Salivary Antibodies against Multiple Environmental Pathogens Found in Individuals Recreating at an Iowa Beach.

Detecting environmental exposures and mitigating their impacts are growing global public health challenges. Antibody tests show great promise and have emerged as fundamental tools for large-scale exposure studies. Here, we apply, demonstrate and validate the utility of a salivary antibody multiplex immunoassay in measuring antibody prevalence and immunoconversions to six pathogens commonly found in the environment. The study aimed to assess waterborne infections in consenting beachgoers recreating at an Iowa riverine beach by measuring immunoglobulin G (IgG) antibodies against select pathogens in serially collected saliva samples. Results showed that nearly 80% of beachgoers had prior exposures to at least one of the targeted pathogens at the beginning of the study. Most of these exposures were to norovirus GI.1 (59.41%), norovirus GII.4 (58.79%) and Toxoplasma gondii (22.80%) and over half (56.28%) of beachgoers had evidence of previous exposure to multiple pathogens. Of individuals who returned samples for each collection period, 6.11% immunoconverted to one or more pathogens, largely to noroviruses (GI.1: 3.82% and GII.4: 2.29%) and T. gondii (1.53%). Outcomes of this effort illustrate that the multiplex immunoassay presented here serves as an effective tool for evaluating health risks by providing valuable information on the occurrence of known and emerging pathogens in population surveillance studies.

Rapid Salivary IgG Antibody Screening for Hepatitis A.

Hepatitis A virus (HAV) is a common infection that is transmitted through the fecal-oral route, shed in the stool of infected individuals, and spread either by direct contact or by ingesting contaminated food or water. Each year, approximately 1.4 million acute cases are reported globally with a major risk factor for exposure being low household socioeconomic status. Recent trends show a decrease in anti-HAV antibodies in the general population, with concomitant increases in the numbers of HAV outbreaks. In line with a recreational water study, this effort aims to assess the prevalence of salivary IgG antibodies against HAV and subsequent incident infections (or immunoconversions) in visitors to a tropical beach impacted by a publicly owned treatment works (POTW). We applied a multiplex immunoassay to serially collected saliva samples gathered from study participants who recreated at Boquerón Beach, Puerto Rico. Analysis of assay results revealed an immunoprevalence rate of 16.17% for HAV with 1.43% of the cohort immunoconverting to HAV. Among those who immunoconverted, 10% reported chronic gastrointestinal symptoms and none experienced diarrhea. Tests on water samples indicated good water quality with low levels of fecal indicator bacteria; however, the collection and analysis of saliva samples afforded the ability to detect HAV infections in beachgoers. This rapid assay serves as a cost-effective tool for examining exposure to environmental pathogens and can provide critical information to policy makers, water quality experts, and risk assessment professionals seeking to improve and protect recreational water and public health.

Visitors to a Tropical Marine Beach Show Evidence of Immunoconversions to Multiple Waterborne Pathogens.

Determining infections from environmental exposures, particularly from waterborne pathogens is a challenging proposition. The study design must be rigorous and account for numerous factors including study population selection, sample collection, storage, and processing, as well as data processing and analysis. These challenges are magnified when it is suspected that individuals may potentially be infected by multiple pathogens at the same time. Previous work demonstrated the effectiveness of a salivary antibody multiplex immunoassay in detecting the prevalence of immunoglobulin G (IgG) antibodies to multiple waterborne pathogens and helped identify asymptomatic norovirus infections in visitors to Boquerón Beach, Puerto Rico. In this study, we applied the immunoassay to three serially collected samples from study participants within the same population to assess immunoconversions (incident infections) to six waterborne pathogens: Helicobacter pylori, Campylobacter jejuni, Toxoplasma gondii, hepatitis A virus, and noroviruses GI. I and GII.4. Further, we examined the impact of sampling on the detection of immunoconversions by comparing the traditional immunoconversion definition based on two samples to criteria developed to capture trends in three sequential samples collected from study participants. The expansion to three samples makes it possible to capture the IgG antibody responses within the survey population to more accurately assess the frequency of immunoconversions to target pathogens. Based on the criteria developed, results showed that when only two samples from each participant were used in the analysis, 25.9% of the beachgoers immunoconverted to at least one pathogen; however, the addition of the third sample reduced immunoconversions to 6.5%. Of these incident infections, the highest levels were to noroviruses followed by T. gondii. Moreover, many individuals displayed evidence of immunoconversions to multiple pathogens. This study suggests that detection of simultaneous infections is possible, with far reaching consequences for the population. The results may lead to further studies to understand the complex interactions that occur within the body as the immune system attempts to ward off these infections. Such an approach is critical to our understanding of medically important synergistic or antagonistic interactions and may provide valuable and critical information to public health officials, water treatment personnel, and environmental managers.

Asymptomatic norovirus infection associated with swimming at a tropical beach: A prospective cohort study.

BACKGROUND: Swimming in fecally-contaminated waterbodies can result in gastrointestinal infections. However, the pathogenic microorganisms responsible are not well understood because sporadic cases of illness are not reported completely, exposure information is often not collected, and epidemiology studies rely on self-reported symptoms. Noroviruses are considered a likely cause because they are found in high densities in sewage, resistant to wastewater treatment and survive in the environment. In this study, saliva samples were collected from subjects at a beach in Puerto Rico and tested for evidence of norovirus-specific IgG responses as an indicator of incident norovirus infection. METHODS: Saliva samples were collected from 1298 participants using an oral swab. Samples were collected on the day of the beach visit (S1); after 10-12 days (S2); and after three weeks (S3). Saliva was tested for IgG responses to GI.1 and GII.4 noroviruses using a microsphere based multiplex salivary immunoassay. Immunoconversion was defined as a four-fold increase in median fluorescence intensity (MFI) from S1 to S2 with the S3 sample at least three times above the S1 MFI. RESULTS: Thirty-four subjects (2.6%) immunoconverted to GI.1 or GII.4 norovirus. Swimmers who immersed their head in water had a higher rate of immunoconversion (3.4%), compared to either non-swimmers (0.0%, p = 0.003) or waders and non-swimmers combined (0.4%, Odds Ratio: 5.07, 95% Confidence Interval:1.48-17.00). Immunoconversion was not associated with gastrointestinal symptoms. CONCLUSIONS: This is the first study to demonstrate an association between swimming at a beach impacted by fecal contamination and asymptomatic norovirus infection. The findings implicate recreational water as potentially important transmission pathway for norovirus infection.

Immunoprevalence to Six Waterborne Pathogens in Beachgoers at Boquerón Beach, Puerto Rico: Application of a Microsphere-Based Salivary Antibody Multiplex Immunoassay.

Waterborne infectious diseases are a major public health concern worldwide. Few methods have been established that are capable of measuring human exposure to multiple waterborne pathogens simultaneously using non-invasive samples such as saliva. Most current methods measure exposure to only one pathogen at a time, require large volumes of individual samples collected using invasive procedures, and are very labor intensive. In this article, we applied a multiplex bead-based immunoassay capable of measuring IgG antibody responses to six waterborne pathogens simultaneously in human saliva to estimate immunoprevalence in beachgoers at Boquerón Beach, Puerto Rico. Further, we present approaches for determining cutoff points to assess immunoprevalence to the pathogens in the assay. For the six pathogens studied, our results show that IgG antibodies against antigens from noroviruses GI.I and GII.4 were more prevalent (60 and 51.6%, respectively) than Helicobacter pylori (21.4%), hepatitis A virus (20.2%), Campylobacter jejuni (8.7%), and Toxoplasma gondii (8%) in the saliva of the study participants. The salivary antibody multiplex immunoassay can be used to examine immunoprevalence of specific pathogens in human populations.

Developing a Salivary Antibody Multiplex Immunoassay to Measure Human Exposure to Environmental Pathogens.

The etiology and impacts of human exposure to environmental pathogens are of major concern worldwide and, thus, the ability to assess exposure and infections using cost effective, high-throughput approaches would be indispensable. This manuscript describes the development and analysis of a bead-based multiplex immunoassay capable of measuring the presence of antibodies in human saliva to multiple pathogens simultaneously. Saliva is particularly attractive in this application because it is noninvasive, cheaper and easier to collect than serum. Antigens from environmental pathogens were coupled to carboxylated microspheres (beads) and used to measure antibodies in very small volumes of human saliva samples using a bead-based, solution-phase assay. Beads were coupled with antigens from Campylobacter jejuni, Helicobacter pylori, Toxoplasma gondii, noroviruses (G I.1 and G II.4) and hepatitis A virus. To ensure that the antigens were sufficiently coupled to the beads, coupling was confirmed using species-specific, animal-derived primary capture antibodies, followed by incubation with biotinylated anti-species secondary detection antibodies and streptavidin-R-phycoerythrin reporter (SAPE). As a control to measure non-specific binding, one bead set was treated identically to the others except it was not coupled to any antigen. The antigen-coupled and control beads were then incubated with prospectively-collected human saliva samples, measured on a high throughput analyzer based on the principles of flow cytometry, and the presence of antibodies to each antigen was measured in Median Fluorescence Intensity units (MFI). This multiplex immunoassay has a number of advantages, including more data with less sample; reduced costs and labor; and the ability to customize the assay to many targets of interest. Results indicate that the salivary multiplex immunoassay may be capable of identifying previous exposures and infections, which can be especially useful in surveillance studies involving large human populations.

Towards Universal Screening for Toxoplasmosis: Rapid, Cost-Effective, and Simultaneous Detection of Anti-Toxoplasma IgG, IgM, and IgA Antibodies by Use of Very Small Serum Volumes.

Rapid, cost-effective, and early determination of the serological status of potentially infected individuals, particularly pregnant women, can be critical in preventing life-threatening infections and subsequent fetal congenital abnormalities. An article in this issue of the Journal of Clinical Microbiology (X. Li, C. Pomares, G. Gonfrier, B. Koh, S. Zhu, M. Gong, J. G. Montoya, and H. Dai, J Clin Microbiol 54:1726-1733, 2016, http://dx.doi.org/10.1128/JCM.03371-15) describes an innovative multiplexed immunoassay that offers a path toward universal screening.

Statistical approaches to developing a multiplex immunoassay for determining human exposure to environmental pathogens.

There are numerous pathogens that can be transmitted through water. Identifying and understanding the routes and magnitude of exposure or infection to these microbial contaminants are critical to assessing and mitigating risk. Conventional approaches of studying immunological responses to exposure or infection such as Enzyme-Linked Immunosorbent Assays (ELISAs) and other monoplex antibody-based immunoassays can be very costly, laborious, and consume large quantities of patient sample. A major limitation of these approaches is that they can only be used to measure one analyte at a time. Multiplex immunoassays provide the ability to study multiple pathogens simultaneously in microliter volumes of samples. However, there are several challenges that must be addressed when developing these multiplex immunoassays such as selection of specific antigens and antibodies, cross-reactivity, calibration, protein-reagent interferences, and the need for rigorous optimization of protein concentrations. In this study, a Design of Experiments (DOE) approach was used to optimize reagent concentrations for coupling selected antigens to Luminex™ xMAP microspheres for use in an indirect capture, multiplex immunoassay to detect human exposure or infection from pathogens that are potentially transmitted through water. Results from Helicobacter pylori, Campylobacter jejuni, Escherichia coli O157:H7, and Salmonella typhimurium singleplexes were used to determine the mean concentrations that would be applied to the multiplex assay. Cut-offs to differentiate between exposed and non-exposed individuals were determined using finite mixed modeling (FMM). The statistical approaches developed facilitated the detection of Immunoglobulin G (IgG) antibodies to H. pylori, C. jejuni, Toxoplasma gondii, hepatitis A virus, rotavirus and noroviruses (VA387 and Norwalk strains) in fifty-four diagnostically characterized plasma samples. Of the characterized samples, the detection rate was 87.5% for H. pylori, and 100% for T. gondii assays and 89% for HAV. Further, the optimized multiplex assay revealed exposure/infection to several other environmental pathogens previously uncharacterized in the samples.

Determining UV inactivation of Toxoplasma gondii oocysts by using cell culture and a mouse bioassay.

The effect of UV exposure on Toxoplasma gondii oocysts has not been completely defined for use in water disinfection. This study evaluated UV-irradiated oocysts by three assays: a SCID mouse bioassay, an in vitro T. gondii oocyst plaque (TOP) assay, and a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay. The results from the animal bioassay show that 1- and 3-log(10) inactivation is achieved with 4 mJ/cm(2) UV and 10 mJ/cm(2) low-pressure UV, respectively. TOP assay results, but not RT-qPCR results, correlate well with bioassay results. In conclusion, a 3-log(10) inactivation of T. gondii oocysts is achieved by 10-mJ/cm(2) low-pressure UV, and the in vitro TOP assay is a promising alternative to the mouse bioassay.

Using quantitative reverse transcriptase PCR and cell culture plaque assays to determine resistance of Toxoplasma gondii oocysts to chemical sanitizers.

Toxoplasma gondii oocysts are highly resistant to many chemical sanitizers. Methods used to determine oocyst infectivity have relied primarily on mouse, chicken, and feline bioassays. Although considered gold standards, they only provide a qualitative assessment of oocyst viability. In this study, two alternative approaches were developed to quantitate viable T. gondii oocysts following treatment with several common sanitizers. The first is a quantitative reverse transcriptase real-time PCR (RT-qPCR) assay targeting the ACT1 and SporoSAG genes to enumerate viable T. gondii oocysts. RT-qPCR C(T) values between Wescodyne(R), acidified ethanol, or heat treated oocysts were not significantly different as compared with untreated controls. By contrast, treatment with formalin or Clorox(R) resulted in a 2-log(10) reduction in C(T) values. An in vitro T. gondii oocyst plaque assay (TOP-assay) was also developed to measure oocyst viability. This assay used a combination of bead milling and bile digestion, followed by culturing the excysted sporozoites in a confluent fibroblast cell monolayer. Results showed that no significant reduction in sporozoite viability was detected in acidified ethanol or Wescodyne(R) treated oocysts while at least a 2-log(10) reduction in plaques formed was observed with Clorox(R) treated oocysts. Moreover, formalin or heat treatment of oocysts resulted in at least a 5-log(10) reduction in plaques formed. This study demonstrates that an mRNA-based PCR viability assay targeting the ACT1 or SporoSAG genes is a relatively rapid technique compared to in vitro and in vivo assays. In addition, the TOP-assay proved very effective and sensitive at quantifying oocyst viability when compared with animal bioassays.

Molecular cloning of a Trypanosoma cruzi cell surface casein kinase II substrate, Tc-1, involved in cellular infection.

In this work, we report the cloning and characterization of the first cell surface casein kinase II (CKII) substrate (Tc-1) of Trypanosoma cruzi, the causative agent of Chagas' disease. Analysis of the gene sequence revealed a 1,653-bp open reading frame coding for 550 amino acid residues. Northern blot analysis showed a 4.5-kb transcript that is expressed in invasive trypomastigotes but not in noninvasive epimastigote forms of T. cruzi. Southern blot analysis indicates that Tc-1 is a single-copy gene. At the amino acid level, Tc-1 displayed 95% and 99% identity to two hypothetical proteins recently reported by the T. cruzi genome project. Analysis of the translated amino acid sequence indicates that the Tc-1 gene has a putative transmembrane domain with multiple cytoplasmic and extracellular CKII phosphosites. Exogenous human CKII was able to phosphorylate serine residues on both recombinant Tc-1 and Tc-1 of intact trypomastigotes. This phosphorylation was inhibited by the CKII inhibitors heparin and 4,5,6,7,-tetrabromo-2-azabenzimidazole. Immunoblots of solubilized trypomastigotes, epimastigotes, and amastigotes probed with anti-recombinant Tc-1 immunoglobulin G revealed a 62-kDa protein that is expressed only in infective trypomastigotes. Immunoprecipitation of labeled surface proteins of trypomastigotes indicated that the 62-kDa protein is a surface protein, and we found that the protein is uniformly distributed on the surface of trypomastigotes by direct immunofluorescence. Antibodies to Tc-1 effectively blocked trypomastigote invasion of host cells and consequently reduced parasite load. Preincubation of either trypomastigotes or myoblasts with CKII inhibitors blocked T. cruzi infection. Thus, for the first time, we describe a cell surface CKII substrate of a protozoan parasite that is phosphorylated by human CKII and that is involved in cellular infection.