Whole Genome Sequencing of Influenza A and B Viruses With the MinION Sequencer in the Clinical Setting: A Pilot Study

Introduction: Whole genome sequencing (WGS) of influenza viruses is important for preparing vaccines and coping with newly emerging viruses. However, WGS is difficult to perform using conventional next-generation sequencers in developing countries, where facilities are often inadequate. In this study, we developed a high-throughput WGS method for influenza viruses in clinical specimens with the MinION portable sequencer.Methods: Whole genomes of influenza A and B viruses were amplified by multiplex RT-PCR from 13 clinical specimens collected in Tokyo, Japan. Barcode tags for multiplex MinION sequencing were added with each multiplex RT-PCR amplicon by nested PCR with custom barcoded primers. All barcoded amplicons were mixed and multiplex sequencing using the MinION sequencer with 1D2 sequencing kit. In addition, multiplex RT-PCR amplicons generated from each clinical specimen were sequenced using the Illumina MiSeq platform to validate the performance of MinION sequencer. The accuracy, recall, and precision rates of MinION sequencing were calculated by comparing the results of variant calling in the Illumina MiSeq platform and MinION sequencer.Results: Whole genomes of influenza A and B viruses were successfully amplified by multiplex RT-PCR from 13 clinical samples. We identified 6 samples as influenza type A virus H3N2 subtype and 7 as influenza B virus Yamagata lineage using the Illumina MiSeq platform. The overall accuracy, recall, and precision rates of the MinION sequencer were, respectively 99.95%, 89.41%, and 97.88% from 1D reads and 99.97%, 93.28%, and 99.86% from 1D2 reads.Conclusion: We developed a novel WGS method for influenza A and B viruses. It is necessary to improve read accuracy and analytical tools in order to better utilize the MinION sequencer for real-time monitoring of genetic rearrangements and for evaluation of newly emerging viruses

A multicenter randomized controlled trial to evaluate the efficacy and safety of nelfinavir in patients with mild COVID-19

Nelfinavir, an orally administered inhibitor of human immunodeficiency virus protease, inhibits the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. We conducted a randomized controlled trial to evaluate the clinical efficacy and safety of nelfinavir in patients with SARS-CoV-2 infection. We included unvaccinated asymptomatic or mildly symptomatic adult patients who tested positive for SARS-CoV-2 infection within 3 days before enrollment. The patients were randomly assigned (1:1) to receive oral nelfinavir (750 mg; thrice daily for 14 days) combined with standard-of-care or standard-of-care alone. The primary endpoint was the time to viral clearance, confirmed using quantitative reverse-transcription PCR by assessors blinded to the assigned treatment. A total of 123 patients (63 in the nelfinavir group and 60 in the control group) were included. The median time to viral clearance was 8.0 (95% confidence interval [CI], 7.0 to 12.0) days in the nelfinavir group and 8.0 (95% CI, 7.0 to 10.0) days in the control group, with no significant difference between the treatment groups (hazard ratio, 0.815; 95% CI, 0.563 to 1.182; P = 0.1870). Adverse events were reported in 47 (74.6%) and 20 (33.3%) patients in the nelfinavir and control groups, respectively. The most common adverse event in the nelfinavir group was diarrhea (49.2%). Nelfinavir did not reduce the time to viral clearance in this setting. Our findings indicate that nelfinavir should not be recommended in asymptomatic or mildly symptomatic patients infected with SARS-CoV-2. The study is registered with the Japan Registry of Clinical Trials (jRCT2071200023). IMPORTANCE The anti-HIV drug nelfinavir suppresses the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. However, its efficacy in patients with COVID-19 has not been studied. We conducted a multicenter, randomized controlled trial to evaluate the efficacy and safety of orally administered nelfinavir in patients with asymptomatic or mildly symptomatic COVID-19. Compared to standard-of-care alone, nelfinavir (750 mg, thrice daily) did not reduce the time to viral clearance, viral load, or the time to resolution of symptoms. More patients had adverse events in the nelfinavir group than in the control group (74.6% [47/63 patients] versus 33.3% [20/60 patients]). Our clinical study provides evidence that nelfinavir, despite its antiviral effects on SARS-CoV-2 in vitro, should not be recommended for the treatment of patients with COVID-19 having no or mild symptoms

Unique Interplay between Sugar and Lipid in Determining the Antigenic Potency of Bacterial Antigens for NKT Cells

Structural and biophysical studies reveal the induced-fit mechanism underlying the stringent specificity of invariant natural killer T cells for unique glycolipid antigens from the pathogen Streptococcus pneumoniae

Acute tenosynovitis following an accidental injection of Bacille Calmette-Guérin (BCG) in a health care worker: A case report

Summary: Case: A 36-year-old female healthcare worker with no past medical history, accidentally injected her flexed right middle finger with live attenuated Mycobacterium bovis bacillus Calmette-Guérin (BCG). Swelling and erythema around the injured area appeared two days after the needlestick injury. She was referred to the hospital and presented approximately nine days after self-inoculation. Surgical debridement was immediately performed. After 38 days, colonies were observed on cultures of the removed tissue on Ogawa's medium. This isolate was identified as M. bovis BCG by polymerase chain reaction (PCR) based on RD1 gene deletion. She had a history of BCG vaccination and her skin lesion appeared immediately after the accidental injection of M. bovis BCG. Therefore, in the differential diagnosis, the possibility that the lesion was an allergic reaction to BCG was considered. The subsequent culture results came back positive for M. bovis BCG and acute tenosynovitis caused by M. bovis BCG was diagnosed. The skin lesion was treated with anti-mycobacterial drugs and resolved. Discussion: The allergic reactions to BCG should be considered in the differential diagnosis of skin lesions following BCG vaccination. It is important to promptly submit a specimen for culture as delayed initiation of appropriate treatment can lead to a poor prognosis. In patients with accidental injection of M. bovis BCG, it is important to consider timely surgical excision and appropriate antimycobacterial therapy

Malignant syphilis requiring differentiation from Kaposi's sarcoma

Malignant syphilis (MS) is a rare variant of secondary syphilis. Also known as rupioid syphilis, MS is characterized by the presence of multiple papules, papulopustules, black lamellate crust that may resemble an oyster shell, or nodules with ulceration lacking central clearing. MS is often associated with immunodeficiency and frequently co-occurs with HIV infection. We here report a case of MS in a patient with HIV infection. HIV infection can cause atypical clinical symptoms of syphilis. In this case, unlike previous cases, cutaneous lesions of MS were limited to the face, making the diagnosis challenging based on clinical findings alone. However, his laboratory findings, appearance of the Jarisch-Herxheimer reaction, and a dramatic response to antibiotic therapy are characteristic of MS, making the diagnosis even more certain. Our case suggests the importance of physicians considering the possibility of MS when observing black-crusted lesions

A Case of COVID-Related MERS (Clinically Mild Encephalitis/Encephalopathy with a Reversible Splenial Lesion) with a Typical Imaging Course and Hyponatremia in Adults—A Case Report and Literature Review

Clinically mild encephalitis/encephalopathy with reversible splenial lesions (MERS) is a mild form of encephalitis/encephalopathy that appears in association with various conditions, including infection. COVID-19 is also known to cause MERS. MERS more commonly occurs in children, and adult cases are relatively rare. Typical head MRI findings include a round lesion in the mid-layer of the corpus callosum with a high signal intensity on diffusion-weighted images. Most improve within a week. Although the exact mechanism by which the cerebral corpus callosum is affected is still unknown, several hypotheses have been proposed, including the involvement of electrolyte abnormalities (e.g., hyponatremia) and inflammatory cytokines (e.g., IL-6). In this report, we describe the first case of COVID-associated MERS with a typical imaging course and hyponatremia, with a review of the relevant literature. When psychiatric symptoms and the disturbance of consciousness appear in COVID patients, MERS should be considered in addition to delirium due to fever and hypoxia

Multicenter Study of the Effectiveness of Antifungal Stewardship Team Intervention for Candidemia in Japan in 2008–2021

Candidemia, linked to high mortality rates, requires prompt antifungal therapy for better outcomes. Treatment is structured as an action bundle, which is beneficial when followed closely. However, the Japanese action bundle lacks detailed guidance on severe complications like endocarditis or ocular issues. To address this, we adjusted the action bundle and assessed outcomes with and without AFT intervention. We strengthened protocols for blood cultures and organ assessments, and the AFT contacted the primary physician when yeast-like fungi were detected in the patient’s blood culture bottles. Analyzing 204 candidemia cases from 2008–2021, we observed increased adherence and reduced mortality post-AFT intervention. Ophthalmology consultations rose significantly, but many patients had only one visit, suggesting inadequate follow-up. If endophthalmitis is diagnosed, a change in the treatment approach may be necessary. There is a possibility that abnormal ocular findings will be detected during subsequent visits, which highlights the need for improvement in ophthalmology follow-up rates as a future challenge for our AFT activities