Diarrhea is a major contributor to morbidity and mortality in children under five years of age, responsible for an estimated half a million deaths annually (Collaborators, 2018; Perin et al., 2022). Infections with diarrheal disease-causing enteric pathogens have been associated with long-term health conditions including environmental enteric dysfunction, physical and cognitive stunting, malnutrition, and poor immune function (Tickell et al., 2019; Kosek and Investigators, 2017; Owino et al., 2016; Kau et al., 2011; Guerrant et al., 2008). Though substantial progress has been made toward reducing the burden of diarrheal diseases, the remaining level of morbidity poses a significant public health problem, especially in low resource settings (Collaborators, 2018; Perin et al., 2022). Water, sanitation, and hygiene (WaSH) interventions have not substantially reduced the overall burden of diarrheal disease (Goddard et al., 2020; Pickering et al., 2019), and currently there are only licensed vaccines available for rotavirus, Cholera, and Salmonella typhi (Cohen and Muhsen, 2019). Characterizing enteric exposures and infections would help inform more targeted and effective intervention methods, but measuring the wide range of bacterial, viral, and protozoan pathogens that can cause diarrheal disease often requires a similarly large range of diagnostic methods (Exum et al., 2016). Detection and identification of enteric pathogens typically involves measuring the presence of the disease-causing pathogens in stool samples. These methods have greatly expanded the understanding of the causation of diarrheal disease, but the direct detection of pathogens can be complicated by sample collection and handling logistics, limited availability of sensitive diagnostics methods, and relatively short windows of pathogen presence, requiring frequent longitudinal sampling to comprehensively identify incident infections (Platts-Mills et al., 2018; Lin et al., 2018).

Serological testing provides a means to address limitations of pathogen detection methods in estimating exposure to enteric pathogens because antibody responses persist for months to years, depending on the isotype and specific pathogen, and can detect not only symptomatic infections and asymptomatic infections, but also enteric exposures that elicit an immune response without the development of established infection (Exum et al., 2016; Simonsen et al., 2008; Arnold et al., 2018; Arnold et al., 2019; Platts-Mills et al., 2013). Serological studies may also serve as an efficient means of characterizing pathogen type-specific incidence, which otherwise generally requires culture of bacterial isolates for typing assays (Platts-Mills et al., 2013). Further, though antibody responses are traditionally measured in blood, there is significant work demonstrating that antibodies can also be reliably measured in salivary oral fluid (Pisanic et al., 2019; Pisanic et al., 2017; Egorov et al., 2021; Egorov et al., 2018; Griffin et al., 2011; Griffin et al., 2015; Wade et al., 2019; Brandtzaeg, 2007; Heaney et al., 2021; Pisanic et al., 2020). The use of a non-invasive sample type such as saliva provides the opportunity to fill gaps in exposure and infection assessments across populations most severely impacted by diarrheal disease, including children, the elderly, immunocompromised individuals, and other populations for which the acceptability or feasibility of blood collection is limited. Likewise, saliva is well-suited for use in low-resource settings where blood collection presents logistical barriers including the need for sample collection by specially trained personnel, infection mitigation protocols, and cold-chain for sample storage and handling (Randad et al., 2020; Figueroa et al., 2018; Wade et al., 2018; Krause et al., 2013).

Conducting serology through multiplexed immunoassays (MIAs) allows for the detection of multiple antigen-specific antibody responses simultaneously, with the range of applications determined by the assay design. For example, several immunogenic targets from a single pathogen may be multiplexed to increase the likelihood that exposure will be captured (Pisanic et al., 2019; Pisanic et al., 2017; Wade et al., 2018; Kaminski et al., 2013; Moss et al., 2004; Priest and Moss, 2020). Conversely, an MIA may include targets from multiple pathogens to characterize disease when individuals present with non-specific symptoms, like diarrhea, abdominal pain, or vomiting, all of which may be caused by bacterial, viral, or protozoan pathogens (Arnold et al., 2019; Egorov et al., 2021; Griffin et al., 2011; Priest et al., 2010; Augustine et al., 2021; Augustine et al., 2017). Further, multiplexing decreases time, labor, material expenses, and required sample volume compared to traditional single-target immunoassays and the specificity of antibody responses provides an opportunity for the piece-wise addition of antigenic targets to existing MIAs (Arnold et al., 2018). For example, enteric pathogen targets could be added into MIAs designed for existing serosurveillance infrastructure, like those that have been developed for assessing seroprevalence to vaccine preventable diseases (VPDs) (Arnold et al., 2018; Njenga et al., 2020). However, current enteric multiplex designs are often limited in scope, containing only peptide- or protein-based antigens from a few genera of pathogens, while missing other immunogenically relevant antigens, such as lipopolysaccharide (LPS), that require more complex assay coupling chemistries. Improved scalability and range of pathogen detection through multiplexing could help inform prevention and treatment guidelines as well as direct public health interventions.

Here, we 1) developed a Luminex xMAP bead-based 50-plex immunoassay designed to quantitatively or semi-quantitatively evaluate IgG and IgA antibody responses to enteric pathogens associated with the greatest contribution to the global diarrheal disease burden. (1)The assay included enteric targets from bacteria (Shigella, Enterotoxigenic Escherichia coli (ETEC), Campylobacter, typhoidal Salmonella, non-typhoidal Salmonella); viruses (norovirus, rotavirus, hepatitis E virus, adenovirus, astrovirus); and protozoa (Cryptosporidium and Giardia).To provide an example of the modularity and flexibility of the multiplexing platform, we also included targets from respiratory viruses including influenza A/B and respiratory syncytial virus (RSV) as well as VPDs of substantial public health interest, including tetanus, measles, and rubella; 2) optimized the antigen-bead coupling process, with substantial consideration given to the modification and coupling of enteric bacterial LPS antigens; 3) assessed assay performance (cross-reactivity, intra- and inter-plate, and inter-operator variability) in both serum and oral fluid samples; and 4) validated a subset of pathogen targets in the MIA against reference immunoassays.