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.

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.

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.

Zoonotic Brugia infection in western Michigan.

A case of zoonotic lymphatic filariasis is reported from a resident of Michigan. Numerous sections of a small, nongravid female worm, measuring approximately 65 microns in diameter, were identified in histological preparations of an excised inguinal lymph node. Based on its location, small size, thin cuticle, small number of muscle cells, and paired uterine tubes, the worm was identified as a Brugia species, undoubtedly of zoonotic origin from the local animal population. The pathological response of the host to the parasite was one of hyperplastic lymph nodes with a focal granulomatous reaction. This is the first such case to be reported from Michigan and further expands the geographical range of states in which zoonotic Brugia infection have been reported.

Zoonotic Brugian lymphadenitis. An unusual case with florid monocytoid B-cell proliferation.

Human infection with a zoonotic Brugia species in the United States is uncommon. Positive identification of the filarial nematode is required for histopathologic diagnosis. Many cases may go unrecognized because of the nonspecific clinical manifestations and the nondiagnostic histologic changes occurring in involved lymph nodes. A case of zoonotic Brugia lymphadenitis is described in a patient from Rhode Island, in which a small nongravid female worm was identified in a lymph node biopsy specimen. The lymph node also showed a spectrum of reaction changes including the presence of florid monocytoid B-cell proliferation, which has not been described in association with zoonotic Brugian filariasis.

In vitro granuloma formation using defined antigenic nidi.

The in vitro granuloma model provides an excellent tool to study granulomatous hypersensitivity. In our studies of granuloma formation in schistosomiasis, we have demonstrated the utility of the model with respect to analysis of human PBL responses, cloned T cells, purified antigens of the parasite, and regulatory mechanisms. We have also presented data which demonstrate the applicability of this model to other antigenic systems. While the suggestion that this model may be suitable for the study of sarcoidosis may be premature, we would propose that the system may be adapted to screen potentially causative antigens. Furthermore, with the identification of the critical antigen(s), the in vitro granuloma model will facilitate studies at the clinical level.

Proliferative responsiveness of lymphocytes from Cryptosporidium parvum-exposed mice to two separate antigen fractions from oocysts.

We assessed lymphoproliferative responsiveness of lymphocytes from the spleen and lymph nodes of inbred neonatal SWR/J H-2q mice at various times postinoculation (PI) using Cryptosporidium parvum oocysts. The lymphocytes were cultured in vitro with a water-soluble (SO) and a water-insoluble (IN) antigen fraction. The IN fraction was prepared by solubilizing particulates, the sediment obtained after centrifuging disrupted oocysts, in urea. Both fractions were characterized using silver stain and enzyme-linked immunoelectrotransfer blot (EITB) with hyperimmune rabbit anti-oocyst serum, monoclonal antibody specific to a 23-kD antigen, and serum from patients with symptoms of cryptosporidiosis. The EITB showed that the antigens in the IN fraction differed both quantitatively and qualitatively from those in the SO fraction. Lymphocytes from lymph nodes of exposed mice cultured with the SO fraction exhibited a significant (P < 0.05) and antigen-specific response compared with those from unexposed mice at days 10, 19, 22, and 28 PI. The response to the IN fraction of lymphocytes from lymph nodes of exposed mice was not as consistent as that to the SO fraction but showed a significant (P < 0.05) and antigen-specific response at days 10 and 19 PI. No significant response occurred when splenic lymphocytes were cultured with SO or IN fractions. These results show that lymphocytes from lymph nodes of mice exposed to Cryptosporidium parvum oocysts proliferate when cultured in vitro with soluble or particulate antigens prepared from oocysts.

Age-specific prevalence of antigenemia in a Wuchereria bancrofti-exposed population.

Antigen detection assays serve as a useful adjunct to blood examinations for studies of filariasis, in terms of the diagnostic and epidemiologic information provided. We examined the utility of the Og4C3 antigen detection enzyme-linked immunosorbent assay for field studies and analyzed the distribution of Wuchereria bancrofti antigenemia in a Haitian population. Using serum samples collected following venipuncture, antigenemia levels were correlated with microfilaremia (P < 0.001). The microfilariae had a pronounced nocturnal periodicity while the sensitivity of the antigen assay was the same whether serum samples were collected during the day or at night. To determine whether the Og4C3 assay could be used in conjunction with fingerprick blood examinations, nocturnal blood surveys were conducted. Of 419 persons surveyed, 207 (49.4%) were antigen-positive with the Og4C3 assay. Serum specimens from all 121 microfilaremic individuals were antigen positive (100% sensitivity). The age prevalence of antigenemia increased from 24.5% for 1-5-year-old children to 70% for persons greater than 50 years of age. These results demonstrate that the Og4C3 assay is a sensitive tool for the detection of infection and raise questions about the expression of protective immunity in populations exposed to infection.

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.

Heightened anti-filarial immune responsiveness in a Haitian pediatric population.

The immunological consequences of exposure to filarial infection were examined by cross-sectional serological studies. Serum samples from 121 pediatric patients (18 months-15 years of age) were analyzed in parallel with a panel of sera from adults residing in the same area of Haiti. Parasite antigen specific IgG and IgE levels were determined by ELISA. IgG levels in children were significantly elevated in humoral immunoreactivity to Brugia pahangi extracts compared to adults. In addition, anti-filarial IgG levels in amicrofilaremic children were significantly greater than in microfilaremic children. In contrast, IgG levels in adults were equivalent independent of microfilaremic status. Anti-filarial IgE levels in sera from both children and adults were low in comparison to that of a subject with tropical pulmonary eosinophilia and were unrelated to clinical status. No correlations were found between humoral responses and age, sex, or degree of parasitemia. Sera from amicrofilaremic children and, to a lesser extent, adults recognize more antigens, particularly those of high molecular weight (greater than 55 kDa), than sera from microfilaremic patients.

Analysis of isotype-specific antifilarial antibody levels in a Haitian pediatric population.

Previous studies of antifilarial antibodies in a pediatric population residing in an area with endemic Wuchereria bancrofti filariasis have demonstrated age related shifts in antifilarial immunity. To further characterize humoral responses in Haitian children, serum samples from 129 patients (3 months-15 years of age) were analyzed by ELISA for isotype-specific antifilarial antibody responses. Age-stratified analysis of geometric mean antibody titers showed significant increases in antibody titers of all isotypes with age in the amicrofilaremic population. Antifilarial IgG1, 2, and 3 levels were higher in amicrofilaremic children than in microfilaremic children, significantly so for IgG2 and IgG3. In contrast, IgG4 antibody levels were higher in microfilaremic subjects than in amicrofilaremic subjects. A multivariate, unconditional, logistic regression model was developed from these data to predict infection status. The model correctly classified 91.6% of the amicrofilaremic subjects, but only 55.6% of the microfilaremic subjects.

Bancroftian filariasis: long-term effects of treatment with diethylcarbamazine in a Haitian population.

Two groups of Wuchereria bancrofti-infected Haitians who had undergone treatment with diethylcarbamazine (DEC) were followed for up to five years after treatment to document the long-term effects of treatment on adult worms and microfilariae and on the recurrence of infection. One group of 69 persons who had received 12 daily treatments had a significant decrease in microfilaria levels until year 4, when a small number of individuals experienced a resurgence of this parasite stage in the peripheral blood. In a second group of 57 persons who had been treated weekly for 12 consecutive weeks, there was a greater reduction in the microfilaria levels following treatment, and for the full four years of followup, these levels remained more depressed than those of the group that received daily treatment. Our results indicate that DEC kills or permanently sterilizes adult W. bancrofti. Furthermore, these results demonstrate conclusively that in Haiti, the use of DEC provides long-term benefits to treated persons, even though they continue to reside in an area with endemic filariasis.

Clearance of Wuchereria bancrofti antigen after treatment with diethylcarbamazine or ivermectin.

The present study was undertaken to assess the relationship between microfilarial clearance and clearance of circulating filarial antigen from the blood of Wuchereria bancrofti-infected persons following chemotherapy with either diethylcarbamazine or ivermectin. Patients received either 12 weekly doses of 6 mg/kg of diethylcarbamazine (DEC), a single dose of 6 mg/kg of DEC, a single dose of 420 microg/kg of ivermectin, or 20 microg/kg of ivermectin, followed by 6 mg/kg of DEC five days later. Microfilarial clearance was marked in all groups, but was significantly less in the single-dose DEC. In contrast, as monitored by the Og4C3 monoclonal anitbody assay, clearance of circulating filarial antigen was highly variable, not only between groups but within each group. As a result, there were few statistically significant differences in antigen clearance between groups. In no instance did the antigen level fall to zero, even in individuals that remained microfilaria negative during two or three years of follow-up. These results suggest that living adult worms persist in almost all persons treated with DEC and/or ivermectin.

Enzyme-linked immunoelectrotransfer blot analysis of a cryptosporidiosis outbreak on a United States Coast Guard cutter.

Symptoms consistent with an outbreak of cryptosporidiosis (diarrhea, vomiting, nausea, and abdominal cramps) occurred on a U.S. Coast Guard cutter within 0-18 days after the cutter filled its tanks with Milwaukee, Wisconsin city water in March 1993. At three-weeks postdocking (PD), the suspected water was removed, and serum samples and stool specimens were collected from 47 of the 58 crew members, as well as questionnaire data on their water consumption and symptoms aboard the cutter. At 10-weeks PD and/or at 28-weeks PD, additional serum specimens were collected. Intensitometric data from enzyme-linked immunoelectrotransfer blot (EITB) were obtained on IgA responses to a 17-kD antigen group, IgM responses to a 27-kD antigen group, and IgG responses to 27-, 17-, and 15-kD antigen groups extracted from oocysts. In addition, IgG responses to crude oocyst antigens were obtained by ELISA. Based on reported symptoms, EITB results, and stool examination, the crew members were classified as confirmed (10), probable (10), suspected (22), and noncases (16). Of the 10 confirmed cases (all symptomatic) and the 10 probable cases (eight symptomatic) whose stools were positive and negative, respectively, for Cryptosporidium oocysts by microscopy, all showed changes in EITB intensities to the antigen groups and were considered EITB positive. The remaining 38 crew members, 22 suspected cases (all symptomatic), and 16 noncases (all asymptomatic), if tested, had negative stool examinations and were considered EITB negative. Of the 10 confirmed cases, only four showed a significant change in IgG responses (P < 0.05) between three-weeks PD and follow-up serum specimens by ELISA. Crew members considered confirmed cases consumed significantly more water (P < or = 0.005) aboard the cutter than noncases. Crew members considered EITB positive consumed more water (P < or = 0.04) than crew members considered EITB negative while there was no significant difference in water consumption (P > or = 0.19) between crew members considered ELISA positive and ELISA negative. Using the EITB, the observation of changes in intensity of IgA responses to the 17-kD antigen group, IgM responses to the 27-kD antigen group, and IgG responses to the 27- 17-, and 15-kD antigen groups from C. parvum oocysts between acute and convalescent serum specimens appears useful for immunodiagnosis of Cryptosporidium infection and for prospective epidemiologic studies designed to monitor infection risk.

A survey of knowledge, attitudes, and perceptions (KAPs) of lymphatic filariasis, elephantiasis, and hydrocele among residents in an endemic area in Haiti.

To assess knowledge, attitudes, and perceptions about bancroftian filariasis, 104 residents of an endemic area in Haiti were interviewed. Questions focused on 1) whether people understood the relationship between infection and disease, 2) recognition of the role that mosquitoes play in transmission, 3) perceived importance of hydrocele and elephantiasis in relation to other recognized diseases, and 4) the willingness of the community to participate in a control program. Fewer than 50% of residents had heard of filariasis and only 6% of those surveyed knew that it was transmitted by mosquitoes. In contrast, all persons knew of the clinical conditions of hydrocele and elephantiasis. Hydrocele was thought to be caused by trauma (60%) or trapped gas (30%); elephantiasis by walking bare foot on soil or water (37%) or by use of ceremonial powder that had been sprinkled on the ground (23%). Of 76 respondents, 53% and 38% thought that hydrocele could be treated through surgery or a drug, respectively, whereas 86 respondents, 85% and 15% believed that either surgery or a drug could be used to treat elephantiasis. In this context, persons were not referring to a specific drug; rather, they believed a drug existed (possibly in some other country) that could cure these conditions. Hydrocele and elephantiasis ranked second to acquired immunodeficiency syndrome as perceived health problems, most likely because residents believed treatment for conditions such as malaria, intestinal worms, anemia, and diarrhea was easily obtained. Responses were influenced by age, sex, and symptoms, but none of these effects were statistically significant except that persons with hydrocele or elephantiasis were more likely to have sought treatment than persons without these conditions (P = 0.0006). The survey results indicate that awareness of the causes of disease, the relationship between infection and disease, and goals of treatment must be heightened through community-based education campaigns to increase the possibility of acceptance and support of control programs.

Comparative efficacy of clearing-dose and single high-dose ivermectin and diethylcarbamazine against Wuchereria bancrofti microfilaremia.

To compare the efficacy and tolerability of various combinations of low- and high-dose ivermectin and diethylcarbamazine (DEC), 59 persons with Wuchereria bancrofti microfilaremia were enrolled in a double-blinded six-arm clinical trial in Leogane, Haiti. On day 1, study participants were treated with low clearing doses of ivermectin, DEC, or placebo; on day 5 they received 200-400 micrograms/kg of ivermectin or 6 mg/kg of DEC. Adverse reactions, which were generally mild, occurred more frequently with ivermectin than with DEC. One year after treatment, the geometric mean microfilarial density returned to 0.9% of pretreatment levels for persons who received a total of 420 micrograms/kg of ivermectin. This rate was significantly lower than 5.6% for persons who were treated with 220 micrograms/kg of ivermectin (P = 0.02) and 9.3% for those receiving 6 or 7 mg/kg of DEC (P = 0.006). Persons treated with a clearing dose of ivermectin followed by 6 mg/kg of DEC also had low microfilarial densities (1.7% of pretreatment levels), suggesting an additive or synergistic effect of the two drugs. The addition of a clearing dose neither reduced the severity of adverse reactions nor improved the efficacy of high-dose ivermectin. Community-based intervention trials are now warranted to determine the feasibility and effectiveness of mass chemotherapy with single high-dose ivermectin for the prevention and control of lymphatic filariasis.

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.

Uptake and development of Wuchereria bancrofti in Culex quinquefasciatus that fed on Haitian carriers with different microfilaria densities.

In studies conducted between 1984 and 1986, the vector competency of Culex quinquefasciatus was assessed after bloodfeeding on 61 Haitian volunteers with different densities of Wuchereria bancrofti microfilariae (mf) and on 11 that were amicrofilaremic. Infected volunteers included persons previously given diethylcarbamazine citrate for 12 consecutive days and some that were untreated. Mosquitoes, derived from field-collected larvae, were released under bed nets and fed upon the volunteers while they slept. The mean mf uptake in mosquitoes that fed on 21 carriers with low to ultralow densities (1-28 mf/ml blood) was 0.2-4 mf/mosquito (mean = 1.7 mf). The observed infectivity rate of greater than 3,000 mosquitoes that fed on high (56-7,500 mf/ml blood; median = 525), low (11-49 mf/ml blood; median = 20), or ultralow (1-10 mf/ml blood; median = 3) density carriers was 4, 11, and 30 times higher, respectively, than the expected rate calculated from the estimated volume of the imbibed bloodmeal. These results indicate that "hidden" carriers (less than 50 mf/ml blood) may serve as a source of infection for mosquitoes, and support the increasing evidence that mosquitoes ingest more microfilariae than expected.

Longitudinal analysis of the development of filarial infection and antifilarial immunity in a cohort of Haitian children.

Longitudinal studies are being conducted in Leogane, Haiti to investigate the relationship between acquisition of filarial infection and development of antifilarial immunity as well as the impact of maternal infection on this relationship. Children (0-24 months of age) residing in Leogane were enrolled and were examined periodically to monitor parasitologic status and to collect serum for antigen and antifilarial antibody determinations. To examine the development of filarial antigenemia and antifilarial antibody responses in this cohort, serum samples were selected from a cross section of the population at two (n = 82) and four years of age (n = 76). Antigen prevalence increased from 6% among two-year-olds to more than 30% among four-year-olds, but in only one four-year-old child were microfilaria detected in a 20-microl smear. The proportion of antigen-positive children born to antigen-positive mothers was higher than the proportion of antigen-positive children born to antigen-negative mothers (9.8% versus 0% for two-year-olds; P = 0.15; and 39.6% versus 22.7% for four-year-olds; P = 0.18). Antifilarial IgG4 levels were significantly higher among antigen-positive children at both two and four years of age (P < 0.001). In analyses of paired samples, antifilarial IgG4 responses increased significantly more among children who acquired infection by four years of age than among children who remained antigen negative, whereas antifilarial IgG1 and IgG2 responses changed equally for antigen-positive and -negative children. Antifilarial antibody levels were not influenced by maternal infection status, but were significantly influenced by age, antigen status, and the neighborhood within the community. These results provide evidence that children acquire infection early in life and suggest that antifilarial antibody responses may peak in early childhood.