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Background: The elimination of lymphatic filariasis (LF) from Brazil by 2020 was not accomplished; however, this goal can be achieved in the upcoming years with the assistance of specific strategies. The surveillance of LF can be performed using molecular xenomonitoring (MX), a noninvasive method used to infer the presence of the parasite in the human population. Herein, São Luís (state of Maranhão) was the first city to be investigated to identify whether LF transmission in Brazil has been interrupted and if there were any new incursions., Methodology/principal Findings: Mosquitoes were collected by aspiration at 901 points distributed among 11 neighborhoods in São Luís with records of patients with microfilaremia. Pools of engorged or gravid Culex quinquefasciatus females were evaluated by WbCx duplex PCR with endogenous control for mosquitoes and target for W. bancrofti for determining the vector infection rate. Among the 10,428 collected mosquitoes, the most abundant species were C. quinquefasciatus (85%) and Aedes aegypti (12%). Significantly larger numbers of mosquitoes were collected from the neighborhoods of Areinha and Coreia (p<0.05). MX performed using PCR validated 705 pools of engorged or gravid females, fifteen of which were positive for Wuchereria bancrofti in two neighborhoods., Conclusions: The high density of engorged C. quinquefasciatus females per home, inadequate sanitation, and detection of W. bancrofti-infected mosquitoes in the city of São Luís represent a warning of the possible upsurge of LF, a disease that is still neglected; this underscores the need for the ostensive monitoring of LF in Brazil., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright: © 2023 Araújo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background & Objectives: An infective stage specific reverse transcriptase-polymerase chain reaction (RT-PCR) assay utilizing the abundant larval transcript-3 (Alt-3) gene of Wuchereria bancrofti was developed at ICMR-VCRC, Puducherry and found to be stage specific, and sensitive upon validation in the laboratory. This study was aimed at independently evaluating this assay for its utility as a monitoring/surveillance tool in the operational programme for elimination of lymphatic filariasis (LF) by four national research laboratories., Methods: Evaluation of the assay was carried out in a multi-centric mode in three phases. In phase I, a workshop was conducted to impart hands-on training to the scientists from the collaborating centres on the RT-PCR assay and in Phase II the assay was evaluated for specificity and sensitivity in detecting the infective (L 3 ) stage larvae of W. bancrofti in its vector, Culex quinquefasciatus, using 50 coded pooled samples. Phase III evaluation was done on wild-caught mosquito vectors from selected endemic areas of Assam and Bhubaneswar States and Andaman Nicobar islands., Results: Phase I data indicated that the assay was able to detect all the pools of mosquito samples contaning L 3 stage larvae of W. bancrofti as positive, even in the presence of other vector stages of the parasite indicating its stage specificity (100%). The assay was found highly sensitive (100%), detecting all the infected pools as positive and specific detecting all uninfected pools as negative. The results of phase II showed inter-laboratory variation. Phase III evaluation from all the centres suggested that the infectivity rate determined for pooled mosquitoes by the RT-PCR assay (0.5%) was comparable to that by dissection method (1.2%) (95% confidence interval overlaps)., Interpretation & Conclusions: Overall, the results from three of the four participating centres indicated that the assay is at least as sensitive and stage specific as the conventional mosquito dissection technique, and hence, may be useful as a xenomonitoring tool for Transmission Assessment Survey in Mass Drug Administration programmes for LF., Competing Interests: None

According to the World Health Organization, lymphatic filariasis (LF), a mosquito-borne neglected tropical disease (NTD), should be eliminated as a public health concern by the end of 2020. To this end, the goals of the Global Programme to Eliminate Lymphatic Filariasis (GPELF) include interrupting transmission through mass drug administration (MDA). After two decades, several countries have implemented MDA and are now ready to confirm whether transmission has been interrupted. The method for detecting the parasites in mosquito vectors known as xenomonitoring is a non-invasive tool for assessing the current transmission status of the filarial nematode Wuchereria bancrofti (which is responsible for 90% of cases) by their vectors. There are several methods available for detection of the worm in mosquito samples, such as dissection or polymerase chain reaction (PCR). However, most of these techniques still produce a considerable number of false-negative results. The present study describes a new duplex PCR protocol, which is an improvement on the traditional PCR methodology, enhanced by introducing the actin gene as an endogenous control gene. After adjusting the mosquito pool size, DNA extraction, and WbCx PCR duplex design, we achieved a reliable and sensitive molecular xenomonitoring protocol. This assay was able to eliminate 5% of false negative samples and detected less than one Wb larvae. This high sensitivity is particularly valuable after MDA, when prevalence declines. This new method could reduce the number of false-negative samples, which will enable us to improve our ability to generate accurate results and aid the monitoring strategies used by LF elimination programmes., (Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Lymphatic filariasis (LF) is targeted for elimination by the year 2020. As of 2017, 67 of the 72 endemic countries have implemented annual Mass Drug Administration (MDA) for interrupting LF transmission. Transmission Assessment Survey (TAS) is the recommended protocol to evaluate the impact of MDA and to decide when to stop MDA in an Evaluation Unit (EU, population ≤2 million). As the human infection levels go down with repeated MDA rounds, it becomes a challenge to select the appropriate survey methods to assess transmission interruption. This study validates a standard protocol for molecular xenomonitoring of infection in vectors (MX) at an EU as a complementary tool for TAS to stop MDA and its utility for post-MDA or post-validation surveillance., Methodology: The study was conducted in Cuddalore district, Tamil Nadu, India, which was found eligible for TAS after 15 annual rounds of MDA (4 with DEC alone and 11 with DEC plus albendazole). The district was divided into two EUs as per the TAS protocol and one EU was randomly selected for the study. A two-stage cluster design vector sampling, developed and validated at a sub-district level, was implemented in 30 randomly selected clusters in the EU. Female Culex quinquefasciatus were collected placing gravid traps overnight (1800-0600 hrs) inside the premises of systematically selected households. Pools of 20-25 blood-fed, semi-gravid and gravid Cx. quinquefasciatus were subjected to real-time quantitative PCR (polymerase chain reaction) assay for detecting Wuchereria bancrofti DNA. Pool infection rate (% of pools positive for W. bancrofti DNA), and the estimated prevalence of W. bancrofti DNA in mosquitoes and its 95% confidence interval were calculated. Additionally, in these 30 clusters, microfilaria (Mf) survey among individuals >5 years old was carried out. School-based TAS was conducted using Immunochromatographic Card Test (ICT) in the EU. Prepared itemized cost-menu for different cost components of MX survey and TAS were estimated and compared., Results: MX survey showed that only 11 (3.1%) of the 358 pools (8850 Cx.quinquefasciatus females), collected from 30 clusters, were found positive for W. bancrofti DNA. The estimated vector infection rate was 0.13% (95% CI: 0.07-0.22%), below the provisional threshold (0.25%) for transmission interruption. Of 1578 children tested in the TAS, only four (0.25%) were positive for filarial antigenemia, and it is well below the critical cut-off (18 positives) for stopping MDA. Among 9804 persons tested in the 30 clusters, only four were found positive for Mf (0.04%; 95% CI: 0.01-0.1%). The Mf-prevalence was <1% threshold for transmission interruption in humans. The estimated costs for TAS and MX per EU were $14,104 USD and $14,259 USD respectively., Conclusions: The result of MX protocol was in good agreement with that of TAS, providing evidence to recommend MX as a complementary tool to TAS to decide on stopping MDA. MX can also be a potential surveillance tool for post-MDA and post-validation phases as it could detect sites with residual infection and risk of resurgence of transmission. MX is economically feasible as its cost is slightly higher than that of TAS., Competing Interests: The authors have declared that no competing interests exist.

Background: Currently, molecular xenomonitoring efforts for lymphatic filariasis rely on PCR or real-time PCR-based detection of Brugia malayi, Brugia timori and Wuchereria bancrofti in mosquito vectors. Most commonly, extraction of DNA from mosquitoes is performed using silica column-based technologies. However, such extractions are both time consuming and costly, and the diagnostic testing which follows typically requires expensive thermal cyclers or real-time PCR instruments. These expenses present significant challenges for laboratories in many endemic areas. Accordingly, in such locations, there exists a need for inexpensive, equipment-minimizing diagnostic options that can be transported to the field and implemented in minimal resource settings. Here we present a novel diagnostic approach for molecular xenomonitoring of filarial parasites in mosquitoes that uses a rapid, NaOH-based DNA extraction methodology coupled with a portable, battery powered PCR platform and a test strip-based DNA detection assay. While the research reported here serves as a proof-of-concept for the backpack PCR methodology for the detection of filarial parasites in mosquitoes, the platform should be easily adaptable to the detection of W. bancrofti and other mosquito-transmitted pathogens., Methodology/principal Findings: Through comparisons with standard silica column-based DNA extraction techniques, we evaluated the performance of a rapid, NaOH-based methodology for the extraction of total DNA from pools of parasite-spiked vector mosquitoes. We also compared our novel test strip-based detection assay to real-time PCR and conventional PCR coupled with gel electrophoresis, and demonstrated that this method provides sensitive and genus-specific detection of parasite DNA from extracted mosquito pools. Finally, by comparing laboratory-based thermal cycling with a field-friendly miniaturized PCR approach, we have demonstrated the potential for the point-of-collection-based use of this entire diagnostic platform that is compact enough to fit into a small backpack., Conclusions/significance: Because this point-of-collection diagnostic platform eliminates reliance on expensive and bulky instrumentation without compromising sensitivity or specificity of detection, it provides an alternative to cost-prohibitive column-dependent DNA extractions that are typically coupled to detection methodologies requiring advanced laboratory infrastructure. In doing so, this field-ready system should increase the feasibility of molecular xenomonitoring within B. malayi-endemic locations. Of greater importance, this backpack PCR system also provides the proof-of-concept framework for the development of a parallel assay for the detection of W. bancrofti., Competing Interests: The authors have declared that no competing interests exist.

Background: Control of lymphatic filariasis (LF) in most of the sub-Saharan African countries is based on annual mass drug administration (MDA) using a combination of ivermectin and albendazole. Monitoring the impact of this intervention is crucial for measuring the success of the LF elimination programmes. This study assessed the status of LF infection in Rufiji district, southeastern Tanzania after twelve rounds of MDA., Methods: Community members aged between 10 and 79 years were examined for Wuchereria bancrofti circulating filarial antigens (CFA) using immunochromatographic test cards (ICTs) and antigen-positive individuals were screened for microfilaraemia. All study participants were examined for clinical manifestation of LF and interviewed for drug uptake during MDA rounds. Filarial mosquito vectors were collected indoor and outdoor and examined for infection with W. bancrofti using a microscope and quantitative real-time polymerase chain reaction (qPCR) techniques., Results: Out of 854 participants tested, nine (1.1%) were positive for CFA and one (0.1%) was found to be microfilaraemic. The prevalence of hydrocele and elephantiasis was 4.8% and 2.9%, respectively. Surveyed drug uptake rates were high, with 70.5% of the respondents reporting having swallowed the drugs in the 2014 MDA round (about seven months before this study). Further, 82.7% of the respondents reported having swallowed the drugs at least once since the inception of MDA programme in 2000. Of the 1054 filarial vectors caught indoors and dissected to detect W. bancrofti infection none was found to be infected. Moreover, analysis by qPCR of 1092 pools of gravid Culex quinquefasciatus collected outdoors resulted in an estimated infection rate of 0.1%. None of the filarial vectors tested with qPCR were found to be infective., Conclusion: Analysis of indices of LF infection in the human population and filarial mosquito vectors indicated a substantial decline in the prevalence of LF and other transmission indices, suggesting that local transmission was extremely low if occurring at all in the study areas. We, therefore, recommend a formal transmission assessment survey (TAS) to be conducted in the study areas to make an informed decision on whether Rufiji District satisfied WHO criteria for stopping MDA.

Introduction: Molecular xenomonitoring (MX)-pathogen detection in the mosquito rather than human-is a promising tool for lymphatic filariasis (LF) surveillance. In the Recife Metropolitan Region (RMR), the last LF focus in Brazil, Culex quinquefasciatus mosquitoes have been implicated in transmitting Wuchereria bancrofti parasites. This paper presents findings on the ideal mosquito collection method, mosquito dispersion, W. bancrofti infection in mosquitoes and W. bancrofti antigen in humans to aid MX development., Methods: Experiments occurred within two densely populated urban areas of Olinda, RMR, in July and August 2015. U.S. Centers for Disease Control and Prevention (CDC) light traps were compared to battery-powered aspirators as collection methods, and mosquito dispersion was measured by mosquito mark release recapture (MMRR). Female Cx. quinquefasciatus were tested by PCR for W. bancrofti infection, and study area residents were screened by rapid tests for W. bancrofti antigen., Results: Aspirators caught 2.6 times more total Cx. quinquefasciatus, including 38 times more blood-fed and 5 times more gravid stages, than CDC light traps. They also collected 123 times more Aedes aegypti. Of the 9,644 marked mosquitoes released, only ten (0.01%) were recaptured, nine of which were < 50m (34.8m median, 85.4m maximum) from the release point. Of 9,169 unmarked mosquitoes captured in the MMR, 38.3% were unfed, 48.8% blood-fed, 5.5% semi-gravid, and 7.3% gravid. PCR on 182 pools (1,556 mosquitoes) found no evidence of W. bancrofti infection in Cx. quinquefasciatus. Rapid tests on 110 of 111 eligible residents were all negative for W. bancrofti antigen., Conclusions: Aspirators were more effective than CDC light traps at capturing Ae. aegypti and all but unfed stages of Cx. quinquefasciatus. Female Cx. quinquefasciatus traveled short (< 86m) distances in this urban area. Lack of evidence for W. bancrofti infection in mosquitoes and antigen in humans in these fine-scale studies does not indicate that LF transmission has ceased in the RMR. A MX surveillance system should consider vector-specific collection methods, mosquito dispersion, and spatial scale but also local context, environmental factors such as sanitation, and host factors such as infection prevalence and treatment history., Competing Interests: The authors have declared that no competing interests exist.

Background: Careful monitoring for recrudescence of Wuchereria bancrofti infection is necessary in communities where mass drug administration (MDA) for the elimination of lymphatic filariasis (LF) as a public health problem has been stopped. During the post-MDA period, transmission assessment surveys (TAS) are recommended by the World Health Organization to monitor the presence of the parasite in humans. Molecular xenomonitoring (MX), a method by which parasite infection in the mosquito population is monitored, has also been proposed as a sensitive method to determine whether the parasite is still present in the human population. The aim of this study was to conduct an MX evaluation in two areas of Bangladesh, one previously endemic district that had stopped MDA (Panchagarh), and part of a non-endemic district (Gaibandha) that borders the district where transmission was most recently recorded., Methodology/principal Findings: Mosquitoes were systematically collected from 180 trap sites per district and mosquito pools were tested for W. bancrofti using real-time PCR. A total of 23,436 intact mosquitoes, representing 31 species, were collected from the two districts, of which 10,344 (41%) were Culex quinquefasciatus, the vector of W. bancrofti in Bangladesh. All of the 594 pools of Cx. quinquefasciatus tested by real-time PCR were negative for the presence of W. bancrofti DNA., Conclusions/significance: This study suggested the absence of W. bancrofti in these districts. MX could be a sensitive tool to confirm interruption of LF transmission in areas considered at higher risk of recrudescence, particularly in countries like Bangladesh where entomological and laboratory capacity to perform MX is available., Competing Interests: The authors have declared that no competing interests exist.

Introduction: Lymphatic filariasis (LF) is a chronic nematode infection transmitted by mosquitoes and in sub-Saharan Africa it is caused by Wuchereria bancrofti. The disease was targeted for global elimination by 2020 using repeated community-wide mass drug administration (MDA) distributed in endemic areas. However, recently, there has been a growing recognition of the potential role of including vector control as a supplement to MDA to achieve elimination goal. This study was carried out to determine mosquito abundance and transmission of bancroftian filariasis on Mafia Islands in Tanzania as a prerequisite for a search for appropriate vector control methods to complement the ongoing MDA campaign., Methods: Mosquitoes were collected indoor and outdoor using Centre for Disease Control (CDC) light and gravid traps, respectively. Collected mosquitoes were identified based on their differential morphological features and Anopheles gambiae complex and An. funestus group were further identified to their respective sibling species by polymerase chain reaction (PCR). Filarial mosquito vectors were then examined for infection with Wuchereria bancrofti by microscopy and PCR technique., Results: Overall, a total of 35,534 filarial mosquito vectors were collected, of which Anopheles gambiae complex, An. funestus group and Culex quinquefasciatus Say accounted for 1.3, 0.5 and 98.2%, respectively. Based on PCR identification, An. gambiae sensu stricto (s.s) and An. funestus s.s sibling species accounted for 88.3% and 99.1% of the identified members of the An. gambiae complex and An. funestus group, respectively. A total of 7,936 mosquitoes were examined for infection with W. bancrofti by microscopy. The infection and infectivity rates were 0.25% and 0.08%, respectively. Using pool screen PCR technique, analysis of 324 mosquito pools (each with 25 mosquitoes) resulted to an estimated infection rate of 1.7%., Conclusion: The study has shown that Cx. quinquefasciatus is the dominant mosquito on Mafia Islands. By using mosquito infectivity as proxy to human infection, the study indicates that W. bancrofti transmission is still ongoing on Mafia Islands after more than a decade of control activities based on MDA.

Background: The monitoring and evaluation of lymphatic filariasis (LF) has largely relied on the detection of antigenemia and antibodies in human populations. Molecular xenomonitoring (MX), the detection of parasite DNA/RNA in mosquitoes, may be an effective complementary method, particularly for detecting signals in low-level prevalence areas where Culex is the primary mosquito vector. This paper investigated the application of a household-based sampling method for MX in Tamil Nadu, India., Methods: MX surveys were conducted in 2010 in two evaluation units (EUs): 1) a hotspot area, defined as sites with community microfilaria prevalence ≥1%, and 2) a larger area that also encompassed the hotspots. Households were systematically selected using a sampling interval proportional to the number of households in the EU. Mosquito pools were collected and analyzed by real-time polymerase chain reaction (qPCR). Two independent samples were taken in each EU to assess reproducibility of results. Follow-up surveys were conducted in 2012., Results: In 2010, the proportion of positive pools in the hotspot EU was 49.3% compared to 23.4% in the overall EU. In 2012, pool positivity was significantly reduced to 24.3% and 6.5%, respectively (p<0.0001). Pool positivity based on independent samples taken from each EU in 2010 and 2012 were not significantly different except for the hotspot EU in 2012 (p = 0.009). The estimated prevalence of infection in mosquitoes, measured by PoolScreen, declined from 2.2-2.7% in 2010 to 0.6-1.2% in 2012 in the hotspot area and from 0.9-1.1% to 0.2-0.3% in the larger area., Conclusions: The household-based sampling strategy for MX led to mostly reproducible results and supported the observed LF infection trends found in humans. MX has the potential to be a cost-effective, non-invasive monitoring and evaluation tool with sensitive detection of infection signals in low prevalence settings. Further investigation and application of this sampling strategy for MX are recommended to support its adoption as a standardized method for global LF elimination programs.

Lymphatic filariasis (LF) is a debilitating disease caused by Wuchereria bancrofti, Brugia malayi and B. timori parasitic worms and transmitted by Culex, Anopheles, Aedes and Mansonia mosquitoes. Mass drug administration (MDA) to reduce the infection levels in the human population is the key component of LF elimination programs. However, the potential of the use of vector control is gaining recognition as a tool that can complement MDA. The method of monitoring the parasites in mosquito vectors is known as xenomonitoring. Monitoring of vectors for filarial larvae is an important assessment tool for LF elimination programs. Xenomonitoring has the advantage of giving a real-time estimate of disease, because the pre-patent period may take months after infection in humans. It is a non-invasive sensitive tool for assessing the presence of LF in endemic areas. The aim of this review is to discuss the prospects, challenges and needs of xenomonitoring as a public health tool, in the post-MDA evaluation activities of national LF elimination programs., (© The Author 2016. Published by Oxford University Press on behalf of Royal Society of Tropical Medicine and Hygiene. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Wuchereria bancrofti, Dirofilaria immitis, and Dirofilaria repens are filarial nematodes transmitted by mosquitoes belonging to Culex, Aedes, and Anopheles genera. Screening by vector dissection is a tiresome technique. We aimed to screen filarial parasites in their vectors by single and multiplex PCR and evaluate the usefulness of multiplex PCR as a rapid xenomonitoring and simultaneous differentiation tool, in area where 3 filarial parasites are coexisting. Female mosquitoes were collected from 7 localities in Assiut Governorate, were microscopically identified and divided into pools according to their species and collection site. Detection of W. bancrofti, D. immitis, and D. repens using single PCR was reached followed by multiplex PCR. Usefulness of multiplex PCR was evaluated by testing mosquito pools to know which genera and species are used by filarial parasites as a vector. An overall estimated rate of infection (ERI) in mosquitoes was 0.6%; the highest was Culex spp. (0.47%). W. bancrofti, D. immitis, and D. repens could be simultaneously and differentially detected in infected vectors by using multiplex PCR. Out of 100 mosquito pools, 8 were positive for W. bancrofti (ERI of 0.33%) and 3 pools each were positive for D. immitis and D. repens (ERI 0.12%). The technique showed 100% sensitivity and 98% specificity. El-Nikhila, El-Matiaa villages, and Sahel Seleem district in Assiut Governorate, Egypt are still endemic foci for filarial parasites. Multiplex PCR offers a reliable procedure for molecular xenomonitoring of filariasis within their respective vectors in endemic areas. Therefore, it is recommended for evaluation of mosquito infection after lymphatic filariasis eradication programs.

Background: Mass drug administration (MDA) programs have dramatically reduced lymphatic filariasis (LF) incidence in many areas around the globe, including American Samoa. As infection rates decline and MDA programs end, efficient and sensitive methods for detecting infections are needed to monitor for recrudescence. Molecular methods, collectively termed 'molecular xenomonitoring,' can identify parasite DNA or RNA in human blood-feeding mosquitoes. We tested mosquitoes trapped throughout the inhabited islands of American Samoa to identify areas of possible continuing LF transmission after completion of MDA., Methodology/principle Findings: Mosquitoes were collected using BG Sentinel traps from most of the villages on American Samoa's largest island, Tutuila, and all major villages on the smaller islands of Aunu'u, Ofu, Olosega, and Ta'u. Real-time PCR was used to detect Wuchereria bancrofti DNA in pools of ≤ 20 mosquitoes, and PoolScreen software was used to infer territory-wide prevalences of W. bancrofti DNA in the mosquitoes. Wuchereria bancrofti DNA was found in mosquitoes from 16 out of the 27 village areas sampled on Tutuila and Aunu'u islands but none of the five villages on the Manu'a islands of Ofu, Olosega, and Ta'u. The overall 95% confidence interval estimate for W. bancrofti DNA prevalence in the LF vector Ae. polynesiensis was 0.20-0.39%, and parasite DNA was also detected in pools of Culex quinquefasciatus, Aedes aegypti, and Aedes (Finlaya) spp., Conclusions/significance: Our results suggest low but widespread prevalence of LF on Tutuila and Aunu'u where 98% of the population resides, but not Ofu, Olosega, and Ta'u islands. Molecular xenomonitoring can help identify areas of possible LF transmission, but its use in the LF elimination program in American Samoa is limited by the need for more efficient mosquito collection methods and a better understanding of the relationship between prevalence of W. bancrofti DNA in mosquitoes and infection and transmission rates in humans.

Molecular xenomonitoring of filariasis is the detection of filarial DNA in mosquitoes by PCR and a useful tool for monitoring transmission. DNA extraction coupled with PCR allows rapid detection of the presence or absence of the filarial parasite in vector mosquitoes compared to traditional method of manual dissection of the mosquito and observation for parasite under a microscope. A Tris-EDTA (TE) buffer-based boiling method of DNA extraction developed earlier by us was employed and explored for its suitability in the detection of Wuchereria bancrofti DNA in pools of Culex quinquefasciatus mosquitoes in real-time PCR assay. In this preliminary study, 1,000 laboratory-reared C. quinquefasciatus were made into 40 pools, each containing 25 mosquitoes spiked with 2mf. DNA from the first 20 pools was extracted using Qiagen DNeasy blood and tissue kit as standard, and the other 20 pools were subjected to TE buffer-based boiling method of DNA extraction. When the results (Ct values) obtained for DNA samples extracted by TE buffer-based boiling method were compared with that of the DNA samples extracted by the standard Qiagen method, they were found to be highly concordant without any significant difference (P = 0.9). Besides being cost- and time-effective, this protocol was found useful in extracting filarial DNA from two other mosquito genus Aedes and Anopheles, species of which have been reported as important vectors of W. bancrofti in other endemic regions of the world. Thus, TE buffer-based boiling method of DNA extraction is useful for the high-throughput detection of W. bancrofti in vector mosquitoes.

Background: Zanzibar has a long history of lymphatic filariasis (LF) caused by the filarial parasite Wuchereria bancrofti, and transmitted by the mosquito Culex quinquefasciatus Say. The LF Programme in Zanzibar has successfully implemented mass drug administration (MDA) to interrupt transmission, and is now in the elimination phase. Monitoring infections in mosquitoes, and assessing the potential role of interventions such as vector control, is important in case the disease re-emerges as a public health problem. Here, we examine Culex mosquito species from the two main islands to detect W. bancrofti infection and to determine levels of susceptibility to the insecticides used for vector control., Methods: Culex mosquitoes collected during routine catches in Vitongoji, Pemba Island, and Makadara, Unguja Island were tested for W. bancrofti infection using PCR. Insecticide bioassays on Culex mosquitoes were performed to determine susceptibility to permethrin, deltamethrin, lambda-cyhalothrin, DDT and bendiocarb. Additional synergism assays with piperonyl butoxide (PBO) were used for lambda-cyhalothrin. Pyrosequencing was used to determine the kdr genotype and sequencing of the mitochondrial cytochrome oxidase I (mtCOI) subunit performed to identify ambiguous Culex species., Results: None of the wild-caught Culex mosquitoes analysed were found to be positive for W. bancrofti. High frequencies of resistance to all insecticides were found in Wete, Pemba Island, whereas Culex from the nearby site of Tibirinzi (Pemba) and in Kilimani, Unguja Island remained relatively susceptible. Species identification confirmed that mosquitoes from Wete were Culex quinquefasciatus. The majority of the Culex collected from Tibirinzi and all from Kilimani could not be identified to species by molecular assays. Two alternative kdr alleles, both resulting in a L1014F substitution were detected in Cx. quinquefasciatus from Wete with no homozygote susceptible detected. Metabolic resistance to pyrethroids was also implicated by PBO synergism assays., Conclusions: Results from the xenomonitoring are encouraging for the LF programme in Zanzibar. However, the high levels of pyrethroid resistance found in the principle LF vector in Pemba Island will need to be taken into consideration if vector control is to be implemented as part of the elimination programme.

We developed a single-step real-time fluorescence resonance energy transfer (FRET) multiplex polymerase chain reaction (PCR) merged with melting curve analysis for the detection of Wuchereria bancrofti and Brugia malayi DNA in blood-fed mosquitoes. Real-time FRET multiplex PCR is based on fluorescence melting curve analysis of a hybrid of amplicons generated from two families of repeated DNA elements: the 188 bp SspI repeated sequence, specific to W. bancrofti, and the 153-bp HhaI repeated sequence, specific to the genus Brugia and two pairs of specific fluorophore-labeled probes. Both W. bancrofti and B. malayi can be differentially detected in infected vectors by this process through their different fluorescence channel and melting temperatures. The assay could distinguish both human filarial DNAs in infected vectors from the DNAs of Dirofilaria immitis- and Plasmodium falciparum-infected human red blood cells and noninfected mosquitoes and human leukocytes. The technique showed 100% sensitivity and specificity and offers a rapid and reliable procedure for differentially identifying lymphatic filariasis. The introduced real-time FRET multiplex PCR can reduce labor time and reagent costs and is not prone to carry over contamination. The test can be used to screen mosquito vectors in endemic areas and therefore should be a useful diagnostic tool for the evaluation of infection rate of the mosquito populations and for xenomonitoring in the community after eradication programs such as the Global Program to Eliminate 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.

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.

Laboratory strain of the Malaysian Culex quinquefasciatus was susceptible to Wuchereria bancrofti. Thirty three percent of the Cx. quinquefasciatus that fed on W. bancrofti patient were infective after 12-14 days. There is a possibility for W. bancrofti to occur in the urban areas of the Malaysia in the near future.

A single step PCR method has been developed for the combined detection of the human filarial parasites, Brugia malayi and Wuchereria bancrofti. Parasites' DNA were isolated from filaria positive blood samples that were collected from endemic areas. The primers used were Hha1 and Ssp I, which amplified the DNA fragments of 322 bp and 188 bp specific to B. malayi and W. bancrofti, respectively. The sensitivity of the assay was tested with blood and mosquito samples having one W. bancrofti in a pool of 10 B. malayi. The assay was further evaluated on field collected blood and mosquito samples. Use of this assay as a diagnostic tool for the detection of filariasis being the most promising aspect of this study, offers scope for detection of both the parasites even at low levels of infection.

A LightCycler real-time polymerase chain reaction (PCR) assay was developed to detect Wuchereria bancrofti DNA in blood-fed mosquitoes. The assay is based on fluorescence melting curve analysis of the PCR product generated from a family of repeated DNA elements: the 182 bp SspI repeat, specific to the genus Wuchereria. According to the melting temperature, W. bancrofti infected-mosquitoes were differentiated from Brugia malayi-infected and non-infected mosquitoes as well as from genomic DNA of Dirofilaria immitis and human DNA. The method proved to be 100% sensitive in all W. bancrofti-infected mosquitoes. Melting curve analysis offers a rapid alternative for the specific detection of W. bancrofti in mosquitoes. It is very accurate and sensitive, allows a high throughput and can be performed on very small samples. The method therefore has great potential for application in epidemiological studies.

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.

A simple, rapid and inexpensive method for the extraction of DNA from filarial vector, Culex quinquefasciatus, useful in Ssp I PCR assay for xenomonitoring of infection with Wuchereria bancrofti is presented. The DNA extracted by this method was found suitable for PCR detection of W. bancrofti infection in pools of 10-30 mosquitoes. The PCR assay employing the simplified DNA extraction method was evaluated for its sensitivity on field caught Cx. quinquefasciatus, in comparison with the conventional dissection and microscopy technique. When assayed on dissection washings of vector mosquitoes the PCR assay detected 45 pools out of 49 dissection positive pools as positive for infection and hence found to be less sensitive than the conventional technique. The reason for detecting four dissection positive pools as negatives by the PCR assay may be due to the loss of a few numbers of parasites (1-3) present in these pools during the transfer of washings of dissected mosquitoes. The PCR assay detected ten out of 72 dissection negative pools as positives, while it did not detect any of the 62 known negative (laboratory reared, uninfected) mosquito pools as positives. When 38 pools (10 mosquitoes/pool) of intact mosquitoes were assessed for infection by each method, the infection rates obtained by the two methods were almost similar (3.35 and 3.01%, respectively, for conventional method and PCR assay). The results thus show that the DNA extraction method, which is simple, rapid, safe and inexpensive, is efficient to generate DNA from vector mosquitoes useful in PCR assay and hence has potential application in xenomonitoring.

Background & Objectives: There is a need to delimit the areas of filariasis transmission in view of the Filariasis Elimination Programme launched in India. Infection rate in vectors is an important parameter in determining transmission and it is conventionally assessed by dissection and microscopy. A PCR assay based on Ssp I repeats of Wuchereria bancrofti has shown potential in the detection of infection in vectors. The aim of the present study was to evaluate the specificity and sensitivity of this assay on W. bancrofti and its vector, Culex quinquefasciatus, prevalent in India., Methods: The DNA from pools of C. quinquefasciatus to which W. bancrofti microfilariae (mf) were added, was extracted by lysing with 0.1 M NaOH and 0.2 per cent sodium dodecyl sulphate (SDS), followed by silica absorption in the presence of guanidinium thiocyanate. The PCR assay of the DNA samples was carried out using NV-1 and NV-2 primers and the species specific SspI band was visualized on agarose gels stained with ethidium bromide., Results: The Ssp I PCR assay was found to be highly species specific, as it did not detect the DNA of a closely related filarial parasite, Brugia malayi. The assay detected as little as 0.04 pg of W. bancrarofti DNA. Minimum number of parasite detectable in pools of mosquitoes was 1 mf. A pool size of 50 mosquitoes was found to be optimum for the PCR assay., Interpretation & Conclusion: The Ssp I PCR assay was found to be highly specific and sensitive in detecting filarial parasite in pools of mosquitoes and therefore has potential application in rapid assessment of transmission of filariasis.

Control programmes have reduced the prevalence of Bancroftian filariasis in Thailand to low levels. Recently, there has been an influx of more than one million Myanmar immigrants into urban centres of Thailand. The prevalence of patent Wuchereria bancrofti infection in these immigrants (2-5%) has prompted concern in the public health community that the potential now exists for a re-emergence of Bancroftian filariasis in Thailand. It is possible that an urban cycle of transmission could become established. The Myanmar immigrants are infected with the nocturnal periodic (urban) type W. bancrofti for which Culex quinquefasciatus serves as the main vector. The Thai strains of Cx. quinquefasciatus have never been reported to transmit Bancroftian filariasis. Our results of feeding experiments demonstrated that the Thai Cx. quinquefasciatus are permissive for the development of Myanmar W. bancrofti to infective third-stage larvae thus establishing the potential for establishing an urban cycle of transmission in Thailand. We also adapted the SspI repeat PCR assay for the identification of infective mosquitoes that was capable of detecting a single infective stage larvae in a pool of 100 mosquitoes.