MinION sequencing is widely used to sequence influenza A virus (IAV) genomes. However, the accuracy and utility of this approach, which uses the latest chemistry to obtain whole viral genome sequences directly from clinical samples, remain insufficiently investigated. We evaluated the sequencing accuracy of combining simultaneous multi-segment one-step RT-PCR and MinION sequencing using various subtypes of 13 IAV isolates. The latest R10.4.1 chemistry significantly improved sequencing accuracy, achieving ≥99.993% identity with the results of Illumina MiSeq and reducing single nucleotide deletion in homopolymer regions. Applying this method to 11 clinical samples enabled rapid subtype identification and acquisition of eight full-length IAV genomes. In four of these samples, subtype identification of hemagglutinin and neuraminidase was achieved within 20 min of starting the sequencing, and a full-length IAV genome was obtained within 7 h of RNA extraction. However, cross-barcode misassignment during demultiplexing might have affected data interpretation, particularly for samples with low viral genome copy numbers. Although careful data analysis is required for multiplex sequencing of clinical samples with low viral genome copy numbers, this approach can be used for the rapid identification of IAV subtypes and accurate acquisition of full IAV genome sequences from clinical samples.

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This study aimed to characterize cardiac injury in individuals diagnosed with severe fever with thrombocytopenia syndrome (SFTS) and to ascertain its relationship with prognosis. A retrospective analysis of data from 324 patients, diagnosed with SFTS between January 2021 and December 2023, was performed. Patients were categorized into survival and non-survival groups. Univariate and multivariate analyses were performed to identify significant indicators and predictive risk factors for mortality. Statistically significant differences in various parameters were found between the 2 groups, including age, history of hypertension, presence of diarrhea, petechiae, neurological abnormalities, and numerous laboratory measurements. These included lymphocyte, monocyte, and platelet counts, as well as liver enzyme levels, kidney function markers, cardiac biomarkers, clotting factors, inflammatory markers, Dabie Banda virus RNA, heart rate, PR interval, QT interval, and incidence of ST depression. Age, history of hypertension, neurological abnormalities, and creatinine, prothrombin time, and QT intervals were independent risk factors for mortality. The incidence of viral myocarditis in patients with SFTS was 64.12%, and the non-survival group demonstrated a higher incidence of cardiac injury, both earlier and more severe. The incidence of viral myocarditis in patients with SFTS was closely associated with prognosis.

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Early and accurate diagnosis of leprosy is important but remains a significant challenge. Loop-mediated isothermal amplification (LAMP) is a process for the amplification of nucleic acids at a constant temperature and has been used to develop field-friendly tests for many diseases. In the present study, we describe the development of a colorimetric LAMP assay targeting Mycobacterium leprae-specific 450 bp conserved region of the repeat sequences known as RLEP. In addition, the amplicons of LAMP were subjected to restriction analysis using the enzyme EcoRV for specificity. This method has the potential to become an accurate and efficient alternative to Sanger sequencing, which is currently used to validate RLEP-amplified products.

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We describe six independent cases of Mesocestoides infection in dogs presenting with diarrhea. Between November 2022 and August 2024, we were contacted by veterinarians regarding the identification of a species of small tapeworm excreted in dog feces. The veterinarians suspected the organism was Echinococcus multilocularis and believed it should be reported to health centers as a notifiable disease. Segmented and unsegmented worms, approximately 600 to 1,400 µm in length, were recovered from fecal samples. Microscopically, the worms had four suckers on the scolex but no rostellum. Subsequent molecular analysis of the mitochondrial cox1 and 12S rDNA genes revealed that all cases involved Mesocestoides vogae. The affected dogs were treated with an anthelmintic, and the diarrhea disappeared immediately. Possibly owing to the heavy infection load, the host animals had developed diarrhea, with the parasite likely expelled before reaching maturity. These small tapeworms with few proglottids could, therefore, confuse veterinarians.

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A measles outbreak occurred in Japan in February 2024 due to a measles virus variant imported from central Asian countries with three mismatches at the PCR reverse primer (MVN1213R) annealing site. To examine and improve the effectiveness of real-time PCR for detecting this variant, we compared the sensitivity of real-time PCR for MVN1213R and a modified primer using control RNAs, clinical isolates, and clinical specimens. The median difference in the cycle threshold value using the modified primer was 2.92 cycles lower (interquartile range, 1.99–3.38) than when using MVN1213R. Thus, PCR primer sets should be modified to effectively detect measles virus mutations.

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The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has repeatedly undergone mutations since its emergence, based on which, it has been assumed that there have been changes in its characteristics, including virulence and antigenicity. In this study, the viral loads in nasopharyngeal samples of patients with SARS-CoV-2 in Gunma Prefecture, Japan, from April 2, 2020, to April 1, 2023, were determined. The number of virus in samples in the Omicron-variant-prevalent period was higher than that in strains detected in samples before Week 50 of 2020; the B.1.1.284-prevalent period, the Alpha-variant-prevalent period, and the Delta-variant-prevalent period. Moreover, among the Omicron variants, the sub-lineage BA.5-prevalent period had more viral copies in the samples than in the BA.1-prevalent and BA.2-prevalent periods. Hence, the new variant may have released more viruses into the nasopharynx during the process of repeated mutations, resulting in widespread infection. The number of viruses detected in the nasopharyngeal samples showed an increasing trend with the evolution of the virus. Therefore, considering that the number of viruses in samples is also a vital factor contributing to the spread of the infection, it is important to determine the viral load in samples.

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