•SARS-CoV-2 RNA detection is performed for clinical diagnosis of COVID-19.•The detection of two different targets on the SARS-CoV-2 genome is recommended.•An internal control is required for ...evaluating the assay qualities.•A multiplex rRT-PCR methodology was developed for SARS-CoV-2 RNA detection.•A multiplex rRT-PCR will enable reducing reagent use and cost, and handling time.
The detection of SARS-CoV-2 RNA by real-time reverse transcription–polymerase chain reaction (rRT-PCR) is used to confirm the clinical diagnosis of COVID-19 by molecular diagnostic laboratories. We developed a multiplex rRT-PCR methodology for the detection of SARS-CoV-2 RNA.
Three genes were used for multiplex rRT-PCR: the Sarbecovirus specific E gene, the SARS-CoV-2 specific N gene, and the human ABL1 gene as an internal control.
Good correlation of Cq values was observed between the simplex and multiplex rRT-PCR methodologies. Low copies (<25 copies/reaction) of SARS-CoV-2 RNA were detected by the novel multiplex rRT-PCR method.
The proposed multiplex rRT-PCR methodology will enable highly sensitive detection of SARS-CoV-2 RNA, reducing reagent use and cost, and time required by clinical laboratory technicians.
The World Health Organization declared the novel coronavirus outbreak (COVID-19) to be a pandemic on March 11, 2020. Large-scale monitoring for capturing the current epidemiological situation of ...COVID-19 in Japan would improve preparation for and prevention of a massive outbreak.
A chatbot-based healthcare system named COOPERA (COvid-19: Operation for Personalized Empowerment to Render smart prevention And care seeking) was developed using the LINE app to evaluate the current Japanese epidemiological situation. LINE users could participate in the system either though a QR code page in the prefectures' websites or a banner at the top of the LINE app screen. COOPERA asked participants questions regarding personal information, preventive actions, and non-specific symptoms related to COVID-19 and their duration. We calculated daily cross correlation functions between the reported number of infected cases confirmed using polymerase chain reaction and the symptom-positive group captured by COOPERA.
We analyzed 206,218 participants from three prefectures reported between March 5 and 30, 2020. The mean age of participants was 44.2 (standard deviation, 13.2) years. No symptoms were reported by 96.93% of participants, but there was a significantly positive correlation between the reported number of COVID-19 cases and self-reported fevers, suggesting that massive monitoring of fever might help to estimate the scale of the COVID-19 epidemic in real time.
COOPERA is the first real-time system being used to monitor trends in COVID-19 in Japan and provides useful insights to assist political decisions to tackle the epidemic.
Human immunodeficiency virus (HIV) infected individuals are at increased risk of developing active tuberculosis (TB). TB incidence remains higher than in non-HIV subjects after antiretroviral therapy ...(ART) initiation. This study was conducted to estimate the prevalence of positive IGRA, reflecting latent tuberculosis infection and/or a history of active TB, in HIV-infected individuals after ART initiation in Japan.
Two IGRAs (Interferon (IFN)-γ release assays), QuantiFERON®-TB Gold Plus (QFT-Plus) and T-Spot®.TB (TSPOT), were used. We also analyzed the TB associated risk factors for the IGRAs results and the role of CD4+ T-cells, CD8+ T-cells and NK cells for producing IFN-γ. We also analyzed the risk factors for positive IGRA responses and the role of CD4+ T-cells, CD8+ T-cells and NK cells for producing IFN-γ.
One hundred eight-four subjects were prospectively enrolled. Median age was 49 years. The positivity rates of QFT-Plus and TSPOT were 7.6% 95%CI 4.6–12.4 and 2.7% 95%CI 1.2–6.2, respectively, with significant difference. TB-associated risk factors and NK cells ≥300/μL were selected as independently significant factors by multivariate logistic regression. The NK cell count revealed significant linear regression with IFN-γ production responding to TB-specific antigens.
The prevalence of positive IGRAs was 2.7%–7.6%. QFT-Plus would be practical for a higher positivity rate and reflect TB risk factors. The innate immune system, referring to IFN-γ production, plays an important role in the immune response to TB-specific antigens even after initiating ART.
Objective A T-SPOT.TB can yield indeterminate results under two test observation conditions: a high response to the nil in negative control wells (high nil-control) or a low response to the mitogen ...in positive control wells (low mitogen-control). The most strongly influential factors for these indeterminate results, however, have yet to be identified. Methods From June 1, 2015, to June 30, 2021, we conducted a 1:1 matched case-control, retrospective study. Patients Patients who underwent a T-SPOT.TB test at Chiba University Hospital. Results The study included 5,956 participants. Indeterminate results were found in 63 participants (1.1%), including high nil-control in 37 and low mitogen-control in 26. Human T-cell leukemia virus type 1 (HTLV-1) positivity was the only influencing factor associated with high nil-control (adjusted odds ratio=98.5, 95% confidence interval: 6.59-1,480). Conclusion Regarding the indeterminate results, all HTLV-1 positive participants had a high nil response and no low mitogen response. It was suspected that abnormally produced interferon γ caused a nonspecific reaction to the negative control well, resulting in a high nil response. Low mitogen-control, conversely, did not appear to have any statistically significant influential factors.
Numerous SARS-CoV-2 variant strains with altered characteristics have emerged since the onset of the COVID-19 pandemic. Remdesivir (RDV), a ribonucleotide analogue inhibitor of viral RNA polymerase, ...has become a valuable therapeutic agent. However, immunosuppressed hosts may respond inadequately to RDV and develop chronic persistent infections. A patient with respiratory failure caused by interstitial pneumonia, who had undergone transplantation of the left lung, developed COVID-19 caused by Omicron BA.5 strain with persistent chronic viral shedding, showing viral fusogenicity. Genome-wide sequencing analyses revealed the occurrence of several viral mutations after RDV treatment, followed by dynamic changes in the viral populations. The C799F mutation in nsp12 was found to play a pivotal role in conferring RDV resistance, preventing RDV-triphosphate from entering the active site of RNA-dependent RNA polymerase. The occurrence of diverse mutations is a characteristic of SARS-CoV-2, which mutates frequently. Herein, we describe the clinical case of an immunosuppressed host in whom inadequate treatment resulted in highly diverse SARS-CoV-2 mutations that threatened the patient's health due to the development of drug-resistant variants.
Although several studies have reported on the clinical and epidemiological characteristics of the patient with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), clinical course of the ...most severe cases requiring treatment in ICU have been insufficiently reported. A 73-year-old man traveling on a cruise ship with history of hypertension and dyslipidemia developed high fever, dyspnea and cough after 7 days of steroid treatment for sudden sensorineural hearing loss, and tested positive for SARS-CoV-2 in sputa polymerase chain reaction (PCR) examination. His respiratory function deteriorated despite treatments with lopinavir/ritonavir, oseltamivir, azithromycin and meropenem at a regional hospital. He was intubated and transferred to the ICU in the tertiary university hospital on day 10 (ICU day 1). Interferon beta-1b subcutaneous injection was initiated immediately to enhance anti-viral therapy, and favipiravir on ICU day 10 upon availability. Progression of organ dysfunctions necessitated inhalation of nitrogen oxide for respiratory dysfunction, noradrenaline for cardiovascular dysfunction and continuous renal replacement therapy for renal dysfunction. His blood samples PCR also tested positive for SARS-CoV-2, indicating viremia, concomitantly with elevated IL-6 levels. VV-ECMO was initiated after sudden exacerbation of respiratory dysfunction on ICU day 7 to maintain oxygenation. The sustained excessive inflammatory cytokines in the present case might have led to the exacerbation of the disease, requiring vigorous organ support therapies to allow for survival and recovery from the rapid progression of multiple organ dysfunctions and severe respiratory failure.
Introduction Coronavirus disease (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2. Although most patients with COVID-19 develop asymptomatic or mild disease, some patients ...develop severe disease. The effectiveness of various therapeutic agents, including antiviral drugs, steroids, and anti-inflammatories for COVID-19, have been being confirmed. The effect of administering steroids in early disease is unclear. This study therefore aimed to evaluate the effectiveness and risk of exacerbation of steroids administered preceding antiviral drugs in patients with COVID-19 pneumonia. Methods This retrospective, single-center, observational study included consecutive patients with COVID-19 between March 2020 and March 2021. Patients were divided into a steroids-first group and antiviral-drugs-first group. Mortality, duration of hospitalization, incidence rate and duration of intensive care unit (ICU) admission, intubation, and extracorporeal membrane oxygenation (ECMO) induction of the two groups were compared. Results A total of 258 patients were admitted during the study period. After excluding patients who received symptomatic treatment only, who were taking immunosuppressive drugs, or who were administered antiviral drugs only, 68 patients were included in the analysis, 16 in the steroids-first group and 52 in the antiviral-drugs-first group. The rate of intubation, ICU admission and ECMO induction were significantly higher in the steroids-first group than in the antiviral-drugs-first group (81.3% vs. 33.3, p<0.001, 75.0% vs. 29.4%, p = 0.001, and 31.3% vs. 7.8%, p = 0.017, respectively). Furthermore, patients who received steroids within ten days after starting antiviral drugs had significantly lower rates of ICU admission, intubation, and ECMO induction. (81.3% vs. 42.9% p = 0.011, 75.0% vs. 37.1% p = 0.012, and 31.3% vs. 8.6% p = 0.039, respectively). Conclusions Administering steroids prior to antiviral drugs soon after symptom onset can aggravate disease severity. When administration of steroids is considered soon after symptom onset, it may be safer to initiate antiviral drugs first.
Vaccine effectiveness against SARS-CoV-2 infections decreases due to waning immunity, and booster vaccination was therefore introduced. We estimated the anti-spike antibody (AS-ab) recovery by ...booster vaccination and analyzed the risk factors for SARS-CoV-2 infections.
The subjects were health care workers (HCWs) in a Chiba University Hospital vaccination cohort. They had received two doses of vaccine (BNT162b2) and a booster vaccine (BNT162b2). We retrospectively analyzed AS-ab titers and watched out for SARS-CoV-2 infection for 90 days following booster vaccination.
AS-ab titer eight months after two-dose vaccinations had decreased to as low as 587 U/mL (median, IQR (interquartile range) 360–896). AS-ab titer had then increased to 22471 U/mL (15761–32622) three weeks after booster vaccination. There were no significant differences among age groups.
A total of 1708 HCWs were analyzed for SARS-CoV-2 infection, and 48 of them proved positive. SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 1.8% and 4.0%, respectively, and were not significant. However, when restricted to those 20–29 years old, SARS-CoV-2 infections in the booster-vaccinated and non-booster groups were 2.9% and 13.6%, respectively (p = 0.04). After multivariate logistic regression, COVID-19 wards (adjusted odds ratio (aOR):2.9, 95% confidence interval (CI) 1.5–5.6) and those aged 20–49 years (aOR:9.7, 95%CI 1.3–71.2) were risk factors for SARS-CoV-2 infection.
Booster vaccination induced the recovery of AS-ab titers. Risk factors for SARS-CoV-2 infection were HCWs of COVID-19 wards and those aged 20–49 years. Increased vaccination coverage, together with implementing infection control, remains the primary means of preventing HCWs from SARS-CoV-2 infection.
Legionella pneumophila (L. pneumophila) is responsible for most Legionnaire's disease cases diagnosed worldwide. The species includes 16 serogroups, but most Legionnaire's disease cases (85.7% in ...Europe, 87.0% in Japan) are caused by L. pneumophila serogroup 1. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can be used to identify the L. pneumophila serogroup. In this study, we compared three sample preparation methods that are compatible with MALDI-TOF MS: the direct colony transfer method (DCTM), on-target extraction method (OTEM), and in-tube extraction method (ITEM). The aim was to improve the low identification rates for L. pneumophila, and establish and validate a simple, rapid and robust MALDI-TOF MS-based method for routine use in microbiological laboratories for assignment of L. pneumophila isolates to serogroups and identification of reliable peak biomarkers. Using ITEM, 100.0% (29/29) of hot spring water samples and clinical isolates were correctly identified at the species level. Augmented reference spectra correctly identified all 29 strains at the species level and 29 isolates at the serogroup level, displaying sensitivity, specificity and accuracy of 100.0% for serogroup assignment. MALDI-TOF MS is a relatively inexpensive method for assignment of L. pneumophila serogroups that can serve as a first-line tool for rapid prospective typing.
A 55-year-old Japanese man was hospitalized with the novel coronavirus disease 2019 (COVID-19). On the 14th day after the start of favipiravir administration, the patient developed a fever with a ...temperature of 38.1°C. His pulse rate also became elevated to 128 bpm, so relative bradycardia was not suspected. Since he was in good overall health and no concomitant symptoms and signs were apparent, we considered it to be drug fever due to favipiravir. After the completion of favipiravir treatment, the patient's temperature normalized within 24 hours. We herein report this case of drug fever caused by favipiravir.
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