Effects of gamma radiation on Brugia malayi infective larvae and their intracellular Wolbachia bacteria.

Prior studies have shown that irradiated filarial larvae are developmentally stunted but capable of inducing partial immunity to filariasis in animals. The mechanisms for these effects are poorly understood. Recent studies suggest that intracellular Wolbachia bacteria are necessary for the normal development, reproduction and survival of filarial nematodes. The purpose of this study was to examine the effects of irradiation on Wolbachia in Brugia malayi infective larvae (L3) and on L3 development. The L3 were exposed to 0, 25, 35, 45, 55, 65 or 75 krad of gamma irradiation from a (137) Cesium source and cultured in vitro at 37 degrees C in NCTC/IMDM medium with 10% FCS for 12 days. Irradiation prevented molting of L3 to the L4 stage in a dose-dependent manner. Electron microscopy studies showed that irradiation damaged Wolbachia (25 krad) or cleared them from worm tissues (45 krad). In addition, majority of the irradiated L3s failed to develop the L4 cuticle. Real-time PCR studies showed that irradiation reduced Wolbachia DNA in worm tissues. Parallel in vivo studies confirmed decreased development of irradiated L3 in jirds, with associated effects on Wolbachia. Jirds injected s.c with normal L3 developed antibodies to Wolbachia surface protein (wsp) shortly after the onset of microfilarial patency. In contrast, jirds injected with irradiated L3 did not develop microfilaremia or antibodies to wsp. Additional studies are needed to test the hypothesis that irradiation retards growth and development of filarial L3 by killing Wolbachia.

Filariasis elimination in Egypt: impact of low microfilaraemics as sources of infection for mosquitoes.

The elimination strategy for lymphatic filariasis aims at reducing blood microfilaraemia to levels at which vector transmission cannot be sustained. We aimed to determine whether patients with pre-treatment low or ultra-low microfilaria (MF) counts could be a reservoir of infection after mass drug administration (MDA) with a combined regimen. Laboratory-reared mosquitoes were fed on 30 volunteers after 2 rounds of MDA. Microfilaria uptake, infectivity rates and number of Wuchereria bancrofti L3 per mosquito were assessed. One year after MDA-1, 6 subjects transmitted MF, but up to 9 months after MDA-2 transmission failed. Six months after MDA-2 > 90% had clear MF smears and either failed to transmit MF or transmitted MF that did not develop to L3. We conclude that the transmission cycle is seriously weakened after MDA-2.

Filariasis elimination in Egypt: impact of low microfilaraemics as sources of infection for mosquitoes

The elimination strategy for lymphatic filariasis aims at reducing blood microfilaraemia to levels at which vector transmission cannot be sustained. We aimed to determine whether patients with pre-treatment low or ultra-low microfilaria [MF] counts could be a reservoir of infection after mass drug administration [MDA] with a combined regimen. Laboratory-reared mosquitoes were fed on 30 volunteers after 2 rounds of MDA. Microfilaria uptake, infectivity rates and number of Wuchereria bancrofti L3 per mosquito were assessed. One year after MDA-1, 6 subjects transmitted MF, but up to 9 months after MDA-2 transmission failed. Six months after MDA-2 > 90% had clear MF smears and either failed to transmit MF or transmitted MF that did not develop to L3. We conclude that the transmission cycle is seriously weakened after MDA-2

Evaluation of a rapid-format antibody test and the tuberculin skin test for diagnosis of tuberculosis in two contrasting endemic settings.

OBJECTIVE AND SETTING: We evaluated a rapid-format antibody card test and the tuberculin skin test for diagnosis of active tuberculosis (TB) in high (Cairo, Egypt) and low (St. Louis, USA) prevalence areas. DESIGN: Prospective study of hospitalized TB patients and controls with other chest diseases. RESULTS: Test performance varied significantly in the two study sites. The antibody test detected 87% of 71 smear-positive pulmonary TB cases (86% of smear-negative pulmonary cases and 48% of TB meningitis cases) in Egypt; specificity was 82%. The tuberculin test was highly sensitive in Egypt in subjects with pulmonary TB (100%) but not in those with meningitis (23%); specificity was 70%. The sensitivity and specificity of the antibody test in St. Louis were 29% and 79%, respectively; 50% of St. Louis TB cases and 15% of controls had positive tuberculin tests. CONCLUSIONS: This convenient antibody card test may have value for diagnosis of patients suspected of having TB in high prevalence areas like Egypt. However, the specificity of the test is too low for it to be useful as a screening test. Our results suggest that neither the antibody test nor the tuberculin test have much diagnostic utility in low prevalence settings like St. Louis.

Long-term efficacy of single-dose combinations of albendazole, ivermectin and diethylcarbamazine for the treatment of bancroftian filariasis.

In a 'blinded' trial (in Sri Lanka, 1996-98) of 47 male asymptomatic microfilaraemic subjects with Wuchereria bancrofti infection, the safety, tolerability and filaricidal efficacy of 3 single-dose combination regimens were compared: albendazole 400 mg with ivermectin 200 micrograms/kg, albendazole 400 mg with diethylcarbamazine citrate (DEC) 6 mg/kg or albendazole 600 mg with ivermectin 400 micrograms/kg. Treated subjects were followed-up for 24 months. This represents the first long-term study using combinations of albendazole with DEC or ivermectin in the above doses against bancroftian filariasis. All subjects had pre-treatment microfilaria (mf) counts over 100/mL. All 3 treatments significantly reduced mf counts, with the albendazole-DEC-treated group showing the lowest mf levels at 18 and 24 months post-treatment. Filarial antigen tests suggested that all 3 treatments had significant activity against adult W. bancrofti; albendazole-DEC combination had the greatest activity according to this test, with antigen levels decreasing to 30.5% of pre-treatment antigen levels, 24 months after therapy. All 3 treatments were clinically safe and well tolerated. These results suggest that a single dose of albendazole 400 mg together with DEC 6 mg/kg is a safe and effective combination for suppression of microfilaraemia of bancroftian filariasis that could be considered for use in filariasis control programmes based on mass treatment of endemic populations.

Detection of Wuchereria bancrofti in mosquitoes by the polymerase chain reaction: a potentially useful tool for large-scale control programmes.

Focally endemic bancroftian filariasis is targeted for elimination in the Nile delta of Egypt. Improved methods are needed for identifying endemic villages to be included in the control programme and for monitoring its success. We have evaluated the performance of a polymerase chain reaction (PCR) assay in estimating Wuchereria bancrofti infection in pools of Culex pipiens (1-25 females) from 2 adjacent villages with high (El Qolzom, 10.8%) and low (Kafr Shorafa, 2.1%) prevalence rates of human filariasis. This assay detects a repeated sequence in W. bancrofti deoxyribonucleic acid (DNA). Mosquitoes resting within houses were captured by aspiration and pooled by house. Houses were classified as positive or negative for human filarial infection based on night blood examinations of residents. The assay detected parasite DNA in mosquitoes from 60% of 25 infected houses and 24% of 25 uninfected houses. PCR processing of mosquitoes caught within houses of unknown filariasis infection status (44 in El Qolzom, 37 in Kafr Shorafa) identified 31.8% and 8.1% of houses, respectively, as containing infected mosquitoes. These results support the validity of the PCR assay for evaluating filarial prevalence in different villages. C. pipiens collected outdoors in dry ice-baited traps and tested by PCR (266 in Qolzom, 82 in Kafr Shorafa) did not contain parasite DNA. Pools of female mosquitoes (296 in Qolzom, 240 in Kafr Shorafa) captured in oviposition traps were also negative. We concluded that the PCR based assay is a powerful epidemiological tool that can be used for evaluating W. bancrofti infection in villages in the Nile delta and for monitoring the application of control programmes in filariasis endemic areas.

A rapid-format antibody card test for diagnosis of onchocerciasis.

Improved methods are needed for field diagnosis of onchocerciasis, to support efforts aimed at elimination of the disease. A rapid-format card test was evaluated that detects IgG4 antibodies to recombinant Onchocerca volvulus antigen Ov16 with serum samples from patients with onchocerciasis and with various types of control serum samples. The sensitivity of the test with serum samples from 106 microfilariae-positive subjects was 90.6%. The test was equally sensitive with serum samples obtained from patients in Africa and Latin America. Specificity was excellent; positive tests were observed for 2 of 38 serum samples from patients with other filarial infections and for 1 of 23 serum samples from patients with nonfilarial helminth infections. The 3 "false-positive" serum samples were from West Africans who could have been coinfected with onchocerciasis. No positive tests were observed with nonendemic serum samples from normal adults, patients with autoimmune disorders, or patients with the hyper-IgE syndrome. This new test holds great promise as a simple tool for diagnosis of onchocerciasis.

The impact of environmental and entomological factors on intervillage filarial focality in the Nile Delta.

In the Nile Delta of Egypt, levels of W. bancrofti infection in humans vary among nearby villages. Ecological and entomological factors that might explain variability between adjacent villages, El Qolzom (QOL) and Kafr Shorafa (KSH) with respective 10.8% and 2.1% microfilaria (MF) prevalence were examined. The epidemiological study covered 127 and 79 houses scattered in QOL and KSH, respectively, and described 25 items relating to housing characters, socio-economic state and human activities. It revealed that QOL is more rural than KSH, and therefore would be more favorable to the vector mosquito population and hence, filarial parasite transmission in QOL. Weekly records (N = 81 and 62 for QOL and KSH, respectively) of ambient temperature, relative humidity and wind speed taken at sunset, over 3 months during summer, revealed no significant variation between villages. Those measured at sunrise revealed significant, although inconsistent, differences at a particular month, but no difference over the whole period. Whether climatological conditions could have influenced mosquito bionomics in the study villages is questionable. Abundance of female Cx. pipiens collected weekly by standard sampling methods using 247 and 240 dry ice-baited CDC trap-nights in QOL and KSH, respectively, oral aspiration from within 346 and 304 respective house-nights, and 65 and 40 respective ovitrap-nights, did not vary significantly over the whole study period. Daily survival and survival to infectivity rates of wild-caught mosquitoes were based on parity and were generally more elevated in QOL than KSH. Monthly records of abundance and survival seemed to favor filaria transmission by mosquitoes in QOL. Autogeny amounted to 6.5 and 20% for QOL and KSH, respectively. Experimental infection of Cx. pipiens from the study villages with W. bancrofti revealed that QOL females were 3.3 times more efficient vectors than KSH ones, mainly because QOL mosquitoes survived longer. The ultimate outcome of observed entomological factors might explain its preponderance in QOL.

A comparison of newer tests for the diagnosis of onchocerciasis.

Samples of human serum, skin and urine, collected in Cameroon, were used to assess the value of some newer methods for the diagnosis of onchocerciasis. Parasite DNA was detected in skin snips and urine by PCR, and parasite antigen was detected in serum and urine by immunoblotting. Serum concentrations of IgG4 antibodies reacting with recombinant Onchocerca volvulus antigens (OC3.6 and OC9.3) were also measured, using an ELISA. The PCR-based tests of skin snips and the serological tests for antigen and antibody tests showed higher sensitivities (90%-100%) than the urine PCR (14%) or the urine antigen test (68%). Although antibody detection is much easier to perform than tests based on PCR or antigen detection, the latter have an advantage in that they are only positive in people with current infections. Thus, antibody testing may be more useful for screening populations for infection or exposure to O. volvulus, whereas PCR and antigen testing are potentially more useful for diagnosis of infections in individuals and for monitoring the success of therapy.

Human antibody responses to Wuchereria bancrofti infective larvae.

Human IgG antibody responses to Wuchereria bancrofti third stage infective larvae (L3) surface and somatic antigens were studied by indirect immunofluorescence (IFA) and immunoblot with endemic Egyptian sera (n = 115) with the aim of identifying targets of protective immunity. Human sera variably recognized 14 major bands in L3 by immunoblot. The statistical significance of group differences in antibody prevalence was assessed by the chi-squared test. Children and young adults (aged 10-20 years) tended to have antibodies to more L3 somatic antigens than older adults, with significant differences for bands at 66, 60 and 5 kDa. Infected subjects had more consistent antibody responses to antigens at 55, 50 and 6 kDa than endemic normal subjects with negative serum filarial antigen tests, who are presumed to be uninfected. A 5 kDa antigen was preferentially recognized by the latter group. Antibodies to L3 surface antigens were equally prevalent in uninfected children (75%) and adults (90%) but less prevalent in people with microfilaremia (38%) than in amicrofilaremic subjects with or without filarial antigenemia (81%) (P < 0.001). IFA-positive sera showed significantly enhanced recognition of antigens at 66, 40 and 14 kDa in immunoblots relative to IFA-negative sera. Additional studies are needed to further characterize antigens identified in this study and to establish whether they are indeed targets of protective immunity in humans.

Paper chromatography hybridization: a rapid method for detection of Onchocerca volvulus DNA amplified by PCR.

Prior studies have shown that Onchocerca volvulus DNA can be detected in skin snips and in black flies after polymerase chain reaction (PCR) with primers specific for repeated "O-150" DNA sequences. We have adapted a paper chromatography hybridization assay (PCHA) to detect amplified O-150 DNA and compared this method to two established methods, namely agarose gel electrophoresis (AGE) and hybridization enzyme-linked immunosorbent assay (ELISA). The minimum amounts of purified O-150 DNA detected by PCHA, AGE, and ELISA were 5, 10, and 2 ng, respectively. The three methods had similar estimated sensitivities for detecting O. volvulus DNA amplified from skin snips from African subjects with onchocerciasis (88%, 84%, and 91%, respectively). No false positive results were observed with skin snips from uninfected control subjects. The paper chromatography hybridization assay detects PCR products in 30 minutes without electricity or special equipment. This technology brings DNA detection a step closer to widespread use in field settings.

Gender-specific gene expression in Brugia malayi.

Brugia malayi is a mosquito-borne filarial nematode that causes lymphatic filariasis and elephantiasis in humans. The purpose of this study was to identify and characterize genes that are expressed differentially in male and female B. malayi in hopes of gaining new insight into the reproductive biology of the parasite. Two approaches were used. A 5' differential display PCR (splice leader differential display PCR, SL DD-PCR) was performed by PCR with splice leader and random primers on cDNA templates, and electronic subtraction was performed on expressed sequence tag (EST) cluster databases developed by the Filarial Genome Project (FGP). Gender-specific expression of candidate clones was confirmed by RT-PCR for six of 22 (27%) clones identified by DD and in seven of 15 (47%) clones identified by electronic subtraction. One clone was identified by both methods. Several female-specific clones had homology to known nematode genes that encode a fatty acid binding protein, a high mobility group protein, an eggshell protein, a glutamate-gated ion channel, and a collagen. However, most of the clones have no significant homology to known genes or proteins in computer databases. This project has confirmed the value of SL DD-PCR and electronic subtraction for analysis of gene expression in filariae. These two complimentary techniques may be generally applicable to the study of gender-specific (and by analogy stage specific) gene expression in other nematodes.

Immune responses to Brugia malayi paramyosin in rodents after DNA vaccination.

Immunization with recombinant Brugia malayi paramyosin protein (BM5) induces partial immunity to this filarial nematode in jirds. The present study examined the effects of intramuscular immunization with plasmid DNA that encodes BM5. DNA-immunized mice produced strong antibody and cell-mediated responses to paramyosin. The protective activity of DNA vaccination with BM5 was tested in jirds. Vaccinated jirds produced strong antibody responses to paramyosin, but adult worm recoveries after challenge were not decreased in vaccinated animals relative to controls. These studies show that DNA vaccination can induce immune responses to filarial antigens in rodents. Further efforts will be needed to achieve the goal of inducing protective immunity to filariasis with this promising new technology.

A longitudinal study of Bancroftian filariasis in the Nile Delta of Egypt: baseline data and one-year follow-up.

We initiated a longitudinal study of Bancroftian filariasis to improve understanding of dynamics and risk factors for infection in villages near Cairo, Egypt. Baseline prevalence rates for microfilaremia and filarial antigenemia for 1,853 subjects more than 9 years of age were 7.7% and 11.2%, respectively. Microfilaria counts, antigen levels, and microfilaremia incidence over a 1-year period were all significantly lower in older people. These findings suggest that humans develop partial immunity to Wuchereria bancrofti over time. One-year incidence rates for microfilaremia and antigenemia were 1.8% and 3.1%, respectively. Filarial antigenemia, IgG4 antibody to recombinant antigen BmM14, and household infection were all significant risk factors for microfilaremia incidence. Microfilaria counts and parasite antigen levels were significantly reduced by diethylcarbamazine therapy, but many infected subjects refused treatment, and most treated people were still infected one year later. Incident infections approximately balanced infections lost to produce an apparent state of dynamic equilibrium.

Human antibody responses to Brugia malayi antigens in brugian filariasis.

Human antibody responses to Brugia malayi antigens were studied with sera from a Brugia endemic area in South India. Patients with clinical filariasis had significantly higher IgE and lower IgG4 levels to adult worm antigens than people with asymptomatic microfilaraemia. Intermediate antibody levels were observed in endemic normals. A majority of sera from each clinical group contained IgG antibodies to surface antigens of infective larvae (L3) by IFAT. IgG immunoblot studies did not reveal group differences in L3 antigen recognition. IgE antibodies bound to a subset of antigens bound by IgG. IgE antibodies in sera from clinical filariasis patients preferentially bound to L3 antigens at 200, 97, 68 and 58 kDa compared with sera from microfilaria carriers. These results are consistent with prior studies of antibody responses in filariasis and add new information on the targets of IgG and IgE antibodies to L3 antigens in brugian filariasis.

Field evaluation of a rapid-format kit for the diagnosis of bancroftian filariasis in Egypt.

The AMRAD-ICT Filariasis Test (ICT-Fil) is a new, rapid-format card test for the detection of bancroftian antigenaemia in human blood. We evaluated the performance of the test under field conditions in Egypt by comparing 1813 endemic and 102 nonendemic participants. Endemic participants were tested for microfilaraemia (thick smear and membrane filtration) and serum antigenaemia (ELISA). The infection rates detected were 2.8% by thick smear, 3.5% by membrane filtration, 8.8% by ELISA and 9.0% by ICT-Fil. The card test detected antigenaemia in 98.0% and 95.3% of microfilaraemia carriers testing positive by thick smear and blood filtration respectively. Nonendemic participants were ICT-Fil negative. Identical results were obtained for 173 out of 184 (94%) endemic participants tested by the serum and whole blood ICT-Fil versions.

Field evaluation of a rapid-format kit for the diagnosis of bancroftian filariasis in Egypt

The AMRAD-ICT Filariasis Test [ICT-Fil] is a new, rapid-format card test for the detection of bancroftian antigenaemia in human blood. We evaluated the performance of the test under field conditions in Egypt by comparing 1813 endemic and 102 nonendemic participants. Endemic participants were tested for microfilaraemia [thick smear and membrane filtration] and serum antigenaemia [ELISA]. The infection rates detected were 2.8% by thick smear, 3.5% by membrane filtration, 8.8% by ELISA and 9.0% by ICT-Fil. The card test detected antigenaemia in 98.0% and 95.3% of microfilaraemia carriers testing positive by thick smear and blood filtration respectively. Nonendemic participants were ICT-Fil negative. Identical results were obtained for 173 out of 184 [94%] endemic participants tested by the serum and whole blood ICT-Fil versions

Efficacy of single dose combinations of albendazole, ivermectin and diethylcarbamazine for the treatment of bancroftian filariasis.

In a 'blind' trial on 50 male asymptomatic microfilaraemic subjects with Wuchereria bancrofti infection, the safety, tolerability and filaricidal efficacy of a single dose of albendazole (alb) 600 mg alone or in combination with ivermectin (iver) 400 micrograms/kg or diethylcarbamazine citrate (DEC) 6 mg/kg was compared with a single dose of the combination DEC 6 mg/kg and iver 400 micrograms/kg over a period of 15 months after treatment. All but one subject, with 67 microfilariae (mf)/mL, had pre-treatment counts > 100 mf/mL. All 4 treatments significantly reduced mf counts, but alb/iver was the most effective regimen for clearing mf from night blood: 9 of 13 subjects (69%) were amicrofilaraemic by membrane filtration 15 months after treatment compared to one of 12 (8%), 3 of 11 (27%), and 3 of 10 (30%) in the groups treated with alb, alb/DEC, and DEC/iver, respectively. Filarial antigen tests suggested that all 4 treatments had significant activity against adult W. bancrofti; alb/DEC had the greatest activity according to this test, with antigen levels decreasing by 77% 15 months after therapy. All 4 regimens were well tolerated and clinically safe, although mild, self-limited systemic reactions were observed in all treatment groups. These results suggest that alb/iver is a safe and effective single dose regimen for suppression of microfilaraemia in bancroftian filariasis that could be considered for control programmes. Additional benefits of this combination are its potent, broad spectrum activity against intestinal helminths and potential relative safety in areas of Africa where DEC cannot be used for filariasis control because of co-endemicity with onchocerciasis or loiasis.