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Background Aedes mosquitoes severely affect the health and wellbeing of human populations by transmitting infectious diseases. In French Polynesia, Aedes aegypti is the main vector of dengue, chikungunya and Zika, and Aedes polynesiensis the primary vector of Bancroftian filariasis and a secondary vector of arboviruses. Tools for assessing the risk of disease transmission or for measuring the efficacy of vector control programmes are scarce. A promising approach to quantify the human-vector contact relies on the detection and the quantification of antibodies directed against mosquito salivary proteins. Methodology/Principal findings An ELISA test was developed to detect and quantify the presence of immunoglobulin G (IgG) directed against proteins from salivary gland extracts (SGE) of Ae. aegypti and Ae. polynesiensis in human populations exposed to either species, through a cross-sectional study. In Tahiti and Moorea islands where Ae. aegypti and Ae. polynesiensis are present, the test revealed that 98% and 68% of individuals have developed IgG directed against Ae. aegypti and Ae. polynesiensis SGE, respectively. By comparison, ELISA tests conducted on a cohort of people from metropolitan France, not exposed to these Aedes mosquitoes, indicated that 97% of individuals had no IgG directed against SGE of either mosquito species. The analysis of additional cohorts representing different entomological Aedes contexts showed no ELISA IgG cross-reactivity between Ae. aegypti and Ae. polynesiensis SGE. Conclusions/Significance The IgG response to salivary gland extracts seems to be a valid and specific biomarker of human exposure to the bites of Ae. aegypti and Ae. polynesiensis. This new immuno-epidemiological tool will enhance our understanding of people exposure to mosquito bites, facilitate the identification of areas where disease transmission risk is high and permit to evaluate the efficacy of novel vector control strategies in Pacific islands and other tropical settings., Author summary In Pacific islands like in most tropical regions, Aedes mosquitoes affect the health of human populations by transmitting diseases like dengue, chikungunya, Zika and filariasis. The biting nuisance of Aedes mosquitoes also impacts local tourism, affecting the sustainability of island economies. Mosquito saliva is injected during the biting process, and the response triggered by the human immune system to proteins contained in mosquito saliva was shown to be a relevant biomarker of exposure to mosquito bites. Using this approach, we have developed an immuno-epidemiological tool to investigate the exposure of people to the bites of Aedes aegypti and Aedes polynesiensis, two significant mosquito vectors of infectious diseases in French Polynesia and other island countries and territories in the Pacific. This novel tool proved specific and reliable. It will improve the assessment of disease transmission risk and be useful for measuring the efficacy of both conventional and innovative vector control strategies.

Background Elimination of lymphatic filariasis (LF) in Samoa continues to be challenging despite multiple annual mass drug campaigns aimed at stopping transmission by reducing the prevalence and density of microfilaraemia. The persistence of transmission may be partly related to the highly efficient Aedes vectors. The assessment of pathogen transmission by mosquito vectors and of vector control relies on the ability to capture mosquitoes efficiently. The aims of this study are to compare trapping methods to capture LF-infected mosquitoes and determine the role in transmission of the species of Aedes mosquitoes in the area. Methods Fasitoo-Tai village was the chosen site because of persistent transmission despite annual mass drug administration. Sampling methods included BioGents Sentinel (BGS) trap, human-baited collections (HBC) and the Centers for Disease Control (CDC) trap. BGS and CDC traps were baited with BG-lure, CO2, and/or octenol. Individual trap locations were geo-located and efficiency of sampling methods was evaluated using a randomized Latin-square design in two locations. Number of mosquitoes collected (male and female), as well as species for each trapping method were determined. Additionally, Ae. polynesiensis and Ae. (Finlaya) spp. females were pooled by trap method and analysed for filarial DNA. Infection prevalence was estimated using the PoolScreen software. Results The BGS trap with any type of bait collected more mosquitoes compared to both the CDC trap and the HBC. The BGS trap baited with BG-lure collected more mosquitoes than with CO2 and octenol. There were no significant differences between trapping methods in terms of proportions of infected females collected. The prevalence of filarial infection in Ae. polynesiensis and Ae. (Finlaya) spp. was estimated at 4.7 % and 0.67 % respectively. Conclusions This study supports the use of the BGS trap for research on and surveillance of the mosquito vectors of LF in Samoa. The BGS trap is a suitable and safer alternative to HBC for sampling Ae. polynesiensis and Ae. (Finlaya) spp., which continue to be the predominant vectors of LF. Of concern was the high prevalence of LF in mosquitoes despite a recent mass drug administration programme. This highlights the urgency for updated policies concerning filariasis elimination in Samoa.

Semi-annual mass DEC chemotherapy combined with vector control at the beginning of the programme, has been administered on the remote island of Maupiti (French Polynesia) since 1955 (except two periods in 1960-67 and 1970-74). The results of two surveys in 1985 and 1989, reporting 0% microfilaraemia, led to the hope that the eradication of lymphatic filariasis had been achieved. We combined parasitological criteria (microfilaraemia by membrane filtration), immunological (antigenaemia and serum levels of specific IgG antibodies) and molecular (PCR-based evaluation of infection in mosquitoes) techniques and found only good control of the parasite: We found residual microfilaraemia in 0.4% of the sample (mean level in carriers: 101.2 mf/ml), antigenaemia in 4.6% (mean level in positive persons: 714.4 units/ml) and specific IgG in 21.6% (including in one very young child). In addition, an infection rate of 1.4% was calculated in the Aedes polynesiensis vector population. These data, obtained in 1997 just before a hurricane, were partially confirmed in 1999 (0.1% of infection rate in the vector). Together with the possibility of some resistance to DEC, various epidemiological factors critical for the eradication of lymphatic filariasis are discussed.

The full-length cDNA sequence encoding Brugia malayi L3 paramyosin has been isolated by immunoscreening a cDNA library with a mouse antiserum raised against Wuchereria bancrofti L3 infective larvae. A recombinant truncated form of paramyosin was expressed as a glutathione S -transferase fusion protein and used to evaluate humoral responses of adults from a W. bancrofti -endemic area in French Polynesia according to their parasitological status. Immunoglobulin G4 (IgG4) preferentially bound to paramyosin in W. bancrofti -parasitized individuals, in contrast to unparasitized individuals, who harbored neither microfilaria nor Og4C3 adult worm circulating antigen. Reduction of the anti-paramyosin IgG4 titer following combined chemotherapy with diethylcarbamazine and ivermectin was significantly correlated with a reduction in the adult worm burden. This indicates that the presence of paramyosin-reactive IgG4 is associated with the presence of parasites and that reduction can be used as an immunological marker for W. bancrofti clearance.

Circulating filarial antigen (CFA), determined with Og4C3 ELISA, is a marker of Wuchereria bancrofti adult worm infection. The reduction of CFA over 2 years was determined in 185 microfilaremic and 111 amicrofilaremic but CFA+ adults given an annual dose of either diethylcarbamazine (DEC) or ivermectin or the two combined. Reduction of CFA level was good with DEC but weak with ivermectin and followed the same pattern in amicrofilaremic and microfilaremic groups. Combinations and DEC alone had a similar impact on CFA level. CFA clearance was observed in amicrofilaremic but not in microfilaremic persons in all DEC-containing treatments. However, the highest clearance rate was observed in persons treated with DEC at 6 mg/kg combined with ivermectin. Continuous reduction of CFA level after repeated treatments shows that elimination of W. bancrofti infection, monitored by CFA clearance, might be achieved within a few years with annual treatments of DEC combined with ivermectin.

Background Effective diagnostic tools are necessary to monitor and evaluate interruption of Lymphatic Filariasis (LF) transmission. Accurate detection of Wuchereria bancrofti (Wb) microfilaria (mf) is essential to measure the impact of community treatment programmes. PCR-based assays are specific, highly sensitive tools allowing the detection of Wuchereria bancrofti DNA in human blood samples. However, current protocols describing the pool screening approach, use samples of less than 60 μl of blood, which limits the sensitivity of the pool-screen PCR assay. The purpose of this study was to improve the pool-screen PCR protocol to enhance its sensitivity and usefulness for population scale studies. Findings DNA extractions were performed with the DNeasy kit, the PCR with the Wb LDR primers and the SYBR-Green dye. Improvements of our pool-screen real-time PCR (qPCR) assay allowed the detection of as little as one Wb microfilaria diluted in a pool of at least 12 blood samples of 60 μl each. Using this assay, mf burdens can be predicted using a standard curve derived from mf spiked dried blood samples. The sensitivity achieved is equivalent to the detection of a single LF positive individual carrying a mf burden as low as 18 mf/ml, in a pool of blood samples from at least 12 individuals. Conclusions Due to its sensitivity, rapidity and cost-effectiveness, we suggest this qPCR pool-screening assay could be used as a diagnostic tool for population- scale filariasis elimination monitoring and evaluation.

A polymerase chain reaction (PCR) assay based on a highly repeated deoxyribonucleic acid (DNA) sequence found in Wuchereria bancrofti (the Ssp I repeat) has been developed to address the shortcomings of traditional diagnostic methods. In this field study in a W. bancrofti endemic region of French Polynesia, 373 human blood samples were collected and 100 μL of blood were screened by the Ssp I PCR assay and 1 μL by membrane filtration. The Ssp I PCR assay detected 99 of 113 blood samples in which microfilariae had been detected by filtration (sensitivity of 88%) with a specificity of 100%. All the samples missed by the Ssp I PCR assay had less than 8 microfilariae per mL of blood. To evaluate the efficacy of screening larger blood samples by PCR, both 100 μL and 500 μL samples from 50 patients with very low-level microfilaraemia were screened by the Ssp I PCR assay; the sensitivity increased from 60% to 84% when using the larger volume of blood. Finally, an enzyme-linked immunosorbent assay-based version of the Ssp I PCR assay was used to screen blood from 12 patients following treatment with diethylcarbamazine, ivermectin, or both. These results showed that the PCR assay closely paralleled the presence or absence of microfilariae in the blood and that no increase in the DNA level was seen immediately following drug treatment.

The age-specific patterns of microfilaremia, Og4C3 antigenemia, anti-Brugia malayi IgG and IgG4 were assessed in 3 villages of low, medium and high transmission level for Wuchereria bancrofti filariasis. The prevalence rates for each of the 4 markers were clearly age dependent and their patterns strongly associated with the transmission level. The antigenemia prevalence rate was consistently higher than the microfilaremia prevalence rate, in all age groups. The prevalences of anti-B. malayi IgG and IgG4 responses were very similar and much higher than those of microfilaremia or antigenemia. Antibody responses reached the plateau at an earlier age and at a higher prevalence with increased intensity of transmission. For all the markers, the prevalence rates were significantly higher in males than in females.

Background Lymphatic filariasis (LF) is a globally significant disease, with 1.3 billion persons in 83 countries at risk. A coordinated effort of administering annual macrofilaricidal prophylactics to the entire at-risk population has succeeded in impacting and eliminating LF transmission in multiple regions. However, some areas in the South Pacific are predicted to persist as transmission sites, due in part to the biology of the mosquito vector, which has led to a call for additional tools to augment drug treatments. Autocidal strategies against mosquitoes are resurging in the effort against invasive mosquitoes and vector borne disease, with examples that include field trials of genetically modified mosquitoes and Wolbachia population replacement. However, critical questions must be addressed in anticipation of full field trials, including assessments of field competitiveness of transfected males and the risk of unintended population replacement. Methodology/Principal Findings We report the outcome of field experiments testing a strategy that employs Wolbachia as a biopesticide. The strategy is based upon Wolbachia-induced conditional sterility, known as cytoplasmic incompatibility, and the repeated release of incompatible males to suppress a population. A criticism of the Wolbachia biopesticide approach is that unintended female release or horizontal Wolbachia transmission can result in population replacement instead of suppression. We present the outcome of laboratory and field experiments assessing the competitiveness of transfected males and their ability to transmit Wolbachia via horizontal transmission. Conclusions/Significance The results demonstrate that Wolbachia-transfected Aedes polynesiensis males are competitive under field conditions during a thirty-week open release period, as indicated by mark, release, recapture and brood-hatch failure among females at the release site. Experiments demonstrate the males to be ‘dead end hosts’ for Wolbachia and that methods were adequate to prevent population replacement at the field site. The findings encourage the continued development and extension of a Wolbachia autocidal approach to additional medically important mosquito species., Author Summary Additional tools are required to mitigate mosquito borne disease in the South Pacific, including human lymphatic filariasis (LF). Wolbachia are obligate intracellular bacteria that occur in a majority of insect species and that cause a form of conditional sterility in mosquitoes. Prior work demonstrates that male Aedes polynesiensis mosquitoes, which are artificially infected with Wolbachia (i.e., transinfected) can effectively sterilize wild type females in the laboratory, suggesting the potential applied use of Wolbachia as a pesticide for this medically important mosquito. As a critical intermediate step toward the development of the Wolbachia pesticide approach, we report on the field competitiveness of transinfected males and the risk of accidental horizontal transmission of Wolbachia from transinfected males. The outcome of laboratory cage trials and a thirty-week open release field trial provide evidence against horizontal transmission of Wolbachia from the transinfected males. Additionally, the field trial provides evidence for the competitiveness of transinfected males for indigenous female mates, as indicated by the failure of brood hatch and a resulting population level impact. No residual Wolbachia was detected in the targeted population during or after the male releases, showing released males to be ‘dead end hosts’ for Wolbachia. We discuss the results in relation to a disease control approach that integrates vector control with existing measures against LF.

Background Entomological methods may provide important tools for monitoring the transmission of filariasis in French Polynesia. In order to standardize our PCR method and refine our protocol to assess filarial infection levels in mosquitoes, we compared dissection of the vector, Aedes polynesiensis, with the poolscreening polymerase chain reaction (PS-PCR) assay. Methods (1) Mosquitoes were collected in human landing catches in five areas in Moorea island, French Polynesia. (2) A fraction of the captured mosquitoes was dissected for Wuchereria bancrofti larvae. (3) Laboratory-reared mosquitoes (uninfected as well as experimentally infected ones) were repeatedly tested to optimize a PS-PCR protocol (DNA extracts from 1–50 pooled mosquitoes were tested with an internal standardized system and primers specific for the Ssp1 repeat sequence. PCR products were analysed by gel electrophoresis). (4) Another fraction of the captured mosquitoes was assayed by PS-PCR according the optimized protocol. Results The prevalence of field-mosquito infection with W. bancrofti ranged from 1 to 8 % by dissection (L1–L3) and point estimates of infection prevalence, as assayed by PS-PCR, ranged from 0.4 to 3.7 %. There was a moderately strong correlation between larval infection rates as determined by dissection and PCR. Discussion Our results suggest that the PS-PCR assay is specific and highly sensitive for detecting parasite DNA. We obtained similar although not identical results with dissections of mosquitoes. PS-PCR appears to be adequate for testing large numbers of mosquitoes in the context of filariasis elimination programs. The role and advantages of using entomologic methods to monitor filariasis programs are discussed.

Despite control programs based on mass drug administration (MDA) of microfilaricidal compounds, Bancroftian lymphatic filariasis remains a problem in French Polynesia. For an alternative strategy to MDA, we investigated the potential role of Wolbachia to control filarial transmission. Wolbachia are intracellular alpha-proteobacteria endosymbionts that infect a broad range of insects and nematodes. These bacteria have a suspected role in the pathogenesis of filariasis. They also may be useful in mosquito control through cytoplasmic incompatibility. To detect and characterize these bacteria in the filarial and mosquito-vectors in French Polynesia, a survey was conducted on field-collected mosquitoes and microfilariae from infected people. Samples were analyzed by a polymerase chain reaction and gene sequencing. The results indicate that these bacteria are widespread. Sequence analysis of the wsp and ftsZ genes positioned the Aedes polynesiensis Wolbachia in cluster A and Wuchereria bancrofti var. pacifica Wolbachia in cluster D. The implications for possible improved treatment and vector control are discussed.

PCR has recently been studied as a promising tool for monitoring the progress of efforts to eliminate lymphatic filariasis. PCR can be used to test concurrently at least 30 pools, with as many as 40 mosquitoes in each pool, for the presence of filarial larvae. The SspI PCR assay for the detection of Wuchereria bancrofti DNA in pools of mosquitoes has been used since 1994 in a variety of laboratories worldwide. During that time, the original assay has been modified in these different laboratories and no standardized assay currently exists. In an effort to standardize and improve the assay, a meeting was held on 15-16 November 2001, at Emory University in Atlanta, with representatives from most of the laboratories currently using the assay. The first round of testing was designed to test the four most promising methods for DNA extraction from pools of mosquitoes. Two of the four methods stood out as clearly the best and these will be now optimised and evaluated in two further rounds of testing.

Successful mass drug administration (MDA) campaigns have brought several countries near the point of Lymphatic Filariasis (LF) elimination. A diagnostic tool is needed to determine when the prevalence levels have decreased to a point that MDA campaigns can be discontinued without the threat of recrudescence. A six-country study was conducted assessing the performance of seven diagnostic tests, including tests for microfilariae (blood smear, PCR), parasite antigen (ICT, Og4C3) and antifilarial antibody (Bm14, PanLF, Urine SXP). One community survey and one school survey were performed in each country. A total of 8,513 people from the six countries participated in the study, 6,443 through community surveys and 2,070 through school surveys. Specimens from these participants were used to conduct 49,585 diagnostic tests. Each test was seen to have both positive and negative attributes, but overall, the ICT test was found to be 76% sensitive at detecting microfilaremia and 93% specific at identifying individuals negative for both microfilariae and antifilarial antibody; the Og4C3 test was 87% sensitive and 95% specific. We conclude, however, that the ICT should be the primary tool recommended for decision-making about stopping MDAs. As a point-of-care diagnostic, the ICT is relatively inexpensive, requires no laboratory equipment, has satisfactory sensitivity and specificity and can be processed in 10 minutes—qualities consistent with programmatic use. Og4C3 provides a satisfactory laboratory-based diagnostic alternative., Author Summary Lymphatic filariasis (LF), a mosquito-borne parasitic disease, is a candidate for elimination largely because of the success of mass drug administration (MDA) campaigns, in which entire at-risk populations are given a once-yearly regimen of single-dose treatment with two medications. As a result, a diagnostic tool is needed to determine when the prevalence of LF has fallen below the threshold for sustained transmission so that MDA programs can be stopped. To determine the best diagnostic tool available, a multi-country study was conducted to assess the performance of seven diagnostic tests on a panel of patient specimens. The selection of the most effective diagnostic test was based on an evaluation of each test's accuracy, technical requirements, programmatic feasibility and reliability, as well as confidence in test performance. This study found advantages and disadvantages to each test. Based on the data and experiences it was determined that the ICT test, a point-of-care rapid card test, is the preferred diagnostic tool for use in defining the end-point of MDA, although the Og4C3 test provides a suitable laboratory-based alternative.

Enzyme-linked immunosorbent assays (Elisas) for anti-Brugia malayi immunoglobulin (Ig) G and IgG4 were evaluated on sera from 1561 subjects in French Polynesia for the serodiagnosis of Wuchereria bancrofti filariasis, compared with the test for Onchocerca gibsoni circulating antigen (Og4C3) as a ‘gold standard’. The sensitivity of the elisa -IgG and elisa -IgG4 assays was 90.8% and 94.5%, and the specificity was 45.9% and 50.7%. The positive predictive values were 41% and 45% respectively for an antigen prevalence rate of 30%. Thus antibody prevalences exceeded by two-fold the antigen prevalence, which itself exceeded by two-fold the prevalence of microfilaraemia.

In Wuchereria bancrofti transmission areas, three groups of individuals have been identified, according to the presence or absence of microfilariae or adult worm derived molecules in the blood compartment. These groups likely reflect individuals with different permissivity/resistance to the complete development of W. bancrofti cycle. The profile of filarial-specific immunoglobulins was analysed in W. bancrofti-exposed individuals in French Polynesia, according to the presence or absence of microfilariae (Mf) and adult worms, measured by Og4C3 circulating antigen. Individuals harbouring adult worms, have higher filarial-specific IgG4 but lower IgG3 and IgE levels, than adult worm-free individuals, independently of the presence of Mf. Low filarial-specific IgG1 and IgG2 levels were associated with the presence of Mf but independent of the presence/absence of adult worms. The filarial antibody responses were associated with the parasitological status of individuals but not with clinical symptoms such as hydroceles or limb lymphangitis or elephantiasis. The reduction of filarial-specific immunoglobulin levels was higher after treatment with diethylcarbamazine, than ivermectin, which likely reflects the better effect of the former on W. bancrofti adult worms. However, reduction of antibody levels was also observed in Mf-and adult worm-negative individuals. This could be due to the overall reduction of W. bancrofti transmission in the island where this study took place.

Og4C3 circulating filarial antigen was detected in the sera of 94.5% (259/274) of microfilaremic patients, 32% (239/751) of persons with presumption of filariasis, and 23% (11/48) of chronic filariasis patients. The antigen level was correlated with the microfilariae (Mf) density and patient age (P < .01). It remained stable in patients treated with microfilaricidal drugs. Og4C3 antigen, undetectable in Mf culture media, was demonstrated to be a rare somatic Mf antigen. It appears to be an excreted or secreted antigen from adult filaria. It could be used as a marker of infection and an indicator of adult worm burden.