Seroepidemiology of Toxoplasma in a coastal region of Haiti: multiplex bead assay detection of immunoglobulin G antibodies that recognize the SAG2A antigen.

Toxoplasma gondii is a globally distributed parasitic protozoan that infects most warm-blooded animals. We incorporated a bead coupled with recombinant SAG2A protein into our Neglected Tropical Disease (NTD) multiplex bead assay (MBA) panel and used it to determine Toxoplasma infection rates in two studies in Haiti. In a longitudinal cohort study of children aged 0-11 years, the infection rate varied with age reaching a maximum of 0·131 infections/year in children aged 3 years [95% confidence interval (CI) 0·065-0·204]. The median time to seroconversion was estimated to be 9·7 years (95% CI 7·6-∞). In a cross-sectional, community-wide survey of residents of all ages, we determined an overall seroprevalence of 28·2%. The seroprevalence age curve from the cross-sectional study also suggested that the force of infection varied with age and peaked at 0·057 infections/year (95% CI 0·033-0·080) at age 2·6 years. Integration of the Toxoplasma MBA into NTD surveys may allow for better estimates of the potential burden of congenital toxoplasmosis in underserved regions.

Factors associated with participation in a campaign of mass treatment against lymphatic filariasis, in Leogane, Haiti.

In October 2000, to interrupt transmission of Wuchereria bancrofti, an intense health-education campaign followed by a mass drug administration (MDA) with diethylcarbamazine and albendazole was undertaken in Leogane, Haiti. Three months after the MDA, which was the first in the study area, a knowledge-attitude-practice (KAP) survey, with a cluster-sample design and probability sampling, was undertaken, to determine the existing knowledge of the local residents, their attitudes toward the MDA, and the possible reasons for non-compliance. Questionnaire-based interviews were used to explore the KAP of 304 subjects (one randomly chosen resident aged > 14 years from each selected household) in 33 communities. Most (93%) of the interviewees were aware of filariasis and 72% knew at least one clinical sign of the disease. Awareness of the MDA was high (91%). The most frequently mentioned sources of information were other people (56%) and radio announcements (33%). More than 80% of the respondents encouraged other people to take the drugs distributed in the MDA and 63% had been treated. The primary reasons given for failing to take the drugs were absenteeism during the distribution (17%), use of contraceptive drugs (12%) and pregnancy (11%). In a multivariate analysis, being male [odds ratio (OR) = 3.3; 95% confidence interval (CI) = 1.5-7.4], knowing that a mosquito transmits the disease (OR = 2.6; CI = 1.2-5.4), and having learned about the MDA through posters and banners (OR = 2.9; CI = 1.2-7.5) were found to be positively associated with taking the drugs. Information from such post-treatment surveys should be useful in developing better health communication for subsequent MDA.

Influence of infection with non-filarial helminths on the specificity of serological assays for antifilarial immunoglobulin G4.

Serological assays based on the detection of immunoglobulin (Ig) G4 antibodies to crude filarial extracts are widely used for epidemiological and diagnostic purposes. We tested 195 samples collected in 1998 from an area of Brazil where filariasis is not endemic and 13 (6.7%) had levels of antifilarial IgG4 antibodies that were defined as positive. Both Strongyloides infection and the presence of Strongyloides antibody responses were associated with higher antifilarial antibody responses. None of the specimens had a positive response to the Brugia malayi recombinant antigen (Bm14). These data suggest that serodiagnostic assays based on the use of crude filarial antigens should be interpreted with caution because of the potential for cross-reactivity with Strongyloides.

Development and standardization of a rapid, PCR-based method for the detection of Wuchereria bancrofti in mosquitoes, for xenomonitoring the human prevalence of bancroftian filariasis.

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.

The Serologic response to Cryptosporidium in HIV-infected persons: implications for epidemiologic research.

Advances in serologic assays for Cryptosporidium parvum have made serology an attractive surveillance tool. The sensitivity, specificity, and predictive value of these new assays for surveillance of immunocompromised populations, however, have not been reported. Using stored serum specimens collected for the San Francisco Men's Health Study, we conducted a case-control study with 11 clinically confirmed cases of cryptosporidiosis. Based on assays using a 27-kDa antigen (CP23), the serum specimens from cases had a median response immunoglobulin (Ig) G level following clinical diagnosis (1,334) and a net response (433, change in IgG level from baseline) that were significantly higher than their respective control values (329 and -32, Wilcoxon p value = 0.01). Receiver operator curves estimated a cutoff of 625 U as the optimal sensitivity (0.86 [0.37, 1.0]) and specificity (0.86 [0.37, 1.0]) for predicting Cryptosporidium infection. These data suggest that the enzyme-linked immunosorbent assay technique can be an effective epidemiologic tool to monitor Cryptosporidium infection in immunocompromised populations.

Detection of serum IgG antibodies specific for Wolbachia surface protein in rhesus monkeys infected with Brugia malayi.

The mechanism of lymphedema development in individuals with lymphatic filariasis is presently poorly understood. To investigate whether Wolbachia, symbiotic bacteria living within filarial nematodes, may be involved in disease progression, Wolbachia-specific immune responses were assayed in a group of Brugia malayi-infected rhesus monkeys. Serum IgG antibodies specific for a major Wolbachia surface protein (WSP) were detected in 2 of 12 infected monkeys. It is interesting that both of these monkeys developed lymphedema after becoming amicrofilaremic. WSP-specific antibody responses were temporally associated with increases in antifilarial IgG1 antibodies as well as lymphedema development. These findings suggest that Wolbachia may be important in understanding disease caused by filarial worms.

Short report: documentation of iodine deficiency in Haitian schoolchildren: implication for lymphatic filariasis elimination in Haiti.

In this study we documented unexpected moderate-to-severe iodine deficiency in Haitian schoolchildren although they live in a coastal community where presumably they have access to iodine-containing seafood. This fact combined with the lack of an iodized salt supply and endemic lymphatic filariasis makes community distribution of diethylcarbamazine-fortified, iodized salt an attractive strategy for elimination of lymphatic filariasis and iodine deficiency disorders in this area of Haiti. Combining lymphatic filariasis elimination with other public health interventions is one strategy to increase its public health benefit and maximize the impact of limited public health resources.

Human T and B cell immunoreactivity to a recombinant 23-kDa Cryptosporidium parvum antigen.

Cryptosporidial infection in humans results in parasite-specific IgG, IgM, and IgA antibody responses, but little is known of the cell-mediated immune responses to cryptosporidial antigens. In a convenience sample of 35 Haitian residents, there was a high level of cryptosporidial exposure (>90%) as determined by immunoblot reactivity of serum against cryptosporidial antigens. An attempt was made to determine if there was a relationship between antibody and T cell-mediated responses to recombinant Cp23 antigen and how this correlated with reactivity to crude sporozoite antigen preparations (SAg). T cell reactivity was greater against SAg (57%) than to Cp23 (34.3%) as measured by [3H]thymidine incorporation. Proliferative responses to Cp23 were significantly correlated with SAg responses. By enzyme-linked immunosorbent assay, most persons had IgG responses to both SAg (91.4%) and to recombinant Cp23 (88.5%). Antibody responses were greater among persons who exhibited T cell responses to SAg and Cp23. This study demonstrates that recombinant Cp23 antigen could be a useful antigen for detection of both antibody and cell-mediated responses in epidemiologic studies.

Cryptosporidium parvum-specific antibody responses among children residing in Milwaukee during the 1993 waterborne outbreak.

A major gastroenteritis outbreak among >400,000 residents of Milwaukee, Wisconsin, in April 1993 was attributed to Cryptosporidium parvum oocysts in drinking water. Plasma specimens obtained from children (6 months to 12 years old) for routine blood lead level surveillance March-May 1993 were assayed by ELISA for levels of IgG antibody against the immunodominant Triton-17 and 27-kDa C. parvum antigens. Over a 5-week period, the seroprevalence for antibodies to the 2 antigens increased from 15% to 82% and from 17% to 87%, respectively, in samples from children living in southern ZIP code areas (n=218), whereas smaller increases (20% to 43% and 22% to 46%, respectively) were noted among samples from children living in northern ZIP code areas (n=335; P<.0001). The results demonstrate that C. parvum infection was much more widespread than previously appreciated and confirm that infection was associated with residence in the area served by the southern water treatment plant.

Enzyme immunoassay detection of antigen-specific immunoglobulin g antibodies in longitudinal serum samples from patients with cryptosporidiosis.

Cryptosporidium parvum is a protozoan parasite that causes diarrheal illness in a wide range of mammalian hosts, including humans. Characteristic serum immunoglobulin G (IgG) antibody responses to antigens in the 27- and 17-kDa size ranges have been shown to develop after infection, and several enzyme-linked immunosorbent assay (ELISA) and Western blot assay formats have been used to measure these IgG levels in human serum. Using a collection of serial samples from laboratory-confirmed cryptosporidiosis patients, we compared the results obtained by using two new ELISAs with those obtained with two different Western blot assays. When assayed with the large-format Western blot, 97% of the 67 patients had a demonstrable antibody response on at least one occasion. The Cp23 ELISA correctly identified 93% of the samples that had a 27-kDa response by Western blot and 100% of the negative samples. The Triton antigen ELISA detected 77% of the samples that had a 17-kDa response by Western blot and 88% of the negative samples. The sensitivity of the Triton antigen assay was higher for samples collected between 16 and 92 days after the onset of symptoms (96%). The minigel-format Western blot did not compare favorably with the large-format blot for the detection of antibodies to the 27-kDa antigen (71% sensitivity). A half-life of about 12 weeks was estimated for antibodies to both the 27- and 17-kDa antigens. We believe the Cp23 and Triton antigen ELISAs will be useful in epidemiologic studies of the prevalence of Cryptosporidium infection in the population.

The immunodominant 17-kDa antigen from Cryptosporidium parvum is glycosylphosphatidylinositol-anchored.

Cryptosporidium parvum is a protozoan parasite of the intestinal epithelium that has caused numerous outbreaks of diarrheal illness in humans. During our studies of the host immune response to C. parvum infection, we noted that two of the immunodominant surface antigens of the sporozoite stage of the parasite readily extract into Triton X-114. We recently cloned the immunodominant 17-kDa surface antigen and suggested that the carboxy-terminal peptide sequence may satisfy the requirements for GPI anchor addition. In the work presented here, we were able to show that the 17-kDa antigen could be metabolically labeled in vitro with tritiated ethanolamine and that the antigen contained myo-inositol. The antigen was cleaved by GPI-PLD but not by PI-PLC and it could be converted to a water soluble form by chemical deglycosylation. We suggest that the 17-kDa antigen is indeed GPI anchored and that the anchor contains an acylated inositol and either a lyso-acyl- or a diacyl-glycerol. We are currently working to determine what role the anchor may play in the human immune response to this antigen.

A community-based trial for the control of lymphatic filariasis and iodine deficiency using salt fortified with diethylcarbamazine and iodine.

To evaluate the effectiveness of salt fortified with diethylcarbamazine (DEC) and iodine for elimination of Bancroftian filariasis and iodine deficiency, all consenting residents of Miton, Haiti (n = 1,932) were given salt fortified with 0.25% diethylcarbamazine and 25 ppm of iodine for one year. Wuchereria bancrofti microfilaria prevalence and intensity, antigenemia, and urinary iodine were measured before and one year after salt distribution began. To measure the effect of DEC-fortified salt on adult worm motility, 15 microfilaria-positive men were examined by ultrasound of the scrotal area. Entomologic surveys were conducted to determine the proportion of W. bancrofti-infected Culex quinquefasciatus. After one year of treatment, the prevalence and intensity of microfilaremia were both reduced by more than 95%, while antigenemia levels were reduced by 60%. The motility of adult worms, as detected by ultrasound, was decreased, but not significantly, by DEC-fortified salt. The proportion of vector mosquitoes carrying infective stage larvae decreased significantly from 2.3% in the nine months before the intervention to 0.2% in the last three-month follow-up period. Iodine deficiency, which had been moderate to severe, was eliminated after one year of iodized salt consumption. The DEC-fortified salt was well accepted by the community and reduced microfilaremia and transmission to low levels in the absence of reported side effects. Based on these results, salt cofortified with DEC and iodine should be considered as a concurrent intervention for lymphatic filariasis and iodine deficiency elimination programs.

Cloning of the immunodominant 17-kDa antigen from Cryptosporidium parvum.

Infection with Cryptosporidium parvum causes a self-limiting diarrheal illness in immunocompetent humans and is associated with the development of a serum IgG antibody response dominated by the 27-kDa and 17-kDa parasite surface antigens. Antibodies against the 27-kDa and 17-kDa antigens may serve as useful markers for past infection in population-based studies of the risk factors associated with Cryptosporidium infection. A recombinant form of the 17-kDa antigen would be useful both in epidemiologic studies and in studies of the role of the humoral response in immunity. We have partially purified and sequenced the immunodominant 17-kDa surface antigen from sporozoites, and we have cloned a 975 bp open reading frame from C. parvum that includes all of the 17-kDa antigen peptide sequences. We show immunologic identity between a recombinant form of the protein and the native 17-kDa antigen. We conclude that the carboxy-terminal fragment of the cloned protein is the authentic 17-kDa antigen.

Evaluation of the whole blood filariasis ICT test for short-term monitoring after antifilarial treatment.

The immunochromatographic (ICT) filariasis test is a rapid screening tool that will be useful for defining the prevalence and distribution of Wuchereria bancrofti as part of the global program to eliminate lymphatic filariasis. To address questions about its usefulness for monitoring control programs, we used the ICT filariasis test to assess residual antigen levels following antifilarial treatment. Our results demonstrate that antigen levels persist in microfilaria-negative persons for up to three years after treatment. Different strategies for monitoring control programs may have to be considered.

An analysis of the safety of the single dose, two drug regimens used in programmes to eliminate lymphatic filariasis.

This review of the safety of the co-administration regimens to be used in programmes to eliminate lymphatic filariasis (albendazole + ivermectin or albendazole + diethylcarbamazine [DEC]) is based on 17 studies conducted in Sri Lanka, India, Haiti, Ghana, Tanzania, Kenya, Ecuador, the Philippines, Gabon, Papua New Guinea, and Bangladesh. The total data set comprises 90,635 subject exposures and includes individuals of all ages and both genders. Results are presented for hospital-based studies, laboratory studies, active surveillance of microfilaria-positive and microfilaria-negative individuals, and passive monitoring in both community-based studies and mass treatment programmes of individuals treated with albendazole (n = 1538), ivermectin (9822), DEC (576), albendazole + ivermectin (7470), albendazole + DEC (69,020), or placebo (1144). The most rigorous monitoring, which includes haematological and biochemical laboratory parameters pre- and post-treatment, provides no evidence that consistent changes are induced by any treatment; the majority of abnormalities appear to be sporadic, and the addition of albendazole to either ivermectin or DEC does not increase the frequency of abnormalities. Both DEC and ivermectin show, as expected, an adverse event profile compatible with the destruction of microfilariae. The addition of albendazole to either single-drug treatment regimen does not appear to increase the frequency or intensity of events seen with these microfilaricidal drugs when used alone. Direct observations indicated that the level of adverse events, both frequency and intensity, was correlated with the level of microfilaraemia. In non microfilaraemic individuals, who form 80-90% of the 'at risk' populations to be treated in most national public health programmes to eliminate lymphatic filariasis (LF), the event profile with the compounds alone or in combination does not differ significantly from that of placebo. Data on the use of ivermectin + albendazole in areas either of double infection (onchocerciasis and LF), or of loiais (with or without concurrent LF) are still inadequate and further studies are needed. Additional data are also recommended for populations infected with Brugia malayi, since most data thus far derive from populations infected with Wuchereria bancrofti.

Assessment of combined ivermectin and albendazole for treatment of intestinal helminth and Wuchereria bancrofti infections in Haitian schoolchildren.

This randomized, placebo-controlled trial investigated the efficacy and nutritional benefit of combining chemotherapeutic treatment for intestinal helminths (albendazole) and lymphatic filariasis (ivermectin). Children were infected with Ascaris (29.2%), Trichuris (42.2%), and hookworm (6.9%), with 54.7% of children having one or more of these parasites. Wuchereria bancrofti microfilaria were found in 13.3% of the children. Children were randomly assigned to treatment with placebo, albendazole, ivermectin, or combined therapy. Combination treatment reduced the prevalence of Trichuris infections significantly more than either drug alone. Combination therapy also significantly reduced the prevalence and density of W. bancrofti microfilaremia compared with placebo or ivermectin alone. Only combination therapy resulted in significantly greater gains in height (hookworm-infected children) or weight (Trichuris-infected children) compared with the placebo group. Combined albendazole and ivermectin was a more efficacious treatment for intestinal helminth and W. bancrofti infections in children and resulted in nutritional benefits not found with either drug alone.

Cyclospora cayetanensis infections in Haiti: a common occurrence in the absence of watery diarrhea.

Stool samples from a population-based cohort of mothers and children living in Leogane, Haiti were tested for Cyclospora cayetanensis from January 1997 through January 1998. Data on gastrointestinal symptoms were also collected. During the winter months of January to March, the infection was detected in 15-20% of the persons sampled. Most infections did not appear to be causing diarrhea and most infected persons had few oocysts detectable in concentrates of stool. The infection appears to have marked seasonality, with highest rates during the driest and coolest time of the year. It may be that in this tropical setting, high summer temperature is the critical environmental factor that influences the seasonality of infection. This study demonstrates that Cyclospora infections in Haiti are common in the general population.

Detection by enzyme immunoassay of serum immunoglobulin G antibodies that recognize specific Cryptosporidium parvum antigens.

Human infection with Cryptosporidium parvum usually elicits characteristic immunoglobulin G (IgG), IgA, and IgM antibody responses against two sporozoite surface antigens with apparent molecular masses of approximately 27 and 17 kDa. We have determined that these two antigens are actually complex families of related antigens. We have developed two new enzyme-linked immunosorbent assays (ELISAs) for the detection and quantitation of serum IgG antibodies against both antigens. The assays utilize a recombinant form of the 27-kDa antigen and a partially purified native fraction isolated from sonicated whole oocysts that contains 17-kDa antigen. An immunoblot assay previously developed in our laboratory served as the reference, or "gold standard," seroassay for the assessment of the new ELISAs. Positive responses with the recombinant-27-kDa-antigen ELISA were correlated with the immunoblot results for the 27-kDa antigen, with a sensitivity and specificity of 90 and 92%, respectively. Similarly, positive responses with the partially purified native-17-kDa-antigen ELISA correlated with the immunoblot results for the 17-kDa antigen, with a sensitivity and specificity of 90 and 94%, respectively. For both ELISAs the median IgG antibody levels for serum sets collected during outbreaks of waterborne C. parvum infection were at least 2.5-fold higher than the levels determined for a nonoutbreak set. Using the immunoblot as the "gold standard," the new ELISAs were more specific and, in the case of the 27-kDa-antigen ELISA, more sensitive than the crude oocyst antigen ELISA currently in use. These assays will be useful in future epidemiologic studies.

The antibody response to 27-, 17-, and 15-kDa Cryptosporidium antigens following experimental infection in humans.

Previous studies have suggested that persons infected with Cryptosporidium parvum develop antibody responses to 27-, 17-, and 15-kDa C. parvum antigens. Studies of volunteers infected with Cryptosporidium species provided an opportunity to evaluate the relationship between antibody reactivity to these antigens and infection outcome. As monitored by immunoblot, increases in specific antibody reactivity were more prevalent among volunteers who developed signs and symptoms of cryptosporidiosis (n = 11) than among asymptomatic infected (n = 7; P = .05) or oocyst-negative volunteers (n = 11; P = .02). Volunteers with preexisting IgG antibody to the 27-kDa antigen excreted fewer oocysts than volunteers without this antibody (P = .003). IgG reactivity to the 17-kDa antigens and IgM reactivity to the 27-kDa antigens were higher at day 0 for asymptomatic infected persons than for those who developed symptoms (P = .03 and P = .04, respectively). These results suggest that characteristic antibody responses develop following C. parvum infection and that persons with preexisting antibodies may be less likely to develop illness.