Detection of Wuchereria bancrofti L3 larvae in mosquitoes: a reverse transcriptase PCR assay evaluating infection and infectivity.

BACKGROUND: Detection of filarial DNA in mosquitoes by PCR cannot differentiate infective mosquitoes from infected mosquitoes. In order to evaluate transmission risk an assay is needed that can specifically detect infective L3 stage parasites. We now report the development of an assay that specifically detects the infective stage of Wuchereria bancrofti in mosquitoes. The assay detects an L3-activated mRNA transcript by reverse-transcriptase PCR (RT-PCR). METHODOLOGY/PRINCIPAL FINDINGS: W. bancrofti cuticle-related genes were selected using bioinformatics and screened as potential diagnostic target genes for L3 detection in mosquitoes. Expression profiles were determined using RT-PCR on RNA isolated from mosquitoes collected daily across a two-week period after feeding on infected blood. Conventional multiplex RT-PCR and real-time multiplex RT-PCR assays were developed using an L3-activated cuticlin transcript for L3 detection and a constitutively expressed transcript, tph-1, for 'any-stage' detection. CONCLUSIONS/SIGNIFICANCE: This assay can be used to simultaneously detect W. bancrofti infective stage larvae and 'any-stage' larvae in pooled vector mosquitoes. This test may be useful as a tool for assessing changes in transmission potential in the context of filariasis elimination programs.

A critical appraisal of molecular xenomonitoring as a tool for assessing progress toward elimination of Lymphatic Filariasis.

We used molecular xenomonitoring (MX, detection of filarial DNA in mosquitoes) to evaluate the impact of mass drug administration (MDA) in sentinel locations in Egypt with high (11.5%) and low (4.1%) baseline microfilaria prevalence rates. Blood-fed Culex pipiens were pooled by household and tested for Wuchereria bancrofti DNA by PCR. There was no significant relationship between the infection status of household residents and parasite DNA status of mosquitoes from the same houses. After 5 MDA rounds, parasite DNA rates in mosquitoes in high- and low-prevalence areas were reduced by 93.8% and 100% to 0.19% (95% CI: 0.076-0.382%) and 0% (95% CI: 0-0.045%), respectively. These changes were consistent with decreases in microfilaria prevalence rates in these sites; they provide insight regarding the minimal mosquito DNA rates necessary for sustained transmission of filariasis in Egypt. We conclude that MX is a powerful tool for monitoring the impact of MDA on filariasis endemicity and transmission.

The effect of compliance on the impact of mass drug administration for elimination of lymphatic filariasis in Egypt.

We studied effects of compliance on the impact of mass drug administration (MDA) with diethylcarbamazine and albendazole for lymphatic filariasis (LF) in an Egyptian village. Baseline microfilaremia (mf) and filarial antigenemia rates were 11.5% and 19.0%, respectively. The MDA compliance rates were excellent (> 85%). However, individual compliance was highly variable; 7.4% of those surveyed after five rounds of MDA denied having ever taken the medications and 52.4% reported that they had taken all five doses. The mf and antigenemia rates were 0.2% and 2.7% in those who reported five doses of MDA and 8.3% and 13.8% in those who reported zero doses. There was no significant difference in residual infection rates among those who had taken two or more doses. These results underscore the importance of compliance for LF elimination programs based on MDA and suggest that two ingested doses of MDA are as effective as five doses for reducing filariasis infection rates.

A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes.

We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.

Effect of yearly mass drug administration with diethylcarbamazine and albendazole on bancroftian filariasis in Egypt: a comprehensive assessment.

BACKGROUND: Egypt was one of the first countries to implement a national programme to eliminate lymphatic filariasis based on WHO's strategy of repeated rounds of mass drug administration (MDA) with diethylcarbamazine and albendazole (target population, 2.5 million in 181 localities). We assessed the effect of five yearly rounds of MDA on filariasis in four sentinel villages in Egypt. METHODS: We studied two areas with different infection rates before MDA: the Qalubyia study area had a low infection rate because of previous treatment with diethylcarbamazine; this was typical of most filariasis-endemic villages in Egypt before MDA. The Giza study area had a high baseline infection rate. We undertook repeated surveys in villages for treatment compliance and tests for microfilaraemia and circulating filarial antigenaemia, antibodies to filarial antigen Bm14 in schoolchildren, and infections in indoor-resting mosquitoes (assessed by PCR). FINDINGS: MDA compliance rates were excellent (>80%). In Giza after MDA, prevalence rates of microfilaraemia and circulating filarial antigenaemia fell from 11.5% to 1.2%, and from 19.0% to 4.8%, respectively (p<0.0001). Corresponding rates in Qalubyia fell from 3.1% to 0% and 13.6% to 3.1%, respectively (p<0.0001). Rates of antifilarial antibody and circulating filarial antigenaemia in schoolchildren (aged about 7-8 years), fell from 18.3% to 0.2% (p<0.0001) and from 10.0% to 0.4% (p<0.0001) in Giza, respectively, and from 1.7% to 0% and 1.7% to 0% (both p=0.13) in Qalubyia, respectively. Mosquito infection rates fell from 3.07% (95% CI 2.38-3.88) to 0.19% (0.08-0.38) in Giza and from 4.37% (3.07-5.99) to 0% (0-0.05) in Qalubyia. INTERPRETATION: MDA greatly affects variables related to infection (microfilaraemia and circulating filarial antigenaemia prevalence rates) and transmission (antifilarial antibodies in young children and mosquito infection rates). Our results suggest that after five rounds of MDA filariasis is likely to have been eliminated in most endemic localities in Egypt.

Effects of combined diethylcarbamazine and albendazole treatment of bancroftian filariasis on parasite uptake and development in Culex pipiens L.

We studied effects of combined diethylcarbamazine (DEC) and albendazole (ALB) treatment on Wuchereria bancrofti microfilaria (MF) uptake and development of infective larvae (L3) in Culex pipiens. Consenting Egyptian adults with microfilaremia (MF > 300/mL) were treated with one or seven daily doses of DEC/ALB. Laboratory-reared mosquitoes were fed on subjects before and after treatment. MF uptake and infectivity (assessed by mosquito dissection) were reduced by 89.6% and 82.9%, respectively, 12 months after single-dose treatment and by 96.2% and 99.7%, respectively, after multi-dose treatment. The L3:mosquito ratio decreased by 88% to 0.082 after single-dose treatment and by 99.8% to 0.001 after multi-dose treatment. If high coverage rates can be achieved for several annual cycles, mass drug administration (MDA) with DEC/ALB has the potential to decrease transmission to unsustainable levels and eliminate filariasis in populations. Multi-dose MDA (especially in the first year) might interrupt transmission with fewer cycles than single-dose treatment.

A randomized clinical trial comparing single- and multi-dose combination therapy with diethylcarbamazine and albendazole for treatment of bancroftian filariasis.

The Global Program for Elimination of Lymphatic Filariasis calls for mass drug administration for endemic populations outside of sub-Saharan Africa with a single dose of diethylcarbamazine (DEC) and albendazole (Alb) annually for 4-6 years. Single-dose DEC/Alb dramatically reduces blood microfilaria (MF) counts, but most treated subjects fail to completely clear MF after a single dose. A more effective regimen might reduce the number of years required for elimination programs. We performed a randomized clinical trial in Egyptian adults with asymptomatic microfilaremia to compare treatment with seven daily doses of oral DEC (6 mg/kg) and Alb (400 mg) with a single dose of the same combination. We also studied the effect of re-treatment with single-dose DEC/Alb 12 months after the first treatment course. Multi-dose DEC/Alb was significantly more effective than single-dose therapy for reducing and clearing microfilaremia (mean reduction in MF/ml relative to pretreatment counts at 12 months, 99.6% versus 85.7%, with complete clearance in 75% versus 23.1%). The two regimens had similar activity against adult filarial worms, as indicated by serial ultrasound assessments. Neither regimen resulted in complete clearance of filarial antigenemia. There was no difference in adverse events, which were mild to moderate. Blood microfilaria and parasite antigen clearance rates increased following re-treatment. Multi-dose DEC/Alb may be a useful option for filariasis elimination programs, especially in the first year (when enthusiasm for mass drug administration and coverage rates are high), to quickly reduce community MF loads and transmission rates.

Test strip detection of Wuchereria bancrofti amplified DNA in wild-caught Culex pipiens and estimation of infection rate by a PoolScreen algorithm.

Bancroftian filariasis is targeted for elimination in the Nile Delta of Egypt. Improved simple methods are needed for monitoring Wuchereria bancrofti infection in the mosquito vector and thereby the success of elimination programmes. We evaluated the performance of the SspI-PCR assay combined with a DNA Detection Test StripTM method and used the PoolScreen algorithm method for estimating mosquito infection rates. A total of 769 indoor-resting Culex pipiens were captured in 79 randomly selected houses from a filaria-endemic village in the Nile Delta of Egypt (24.4% antigenaemia and 8.6% microfilaraemia). Collected mosquitoes were pooled by house, and assayed by the SspI-PCR. Amplified parasite DNA was detected by both electrophoresis of agarose gel stained with ethidium bromide (EtBr) and by test strips. PCR based on EtBr and test strip methods identified 43 (54.4%) and 45 (56.9%) houses, respectively, as being filaria positive. The minimum mosquito infection rate, assuming one infected female/pool was 6.85% by the PCR test strips. Mosquito infection rate calculated by the PoolScreen2 algorithm software amounted to 8.1% [95% confidence interval 5.85, 10.47]. Because it is faster and safer, the PCR test strip is a practical tool, especially when combined with the PoolScreen algorithm method, for xenomonitoring the success of elimination programmes.

Culex pipiens pipiens: characterization of immune peptides and the influence of immune activation on development of Wuchereria bancrofti.

Stimulating or augmenting the innate immune response of insect vectors has been shown to impede or disrupt the development and transmission of eukaryotic pathogens; however, the majority of such studies have utilized model systems and not natural parasite-vector systems. The Culex pipiens complex of mosquitoes functions as a primary urban vector of Wuchereria bancrofti, a causative agent of lymphatic filariasis. To test the effects of immune activation on this vector-parasite interaction, Culex pipiens pipiens from the filariasis-endemic Nile Delta were subjected to bacteria inoculation and subsequently fed a blood meal containing W. bancrofti. No difference was seen between parasite development in these mosquitoes as compared to non-inoculated controls. A set of expressed sequence tags from blood-fed midgut and bacteria-inoculated Cx. p. pipiens reveals transcripts for the immune peptides cecropin, gambicin and defensin--all of which have been reported to have antiparasitic effects. Sequences and transcriptional profiles for these peptides are reported. The discrepancy between these results and those reported for the model parasite, Brugia malayi, in the mosquito Aedes aegypti are discussed.

Relationships between Wuchereria bancrofti microfilaria counts in human blood and parasite uptake and maturation in Culex pipiens, with observations on the effects of diethylcarbamazine treatment on these parameters.

This study examined relationships between blood microfilaria (MF) counts and parasite uptake and maturation in Culex pipiens fed on Egyptian volunteers with bancroftian filariasis. Uptake of MF and production of infective larvae (L3) were more closely correlated with MF counts in finger prick blood than in venous blood. Only a minority of ingested MF developed into L3. Few MF were ingested, and very few L3 were produced by mosquitoes that fed on infected subjects who were amicrofilaremic by 50 microL thick blood smear; the contribution of such carriers to filariasis transmission in Egypt is probably negligible. These results suggest that filariasis elimination programs should aim to achieve MF smear rates of zero. Single-dose diethylcarbamazine therapy reduced MF counts by 87.9% 6-7 months after treatment; similar reductions were observed for MF uptake, MF/mosquito, infectivity, and L3/mosquito. Thus, single-dose diethylcarbamazine had a major impact on MF ingestion and L3 production by mosquitoes.

The impact of single-dose diethylcarbamazine treatment of bancroftian filariasis in a low-endemicity setting in Egypt.

This study was designed to evaluate the effect of a single dose of diethylcarbamazine (DEC, 6 mg/kg) on Wuchereria bancrofti infections in a low-endemicity setting in Egypt (microfilaremia, or MF, 3.7%, median MF 34/mL). Subjects with MF or filarial antigenemia were treated and restudied 1 year later. Treatment with DEC dramatically reduced blood MF counts, with clearance in 69% of subjects. Treatment also reduced filarial antigen levels, but low clearance rates suggest that some adult worms survived treatment in most patients. Mass treatment was administered in one village; 27 months later, MF prevalence had decreased 84% (from 4.9% to 0.8%). These results show that single-dose DEC treatment can have a major effect on MF prevalence rates and levels in low-endemicity settings. Although the World Health Organization advocates repeated multidrug regimens for filariasis elimination, mass treatment with DEC alone may be sufficient to interrupt transmission in areas with low infection intensities and prevalence rates.