Ramakrishna U. Rao

A multicenter evaluation of diagnostic tools to define endpoints for programs to eliminate bancroftian filariasis

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.

IN VITRO EFFECTS OF ANTIBIOTICS ON BRUGIA MALAYI WORM SURVIVAL AND REPRODUCTION

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 μg/ml for Doxy, 20 μg/ml for Tet, 40 μg/ml for Rif, and 100 μg/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.

Detection of Brugia Parasite DNA in Human Blood by Real-Time PCR

ABSTRACT 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.

Transmission assessment surveys (TAS) to define endpoints for lymphatic filariasis mass drug administration: a multicenter evaluation.

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.

Brugia malayi: effects of antibacterial agents on larval viability and development in vitro

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 microg/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 microg/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.

A Comprehensive Assessment of Lymphatic Filariasis in Sri Lanka Six Years after Cessation of Mass Drug Administration

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 <2%, antibody in school children <2%, and filarial DNA in mosquitoes <0.25%. Community Mf and CFA prevalence rates ranged from 0–0.9% and 0–3.4%, respectively. Infection rates were significantly higher in males and lower in people who denied prior treatment. Antibody rates in school children exceeded 2% in 10 study sites; the area that had the highest community and school CFA rates also had the highest school antibody rate (6.9%). Filarial DNA rates in mosquitoes exceeded 0.25% in 10 PHI areas. Conclusions Comprehensive surveillance is feasible for some national filariasis elimination programs. Low-level persistence of LF was present in all study sites; several sites failed to meet provisional endpoint criteria for LF elimination, and follow-up testing will be needed in these areas. TAS was not sensitive for detecting low-level persistence of filariasis in Sri Lanka. We recommend use of antibody and MX testing as tools to complement TAS for post-MDA surveillance.

Brugia malayi: Effects of nitazoxanide and tizoxanide on adult worms and microfilariae of filarial nematodes.

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 microg/ml killed adult worms. NTZ or TZ (5 microg/ml) significantly reduced microfilaria release. These compounds blocked worms 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.

Using Existing Drugs as Leads for Broad Spectrum Anthelmintics Targeting Protein Kinases

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.

A qPCR-based multiplex assay for the detection of Wuchereria bancrofti, Plasmodium falciparum and Plasmodium vivax DNA

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.

The Discovery of Wolbachia in Arthropods and Nematodes – A Historical Perspective

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