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-infinity). 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.

Human babesiosis, a blood-borne infection caused by several species of Babesia, including B. microti, is an emerging disease that is endemic in the Northeast, upper Midwest, and Pacific Northwest regions of the United States. Risk factors for babesiosis include exposure to the infected tick vector and blood transfusions from infected donors. In this work, we cloned and expressed two of the immunodominant antigens from B. microti and used them in a multiplex bead format assay (MBA) to detect parasite-specific IgG responses in human sera. The MBA using recombinant B. microti secreted antigen 1 (BmSA1) protein was more specific (100%) and slightly more sensitive (98.7%) than the assay using a truncated recombinant BMN1-17 construct (97.6% and 97.4%, respectively). Although some antibody reactivity was observed among sera from confirmed-malaria patients, only one Plasmodium falciparum sample was simultaneously positive for IgG antibodies to both antigens. Neither antigen reacted with sera from babesiosis patients who were infected with Babesia species other than B. microti. Both positive and negative MBA results were reproducible between assays and between instruments. Additional studies of these recombinant antigens and of the multiplex bead assay using blood samples from clinically defined babesiosis patients and from blood donors are needed to more clearly define their usefulness as a blood screening assay.

Giardiasis and cryptosporidiosis are common enteric parasitic diseases that have similar routes of transmission. In this work, we have identified epitopes within the Giardia variant-specific surface protein (VSP) sequences that are recognized by IgG antibodies from 13/14 (93%) stool-confirmed giardiasis patient sera. The conserved epitopes are shared among VSPs from both of the assemblages that commonly infect humans, and they are likely to be structural as both sodium dodecylsulfate treatment and dithiothreitol reduction decrease antibody recognition. In a multiplex bead assay (MBA), we have used three VSP fragments from an Assemblage A Giardia strain, three VSP fragments from Assemblage B strains, and the alpha-1 giardin structural antigen to detect IgG antibodies to Giardia and have used the recombinant 17- and 27-kDa antigens to simultaneously detect IgG antibodies to Cryptosporidium. The MBA differentiated between sera from Giardia and Cryptosporidium outbreaks and also identified a giardiasis outbreak that may have included cryptosporidiosis cases. Approximately 40% of cryptosporidiosis outbreak samples had high MBA responses for both the 27- and 17-kDa antigens, while <10% of non-outbreak and giardiasis outbreak samples were high responders. At least 60% of giardiasis outbreak samples were positive for antibodies to multiple Giardia antigens, while ≤12% of non-outbreak samples and samples from US and British Columbia cryptosporidiosis outbreaks met our definition for Giardia seropositivity. MBA using multiple parasite antigens may prove useful in the epidemiologic analysis of future waterborne or foodborne outbreaks of diarrheal disease.

Cryptosporidium infection is commonly observed among children and the immune compromised in developing countries, but large-scale outbreaks of disease among adults have not been reported. In contrast, outbreaks of cryptosporidiosis in the US and Canada are increasingly common among patients of all ages. Thus, it seems likely that residents of highly endemic regions acquire some level of immunity while residents of the developed world do not. A new immunodominant Cryptosporidium parvum antigen in the 15/17-kDa size range was identified as the 60S acidic ribosomal protein P2 (CpP2). We developed a recombinant protein-based ELISA for serologic antibody population surveillance that was 89% sensitive and 92% specific relative to the large format Western blot. The human IgG response is directed almost exclusively towards the highly-conserved, carboxy-terminal 15 amino acids of the protein. Although IgG antibody cross reactivity was documented using sera from acute babesiosis patients, the development of an anti-CpP2 antibody response in our Peru study population correlated better with Cryptosporidium infection than with infection by any other parasitic protozoan. In Haiti, antibody prevalence to the CpP2 protein plateaus at 11-20 years of age. Because anti-CpP2 IgG antibodies were only found among residents of developing world countries where Cryptosporidium infection occurs early and often, we propose that this response may be a proxy for the intensity of infection and for acquired immunity.