Rift Valley fever virus (RVFV) is endemic in sub-Saharan Africa (SSA), with outbreaks reported in the Arabian Peninsula and throughout SSA. The natural reservoir for RVFV are ruminants, with livestock populations exceeding 50% exposure rates in some areas of SSA. Transmission to humans can occur through exposure to infected livestock products or multiple species of mosquito vectors. In 2013 and 2014, cross-sectional surveys occurred in two districts of Nacala-a-Velha and Mecubri in northern Mozambique, and participants provided blood samples for later serological assays. IgG against the N protein of RVFV was detected through multiplex bead assay (MBA). Of the 2,278 persons enrolled between the two surveys and study sites, 181 (7.9%, 95% confidence interval (CI): 6.9%-9.1%) were found to be IgG seropositive with increasing seroprevalence with older age and significantly higher seroprevalence in Nacala-a-Velha (10.5%, 8.8%-12.5%) versus Mecubri (5.7%, 4.5%-7.1%). Seroprevalence estimates were not significantly different between the 2013 and 2014 surveys. Significant spatial clustering of IgG positive persons were consistent among surveys and within the two districts, pointing toward the consistency of serology data for making population-level assumptions regarding RVFV seroprevalence. A subset of persons (n=539) provided samples for both the 2013 and 2014 surveys, and a low percentage (0.81%) of these were found to seroconvert between these two surveys. Including the RVFV N protein in an MBA antigen panel could assist elucidate RVFV exposure in SSA. IMPORTANCE Due to sporadic transmission, human contact with Rift Valley Fever Virus (RVFV) is difficult to ascertain at a population level. Detection of antibodies against RVFV antigens assist in estimating exposure as antibodies remain in the host long after the virus has been cleared. In this study, we show that antibodies against RVFV N protein can be detected from dried blood spot (DBS) samples being assayed by multiplex bead assay. DBS from two districts in northern Mozambique were tested for IgG against the N protein, and 7.9% of all enrolled persons were seropositive. Older persons, males, and persons residing closer to the coast had higher RVFV N protein seroprevalence. Spatial clustering of IgG positive persons was noted in both districts. These results show low exposure rates to RVFV in these two northern districts in Mozambique, and the ability to perform serology for the RVFV N protein from dried blood samples.

For more than 35 years, various assay formats have been used to detect Cryptosporidium-specific antibodies in human and animal sera. Cryptosporidium parvum 17- and 27-kDa antigens, identified from invasive sporozoites, have been used in serologic antibody assays to identify individuals infected in outbreaks of diarrheal disease caused by this protozoan parasite and to monitor exposures in communities. During infection, immunoglobulin (Ig) A, IgM, and IgG responses are elicited by these immunodominant antigens, and the parasite-specific Ig responses diminish following the resolution of infection. Using the recombinant forms of the 17- and 27-kDa C. parvum antigens and the relatively recently developed multiplex bead assay (MBA), data from serologic antibody responses can be economically and efficiently acquired, especially when the Cryptosporidium assays are integrated with assays for antibody responses to antigens from other pathogens monitored in community-wide or nation-wide serosurveys. Here we describe the coupling of the C. parvum recombinant antigens to carboxylated polystyrene beads, the data acquisition and analysis of IgG antibodies bound to the coupled beads, and the quality control methods required for data validation using the Luminex/MBA system.

Background: Cyclospora cayetanensis is a food-borne intestinal human parasite that causes outbreaks of diarrhea. There is a need for efficient laboratory methods for strain-level characterization to assist in outbreak investigations. By using next generation sequencing, genomic sequences can be obtained and compared to identify potential genotyping markers. However, there is no method available to propagate this parasite in the laboratory. Therefore, genomic DNA must be extracted from oocysts purified from human stool. The objective of this study was to apply optimized methods to purify C. cayetanensis oocysts and extract DNA in order to obtain high-quality whole genome sequences with minimum contamination of DNA from other organisms. Results: Oocysts from 21 human stool specimens were separated from other stool components using discontinuous density gradient centrifugation and purified further by flow cytometry. Genomic DNA was used to construct Ovation Ultralow libraries for Illumina sequencing. MiSeq sequencing reads were taxonomically profiled for contamination, de novo assembled, and mapped to a draft genome available in GenBank to assess the quality of the resulting genomic sequences. Following all purification steps, the majority (81-99%) of sequencing reads were from C. cayetanensis. They could be assembled into draft genomes of around 45 MB in length with GC-content of 52%. Conclusions: Density gradients performed in the presence of a detergent followed by flow cytometry sorting of oocysts yielded sufficient genomic DNA largely free from contamination and suitable for whole genome sequencing of C. cayetanensis. The methods described here will facilitate the accumulation of genomic sequences from various samples, which is a prerequisite for the development of typing tools to aid in outbreak investigations.

The index case of chikungunya virus (CHIKV) in Haiti was reported during early 2014; the vector, the pervasive Aedes aegypti mosquito, promoted rapid spread throughout the country. During December 2014-February 2015, we collected blood samples from 4,438 persons at 154 sites (62 urban, 92 rural) throughout Haiti and measured CHIKV IgG by using a multiplex bead assay. Overall CHIKV seroprevalence was 57.9%; differences between rural (mean 44.9%) and urban (mean 78.4%) areas were pronounced. Logistic modeling identified the urban environment as a strong predictor of CHIKV exposure (adjusted odds ratio 3.34, 95% CI 2.38-4.69), and geographic elevation provided a strong negative correlation. We observed no correlation between age and antibody positivity or titer. Our findings demonstrated through serologic testing the recent and rapid dissemination of the arbovirus throughout the country. These results show the utility of serologic data to conduct epidemiologic studies of quickly spreading mosquitoborne arboviruses.

BACKGROUND: Evidence from recent studies assessing the impact of school water, sanitation and hygiene (WASH) interventions on child health has been mixed. Self-reports of disease are subject to bias, and few WASH impact evaluations employ objective health measures to assess reductions in disease and exposure to pathogens. We utilized antibody responses from dried blood spots (DBS) to measure the impact of a school WASH intervention on infectious disease among pupils in Mali. METHODOLOGY/PRINCIPAL FINDINGS: We randomly selected 21 beneficiary primary schools and their 21 matched comparison schools participating in a matched-control trial of a comprehensive school-based WASH intervention in Mali. DBS were collected from 20 randomly selected pupils in each school (n = 807). We analyzed eluted IgG from the DBS using a Luminex multiplex bead assay to 28 antigens from 17 different pathogens. Factor analysis identified three distinct latent variables representing vector-transmitted disease (driven primarily by dengue), food/water-transmitted enteric disease (driven primarily by Escherichia coli and Vibrio cholerae), and person-to-person transmitted enteric disease (driven primarily by norovirus). Data were analyzed using a linear latent variable model. Antibody evidence of food/water-transmitted enteric disease (change in latent variable mean (beta) = -0.24; 95% CI: -0.53, -0.13) and person-to-person transmitted enteric disease (beta = -0.17; 95% CI: -0.42, -0.04) was lower among pupils attending beneficiary schools. There was no difference in antibody evidence of vector-transmitted disease (beta = 0.11; 95% CI: -0.05, 0.33). CONCLUSIONS/SIGNIFICANCE: Evidence of enteric disease was lower among pupils attending schools benefitting from school WASH improvements than students attending comparison schools. These findings support results from the parent study, which also found reduced incidence of self-reported diarrhea among pupils of beneficiary schools. DBS collection was feasible in this resource-poor field setting and provided objective evidence of disease at a low cost per antigen analyzed, making it an effective measurement tool for the WASH field. TRIAL REGISTRATION: The trial was registered at ClinicalTrials.gov (NCT01787058).

Blood samples from 805 students attending 42 elementary schools in Mopti, Sikasso, and Koulikoro regions, and Bamako district in Mali participated in a school water, sanitation, and hygiene intervention. Immunoglobulin (Ig) G responses to several antigens/pathogens were assessed by a multiplex bead assay (MBA), and the recombinant Taenia solium T24H antigen was included. Of all students tested, 8.0% were positive to rT24H, but in some schools 25-30%. A cluster of 12 widespread school locations showed not only a relative risk of 3.23 for T. solium exposure and significantly higher IgG responses (P < 0.001) but also significantly lower elevation (P = 0.04) (m, above sea level) compared with schools outside the cluster. All schools at elevations < 425 m showed significantly higher IgG responses (P = 0.017) than schools at elevations >/= 425 m. The MBA is an excellent serological platform that provides cost-effective opportunities to expand testing in serosurveys.

BACKGROUND: Serological antibody levels are a sensitive marker of pathogen exposure, and advances in multiplex assays have created enormous potential for large-scale, integrated infectious disease surveillance. Most methods to analyze antibody measurements reduce quantitative antibody levels to seropositive and seronegative groups, but this can be difficult for many pathogens and may provide lower resolution information than quantitative levels. Analysis methods have predominantly maintained a single disease focus, yet integrated surveillance platforms would benefit from methodologies that work across diverse pathogens included in multiplex assays. METHODS/PRINCIPAL FINDINGS: We developed an approach to measure changes in transmission from quantitative antibody levels that can be applied to diverse pathogens of global importance. We compared age-dependent immunoglobulin G curves in repeated cross-sectional surveys between populations with differences in transmission for multiple pathogens, including: lymphatic filariasis (Wuchereria bancrofti) measured before and after mass drug administration on Mauke, Cook Islands, malaria (Plasmodium falciparum) before and after a combined insecticide and mass drug administration intervention in the Garki project, Nigeria, and enteric protozoans (Cryptosporidium parvum, Giardia intestinalis, Entamoeba histolytica), bacteria (enterotoxigenic Escherichia coli, Salmonella spp.), and viruses (norovirus groups I and II) in children living in Haiti and the USA. Age-dependent antibody curves fit with ensemble machine learning followed a characteristic shape across pathogens that aligned with predictions from basic mechanisms of humoral immunity. Differences in pathogen transmission led to shifts in fitted antibody curves that were remarkably consistent across pathogens, assays, and populations. Mean antibody levels correlated strongly with traditional measures of transmission intensity, such as the entomological inoculation rate for P. falciparum (Spearman's rho = 0.75). In both high- and low transmission settings, mean antibody curves revealed changes in population mean antibody levels that were masked by seroprevalence measures because changes took place above or below the seropositivity cutoff. CONCLUSIONS/SIGNIFICANCE: Age-dependent antibody curves and summary means provided a robust and sensitive measure of changes in transmission, with greatest sensitivity among young children. The method generalizes to pathogens that can be measured in high-throughput, multiplex serological assays, and scales to surveillance activities that require high spatiotemporal resolution. Our results suggest quantitative antibody levels will be particularly useful to measure differences in exposure for pathogens that elicit a transient antibody response or for monitoring populations with very high- or very low transmission, when seroprevalence is less informative. The approach represents a new opportunity to conduct integrated serological surveillance for neglected tropical diseases, malaria, and other infectious diseases with well-defined antigen targets.

Malaria serology through assaying for IgG against Plasmodium spp. antigens provides evidence into the infection history for an individual. The multiplex bead assay (MBA) allows for detection of IgG against multiple Plasmodium spp., and can be especially useful in many regions where Plasmodium falciparum is of primary clinical focus, but other species are co-endemic. Dried blood spots were collected from 805 Malian children attending 42 elementary schools in the regions of Mopti, Sikasso, Koulikoro, and Bamako capital district, and IgG assayed by MBA. As southern Mali is known to be holoendemic for P. falciparum, merozoite surface protein 1 19-kDa subunit (MSP-142) and apical membrane antigen 1 (AMA-1) antigens were included for serology against this parasite. Responses to these antigens both provided high estimates for lifetime exposure, with 730 (90%) children with IgG antibodies for MSP-142, 737 (91%) for AMA-1, and 773 (96%) positive for either or both. Also included was the antigen Plasmodium vivax MSP-119, against which 140 (17.4%) children were found to have antibodies. Increases in antibody titers with older age were clearly seen with the P. falciparum antigens, but not with the P. vivax antigen, likely indicating more of a sporadic, rather than sustained transmission for this species. The MBA provides effective opportunities to evaluate malaria transmission through serological analysis for multiple Plasmodium species.

OBJECTIVE: To differentiate exposure to the newly introduced chikungunya virus from exposure to endemic dengue virus and other pathogens in Haiti. METHODS: We used a multiplex bead assay to detect immunoglobulin G (IgG) responses to a recombinant chikungunya virus antigen, two dengue virus-like particles and three recombinant Plasmodium falciparum antigens. Most (217) of the blood samples investigated were collected longitudinally, from each of 61 children, between 2011 and 2014 but another 127 were collected from a cross-sectional sample of children in 2014. FINDINGS: Of the samples from the longitudinal cohort, none of the 153 collected between 2011 and 2013 but 78.7% (48/61) of those collected in 2014 were positive for IgG responses to the chikungunya virus antigen. In the cross-sectional sample, such responses were detected in 96 (75.6%) of the children and occurred at similar prevalence across all age groups. In the same sample, responses to malarial antigen were only detected in eight children (6.3%) but the prevalence of IgG responses to dengue virus antigens was 60.6% (77/127) overall and increased steadily with age. Spatial analysis indicated that the prevalence of IgG responses to the chikungunya virus and one of the dengue virus-like particles decreased as the sampling site moved away from the city of Leogane and towards the ocean. CONCLUSION: Serological evidence indicates that there had been a rapid and intense dissemination of chikungunya virus in Haiti. The multiplex bead assay appears to be an appropriate serological platform to monitor the seroprevalence of multiple pathogens simultaneously.

Dried blood spots (DBS) were collected from 805 children attending 42 elementary schools in the regions of Mopti, Sikasso, Koulikoro, and the regional capital of Bamako in Mali as part of an evaluation of a school health intervention. Eluted immunoglobulin (Ig) G from the DBS was assessed by a multiplex bead assay (MBA) for two filariasis antigens, Wuchereria bancrofti, Wb123, and Brugia malayi, Bm14, to determine the effectiveness of mass drug administration (MDA) programs to eliminate lymphatic filariasis (LF). The prevalence of positive IgG responses in the children to each antigen was less than 1%, indicating effectiveness of the MDA against LF. The MBA is an excellent serological platform that provides cost-effective opportunities to evaluate public health activities beyond the survey targets.

Because the lack of typing tools for Cyclospora cayetanensis has hampered outbreak investigations, we sequenced its genome and developed a genotyping tool. We observed 2 to 10 geographically segregated sequence types at each of 5 selected loci. This new tool could be useful for case linkage and infection/contamination source tracking.

BACKGROUND: Cyclospora cayetanensis is an apicomplexan that causes diarrhea in humans. The investigation of foodborne outbreaks of cyclosporiasis has been hampered by a lack of genetic data and poor understanding of pathogen biology. In this study we sequenced the genome of C. cayetanensis and inferred its metabolism and invasion components based on comparative genomic analysis. RESULTS: The genome organization, metabolic capabilities and potential invasion mechanism of C. cayetanensis are very similar to those of Eimeria tenella. Propanoyl-CoA degradation, GPI anchor biosynthesis, and N-glycosylation are some apparent metabolic differences between C. cayetanensis and E. tenella. Unlike Eimeria spp., there are no active LTR-retrotransposons identified in C. cayetanensis. The similar repertoire of host cell invasion-related proteins possessed by all coccidia suggests that C. cayetanensis has an invasion process similar to the one in T. gondii and E. tenella. However, the significant reduction in the number of identifiable rhoptry protein kinases, phosphatases and serine protease inhibitors indicates that monoxenous coccidia, especially C. cayetanensis, have limited capabilities or use a different system to regulate host cell nuclear activities. C. cayetanensis does not possess any cluster of genes encoding the TA4-type SAG surface antigens seen in E. tenella, and may use a different family of surface antigens in initial host cell interactions. CONCLUSIONS: Our findings indicate that C. cayetanensis possesses coccidia-like metabolism and invasion components but unique surface antigens. Amino acid metabolism and post-translation modifications of proteins are some major differences between C. cayetanensis and other apicomplexans. The whole genome sequence data of C. cayetanensis improve our understanding of the biology and evolution of this major foodborne pathogen and facilitate the development of intervention measures and advanced diagnostic tools.

Collection of surveillance data is essential for monitoring and evaluation of public health programs. Integrated collection of household-based health data, now routinely carried out in many countries through demographic health surveys and multiple indicator surveys, provides critical measures of progress in health delivery. In contrast, biomarker surveys typically focus on single or related measures of malaria infection, HIV status, vaccination coverage, or immunity status for vaccine-preventable diseases (VPD). Here we describe an integrated biomarker survey based on use of a multiplex bead assay (MBA) to simultaneously measure antibody responses to multiple parasitic diseases of public health importance as part of a VPD serological survey in Cambodia. A nationally-representative cluster-based survey was used to collect serum samples from women of child-bearing age. Samples were tested by MBA for immunoglobulin G antibodies recognizing recombinant antigens from Plasmodium falciparum and P. vivax, Wuchereria bancrofti, Toxoplasma gondii, Taenia solium, and Strongyloides stercoralis. Serologic IgG antibody results were useful both for generating national prevalence estimates for the parasitic diseases of interest and for confirming the highly focal distributions of some of these infections. Integrated surveys offer an opportunity to systematically assess the status of multiple public health programs and measure progress toward Millennium Development Goals.

INTRODUCTION: To monitor progress toward maternal and neonatal tetanus elimination (MNTE) in Cambodia, we conducted a nationwide serosurvey of tetanus immunity in 2012. METHODS: Multi-stage cluster sampling was used to select 2,154 women aged 15-39 years. Tetanus toxoid antibodies in sera were measured by gold-standard double antigen ELISA (DAE) and a novel multiplex bead assay (MBA). Antibody concentrations ≥0.01 IU/ml by DAE, or equivalent for MBA, were considered seroprotective. RESULTS: Estimated tetanus seroprotection was 88% (95% CI: 86%-89%); 64% (95% CI: 61%-67%) of women had antibody levels ≥1.0 IU/ml. Seroprotection was significantly lower (p <0.001) among women aged 15-19 years (63%) and 20-24 years (87%) compared with ≥25 years (96%), nulliparous compared with parous (71% vs. 97%), and living in the west compared with other regions (82% vs. 89%). The MBA showed high sensitivity (99% [95% CI: 98%-99%]) and specificity (92% [95% CI: 88%-95%]) compared with DAE. CONCLUSIONS: Findings were compatible with MNTE in Cambodia (≥80% protection). Tetanus immunity gaps should be addressed through strengthened routine immunization and targeted vaccination campaigns. Incorporating tetanus testing in national serosurveys using MBAs, which can measure immunity to multiple pathogens simultaneously, may be beneficial for monitoring MNTE.

The parasite Cyclospora cayetanensis causes foodborne diarrheal illness. Here, we report draft genome sequences obtained from C. cayetanensis oocysts purified from a human stool sample. The genome assembly consists of 865 contigs with a total length of 44,563,857 bases. These sequences can facilitate the development of subtyping tools to aid outbreak investigations.

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.

BACKGROUND: Efforts to monitor malaria transmission increasingly use cross-sectional surveys to estimate transmission intensity from seroprevalence data using malarial antibodies. To date, seroconversion rates estimated from cross-sectional surveys have not been compared to rates estimated in prospective cohorts. Our objective was to compare seroconversion rates estimated in a prospective cohort with those from a cross-sectional survey in a low-transmission population. METHODS AND FINDINGS: The analysis included two studies from Haiti: a prospective cohort of 142 children ages ≤11 years followed for up to 9 years, and a concurrent cross-sectional survey of 383 individuals ages 0-90 years old. From all individuals, we analyzed 1,154 blood spot specimens for the malaria antibody MSP-119 using a multiplex bead antigen assay. We classified individuals as positive for malaria using a cutoff derived from the mean plus 3 standard deviations in antibody responses from a negative control set of unexposed individuals. We estimated prospective seroconversion rates from the longitudinal cohort based on 13 incident seroconversions among 646 person-years at risk. We also estimated seroconversion rates from the cross-sectional survey using a reversible catalytic model fit with maximum likelihood. We found the two approaches provided consistent results: the seroconversion rate for ages ≤11 years was 0.020 (0.010, 0.032) estimated prospectively versus 0.023 (0.001, 0.052) in the cross-sectional survey. CONCLUSIONS: The estimation of seroconversion rates using cross-sectional data is a widespread and generalizable problem for many infectious diseases that can be measured using antibody titers. The consistency between these two estimates lends credibility to model-based estimates of malaria seroconversion rates using cross-sectional surveys. This study also demonstrates the utility of including malaria antibody measures in multiplex assays alongside targets for vaccine coverage and other neglected tropical diseases, which together could comprise an integrated, large-scale serological surveillance platform.

Haitian children were monitored longitudinally in a filariasis study. Included were stool samples examined for Giardia intestinalis and Entamoeba histolytica cysts, and serum specimens analyzed for immunoglobulin G (IgG) responses to eight recombinant antigens from G. intestinalis (variant-specific surface protein [VSP1-VSP5]), E. histolytica (lectin adhesion molecule [LecA]), and Cryptosporidium parvum (17- and 27-kDa) using a multiplex bead assay. The IgG responses to VSP antigens peaked at 2 years of age and then diminished and were significantly lower (P < 0.002) in children > 4.5 years than in children < 4.5 years. The IgG responses to Cryptosporidium tended to increase with age. The IgG responses to LecA and VSP antigens and the prevalence of stools positive for cysts were significantly higher (P < 0.037 and P < 0.035, respectively) in the rainy season than in the dry season. The multiplex bead assay provides a powerful tool for analyzing serologic responses to multiple pathogens.

Antifilarial antibody testing has been established as a sensitive and specific method of diagnosing lymphatic filariasis. However, the development of serological responses to specific filarial antigens and their relationship to acquisition of infection is poorly understood. In order to evaluate whether the development of antigen specific antifilarial antibodies precedes microfilaremia and antigenemia, we compared the antibody responses of serum samples collected between 1990 and 1999 from a cohort of 142 Haitian children followed longitudinally. Antigen status was determined using the Og4C3 ELISA and the presence of microfilaremia was detected using microscopy. Antibody responses to Wb123, a Wuchereria bancrofti L3 antigen, were measured using a Luciferase Immunoprecipitation System (LIPS) assay. Antibody responses to Bm14 and Bm33, Brugia malayi antigens and to a major surface protein (WSP) from Wolbachia were analyzed using a multiplex bead assay. Over follow-up, 80 (56%) of the children became antigen-positive and 30 (21%) developed microfilaremia. Detectable antibody responses to Bm14, Bm33, Wb123, and WSP developed in 95%, 100%, 92%, and 29% of children, respectively. With the exception of WSP, the development of antibody responses generally preceded detection of filarial antigen. Our results show that antifilarial antibody responses can serve as an important epidemiological indicator in a sentinel population of young children and thus, may be valuable as tool for surveillance in the context of lymphatic filariasis elimination programs.

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.

BACKGROUND: Defining endpoints for trachoma programs can be a challenge as clinical signs of infection may persist in the absence of detectable bacteria. Antibody-based tests may provide an alternative testing strategy for surveillance during terminal phases of the program. Antibody-based assays, in particular ELISAs, have been shown to be useful to document C. trachomatis genital infections, but have not been explored extensively for ocular C. trachomatis infections. METHODOLOGY/PRINCIPAL FINDINGS: An antibody-based multiplex assay was used to test two C. trachomatis antigens, pgp3 and CT694, for detection of trachoma antibodies in bloodspots from Tanzanian children (n=160) collected after multiple rounds of mass azithromycin treatment. Using samples from C. trachomatis-positive (by PCR) children from Tanzania (n=11) and control sera from a non-endemic group of U.S. children (n=122), IgG responses to both pgp3 and CT694 were determined to be 91% sensitive and 98% specific. Antibody responses of Tanzanian children were analyzed with regard to clinical trachoma, PCR positivity, and age. In general, children with more intense ocular pathology (TF/TI=2 or most severe) had a higher median antibody response to pgp3 (p=0.0041) and CT694 (p=0.0282) than those with normal exams (TF/TI=0). However, 44% of children with no ocular pathology tested positive for antibody, suggesting prior infection. The median titer of antibody responses for children less than three years of age was significantly lower than those of older children. (p<0.0001 for both antigens). CONCLUSIONS/SIGNIFICANCE: The antibody-based multiplex assay is a sensitive and specific additional tool for evaluating trachoma transmission. The assay can also be expanded to include antigens representing different diseases, allowing for a robust assay for monitoring across NTD programs.

An expanded global focus on the control and elimination of neglected tropical diseases (NTDs) has called attention to the need to develop and validate surveillance strategies that are cost effective and can be integrated across diseases. Here, we describe a multiplex tool for the sensitive detection of antibody responses to NTDs as well as vaccine preventable diseases, malaria, and waterborne and zoonotic infections. The assay platform is robust, can be performed with either serum or dried blood spots and can be adapted to local epidemiological conditions and public health priorities. Multiplex assays open the door to conducting routine serosurveillance for NTDs through demographic health surveillance or malaria indicator surveys.

A multiplex bead assay (MBA) was used to analyze serum samples collected longitudinally from children enrolled in a drug trial for treatment of filariasis in Leogane, Haiti. Recombinant antigens Bm14 and Bm33 from Brugia malayi, third polar tube protein (PTP3) from Encephalitozoon cuniculi, and merozoite surface protein-1(19) (MSP-1(19)) from Plasmodium falciparum were coupled to carboxylated polystyrene microspheres. IgG responses to PTP3 and MSP-1(19) were not affected by albendazole (ALB), diethylcarbamazine (DEC), or combination of diethylcarbamazine and albendazole (DEC/ALB). However, IgG and IgG4 responses to Bm14 and Bm33 were significantly decreased (P < 0.001) by DEC and DEC/ALB treatment. Antibody responses to Bm14 and Bm33 decreased after DEC treatment (but not placebo) among children who were negative for microfilaremia and antigenemia at baseline, suggesting that these children harbored early stages of infection. The MBA is an excellent serologic technique for multiple antigens that offers substantial advantages over single-antigen based enzyme-linked immunosorbent assay in mass drug administration studies for monitoring changes in antibody levels.

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.