demonstrated inhibitory activity against JEV, ZIKV, and DENV replication and reduced viral RNA levels in a dose-responsive manner, with non-cytotoxic concentration ranging from 0.1 to 10 mg/mL. HD and AC had low cytotoxicity to Vero cells, with CC50 values of 33.7 ± 1.6 and 30.3 ± 1.7 mg/mL (mean ± SD), respectively. The anti-flavivirus activity of HD and AC was also consistent in human cell lines, including human glioblastoma (T98G), human chronic myeloid leukemia (K562), and human embryonic kidney (HEK-293T) cells. Viral-infected, HD-treated cells demonstrated downregulation of cytokines including CCR1, CCL26, CCL15, CCL5, IL21, and IL17C. In contrast, CCR1, CCL26, and AIMP1 were elevated following AC treatment in viral-infected cells. Overall, HD and AC plant extracts demonstrated flavivirus replication inhibitory activity, and together with immunoregulatory cytokine signatures, these results suggest that HD and AC possess bioactive compounds that may further be refined as promising candidates for clinical applications.

Predicting malaria outbreaks from sea surface temperature variability up to 9 months ahead in Limpopo, South Africa, using machine learning.

Malaria is the cause of nearly half a million deaths worldwide each year, posing a great socioeconomic burden. Despite recent progress in understanding the influence of climate on malaria infection rates, climatic sources of predictability remain poorly understood and underexploited. Local weather variability alone provides predictive power at short lead times of 1-2 months, too short to adequately plan intervention measures. Here, we show that tropical climatic variability and associated sea surface temperature over the Pacific and Indian Oceans are valuable for predicting malaria in Limpopo, South Africa, up to three seasons ahead. Climatic precursors of malaria outbreaks are first identified via lag-regression analysis of climate data obtained from reanalysis and observational datasets with respect to the monthly malaria case count data provided from 1998-2020 by the Malaria Institute in Tzaneen, South Africa. Out of 11 sea surface temperature sectors analyzed, two regions, the Indian Ocean and western Pacific Ocean regions, emerge as the most robust precursors. The predictive value of these precursors is demonstrated by training a suite of machine-learning classification models to predict whether malaria case counts are above or below the median historical levels and assessing their skills in providing early warning predictions of malaria incidence with lead times ranging from 1 month to a year. Through the development of this prediction system, we find that past information about SST over the western Pacific Ocean offers impressive prediction skills (~80% accuracy) for up to three seasons (9 months) ahead. SST variability over the tropical Indian Ocean is also found to provide good skills up to two seasons (6 months) ahead. This outcome represents an extension of the effective prediction lead time by about one to two seasons compared to previous prediction systems that were more computationally costly compared to the machine learning techniques used in the current study. It also demonstrates the value of climatic information and the prediction framework developed herein for the early planning of interventions against malaria outbreaks.

A comparative study of dengue virus vectors in major parks and adjacent residential areas in Ho Chi Minh City, Vietnam.

The primary dengue virus vectors, Aedes aegypti and Aedes albopictus, are primarily daytime biting mosquitoes. The risk of infection is suspected to be considerable in urban parks due to visitor traffic. Despite the importance of vector control for reducing dengue transmission, little information is available on vector populations in urban parks. The present study characterized mosquito habitats and estimated vector densities in the major urban parks in Ho Chi Minh City, Vietnam and compared them with those in adjacent residential areas. The prevalences of habitats where Aedes larvae were found were 43% and 9% for the parks and residential areas, respectively. The difference was statistically significant (prevalence ratio [PR]: 5.00, 95% CI: 3.85-6.49). The prevalences of positive larval habitats were significantly greater in the parks for both species than the residential areas (PR: 1.52, 95% CI: 1.04-2.22 for A. aegypti, PR: 10.10, 95% CI: 7.23-14.12 for A. albopictus). Larvae of both species were positively associated with discarded containers and planters. Aedes albopictus larvae were negatively associated with indoor habitats, but positively associated with vegetation shade. The adult density of A. aegypti was significantly less in the parks compared with the residential areas (rate ratio [RR]; 0.09, 95% CI: 0.05-0.16), while the density of A. albopictus was significantly higher in the parks (RR: 9.99, 95% CI: 6.85-14.59). When the species were combined, the density was significantly higher in the parks (RR: 2.50, 95% CI: 1.92-3.25). The urban parks provide suitable environment for Aedes mosquitoes, and A. albopictus in particular. Virus vectors are abundant in the urban parks, and the current vector control programs need to have greater consideration of urban parks.

Direct Viral RNA Detection of SARS-CoV-2 and DENV in Inactivated Samples by Real-Time RT-qPCR: Implications for Diagnosis in Resource Limited Settings with Flavivirus Co-Circulation.

The RT-qPCR method remains the gold standard and first-line diagnostic method for the detection of SARS-CoV-2 and flaviviruses, especially in the early stage of viral infection. Rapid and accurate viral detection is a starting point in the containment of the COVID-19 pandemic and flavivirus outbreaks. However, the shortage of diagnostic reagents and supplies, especially in resource-limited countries that experience co-circulation of SARS-CoV-2 and flaviviruses, are limitations that may result in lesser availability of RT-qPCR-based diagnostic tests. In this study, the utility of RNA-free extraction methods was assessed for the direct detection of SARS-CoV-2 and DENV-2 in heat-inactivated or chemical-inactivated samples. The findings demonstrate that direct real-time RT-qPCR is a feasible option in comparison to conventional real-time RT-qPCR based on viral genome extraction-based methods. The utility of heat-inactivation and direct real-time RT-qPCR for SARS-CoV-2, DENV-2 viral RNA detection was demonstrated by using clinical samples of SARS-CoV-2 and DENV-2 and spiked cell culture samples of SARS-CoV-2 and DENV-2. This study provides a simple alternative workflow for flavivirus and SARS-CoV-2 detection that includes heat inactivation and viral RNA extraction-free protocols, with aims to reduce the risk of exposure during processing of SARS-CoV-2 biological specimens and to overcome the supply-chain bottleneck, particularly in resource limited settings with flavivirus co-circulation.

Searching for a sign of exotic Aedes albopictus (Culicidae) introduction in major international seaports on Kyushu Island, Japan.

BACKGROUND: The Asian tiger mosquito, Aedes albopictus, has spread around the world. The migration was mainly mediated by maritime transportations. This species is known as an efficient vector for arboviruses, and it was responsible for the recent dengue outbreak in Tokyo, Japan. As the vector competence varies among geographical populations, and insecticide resistant populations have emerged, it is important to reveal their movements. The present study uses molecular techniques to search for a sign of introduction of an exotic population in three major international seaports on Kyushu Island. METHODOLOGY/PRINCIPAL FINDINGS: Adults of Ae. albopictus were sampled around the international seaports of Fukuoka, Kitakyushu, and Nagasaki. Pairwise fixation indexes were estimated between the sampled populations based on 13 microsatellite markers. There was no clear genetic differentiation between distant and port populations in Kitakyushu and Nagasaki. However, the analysis found one distinct group near the container terminal in Fukuoka, which handles international freight containers mainly from adjacent countries. DNA samples were also obtained from Goto, Tsushima, Honshu, Ryukyu, Thailand, and the Philippines; and a cluster analysis and discriminant analysis revealed that the distinct group in Fukuoka did not belong to these groups. Combined with the results of phylogenetic analysis based on CO1, these results implied that this group originated from one Asian temperate region outside of Japan. Neutrality test and mismatch distribution analysis suggested that the establishment of this group was not recent. CONCLUSIONS/SIGNIFICANCE: The present study found a sign of Ae. albopictus introduction from a temperate region of Asia through maritime freight container transportation. The genetically distinct group found in Fukuoka likely originated from a temperate region outside of Japan. Maritime container transportation may introduce to Japan mosquitoes with greater vector competence/insecticide resistance. This is the first study to describe the spatial population structure of Ae. albopictus in Japan using molecular techniques.

Oviposition of Aedes aegypti and Aedes albopictus on Ovitraps in Dry and Rainy Seasons in Southern Vietnam.

Ovitraps were set inside and outside of 15 households in December 2012 (dry season) and August 2013 (rainy season) in 2 communes (An Thanh and Phu Hoa) of Binh Duong Province in southern Vietnam. Eggs laid in the ovitraps were collected after 4 days, dried, and soaked in water. Hatched larvae were transferred to cups and reared to adulthood to identify the species. The rate of positive ovitraps did not differ between December and August for Aedes aegypti, but it was lower in December for Ae. albopictus. The number of eggs laid per ovitrap by Ae. aegypti did not differ between December and August, while that for Ae. albopictus decreased significantly in December in both communes. Moreover, Ae. albopictus laid eggs in ovitraps placed outside the households. Therefore, it is necessary for future studies to investigate whether the major source containers for oviposition had dried in the dry season or Ae. albopictus entered diapause, leading to these observations.

Presence of dogs and proximity to a wildlife reserve increase household level risk of tungiasis in Kwale, Kenya.

INTRODUCTION: Tungiasis is a ectopic skin disease caused by some species of fleas in the Tunga genus, most notably T. penetrans. The disease afflicts poor and marginalized communities in developing countries. Transmission of tungiasis comprises a complex web of factors including domesticated animals and wildlife. This research explores animal and environmental risk factors for tungiasis in an area adjacent to a wildlife reserve in Kwale, Kenya. METHODS: A two-stage complex sampling strategy was used. Households were selected from three areas in and around Kwale Town, Kenya, an area close to the Kenyan Coast. Households were listed as positive if at least one member had tungiasis. Each household was administered a questionnaire regarding tungiasis behaviors, domesticated animal assets, and wild animal species that frequent the peridomiciliary area. Associations of household tungiasis were tests with household and environmental variables using regression methods. RESULTS: The study included 319 households. Of these, 41 (12.85%) were found to have at least one person who had signs of tungiasis. There were 295 (92.48%) households that possessed at least one species of domesticated animal. It was reported that wildlife regularly come into the vicinity of the home 90.59% of households. Presence of dogs around the home (OR 3.85; 95% CI 1.84; 8.11) and proximity to the park were associated with increased risk for tungiasis infestation in humans in a multivariate regression model. CONCLUSIONS: Human tungiasis is a complex disease associated with domesticated and wild animals. Canines in particular appear to be important determinants of household level risk.

Long-Lasting Insecticidal Nets Incorporating Piperonyl Butoxide Reduce the Risk of Malaria in Children in Western Kenya: A Cluster Randomized Controlled Trial.

Malaria vectors have acquired an enzyme that metabolizes pyrethroids. To tackle this problem, we evaluated long-lasting insecticidal nets incorporating piperonyl butoxide (PBO-LLINs) with a community-based cluster randomized control trial in western Kenya. The primary endpoints were anopheline density and Plasmodium falciparum polymerase chain reaction (PCR)-positive prevalence (PCRpfPR) of children aged 7 months to 10 years. Four clusters were randomly selected for each of the treatment and control arms (eight clusters in total) from 12 clusters, and PBO-LLINs and standard LLINs were distributed in February 2011 to 982 and 1,028 houses for treatment and control arms, respectively. Entomological surveys targeted 20 houses in each cluster, and epidemiological surveys targeted 150 children. Cluster-level permutation tests evaluated the effectiveness using the fitted values from individual level regression models adjusted for baseline. Bootstrapping estimated 95% confidence intervals (CIs). The medians of anophelines per house were 1.4 (interquartile range [IQR]: 2.3) and 3.4 (IQR: 3.7) in the intervention and control arms after 3 months, and 0.4 (IQR: 0.2) and 1.6 (IQR: 0.5) after 10 months, respectively. The differences were -2.5 (95% CI: -6.4 to -0.6) and -1.3 (95% CI: -2.0 to -0.7), respectively. The datasets of 861 and 775 children were analyzed in two epidemiological surveys. The median PCRpfPRs were 25% (IQR: 11%) in the intervention arm and 52% (IQR: 11%) in the control arm after 5 months and 33% (IQR: 11%) and 45% (IQR: 5%) after 12 months. The PCRpfPR ratios were 0.67 (95% CI: 0.38, 0.91) and 0.74 (95% CI: 0.53, 0.90), respectively. We confirmed the superiority of PBO-LLINs.

Exploring rural hospital admissions for diarrhoeal disease, malaria, pneumonia, and asthma in relation to temperature, rainfall and air pollution using wavelet transform analysis.

BACKGROUND: Climate variables impact human health and in an era of climate change, there is a pressing need to understand these relationships to best inform how such impacts are likely to change. OBJECTIVES: This study sought to investigate time series of daily admissions from two public hospitals in Limpopo province in South Africa with climate variability and air quality. METHODS: We used wavelet transform cross-correlation analysis to monitor coincidences in changes of meteorological (temperature and rainfall) and air quality (concentrations of PM2.5 and NO2) variables with admissions to hospitals for gastrointestinal illnesses including diarrhoea, pneumonia-related diagnosis, malaria and asthma cases. We were interested to disentangle meteorological or environmental variables that might be associated with underlying temporal variations of disease prevalence measured through visits to hospitals. RESULTS: We found preconditioning of prevalence of pneumonia by changes in air quality and showed that malaria in South Africa is a multivariate event, initiated by co-occurrence of heat and rainfall. We provided new statistical estimates of time delays between the change of weather or air pollution and increase of hospital admissions for pneumonia and malaria that are addition to already known seasonal variations. We found that increase of prevalence of pneumonia follows changes in air quality after a time period of 10 to 15 days, while the increase of incidence of malaria follows the co-occurrence of high temperature and rainfall after a 30-day interval. DISCUSSION: Our findings have relevance for early warning system development and climate change adaptation planning to protect human health and well-being.

New mosquito repellency bioassay for evaluation of repellents and pyrethroids using an attractive blood-feeding device.

BACKGROUND: With the increasing threat of the worldwide spread of mosquito-borne infectious diseases, consumer interest in anti-mosquito textiles that protect against mosquito bites is also increasing. Accordingly, repellent- or insecticide-treated textiles are gaining popularity. The standardization of commercial textile products is, therefore, indispensable for an authentic and objective evaluation of these products. Here we report a textile testing method using an artificial blood-feeding system that does not involve human volunteers or live animals, which aligns with the policy of protecting human and animal welfare. METHODS: The attractive blood-feeding device (ABFD) was designed using the Hemotek® membrane feeding system. The repellency of DEET, icaridin and permethrin was assayed using unfed female adults of Aedes albopictus (Skuse) under two different test conditions, namely choice and no-choice tests. The choice test consisted of two feeding units, one chemically treated and untreated, that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the overall blood-feeding rates recorded. The no-choice test consisted of two feeding units treated with the same chemical that were installed on the ABFD; mosquitoes attracted to and resting on the feeding units were counted and the blood-feeding rates were recorded. A control test was conducted using two feeding units, both sides of which were untreated. RESULTS: In the choice test, high repellency (> 95% inhibition of resting on the treated surface) of 1% DEET and 2% icaridin was observed, whereas 2% permethrin was not an effective repellent. Also, high blood-feeding inhibition (> 95%) was observed for 2% DEET and 2% icaridin. In the no-choice test, high repellency was observed for 1% DEET and 2% icaridin, whereas the repellency of 2% permethrin was low. Also, high blood-feeding inhibition was observed for 2% DEET, 4% icaridin and 2% permethrin. CONCLUSIONS: The accuracy and reproducibility of the developed method demonstrate that the ABFD may be widely used for fundamental experiments in the field of mosquito physiology, for the development of new repellent chemicals and in evaluation studies of mosquito repellent products, such as anti-mosquito textiles. The further development of the membrane and feeding unit systems will enable a more practical evaluation of mosquito repellents and blood-feeding inhibitors, such as pyrethroids.

Tracking Progress Towards the Sustainable Development Goals in Four Rural Villages in Limpopo, South Africa.

Background: Measuring national progress towards the Sustainable Development Goals (SDGs) enables the identification of gaps which need to be filled to end poverty, protect the planet and improve lives. Progress is typically calculated using indicators stemming from published methodologies. South Africa tracks progress towards the SDGs at a national scale, but aggregated data may mask progress, or lack thereof, at local levels. Objective: To assess the progress towards achievement of the SDGs in four low-income, rural villages (Giyani) in South Africa and to relate the findings to national SDG indicators. Methods: Using data from a cross-sectional environmental health study, the global indicator framework for the SDGs was applied to calculate indicators for Giyani. Local progress towards SDG achievement was compared with national progress, to contextualize and supplement national scale tracking. Findings: Village scores were mostly in line with country scores for those indices which were computable, given the available data. Low data availability prevented a complete local progress assessment. Higher levels of poverty prevail in the study villages compared to South Africa as a whole (17.7% compared to 7.4%), high unemployment (49.0% compared to 27.3%) and lack of access to information via the Internet (only 4.2% compared to 61.8%) were indicators in the villages identified as falling far short of the South African averages. Conclusions: Understanding progress towards the SDGs at a local scale is important when trying to unpack national progress. It shines a light upon issues that are not picked up by national composite assessments yet require most urgent attention. Gaps in data required to measure progress towards targets represents a serious stumbling block, preventing the creation of a true reflection of local and national scale progress.

A preliminary study on designing a cluster randomized control trial of two new mosquito nets to prevent malaria parasite infection.

BACKGROUND: Although long-lasting insecticidal nets (LLINs) are the most effective tool for preventing malaria parasite transmission, the nets have some limitations. For example, the increase of LLIN use has induced the rapid expansion of mosquito insecticide resistance. More than two persons often share one net, which increases the infection risk. To overcome these problems, two new mosquito nets were developed, one incorporating piperonyl butoxide and another covering ceilings and open eaves. We designed a cluster randomized controlled trial (cRCT) to evaluate these nets based on the information provided in the present preliminary study. RESULTS: Nearly 75% of the anopheline population in the study area in western Kenya was Anopheles gambiae s. l., and the remaining was Anopheles funestus s. l. More female anophelines were recorded in the western part of the study area. The number of anophelines increased with rainfall. We planned to have 80% power to detect a 50% reduction in female anophelines between the control group and each intervention group. The between-cluster coefficient of variance was 0.192. As the number of clusters was limited to 4 due to the size of the study area, the estimated cluster size was 7 spray catches with an alpha of 0.05. Of 1619 children tested, 626 (48%) were Plasmodium falciparum positive using a rapid diagnostic test (RDT). The prevalence was higher in the northwestern part of the study area. The number of children who slept under bed nets was 929 (71%). The P. falciparum RDT-positive prevalence (RDTpfPR) of net users was 45%, and that of non-users was 55% (OR 0.73; 95% CI 0.56, 0.95). Using 45% RDTpfPR of net users, we expected each intervention to reduce prevalence by 50%. The intracluster correlation coefficient was 0.053. With 80% power and an alpha of 0.05, the estimated cluster size was 116 children. Based on the distribution of children, we modified the boundaries of the clusters and established 300-m buffer zones along the boundaries to minimize a spillover effect. CONCLUSIONS: The cRCT study design is feasible. As the number of clusters is limited, we will apply a two-stage procedure with the baseline data to evaluate each intervention.

Highly Sensitive and Rapid Quantitative Detection of Plasmodium falciparum Using an Image Cytometer.

The gold standard for malaria diagnosis is microscopic examination of blood films by expert microscopists. It is important to detect submicroscopic and asymptomatic Plasmodium infections in people, therefore the development of highly sensitive devices for diagnosing malaria is required. In the present study, we investigated whether an imaging cytometer was useful for the highly sensitive quantitative detection of parasites. Whole blood samples were prepared from uninfected individuals spiked with Plasmodium falciparum-infected erythrocytes. Thereafter, erythrocytes were purified using a push column comprising of a syringe filter unit with SiO2-nanofiber filters. After adding the erythrocytes, stained with nuclear stain, to a six-well plate, quantitative detection of the parasites was performed using an image cytometer, CQ1. Imaging of 2.6 × 106 erythrocytes was completed in 3 min, and the limit of detection indicated parasitemia of 0.00010% (≈5 parasites/µL of blood). In addition to rapid, highly sensitive, and quantitative detection, the ease of application and economic costs, image cytometry could be efficiently applied to diagnose submicroscopic parasites in infected people from endemic countries.

Protective effects of Olyset® Net on Plasmodium falciparum infection after three years of distribution in western Kenya.

BACKGROUND: Several types of insecticides, treating technologies and materials are available for long-lasting insecticide-treated nets (LLINs). The variations may result in different efficacies against mosquitoes and correspondingly infection risks for the Plasmodium falciparum malaria parasite. This cross-sectional study investigated whether infection risk varied among children who slept under different LLIN brands in rural villages of western Kenya. METHODS: Children sleeping under various types of LLINs were tested for P. falciparum infection using a diagnostic polymerase chain reaction (PCR) assay. Data were collected for other potential factors associated with infection risk: sleeping location (with bed/without bed), number of persons sharing the same net, dwelling wall material, gap of eaves (open/close), proportional hole index, socio-economic status, and density of indoor resting anophelines. Bed-net efficacy against the Anopheles gambiae susceptible strain was estimated using the WHO cone test and the tunnel test. The residual insecticide content on nets was measured. RESULTS: Seven LLIN brands were identified, and deltamethrin-based DawaPlus® 2.0 was the most popular (48%) followed by permethrin-based Olyset® Net (28%). The former LLIN was distributed in the area about six months before the present study was conducted, and the latter net was distributed at least three years before. Of 254 children analysed, P. falciparum PCR-positive prevalence was 58% for DawaPlus® 2.0 users and 38% for Olyset® users. The multiple regression analysis revealed that the difference was statistically significant (adjusted OR: 0.67, 95% credible interval: 0.45-0.97), whereas the confounders were not statistically important. Among randomly selected net samples, all DawaPlus® 2.0 (n = 20) and 95% of Olyset® (n = 19) passed either the cone test or the tunnel test. CONCLUSIONS: Olyset® was more effective in reducing infection risk compared with DawaPlus® 2.0. Although the data from the present study were too limited to explain the mechanism clearly, the results suggest that the characteristics of the former brand are more suitable for the conditions, such as vector species composition, of the study area.