Search

Semi-annual mass DEC chemotherapy combined with vector control at the beginning of the programme, has been administered on the remote island of Maupiti (French Polynesia) since 1955 (except two periods in 1960-67 and 1970-74). The results of two surveys in 1985 and 1989, reporting 0% microfilaraemia, led to the hope that the eradication of lymphatic filariasis had been achieved. We combined parasitological criteria (microfilaraemia by membrane filtration), immunological (antigenaemia and serum levels of specific IgG antibodies) and molecular (PCR-based evaluation of infection in mosquitoes) techniques and found only good control of the parasite: We found residual microfilaraemia in 0.4% of the sample (mean level in carriers: 101.2 mf/ml), antigenaemia in 4.6% (mean level in positive persons: 714.4 units/ml) and specific IgG in 21.6% (including in one very young child). In addition, an infection rate of 1.4% was calculated in the Aedes polynesiensis vector population. These data, obtained in 1997 just before a hurricane, were partially confirmed in 1999 (0.1% of infection rate in the vector). Together with the possibility of some resistance to DEC, various epidemiological factors critical for the eradication of lymphatic filariasis are discussed.

The full-length cDNA sequence encoding Brugia malayi L3 paramyosin has been isolated by immunoscreening a cDNA library with a mouse antiserum raised against Wuchereria bancrofti L3 infective larvae. A recombinant truncated form of paramyosin was expressed as a glutathione S -transferase fusion protein and used to evaluate humoral responses of adults from a W. bancrofti -endemic area in French Polynesia according to their parasitological status. Immunoglobulin G4 (IgG4) preferentially bound to paramyosin in W. bancrofti -parasitized individuals, in contrast to unparasitized individuals, who harbored neither microfilaria nor Og4C3 adult worm circulating antigen. Reduction of the anti-paramyosin IgG4 titer following combined chemotherapy with diethylcarbamazine and ivermectin was significantly correlated with a reduction in the adult worm burden. This indicates that the presence of paramyosin-reactive IgG4 is associated with the presence of parasites and that reduction can be used as an immunological marker for W. bancrofti clearance.

Circulating filarial antigen (CFA), determined with Og4C3 ELISA, is a marker of Wuchereria bancrofti adult worm infection. The reduction of CFA over 2 years was determined in 185 microfilaremic and 111 amicrofilaremic but CFA+ adults given an annual dose of either diethylcarbamazine (DEC) or ivermectin or the two combined. Reduction of CFA level was good with DEC but weak with ivermectin and followed the same pattern in amicrofilaremic and microfilaremic groups. Combinations and DEC alone had a similar impact on CFA level. CFA clearance was observed in amicrofilaremic but not in microfilaremic persons in all DEC-containing treatments. However, the highest clearance rate was observed in persons treated with DEC at 6 mg/kg combined with ivermectin. Continuous reduction of CFA level after repeated treatments shows that elimination of W. bancrofti infection, monitored by CFA clearance, might be achieved within a few years with annual treatments of DEC combined with ivermectin.

Background Effective diagnostic tools are necessary to monitor and evaluate interruption of Lymphatic Filariasis (LF) transmission. Accurate detection of Wuchereria bancrofti (Wb) microfilaria (mf) is essential to measure the impact of community treatment programmes. PCR-based assays are specific, highly sensitive tools allowing the detection of Wuchereria bancrofti DNA in human blood samples. However, current protocols describing the pool screening approach, use samples of less than 60 μl of blood, which limits the sensitivity of the pool-screen PCR assay. The purpose of this study was to improve the pool-screen PCR protocol to enhance its sensitivity and usefulness for population scale studies. Findings DNA extractions were performed with the DNeasy kit, the PCR with the Wb LDR primers and the SYBR-Green dye. Improvements of our pool-screen real-time PCR (qPCR) assay allowed the detection of as little as one Wb microfilaria diluted in a pool of at least 12 blood samples of 60 μl each. Using this assay, mf burdens can be predicted using a standard curve derived from mf spiked dried blood samples. The sensitivity achieved is equivalent to the detection of a single LF positive individual carrying a mf burden as low as 18 mf/ml, in a pool of blood samples from at least 12 individuals. Conclusions Due to its sensitivity, rapidity and cost-effectiveness, we suggest this qPCR pool-screening assay could be used as a diagnostic tool for population- scale filariasis elimination monitoring and evaluation.

A polymerase chain reaction (PCR) assay based on a highly repeated deoxyribonucleic acid (DNA) sequence found in Wuchereria bancrofti (the Ssp I repeat) has been developed to address the shortcomings of traditional diagnostic methods. In this field study in a W. bancrofti endemic region of French Polynesia, 373 human blood samples were collected and 100 μL of blood were screened by the Ssp I PCR assay and 1 μL by membrane filtration. The Ssp I PCR assay detected 99 of 113 blood samples in which microfilariae had been detected by filtration (sensitivity of 88%) with a specificity of 100%. All the samples missed by the Ssp I PCR assay had less than 8 microfilariae per mL of blood. To evaluate the efficacy of screening larger blood samples by PCR, both 100 μL and 500 μL samples from 50 patients with very low-level microfilaraemia were screened by the Ssp I PCR assay; the sensitivity increased from 60% to 84% when using the larger volume of blood. Finally, an enzyme-linked immunosorbent assay-based version of the Ssp I PCR assay was used to screen blood from 12 patients following treatment with diethylcarbamazine, ivermectin, or both. These results showed that the PCR assay closely paralleled the presence or absence of microfilariae in the blood and that no increase in the DNA level was seen immediately following drug treatment.

The age-specific patterns of microfilaremia, Og4C3 antigenemia, anti-Brugia malayi IgG and IgG4 were assessed in 3 villages of low, medium and high transmission level for Wuchereria bancrofti filariasis. The prevalence rates for each of the 4 markers were clearly age dependent and their patterns strongly associated with the transmission level. The antigenemia prevalence rate was consistently higher than the microfilaremia prevalence rate, in all age groups. The prevalences of anti-B. malayi IgG and IgG4 responses were very similar and much higher than those of microfilaremia or antigenemia. Antibody responses reached the plateau at an earlier age and at a higher prevalence with increased intensity of transmission. For all the markers, the prevalence rates were significantly higher in males than in females.

Background Entomological methods may provide important tools for monitoring the transmission of filariasis in French Polynesia. In order to standardize our PCR method and refine our protocol to assess filarial infection levels in mosquitoes, we compared dissection of the vector, Aedes polynesiensis, with the poolscreening polymerase chain reaction (PS-PCR) assay. Methods (1) Mosquitoes were collected in human landing catches in five areas in Moorea island, French Polynesia. (2) A fraction of the captured mosquitoes was dissected for Wuchereria bancrofti larvae. (3) Laboratory-reared mosquitoes (uninfected as well as experimentally infected ones) were repeatedly tested to optimize a PS-PCR protocol (DNA extracts from 1–50 pooled mosquitoes were tested with an internal standardized system and primers specific for the Ssp1 repeat sequence. PCR products were analysed by gel electrophoresis). (4) Another fraction of the captured mosquitoes was assayed by PS-PCR according the optimized protocol. Results The prevalence of field-mosquito infection with W. bancrofti ranged from 1 to 8 % by dissection (L1–L3) and point estimates of infection prevalence, as assayed by PS-PCR, ranged from 0.4 to 3.7 %. There was a moderately strong correlation between larval infection rates as determined by dissection and PCR. Discussion Our results suggest that the PS-PCR assay is specific and highly sensitive for detecting parasite DNA. We obtained similar although not identical results with dissections of mosquitoes. PS-PCR appears to be adequate for testing large numbers of mosquitoes in the context of filariasis elimination programs. The role and advantages of using entomologic methods to monitor filariasis programs are discussed.

Despite control programs based on mass drug administration (MDA) of microfilaricidal compounds, Bancroftian lymphatic filariasis remains a problem in French Polynesia. For an alternative strategy to MDA, we investigated the potential role of Wolbachia to control filarial transmission. Wolbachia are intracellular alpha-proteobacteria endosymbionts that infect a broad range of insects and nematodes. These bacteria have a suspected role in the pathogenesis of filariasis. They also may be useful in mosquito control through cytoplasmic incompatibility. To detect and characterize these bacteria in the filarial and mosquito-vectors in French Polynesia, a survey was conducted on field-collected mosquitoes and microfilariae from infected people. Samples were analyzed by a polymerase chain reaction and gene sequencing. The results indicate that these bacteria are widespread. Sequence analysis of the wsp and ftsZ genes positioned the Aedes polynesiensis Wolbachia in cluster A and Wuchereria bancrofti var. pacifica Wolbachia in cluster D. The implications for possible improved treatment and vector control are discussed.

PCR has recently been studied as a promising tool for monitoring the progress of efforts to eliminate lymphatic filariasis. PCR can be used to test concurrently at least 30 pools, with as many as 40 mosquitoes in each pool, for the presence of filarial larvae. The SspI PCR assay for the detection of Wuchereria bancrofti DNA in pools of mosquitoes has been used since 1994 in a variety of laboratories worldwide. During that time, the original assay has been modified in these different laboratories and no standardized assay currently exists. In an effort to standardize and improve the assay, a meeting was held on 15-16 November 2001, at Emory University in Atlanta, with representatives from most of the laboratories currently using the assay. The first round of testing was designed to test the four most promising methods for DNA extraction from pools of mosquitoes. Two of the four methods stood out as clearly the best and these will be now optimised and evaluated in two further rounds of testing.

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.

Enzyme-linked immunosorbent assays (Elisas) for anti-Brugia malayi immunoglobulin (Ig) G and IgG4 were evaluated on sera from 1561 subjects in French Polynesia for the serodiagnosis of Wuchereria bancrofti filariasis, compared with the test for Onchocerca gibsoni circulating antigen (Og4C3) as a ‘gold standard’. The sensitivity of the elisa -IgG and elisa -IgG4 assays was 90.8% and 94.5%, and the specificity was 45.9% and 50.7%. The positive predictive values were 41% and 45% respectively for an antigen prevalence rate of 30%. Thus antibody prevalences exceeded by two-fold the antigen prevalence, which itself exceeded by two-fold the prevalence of microfilaraemia.

In Wuchereria bancrofti transmission areas, three groups of individuals have been identified, according to the presence or absence of microfilariae or adult worm derived molecules in the blood compartment. These groups likely reflect individuals with different permissivity/resistance to the complete development of W. bancrofti cycle. The profile of filarial-specific immunoglobulins was analysed in W. bancrofti-exposed individuals in French Polynesia, according to the presence or absence of microfilariae (Mf) and adult worms, measured by Og4C3 circulating antigen. Individuals harbouring adult worms, have higher filarial-specific IgG4 but lower IgG3 and IgE levels, than adult worm-free individuals, independently of the presence of Mf. Low filarial-specific IgG1 and IgG2 levels were associated with the presence of Mf but independent of the presence/absence of adult worms. The filarial antibody responses were associated with the parasitological status of individuals but not with clinical symptoms such as hydroceles or limb lymphangitis or elephantiasis. The reduction of filarial-specific immunoglobulin levels was higher after treatment with diethylcarbamazine, than ivermectin, which likely reflects the better effect of the former on W. bancrofti adult worms. However, reduction of antibody levels was also observed in Mf-and adult worm-negative individuals. This could be due to the overall reduction of W. bancrofti transmission in the island where this study took place.

Og4C3 circulating filarial antigen was detected in the sera of 94.5% (259/274) of microfilaremic patients, 32% (239/751) of persons with presumption of filariasis, and 23% (11/48) of chronic filariasis patients. The antigen level was correlated with the microfilariae (Mf) density and patient age (P < .01). It remained stable in patients treated with microfilaricidal drugs. Og4C3 antigen, undetectable in Mf culture media, was demonstrated to be a rare somatic Mf antigen. It appears to be an excreted or secreted antigen from adult filaria. It could be used as a marker of infection and an indicator of adult worm burden.