The most common diagnostic method used for coronavirus disease-2019 (COVID-19) is real-time reverse transcription polymerase chain reaction (PCR). However, it requires complex and labor-intensive procedures and involves excessive positive results derived from viral debris. We developed a method for the direct detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in nasopharyngeal swabs, which uses matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-ToF MS) to identify specific peptides from the SARS-CoV-2 nucleocapsid phosphoprotein (NP). SARS-CoV-2 viral particles were separated from biological molecules in nasopharyngeal swabs by an ultrafiltration cartridge. Further purification was performed by an anion exchange resin, and purified NP was digested into peptides using trypsin. The peptides from SARS-CoV-2 that were inoculated into nasopharyngeal swabs were detected by MALDI-ToF MS, and the limit of detection was 10 6.7 viral copies. This value equates to 10 7.9 viral copies per swab and is approximately equivalent to the viral load of contagious patients. Seven NP-derived peptides were selected as the target molecules for the detection of SARS-CoV-2 in clinical specimens. The method detected between two and seven NP-derived peptides in 19 nasopharyngeal swab specimens from contagious COVID-19 patients. These peptides were not detected in four specimens in which SARS-CoV-2 RNA was not detected by PCR. Mutated NP-derived peptides were found in some specimens, and their patterns of amino acid replacement were estimated by accurate mass. Our results provide evidence that the developed MALDI-ToF MS-based method in a combination of straightforward purification steps and a rapid detection step directly detect SARS-CoV-2-specific peptides in nasopharyngeal swabs and can be a reliable high-throughput diagnostic method for COVID-19.

Escherichia albertii is an emerging foodborne pathogen. The source of infection in most foodborne outbreaks is unknown, as it is difficult to isolate E. albertii from suspected foods or water. E. albertii has a broad host range among birds and can be isolated from chicken meat. In this study, PCR assay, enrichment and isolation conditions for detecting E. albertii in chicken meat were evaluated. The growth of 48 E. albertii strains isolated in Japan between 1994 and 2018 was evaluated in modified EC broth (mEC) and mEC supplemented with novobiocin (NmEC) and on different media containing carbohydrates. In addition, the enzyme for nested PCR, the enrichment condition, the most probable number (MPN) method, and agar media were also evaluated for chicken meat. To distinguish E. albertii from presumptive non-E. albertii bacteria, desoxycholate hydrogen sulfide lactose agar (DHL), MacConkey agar (MAC), and these agars supplemented with rhamnose and xylose (RX-DHL and RX-MAC, respectively) were used. All E. albertii strains grew in mEC and NmEC at both 36°C and 42°C and did not utilize rhamnose, sucrose, or xylose. Both the first PCR and nested PCR, using TaKaRa Ex Taq which was 10–100 times superior to the other enzymes, showed positive results in enrichment culture of 25 g of chicken meat inoculated with >20 CFU of E. albertii in mEC and NmEC at 42°C for 22 ± 2 h. Thus, the first PCR was sensitive enough to detect E. albertii in chicken meat. The MPN values in mEC and NmEC were 0.5- and 2.3-fold of inoculated bacterial concentration, respectively. E. albertii in chicken meat was more efficiently isolated with enrichment in NmEC (70.1-100%) and plating onto RX-DHL (85.4%) and RX-MAC (100%) compared to enrichment in mEC (53.5-83.3%) and plating onto DHL (70.1%) and MAC (92.4%), respectively. Thus, optimized conditions for the surveillance of E. albertii contamination in food and investigations of E. albertii outbreaks, including the infectious dose, were clarified.

Escherichia albertii  is an emerging foodborne pathogen. Owing to its distribution in river water,  it is important to determine the presence of  E. albertii  in aquaculture-related foods. In this study, we investigated the distribution of  E. albertii  in retail oyster samples.  A total of  427 raw oyster samples (385 Pacific oysters, and 42 Japanese rock oysters) were enriched in  modified Escherichia coli  broth (mEC) or mEC supplemented with novobiocin (NmEC) at 42 °C. The cultures were used for  E. albertii -specific nested PCR assay, as well as for  E. albertii  isolation using  deoxycholate hydrogen sulfide lactose agar  (DHL), DHL supplemented with rhamnose and xylose (RX-DHL), and MacConkey agar supplemented with rhamnose and xylose (RX-MAC). The population of  E. albertii  in nested PCR-positive samples was  determined using the  most probable number  (MPN) method.  E. albertii  isolates were subjected to biochemical and genetic characterization.  E. albertii   was detected in 5 of 315 (1.6%) Pacific oyster samples  (one piece each), 2 of 70 (2.9 %)  Pacific oyster samples  (25 g each), and 2 of 42 (4.8 %) Japanese rock oyster samples  procured from four geographically distant regions. A total of 64  E. albertii  strains were isolated from eight of the nine nested PCR assay-positive oyster samples, and  the MPN value was under the detection limit (< 3 MPN/10 g).  A specific season or month for detecting  E. albertii  was not observed in this study, suggesting that the pathogen is present in seawater.   All the  E. albertii  isolates, except one, were positive for the virulence factor  eae,  indicating that these isolates have  the potential to infect humans.

Aerosols or saliva containing severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) can contaminate living environments, and viruses can be indirectly transmitted. To understand the survival potential of the virus, the viral titers of bovine coronavirus (BCoV), as a model virus, and SARS-CoV-2 were measured on porous and non-porous surfaces. The amount of infectious BCoV recovered remained relatively high on non-porous substrates. However, it quickly decreased on several non-porous surfaces such as nitrile rubber. The time taken to reach the limit of detection on non-woven masks, as a porous substrate, was longer than that of non-porous substrates. On porous substrates other than non-woven masks, the amount of virus recovered quickly decreased, and then remained at a low level. Representative substrates were tested with SARS-CoV-2. The decrease in the amount of infectious virus recovered was similar to that of BCoV, although that of SARS-CoV-2 was more rapid. RNA derived from SARS-CoV-2 was also detected using real-time PCR, and it remained on surfaces much longer than infectious virus, on all substrates. Therefore, it is important to measure the viral titer to avoid the overestimation of infectious virus contamination in the environments. Our results suggest that the surface structure was not directly related to viral survivability.

No abstract