CFTR gene variants, air pollution, and childhood asthma in a California Medicaid population.

Carriers of the cystic fibrosis transmembrane conductance regulator (CFTR) gene ("carriers") have been found to have an increased risk of persistent asthma. However, it is unclear at what level of CFTR function this risk exists and whether it is modified by asthmogens, such as air pollution. We conducted a retrospective cohort study of children born in California between July 2007 and December 2013, linking CFTR genotype data from the California newborn screening program to Medicaid claims records through March 17, 2020 to identify asthma cases, and to air pollution data from CalEnviroScreen 3.0 to identify levels of particulate matter with diameter 2.5 microns or smaller (PM2.5 ). Log-binomial regression models for asthma risk were fitted, adjusting for race/ethnicity and sex. Compared to population controls, carriers had higher risk of asthma (adjusted risk ratio (aRR) = 1.29, 95% confidence interval (CI): 0.98, 1.69; p < 0.1). Other non-CF-causing variants on the second allele did not appear to further increase risk. Genotypes with the greatest asthma risk were F508del with an intron 10 T7 or (TG)11T5 in trans (aRR=1.52, 95% CI: 1.10, 2.12). This association was higher among children living in areas at or above (aRR = 1.80) versus below (aRR = 1.37) the current national air quality standard for PM2.5 , though this difference was not statistically significant (pinteraction > 0.2). These results suggest carriers with CFTR functional levels between 25% and 45% of wildtype are at increased risk of asthma. Knowledge of CFTR genotype in asthmatics may be important to open new CFTR-related treatment options for these patients.

The effectiveness of older insecticide-treated bed nets (ITNs) to prevent malaria infection in an area of moderate pyrethroid resistance: results from a cohort study in Malawi.

BACKGROUND: A previous cohort study in Malawi showed that users of new insecticide-treated bed nets (ITNs) were significantly protected against malaria compared to non-users, despite moderate levels of pyrethroid resistance among the primary mosquito vectors. The present study investigated whether ITNs that were 1-2 years old continued to protect users in the same area with moderate pyrethroid resistance. METHODS: One year following a baseline cross-sectional malaria parasitaemia prevalence survey and universal distribution of deltamethrin ITNs (May 2012), a fixed cohort of 1223 children aged 6-59 months was enrolled (April 2013). Children were tested for parasitaemia at monthly scheduled visits and at unscheduled sick visits from May to December 2013 using rapid diagnostic tests. ITN use the prior night and the condition of ITNs (based on presence of holes) was assessed by caregiver self-report. The incidence rate ratio (RR) comparing malaria infection among users and non-users of ITNs was modelled using generalized estimating equations adjusting for potential confounders and accounting for repeated measures on each child. The protective efficacy (PE) of ITN use was calculated as 1 - RR. RESULTS: In this cohort, self-reported ITN use remained consistently high (> 95%) over the study period. Although users of ITNs were slightly more protected compared to non-users of ITNs, the difference in incidence of infection was not statistically significant (RR 0.83, 95% confidence interval [CI] 0.54-1.27). Among ITN users, malaria incidence was significantly lower in users of ITNs with no holes (of any size) compared to users of ITNs with ≥ 1 hole (RR 0.82, 95% CI 0.69-0.98). CONCLUSIONS: There was no significant PE of using 1-2 year-old ITNs on the incidence of malaria in children in an area of moderate pyrethroid resistance, but among ITN users, the authors found increased protection by ITNs with no holes compared to ITNs with holes. Given the moderate levels of pyrethroid resistance in the primary malaria vector and recent evidence of added benefits of ITNs with synergists or non-pyrethroid insecticides, next-generation ITNs may be a useful strategy to address pyrethroid resistance and should be further explored in Malawi.

The genetic structure of Aedes aegypti populations is driven by boat traffic in the Peruvian Amazon.

In the Americas, as in much of the rest of the world, the dengue virus vector Aedes aegypti is found in close association with human habitations, often leading to high population densities of mosquitoes in urban settings. In the Peruvian Amazon, this vector has been expanding to rural communities over the last 10-15 years, but to date, the population genetic structure of Ae. aegypti in this region has not been characterized. To investigate the relationship between Ae. aegypti gene flow and human transportation networks, we characterized mosquito population structure using a panel of 8 microsatellite markers and linked results to various potential mechanisms for long-distance dispersal. Adult and immature Ae. aegypti (>20 individuals per site) were collected from Iquitos city and from six neighboring riverine communities, i.e., Nauta, Indiana, Mazan, Barrio Florida, Tamshiaco, and Aucayo. FST statistics indicate significant, but low to moderate differentiation for the majority of study site pairs. Population structure of Ae. aegypti is not correlated with the geographic distance between towns, suggesting that human transportation networks provide a reasonable explanation for the high levels of population mixing. Our results indicate that Ae. aegypti gene flow among sub-populations is greatest between locations with heavy boat traffic, such as Iquitos-Tamshiaco and Iquitos-Indiana-Mazan, and lowest between locations with little or no boat/road traffic between them such as Barrio Florida-Iquitos. Bayesian clustering analysis showed ancestral admixture among three genetic clusters; no single cluster was exclusive to any site. Our results are consistent with the hypothesis that human transportation networks, particularly riverways, are responsible for the geographic spread of Ae. aegypti in the Peruvian Amazon. Our findings are applicable to other regions of the world characterized by networks of urban islands connected by fluvial transport routes.

Hemodialyzer Reuse and Gram-Negative Bloodstream Infections.

BACKGROUND: Clusters of bloodstream infections caused by Burkholderia cepacia and Stenotrophomonas maltophilia are uncommon, but have been previously identified in hemodialysis centers that reprocessed dialyzers for reuse on patients. We investigated an outbreak of bloodstream infections caused by B cepacia and S maltophilia among hemodialysis patients in clinics of a dialysis organization. STUDY DESIGN: Outbreak investigation, including matched case-control study. SETTING & PARTICIPANTS: Hemodialysis patients treated in multiple outpatient clinics owned by a dialysis organization. PREDICTORS: Main predictors were dialyzer reuse, dialyzer model, and dialyzer reprocessing practice. OUTCOMES: Case patients had a bloodstream infection caused by B cepacia or S maltophilia; controls were patients without infection dialyzed at the same clinic on the same day as a case; results of environmental cultures and organism typing. RESULTS: 17 cases (9 B cepacia and 8 S maltophilia bloodstream infections) occurred in 5 clinics owned by the same dialysis organization. Case patients were more likely to have received hemodialysis with a dialyzer that had been used more than 6 times (matched OR, 7.03; 95% CI, 1.38-69.76) and to have been dialyzed with a specific reusable dialyzer (Model R) with sealed ends (OR, 22.87; 95% CI, 4.49-∞). No major lapses during dialyzer reprocessing were identified that could explain the outbreak. B cepacia was isolated from samples collected from a dialyzer header-cleaning machine from a clinic with cases and was indistinguishable from a patient isolate collected from the same clinic, by pulsed-field gel electrophoresis. Gram-negative bacteria were isolated from 2 reused Model R dialyzers that had undergone the facility's reprocessing procedure. LIMITATIONS: Limited statistical power and overmatching; few patient isolates and dialyzers available for testing. CONCLUSIONS: This outbreak was likely caused by contamination during reprocessing of reused dialyzers. Results of this and previous investigations demonstrate that exposing patients to reused dialyzers increases the risk for bloodstream infections. To reduce infection risk, providers should consider implementing single dialyzer use whenever possible.

Home visits to assess the reliability of caregiver-reported use of insecticide-treated bednets by children in Machinga District, Malawi.

A malaria cohort study was conducted among young children in Machinga District, Malawi, following distribution of insecticide-treated bednets (ITNs) in May 2012. To assess ITN use, two independently sampled subsets of children (211 during survey 1 [December 2012-January 2013] and 325 during survey 2 [September-October 2013]) were randomly selected to compare the proportions of positive and negative agreement between caregiver verbal reports at monthly interviews with visual observation of the ITN at home visits. Caregiver-reported ITN use was consistently high during both surveys (98.1% and 96.0%, respectively; P = 0.17). Home visit-based ITN use fell significantly (P < 0.001) from survey 1 (98.6%) to survey 2 (88.6%). The proportions of positive agreement between caregiver report and home visit in the first and second surveys were 98.8% (95% confidence interval [CI] 97.6-99.8%) and 93.3% (95% CI 91.2-95.3%), respectively. The proportions of negative agreement in the first and second surveys were 28.6% (95% CI 0-75.0%) and 20.0% (95% CI 0.1-35.0%), respectively. ITN use by children was high in Machinga District, and caregiver reports and home visits with visual confirmation of the net demonstrated a high level of agreement for use of ITNs, but a low level of agreement when ITNs were not used.

A cohort study of the effectiveness of insecticide-treated bed nets to prevent malaria in an area of moderate pyrethroid resistance, Malawi.

BACKGROUND: Insecticide-treated bed nets (ITNs) are the cornerstone of malaria control in sub-Saharan Africa but their effectiveness may be compromised by the spread of pyrethroid resistance among malaria vectors. The objective of this investigation was to assess the effectiveness of ITNs to prevent malaria in an area of Malawi with moderate pyrethroid resistance. METHODS: One deltamethrin ITN was distributed in the study area for every two individuals in each household plus one extra ITN for households with an odd number of residents. A fixed cohort of 1,199 children aged six to 59 months was seen monthly for one year and at sick visits to measure malaria infection and use of ITNs. Insecticide resistance among malaria vectors was measured. The effect of ITN use on malaria incidence was assessed, adjusting for potential confounders using generalized estimating equations accounting for repeated measures. RESULTS: There were 1,909 infections with Plasmodium falciparum over 905 person-years at risk (PYAR), resulting in an observed incidence of 2.1 infections per person-year (iPPY). ITNs were used during 97% of the PYAR. The main vector was Anopheles funestus: mortality in WHO tube assays after exposure to 0.05% deltamethrin was 38% (95% confidence interval (CI) 29-47), and resistance was due to elevated oxidase enzymes. After adjusting for potential confounders, the incidence of malaria infection among ITN users was 1.7 iPPY (95% CI 1.5-2.1) and among non-bed net users was 2.6 iPPY (95% CI 2.0-3.3). Use of ITNs reduced the incidence of malaria infection by 30% (rate ratio 0.7; 95% CI, 0.5-0.8) compared to no bed nets. CONCLUSION: ITNs significantly reduced the incidence of malaria infection in children in an area with moderate levels of pyrethroid resistance and considerable malaria transmission. This is the first study to show that ITNs provide protection in areas where pyrethroid-resistant An. funestus is the major malaria vector. Malaria control programmes should continue to distribute and promote ITNs in areas with low to moderate pyrethroid resistance; however, insecticide resistance may intensify further and it is not known whether ITNs will remain effective at higher levels of resistance. There is an urgent need to identify or develop new insecticides and technologies to limit the vulnerability of ITNs to insecticide resistance.

Serological markers for monitoring historical changes in malaria transmission intensity in a highly endemic region of Western Kenya, 1994-2009.

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.

Standardizing operational vector sampling techniques for measuring malaria transmission intensity: evaluation of six mosquito collection methods in western Kenya.

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.

Linking oviposition site choice to offspring fitness in Aedes aegypti: consequences for targeted larval control of dengue vectors.

BACKGROUND: Current Aedes aegypti larval control methods are often insufficient for preventing dengue epidemics. To improve control efficiency and cost-effectiveness, some advocate eliminating or treating only highly productive containers. The population-level outcome of this strategy, however, will depend on details of Ae. aegypti oviposition behavior. METHODOLOGY/PRINCIPAL FINDINGS: We simultaneously monitored female oviposition and juvenile development in 80 experimental containers located across 20 houses in Iquitos, Peru, to test the hypothesis that Ae. aegypti oviposit preferentially in sites with the greatest potential for maximizing offspring fitness. Females consistently laid more eggs in large vs. small containers (ß = 9.18, p<0.001), and in unmanaged vs. manually filled containers (ß = 5.33, p<0.001). Using microsatellites to track the development of immature Ae. aegypti, we found a negative correlation between oviposition preference and pupation probability (ß = -3.37, p<0.001). Body size of emerging adults was also negatively associated with the preferred oviposition site characteristics of large size (females: ß = -0.19, p<0.001; males: ß = -0.11, p = 0.002) and non-management (females: ß = -0.17, p<0.001; males: ß = -0.11, p<0.001). Inside a semi-field enclosure, we simulated a container elimination campaign targeting the most productive oviposition sites. Compared to the two post-intervention trials, egg batches were more clumped during the first pre-intervention trial (ß = -0.17, P<0.001), but not the second (ß = 0.01, p = 0.900). Overall, when preferred containers were unavailable, the probability that any given container received eggs increased (ß = 1.36, p<0.001). CONCLUSIONS/SIGNIFICANCE: Ae. aegypti oviposition site choice can contribute to population regulation by limiting the production and size of adults. Targeted larval control strategies may unintentionally lead to dispersion of eggs among suitable, but previously unoccupied or under-utilized containers. We recommend integrating targeted larval control measures with other strategies that leverage selective oviposition behavior, such as luring ovipositing females to gravid traps or egg sinks.

Microsatellite-based parentage analysis of Aedes aegypti (Diptera: Culicidae) using nonlethal DNA sampling.

To track Aedes aegypti (L.) egg-laying behavior in the field in Iquitos, Peru, we developed methods for 1) sampling DNA from live mosquitoes and 2) high through-put parentage analysis using microsatellite markers. We were able to amplify DNA extracted from a single hind leg, but not from the pupal exuvia. Removal of a leg from teneral females caused no significant changes in female behavioral or life history traits (e.g., longevity, blood feeding frequency, fecundity, egg hatch rate, gonotrophic cycle length, or oviposition behavior). Using a panel of nine microsatellite markers and an exclusion-based software program, we matched offspring to parental pairs in 10 Ae. aegypti test families in which parents originated from natural development sites in Iquitos. By mating known individuals in the laboratory, retaining the male, sampling the female's DNA before release, and collecting offspring in the field, the technique we developed can be used to genotype large numbers of Ae. aegypti, reconstruct family relationships, and track the egg-laying behavior of individual Ae. aegypti in nature.

Oviposition site selection by the dengue vector Aedes aegypti and its implications for dengue control.

BACKGROUND: Because no dengue vaccine or antiviral therapy is commercially available, controlling the primary mosquito vector, Aedes aegypti, is currently the only means to prevent dengue outbreaks. Traditional models of Ae. aegypti assume that population dynamics are regulated by density-dependent larval competition for food and little affected by oviposition behavior. Due to direct impacts on offspring survival and development, however, mosquito choice in oviposition site can have important consequences for population regulation that should be taken into account when designing vector control programs. METHODOLOGY/PRINCIPAL FINDINGS: We examined oviposition patterns by Ae. aegypti among 591 naturally occurring containers and a set of experimental containers in Iquitos, Peru. Using larval starvation bioassays as an indirect measure of container food content, we assessed whether females select containers with the most food for their offspring. Our data indicate that choice of egg-laying site is influenced by conspecific larvae and pupae, container fill method, container size, lid, and sun exposure. Although larval food positively influenced oviposition, our results did not support the hypothesis that females act primarily to maximize food for larvae. Females were most strongly attracted to sites containing immature conspecifics, even when potential competitors for their progeny were present in abundance. CONCLUSION/SIGNIFICANCE: Due to strong conspecific attraction, egg-laying behavior may contribute more to regulating Ae. aegypti populations than previously thought. If highly infested containers are targeted for removal or larvicide application, females that would have preferentially oviposited in those sites may instead distribute their eggs among other suitable, previously unoccupied containers. Strategies that kill mosquitoes late in their development (i.e., insect growth regulators that kill pupae rather than larvae) will enhance vector control by creating "egg sinks," treated sites that exploit conspecific attraction of ovipositing females, but reduce emergence of adult mosquitoes via density-dependent larval competition and late acting insecticide.

Sampling considerations for designing Aedes aegypti (Diptera:Culicidae) oviposition studies in Iquitos, Peru: substrate preference, diurnal periodicity, and gonotrophic cycle length.

When devising methods to sample Aedes aegypti (L.) eggs from naturally-occurring containers to investigate selective oviposition, failure to take into account certain aspects of Ae. aegypti behavior can bias study inferences. In Iquitos, Peru, we tested three assumptions related to designing Ae. aegypti oviposition field studies, as follows: 1) lining containers with paper as an oviposition substrate does not affect oviposition; 2) diurnal egg-laying activity peaks in the late afternoon or early evening, and there is little oviposition during midday; and 3) the gonotrophic cycle length of wild females averages from 3 to 4 d. When wild females were presented with containers lined and unlined with paper toweling, the presence of paper increased oviposition in plastic and metal containers, but had no effect in cement containers. Recording the number of eggs laid by Ae. aegypti every 2 h throughout the day delineated a bimodal diurnal oviposition pattern, with a small morning peak, decreased activity during midday, and a predominant peak in the late afternoon and evening from 16:00 to 20:00 h. Daily monitoring of captive individual Fo females revealed that the gonotrophic cycle length was typically 3-4 d for the Iquitos population. These findings will be used to adjust field study design to 1) account for sampling eggs using paper toweling, and 2) determine the time of day and number of days over which to sample Ae. aegypti eggs. We explored how failure to consider these behaviors could potentially bias field assessments of oviposition preferences.

Shifting priorities in vector biology to improve control of vector-borne disease.

Vector control remains the primary measure available to prevent pathogen transmission for the most devastating vector-borne diseases (VBDs): malaria, dengue, trypanosomiasis, filariasis, leishmaniasis, and Chagas disease. Current control strategies, however, are proving insufficient and the remarkable advances in the molecular biology of disease vectors over the last two decades have yet to result in tangible tools that effectively reduce VBD incidence. Here we argue that vector biologists must fundamentally shift their approach to VBD research. We propose an agenda highlighting the most critical avenues to improve the effectiveness of vector control. Research priorities must be diversified to support simultaneous development of multiple, alternative control strategies. Knowledge across relevant diseases and disciplines should be better integrated and disease prevention efforts extended beyond the academic sector to involve private industry, ministries of health, and local communities. To obtain information of more immediate significance to public health, the research focus must shift from laboratory models to natural pathogen-transmission systems. Identification and characterization of heterogeneities inherent to VBD systems should be prioritized to allow development of local, adaptive control strategies that efficiently make use of limited resources. Importantly, increased involvement of disease-endemic country (DEC) scientists, institutes, and communities will be key to enhance and sustain the fight against VBD.

SSCP analysis of scnDNA for genetic profiling of Aedes aegypti.

We characterized genetic profiling markers for Aedes aegypti using single-strand conformation polymorphism (SSCP) analysis of single copy nuclear genes (scnDNA). Nucleotide variations at 18 loci were evaluated in 173 wild Ae. aegypti collected from a single population in northwestern Thailand. We identified seven scnDNAs with polymorphisms sufficient to determine a unique genetic profile for each mosquito examined. Six markers were derived from previously mapped cDNA loci. One marker was developed from a non-coding region of a gene. The number of alleles at each scnDNA locus ranged from 3 to 9. The described scnDNAs can be used to quickly fingerprint large numbers of Ae. aegypti to track the behavior of individual mosquitoes in the field.