Congenital transmission of Toxoplasma gondii can occur when a woman becomes infected for the first time during or just before pregnancy. Toxoplasma gondii in the fetus can lead to miscarriage, stillbirth, ocular or neurological abnormalities at birth, or progressive visual, hearing, motor, and cognitive deficiencies. The national seroprevalence of T. gondii infection in Nigeria was previously unknown. The 2018 Nigeria HIV/AIDS Indicator and Impact Survey collected demographic, socioeconomic, and HIV-related data and stored blood specimens with consent for future analysis for other pathogens of public health importance. We evaluated toxoplasmosis seropositivity and risk factors in a sample of 44,269 women of reproductive age (WRA) between 15 and 44 years. The national T. gondii seroprevalence among WRA was 26.8% (95% CI: 25.8-27.7%). We found that WRA from all 36 states and the Federal Capital Territory had T. gondii exposure. Seroprevalence was higher in 25- to 44-year-olds than in 15- to 24-year-olds. A similar proportion of pregnant and nonpregnant women were seropositive. Increased odds of seropositivity were associated with unimproved toilet facilities and drinking water sources, being in a higher wealth quintile, and primary and secondary education compared with no education. Decreased odds of seropositivity were associated with living in an urban area and owning livestock. This study provides the first-ever national seroprevalence estimate for WRA in Nigeria. Although information on known risk factors for toxoplasmosis (e.g., consumption of undercooked meat, cat ownership) was not collected, future studies could further investigate potential risk factors to inform the development of effective toxoplasmosis prevention measures.

Hypoplastic left heart syndrome (HLHS) is a severe congenital heart defect (CHD) characterized by hypoplasia of the left ventricle and aorta along with stenosis or atresia of the aortic and mitral valves. HLHS represents only ∼4%-8% of all CHDs but accounts for ∼25% of deaths. HLHS is an isolated defect (i.e., iHLHS) in 70% of families, the vast majority of which are simplex. Despite intense investigation, the genetic basis of iHLHS remains largely unknown. We performed exome sequencing on 331 families with iHLHS aggregated from four independent cohorts. A Mendelian-model-based analysis demonstrated that iHLHS was not due to single, large-effect alleles in genes previously reported to underlie iHLHS or CHD in >90% of families in this cohort. Gene-based association testing identified increased risk for iHLHS associated with variation in CAPN2 (p = 1.8 × 10(-5)), encoding a protein involved in functional adhesion. Functional validation studies in a vertebrate animal model (Xenopus laevis) confirmed CAPN2 is essential for cardiac ventricle morphogenesis and that in vivo loss of calpain function causes hypoplastic ventricle phenotypes and suggest that human CAPN2(707C>T) and CAPN2(1112C>T) variants, each found in multiple individuals with iHLHS, are hypomorphic alleles. Collectively, our findings show that iHLHS is typically not a Mendelian condition, demonstrate that CAPN2 variants increase risk of iHLHS, and identify a novel pathway involved in HLHS pathogenesis.

Background The Macrolides Oraux pour Réduire les Décès avec un Oeil sur la Résistance (MORDOR) trial in Niger, Malawi, and Tanzania found that biannual mass distribution of azithromycin to children younger than 5 years led to a 13.5% reduction in all-cause mortality. Additional endpoints in the trial have attempted to elucidate the mechanisms for mortality reduction. In this pre-specified secondary analysis, we assessed the effect of azithromycin compared with placebo on IgG- based measures of infectious disease exposure with a multiplex bead assay that included antigens to malaria parasites (Plasmodium falciparum, P. vivax, P. malariae, P. ovale), bacterial pathogens (Campylobacter spp., enterotoxigenic Escherichia coli, Vibrio cholerae, Salmonella enterica, Streptococcus pyogenes) and protozoans (Cryptosporidium parvum, Giardia duodenales).Methods and Findings Thirty communities in rural Niger were randomized 1:1 to biannual distributions of azithromycin or placebo among children ages 1-59 months. The analysis included 5,642 blood specimens collected from 3,814 children ages 1-59 months, measured at 6, 12, 24, and 36 months of follow-up in a repeated cross-sectional design. Campylobacter spp. seroprevalence averaged over all study visits was lower in azithromycin communities compared to placebo (91% vs 94%, difference = –3%, 95% CI: –5%, –1%; P=0.03), which corresponded to a 29% lower seroconversion rate (1.30 versus 1.84 seroconversions per year, hazard ratio = 0.71, 95% CI: 0.56, 0.89; P=0.004). Antibody-based measures of infection with P. falciparum and group A streptococcus were consistently lower in azithromycin communities, but were not statistically different from placebo, and there were no other differences across pathogens. Strengths of the study included masking of participants, investigators, and analysts, high treatment coverage, large sample size, and objective outcomes. Principal limitations included the timing of blood collection with respect to treatment (approximately 6 months later, which could have missed transient effects in the weeks immediately following treatment), and the durability of IgG response following clearance of infection. Both limitations would lead the trial to under-estimate effects on antibody-based measures of infection.Conclusions The reduction in Campylobacter spp. despite these limitations suggests an effect on carriage, findings which align with an independent metagenomic analysis of rectal swabs collected in the same villages and with previously reported reductions in dysentery-associated mortality. Given significant sequelae of Campylobacter infection among preschool aged children, our results support at least one possible mechanism through which biannual mass distribution of azithromycin likely reduced mortality in this study population.Competing Interest StatementThis work was supported by the Bill & Melinda Gates Foundation (award no. OPP1032340 to TML) and was supported in part by an unrestricted grant from Research to Prevent Blindness and by the National Institute of Allergy and Infectious Diseases (award no. K01-AI119180 to BFA). The Gates Foundation approved the study design, but had no role in data collection, data analysis, data interpretation, or writing of the report. The authors declare no competing interests. The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services. Clinical TrialNCT02048007Funding StatementThis work was supported by the Bill & Melinda Gates Foundation (award no. OPP1032340 to TML) and was supported in part by an unrestricted grant from Research to Prevent Blindness and by the National Institute of Allergy and Infectious Diseases (award no. K01-AI119180 to BFA). The Gates Foundation approved the study design, but had no role in data collection, data nalysis, data interpretation, or writing of the report.Author DeclarationsI confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.YesThe details of the IRB/oversight body that provided approval or exemption for the research described are given below:The trial protocol was reviewed and approved by the Committee on Human Research at the University of California, San Francisco, and the Niger Ministry of Health's Ethical Committee. Parents or guardians of enrolled children provided oral consent before each azithromycin or placebo treatment and at each specimen collection visit. Parents or guardians were instructed to report any adverse event within 7 days of treatment by contacting their village representative, who then reported events to the site coordinator and UCSF. An independent Data and Safety Monitoring Committee provided additional oversight. CDC researchers had access to de-identified samples for analysis (no personally identifying information).All necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).Yes I have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesData and computational notebooks used to conduct the analyses are available through the Open Science Framework (https://osf.io/954bt) and Dryad (xx DOI forthcoming xx). Analyses used R statistical software, version 4.0.2. https://osf.io/954bt

Identifying populations with active transmission and monitoring changes in transmission is centrally important in guiding schistosomiasis control programs. Traditionally, human Schistosoma mansoni infections have been detected in stool using microscopy, which is logistically difficult at program scale and has low sensitivity when people have low infection burdens. We compared serological measures of transmission based on antibody response to schistosomiasis soluble egg antigen (SEA) with stool-based measures of infection among 3,663 preschool-age children in an area endemic for S. mansoni in western Kenya. Serological measures of transmission closely aligned with stool-based measures of infection, and serological measures provided better resolution for between-community differences at lower levels of infection. Serology enabled fine- scale measures of heterogeneity in force of infection both geographically and by age. Our results show that serologic surveillance platforms represent an important new opportunity to guide and monitor schistosomiasis control programs.Competing Interest StatementThe authors have declared no competing interest.Funding StatementThis work was supported by the National Institutes of Allergy and Infectious Diseases (K01 AI119180 to BFA) and the Bill & Melinda Gates Foundation (OPP1022543 to PJL). The findings and conclusions in this article are those of the authors and do not necessarily represent the official position of the Centers for Disease Control and Prevention. Use of trade names is for identification only and does not imply endorsement by the Public Health Service or by the U.S. Department of Health and Human Services.Author DeclarationsAll relevant ethical guidelines have been followed; any necessary IRB and/or ethics committee approvals have been obtained and details of the IRB/oversight body are included in the manuscript.YesAll necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived.YesI understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance).YesI have followed all appropriate research reporting guidelines and uploaded the relevant EQUATOR Network research reporting checklist(s) and other pertinent material as supplementary files, if applicable.YesData and replication files required to conduct the analyses are available through GitHub and the Open Science Framework: https://osf.io/rnme8. Community location data required to replicate the geostatistical model fits is not publicly available to protect participant confidentiality but is available from the corresponding author upon request, pending appropriate human subjects review and approval. https://osf.io/rnme8

Accurate, cost-effective measurement of the burden of co-endemic infections would enable public health managers to identify opportunities for implementation of integrated control programs. Dried blood spots (DBS) collected during a cross-sectional lymphatic filariasis sentinel site survey in the Kenyan coastal counties of Lamu, Tana River, Kilifi, Kwale, and Taita-Taveta were used for the integrated detection of serologic IgG antibodies against antigens from several parasitic infections (Wuchereria bancrofti, Schistosoma mansoni, Plasmodium spp, Ascaris lumbricoides, and Strongyloides stercoralis) as well as markers for immunity to vaccine-preventable diseases (measles, diphtheria, and tetanus) on a multiplex bead assay (MBA) platform. High heterogeneity was observed in antibody responses by pathogen and antigen across the sentinel sites. Antibody seroprevalence against Wb123, Bm14, and Bm33 recombinant filarial antigens were generally higher in Ndau Island (p<0.0001), which also had the highest prevalence of filarial antigenemia compared to other communities. Antibody responses to the Plasmodium species antigens CSP and MSP-119 were higher in Kilifi and Kwale counties, with Jaribuni community showing higher overall mean seroprevalence (p<0.0001). Kimorigo community in Taita-Taveta County was the only area where antibody responses against Schistosoma mansoni Sm25 recombinant antigen were detected. Seroprevalence rates to Strongyloides antigen NIE ranged between 3% and 26%, and there was high heterogeneity in immune responses against an Ascaris antigen among the study communities. Differences were observed between communities in terms of seroprevalence to vaccine-preventable diseases. Seroprotection to tetanus was lower in all 3 communities in Kwale County compared to the rest of the communities. This study has demonstrated that the MBA platform holds promise for rapid integrated monitoring of trends of infections of public health importance in endemic areas, and assessing the effectiveness of control and elimination programs.Author Summary Establishment of successful private-public partnerships in the recent past has led to an increase in resources available for control and elimination of malaria and Neglected Tropical Diseases (NTDs). Implementation of control and elimination programs and their subsequent monitoring and evaluation would be greatly facilitated by development of new tools and strategies for rapid identification of areas of transmission so that interventions could be prioritized to regions where they were most needed. Since development of antibody responses in a host depend on exposure to an infectious agent, assessment of such serologic markers provides a sensitive way to measure differences between populations in pathogen exposure. Our study applied a state-of-the-art multiplex bead assay platform to perform integrated measurement of antibody responses to multiple parasitic diseases and immunizing antigens for vaccine-preventable diseases (VPDs) in ten lymphatic filariasis sentinel sites across the Kenyan coastal region. A community-level analysis of age-specific and overall mean seroprevalence fit using a flexible model ensemble provided an improved understanding about the distributions of the various parasitic infections and seroprotection to VPDs. This study provides an important proof of concept for how we could dramatically increase the value of existing surveillance activities using small volumes of blood collected on filter paper and analyzed using a single multiplex laboratory assay and novel data analysis techniques.

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles revealed significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest multiplex antibody assays are a promising approach to measure population-level enteropathogen transmission in serologic surveillance.

Background: We estimated SARS-CoV-2 Delta and Omicron-specific effectiveness of 2 and 3 mRNA COVID-19 vaccine doses in adults against symptomatic illness in US outpatient settings. Method(s): Between October 1, 2021, and February 12, 2022, research staff consented and enrolled eligible participants who had fever, cough, or loss of taste or smell and sought outpatient medical care or clinical SARS-CoV-2 testing within 10 days of illness onset. Using the test-negative design, we compared the odds of receiving 2 or 3 mRNA COVID-19 vaccine doses among SARS-CoV-2 cases versus controls using logistic regression. Regression models were adjusted for study site, age, onset week, and prior SARS-CoV-2 infection. Vaccine effectiveness (VE) was calculated as (1 - adjusted odds ratio) x 100%. Result(s): Among 3847 participants included for analysis, 574 (32%) of 1775 tested positive for SARS-CoV-2 during the Delta predominant period and 1006 (56%) of 1794 participants tested positive during the Omicron predominant period. When Delta predominated, VE against symptomatic illness in outpatient settings was 63% (95% CI: 51% to 72%) among mRNA 2-dose recipients and 96% (95% CI: 93% to 98%) for 3-dose recipients. When Omicron predominated, VE was 21% (95% CI: -6% to 41%) among 2-dose recipients and 62% (95% CI: 48% to 72%) among 3-dose recipients. Conclusion(s): In this adult population, 3 mRNA COVID-19 vaccine doses provided substantial protection against symptomatic illness in outpatient settings when the Omicron variant became the predominant cause of COVID-19 in the U.S. These findings support the recommendation for a 3rd mRNA COVID-19 vaccine dose. Copyright The copyright holder for this preprint is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. This article is a US Government work. It is not subject to copyright under 17 USC 105 and is also made available for use under a CC0 license.

BACKGROUND: Infectious diseases continue to burden populations in Malaysia, especially among rural communities where resources are limited and access to health care is difficult. Current epidemiological trends of several neglected tropical diseases in these populations are at present absent due to the lack of habitual and efficient surveillance. To date, various studies have explored the utility of serological multiplex beads to monitor numerous diseases simultaneously. We therefore applied this platform to assess population level exposure to six infectious diseases in Sabah, Malaysia. Furthermore, we concurrently investigated demographic and spatial risk factors that may be associated with exposure for each disease. METHODS: This study was conducted in four districts of Northern Sabah in Malaysian Borneo, using an environmentally stratified, population-based cross-sectional serological survey targeted to determine risk factors for malaria. Samples were collected between September to December 2015, from 919 villages totaling 10,100 persons. IgG responses to twelve antigens of six diseases (lymphatic filariasis- Bm33, Bm14, BmR1, Wb123; strongyloides- NIE; toxoplasmosis-SAG2A; yaws- Rp17 and TmpA; trachoma- Pgp3, Ct694; and giardiasis- VSP3, VSP5) were measured using serological multiplex bead assays. Eight demographic risk factors and twelve environmental covariates were included in this study to better understand transmission in this community. RESULTS: Seroprevalence of LF antigens included Bm33 (10.9%), Bm14+ BmR1 (3.5%), and Wb123 (1.7%). Seroprevalence of Strongyloides antigen NIE was 16.8%, for Toxoplasma antigen SAG2A was 29.9%, and Giardia antigens GVSP3 + GVSP5 was 23.2%. Seroprevalence estimates for yaws Rp17 was 4.91%, for TmpA was 4.81%, and for combined seropositivity to both antigens was 1.2%. Seroprevalence estimates for trachoma Pgp3 + Ct694 were 4.5%. Age was a significant risk factors consistent among all antigens assessed, while other risk factors varied among the different antigens. Spatial heterogeneity of seroprevalence was observed more prominently in lymphatic filariasis and toxoplasmosis. CONCLUSIONS: Multiplex bead assays can be used to assess serological responses to numerous pathogens simultaneously to support infectious disease surveillance in rural communities, especially where prevalences estimates are lacking for neglected tropical diseases. Demographic and spatial data collected alongside serosurveys can prove useful in identifying risk factors associated with exposure and geographic distribution of transmission.

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.

BACKGROUND: Integrated surveillance for multiple diseases can be an efficient use of resources and advantageous for national public health programs. Detection of IgG antibodies typically indicates previous exposure to a pathogen but can potentially also serve to assess active infection status. Serological multiplex bead assays have recently been developed to simultaneously evaluate exposure to multiple antigenic targets. Haiti is an island nation in the Caribbean region with multiple endemic infectious diseases, many of which have a paucity of data for population-level prevalence or exposure. METHODS: A nationwide serosurvey occurred in Haiti from December 2014 to February 2015. Filter paper blood samples (n = 4,438) were collected from participants in 117 locations and assayed for IgG antibodies on a multiplex bead assay containing 15 different antigens from 11 pathogens: Plasmodium falciparum, Toxoplasma gondii, lymphatic filariasis roundworms, Strongyloides stercoralis, chikungunya virus, dengue virus, Chlamydia trachomatis, Treponema pallidum, enterotoxigenic Escherichia coli, Entamoeba histolytica, and Cryptosporidium parvum. RESULTS: Different proportions of the Haiti study population were IgG seropositive to the different targets, with antigens from T. gondii, C. parvum, dengue virus, chikungunya virus, and C. trachomatis showing the highest rates of seroprevalence. Antibody responses to T. pallidum and lymphatic filariasis were the lowest, with <5% of all samples IgG seropositive to antigens from these pathogens. Clear trends of increasing seropositivity and IgG levels with age were seen for all antigens except those from chikungunya virus and E. histolytica. Parametric models were able to estimate the rate of seroconversion and IgG acquisition per year for residents of Haiti. CONCLUSIONS: Multiplex serological assays can provide a wealth of information about population exposure to different infectious diseases. This current Haitian study included IgG targets for arboviral, parasitic, and bacterial infectious diseases representing multiple different modes of host transmission. Some of these infectious diseases had a paucity or complete absence of published serological studies in Haiti. Clear trends of disease burden with respect to age and location in Haiti can be used by national programs and partners for follow-up studies, resource allocation, and intervention planning.

The MORDOR trial in Niger, Malawi, and Tanzania found that biannual mass distribution of azithromycin to children younger than 5 years led to a 13.5% reduction in all-cause mortality (NCT02048007). To help elucidate the mechanism for mortality reduction, we report IgG responses to 11 malaria, bacterial, and protozoan pathogens using a multiplex bead assay in pre-specified substudy of 30 communities in the rural Niger placebo-controlled trial over a three-year period (n = 5642 blood specimens, n = 3814 children ages 1-59 months). Mass azithromycin reduces Campylobacter spp. force of infection by 29% (hazard ratio = 0.71, 95% CI: 0.56, 0.89; P = 0.004) but serological measures show no significant differences between groups for other pathogens against a backdrop of high transmission. Results align with a recent microbiome study in the communities. Given significant sequelae of Campylobacter infection among preschool aged children, our results support an important mechanism through which biannual mass distribution of azithromycin likely reduces mortality in Niger.

The recent introduction of large-scale, population-based serologic surveys in several nations where human African trypanosomiasis (HAT) remains endemic could provide an opportunity to better map the remaining disease foci and to identify asymptomatic, seropositive individuals who are infected with the more chronic form of the parasite, Trypanosoma brucei gambiense (gHAT). We have incorporated a soluble form of variant surface glycoprotein 117 and a recombinant invariant surface glycoprotein 65.1 into a multiplex bead assay (MBA) method that is commonly used for the detection of IgG antibody responses to other neglected tropical diseases. A positive result was defined as reactivity to both antigens. MBA sensitivity and specificity for gHAT infection were 92% and 96%, respectively. Assay specificity for the acute form of disease caused by T.b. rhodesiense (rHAT) was 94%, but the sensitivity was only 63.6%. In the future, additional antigens could be incorporated into the multiplex assay to improve rHAT sensitivity.

BACKGROUND: Serological surveys with multiplex bead assays can be used to assess seroprevalence to multiple pathogens simultaneously. However, multiple methods have been used to generate cut-off values for seropositivity and these may lead to inconsistent interpretation of results. A literature review was conducted to describe the methods used to determine cut-off values for data generated by multiplex bead assays. METHODOLOGY/PRINCIPAL FINDINGS: A search was conducted in PubMed that that included articles published from January 2010 to January 2020, and 291 relevant articles were identified that included the terms "serology", "cut-offs", and "multiplex bead assays". After application of exclusion of articles not relevant to neglected tropical diseases (NTD), vaccine preventable diseases (VPD), or malaria, 55 articles were examined based on their relevance to NTD or VPD. The most frequently applied approaches to determine seropositivity included the use of presumed unexposed populations, mixture models, receiver operating curves (ROC), and international standards. Other methods included the use of quantiles, pre-exposed endemic cohorts, and visual inflection points. CONCLUSIONS/SIGNIFICANCE: For disease control programmes, seropositivity is a practical and easily interpretable health metric but determining appropriate cut-offs for positivity can be challenging. Considerations for optimal cut-off approaches should include factors such as methods recommended by previous research, transmission dynamics, and the immunological backgrounds of the population. In the absence of international standards for estimating seropositivity in a population, the use of consistent methods that align with individual disease epidemiological data will improve comparability between settings and enable the assessment of changes over time.

We validated a multiplex bead assay for diphtheria toxoid IgG antibodies against the Vero cell toxin neutralization test using 1300 specimens (correlation = 0.88). At the ≥0.01 IU/mL cutoff for minimal seroprotection, sensitivity was 95% and specificity was 83%. Agreement for three categories (<0.01, 0.01-<0.1, ≥0.1 IU/mL) was 81% (kappa = 0.71).

Serosurveys are important tools for estimating population immunity and providing immunization activity guidance. The measles and rubella multiplex bead assay (MBA) offers multiple advantages over standard serological assays and was validated by comparison with the enzyme-linked immunosorbent assay (ELISA) and the measles plaque reduction neutralization (PRN) assay. Results from a laboratory-produced purified measles whole virus antigen MBA (MeV WVA(L)) correlated better with ELISA and PRN than results from the baculovirus-expressed measles nucleoprotein (N) MBA. Therefore, a commercially produced whole virus antigen (MeV WVA(C)) was evaluated. Serum IgG antibody concentrations correlated significantly with a strong linear relationship between the MeV WVA(C) and MeV WVA(L) MBAs (R=0.962, R(2)=0.926). IgG concentrations from the MeV WVA(C) MBA showed strong correlation with PRN titers (R=0.846) with a linear relationship comparable to values obtained with the MeV WVA(L) MBA and PRN assay (R(2)=0.716 and R(2)=0.768, respectively). Receiver-operating characteristic (ROC) curve analysis of the MeV WVA(C) using PRN titer as the comparator resulted in a seroprotection cutoff of 153 mIU/ml, similar to the established correlate of protection of 120 mIU/ml, with a sensitivity of 98% and a specificity of 84%. IgG concentrations correlated strongly between the rubella WVA MBA and ELISA (R=0.959 and R(2)=0.919). ROC analysis of the rubella MBA using ELISA as the comparator yielded a cutoff of 9.36 IU/ml, similar to the accepted cutoff of 10 IU/ml for seroprotection, with a sensitivity of 99% and a specificity of 100%. These results support use of the MBA for multi-antigen serosurveys assessing measles and rubella population immunity.

Increased levels of guinea worm (GW) disease transmission among dogs in villages along the Chari River in Chad threaten the gains made by the GW Eradication Program. Infected dogs with preemergent worm blisters are difficult to proactively identify. If these dogs are not contained, blisters can burst upon submersion in water, leading to the contamination of the water supply with L1 larvae. Guinea worm antigens previously identified using sera from human dracunculiasis patients were coupled to polystyrene beads for multiplex bead assay analysis of 41 non-endemic (presumed negative) dog sera and 39 sera from GW-positive dogs from Chad. Because commercially available anti-dog IgG secondary antibodies did not perform well in the multiplex assay, dog IgGs were partially purified, and a new anti-dog IgG monoclonal antibody was developed. Using the new 4E3D9 monoclonal secondary antibody, the thioredoxin-like protein 1-glutathione-S-transferase (GST), heat shock protein (HSP1)-GST, and HSP2-GST antigen multiplex assays had sensitivities of 69-74% and specificities of 73-83%. The domain of unknown function protein 148 (DUF148)-GST antigen multiplex assay had a sensitivity of 89.7% and a specificity of 85.4%. When testing samples collected within 1 year of GW emergence (n = 20), the DUF148-GST assay had a sensitivity of 90.0% and a specificity of 97.6% with a receiver-operating characteristic area under the curve of 0.94. Using sera from two experimentally infected dogs, antibodies to GW antigens were detected within 6 months of exposure. Our results suggest that, when used to analyze paired, longitudinal samples collected 1-2 months apart, the DUF148/GST multiplex assay could identify infected dogs 4-8 months before GW emergence.

The success of the Guinea Worm (GW) Eradication Program over the past three decades has been tempered by the persistence of GW disease in a few African nations and the potential for a future resurgence in cases. Domestic dogs are now a major concern as a disease reservoir as large numbers of cases of canine GW disease are now reported each year, mainly along the Chari River in Chad. As a first step toward the development of a serologic assay for dogs, archived human plasma samples from dracunculiasis-positive donors from Togo were used to select adult female GW antigens for peptide sequencing and cloning. Eight protein sequences of interest were expressed as recombinant glutathione-S-transferase (GST) fusion proteins, and the most promising proteins were coupled to carboxylated microspheres for use in multiplex assays. A thioredoxin-like protein (TRXL1) and a domain of unknown function (DUF148) were assessed for total IgG and IgG(4) reactivities using a panel of specimens from GW cases, uninfected donors, and individuals infected with various nematode worms, including Onchocerca volvulus. Both the DUF148-GST and the TRXL1-GST assays cross-reacted with O. volvulus sera, but the latter assay was always the more specific. The IgG(4) and total IgG TRXL1-GST assays both had sensitivities > 87% and specificities > 90%. Maximum specificity (> 96%) was obtained with the total IgG assay when reactivity to both antigens was used to define a positive case. Given the good performance of the human assay, we are now working to modify the assay for dog assessments.

Schistosomiasis is among the most common parasitic diseases in the world, with over 142 million people infected in low- and middle-income countries. Measuring population-level transmission is centrally important in guiding schistosomiasis control programs. Traditionally, human Schistosoma mansoni infections have been detected using stool microscopy, which is logistically difficult at program scale and has low sensitivity when people have low infection burdens. We compared serological measures of transmission based on antibody response to S. mansoni soluble egg antigen (SEA) with stool-based measures of infection among 3,663 preschool-age children in an area endemic for S. mansoni in western Kenya. We estimated force of infection among children using the seroconversion rate and examined how it varied geographically and by age. At the community level, serological measures of transmission aligned with stool-based measures of infection (ρ = 0.94), and serological measures provided more resolution for between-community differences at lower levels of infection. Force of infection showed a clear gradient of transmission with distance from Lake Victoria, with 94% of infections and 93% of seropositive children in communities <1.5 km from the lake. Force of infection increased through age 3 y, by which time 65% (95% CI: 53%, 75%) of children were SEA positive in high-transmission communities-2 y before they would be reached by school-based deworming programs. Our results show that serologic surveillance platforms represent an important opportunity to guide and monitor schistosomiasis control programs, and that in high-transmission settings preschool-age children represent a key population missed by school-based deworming programs.

In Haiti, measles, rubella, and maternal and neonatal tetanus have been eliminated, but a diphtheria outbreak is ongoing as of 2019. We conducted a national representative, household-based, two-stage cluster survey among children aged 5-7 years in 2017 to assess progress toward maintenance of control and elimination of selected vaccine-preventable diseases (VPDs). We stratified Haiti into west region (west department, including the capital city) and non-west region (all other departments). We obtained vaccination history and dried blood spots, and measured antibody concentrations to VPDs on a multiplex bead assay. Among 1,146 children, national seropositivity was 83% (95% CI: 80-86%) for tetanus, 83% (95% CI: 81-85%) for diphtheria, 87% (95% CI: 85-89%) for measles, and 84% (95% CI: 81-87%) for rubella. None of the children had long-term immunity to tetanus or diphtheria (IgG concentration >/= 1 international unit/mL). Seropositivity in the west region was lower than that in the non-west region. Vaccination coverage was 68% (95% CI: 61-74%) for >/= 3 doses of tetanus- and diphtheria-containing vaccine (DTP3), 84% (95% CI: 80-87%) for one dose of measles-rubella (MR1) vaccine, and 20% (95% CI: 16-24%) for MR2. The seroprevalence of measles, rubella, and diphtheria antibodies is lower than population immunity levels needed to prevent disease transmission, particularly in the west region; reintroduction of these diseases could lead to an outbreak. To maintain VPD control and elimination, Haiti should achieve DTP3 and MR2 coverage >/= 95%, and include tetanus and diphtheria booster doses in the routine immunization schedule.

Accurate and cost-effective identification of areas where co-endemic infections occur would enable public health managers to identify opportunities for implementation of integrated control programs. Dried blood spots collected during cross-sectional lymphatic filariasis surveys in coastal Kenya were used for exploratory integrated detection of IgG antibodies against antigens from several parasitic infections (Wuchereria bancrofti, Schistosoma mansoni, Plasmodium spp., Ascaris lumbricoides, and Strongyloides stercoralis) as well as for detection of responses to immunizing agents used against vaccine-preventable diseases (VPDs) (measles, diphtheria, and tetanus) using a multiplex bead assay (MBA) platform. High heterogeneity was observed in antibody responses by pathogen and antigen across the sentinel sites. Antibody seroprevalence against filarial antigens were generally higher in Ndau Island (P < 0.0001), which also had the highest prevalence of filarial antigenemia compared with other communities. Antibody responses to the Plasmodium species antigens CSP and MSP-119 were higher in Kilifi and Kwale counties, with Jaribuni community showing higher overall mean seroprevalence (P < 0.0001). Kimorigo community in Taita-Taveta County was the only area where antibody responses against S. mansoni Sm25 recombinant antigen were detected. Seroprevalence rates to Strongyloides antigen NIE ranged between 3% and 26%, and there was high heterogeneity in immune responses against an Ascaris antigen among the study communities. Differences were observed between communities in terms of seroprevalence to VPDs. Seroprotection to tetanus was generally lower in Kwale County than in other counties. This study has demonstrated that MBA holds promise for rapid integrated monitoring of trends of infections of public health importance in endemic areas.

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.

Little is known about enteropathogen seroepidemiology among children in low-resource settings. We measured serological IgG responses to eight enteropathogens (Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, enterotoxigenic Escherichia coli, Vibrio cholerae, Campylobacter jejuni, norovirus) in cohorts from Haiti, Kenya, and Tanzania. We studied antibody dynamics and force of infection across pathogens and cohorts. Enteropathogens shared common seroepidemiologic features that enabled between-pathogen comparisons of transmission. Overall, exposure was intense: for most pathogens the window of primary infection was <3 years old; for highest transmission pathogens primary infection occurred within the first year. Longitudinal profiles demonstrated significant IgG boosting and waning above seropositivity cutoffs, underscoring the value of longitudinal designs to estimate force of infection. Seroprevalence and force of infection were rank-preserving across pathogens, illustrating the measures provide similar information about transmission heterogeneity. Our findings suggest antibody response can be used to measure population-level transmission of diverse enteropathogens in serologic surveillance.

In this prospective cohort study of Bangladeshi children, greater fecal IgA, but not plasma IgG, directed against the Cryptosporidium sporozoite-expressed antigen Cp23 at 12 months of age was associated with delayed time to subsequent cryptosporidiosis. This finding suggests a protective role for mucosal antibody-mediated immunity in naturally exposed children.

BACKGROUND: Due to their close relationship with the environment, Alaskans are at risk for zoonotic pathogen infection. One way to assess a population's disease burden is to determine the seroprevalence of pathogens of interest. The objective of this study was to determine the seroprevalence of 11 zoonotic pathogens in people living in Alaska. METHODS: In a 2007 avian influenza exposure study, we recruited persons with varying wild bird exposures. Using sera from this study, we tested for antibodies to Cryptosporidium spp., Echinococcus spp., Giardia intestinalis, Toxoplasma gondii, Trichinella spp., Brucella spp., Coxiella burnetii, Francisella tularensis, California serogroup bunyaviruses, and hepatitis E virus (HEV). RESULTS: Eight hundred eighty-seven persons had sera tested, including 454 subsistence bird hunters and family members, 160 sport bird hunters, 77 avian wildlife biologists, and 196 persons with no wild bird exposure. A subset (n = 481) of sera was tested for California serogroup bunyaviruses. We detected antibodies to 10/11 pathogens. Seropositivity to Cryptosporidium spp. (29%), California serotype bunyaviruses (27%), and G. intestinalis (19%) was the most common; 63% (301/481) of sera had antibodies to at least one pathogen. Using a multivariable logistic regression model, Cryptosporidium spp. seropositivity was higher in females (35.7% vs. 25.0%; p = 0.01) and G. intestinalis seropositivity was higher in males (21.8% vs. 15.5%; p = 0.02). Alaska Native persons were more likely than non-Native persons to be seropositive to C. burnetii (11.7% vs. 3.8%; p = 0.005) and less likely to be seropositive to HEV (0.4% vs. 4.1%; p = 0.01). Seropositivity to Cryptosporidium spp., C. burnetii, HEV, and Echinococcus granulosus was associated with increasing age (p </= 0.01 for all) as was seropositivity to >/=1 pathogen (p < 0.0001). CONCLUSION: Seropositivity to zoonotic pathogens is common among Alaskans with the highest to Cryptosporidium spp., California serogroup bunyaviruses, and G. intestinalis. This study provides a baseline for use in assessing seroprevalence changes over time.

BACKGROUND: Multiplex bead assays (MBA) that measure IgG antibodies to the carboxy-terminal 19-kDa sub-unit of the merozoite surface protein 1 (MSP119) are currently used to determine malaria seroprevalence in human populations living in areas with both stable and unstable transmission. However, the species specificities of the IgG antibody responses to the malaria MSP119 antigens have not been extensively characterized using MBA. METHODS: Recombinant Plasmodium falciparum (3D7), Plasmodium malariae (China I), Plasmodium ovale (Nigeria I), and Plasmodium vivax (Belem) MSP119 proteins were covalently coupled to beads for MBA. Threshold cut-off values for the assays were estimated using sera from US citizens with no history of foreign travel and by receiver operator characteristic curve analysis using diagnostic samples. Banked sera from experimentally infected chimpanzees, sera from humans from low transmission regions of Haiti and Cambodia (N = 12), and elutions from blood spots from humans selected from a high transmission region of Mozambique (N = 20) were used to develop an antigen competition MBA for antibody cross-reactivity studies. A sub-set of samples was further characterized using antibody capture/elution MBA, IgG subclass determination, and antibody avidity measurement. RESULTS: Total IgG antibody responses in experimentally infected chimpanzees were species specific and could be completely suppressed by homologous competitor protein at a concentration of 10 mug/ml. Eleven of 12 samples from the low transmission regions and 12 of 20 samples from the high transmission area had antibody responses that were completely species specific. For 7 additional samples, the P. falciparum MSP119 responses were species specific, but various levels of incomplete heterologous competition were observed for the non-P. falciparum assays. A pan-malaria MSP119 cross-reactive antibody response was observed in elutions of blood spots from two 20-30 years old Mozambique donors. The antibody response from one of these two donors had low avidity and skewed almost entirely to the IgG3 subclass. CONCLUSIONS: Even when P. falciparum, P. malariae, P. ovale, and P. vivax are co-endemic in a high transmission setting, most antibody responses to MSP119 antigens are species-specific and are likely indicative of previous infection history. True pan-malaria cross-reactive responses were found to occur rarely.

Antibodies are unique among biomarkers in their ability to identify persons with protective immunity to vaccine-preventable diseases and to measure past exposure to diverse pathogens. Most infectious disease surveillance maintains a single-disease focus, but broader testing of existing serologic surveys with multiplex antibody assays would create new opportunities for integrated surveillance. In this perspective, we highlight multiple areas for potential synergy where integrated surveillance could add more value to public health efforts than the current trend of independent disease monitoring through vertical programs. We describe innovations in laboratory and data science that should accelerate integration and identify remaining challenges with respect to specimen collection, testing, and analysis. Throughout, we illustrate how information generated through integrated surveillance platforms can create new opportunities to more quickly and precisely identify global health program gaps that range from undervaccination to emerging pathogens to multilayered health disparities that span diverse communicable diseases.

The potential consequences of parasitic infections on a person's immune responsiveness to unrelated antigens are often conjectured upon in relationship to allergic responses and autoimmune diseases. These considerations sometimes extend to whether parasitic infection of pregnant women can influence the outcomes of responses by their offspring to the immunizations administered during national Expanded Programs of Immunization. To provide additional data to these discussions, we have enrolled 99 close-to-term pregnant women in western Kenya and determined their Schistosoma mansoni and Plasmodium falciparum infection status. At 2 years of age, when the initial immunization schedule was complete, we determined their children's IgG antibody levels to tetanus toxoid, diphtheria toxoid, and measles nucleoprotein (N-protein) antigens using a multiplex assay. We also monitored antibody responses during the children's first 2 years of life to P. falciparum MSP119 (PfMSP119), S. mansoni Soluble Egg Antigen (SEA), Ascaris suum hemoglobin (AsHb), and Strongyloides stercoralis (SsNIE). Mothers' infections with either P. falciparum or S. mansoni had no impact on the level of antibody responses of their offspring or the proportion of offspring that developed protective levels of antibodies to either tetanus or diphtheria antigens at 2 years of age. However, children born of S. mansoni-positive mothers and immunized for measles at 9 months of age had significantly lower levels of anti-measles N-protein antibodies when they were 2 years old (p = 0.007) and a lower proportion of these children (62.5 vs. 90.2%, OR = 0.18, 95% CI = 0.04-0.68, p = 0.011) were considered positive for measles N-protein antibodies. Decreased levels of measles antibodies may render these children more susceptible to measles infection than children whose mothers did not have schistosomiasis. None of the children demonstrated responses to AsHb or SsNIE during the study period. Anti-SEA and anti-PfMSP119 responses suggested that 6 and 70% of the children acquired schistosomes and falciparum malaria, respectively, during the first 2 years of life.

Giardia duodenalis and Cryptosporidium spp. are common intestinal protozoa that can cause diarrhoeal disease. Although cases of infection with Giardia and Cryptosporidium have been reported in Alaska, the seroprevalence and correlates of exposure to these parasites have not been characterised. We conducted a seroprevalence survey among 887 residents of Alaska, including sport hunters, wildlife biologists, subsistence bird hunters and their families and non-exposed persons. We tested serum using a multiplex bead assay to evaluate antibodies to the Giardia duodenalis variant-specific surface protein conserved structural regions and to the Cryptosporidium parvum 17- and 27-kDa antigens. Approximately one third of participants in each group had evidence of exposure to Cryptosporidium. Prevalence of Giardia antibody was highest among subsistence hunters and their families (30%), among whom positivity was associated with lack of community access to in-home running water (adjusted prevalence ratio [aPR] 1.15, 95% confidence interval (CI) 1.02-1.28) or collecting rain, ice, or snow to use as drinking water (aPR 1.09, 95% CI 1.01-1.18). Improving in-home water access for entire communities could decrease the risk of exposure to Giardia.

Cryptosporidium is a leading cause of diarrhea among Kenyan infants. Ceramic water filters (CWFs) are used for household water treatment. We assessed the impact of CWFs on diarrhea, cryptosporidiosis prevention, and water quality in rural western Kenya. A randomized, controlled intervention trial was conducted in 240 households with infants 4-10 months old. Twenty-six weekly household surveys assessed infant diarrhea and health facility visits. Stool specimens from infants with diarrhea were examined for Cryptosporidium. Source water, filtered water, and filter retentate were tested for Cryptosporidium and/or microbial indicators. To estimate the effect of CWFs on health outcomes, logistic regression models using generalized estimating equations were performed; odds ratios (ORs) and 95% confidence intervals (CIs) are reported. Households reported using surface water (36%), public taps (29%), or rainwater (17%) as their primary drinking water sources, with no differences in treatment groups. Intervention households reported less diarrhea (7.6% versus 8.9%; OR: 0.86 [0.64-1.16]) and significantly fewer health facility visits for diarrhea (1.0% versus 1.9%; OR: 0.50 [0.30-0.83]). In total, 15% of intervention and 12% of control stools yielded Cryptosporidium (P = 0.26). Escherichia coli was detected in 93% of source water samples; 71% of filtered water samples met World Health Organization recommendations of < 1 E. coli/100 mL. Cryptosporidium was not detected in source water and was detected in just 2% of filter rinses following passage of large volumes of source water. Water quality was improved among CWF users; however, the short study duration and small sample size limited our ability to observe reductions in cryptosporidiosis.

BACKGROUND: Universal coverage with long-lasting insecticidal nets (LLINs) is a primary control strategy against Plasmodium falciparum malaria. However, its impact on the three other main species of human malaria and lymphatic filariasis (LF), which share the same vectors in many co-endemic areas, is not as well characterized. The recent development of multiplex antibody detection provides the opportunity for simultaneous evaluation of the impact of control measures on the burden of multiple diseases. METHODOLOGY/PRINCIPAL FINDINGS: Two cross-sectional household surveys at baseline and one year after a LLIN distribution campaign were implemented in Mecuburi and Nacala-a-Velha Districts in Nampula Province, Mozambique. Both districts were known to be endemic for LF; both received mass drug administration (MDA) with antifilarial drugs during the evaluation period. Access to and use of LLINs was recorded, and household members were tested with P. falciparum rapid diagnostic tests (RDTs). Dried blood spots were collected and analyzed for presence of antibodies to three P. falciparum antigens, P. vivax MSP-119, P. ovale MSP-119, P. malariae MSP-119, and three LF antigens. Seroconversion rates were calculated and the association between LLIN use and post-campaign seropositivity was estimated using multivariate regression. The campaign covered 68% (95% CI: 58-77) of the population in Nacala-a-Velha and 46% (37-56) in Mecuburi. There was no statistically significant change in P. falciparum RDT positivity between the two surveys. Population seropositivity at baseline ranged from 31-81% for the P. falciparum antigens, 3-4% for P. vivax MSP-119, 41-43% for P. ovale MSP-119, 46-56% for P. malariae MSP-119, and 37-76% for the LF antigens. The seroconversion rate to the LF Bm33 antigen decreased significantly in both districts. The seroconversion rate to P. malariae MSP-119 and the LF Wb123 and Bm14 antigens each decreased significantly in one of the two districts. Community LLIN use was associated with a decreased risk of P. falciparum RDT positivity, P. falciparum CSP and LSA-1 seropositivity, and P. malariae MSP-119 seropositivity, but not LF antigen seropositivity. CONCLUSIONS/SIGNIFICANCE: The study area noted significant declines in LF seropositivity, but these were not associated with LLIN use. The MDA could have masked any impact of the LLINs on population LF seropositivity. The LLIN campaign did not reach adequately high coverage to decrease P. falciparum RDT positivity, the most common measure of P. falciparum burden. However, the significant decreases in the seroconversion rate to the P. malariae antigen, coupled with an association between community LLIN use and individual-level decreases in seropositivity to P. falciparum and P. malariae antigens show evidence of impact of the LLIN campaign and highlight the utility of using multiantigenic serological approaches for measuring intervention impact.