Akihiko Hata

Wastewater-based monitoring of SARS-CoV-2 and other pathogens is a widely adopted tool for assessing epidemic dynamics. While quantitative assays are commonly used to estimate infection levels in catchment populations, phylogenetic information—such as identifying circulating variants—is also crucial for public health. However, applying the widely used ARTIC protocol for full-genome sequencing to wastewater samples has proven challenging, likely due to the limited specificity and sensitivity of multiplex RT-PCR in such complex matrices. In this study, we developed and optimized a semi-nested RT-PCR assay targeting the full S-protein coding region (~4000 bases) for phylogenetic characterization of SARS-CoV-2 in wastewater. By reducing multiplexing and using single-plex reactions for both RT and PCR steps, we successfully amplified ~2000 bp fragments. Amplicons were sequenced using the Flongle Flow Cell platform. The optimized method—consisting of reverse transcription with specific primers followed by three parallel single-plex semi-nested PCRs—yielded over 1,000 SARS-CoV-2-like reads per primer set in 30 out of 39 wastewater samples in treatment plants in Japan, including those with <10 copies per analyte. Variant proportions were estimated using a newly developed approach based on single-nucleotide variant pattern matrix, revealing the presence of multiple co-circulating variants, including XBB lineages, JN.1, and notably BA.2.75, which was undetected in domestic clinical surveillance. These results highlight the effectiveness of our approach for detecting temporal shifts in SARS-CoV-2 variants, even at low RNA concentrations.

A variety of animal DNA in surface water was identified by a metabarcoding approach. They corresponded to host-specific Bacteroidales markers, highlighting the potential of this approach for comprehensively identifying pollution sources.

The hollow fiber ultrafiltration (HFUF)-based microbial concentration method is widely applied for monitoring pathogenic viruses and microbial indicators in environmental water samples. However, the HFUF-based method can co-concentrate substances that interfere with downstream molecular processes—nucleic acid extraction, reverse transcription (RT), and PCR. These inhibitory substances are assumed to be hydrophobic and, therefore, expected to be excluded by a simple surfactant treatment before the silica membrane-based RNA extraction process. In this study, the efficacy and limitations of the sodium deoxycholate (SD) treatment were assessed by quantifying a process control and indigenous viruses using 42 surface water samples concentrated with HFUF. With some exceptions, which tended to be seen in samples with high turbidity (> 4.0 NTU), virus recovery by the ultrafiltration method was sufficiently high (> 10%). RNA extraction-RT-quantitative PCR (RT-qPCR) efficiency of the process control was insufficient (10%) for 30 of the 42 HFUF concentrates without any pretreatments, but it was markedly improved for 21 of the 30 inhibitory concentrates by the SD treatment. Detection rates of indigenous viruses were also improved and no substantial loss of viral RNA was observed. The SD treatment was particularly effective in mitigating RT-qPCR inhibition, although it was not effective in improving RNA extraction efficiency. The methodology is simple and easily applied. These findings indicate that SD treatment can be a good alternative to sample dilution, which is widely applied to mitigate the effect of RT-qPCR inhibition, and can be compatible with other countermeasures.

集団感染症が報告されていない平常時において, 小学校やスポーツクラブなどのプールを対象に糞便汚染指標細菌・ウイルス, 腸管系ウイルスやレジオネラ属菌の検出を試み, 微生物汚染の実態調査と汚染要因の検証, 汚染指標の有効性評価を行った。利用状況等の条件によっては指標微生物の検出率が高くなった。大腸菌, F特異大腸菌ファージは培養法で検出され, 現行の管理手法では完全な不活化は困難だと考えられた。Pepper mild mottle virus, cross-assembly phageや, ヒト特異的なバクテロイデス属菌の遺伝子マーカーであるHF183は高頻度で検出され, 特にcross-assembly phage, HF183は水質管理下のプールにおいてもヒト糞便汚染指標としての有効性が示唆された。腸管系ウイルスは不検出であったが, 感染者の利用により, 感染力を保持したウイルスが滞留する可能性が示された。