Biomedical subjects

Gary J Weil

Publications and source records attributed to Gary J Weil.

18 recordsLinked to original sources

PubMed2006

Diagnostic tools for filariasis elimination programs.

The ambitious and exciting Global Programme to Eliminate Lymphatic Filariasis (GPELF) is largely based on a strategy of mass drug administration (MDA) of repeated rounds of antifilarial medications to endemic populations around the world. Diagnostic tools are important to GPELF because they affect decisions regarding where to distribute MDA, how to measure its effects, how to define targets and endpoints for stopping MDA, and how to monitor populations for possible resurgence of filariasis transmission following suspension of MDA. This article reviews available diagnostic tests for filariasis and their potential use as tools for different phases of filariasis elimination programs.

Animals↗

PubMed2006

Detection of Brugia parasite DNA in human blood by real-time PCR.

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.

Animals↗

PubMed2006

Brugia malayi: effects of radiation and culture on gene expression in infective larvae.

Third-stage infective larvae (L3i) of Brugia malayi are developmentally arrested in mosquitoes but must quickly adapt to a new environment when they enter mammalian hosts to initiate infections. These changes can be studied by in vitro culture of L3 (L3c) under conditions that permit molting of L3-L4. Irradiated L3 (L3ir) have stunted growth and limited lifespan in mammalian hosts, and they induce high levels of immunity to challenge infections in animal models. This study explored differences in gene expression in L3i, L3c and L3ir by expressed sequence tag EST generation and qRT-PCR. 2506 ESTs generated from cDNA libraries constructed from L3i, L3c and L3ir were grouped into 1309 gene clusters. Despite extensive prior sampling from B. malayi (>22,000 ESTs in dbEST), 73% of the L3 clusters described here are novel. Sixty-three percentage of the clusters have homology to proteins from other species including 187 specific to nematodes and 141 that have to date only been described in non-nematode species. The transcript levels of 62 candidates for up- or down-regulation in L3i, L3c and L3ir based on EST frequencies were evaluated by qRT-PCR. Twenty-eight were confirmed to have > or = 3-fold differences in expression. Genes coding for proteins believed to be involved in establishment of infection, host adaptation and targets of protective immunity were confirmed to have higher expression in L3i than in L3c. Some of the genes that were down-regulated in L3c were highly expressed in L3ir. This study provides an improved description of the adaptations that accompany the transition from L3i to L3c and the special ability of L3ir to induce protective immunity.

Aedes↗

PubMed2006

Field applicability of a rapid-format anti-Ov-16 antibody test for the assessment of onchocerciasis control measures in regions of endemicity.

BACKGROUND: A previously developed, specific, rapid-format immunochromatographic card test that detects immunoglobulin G4 to the recombinant Onchocerca volvulus antigen Ov-16 was modified to detect antibodies in whole blood. METHODS: Ov-16 card test results were assessed in 1511 subjects > or =2 years of age in 7 West African villages with varying histories of onchocerciasis control measures. RESULTS: In villages in which control measures had been implemented, anti-Ov-16 antibody prevalence rates ranged from 5.2% to 65.1%. Antibody prevalence rates were close to zero among subjects born after effective control measures had been implemented. In 2 villages without a history of control measures where onchocerciasis was endemic, microfilariae (MF) prevalence rates were 82.8% and 65.1%, and antibody prevalence rates were 73.1% and 62.1%. In these 2 villages, the sensitivity of the Ov-16 card test was 81.1% and 76.5%, the specificity was 100%, and the positive predictive value was 91.8% and 80.5%. MF and antibody prevalence rates were correlated (Spearman's r=0.815; P<.038). CONCLUSIONS: The Ov-16 card test is field applicable, exhibits high sensitivity and specificity for O. volvulus infection, and has great potential as a tool for surveillance and for evaluating the success of onchocerciasis control measures.

Adolescent↗

PubMed2006

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

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

Albendazole↗

PubMed2006

Bancroftian filariasis: effect of repeated treatment with diethylcarbamazine and albendazole on microfilaraemia, antigenaemia and antifilarial antibodies.

Diethylcarbamazine/albendazole (DEC/ALB) therapy is widely used in mass drug administration (MDA) programmes aimed at eliminating lymphatic filariasis. We studied the effects of repeated annual treatments with DEC/ALB on Wuchereria bancrofti microfilaraemia, filarial antigenaemia and IgG4 antibodies to Bm14 antigen. Fifty-seven subjects with asymptomatic microfilaraemia were treated with one or seven daily doses of DEC/ALB at time zero. All subjects were re-treated with single-dose DEC/ALB 12, 24 and 36 months later. The two treatment groups had comparable pre-treatment microfilaria counts. Multidose treatment cleared microfilaraemia more effectively than single-dose treatment. Filarial antigen levels decreased equally in both treatment groups. Total antigen clearance was observed in 29.6%, 52.0%, 63.6% and 79.5% of subjects at 12, 24, 36 and 48 months. These clearance rates are much higher than those observed in prior treatment trials with DEC or ivermectin. Antibody levels increased 4 weeks after treatment and then slowly decreased in most subjects. Antibody tests turned negative in 20%, 35%, 39.4% and 52.5% of treated subjects at 12, 24, 36 and 48 months post treatment. These results show that the studied parameters decline at different rates and to differing degrees following DEC/ALB treatment. These findings have important implications regarding strategies for monitoring the effects of MDA in populations.

Adolescent↗

PubMed2006

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

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

Animals↗

PubMed2005

Brugia malayi: effects of gamma radiation on adult worms and their intracellular Wolbachia bacteria.

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.

Aedes↗

PubMed2005

Profiling of gender-regulated gene transcripts in the filarial nematode Brugia malayi by cDNA oligonucleotide array analysis.

Microarray technology permits high-throughput comparisons of gene expression in different parasite stages or sexes and has been used widely. We report the first use of this technology for analysis of gene expression in filarial male and female worms. The slide array (comprised of 65-mer oligos representing 3569 EST clusters) was spotted with sequences selected from the extensive Brugia malayi EST database (). Arrays were hybridized with Cy dye labeled male and female cDNA. The experimental design included both biological and technical (dye-flip) replicates. The data were normalized for background and probe intensity, and the relative abundance of hybridized cDNA for each spot was determined. Genes showing two-fold or greater differences with P<0.05 were considered gender-regulated candidates. One thousand one hundred and seventy of 2443 clusters (48%) with signals above threshold in at least one sex were considered as gender-regulated gene candidates. This included 520 and 650 clusters up-regulated in male and female worms, respectively. Fifty of 53 (94%) gender-regulated candidate genes identified by microarray analysis were confirmed by real-time RT-PCR. Approximately 61% of gender-regulated genes had significant similarity to known genes in other organisms such as Caenorhabditis elegans. Many C. elegans homologues of these genes have been reported to have reproductive phenotypes (sterility or abnormal embryo development) by RNA interference. This study has provided the first broad view of gender-regulated gene expression in B. malayi; this should lead to improved understanding of reproduction in filarial nematodes. More generally, this approach holds great promise as a means of studying stage-specific or tissue-specific gene expression in parasitic nematodes.

Animals↗

PubMed2005

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

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

Adult↗

PubMed2004

Antibody responses to Brugia malayi antigens induced by DNA vaccination.

BACKGROUND: DNA vaccination is a convenient means of immunizing animals with recombinant parasite antigens. DNA delivery methods are believed to affect the qualitative nature of immune responses to DNA vaccines in ways that may affect their protective activity. However, relatively few studies have directly compared immune responses to plasmids encoding the same antigens after injection by different routes. Therefore, the purpose of this study was to explore the influence of the route of administration on antibody responses to plasmids encoding antigens from the filarial nematode parasite Brugia malayi. METHODS: Four B. malayi genes and partial genes encoding paramyosin (BM5), heat shock protein (BMHSP-70), intermediate filament (BMIF) and a serodiagnostic antigen (BM14) were inserted in eukaryotic expression vectors (pJW4303 and pCR trade mark 3.1). BALB/c mice were immunized with individual recombinant plasmids or with a cocktail of all four plasmids by intramuscular injection (IM) or by gene gun-intradermal inoculation (GG). Antibody responses to recombinant antigens were measured by ELISA. Mean IgG1 to IgG2a antibody ratios were used as an indicator of Th1 or Th2 bias in immune responses induced with particular antigens by IM or GG immunization. The statistical significance of group differences in antibody responses was assessed by the non-parametric Kruskal-Wallis test. RESULTS: Mice produced antibody responses to all four filarial antigens after DNA vaccination by either the IM or GG route. Antibody responses to BM5 paramyosin were strongly biased toward IgG1 with lower levels of IgG2a after GG vaccination, while IM vaccination produced dominant IgG2a antibody responses. Antibody responses were biased toward IgG1 after both IM and GG immunization with BMIF, but antibodies were biased toward IgG2a after IM and GG vaccination with BMHSP-70 and BM14. Animals injected with a mixture of four recombinant plasmid DNAs produced antibodies to all four antigens. CONCLUSIONS: Our results show that monovalent and polyvalent DNA vaccination successfully induced antibody responses to a variety of filarial antigens. However, antibody responses to different antigens varied in magnitude and with respect to isotype bias. The isotype bias of antibody responses following DNA vaccination can be affected by route of administration and by intrinsic characteristics of individual antigens.

Journal Article↗

PubMed2004

Quantitative analysis of gender-regulated transcripts in the filarial nematode Brugia malayi by real-time RT-PCR.

Improved understanding of the biology of reproduction in filarial worms may lead to identification of new targets for drugs or vaccines. Real-time RT-PCR is increasingly being adopted for RNA quantification and genetic analysis. Candidate gender-regulated genes were selected from genes identified in prior studies by differential display RT-PCR and by electronic selection of the Brugia malayi expression sequence tag (EST) database for clusters with possible gender-specific expression (four or more transcripts in male cDNA library ESTs but none in female ESTs or vice versa). Expression of candidate genes in male and female worms was compared by real-time reverse transcription PCR with sequence-specific primers. Double stranded DNA product was measured by SYBR Green I fluorescence; melting curves and agarose gel electrophoresis were used to verify the specificity of results. Relative gene expression results were normalized by parallel studies with internal control genes that were shown to be equally expressed in male and female worms (beta actin 2B, histone H3, NADH dehydroxygenase subunit 1) and calculated by the comparative C(t) method. Nineteen of 31 candidate genes were verified to have reproducible, gender-biased expression with fold differences between 5 and >30,000. These included several well-known genes (for example, genes encoding major sperm protein and a microfilaria sheath protein) and many novel genes. This paper reports the first large scale use of real time RT-PCR to quantitate and study gene expression in a nematode parasite. Our results represent an important step toward improved understanding of the molecular biology of reproduction in filarial nematodes.

Animals↗

PubMed2004

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

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

Adult↗

PubMed2004

Duplex Doppler sonographic assessment of the effects of diethylcarbamazine and albendazole therapy on adult filarial worms and adjacent host tissues in Bancroftian filariasis.

We used duplex Doppler sonography to assess effects of diethylcarbamazine and albendazole therapy (DEC/ALB) on adult Wuchereria bancrofti in vivo. The study was performed in clinically normal Egyptian adults with blood microfilaria counts > 80/mL. Motile adult worms were observed before treatment in dilated scrotal lymphatic vessels in 28 of 36 men (78%) and over the proximal extremities in 5 of 22 women (23%). Most worm nests were inactivated in the months following treatment (90% at 12 months). Circulating filarial antigen levels (a marker for living adult worms) also fell dramatically following treatment. Some men had intrascrotal calcifications and/or non-palpable hydroceles detectable by ultrasound before they were treated. New hydroceles and intrascrotal calcifications appeared after treatment in many cases. However, most of these were transient and of no clinical significance. Prevelance rates for hydrocele and intrascrotal calcifications 24 months after treatment were essentially the same as those prior to treatment. These results show that DEC/ALB is highly active against adult W. bancrofti. They also suggest that host responses to dying adult worms are important in the pathogenesis of filarial hydroceles.

Adult↗

PubMed2003

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

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

Animals↗

PubMed2002

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.

Animals↗

PubMed2002

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

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

Adolescent↗

PubMedDate not supplied

An embryo-associated fatty acid-binding protein in the filarial nematode Brugia malayi.

Brugia malayi is a filarial nematode parasite that causes lymphatic filariasis, a disease that affects millions of people in the tropics. Sexual reproduction of filarial worms occurs within the lymphatic vessels of the human host and is crucial for transmission of the parasite to the mosquito vector. We have previously identified several B. malayi genes that exhibit apparent gender-specific expression. One of these had significant sequence similarity to the Ascaris suum embryo-associated fatty acid binding protein, As-p18. The full length cDNA for the B. malayi female-associated fatty acid binding protein (Bm-FAB-1) encodes a 17.8 kDa protein (excluding a signal peptide) with 70% sequence identity with mature As-p18 and significant similarity to Caenorhabditis elegans and mammalian fatty acid-binding proteins (FABPs). Antibodies raised to Bm-FAB-1 bound to developing embryos within female worms, especially around early embryo cells and the surfaces of immature worms within eggs. Functional studies showed that recombinant Bm-FAB-1 binds to several long chain fatty acids including oleate, but not retinol. Taken together, these results demonstrate that Bm-FAB-1 is a member of an unusual nematode-specific, secreted lipid binding protein family. The existence of a novel class of lipid binding proteins in nematode embryos raises the possibility that drugs targeting these proteins could be developed with broad activity against nematode parasites of medical and veterinary importance.

Amino Acid Sequence↗