bioRxiv Science⌕ Search
Home/Archive/bioRxiv

bioRxiv · 10.64898/2026.01.05.697665

Stool antigen-based enzyme immunoassays: Performance evaluations and automation

Bao, J. R.·Jones, R. S.
View original source ↗Export metadata ↓

Abstract

Three enzyme immunoassays (EIAs) were evaluated on two platforms each for detecting Cryptosporidium, Giardia, and Campylobacter in stool samples compared to gold standard methods. The Cryptosporidium EIAs run on stool specimens in three preservative media showed 100% agreement with direct fluorescent antibody (DFA) testing and high correlation with the microscopic method. The Giardia EIAs demonstrated 100% sensitivity but only moderate correlation with the microscopic method. The Campylobacter EIAs had 93.5% sensitivity (out of 65 positives) and 100% specificity compared to the culture method. Automation on a DS2 system for these three EIAs, along with five others, yielded acceptable performance (92.5-100% accuracy) compared to manual methods. The automation saved labor time and improved operational efficiency but may not be cost-effective for low-volume runs due to the labor required for automation not scaling proportionally with sample numbers. In conclusion, EIAs are preferred for detecting protozoan parasites in stool, with the Cryptosporidium EIA showing potential as a semi-quantitative assay and a reference method. Automation benefits high-throughput laboratories but may not be as advantageous for low-volume laboratories. IMPORTANCEEnzyme immunoassay (EIA) is a widely used method in clinical laboratories to detect pathogens in stool samples related to diarrhea diseases. This study evaluated the performance of three EIAs for detecting Cryptosporidium, Giardia, and Campylobacter antigens compared to their gold standard methods. The Cryptosporidium EIAs matched the direct fluorescent antibody (DFA) testing and had a high correlation with microscopic findings (99.7%). The Giardia EIAs showed 100% sensitivity but lower specificity (58%) and moderate correlation with microscopic results (87.4%). The Campylobacter EIA had 93.5% sensitivity (n=65 positive samples) with few discrepancies. Automating these three and five other EIAs using a DS2 system (Dynex) yielded good accuracy (92.5-100%) and 100% precision compared to manual methods. While automation saved hands-on time for high-volume assays, it may not be cost-effective for low-volume laboratories.

Source connections

Explore related subjects

microbiologyPreprint

Keep this discovery

Explore connections, maps & timelines

BibTeXRIS

Bao, J. R., Jones, R. S.. 2026-01-05. Stool antigen-based enzyme immunoassays: Performance evaluations and automation. https://doi.org/10.64898/2026.01.05.697665

Cite the original work for its findings. Save a collection to share your selection of sources.

KEEP EXPLORING

Related preprints

bioRxiv2026

Reverse Transcription Loop-Mediated Isothermal Amplification Combined with Lateral Flow Assay (RT-LAMP-LFA) for Rapid Detection of the 2026 Bundibugyo Virus (BDBV) Outbreak Strain

Making accurate diagnosis is a critical first step in patient management and control for emerging and re-emerging disease outbreaks. Bundibugyo virus (BDBV) is a filovirus that causes Ebola disease, and the 2026 outbreak highlights the need for rapid diagnostic approaches that can be deployed in settings with limited laboratory infrastructure. Here, we developed a reverse transcription loop-mediated isothermal amplification-lateral flow assay (RT-LAMP-LFA) for rapid detection of the 2026 BDBV outbreak strain. LAMP primers were designed based on the genome sequence of the 2026 outbreak strain, and the conservation of the primer binding sites among all available 2026 outbreak genomes was confirmed in silico. LAMP primers were evaluated for analytical specificity and sensitivity using in vitro-transcribed RNA (ivtRNA) from BDBV and other filoviruses, as well as RNA derived from arboviruses and mammalian cells. Amplification products were detected using a Chromatography-Printed Array Strip (C-PAS) lateral flow platform. The newly designed primers specifically detected the presence of BDBV RNA without nonspecific amplification of the other viral or cellular RNA samples tested. Consistent amplification was observed down to 103 copies per reaction. In addition, BDBV viral RNA was successfully amplified, and the amplification products were detected using C-PAS, with the total workflow being completed within 25 min. These findings demonstrate the analytical specificity and sensitivity of the developed RT-LAMP-LFA assay and its compatibility with rapid visual lateral flow detection, supporting its potential application as a point-of-care approach for BDBV detection.

microbiology↗
bioRxiv2026

Divergent methane production and microbial community responses to carbon enrichment in freshwater wetland sediments

Methane (CH4) accumulation in wetland sediments reflects interactions among organic matter, sediment chemistry, and microbial communities. We combined depth-resolved field characterization across four wetland habitats in northern Israel with complementary incubations to examine methane accumulation and responses to labile-carbon enrichment. Sediment cores were characterized using Rock-Eval analysis, porewater chemistry, oxygen profiling at three sites, and mcrA amplicon sequencing. High CH4 concentrations occurred under contrasting conditions: Ein Nimfit (EN) contained approximately 12% total organic carbon and reached 2.1 mM CH4, whereas Ein Afek site EA2 contained approximately 5% carbon and accumulated 0.7-1.7 mM CH4. Both showed sulfate depletion, but EN exhibited pronounced sulfide accumulation, whereas EA2 showed greater dissolved iron and nutrient enrichment. Their classified methanogenic assemblages also differed. EA1 remained methane poor despite increasing carbon content with depth, while EA3 combined low carbon content and CH4 accumulation with detectable methanogens and lower methanogenic diversity. In a 36-day bottle experiments using sediments from two of the four wetland habitats, sucrose-amended EA3 exhibited a transient maximum of 18.8 mmol collected CH4 kg-1 wet sediment during Days 22-29, whereas EN reached its highest collected amount during Days 29-36. Methane proportions increased before the large output increase in EA3 but remained comparatively stable and high in EN. Soluble chemical oxygen demand trajectories and microbial community changes also differed. These findings identify distinct biogeochemical settings supporting methane accumulation and demonstrate methanogenic potential in low-CH4 sediment, emphasizing the complementary information provided by field profiles, microbial characterization, and exploratory enrichment experiments.

microbiology↗
bioRxiv2026

The absence of microbiome in germ-free mice exacerbates the pathogenicity of mouse-adapted SARS-CoV-2 MA10

Germ-free mice, which are devoid of microbiota, provide a powerful system to define the contribution of the microbiome to host responses during infection. Severe COVID-19 outcomes are associated with multiple comorbidities that are themselves characterized by microbiome dysbiosis; however, the causal role of the microbiota in SARS-CoV-2 pathogenesis remains poorly understood. Here, we use germ-free mouse models to evaluate the impact of the microbiome on disease severity following infection with mouse-adapted SARS-CoV-2 MA10. Comparison with conventionally housed mice revealed that the absence of the microbiome markedly exacerbates SARS-CoV-2-induced disease in both C57BL/6 and Swiss Webster backgrounds. Germ-free mice exhibited greater weight loss, impaired lung function, worsened pulmonary pathology, and pronounced alterations in circulating cytokine and chemokine profiles compared with conventional controls. These findings show that resident microbiota limit SARS-CoV-2 pathogenicity and establish germ-free COVID-19 models as a powerful platform to identify protective microbial taxa and microbiota-based interventions to reduce severe disease.

microbiology↗