A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes

Background: Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript. Methodology/Principal Findings: Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayilife cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes. Conclusions/Significance: This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites

The Impact of Repeated Rounds of Mass Drug Administration with Diethylcarbamazine Plus Albendazole on Bancroftian Filariasis in Papua New Guinea

Lymphatic filariasis (LF) is a deforming and disabling disease that is caused by parasitic worms that are transmitted by mosquitoes. While a number of countries have initiated LF elimination programs based on mass drug administration (MDA), relatively little good information is available on the impact of MDA on filariasis prevalence and incidence rates in populations. This study assessed the impact of three rounds of MDA (with single doses of diethylcarbamazine and albendazole) on filariasis infection rates in villages in Papua New Guinea, which has the largest filariasis problem in the Pacific region. MDA dramatically reduced rates for all filariasis infection markers tested. These included microfilaremia (parasites in blood that are necessary for transmission of the infection), filarial antigenemia (a marker for adult worm infection), anti-filarial antibodies (which indicate infection or heavy exposure to the parasite), and parasites in mosquitoes that transmit the infection. In addition to curing existing infections, MDA also reduced new infection rates in the study population to very low levels. These results suggest that it should be possible to eliminate LF in Papua New Guinea if MDA can be effectively delivered to endemic populations

Detection of Wuchereria bancrofti L3 Larvae in Mosquitoes: A Reverse Transcriptase PCR Assay Evaluating Infection and Infectivity

Lymphatic filariasis is a disabling and disfiguring disease caused by a parasite that is transmitted by a mosquito. The life cycle of the parasite requires two hosts: the mosquito vector and the human host. Part of the developmental life cycle of the parasite occurs in the mosquito and the other part in the human host. The parasite develops through four stages in the mosquito, only the last of which is infectious to humans. The third larval stage (L3) is the infective stage that initiates human infections when infective mosquitoes bite humans. There is currently a global program attempting to eliminate this disease by administering drugs to affected communities with the goal of interrupting transmission of the parasite. The new diagnostic tool described in this paper uses molecular techniques to specifically detect the infective stage of the parasite in mosquitoes. Many mosquitoes can be tested at one time to assess the risk of ongoing transmission of filariasis in communities. In addition, this new L3-detection assay can simultaneously detect whether the mosquitoes contain ‘any-stage’ of the parasite. This provides information on infection rates in humans in the community. Both pieces of information can be used in assessing the progress of disease elimination efforts

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

Application of a Household-Based Molecular Xenomonitoring Strategy to Evaluate the Lymphatic Filariasis Elimination Program in Tamil Nadu, India

Lymphatic filariasis (LF) is one of the world’s foremost debilitating infectious diseases with nearly 800 million people at risk of infection. Given that LF is a mosquito-borne disease, the use of molecular xenomonitoring (MX) to detect parasite DNA/RNA in mosquitoes can serve as a valuable tool for LF monitoring and evaluation, particularly in Culexvector areas. We investigated using MX in a low-level prevalence district of Tamil Nadu, India by applying a household-based sampling strategy to determine trap location sites. Two independent mosquito samples were collected in each of a higher human infection hotspot area (sites with community microfilaria prevalence �1%) and across a larger evaluation area that also encompassed the hotspots. Pooled results showed mostly reproducible outcomes in both settings and a significant higher pool positivity in the hotspot area. A follow-up survey conducted two years later reconfirmed these findings while also showing a reduction in pool positivity and estimated prevalence of infection in mosquitoes in both settings. The utilization of a household-based sampling strategy for MX proved effective and should be further validated in wider epidemiological settings

A Reverse Transcriptase-PCR Assay for Detecting Filarial Infective Larvae in Mosquitoes

The Global Programme for the Elimination of Lymphatic Filariasis (GPELF) was launched in the year 1998 with the goal of eliminating lymphatic filariasis by 2020. As the success of mass drug administration (MDA) in the global program drives the rates of infection in endemic populations to very low levels, the development of new, highly sensitive methods are required for monitoring transmission by screening mosquitoes for the presence of L3 infective larvae. The current method of mosquito dissection to identify L3 larvae is laborious and insensitive and is not amenable to screening large numbers of mosquitoes. Existing molecular assays for the detection of filarial parasite DNA in mosquitoes are sensitive and can easily screen large numbers of vectors. However, current PCR-based methods cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. This paper reports the first development of a molecular L3-detection assay for a filarial parasite in mosquitoes based on RT-PCR detection of an L3-activated gene transcript. This strategy of detecting stage-specific messenger RNA from filarial parasites may also prove useful for detecting infective stages of other vector-borne pathogens

A Community-Based Study of Factors Associated with Continuing Transmission of Lymphatic Filariasis in Leogane, Haiti

Seven rounds of mass drug administration (MDA) have been administered in Leogane, Haiti, an area hyperendemic for lymphatic filariasis (LF). Sentinel site surveys showed that the prevalence of microfilaremia was reduced to <1% from levels as high as 15.5%, suggesting that transmission had been reduced. A separate 30-cluster survey of 2- to 4-year-old children was conducted to determine if MDA interrupted transmission. Antigen and antifilarial antibody prevalence were 14.3% and 19.7%, respectively. Follow-up surveys were done in 6 villages, including those selected for the cluster survey, to assess risk factors related to continued LF transmission and to pinpoint hotspots of transmission. One hundred houses were mapped in each village using GPS-enabled PDAs, and then 30 houses and 10 alternates were chosen for testing. All individuals in selected houses were asked to participate in a short survey about participation in MDA, history of residence in Leogane and general knowledge of LF. Survey teams returned to the houses at night to collect blood for antigen testing, microfilaremia and Bm14 antibody testing and collected mosquitoes from these communities in parallel. Antigen prevalence was highly variable among the 6 villages, with the highest being 38.2% (Dampus) and the lowest being 2.9% (Corail Lemaire); overall antigen prevalence was 18.5%. Initial cluster surveys of 2- to 4-year-old children were not related to community antigen prevalence. Nearest neighbor analysis found evidence of clustering of infection suggesting that LF infection was focal in distribution. Antigen prevalence among individuals who were systematically noncompliant with the MDAs, i.e. they had never participated, was significantly higher than among compliant individuals (p<0.05). A logistic regression model found that of the factors examined for association with infection, only noncompliance was significantly associated with infection. Thus, continuing transmission of LF seems to be linked to rates of systematic noncompliance