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ABSTRACTWHO has targeted yaws for global eradication by 2020. The program goals are to interrupt the transmission in countries where yaws is endemic and to certify countries as yaws free where yaws was endemic in the past. No new rapid plasmin reagin (RPR) seroreactivity in young children is required for certification of elimination at a country level. We sought to evaluate whether antibody responses to specific treponemal antigens measured in a high-throughput multiplex bead array (MBA) assay differentiate past versus current infection and whether a nontreponemal lipoidal antigen test can be incorporated into the MBA. Serum and dried blood spot specimens collected for yaws surveillance projects in Ghana, Vanuatu, and Papua New Guinea (PNG) were run on MBA to measure antibodies against recombinant p17 (rp17) and treponemal membrane protein A (TmpA) treponemal antigens. Results were compared to standard treponemal laboratory (TPPA or TPHA [TPP(H)A]) and quantitative RPR test data. Of 589 specimens, 241 were TPP(H)A+/RPR+, 88 were TPP(H)A+/RPR-, 6 were TPP(H)A-/RPR+, and 254 were negative for both tests. Compared to TPP(H)A, reactive concordance of rp17 was 93.7%, while reactive concordance of TmpA was only 81.9%. TmpA-specific reactivity showed good correlation with RPR titers (R2= 0.41; P< 0.0001). IgG responses to the lipoidal antigen used in RPR testing (cardiolipin) were not detected in the MBA. Our results suggest that TmpA can be used as a treponemal antigen marker for recent or active infection and potentially replace RPR in a high-throughput multiplex tool for large-scale yaws surveillance.

ABSTRACTCryptosporidiuminfection is commonly observed among children and immunocompromised individuals in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the United States and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of regions where Cryptosporidiumis highly endemic acquire some level of immunity, while residents of the developed world do not. A new immunodominant Cryptosporidium parvumantigen in the 15- to 17-kDa size range was identified as the Cryptosporidium parvum60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based enzyme-linked immunosorbent assay for serologic population surveillance for antibodies that was 89% sensitive and 92% specific relative to the results of the large-format Western blot assay. The human IgG response is directed almost exclusively toward the highly conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross-reactivity was documented with sera from patients with acute babesiosis, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidiuminfection than with infection by any other parasitic protozoan. In Haiti, the prevalence of antibodies to CpP2 plateaus at 11 to 20 years of age. Because anti-CpP2 IgG antibodies were found only among residents of countries in the developing world where Cryptosporidiuminfection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity.

ABSTRACTCryptosporidiumspecies are ubiquitous in the environment and are frequently detected in the stools of children who live where sanitation conditions are poor. To better characterize the immune response to these parasites, we monitored immunoglobulin G (IgG) antibody levels in a cohort of children from Lima, Peru. Two new enzyme-linked immunosorbent assays based on the C. parvum(bovine, subtype IIa) Iowa strain 17-kDa and 27-kDa antigens were used to measure IgG antibody levels in longitudinal serum samples. Antibody responses were detected during infections with C. parvum, C. felis, and C. meleagridisand with four different subtypes of C. hominis. We also noted that the magnitude of the antibody response was related to the number of previous infections and that older children generally had higher levels of antibodies to the two C. parvumantigens. Antibody responses were not associated with infections with either Cyclosporasp. or Giardiasp. We believe the antibody assays will be important tools for monitoring the success of future public health interventions.

Symbiotic Wolbachia organisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-Wolbachia immune responses and chronic filarial disease in humans, antibody responses to Wolbachia surface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where Wuchereria bancrofti infection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.

ABSTRACTSymbiotic Wolbachiaorganisms of filarial nematodes have received much attention as possible chemotherapy targets and disease-causing organisms. In order to further investigate the association between anti-Wolbachiaimmune responses and chronic filarial disease in humans, antibody responses to Wolbachiasurface protein (WSP) were assayed in serum samples collected from 232 individuals living in Leogane, Haiti, an area where Wuchereria bancroftiinfection is endemic, and from 67 North Americans with no history of lymphatic filariasis. As opposed to antifilarial antibody responses, which were largely influenced by the patient's infection status, the prevalence and levels of anti-WSP immunoglobulin G (IgG) antibodies among individuals with lymphedema or hydrocele were significantly greater than those in gender- and infection-matched individuals without disease. In at least one case, the anti-WSP IgG response was coincident with the onset of lymphedema development, and among anti-WSP-positive women with lymphedema, anti-WSP IgG levels were negatively correlated with the duration of lymphedema. The presence of anti-WSP IgG was also associated with the severity of inguinal adenopathy among men with hydrocele. In addition to the presence of anti-WSP antibodies among Haitians, 15 of 67 (22%) serum samples collected from individuals from North America, where filariasis is not endemic, were also positive for anti-WSP antibodies. In comparison to those from Haitians, anti-WSP antibodies from North Americans primarily recognized a distinct region of WSP located within the highly conserved second transmembrane domain. The results of this study demonstrate that anti-WSP antibody responses are associated with the presence of chronic filarial morbidity and not filarial infection status in humans and suggest that WSP should be further studied as a potential trigger for the development of filarial disease.

Our understanding of the pathogenesis of filarial lymphedema, although evolving, is still limited. Recurrent bacterial infections play a major role in the progression of lymphedema to elephantiasis, but the host and parasite factors that trigger disease development are not known. Field studies in Haiti show that lymphedema and host responses to parasite antigens cluster in families, consistent with the hypothesis that host genes influence lymphedema susceptibility. The recent recognition that filarial parasites harbor the endosymbiotic bacteria, Wolbachia,also raises questions about the potential contribution of the inflammatory response to Wolbachiaantigens to lymphedema development. In this review, we discuss potential risk factors for lymphedema and try to integrate these in a model of pathogenesis.

ABSTRACTHuman infection with Cryptosporidium parvumusually 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. parvuminfection 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.

In this article, the authors describe how to detect blood-borne and skin-dwelling filarial infections parasitologically. Considerable space is devoted to identifying and separating microfilarial from nonparasite objects, and the evaluation of morphologic features, including size, shape, and internal anatomy is stressed.

ABSTRACTIn a retrospective analysis, we assessed the usefulness of two serologic enzyme-linked immunosorbent assays as epidemiologic tools for the detection of cryptosporidiosis episodes in children from a Peruvian community. The incidence rate determined by the serologic assay was higher than the rate determined by stool microscopy (0.77 versus 0.41 infection/child-year of surveillance).

To characterize filarial antigens that may beassociated with the development of chronic lymphatic dysfunction in persons with lymphatic filariasis, T cell responsiveness to Brugia pahangi adult worm extracts and SDS-PAGE antigen fractions were examined among Haitians from an area in which Wuchereria bancrofti is endemic. Greater T cell proliferation and interleukin-10 (IL-10) production were observed in amicrofilaremic patients with hydrocele or elephantiasis than in amicrofilaremic or microfilaremic asymptomatic persons. Antigen fractions that stimulated the highest proliferative responses (in the 25-49 kDa range) and IL-10 production were not identical. Further separation of an immunodominant 30- to 38-kDa fraction by ion exchange high-pressure liquid chromatography identified several subfractions, including a 32-kDa protein band, that elicited T cell responses from patients with elephantiasis or hydrocele. By immunoblot, these patients also had markedly greater humoral reactivity to parasite antigens of ∼52,43, 32, and 30 kDa.

This study assessed Wuchereria bancrojti-infected patients who received diethylcarbamazine (DEC) to determined if drug levels were comparable between individuals who continued to harbor circulating microfilariae and those whose blood became clear of parasites, between those with high and low microfilaria counts, and between infected and noninfected persons. Haitian volunteers undergoing treatment for W. bancrofti and two nonendemic controls were enrolled. DEC levels in serum and urine samples were determined using a gas chromatographic method. No correlation was found between pre- and posttreatment microfilarial levelsand drug levels. Drug levels were comparable in persons with or without residual microfilaria and in infected and uninfected persons. These results indicate that incomplete drug regimens, differences in serum drug levels, and inadequate drug dosage are not the primary causes of treatment failure and suggest there is a degree of parasite tolerance for DEC.

The relationship between chronic obstructive disease and antifilarial immune responsiveness was studied in the Haitian community of Leogane, where Wuchereria bancrofti is endemic. Differences in sex ratios and in the prevalence of microfilaremia were observed between patients with hydrocele and those with lymphedema or elephantiasis of the lower limb. Only 2 of 84 patients with limb involvement (74 women, 10 men) were microfilaremic compared with 25 of 42 men with hydrocele. Microfilaria-positive men with hydrocele had significantly lower IgG2 and proliferative responses to filarial antigen than did amicrofilaremic men with hydrocele or individuals with lymphedema or elephantiasis. Parasite-specific cellular responses of amicrofilaremic individuals with obstructive disease were greater, although not significantly so, than those of amicrofilaremic asymptomatic members of the community. These results are compatible with the hypothesis that development of obstructive disease of the lymphatics has an immune component in amicrofilaremic persons.

Hydrocele and elephantiasis, major clinical manifestations of bancroftian filariasis, are thought to share a common pathogenesis. The characteristics of 121 patients with hydrocele or elephantiasis in Leogane, Haiti, were compared: 39% of 57 men with hydrocele and 3% of 64 persons with lymphedema of the leg were microfilaria-positive (P < .001). Circulating filarial antigen, presumably from the adult worm, was detected in 15 (43%) microfilaria-negative men with hydrocele and 9 (15%) microfilaria-negative persons with leg edema (P = .004). Microfilaria-positive men had lower levels of filaria-specific IgG1 and hydroceles of significantly smaller volume and shorter duration than did microfilaria-negative men; hydrocele volume was inversely associated with microfilarial density (P = .001). In contrast, filarial antigen but not microfilariae was associated with filaria-specific IgG4 and decreased lymphocyte proliferation. Antigen status was not associated with severity of leg edema. In this filariasis-endemic area, men with hydrocele are more immunologically and parasitologically heterogeneous than are persons with elephantiasis.

Patent filarial infections are associated with elevated levels of parasite-specific IgG4. This study investigated the shifts of filarial-specific human IgG and IgG4 antibodies after diethylcarbamazine and ivermectin treatment of bancroftian filariasis. Thirty adult Haitians were treated first with a l-mg clearing dose of ivermectin and then with either one or two 200µg/kg doses of ivermectin or with 12 daily 6-mg/kg doses of diethylcarbamazine. Posttreatment levels of antifilarial IgG4 were dependent on both treatment group and time of follow-up, IgG4 increased markedly to a maximum by day 30 in all treatment groups and then began to decrease; the greatest decrease was among diethylcarbamazine-treated patients. Posttreatment microfilaremia was inversely correlated with the decrease in IgG4; thus, shifts in IgG4 were associated with treatment response for all groups. Antifilarial IgG levelswere not correlated with drug treatment and did not change to the same degree as did IgG4 responses.

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.

This study assessed changes in circulating parasite antigen levels after diethylcarbamazine (DEC) and ivermectin treatment of bancroftian filariasis to determine effects of these drugs on adult Wuchereria bancrofti in vivo. Thirty adult Haitians with microfilaremia were treated with 1 mg of ivermectin to reduce counts of micro filariae. Later, subjects were treated with either one or two 200 μg/kg doses of ivermectin or with 12 daily 6 mg/kg doses of DEC. Macrofilaricidal activity of these drugs was indirectly monitored by measuring circulating W. bancrofti antigen by EIA. Antigen levels fell by 75% after DEC and by 34% after ivermectin. These results suggest that low-dose ivermectin treatment followed by a standard course of DEC is a more effective macrofilaricidal regimen for W. bancrofti than either of the multidose ivermectin regimens used in this study.

Previous reports have demonstrated age-related shifts in antifilarial humoral immune responses in 6- to 10-year-old Haitian children; the responses consisted of elevated parasite-specific IgG 2 and IgG3 in amicrofilaremic children and elevated IgG4 in microfilaremic children. In this study, the cell-mediated immune responses to soluble adult and microfilarial extracts of Brugia pahangi, determined by use of a microblastogenesis assay, were examined. Capillary blood samples were collected by finger prick from 176 Haitian children in an area with endemic Wuchereria bancrofti. Antigen-specific cellular responsiveness varied as a function of infection status but not age or sex; amicrofilaremic children had significantly greater responses to adult antigens than did microfilaremic children. Significant responses were detected in children <2 years of age; thus, correlations observed between filarial antigen-specific responses and infection status are established early in life.

Antifilarial IgG4 is a marker of active filarial infection; however, 10% of microfilaremic persons may have low levels of antifilarial IgG4. To gain insight into how persons with microfilaremia with low antifilarial IgG4 levels (<10 µ/mL) differ from those with high levels (>170 µg/mL), total IgG4 and IgE and filaria-specific IgE, IgG1, and IgG2 were measured by ELISA in serum samples collected from 85 microfilaremic Haitians. Persons with lower levels of antifilarial IgG4 had significantly lower total IgG4 and total IgE (P < .01), lower levels of antifilarial IgG1, IgG2, and IgE (P < .01, = .03, and < .01, respectively), and higher antigenemia and microfilaremia (P < .01) than did persons with higher levels of antifilarial IgG4. Increased antigen loads in microfilaremic persons may be associated with a down-regulation of antibody production, which extends to all isotypes.