Various diagnostic methods are used in infection diagnostics. The key parameters for these tests are specificity and sensitivity, as well as usability and time to results. The general trend in infection testing has been towards tests based on gene amplification, such as polymerase chain reaction (PCR). The testing is often central laboratory-based, where obtaining the results can take several hours or days after collecting the sample. In managing acute infections, such as influenza, the time to results is critical for proper medication and isolation. Gene amplification methods can be analytically highly specific and sensitive, but may not indicate the current state of the disease. Methods multiplying genetic material can react to past infections or contamination, complicating or misleading the diagnosis. During the COVID-19 pandemic, utilization of rapid antigen tests experienced a renaissance. These tests are based on the ability of microbe-specific antibodies to bind to structural parts of microbes. The detected parts are produced only in the acute phase of the disease when the microbe is replicating. During the pandemic, these rapid on-site tests enabled quick diagnosis, treatment, and early isolation measures.

This thesis studied the applicability of antibody-based two-photon excitation fluorometry (separation-free mariPOC technology) for the detection of microbial antigens from feces and urine, and the utility of rapid antigen detection as part of diagnostic process. In Study I, sample pretreatment methods and antigen tests were developed for the mariPOC platform. Feces and urine showed elevated fluores-cence levels. However, the methodology has a unique property to compensate matrix effects enabling accurate results. Special features of these matrices were ta-ken into account in the immunoassay, instrumentation, and data algorithm designs. As a result, feces and urine proved well suited for the separation-free method.

In Studies II and III, Clostridioides difficile and SARS-CoV-2 antigen tests were developed, respectively, and the performance and usability were evaluated in comparison to routine practices. According to the results, the sensitivity of the tests met their intended purpose, and the specificity was state-of-the-art. It is concluded that microbial antigens are clinically accurate markers of ongoing infection and hence suited for the frontline management of infectious diseases. Antigen tests would be useful in the control of the spread of infectious diseases during epidemics and/or pandemics.