A rapid and simple alternative test to real-time reverse transcription polymerase chain reaction (RT-PCR) for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is required to help curb the spread of this infection. In this study, we compared the RT-PCR method with the chemiluminescent enzyme immunoassay (CLEIA) and reverse transcription loop mediated isothermal amplification (RT-LAMP) methods. The results for the number of SARS-CoV-2 RNA copies and the CLEIA antigen quantification values were highly correlated. The detection limit for antigen quantification was 42.8 RNA copies for saliva samples and 23.4 copies for nasopharyngeal swab (NPS) samples.The number of RNA copies and RT-LAMP threshold time (Tt) values were inversely correlated for both purified RNA and purification-free crude RNA. RT-LAMP with purified RNA detected low copy numbers of RNA (5-50 copies) whereas fewer than 250 RNA copies could not be detected using crude RNA. CLEIA antigen quantification is potentially useful for large scale screening because it is compatible with high throughput testing. RT-LAMP with crude RNA samples is applicable to rapid point-of-care testing because it can directly use the patient specimen. It is important to select a diagnostic method that is simple and rapid compared to RT-PCR, depending on the situation.

Fig. S1. Number of third-generation cephalosporin-susceptible and resistant pneumococcal meningitis cases. Susceptible: third-generation cephalosporin-susceptible; resistant: third-generation cephalosporin-resistant; PCV7 was introduced in 2010, and PCV13 was introduced 2013. The patients have been decreasing since introduction of vaccination.Table S1. Characteristics of patients infected with third-generation cephalosporin-susceptible and third-generation cephalosporin-resistant pneumococcal isolates.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the cause of the current coronavirus disease 2019 (COVID-19) pandemic and associated respiratory infections, has been detected in the feces of patients. Therefore, determining SARS-CoV-2 RNA levels in sewage may help to predict the number of infected people within the area. In this study, we quantified SARS-CoV-2 RNA copy number using reverse transcription quantitative real-time PCR with primers and probes targeting the N gene, which allows the detection of both wild-type and variant strain of SARS-CoV-2 in sewage samples from two wastewater treatment plants (WWTPs) in Kobe City, Japan, during the fourth and fifth pandemic waves of COVID-19 between February 2021 and October 2021. The wastewater samples were concentrated via centrifugation, yielding a pelleted solid fraction and a supernatant, which was subjected to polyethylene glycol (PEG) precipitation. The SARS-CoV-2 RNA was significantly and frequently detected in the solid fraction than in the PEG-precipitated fraction. In addition, the copy number in the solid fraction was highly correlated with the number of COVID-19 cases in the WWTP basin (WWTP-A: r = 0.8205, p < 0.001; WWTP-B: r = 0.8482, p < 0.001). The limit of capturing COVID-19 cases per 100,000 people was 0.75 cases in WWTP-A and 1.20 cases in WWTP-B, respectively. Quantitative studies of RNA in sewage can be useful for administrative purposes related to public health, including issuing warnings and implementing preventive measures within sewage basins.