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The sequence diversity of natural and laboratory populations of Brugia pahangi and Brugia malayi was assessed with Illumina resequencing followed by mapping in order to identify single nucleotide variants and insertions/deletions. In natural and laboratory Brugia populations, there is a lack of sequence diversity on chromosome X relative to the autosomes (πX/πA = 0.2), which is lower than the expected (πX/πA = 0.75). A reduction in diversity is also observed in other filarial nematodes with neo-X chromosome fusions in the genera Onchocerca and Wuchereria, but not those without neo-X chromosome fusions in the genera Loa and Dirofilaria. In the species with neo-X chromosome fusions, chromosome X is abnormally large, containing a third of the genetic material such that a sizable portion of the genome is lacking sequence diversity. Such profound differences in genetic diversity can be consequential, having been associated with drug resistance and adaptability, with the potential to affect filarial eradication., Author summary Almost a billion people receive >7.7 billion doses of treatment aimed at eliminating lymphatic filariasis, which is caused by three filarial nematodes: Wuchereria bancrofti, Brugia malayi, and Brugia timori. Drug resistance and adaptation are both associated with pathogen success as well as higher levels of genetic diversity. In an examination of genetic diversity in Brugia malayi and Brugia pahangi, we observed a lack of genetic diversity over a third of the genome that is found on chromosome X. These species have neo-X chromosomes where a chromosome X fused with an autosome. Using publicly-available published data, the other filarial nematodes of greatest human significance are also found to have a similar lack of genetic diversity on their neo-X chromosomes. The two filarial nematodes with publicly-available data that lack neo-X chromosomes did not have this lack of genetic diversity. This lack of sequence diversity in B. malayi, W. bancrofti, and O. volvulus could have profound effects on all traits encoded on chromosome X.

Background Sri Lanka’s Anti-Filariasis Campaign conducted 5 annual rounds of mass drug administration (MDA) with diethylcarbamazine (DEC) plus albendazole to eliminate lymphatic filariasis (LF) in all endemic districts between 2002 and 2006. Post-MDA surveillance has consistently documented Wuchereria bancrofti microfilaremia (Mf) rates below 1% in all sentinel and spot check sites since that time, and all implementation units easily satisfied WHO’s target for school-based transmission assessment surveys (school-TAS) in 2013. However, more detailed studies have identified foci of persistent infection in the large coastal evaluation unit (EU) (population about 0.6 million) in Galle district. Therefore, the purpose of this study was to assess the sensitivity and feasibility of community-based TAS in adults (adult-TAS) and to compare results obtained by adult-TAS with prior school-TAS and molecular xenomonitoring (MX, molecular detection of filarial DNA in systematically sampled mosquitoes) results in this known problem area. Methodology and principal findings Two cluster surveys were performed in independent samples of 30 evaluation areas (EAs) in the coastal Galle EU in 2015. Each survey tested approximately 1,800 adults for circulating filarial antigenemia (CFA) with the Alere Filariasis Test Strip. The CFA prevalence for all persons tested (N = 3,612) was 1.8% (CI 1.4–2.2), and this was significantly higher than the CFA rate of 0.4% obtained by school-TAS in 2013. CFA prevalences in the two samples were similar [1.5% (CI 1.0–2.2), and 2.0% (CI 1.4–2.7)]. Antigenemia prevalence in sampled EUs was highly variable (range 0–11%), and it exceeded 5% in 6 EAs. The 30 EAs sampled in one of our adult-TAS surveys had recently been assessed for persistent filariasis by molecular xenomonitoring (MX). CFA prevalence in adults and filarial DNA prevalence in mosquitoes in these EAs were significantly correlated (r = 0.43; P = 0.02). Conclusions Community based adult-TAS provided a reproducible measure of persistent W. bancrofti infection in a large evaluation unit in Sri Lanka that has low-level persistence of LF following multiple rounds of MDA. In addition, adult-TAS and MX results illustrate the focality of persistent LF in this setting. Adult-TAS may be more sensitive than school-TAS for this purpose. Adult-TAS and MX are potential options for post-MDA and post-validation surveillance programs to identify problem areas that require mop-up activities. Adult-TAS should also be useful for remapping areas with uncertain LF endemicity for possible inclusion in national LF elimination programs., Author summary Lymphatic filariasis (LF, also commonly known as “elephantiasis) is a leading cause of disability in the developing world. It is caused by parasitic worms that are transmitted by mosquitoes. The Sri Lankan Anti-Filariasis Campaign provided five annual rounds of MDA with diethylcarbamazine and albendazole in all infected areas between 2002 and 2006, and this reduced infection prevalence to very low levels in all sentinel and spot check sites. Sri Lanka conducted transmission assessment surveys (TAS, surveys for filarial antigenemia in primary school children) based on WHO guidelines in 2012–2013, and all evaluation units (EUs) in formerly endemic areas easily passed this test. However, other types of post-MDA surveys (antigenemia and microfilaremia assessments in communities, anti-filarial antibody assessment in primary grade school children and mosquito monitoring for filarial DNA) have shown evidence of persistent LF in Sri Lanka, with the strongest signals in a coastal EU in Galle district. Results from this study show that adult-TAS efficiently detected residual filarial infections in this EU that had passed school-TAS. Adult-TAS results were highly correlated with results from prior surveys that used molecular xenomonitoring (MX) to detect filarial parasite DNA in pools of mosquitoes collected in the same study areas. Thus adult-TAS and MX should be considered as an alternative surveillance approaches for verifying that LF has been eliminated following MDA and for identifying areas that require additional intervention.

The Sri Lankan Anti-Filariasis campaign distributed five rounds of mass drug administration (MDA with diethylcarbamazine plus albendazole) to some 10 million people in eight districts between 2002 and 2006. Sri Lanka was recognized by the WHO for having eliminated lymphatic filariasis (LF) as a public health problem in 2016. However, recent studies by our group documented pockets with persistent LF in coastal Sri Lanka, especially in Galle district. The present study was performed to reexamine an area previously identified as a potential hotspot for persistent LF (Balapitiya Public Health Inspector area, population 17,500). A community survey documented high rates for circulating filarial antigenemia (3%, confidence interval [CI]: 1.8–4.9) and microfilaremia (1%, CI: 0.5–2.5%). Circulating filarial antigenemia rates were 2.8-fold higher in males than females. High prevalence was also observed for anti-filarial antibodies in young children (5.7%, CI: 3.7–8.4%) and for filarial DNA in vector mosquitoes (5.2%, CI: 4.2–6.3%). Spatial data showed that persistent LF was dispersed across the entire study area. Other studies showed that persistent LF was not limited to Balapitiya and not solved by additional rounds of MDA. Molecular xenomonitoring studies conducted in 2016 in 22 of 168 Public Health Midwife areas in the coastal Galle evaluation unit (approximate population 600,000) found that 179 of 660 (27%) pools of Culex collected from all areas were positive for Wuchereria bancrofti DNA by quantitative polymerase chain reaction; the estimated infection rate in mosquitoes was 1.26%, CI: 1.0–1.5%. Interventions other than routine MDA will be required to remove LF hotspots in Balapitiya and in other areas in coastal Sri Lanka.

Background The Global Programme to Eliminate Lymphatic Filariasis (LF) emphasizes hygiene, exercise, and other measures to reduce morbidity and disability related to LF. We recently reported that a portable, three-dimensional, infrared imaging system (3DIS) provides accurate limb volume measurements in patients with filarial lymphedema. To assess the practical utility of repeated 3DIS measurements for longitudinal lymphedema management, we examined intraday and day-to-day leg volume changes in adults with filarial lymphedema in southern Sri Lanka. Methodology and principal findings We assessed 41 participants with lower extremity lymphedema (stages 1–6) in their homes in the mornings (6:00–9:00 AM) and afternoons (2:00–6:00 PM) of three days within one calendar week. Two examiners performed replicate 3DIS volume measurements at each visit. Median coefficient of variation among replicate volume measurements was 1.7% (IQR 1.1% - 2.3%) for left legs and 2.2% (IQR 1.6% - 2.8%) for right legs. Median intraday volume increase was 3.0%. Range among daily volume measurements tended to be lower for afternoon measurements (median 2.25%, IQR 1.4%– 5.4%) than for morning measurements (median 3.0%, IQR 1.4% - 8.4%). Conclusions and significance Limb volume measurements by 3DIS are accurate and reproducible, and this technique is feasible for use in patients’ homes. We have developed practical suggestions for optimal outcomes with 3DIS. Duplicate measurements should be performed and repeat assessments should be done at approximately the same time of day to minimize bias. Duplicate measures that vary by more than 8% should prompt review of scanning technique with a repeat measurement. With proper training and attention to technique, 3DIS can be a valuable tool for healthcare workers who work with lymphedema patients., Author summary Morbidity Management and Disability Prevention (MMDP) is a critical but neglected component of the Global Programme to Eliminate Lymphatic Filariasis. Lack of infrastructure, trained practitioners, political will, and resources are challenges to effective implementation of MMDP services. This is compounded by the paucity of treatment options for limb lymphedema. Currently daily washing, elevation, and exercise are the only therapy available to most patients with filarial limb lymphedema, and new treatments are sorely needed. We previously reported that portable three-dimensional infrared imaging provides rapid, accurate, and reproducible limb measurements in patients with filarial lymphedema. Here, we show that similarly accurate and reproducible limb measurements are possible outside the clinic (in or outside patients’ homes). Using this technique we quantify how much limb volume varies from day to day and during the day for patients with filarial lymphedema, and provide recommendations for the optimal use of this tool in longitudinal monitoring of limb volume in research or in the clinic.

We explored the practicality of integrating surveillance for soil-transmitted helminthiasis (STH, assessed by Kato-Katz) with transmission assessment surveys for lymphatic filariasis (LF) in two evaluation units (EUs) in Gampaha district, Sri Lanka (population 2.3 million). The surveys were performed 6 years after five annual rounds of mass drug administration with diethylcarbamazine and albendazole. Each transmission assessment survey tested children (N = 1,462 inland EU; 1,642 coastal EU) sampled from 30 primary schools. Low filarial antigenemia rates (0% and 0.1% for the inland and coastal EUs) suggest that LF transmission is very low in this district. The STH rates and stool sample participation rates were 0.8% and 61% (inland) and 2.8% and 58% (coastal). Most STH detected were low or moderate intensity Trichuris trichiura infections. The added cost of including STH testing was ∼$5,000 per EU. These results suggest that it is feasible to integrate school-based surveillance for STH and LF.

The purpose of this study was to develop real-time multiplex quantitative PCR (qPCR) assays for the simultaneous detection of Wuchereria bancrofti (Wb), Plasmodium falciparum (Pf) and P. vivax (Pv) in mosquitoes. We optimized the assays with purified DNA samples and then used these assays to test DNA samples isolated from Anopheles punctulatus mosquitoes collected in villages in Papua New Guinea where these infections are co-endemic. Singleplex assays detected Wb, Pf and Pv DNA in 32%, 19% and 15% of the mosquito pools, respectively, either alone or together with other parasites. Multiplex assay results agreed with singleplex results in most cases. Overall parasite DNA rates in mosquitoes, estimated by PoolScreen 2 software, for Wb, Pf and Pv were 4.9%, 2.7% and 2.1%, respectively. Parasite DNA rates were consistently higher in blood-fed mosquitoes than in host-seeking mosquitoes. Our results show that multiplex qPCR can be used to detect and estimate prevalence rates for multiple parasite species in arthropod vectors. We believe that multiplex molecular xenodiagnosis has great potential as a tool for non-invasively assessing the distribution and prevalence of vector-borne pathogens such as W. bancrofti and Plasmodium spp. in human populations and for assessing the impact of interventions aimed at controlling or eliminating these diseases.

Background Filarial antigen tests are key tools for mapping the distribution of bancroftian filariasis and for detecting areas with persistent infections following mass drug administration (MDA). A recent study showed that the new Alere Filariasis Test Strip (FTS) has better analytical sensitivity than the BinaxNOW Filariasis card test (Card Test) for detecting circulating filarial antigen, and the FTS detected more positive results than the Card Test in a field study performed in a highly endemic area in Liberia. Methods The present study compared the performance of the FTS and the Card Test in community surveys that were conducted in southern Sri Lanka and in Indonesia (Central Java) in areas with low-level persistence of LF following multiple rounds of MDA with diethylcarbamazine plus albendazole. The studies were performed in densely populated semi-urban areas where Wuchereria bancrofti is transmitted by Culex quinquefasciatus. Results Antigenemia rates by FTS were 138 % higher in the Sri Lanka study (43/852 vs. 18/852) and 21 % higher in the Indonesia study (50/778 vs. 41/778) than antigenemia rates by Card Test. Antigenemia rates were significantly higher in males than in females and higher in adults than in children in both study sites. Although overall antigenemia rates and test scores were significantly higher by FTS than by Card Test in both study areas, rates in young children were similar with both tests in both areas. Conclusions These results extend the previously reported superior sensitivity of the FTS to areas with low residual infection rates following MDA, and this could affect mapping and post-MDA survey results in adults. However, our findings suggest that results of transmission assessment surveys (TAS) performed in school-aged children are likely to be similar with both tests.

Brugian filariasis (caused by the nematodes Brugia malayi and B. timori ) is an important cause of disability in Southeast Asia. Improved diagnostic tests are needed for filariasis elimination programs (to identify areas of endemicity and to monitor progress) and for diagnosis of the disease in infected individuals. We have developed and evaluated two real-time PCR assays for detecting Brugia DNA in human blood and compared the results of these assays to those of “gold standard” assays. One assay uses a TaqMan probe (TaqM) to amplifiy a 320-bp “HhaI repeat” DNA sequence. The other assay uses a minor groove binding probe (MGB) and modified nucleotides in primers (Eclipse MGB) to amplify a 120-bp fragment of the HhaI repeat. This assay detects 22 copies of the target sequence, and it is more sensitive than the TaqM assay. Both assays were evaluated with human blood samples from two different areas of endemicity. The MGB assay was as sensitive as membrane filtration and microscopy for the detection of B. malayi infection in 57 blood samples recovered at night from patients in Sulawesi, Indonesia. The MGB assay also detected parasite DNA in 17 of 31 (55%) of microfilaria-negative day blood samples from these subjects. This test was more sensitive than the conventional and the TaqM PCRs (and was almost as sensitive as night blood membrane filtration) for the detection of infection in 52 blood samples recovered at night from individuals in an area of B. timori endemicity on Alor Island, Indonesia, where microfilaria-positive individuals had low densities after mass treatment. Thus, the Eclipse MGB real-time PCR assay is a sensitive means of detecting Brugia parasite DNA in human blood.

Recent studies have suggested that intracellular Wolbachia bacteria are necessary for reproduction and survival of adult filarial worms. We now report results of in vitro studies of effects of antibacterial antibiotics (tetracycline, rifampicin, chloramphenicol, azithromycin, and doxycycline) on Brugia malayi infective larvae (L3) motility and molting. All of the antibiotics tested except chloramphenicol decreased L3 motility by 50% or more at 10 days, with minimal effective concentrations (MECs) of 20–100 μg/ml. Tetracyclines, rifampicin, and chloramphenicol inhibited L3 to L4 molting by 12 days in a concentration- and time-dependent manner, with MECs in the range of 1–20 μg/ml. These studies show that antibiotics active against Rickettsiaceae inhibit B. malayi L3 molting at low concentrations in vitro; higher concentrations kill the larvae. While it is possible that antibiotics directly affect filarial L3, we believe it is more likely that the effects seen are indirect effects related to bacterial killing. Index Descriptors and Abbreviations: Brugia malayi; Filarioidea; Rickettsiaceae; Wolbachia; antibiotics; NI-medium; NCTC-135 and IMDM, Iscove’s Modified Dulbecco’s Medium; FCS, fetal calf serum; Tet, tetracycline; Rif, rifampicin; Chlor, chloramphenicol; Azith, azithromycin; Doxy, doxycycline; IM, incomplete molting larvae; MEC, minimum effective concentration; PCR, polymerase chain reaction.

Background Lymphatic filariasis (LF) is targeted for global elimination through treatment of entire at-risk populations with repeated annual mass drug administration (MDA). Essential for program success is defining and confirming the appropriate endpoint for MDA when transmission is presumed to have reached a level low enough that it cannot be sustained even in the absence of drug intervention. Guidelines advanced by WHO call for a transmission assessment survey (TAS) to determine if MDA can be stopped within an LF evaluation unit (EU) after at least five effective rounds of annual treatment. To test the value and practicality of these guidelines, a multicenter operational research trial was undertaken in 11 countries covering various geographic and epidemiological settings. Methodology The TAS was conducted twice in each EU with TAS-1 and TAS-2 approximately 24 months apart. Lot quality assurance sampling (LQAS) formed the basis of the TAS survey design but specific EU characteristics defined the survey site (school or community), eligible population (6–7 year olds or 1st–2nd graders), survey type (systematic or cluster-sampling), target sample size, and critical cutoff (a statistically powered threshold below which transmission is expected to be no longer sustainable). The primary diagnostic tools were the immunochromatographic (ICT) test for W. bancrofti EUs and the BmR1 test (Brugia Rapid or PanLF) for Brugia spp. EUs. Principal Findings/Conclusions In 10 of 11 EUs, the number of TAS-1 positive cases was below the critical cutoff, indicating that MDA could be stopped. The same results were found in the follow-up TAS-2, therefore, confirming the previous decision outcome. Sample sizes were highly sex and age-representative and closely matched the target value after factoring in estimates of non-participation. The TAS was determined to be a practical and effective evaluation tool for stopping MDA although its validity for longer-term post-MDA surveillance requires further investigation., Author Summary Lymphatic filariasis (LF) is targeted for global elimination through a strategy of repeated annual mass drug administration (MDA) to entire at-risk populations. A transmission assessment survey (TAS) is designed to evaluate whether transmission of LF is presumed to have reached a level low enough that it cannot be sustained in the absence of drug intervention and, therefore, MDA can be stopped. This multicenter operational research trial examines the value and practicality of the TAS guidelines through its implementation in 11 countries of diverse geographical and epidemiologic profiles. The field experiences support the TAS survey design methodology with particular respect to school and cluster-based sampling strategies. We found that sample sizes were age and sex representative and met the target values after factoring in estimates of non-participation rates. In 10 of 11 countries, the TAS found the number of positive cases in the evaluation unit to be no more than the statistically powered critical threshold. These results were corroborated in a follow-up TAS approximately 24 months later. We conclude the TAS is a valuable and effective tool for stopping MDA but its utility for longer-term post-MDA surveillance needs further empirical evidence and may be best supported with complementary tools and methods.

Background Sri Lanka’s Anti Filariasis Campaign distributed 5 rounds of mass drug administration (MDA with DEC plus albendazole) to all endemic regions in the country from 2002–2006. Post-MDA surveillance results have generally been encouraging. However, recent studies have documented low level persistence of Wuchereria bancrofti in Galle district based on comprehensive surveys that include molecular xenomonitoring (MX, detection of filarial DNA in mosquitoes) results. The purposes of this study were to demonstrate the use of MX in large evaluation units (EUs) and to field test different mosquito sampling schemes. Methodology/Principal Findings Galle district (population 1.1 million) was divided into two EUs. These included a coastal EU with known persistent LF and an inland EU with little persistent LF. Mosquitoes were systematically sampled from ~300 trap locations in 30 randomly selected clusters (health administrative units) per EU. Approximately 28,000 Culex quinquefasciatus were collected with gravid traps and tested for filarial DNA by qPCR. 92/625 pools (14.7%) from the coastal EU and 8/583 pools (1.4%) from the inland EU were positive for filarial DNA. Maximum likelihood estimates (MLE) for filarial DNA rates were essentially the same when the same number of mosquito pools were collected and tested from 75, 150, or 300 trap sites (range 0.61–0.78% for the coastal EU and 0.04–0.07% for the inland EU). The ability to use a smaller number of trap sites reduces the cost and time required for mosquito sampling. Conclusions/Significance These results suggest there is widespread persistence of W. bancrofti infection in the coastal Galle EU 8 years after the last round of MDA in 2006, and this is consistent with other data from the district. This study has shown that MX can be used by national programs to assess and map the persistence of W. bancrofti at the level of large EUs in areas with Culex transmission., Author Summary Lymphatic filariasis (LF) is a disabling neglected tropical disease that affects millions of people in 73 countries. The Global Program to Eliminate Lymphatic Filariasis (GPELF) is using mass drug administration (MDA) to reduce infections in humans below levels required for sustained transmission by mosquitoes. Prior studies have shown that molecular xenomonitoring (MX, detection of filarial DNA in insect vectors) is useful for detecting low-level persistence of W. bancrofti infection following MDA, but technical challenges have prevented national programs from adopting this approach on a large scale. This paper reports the first use of MX for post-MDA surveillance in large evaluation units. CDC gravid traps were effective for collecting large numbers of Culex quinquefasciatus mosquitoes, and filarial DNA was detected by qPCR. We found that MX is more sensitive than other surveillance methods for detecting persistent W. bancrofti in communities and that there is widespread, low-level persistence of infection in coastal Galle district (Sri Lanka) 8 years after the last round of MDA. The systematic mosquito sampling protocol used in this study should be feasible for use by national filariasis elimination programs to assess and map the persistence of W. bancrofti in regions where the infection is transmitted by Culex mosquitoes.

There is an urgent need for safe and effective antifilarials. Prior studies have shown that the nitazoxanide (NTZ) exhibits broad activity against anaerobic bacteria, protozoa, and certain intestinal helminths. We examined the effects of NTZ and tizoxanide (TZ) on Brugia malayi nematodes in vitro and in vivo. In vitro, NTZ and TZ reduced worm motility and viability in a dose-dependent manner. Worm viability was reduced by 50% with both compounds at 2.5 and 20 μg/ml killed adult worms. NTZ or TZ (5 μg/ml) significantly reduced microfilaria release. These compounds blocked worm’s embryogenesis, and decreased microfilarial motility and viability. Treated worms had damaged cuticles and abnormal mitochondria. Wolbachia were not cleared by NTZ or TZ treatment. Neither NTZ nor TZ cleared adult worms or microfilariae in infected gerbils. These results show that NTZ and TZ have potent effects on B. malayi nematodes in vitro. However, they were not effective in vivo.

Xenomonitoring (detection of filarial larvae or their DNA in mosquitoes) is a sensitive marker for assessing the endemicity of filariasis and a useful tool for evaluating elimination programs. To examine the fate of microfilariae (mf) and filarial DNA in vector competent and non-competent mosquito strains, we compared the detection of Brugia malayi parasites by dissection and by quantitative real-time polymerase chain reaction (PCR) in three different mosquito strains. We conclude that PCR is much more sensitive than dissection for detecting filarial larvae, especially their remnants in mosquitoes. However, parasite DNA can be detected in both vector and non-vector mosquitoes for two weeks or longer after they ingest mf-positive blood. Thus, although xenomonitoring with vector and non-vector mosquito species may be a sensitive method for indirectly detecting filarial parasites in human populations, positive test results for parasite DNA in mosquitoes do not necessarily prove that transmission is ongoing in the study area.

Collaborative studies between Marshall Hertig, an entomologist, and Samuel Wolbach, a pathologist, on the presence and identification of microorganisms in arthropods, resulted in the discovery of Wo

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.

Background The Sri Lankan Anti-Filariasis Campaign conducted 5 rounds of mass drug administration (MDA) with diethycarbamazine plus albendazole between 2002 and 2006. We now report results of a comprehensive surveillance program that assessed the lymphatic filariasis (LF) situation in Sri Lanka 6 years after cessation of MDA. Methodology and Principal Findings Transmission assessment surveys (TAS) were performed per WHO guidelines in primary school children in 11 evaluation units (EUs) in all 8 formerly endemic districts. All EUs easily satisfied WHO criteria for stopping MDA. Comprehensive surveillance was performed in 19 Public Health Inspector (PHI) areas (subdistrict health administrative units). The surveillance package included cross-sectional community surveys for microfilaremia (Mf) and circulating filarial antigenemia (CFA), school surveys for CFA and anti-filarial antibodies, and collection of Culex mosquitoes with gravid traps for detection of filarial DNA (molecular xenomonitoring, MX). Provisional target rates for interruption of LF transmission were community CFA, Author Summary Lymphatic Filariasis (LF, also known as “elephantiasis”) is a disabling and deforming disease that is caused by parasitic worms that are transmitted by mosquitoes. The Sri Lankan Anti-Filariasis Campaign provided five annual rounds of mass drug administration (MDA) with diethylcarbamazine and albendazole between 2002 and 2006 in all endemic areas (districts or implementation units), and this reduced infection rates to very low levels in all sentinel and spot check sites. Transmission Assessment Surveys (TAS, surveys for filarial antigenemia in primary school children) performed in 2012–2013 (about 6 years after the last round of MDA) showed that all 11 evaluation units in formerly endemic areas easily satisfied a key World Health Organization target for LF elimination programs. More comprehensive surveillance was performed with other tests to assess LF parameters in 19 study sites in the same eight districts. We detected evidence of persistent LF in all districts and evidence of ongoing transmission in several areas. Exposure monitoring (screening for anti-filarial antibodies in primary school children) and molecular xenomonitoring (detecting filarial DNA in mosquito vectors) were much more sensitive than TAS for detecting low level persistence of filariasis in Sri Lanka. These methods are complementary to TAS, and they are feasible for use by some national filariasis elimination programs. Results from this study suggest that TAS alone may not be sufficient for assessing the success of filariasis elimination programs.

The parasitic nematodes Wuchereria bancrofti, Brugia malayi and B. timori cause a dreadful disease in humans known as lymphatic filariasis, which afflicts more than 120 million people worldwide. As per recent epidemiologic estimates on prevalence of W. bancrofti and B. malayi, about 428 million people are at risk, with 28 million microfilaria carriers and 21 million clinical cases spread out in 13 States and 5 Union Territories of India. The Indian subcontinent that comprises Bangladesh, India, Maldives, Nepal and Sri Lanka harbours 50 per cent of the world's lymphatic filarial disease burden. Recently, an endobacterium of Wolbachia species that belongs to the family Rickettsiaceae was found in all life cycle stages of these nematodes and the transmission is exclusively vertical through the embryonic stages of the female worms. People with filariasis have been exposed to these Wolbachia bacteria or their proteins by the natural killing of parasites. Wolbachia have also been identified occasionally in body fluids of infected patients. Evidence suggests that these Wolbachia are mutualistic symbionts and can be cured from the nematodes by several antibiotics having antirickettsial properties. Treatment of nematodes with tetracyclines affect Wolbachia and they get cleared from worm tissues; and this elimination causes reproductive abnormalities in worms and affect worm's embryogenesis, resulting in sterility. Although it is impractical, prolonged treatment with doxycycline significantly reduces the numbers of microfilaria in circulation, which is an important strategy to control transmission of filariasis by mosquito vectors. In this review, the current knowledge of Wolbachia as a drug target and potential ways to reduce the infection through anti-Wolbachia treatments is discussed.

Prior studies have shown that intracellular Wolbachia endobacteria are necessary for the normal development, reproduction, and survival of filarial nematodes. The purpose of this study was to examine effects of gamma radiation on Wolbachia and reproduction in Brugia malayi adult worms. Worms were exposed to 0, 10, 25, 45, 75, and 105 krad of gamma radiation from a 137cesium source and cultured in vitro for 10 days. Irradiation reduced production of microfilariae in a dose-dependent manner. Embryograms of irradiated female worms showed dose-related abnormalities with arrested development at the early embryo stage. Irradiation reduced the viability of adult worms in a dose-dependent manner, but no lethal effect was observed. Electron microscopy studies showed that irradiation cleared Wolbachia from worm tissues. Real-time polymerase chain reaction studies demonstrated greatly reduced Wolbachia DNA in irradiated worms. These effects are essentially the same as those observed in adult worms treated with doxycycline. These studies suggest that effects of irradiation on reproduction in Brugia malayi may be caused by effects of irradiation on Wolbachia.

Prokaryotes and various eukaryotes have remarkable ability to survive under adverse physiologic conditions and protect themselves from environmental stresses. An important mechanism by which they accomplish this is by synthesizing rigid and biochemically inert structures around them. In general, these structures are highly stable and resistant to mechanical and chemical insults. Biochemically, they are composed of complex carbohydrates, such as chitin and heavily crosslinked scaffold of proteins to form complex structures, such as sheath, cuticle, and epicuticle. Transglutaminases (TGases) are a family of enzymes that share catalytic function with thioredoxin and protein disulphide isomerases (PDI) and catalyze protein crosslink reaction by establishing epsilon-(gamma-glutamyl)lysine isopeptide bonds. The isopeptide bonds thus formed are of great physiologic significance because once formed, they cannot be hydorlysed by any known enzymes of the eukaryote system and exhibit high resistance to reducing agents, detergents, and chaotropic agents. Therefore, it is likely that protective structures viz., sheath, cuticle, epicuticle, and viral core proteins synthesized by microorganisms involve active participation of TGases. In this review, we briefly describe the current knowledge of non-mammalian TGases and their possible role in growth, development, and survival of small organisms. Special reference is made to filarial nematode and bacterial TGases since they are the most well-characterized and studied enzymes among non-mammalian TGases.

Recent studies have suggested that intracellular Wolbachia spp. endobacteria are necessary for the reproduction and survival of filarial nematodes. The effects of antibiotics that are active against related bacteria on adult worms and microfilariae (Mf) of Brugia malayi in vitro were investigated. Antibiotics tested were doxycycline (Doxy), tetracycline (Tet), rifampicin (Rif), azithromycin (Azith), and chloramphenicol (Chlor). Doxy, Tet, Rif, and Azith reduced release of Mf by adult female worms. The minimum effective concentrations that reduced Mf release by 50% were 5 microg/ml for Doxy, 20 microg/ml for Tet, 40 microg/ml for Rif, and 100 microg/ml for Azith. The same drugs (at higher concentrations) killed adult worms and Mf. Embryograms showed that Tets blocked embryogenesis in female worms. Electron microscopy (EM) showed that the Tets, Rif, and Azith cleared Wolbachia spp. from adult worms and damaged developing embryos. These studies show that antibiotics active against Rickettsiaceae affect adult B. malayi worms and Mf in vitro. Additional studies will be needed to elucidate the mechanisms of action of these antibiotics on Wolbachia and filarial worms.

As one of the largest protein families, protein kinases (PKs) regulate nearly all processes within the cell and are considered important drug targets. Much research has been conducted on inhibitors for PKs, leading to a wealth of compounds that target PKs that have potential to be lead anthelmintic drugs. Identifying compounds that have already been developed to treat neglected tropical diseases is an attractive way to obtain lead compounds inexpensively that can be developed into much needed drugs, especially for use in developing countries. In this study, PKs from nematodes, hosts, and DrugBank were identified and classified into kinase families and subfamilies. Nematode proteins were placed into orthologous groups that span the phylum Nematoda. A minimal kinome for the phylum Nematoda was identified, and properties of the minimal kinome were explored. Orthologous groups from the minimal kinome were prioritized for experimental testing based on RNAi phenotype of the Caenorhabditis elegans ortholog, transcript expression over the life-cycle and anatomic expression patterns. Compounds linked to targets in DrugBank belonging to the same kinase families and subfamilies in the minimal nematode kinome were extracted. Thirty-five compounds were tested in the non-parasitic C. elegans and active compounds progressed to testing against nematode species with different modes of parasitism, the blood-feeding Haemonchus contortus and the filarial Brugia malayi. Eighteen compounds showed efficacy in C. elegans, and six compounds also showed efficacy in at least one of the parasitic species. Hypotheses regarding the pathway the compounds may target and their molecular mechanism for activity are discussed., Author Summary Parasitic nematode infection is a large global health and economic problem, infecting around 2 billion people and costing $100 billion in crops and livestock. People in developing countries often live on one dollar per day, so treatments cannot be expensive, therefore using pre-existing drugs as lead compounds provides an economical way to begin to develop affordable treatments. Protein kinases were chosen as the focus of this work due to the large number of pre-existing drugs that target them and their important role in regulating almost all activities in the cell. Herein we describe a set of protein kinases conserved in diverse nematode species and experimental screening results of pre-existing drugs that target these kinases. The compounds that show in vitro efficacy in both C. elegans and parasitic nematodes, H. contortus or B. malayi have potential to be optimized further. These compounds have potential to provide accessible treatment to people in developing countries, as well as improving the health of livestock and boosting food production globally.

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.

Background Most filarial nematodes contain Wolbachia symbionts. The purpose of this study was to examine the effects of doxycycline on gene expression in Wolbachia and adult female Brugia malayi. Methods Brugia malayi infected gerbils were treated with doxycycline for 6-weeks. This treatment largely cleared Wolbachia and arrested worm reproduction. RNA recovered from treated and control female worms was labeled by random priming and hybridized to the Version 2- filarial microarray to obtain expression profiles. Results and discussion Results showed significant changes in expression for 200 Wolbachia (29% of Wolbachia genes with expression signals in untreated worms) and 546 B. malayi array elements after treatment. These elements correspond to known genes and also to novel genes with unknown biological functions. Most differentially expressed Wolbachia genes were down-regulated after treatment (98.5%). In contrast, doxycycline had a mixed effect on B. malayi gene expression with many more genes being significantly up-regulated after treatment (85% of differentially expressed genes). Genes and processes involved in reproduction (gender-regulated genes, collagen, amino acid metabolism, ribosomal processes, and cytoskeleton) were down-regulated after doxycycline while up-regulated genes and pathways suggest adaptations for survival in response to stress (energy metabolism, electron transport, anti-oxidants, nutrient transport, bacterial signaling pathways, and immune evasion). Conclusions Doxycycline reduced Wolbachia and significantly decreased bacterial gene expression. Wolbachia ribosomes are believed to be the primary biological target for doxycycline in filarial worms. B. malayi genes essential for reproduction, growth and development were also down-regulated; these changes are consistent with doxycycline effects on embryo development and reproduction. On the other hand, many B. malayi genes involved in energy production, electron-transport, metabolism, anti-oxidants, and others with unknown functions had increased expression signals after doxycycline treatment. These results suggest that female worms are able to compensate in part for the loss of Wolbachia so that they can survive, albeit without reproductive capacity. This study of doxycycline induced changes in gene expression has provided new clues regarding the symbiotic relationship between Wolbachia and B. malayi.

Background A granulomatous inflammatory response develops in jirds infected subcutaneously or intraperitoneally with filarial nematodes namely Brugia pahangi and B. malayi. Previous studies by light and electron microscopy have shown cellular inflammatory responses in and around these granulomas. Furthermore, the cellular inflammatory responses of granulomas found in the lymphatics and peritoneal cavity appear to be similar. The purpose of this study was to determine the cytokine profiles of granulomas in the peritoneal cavity of B. pahangi-infected jirds and to determine whether the granulomas release any proinflammatory cytokines ex vivo. Methods A semiautomated quantitative polymerase chain reaction (Q-PCR) was performed on cDNA prepared from the granulomas of infected jirds to study the species-specific mRNA expression of IL-2, IL-4, IFN-γ, IL-5, and IL-10. Genomic DNA was extracted from the granulomas, and parasite DNA was detected by Q-PCR by amplifying the HhaI repeat sequence. The levels of the inflammation-causing cytokines IL-6 and TNFα that were secreted by the granulomas were measured by cell-based assays. Results Florid granulomas showed higher levels of IFN-γ than other cytokines, linking this Th1 cytokine to the granulomatous inflammation that develops in jirds and humans. IL-4 expression was much lower than that of IFN-γ but higher than that of IL-10. A low level of IL-5 mRNA expression was detectable in all granulomas as was the level of IL-2 expression. The levels of the inflammatory cytokines IL-6 and TNFα, secreted by intact granulomas, spontaneously increased by 48 h after culture. Parasite antigen stimulation and subsequent release of IL-6 and TNFα by the granulomas indicated a moderate increase in the levels of these two cytokines. The amplification of the Brugia HhaI repeat DNA and Wolbachia 16S rDNA indicated worm components and bacterial components in the granulomatous tissue. Conclusion Granuloma development in filarial infections is a complex process involving cellular reactions responding to parasite/bacteria and their components. The interactions between worm-derived granulomas and their hosts are dynamic and multifaceted. The data collected thus far suggest that the expression profiles of many of the measured cytokines in the lymphoid tissues of Brugia-infected jirds are different from those of the cytokines in granulomas. Moreover, granulomas have the ability to secrete the inflammatory cytokines IL-6 and TNFα.

Background Diagnostics provide a means to measure progress toward disease elimination. Many countries in Africa are approaching elimination of onchocerciasis after successful implementation of mass drug administration programs as well as vector control. An understanding of how markers for infection such as skin snip microfilaria and Onchocerca volvulus-specific seroconversion perform in near-elimination settings informs how to best use these markers. Methods All-age participants from 35 villages in Togo were surveyed in 2013 and 2014 for skin snip Onchocerca volvulus microfilaria and IgG4 antibody response by enzyme-linked immunosorbent assay (ELISA) to the Onchocerca volvulus-specific antigen Ov16. A Gaussian mixture model applying the expectation-maximization (EM) algorithm was used to determine seropositivity from Ov16 ELISA data. For a subset of participants (n = 434), polymerase chain reaction (PCR) was performed on the skin snips taken during surveillance. Results Within the 2,005 participants for which there was Ov16 ELISA data, O. volvulus microfilaremia prevalence and Ov16 seroprevalence were, 2.5 and 19.7 %, respectively, in the total population, and 1.6 and 3.6 % in children under 11. In the subset of 434 specimens for which ELISA, PCR, and microscopy data were generated, it was found that in children under 11 years of age, the anti-Ov16 IgG4 antibody response demonstrate a sensitivity and specificity of 80 and 97 %, respectively, against active infections as determined by combined PCR and microscopy on skin snips. Further analysis was performed in 34 of the 35 villages surveyed. These villages were stratified by all-age seroprevalence into three clusters: < 15 %; 15–20 %; and > 20 %. Age-dependence of seroprevalence for each cluster was best reflected by a two-phase force-of-infection (FOI) catalytic model. In all clusters, the lower of the two phases of FOI was associated with a younger age group, as reflected by the seroconversion rates for each phase. The age at which transition from lower to higher seroconversion, between the two phases of FOI, was found to be highest (older) for the cluster of villages with