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This study aimed to investigate the optimal conditions for Papanicolaou (Pap) smear to increase the success rate of target cell isolation through manual microdissection (MMD) and prevent cell spread. Pap smears were prepared using an HPV42-positive SurePath™ liquid-based cytology case, and 46 and 50 koilocytes were used in wet and dried Pap smears, respectively, to verify the success rate of target cell isolation using MMD based on the HPV detection rate. During MMD, the microscopic examination of both specimens revealed that cells in dried smears could be easily identified; however, cell debris remained in the surrounding area after MMD. Although it was difficult to observe cells in wet smears, there was no cell debris. When the needle tip was immersed in DNA lysate after cell isolation through MMD, a difference in cell solubility was found between dry and wet smears. HPV42 was detected in 94.7% and 97.4% of dried and wet Pap smears, respectively, via polymerase chain reaction genotyping using lysed cell solution; the detection rates were not significantly different. The isolation of target cells from wet Pap smears using MMD reduced the risk of contamination and increased the success rate of HPV detection. This study might facilitate the identification of new CIN-derived HPV-infected cells using MMD with wet Pap smears.

To better understand the evolution of the SARS-CoV-2 Omicron subvariants, we performed molecular evolutionary analyses of the spike ( S ) protein gene/S protein using advanced bioinformatics technologies. First, time-scaled phylogenetic analysis estimated that a common ancestor of the Wuhan, Alpha, Beta, Delta variants, and Omicron variants/subvariants diverged in May 2020. After that, a common ancestor of the Omicron variant generated various Omicron subvariants over one year. Furthermore, a chimeric virus between the BM.1.1.1 and BJ.1 subvariants, known as XBB, diverged in July 2021, leading to the emergence of the prevalent subvariants XBB.1.5 and XBB.1.16. Next, similarity plot (SimPlot) data estimated that the recombination point (breakpoint) corresponded to nucleotide position 1373. As a result, XBB.1.5 subvariants had the 5' nucleotide side from the breakpoint as a strain with a BJ.1 sequence and the 3' nucleotide side as a strain with a BM.1.1.1 sequence. Genome network data showed that Omicron subvariants were genetically linked with the common ancestors of the Wuhan and Delta variants, resulting in many amino acid mutations. Selective pressure analysis estimated that the prevalent subvariants, XBB.1.5 and XBB.1.16, had specific amino acid mutations, such as V445P, G446S, N460K, and F486P, located in the RBD when compared with the BA.4 and BA.5 subvariants. Moreover, some representative immunogenicity-associated amino acid mutations, including L452R, F486V, R493Q, and V490S, were also found in these subvariants. These substitutions were involved in the conformational epitopes, implying that these mutations affect immunogenicity and vaccine evasion. Furthermore, these mutations were identified as positive selection sites. These results suggest that the S gene/S protein Omicron subvariants rapidly evolved, and mutations observed in the conformational epitopes may reduce the effectiveness of the current vaccine, including bivalent vaccines such as mRNA vaccines containing the BA.4/BA.5 subvariants.

The identification of pathogens associated with respiratory symptoms other than the novel coronavirus disease 2019 (COVID-19) can be challenging. However, the diagnosis of pathogens is crucial for assessing the clinical outcome of patients. We comprehensively profiled pathogens causing non-COVID-19 respiratory symptoms during the 7th prevalent period in Gunma, Japan, using deep sequencing combined with a next-generation sequencer (NGS) and advanced bioinformatics technologies. The study included nasopharyngeal swabs from 40 patients who tested negative for severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) using immuno-chromatography and/or quantitative reverse transcription polymerase chain reaction (qRT-PCR) methods. Comprehensive pathogen sequencing was conducted through deep sequencing using NGS. Additionally, short reads obtained from NGS were analyzed for comprehensive pathogen estimation using MePIC (Metagenomic Pathogen Identification Pipeline for Clinical Specimens) and/or VirusTap. The results revealed the presence of various pathogens, including respiratory viruses and bacteria, in the present subjects. Notably, human adenovirus (HAdV) was the most frequently detected virus in 16 of the 40 cases (40.0%), followed by coryneforms, which were the most frequently detected bacteria in 21 of the 40 cases (52.5%). Seasonal human coronaviruses (NL63 type, 229E type, HKU1 type, and OC43 type), human bocaviruses, and human herpesviruses (human herpesvirus types 1-7) were not detected. Moreover, multiple pathogens were detected in 50% of the subjects. These results suggest that various respiratory pathogens may be associated with non-COVID-19 patients during the 7th prevalent period in Gunma Prefecture, Japan. Consequently, for an accurate diagnosis of pathogens causing respiratory infections, detailed pathogen analyses may be necessary. Furthermore, it is possible that various pathogens, excluding SARS-CoV-2, may be linked to fever and/or respiratory infections even during the COVID-19 pandemic.

Despite the increasing evidence of the clinical impact of Pseudomonas -derived cephalosporinase (PDC) sequence polymorphisms, the molecular evolution of its encoding gene, bla PDC , remains elusive. To elucidate this, we performed a comprehensive evolutionary analysis of bla PDC . A Bayesian Markov Chain Monte Carlo phylogenetic tree revealed that a common ancestor of bla PDC diverged approximately 4660 years ago, leading to the formation of eight clonal variants (clusters A-H). The phylogenetic distances within clusters A to G were short, whereas those within cluster H were relatively long. Two positive selection sites and many negative selection sites were estimated. Two PDC active sites overlapped with negative selection sites. In docking simulation models based on samples selected from clusters A and H, piperacillin was bound to the serine and the threonine residues of the PDC active sites, with the same binding mode for both models. These results suggest that, in P. aeruginosa , bla PDC is highly conserved, and PDC exhibits similar antibiotic resistance functionality regardless of its genotype.

DNA gyrase plays important roles in genome replication in various bacteria, including Pseudomonas aeruginosa . The gyrA gene encodes the gyrase subunit A protein (GyrA). Mutations in GyrA are associated with resistance to quinolone-based antibiotics. We performed a detailed molecular evolutionary analyses of the gyrA gene and associated resistance to the quinolone drug, ciprofloxacin, using bioinformatics techniques. We produced an evolutionary phylogenetic tree using the Bayesian Markov Chain Monte Carlo (MCMC) method. This tree indicated that a common ancestor of the gene was present over 760 years ago, and the offspring formed multiple clusters. Quinolone drug-resistance-associated amino-acid substitutions in GyrA, including T83I and D87N, emerged after the drug was used clinically. These substitutions appeared to be positive selection sites. The molecular affinity between ciprofloxacin and the GyrA protein containing T83I and/or D87N decreased significantly compared to that between the drug and GyrA protein, with no substitutions. The rate of evolution of the gene before quinolone drugs were first used in the clinic, in 1962, was significantly lower than that after the drug was used. These results suggest that the gyrA gene evolved to permit the bacterium to overcome quinolone treatment.

Human papillomavirus (HPV) testing using self-collected vaginal specimens is the preferred choice to increase screening uptake. Although the HPV testing results of these samples depend on the cells that naturally exfoliate from the cervical lesion and drift into the vagina, the mechanism of when and how these exfoliated cells mix with the self-collected sample remains unclear. Hence, the study aimed to clarify the relationship between the vaginal drift of HPV-infected cells exfoliated from the cervix, and the menstrual cycle. A total of 180 scraped samples of the cervix and vagina were examined. The exfoliated cells were classified into two categories according to the HPV genotyping results of each sample: sufficient accumulation (same HPV types in cervical and vaginal samples) and insufficient accumulation (fewer HPV types in vaginal samples than in cervical samples, or HPV positivity in cervical samples and HPV negativity in vaginal samples). A moderately strong statistically significant association was observed between exfoliated cell accumulation and the menstrual cycle, and insufficient accumulation was statistically significantly increased at the early proliferative phases. Self-collection of vaginal samples at the early proliferation phase indicates insufficient sample quantities or lower viral load, thereby affecting HPV genotyping.

Many genotypes of human papillomaviruses (HPVs) may lead to morphological changes in cells, resulting in various atypical cells, such as multinucleated cells (MNCs) and koilocytes, in the cervix. However, the relationships between the profiles of HPV genotypes and MNCs are not exactly known. Thus, this study comprehensively profiles the HPV genotypes in MNCs using a microdissection method. HPV genotypes and MNCs were detected in 651 cases with an abnormal Pap smear by liquid-based cytology. Specific HPV genotypes were also detected, including HPV16, 34, and 56, which might be associated with MNCs. This result suggests that the high-risk HPV genotypes, such as HPV16 and 56, are associated with the atypical changes in MNC morphology from normal cervical cells. The results also show that MNCs may be a predictor of squamous intraepithelial lesion.

Human papillomavirus (HPV) infection is thought to be strongly associated with the precarcinomatous state cervical intraepithelial neoplasia (CIN) and cervical carcinoma. To accurately assess the correlation between HPV detection profiles and CIN, the uniplex E6/E7 polymerase chain reaction (PCR) method was used. We detected HPV (37 genotypes) in 267 CIN cases. The detection of a single high-risk HPV genotype occurred in 69.7% of CIN1 and worse than CIN1 (CIN1+) cases whereas other types were detected in 11.6% of cases. Codetection of high-risk HPV genotypes occurred in 4.9% of CIN1+ cases. The high-risk genotype HPV16 was the most frequently detected genotype in CIN1+ lesions; the genotype HPV34 (not a high-risk type) was detected in some CIN3 cases. Furthermore, HPV codetection may not be associated with CIN grades. These results suggest that various HPV genotypes are associated with CIN across all analyzed cases.

Favipiravir was initially developed as an antiviral drug against influenza and is currently used in clinical trials against severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection (COVID-19). This agent is presumably involved in RNA chain termination during influenza virus replication, although the molecular interactions underlying its potential impact on the coronaviruses including SARS-CoV-2, SARS-CoV, and Middle East respiratory syndrome coronavirus (MERS-CoV) remain unclear. We performed in silico studies to elucidate detailed molecular interactions between favipiravir and the SARS-CoV-2, SARS-CoV, MERS-CoV, and influenza virus RNA-dependent RNA polymerases (RdRp). As a result, no interactions between favipiravir ribofuranosyl-5'-triphosphate (F-RTP), the active form of favipiravir, and the active sites of RdRps (PB1 proteins) from influenza A (H1N1)pdm09 virus were found, yet the agent bound to the tunnel of the replication genome of PB1 protein leading to the inhibition of replicated RNA passage. In contrast, F-RTP bound to the active sites of coronavirus RdRp in the presence of the agent and RdRp. Further, the agent bound to the replicated RNA terminus in the presence of agent, magnesium ions, nucleotide triphosphate, and RdRp proteins. These results suggest that favipiravir exhibits distinct mechanisms of action against influenza virus and various coronaviruses.