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Objectives: The accurate detection of severe acute respiratory syndrome–coronavirus 2 (SARS-CoV-2) is essential for the diagnosis of coronavirus disease 2019 (COVID-19). We compared the quantitative RT-PCR results between nasopharyngeal swabs and saliva specimens. Methods: A COVID-19 outbreak occurred on a cruise ship at Nagasaki port, Japan. We obtained 123 nasopharyngeal swabs and saliva each from asymptomatic or mild patients in the late phase of infection. Results: The intervals from the diagnosis to the sampling were 25.5 days for nasopharyngeal swabs and 28.9 days for saliva. The positive rate was 19.5% (24/123) for nasopharyngeal swabs and 38.2% (47/123) for saliva (P = 0.48). The quantified viral copies (mean ± SEM copies/5 μl) were 9.3±2.6 in nasopharyngeal swabs and 920±850 in saliva (P = 0.0006). Conclusions: The advantages of saliva specimens include positive rate improvement and accurate viral load detection. Saliva may be used as a reliable sample for SARS-CoV-2 detection., PLoS ONE, 16(6), art. no. e0252964; 2021

Objectives: The accurate prevalence of acquired immunodeficiency syndrome (AIDS)-defining illnesses (ADIs) in human immunodeficiency virus (HIV)-infected patients has not been well investigated. Hence, a longitudinal nationwide surveillance study analyzing the current status and national trend of opportunistic complications in HIV-infected patients in Japan is warranted. Methods: A nationwide surveillance of opportunistic complications in HIV-infected patients from 1995 to 2017 in Japan was conducted. An annual questionnaire was sent to 383 HIV/AIDS referral hospitals across Japan to collect information (CD4+ lymphocyte count, time of onset, outcome, and antiretroviral therapy [ART] status) of patients diagnosed with any of 23 ADIs between 1995 and 2017. Results: The response and case capture rates of the questionnaires in 2017 were 53% and 76%, respectively. The number of reported cases of opportunistic complications peaked in 2011 and subsequently declined. Pneumocystis pneumonia (38.7%), cytomegalovirus infection (13.6%), and candidiasis (12.8%) were associated with the cumulative incidence of ADIs between 1995 and 2017. The mortality rate in HIV-infected patients with opportunistic complications substantially decreased to 3.6% in 2017. The mortality rate was significantly higher in HIV patients who received ART within 14 days of diagnosis of complications than in those who received ART 15 days after diagnosis (13.0% vs. 3.2%, p < 0.01). Conclusions: We have demonstrated a 23-year trend of a newly diagnosed AIDS status in Japan with high accuracy. The current data reveal the importance of Pneumocystis pneumonia as a first-onset illness and that early initiation of ART results in poor outcomes in HIV patients in Japan., PLoS ONE, 16(8), art. no. e0256452; 2021

Objectives: A few studies on antibody testing have focused on asymptomatic or mild coronavirus disease 2019 (COVID-19) patients with low initial anti-severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibody responses. Anti-SARS-CoV-2 antibody-testing performance was evaluated using blood samples from asymptomatic or mild COVID-19 patients. Methods: Blood samples were collected from 143 COVID-19 patients during an outbreak on a cruise ship 3 weeks after diagnosis. Simultaneously, a follow-up SARS-CoV-2 genetic test was performed. Samples stored before the COVID-19 pandemic were also used to evaluate the lateral flow immunochromatographic assay (LFA) and electrochemiluminescence immunoassay (ECLIA). Titers of anti-SARS-CoV-2 IgM and IgG antibodies against the nucleocapsid and spike proteins were measured using the enzyme-linked immunosorbent assay to confirm which antibodies were influenced on LFA- and ECLIA- false-negative result in crew-member samples. Results: Sensitivity, specificity, positive-predictive, and negative-predictive values of LFA-detected IgM antibodies were 0.231, 1.000, 1.000, and 0.613, respectively; those of LFA-detected IgG antibodies were 0.483, 0.989, 0.972, and 0.601, respectively; and those of ECLIA-detected total antibodies were 0.783, 1.000, 1.000, and 0.848, respectively. All antibody titers measured using ELISA were significantly lower in blood samples with negative results than in those with positive results in both LFA and ECLIA. In the patients with negative results from the follow-up genetic testing, IgM-, IgG-, and total-antibody positivity rates were 22.9%, 47.6%, and 72.4%, respectively. Conclusions: These findings suggest that anti-SARS-CoV-2 antibody testing has lower performance in asymptomatic or mild COVID-19 patients than required in the guidelines., PLoS ONE, 16(9), art. no. e0257452; 2021

The alteration of the microbial community in the upper respiratory tract (URT) can contribute to the colonization and invasion of respiratory pathogens. However, there are no studies regarding whether the characteristics of the URT microbiota can be affected by infections in lower respiratory tract (LRT). To elucidate the microbial profiles of the URT during pneumonia, the oral, nasal, and lung microbiota was evaluated at the early phase in a murine pneumonia model by direct intratracheal inoculation of Klebsiella pneumoniae. The meta 16S rRNA sequencing of bronchoalveolar lavage fluid after K. pneumoniae inoculation presented alterations in the beta diversity of the microbes, but not in the alpha diversity. At this point, a significant increase in microbial alpha diversity was observed in the oral cavity, but not in the nasal cavity. The significant increase was observed in the family Carnobacteriaceae and family Enterococcaceae. These results suggest that characterizing the microbial community of the respiratory tract may not just involve a simple downstream relationship from the URT to the LRT. The health status of the LRT may influence the oral microbiota. Thus, evaluation of the oral microbiota may contribute towards monitoring lung health; the oral microbiota may act as a diagnostic marker of pneumonia., PLoS ONE, 14(9), art.no.e0222589; 2019

Vacuolar proton-translocating ATPase (V-ATPase) is located in fungal vacuolar membranes.It is involved in multiple cellular processes, including the maintenance of intracellular ion homeostasis by maintaining acidic pH within the cell. The importance of V-ATPase in virulence has been demonstrated in several pathogenic fungi, including Candida albicans. However, it remains to be determined in the clinically important fungal pathogen Candida glabrata.Increasing multidrug resistance of C. glabrata is becoming a critical issue in the clinical setting. In the current study, we demonstrated that the plecomacrolide V-ATPase inhibitor bafilomycin B1 exerts a synergistic effect with azole antifungal agents, including fluconazole and voriconazole, against a C. glabrata wild-type strain. Furthermore,the deletion of the VPH2 gene encoding an assembly factor of V-ATPase was sufficient to interfere with V-ATPase function in C. glabrata, resulting in impaired pH homeostasis in the vacuole and increased sensitivity to a variety of environmental stresses, such as alkaline conditions (pH 7.4), ion stress (Na+, Ca2+, Mn2+, and Zn2+ stress),exposure to the calcineurin inhibitor FK506 and antifungal agents (azoles and amphotericin B), and iron limitation. In addition, virulence of C. glabrata Δvph2 mutant in a mouse model of disseminated candidiasis was reduced in comparison with that of the wild-type and VPH2-reconstituted strains.These findings support the notion that V-ATPase is a potential attractive target for the development of effective antifungal strategies., PLoS ONE, 14(1), art.no.e0210883; 2019

The pathogenic fungus Candida glabrata is often resistant to azole antifungal agents. Drug efflux through azole transporters, such as Cdr1 and Cdr2, is a key mechanism of azole resistance and these genes are under the control of the transcription factor Pdr1. Recently, the monoamine oxidase A (MAO-A) inhibitor clorgyline was shown to inhibit the azole efflux pumps, leading to increased azole susceptibility in C. glabrata. In the present study, we have evaluated the effects of clorgyline on susceptibility of C. glabrata to not only azoles, but also to micafungin and amphotericin B, using wild-type and several mutant strains. The addition of clorgyline to the culture media increased fluconazole susceptibility of a C. glabrata wild-type strain, whereas micafungin and amphotericin B susceptibilities were markedly decreased. These phenomena were also observed in other medically important Candida species, including Candida albicans, Candida parapsilosis, Candida tropicalis, and Candida krusei. Expression levels of CDR1, CDR2 and PDR1 mRNAs and an amount of Cdr1 protein in the C. glabrata wild-type strain were highly increased in response to the treatment with clorgyline. However, loss of Cdr1, Cdr2, Pdr1, and a putative clorgyline target (Fms1), which is an ortholog of human MAO-A, or overexpression of CDR1 did not affect the decreased susceptibility to micafungin and amphotericin B in the presence of clorgyline. The presence of other azole efflux pump inhibitors including milbemycin A4 oxime and carbonyl cyanide 3-chlorophenylhydrazone also decreased micafungin susceptibility in C. glabrata wild-type, Δcdr1, Δcdr2, and Δpdr1 strains. These findings suggest that azole efflux pump inhibitors increase azole susceptibility but concurrently induce decreased susceptibility to other classes of antifungals independent of azole transporter functions., PLOS ONE, 12(7), e0180990; 2017

Background: Macrolides have antibiotic and immunomodulatory activities, which may have a favorable effect on the clinical outcome of patients with infections, including influenza. This study aimed to evaluate the effects of combination therapy with an anti-influenza agent, oseltamivir, and a single-dose formulation of azithromycin (AZM), which has been used for influenza-related secondary pneumonia, on influenza patients. The primary endpoint was a change in the expression levels of inflammatory cytokines. Secondary endpoints were the time required for resolution of influenza-related symptoms, incidence of complications, and adverse reactions. Methods: Patients with seasonal influenza were enrolled in this multicenter, open-label, randomized study. Patients were stratified according to the presence of a high risk factor and were randomized to receive combination therapy with oseltamivir plus an extended-release formulation of AZM (combo-group) or oseltamivir monotherapy (mono-group). Results: We enrolled 107 patients and randomized them into the mono-group (56 patients) or the combo-group (51 patients). All patients were diagnosed with influenza A infection, and none of the patients had comorbid pneumonia. Statistically significant differences were not observed in the expression levels of inflammatory cytokines and chemokines between the 2 groups. The maximum temperature in the combo-group was lower than that in the mono-group on day 3 through day 5 (p = 0.048), particularly on day 4 (p = 0.037). Conclusion: To our knowledge, this is the first prospective, randomized, clinical trial of oseltamivir and AZM combination therapy for influenza. Although the difference in inflammatory cytokine expression level was not statistically significant, combination therapy showed an early resolution of some symptoms. Name of registry: University hospital Medical Information Network (UMIN). Trial Registration no.: UMIN000005371., PLoS ONE, 9(3), e91293; 2014