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This study aimed to investigate the bacterial characteristics of pneumococcal isolates obtained from a tertiary care hospital in Japan. We analyzed the antimicrobial susceptibility, possession of macrolide resistance genes, pneumococcal serogroup/serotype, and sequence type (ST) of pneumococcal isolates from patients aged 15 years or older between 2011 and 2020 at Nagasaki University Hospital. Of the 73 isolates analyzed, 86.3% showed resistance to macrolides, and 28.8%, 46.6%, and 11.0% harbored mefA, ermB, and both, respectively. Of the isolates possessing ermB, 97.6% showed high levels of macrolide resistance [minimal inhibitory concentration (MIC) range, > 16 µg/mL]. Solithromycin (MIC range, 0.03–0.25 µg/mL), regardless of the presence of macrolide resistance genes, and lascufloxacin (MIC range, 0.06–0.5 µg/mL) showed potent in vitro activity against pneumococci. Serotype 19A was the most prevalent (six isolates), followed by serotypes 10A, 15A, and 15B/C (five isolates each). Four serotypes (11A, 19A, 22F, and 23B) and five STs (36, 99, 433, 558, and 3111) were significantly correlated with the presence of macrolide resistance genes. All four isolates with serotype 11A/ST99 and three isolates with serotype 19A/ST3111 harbored both mefA and ermB. No macrolide resistance genes were detected in either of the two isolates with serotype 22F/ST433, while all ten isolates with serogroup 15 (serotypes 15A and 15B/C, five isolates each) possessed ermB alone. Our study revealed the bacterial characteristics of the pneumococcal isolates obtained from our hospital. In vitro activity of solithromycin and lascufloxacin against these isolates was confirmed.

Objectives: In the present study, we aimed to clarify the mechanisms by which periodontal pathogens, particularly Prevotella intermedia, induce severe neutrophilic inflammation. In addition, we aimed to test the efficacy of macrolides, which has not been resolved in the neutrophilic inflammation induced by P. intermedia. Methods: NCl-H292 human airway epithelial cells were pre-incubated with clarithromycin for 2 h before incubation with P. intermedia supernatants. Then, C-X-C motif chemokine ligand 8 (CXCL8) transcription and interleukin (IL)-8 production were measured. To elucidate the signaling pathway, mitogen-activated protein kinase inhibitors were added to the cell culture, and the cells were subjected to Western blotting. Results:P. intermedia supernatants promoted CXCL8 transcription and IL-8 production, and the reactions were significantly suppressed by clarithromycin pretreatment. Only trametinib, the selective mitogen-activated extracellular signal-regulated kinase inhibitor, downregulated CXCL8 transcription and IL-8 production. Furthermore, Western blotting revealed that stimulation with P. intermedia supernatants specifically induces extracellular signal-regulated kinases (ERK) 1/2 phosphorylation, which is suppressed by clarithromycin pretreatment. Notably, the interference analysis revealed that ERK3 might be dispensable for IL-8 production under the stimulation of P. intermedia supernatants. Conclusions: Our results provide new insight into the mechanism underlying P. intermedia-induced production of IL-8 from human airway epithelial cells. Furthermore, macrolides might have therapeutic potential in regulating periodontal pathogen-induced neutrophilic inflammation in the lungs.

Candida vulturna is a recently discovered and not widely documented ascomycetous yeast phylogenetically related to the outbreak-causing and multidrug-resistant Candida auris. A middle-aged Japanese man with no discernible immunodeficiency was admitted to hospital with ileal diverticulitis. Following laparoscopic right hemicolectomy against abscess formation on postoperative day (POD) 7, continuous fungemia occurred due to Candida haemulonii, identified using a conventional method by confirming the biochemical phenotype. Micafungin was initiated; however, the fungus was persistently isolated from blood cultures. Eventually, the antifungal agent was changed to a combination of liposomal amphotericin B (L-AMB) and caspofungin (CPFG), which cleared the infection, and no pathogens were detected in the blood cultures on POD 31. Contrast-enhanced computed tomography showed septic emboli in the lungs and spleen; however, no evidence of vasculitis was observed. Moreover, sequential echocardiography did not reveal any signs of infectious endocarditis. Finally, CPFG and L-AMB were administered to the patient for 7 and 9 weeks, respectively, during which the patient's symptoms did not relapse. The strain was later genetically identified as C. vulturna. This case report illustrates a clinical presentation of C. vulturna and provides the diagnostic approach and treatment methods for this pathogen.

ABSTRACTInvasive aspergillosis (IA) and mucormycosis are life-threatening diseases, especially among immunocompromised patients. Drug-resistant Aspergillus fumigatus strains have been isolated worldwide, which can pose a serious clinical problem. As IA mainly occurs in patients with compromised immune systems, the ideal therapeutic approach should aim to bolster the immune system. In this study, we focused on Vγ9Vδ2 T cells that exhibit immune effector functions and examined the possibility of harnessing this unconventional T cell subset as a novel therapeutic modality for IA. A potent antifungal effect was observed when A. fumigatus (Af293) hyphae were challenged by Vγ9Vδ2 T cells derived from peripheral blood. In addition, Vγ9Vδ2 T cells exhibited antifungal activity against hyphae of all Aspergillus spp., Cunninghamella bertholletiae, and Rhizopus microsporus but not against their conidia. Furthermore, Vγ9Vδ2 T cells also exhibited antifungal activity against azole-resistant A. fumigatus, indicating that Vγ9Vδ2 T cells could be used for treating drug-resistant A. fumigatus. The antifungal activity of Vγ9Vδ2 T cells depended on cell-to-cell contact with A. fumigatus hyphae, and degranulation characterized by CD107a mobilization seems essential for this activity against A. fumigatus. Vγ9Vδ2 T cells could be developed as a novel modality for treating IA or mucormycosis.IMPORTANCEInvasive aspergillosis (IA) and mucormycosis are often resistant to treatment with conventional antifungal agents and have a high mortality rate. Additionally, effective antifungal treatment is hindered by drug toxicity, given that both fungal and human cells are eukaryotic, and antifungal agents are also likely to act on human cells, resulting in adverse effects. Therefore, the development of novel therapeutic agents specifically targeting fungi is challenging. This study demonstrated the antifungal activity of Vγ9Vδ2 T cells against various Aspergillus spp. and several Mucorales in vitro and discussed the mechanism underlying their antifungal activity. We indicate that adoptive immunotherapy using Vγ9Vδ2 T cells may offer a new therapeutic approach to IA.

Abstract Background A test-based strategy against coronavirus disease 2019 (COVID-19) is one of the measures to assess the need for isolation and prevention of infection. However, testing with high sensitivity methods, such as quantitative RT-PCR, leads to unnecessary isolation, whereas the lateral flow antigen test shows low sensitivity and false negative results. The purpose of this study was to evaluate the performance of the LumiraDx SARS-CoV-2 Ag test (Lumira Ag), a rapid microfluidic immunofluorescence method, in assessing infectivity. Methods This study was performed from March 2022 to July 2022. A pair of nasopharyngeal swab samples were obtained from each patient with mild COVID-19. One swab was used for Lumira Ag testing, and the other for quantitative RT-PCR testing and virus culture. Results A total of 84 patients were included in the study. Among them, PCR, Lumira Ag test, and virus culture indicated positivity for 82, 66, and 24 patients, respectively. When comparing the Lumira Ag test to virus culture, its sensitivity was 100.0% (24/24), specificity, 30.0% (18/60); positive predictive value, 36.3% (24/66); and negative predictive value (NPV), 100.0% (18/18). The positive sample for virus culture was observed until the ninth day from the onset of symptoms, while the Lumira Ag test was observed until day 11. Conclusions The Lumira Ag test showed high sensitivity and NPV (100% each) compared to virus culture. A test-based strategy using the Lumira Ag test can effectively exclude COVID-19 infectiousness.

Abstract Background Current microbiological tests fail to identify the causative microorganism in more than half of all pneumonia cases. We explored biomarkers that could be used for differentiating between bacterial and viral pneumonia in patients with community-acquired pneumonia (CAP). Methods In this prospective cohort study conducted in Japan, data obtained from adult patients with bacterial pneumonia, including bacterial and viral coinfections (bacterial pneumonia [BP] group), and purely viral pneumonia (VP group) at diagnosis were analyzed using multivariate logistic regression analysis to identify predictors of bacterial pneumonia. Furthermore, a decision tree was developed using the predictors. Results A total of 210 patients were analyzed. The BP and VP groups comprised 108 and 18 patients, respectively. The other 84 patients had no identified causative microorganism. The two groups shared similar characteristics, including disease severity; however, a significant difference (p

Although antibiotics are most frequently prescribed for respiratory tract infections, effective interventions for their proper use by physicians have not been fully established. We assessed the impact of educational films on the rates of antibiotic prescriptions for respiratory tract infections using fictitious scenarios. In this nationwide web-based survey prospective study, a total of 1100 physicians were included. The physicians were required to view educational short films and determine the need for prescribing antibiotics in 10 fictitious scenarios involving adults diagnosed with different acute respiratory tract infectious diseases. The antibiotic prescription rates for each scenario were compared before and after viewing the educational short film. The rates of antibiotic prescription significantly decreased after viewing the educational film, especially in cases with a narrowly defined common cold (from 51% to 15%), mild pharyngolaryngitis (from 71% to 25%), and acute bronchitis without chronic respiratory underlying diseases (from 63% to 23%). Alternatively, a slight decrease in rates was observed in cases with moderate or severe rhinosinusitis (from 94% to 79%), moderate or severe acute pharyngitis (from 88% to 69%), and acute bronchitis with chronic lung disease (from 70% to 58%), for which antibiotics are recommended. Educational short films may encourage the proper use of antibiotics for respiratory tract infections; however, the possibility of undertreatment in patients requiring antibiotics must be considered.

A 63-year-old male received a third allogeneic hematopoietic stem cell transplantation with voriconazole prophylaxis for relapsed acute myeloid leukemia. He developed septic arthritis without any typical skin lesions due to fungal infection on day 42. Treatment with liposomal amphotericin B was initiated following surgical debridement; however, he died of progressive fungal infection. Ribosomal DNA sequencing identified Fusarium solani species complex (FSSC) harboring voriconazole resistance. This clinical course indicates that breakthrough invasive fusariosis (azole-resistant FSSC infection) needs to be considered as a pathogen when patients with hematological malignancies develop septic arthritis without typical skin lesions during voriconazole prophylaxis.

Saliva is a key component of mucosal immunity, which protects the oral cavity from viral infections. However, salivary immune response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), in terms of immunoglobulin dynamics and recognition, have not been investigated sufficiently. In this study, saliva samples were collected from individuals that received SARS-CoV-2 vaccine, and immunoglobulin G (IgG), IgM, and IgA against whole spike protein and S1 protein were measured. IgA against whole spike protein increased significantly following vaccination, while IgA against S1 protein did not. Of note, the IgA response was evident two weeks after the first vaccine dose and continued to rise thereafter. On the contrary, IgG antibodies against S1 increased significantly at four weeks after vaccination. These results reveal the dynamics and recognition antigens of immunoglobulins in saliva, indicating the function of IgA in the mucosal immune system. These findings may pave the way for further studies on mucosal immune response induced by vaccination.

Abstract We investigated the clinical features of bloodstream infections (BSIs) caused by Klebsiella pneumoniae harboring rmpA and molecular characteristics of the bacteria. We retrospectively investigated adult patients with K. pneumoniae BSI from January 2010 to March 2021 at Nagasaki University Hospital. A matched case–control study in a 1:3 ratio was conducted to clarify the clinical and bacterial characteristics of BSI caused by rmpA-positive K. pneumoniae compared with those caused by rmpA-negative isolates. Antimicrobial susceptibility testing and multilocus sequence typing (MLST) were performed for rmpA-positive isolates. The rmpA was detected in 36 (13.4%) of the 268 isolates. Of these 36 isolates, 31 (86.1%) harbored iucA and 35 (97.2%) each possessed peg-344 and iroB; capsular types were identified as K1 in 9 (25.0%) and K2 in 10 isolates (27.8%). Contrarily, of the 108 rmpA-negative isolates, which were matched for case–control studies, 5 isolates (4.6%) harbored iucA and 1 (0.9%) each possessed peg-344 and iroB; 2 (1.9%) and 3 isolates (2.8%) had K1 and K2 capsular types, respectively. Among the rmpA-positive isolates, ST23/K1 (eight isolates) was the most frequent, followed by ST412/non-K1/K2 (seven isolates), ST86/K2 (five isolates), and ST268/non-K1/K2 (four isolates). In a multivariate analysis using clinical factors, liver abscess positively correlated with rmpA-positive isolates, whereas biliary tract infection and use of anticancer drugs negatively correlated with rmpA-positive isolates in patients with K. pneumoniae BSI. Considering the correlation between rmpA-positive isolates and clinical features, rmpA can be used as a marker for understanding the pathophysiology of K. pneumoniae BSI.

Abstract Background We conducted a multicenter study to evaluate the performance of a novel fully automated molecular point-of-care test using transcription-reverse transcription concerted reaction that can detect influenza A and B within 15 min in nasopharyngeal swabs and gargle samples (TRCsatFLU). Methods Patients who visited or were hospitalized at eight clinics and hospitals with influenza-like illnesses between December 2019 and March 2020 participated in this study. We collected nasopharyngeal swabs from all patients and gargle samples from patients whom the physician judged fit to perform gargling. The result of TRCsatFLU was compared to a conventional reverse transcription-polymerase chain reaction (RT-PCR). If the results of TRCsatFLU and conventional RT-PCR were different, the samples were analyzed by sequencing. Results We evaluated 233 nasopharyngeal swabs and 213 gargle samples from 244 patients. The average age of the patients was 39.3 ± 21.2. Of the patients, 68.9% visited a hospital within 24 h of symptom onset. The most common symptoms were fever (93.0%), fatigue (79.5%), and nasal discharge (64.8%). All patients in whom the gargle sample was not collected were children. Influenza A or B was detected in 98 and 99 patients in nasopharyngeal swabs and gargle samples using TRCsatFLU, respectively. Four and five patients in nasopharyngeal swabs and gargle samples, respectively, with different TRCsatFLU and conventional RT-PCR results. Influenza A or B was detected using sequencing in all samples with different results. Based on the combined conventional RT-PCR and sequencing results, the sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of TRCsatFLU for influenza detection in nasopharyngeal swabs were 0.990, 1.000, 1.000, and 0.993, respectively. In the gargle samples, the sensitivity, specificity, PPV, and NPV of the TRCsatFLU for detecting influenza were 0.971, 1.000, 1.000, and 0.974, respectively. Conclusions The TRCsatFLU showed great sensitivity and specificity for the detection of influenza in nasopharyngeal swabs and gargle samples. Trial registration : This study was registered in the UMIN Clinical Trials Registry (reference number: UMIN000038276) on October 11, 2019. Before sample collection, written informed consent for the participation and publication of this study was obtained from all participants.

Abstract Background SARS-CoV-2 seroepidemiological studies are used to guide public health decision making and to prepare for emerging infectious diseases. Disease occurrence estimates are limited in the Philippines, the country with the highest reported number of coronavirus disease-related deaths in the Western Pacific region. We aimed to estimate SARS-CoV-2 seroprevalence and infection rate among outpatient clinic attendees in Metro Manila prior to the implementation of the national coronavirus disease vaccination program. Methods We conducted repeated cross-sectional surveys at the animal bite clinic in San Lazaro Hospital, Manila, the Philippines across four periods, 3 months apart, between May 2020 and March 2021. Multivariable logistic regression was used to assess associations between different characteristics and infection status including seropositivity. Results In total 615 participants were enrolled, ranging from 115 to 174 per period. Seroprevalence quadrupled between the first (11.3%) and second (46.8%) periods and plateaued thereafter (third—46.0%, fourth—44.6%). Among seropositive participants, total antibody concentration was comparable throughout the first to third periods but declined between the third and fourth periods. Infection prevalence was comparable across enrollment periods (range 2.9–9.5%). Post-secondary education [aOR 0.42 (95% CI 0.26, 0.67)] was protective, and frontline work [aOR 1.81 (95% CI 1.18, 2.80)] was associated with increased odds of seropositivity. Frontline work status [aOR 2.27 (95% CI 1.10, 4.75)] and large household size [aOR 2.45 (95% CI 1.18, 5.49)] were associated with increased odds of infection. Conclusions The quadrupling of seroprevalence over 3 months between the first and second enrollment periods coincided with the high burden of infection in Metro Manila in early 2020. Our findings suggest a limit to the rise and potential decline of population-level SARS-CoV-2 infection-induced immunity without introduction of vaccines. These results may add to our understanding of how immunity develops against emerging infectious diseases including coronaviruses.

ABSTRACT 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.

ABSTRACT Over the past 2 decades, the prevalence of macrolide-resistant Streptococcus pneumoniae (MRSP) has increased considerably, due to widespread macrolide use. Although macrolide usage has been proposed to be associated with treatment failure in patients with pneumococcal diseases, macrolides may be clinically effective for treating these diseases, regardless of the susceptibility of the causative pneumococci to macrolides. As we previously demonstrated that macrolides downregulate the transcription of various genes in MRSP, including the gene encoding the pore-forming toxin pneumolysin, we hypothesized that macrolides affect the proinflammatory activity of MRSP. Using HEK-Blue cell lines, we found that the supernatants from macrolide-treated MRSP cultures induced decreased NF-κB activation in cells expressing Toll-like receptor 2 and nucleotide-binding oligomerization domain 2 compared to the supernatants from untreated MRSP cells, suggesting that macrolides inhibit the release of these ligands from MRSP. Real-time PCR analysis revealed that macrolides significantly downregulated the transcription of various genes encoding peptidoglycan synthesis-, lipoteichoic acid synthesis-, and lipoprotein synthesis-related molecules in MRSP cells. The silkworm larva plasma assay demonstrated that the peptidoglycan concentrations in the supernatants from macrolide-treated MRSP cultures were significantly lower than those from untreated MRSP cultures. Triton X-114 phase separation revealed that lipoprotein expression decreased in macrolide-treated MRSP cells compared to the lipoprotein expression in untreated MRSP cells. Consequently, macrolides may decrease the expression of bacterial ligands of innate immune receptors, resulting in the decreased proinflammatory activity of MRSP. IMPORTANCE To date, the clinical efficacy of macrolides in pneumococcal disease is assumed to be linked to their ability to inhibit the release of pneumolysin. However, our previous study demonstrated that oral administration of macrolides to mice intratracheally infected with macrolide-resistant Streptococcus pneumoniae resulted in decreased levels of pneumolysin and proinflammatory cytokines in bronchoalveolar lavage fluid samples compared to the levels in samples from untreated infected control mice, without affecting the bacterial load in the fluid. This finding suggests that additional mechanisms by which macrolides negatively regulate proinflammatory cytokine production may be involved in their efficacy in vivo. Furthermore, in this study, we demonstrated that macrolides downregulated the transcription of various proinflammatory-component-related genes in S. pneumoniae, which provides an additional explanation for the clinical benefits of macrolides.

ABSTRACT BD Phoenix CPO Detect panels can identify and classify carbapenemase-producing organisms (CPOs) simultaneously with antimicrobial susceptibility testing (AST) for Gram-negative bacteria. Detection and classification of carbapenemase producers were performed using the BD Phoenix CPO Detect panels NMIC/ID-441 for Enterobacterales, NMIC/ID-442 for nonfermenting bacteria, and NMIC-440 for both. The results were compared with those obtained using comparator methods. A total of 133 strains (32 Klebsiella pneumoniae, 37 Enterobacter cloacae complex, 33 Pseudomonas aeruginosa, and 31 Acinetobacter baumannii complex strains), including 60 carbapenemase producers (54 imipenemases [IMPs] and 6 OXA type), were analyzed. Using panels NMIC-440 and NMIC/ID-441 or NMIC/ID-442, all 54 IMP producers were accurately identified as CPOs (positive percent agreement [PPA], 100.0%; 54/54). Among the 54 IMP producers identified as CPOs using panels NMIC-440 and NMIC/ID-441, 12 and 14 Enterobacterales were not resistant to carbapenem, respectively. Among all 54 IMP producers, 48 (88.9%; 48/54) were correctly classified as Ambler class B using panel NMIC-440. Using panels NMIC-440 and NMIC/ID-442, all four OXA-23-like carbapenemase-producing A. baumannii complex strains (100.0%, 4/4) were correctly identified as CPOs, and three (75.0%, 3/4) were precisely classified as class D using panel NMIC-440. Both carbapenemase producers harboring the blaISAba1-OXA-51-like gene were incorrectly identified as non-CPOs using panels NMIC-440 and NMIC/ID-442. For detecting carbapenemase producers, the overall PPA and negative percent agreement (NPA) between panel NMIC-440 and the comparator methods were 96.7% (58/60) and 71.2% (52/73), respectively, and the PPA and NPA between panels NMIC/ID-441 or NMIC/ID-442 and the comparator methods were 96.7% (58/60) and 74.0% (54/73), respectively. BD Phoenix CPO Detect panels can successfully screen carbapenemase producers, particularly IMP producers, regardless of the presence of carbapenem resistance and can be beneficial in routine AST workflows. IMPORTANCE Simple and efficient screening methods of detecting carbapenemase producers are required. BD Phoenix CPO Detect panels effectively screened carbapenemase producers, particularly IMP producers, with a high overall PPA. As the panels enable automatic screening for carbapenemase producers simultaneously with AST, the workflow from AST to confirmatory testing for carbapenemase production can be shortened. In addition, because carbapenem resistance varies among carbapenemase producers, the BD Phoenix CPO Detect panels, which can screen carbapenemase producers regardless of carbapenem susceptibility, can contribute to the accurate detection of carbapenemase producers. Our results report that these panels can help streamline the AST workflow before confirmatory testing for carbapenemase production in routine microbiological tests.

BackgroundExtended-spectrum β-lactamase–producing Enterobacterales (ESBL-E) is a great public health concern globally not only in hospitals but also in the community. To our knowledge, there have been few studies on the prevalence of ESBL-E and much less about carbapenem-resistant Enterobacterales (CRE) among children in the community, and there is no such study in Japan despite such situations. This study aimed to clarify their carriage status among Japanese infants in the community by taking the opportunity of the 4-month health checkup.MethodsThis prospective analysis was conducted from April 2020 to March 2021 in Shimabara City, Nagasaki Prefecture, Japan. The research-related items were mailed to all subjects with official documents for the checkup. The fecal samples were obtained from the diaper by guardians beforehand and were collected with the questionnaire and then screened for ESBL-E and CRE by a clinical laboratory company with selective agars followed by identification and confirmation. Only the positive samples were analyzed about resistant genotypes.ResultsOne hundred fifty infants aged 4–5 months, over half of the subjects, participated in this study. The overall ESBL-E carriage rate was 19.3% (n = 29), and no CRE carrier was detected among them. All identified ESBL-E were E. coli except for one K. pneumoniae. A significantly higher carriage rate was recorded among the infants born at “Hospital A” (25.0%) than the others (11.3%). Enterobacterales producing CTX-M-9 ± TEM were broadly distributed among the positive samples (65.5%), whereas the CTX-M-1 group was exclusively detected among those from “Hospital A”. Recursive partitioning analysis suggested that delivery facilities might be an important factor for ESBL-E colonization, although the effect could be decreased as they grow. In contrast, no significant effect was observed for other factors such as parent(s) as healthcare worker(s), having a sibling(s), and the mode of delivery.ConclusionThis study revealed the ESBL-E and CRE carriage status of Japanese infants in the community for the first time, although the setting is somewhat limited. Our findings indicated that environmental factors, especially delivery facilities, influenced ESBL-E colonization among infants aged 4–5 months, implying the need for strengthening countermeasures against antimicrobial resistance at delivery facilities and communities outside the hospitals.

Abstract Background This study investigated the diagnostic utility of the BioFire FilmArray Pneumonia Panel (PN panel), an automated and multiplexed nucleic acid detection system that rapidly detects 26 pathogens (18 bacteria and eight viruses) and seven antimicrobial resistance markers in a single assay. Methods We analyzed the targets in lower respiratory tract specimens using the PN panel and compared the detection results with those of bacterial culture methods and antimicrobial susceptibility testing. Results Of the 57 samples analyzed, the PN panel detected 97 targets (84 bacteria, four viruses, and nine antimicrobial resistance markers). Detection of bacteria and antimicrobial resistance was three times greater than that of the bacterial culture (25 bacteria and two resistant isolates) against the targets available in the panel. The overall positive and negative percent agreements between the PN panel and culture methods for bacterial detection were 100.0% and 92.9%, respectively. Multiple pathogens were detected by the PN panel in 24 samples (42.1%), ranging from two pathogens in 11 samples (19.3%) to six pathogens in one sample (1.8%). The PN panel semiquantitatively detected higher copies (≥ 106 copies/mL) of bacterial targets if the bacteria were positive by the culture method. In contrast, the semiquantitative values obtained by the panel varied (104 to 107 ≤ copies/mL) among bacteria that were negative by the culture method. Conclusions The PN panel enhanced the detection of pathogens and antimicrobial resistance markers in lower respiratory tract specimens.

Integrating RAISING and CLOVA, an effective method for detection and monitoring clonal integration of viruses and viral vectors is presented.

The IgG antibody titer against SARS-CoV-2 receptor binding protein (RBD) after mRNA vaccine were compared between those with and without previous infection (PI) for up to 48 weeks. Though sustained higher IgG-RBD were observed in the PI group after two doses of vaccines, both groups benefited from the booster shots of the third vaccine. This data supports the necessity of the booster shots to those with PI.

Abstract Introduction We investigated the performance of the cobas® 6800 system and cobas SARS-CoV-2 & Influenza A/B, a fully automated molecular testing system for influenza viruses and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This enabled an assay in a batch of 96 samples in approximately 3 h. Methods An assay was performed using the cobas SARS-CoV-2 & Influenza A/B on the cobas 6800 system for samples collected in four facilities between November 2019 and March 2020 in our previous study. The results were compared with those obtained using the reference methods. Results Of the 127 samples analyzed, the cobas SARS-CoV-2 & Influenza A/B detected influenza A virus in 75 samples, of which 73 were positive using the reference methods. No false negative results were observed. The overall positive and negative percent agreement for influenza A virus detection were 100.0% and 96.3%, respectively. There were no positive results for the influenza B virus or SARS-CoV-2. Conclusion The cobas 6800 system and cobas SARS-CoV-2 & Influenza A/B showed high accuracy for influenza A virus detection and can be useful for clinical laboratories, especially those that routinely assay many samples.

In April 2020, a coronavirus disease (COVID-19) outbreak occurred on the cruise ship Costa Atlantica in Nagasaki, Japan. Our outbreak investigation included 623 multinational crewmembers onboard on April 20. Median age was 31 years; 84% were men. Each crewmember was isolated or quarantined in a single room inside the ship, and monitoring of health status was supported by a remote health monitoring system. Crewmembers with more severe illness were hospitalized. The investigation found that the outbreak started in late March and peaked in late April, resulting in 149 laboratory-confirmed and 107 probable cases of infection with severe acute respiratory syndrome coronavirus 2. Six case-patients were hospitalized for COVID-19 pneumonia, including 1 in severe condition and 2 who required oxygen administration, but no deaths occurred. Although the virus can spread rapidly on a cruise ship, we describe how prompt isolation and quarantine combined with a sensitive syndromic surveillance system can control a COVID-19 outbreak.

BackgroundHuman T-cell leukemia virus type-1 (HTLV-1) is transmitted vertically from an infected mother to her child via breastfeeding during infancy or horizontally via sexual contact. However, little information is available on the HTLV-1 seroconversion rate in pregnant mothers and the impact of new HTLV-1 infection on mothers and babies during the perinatal period.MethodsFrom the database of a prefecture-wide antenatal adult T-cell leukemia prevention program in Nagasaki, Japan, we extracted data on 57,323 pregnant women who were screened for anti-HTLV-1 antibody during 2011–2018. Data on the 16,863 subjects whose HTLV-1 proviral load (PVL) was measured more than twice were included in our analyses.ResultsIn total, 133 (0.79%) pregnant women were HTLV-1-positive during their first pregnancy and nine (0.05%) seroconverted before or during subsequent pregnancies (between pregnancies). The median PVL (per 100 peripheral blood mononuclear cells) was significantly lower in the seroconverted mothers (0.10%) than in the initially seropositive mothers (0.15%). A repeated measures correlation analysis for the individual PVLs of the HTLV-1-positive pregnant women showed that PVL increased with parity number (rrm = 0.25) with no perinatal problems.ConclusionThe HTLV-1 seroconversion rate between pregnancies was 0.05%, and their HTLV-1 PVL increased annually but no perinatal problems were noted.

Introduction The COVID-19 pandemic has been a major concern worldwide; however, easily accessible treatment options for patients with mild COVID-19 remain limited. Since the oral intake of Lactococcus lactis strain plasma (LC-Plasma) enhances both the innate and acquired immune systems through the activation of plasmacytoid dendritic cells (pDCs), we hypothesised that the oral intake of LC-Plasma could aid the relief or prevention of symptoms in patients with asymptomatic or mild COVID-19.Methods and analysis This is an exploratory, multicentre, double-blinded, randomised, placebo-controlled trial. This study was initiated in December 2021 and concludes in April 2023. The planned number of enrolled subjects is 100 (50 subjects×2 groups); subject enrolment will be conducted until October 2022. Patients with asymptomatic or mild COVID-19 will be enrolled and randomly assigned in a 1:1 ratio to group A (oral intake of LC-Plasma-containing capsule, 200 mg/day, for 14 days) or group B (oral intake of placebo capsule, for 14 days). The primary endpoint is the change in subjective symptoms measured by the severity score. Secondary endpoints include SARS-CoV-2 viral loads, biomarkers for pDC activation, serum SARS-CoV-2-specific antibodies, serum cytokines, interferon and interferon-inducible antiviral effectors and the proportion of subjects with emergency room visits to medical institutions or who are hospitalised.Ethics and dissemination The study protocol was approved by the Clinical Research Review Board of Nagasaki University, in accordance with the Clinical Trials Act of Japan. The study will be conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act, and other current legal regulations in Japan. Written informed consent will be obtained from all the participants. The results of this study will be reported in journal publications.Trial registration number Japan Registry of Clinical Trials (registration number: jRCTs071210097).

Studies indicated potential harm from empirical broad-spectrum therapy. A recent study of hospitalizations for community-acquired pneumonia suggested that empirical anti-methicillin-resistant Staphylococcus aureus (MRSA) therapy was associated with an increased risk of death and other complications. However, limited evidence supports empirical anti-MRSA therapy for older patients with aspiration pneumonia. In a nationwide Japanese database, patients aged ≥65 years on admission with aspiration pneumonia were analyzed. Patients were divided based on presence of respiratory failure and further sub-categorized based on their condition within 3 days of hospital admission, either receiving a combination of anti-MRSA agents and other antibiotics, or not using MRSA agents. An inverse probability weighting method with estimated propensity scores was used. Out of 81,306 eligible patients, 55,098 had respiratory failure, and 26,208 did not. In the group with and without respiratory failure, 0.93% and 0.42% of the patients, respectively, received anti-MRSA agents. In patients with respiratory failure, in-hospital mortality (31.38% vs. 19.03%, p < 0.001), 30-day mortality, and 90-day mortality were significantly higher, and oxygen administration length was significantly longer in the anti-MRSA agent combination group. Anti-MRSA agent combination use did not improve the outcomes in older patients with aspiration pneumonia and respiratory failure, and should be carefully and comprehensively considered.

Abstract Background The recent increase in cases of azole-resistant Aspergillus fumigatus (ARAf) infections is a major clinical concern owing to its treatment limitations. Patient-derived ARAf occurs after prolonged azole treatment in patients with aspergillosis and involves various cyp51A point mutations or non-cyp51A mutations. The prognosis of patients with chronic pulmonary aspergillosis (CPA) with patient-derived ARAf infection remains unclear. In this study, we reported the case of a patient with ARAf due to HapE mutation, as well as the virulence of the isolate. Case presentation A 37-year-old male was presented with productive cough and low-grade fever. The patient was diagnosed with CPA based on the chronic course, presence of a fungus ball in the upper left lobe on chest computed tomography (CT), positivity for Aspergillus-precipitating antibody and denial of other diseases. The patient underwent left upper lobe and left S6 segment resection surgery because of repeated haemoptysis during voriconazole (VRC) treatment. The patient was postoperatively treated with VRC for 6 months. Since then, the patient was followed up without antifungal treatment but relapsed 4 years later, and VRC treatment was reinitiated. Although an azole-resistant isolate was isolated after VRC treatment, the patient did not show any disease progression in either respiratory symptoms or radiological findings. The ARAf isolated from this patient showed slow growth, decreased biomass and biofilm formation in vitro, and decreased virulence in the Galleria mellonella infection model compared with its parental strain. These phenotypes could be caused by the HapE splice site mutation. Conclusions This is the first to report a case demonstrating the clinical manifestation of a CPA patient infected with ARAf with a HapE splice site mutation, which was consistent with the in vitro and in vivo attenuated virulence of the ARAf isolate. These results imply that not all the ARAf infections in immunocompetent patients require antifungal treatment. Further studies on the virulence of non-cyp51A mutations in ARAf are warranted.

Abstract Objectives The aim of this trial is to evaluate the antiviral efficacy, clinical efficacy, and safety of nelfinavir in patients with asymptomatic and mild COVID-19. Trial design The study is designed as a multicenter, open-label, blinded outcome assessment, parallel group, investigator-initiated, exploratory, randomized (1:1 ratio) controlled clinical trial. Participants Asymptomatic and mild COVID-19 patients will be enrolled in 10 university and teaching hospitals in Japan. The inclusion and exclusion criteria are as follows: Inclusion criteria: (1) Japanese male or female patients aged ≥ 20 years (2) SARS-CoV-2 detected from a respiratory tract specimen (e.g., nasopharyngeal swab or saliva) using PCR, LAMP, or an antigen test within 3 days before obtaining the informed consent (3) Provide informed consent Exclusion criteria: (1) Symptoms developed ≥ 8 days prior to enrolment (2) SpO2 < 96 % (room air) (3) Any of the following screening criteria: a) ALT or AST ≥ 5 × upper limit of the reference range b) Child-Pugh class B or C c) Serum creatinine ≥ 2 × upper limit of the reference range and creatinine clearance < 30 mL/min (4) Poorly controlled diabetes (random blood glucose ≥ 200 mg/dL or HbA1c ≥ 7.0%, despite treatment) (5) Unsuitable serious complications based on the assessment of either the principal investigator or the sub-investigator (6) Hemophiliac or patients with a marked hemorrhagic tendency (7) Severe diarrhea (8) Hypersensitivity to the investigational drug (9) Breastfeeding or pregnancy (10) With childbearing potential and rejecting contraceptive methods during the study period from the initial administration of the investigational drug (11) Receiving rifampicin within the previous 2 weeks (12) Participated in other clinical trials and received drugs within the previous 12 weeks (13) Undergoing treatment for HIV infection (14) History of SARS-CoV-2 vaccination or wishes to be vaccinated against SARS-CoV-2 (15) Deemed inappropriate (for miscellaneous reasons) based on the assessment of either the principal investigator or the sub-investigator Intervention and comparator Patients who meet the inclusion criteria and do not meet any of the exclusion criteria will be randomized to either the nelfinavir group or the symptomatic treatment group. The nelfinavir group will be administered 750 mg of nelfinavir orally, three times daily for 14 days (treatment period). However, if a participant tests negative on two consecutive PCR tests of saliva samples, administration of the investigational drug for that participant can be discontinued at the discretion of the investigators. The symptomatic treatment group will not be administered the investigational drug, but all other study procedures and conditions will be the same for both groups for the duration of the treatment period. After the treatment period of 14 days, each group will be followed up for 14 days (observational period). Main outcomes The primary endpoint is the time to negative conversion of SARS-CoV-2. During the study period from Day 1 to Day 28, two consecutive negative PCR results of saliva samples will be considered as the negative conversion of the virus. The secondary efficacy endpoints are as follows: For patients with both asymptomatic and mild disease: area under the curve of viral load, half decay period of viral load, body temperature at each time point, all-cause mortality, incidence rate of pneumonia, percentage of patients with newly developed pneumonia, rate of oxygen administration, and the percentage of patients who require oxygen administration. For asymptomatic patients: incidence of symptomatic COVID-19, incidence of fever (≥ 37.0 °C for two consecutive days), incidence of cough For patients with mild disease: incidence of defervescence (< 37.0 °C), incidence of recovery from clinical symptoms, incidence of improvement of each symptom The secondary safety endpoints are adverse events and clinical examinations. Randomization Patients will be randomized to either the nelfinavir group or the symptomatic treatment group using the electric data capture system (1:1 ratio, dynamic allocation based on severity [asymptomatic], and age [< 60 years]). Blinding (masking) Only the assessors of the primary outcome will be blinded (blinded outcome assessment). Numbers to be randomized (sample size) The sample size was determined based on our power analysis to reject the null hypothesis, S (t | z =1) = S (t | z = 0) where S is a survival function, t is time to negative conversion, and z denotes randomization group, by the log-rank test with a two-sided p value of 0.05. We estimated viral dynamic parameters by fitting a nonlinear mixed-effects model to reported viral load data, and simulated our primary endpoint from viral-load time-courses that were realized from sets of viral dynamics parameters sampled from the estimated probability distribution of the parameters (sample size: 2000; 1000 each for randomization group). From this estimation of the hazard ratio between the randomization groups for the event of negative conversion using this simulation dataset, the required number of events for rejecting our null hypothesis with a power of 0.80 felled 97.345 by plugging the estimated hazard ratio, 1.79, in Freedman’s equation. Therefore, we decided the required number of randomizations to be 120 after consideration of the frequency of censoring and the anticipated rate of withdrawal caused by factors such as withdrawal of consent. Trial Status Protocol version 6.0 of February 12, 2021. Recruitment started on July 22, 2020 and is anticipated to be completed by March 31, 2022. Trial registration This trial was registered in Japan Registry of Clinical Trials (jRCT) ( jRCT2071200023 ) on 21 July 21, 2020. Full protocol The full protocol is attached as an additional file, accessible from the Trials website (Additional file 1). In the interest in expediting dissemination of this material, the familiar formatting has been eliminated; this Letter serves as a summary of the key elements of the full protocol. The study protocol has been reported in accordance with the Standard Protocol Items: Recommendations for Clinical Interventional Trials (SPIRIT) guidelines (Additional file 2).

Abstract Background The hospital-adapted A1 group of Enterococcus faecium remains an organism of significant concern in the context of drug-resistant hospital-associated infections. How this pathogen evolves and disseminates remains poorly understood. Methods A large, globally representative collection of short-read genomic data from the hospital-associated A1 group of Enterococcus faecium was assembled (n = 973). We analysed, using a novel analysis approach, global diversity in terms of both the dynamics of the accessory genome and homologous recombination among conserved genes. Results Two main modes of genomic evolution continue to shape E. faecium: the acquisition and loss of genes, including antimicrobial resistance genes, through mobile genetic elements including plasmids, and homologous recombination of the core genome. These events lead to new clones emerging at the local level, followed by the erosion of signals of clonality through recombination, and in some identifiable cases producing new clonal clusters. These patterns lead to new, emerging lineages which are able to spread globally over relatively short timeframes. Conclusions The ability of A1 E. faecium to continually present new combinations of genes for potential selection suggests that controlling this pathogen will remain challenging but establishing a framework for understanding genomic evolution is likely to aid in tracking the threats posed by newly emerging lineages.

Outbreaks of invasive infections, with high mortality rates, caused by multidrug-resistant Candida auris have been reported worldwide. Although hotspot mutations in FKS1 are an established cause of echinocandin resistance, the actual contribution of these mutations to echinocandin resistance remains unknown. Here, we sequenced the FKS1 gene of a caspofungin-resistant clinical isolate (clade I) and identified a novel resistance mutation (G4061A inducing R1354H). We applied the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to generate a recovered strain (H1354R) in which only this single nucleotide mutation was reverted to its wild-type sequence. We also generated mutant strains with only the R1354H mutation introduced into C. auris wild-type strains (clade I and II) and analyzed their antifungal susceptibility. Compared to their parental strains, the R1354H mutants exhibited a 4- to 16-fold increase in caspofungin minimum inhibitory concentration (MIC) while the H1354R reverted strain exhibited a 4-fold decrease in caspofungin MIC. In a mouse model of disseminated candidiasis, the in vivo therapeutic effect of caspofungin was more closely related to the FKS1 R1354H mutation and the virulence of the strain than its in vitro MIC. The CRISPR-Cas9 system could thus aid in elucidating the mechanism underlying drug resistance in C. auris.

Clostridioides difficile infection (CDI) is a major healthcare-associated infection that leads to a significant health economic burden in Japan. Using a decision tree model, we evaluated the budget impact of adopting a one-step nucleic acid amplification test (NAAT) alone pathway compared to a two-step diagnostic algorithm with glutamate dehydrogenase (GDH) and toxin antigen, followed by NAAT. The analysis was conducted from the government payer’s perspective for 100,000 symptomatic, hospitalized adults requiring a CDI diagnostic test. One-way sensitivity analysis was conducted for all data inputs. The NAAT alone strategy costed JPY 225,886,360 (USD 2,424,714) more, but was more effective, resulting in 1749 more patients accurately diagnosed and 91 fewer deaths compared to the two-step algorithm. Additionally, the NAAT alone pathway costed JPY 26,146 (USD 281) less per true positive CDI diagnosed. The total budget impact, and cost per CDI diagnosed was most sensitive to GDH sensitivity in one-way sensitivity analysis, where a lower GDH sensitivity resulted in greater cost savings with the NAAT alone pathway. Findings from this budget impact analysis can guide the adoption of a NAAT alone pathway for CDI diagnosis in Japan.

A 60-year-old Japanese woman presented with subacute progressive muscle pain and weakness in her proximal extremities. She was diagnosed with influenza A (H3N2) infection a week before the onset of muscle pain. At the time of admission, she exhibited weakness in the proximal muscles of the upper and lower limbs, elevated serum liver enzymes and creatinine kinase, and myoglobinuria. She did not manifest renal failure and cardiac abnormalities, indicating myocarditis. Electromyography revealed myogenic changes, and magnetic resonance imaging of the upper limb showed abnormal signal intensities in the muscles, suggestive of myopathy. Muscle biopsy of the biceps revealed numerous necrotic regeneration fibers and mild inflammatory cell infiltration, suggesting immune-mediated necrotizing myopathy (IMNM). Necrotized muscle cells were positive for human influenza A (H3N2). Autoantibody analysis showed the presence of antibodies against the signal recognition particle (SRP), and the patient was diagnosed with anti-SRP-associated IMNM. She was resistant to intravenous methylprednisolone pulse therapy but recovered after administration of oral systemic corticosteroids and immunoglobulins. We speculate that the influenza A (H3N2) infection might have triggered her IMNM. Thus, IMNM should be considered as a differential diagnosis in patients with proximal muscle weakness that persists after viral infections.

Abstract The treatment duration for candidemia with septic pulmonary embolism should be determined based on the clearance of fungus from the bloodstream and improvement of symptoms. The remaining lung nodules may not necessarily indicate persistent infection.

Abstract We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.

Objectives: This study evaluated the serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae isolates from adults (aged ≥20 years) with invasive pneumococcal disease (IPD) in Japan. Methods: This prospective observational study was conducted in 49 participating Japanese hospitals. S. pneumoniae isolates were serotyped and tested for antimicrobial susceptibility at a central laboratory. Information regarding patient characteristics, underlying disease, IPD clinical syndromes, and treatment was collected through medical chart review. Results: The final analysis included 177 patients enrolled from 45 hospitals between September 2016 and April 2018 (bacteraemic pneumonia, 110; bacteraemia without identified focus, 29; meningitis, 19). Most patients (70.1%) were aged ≥65 years and most had underlying disease (79.1%). The proportion of isolates from serotypes contained in the pneumococcal polysaccharide vaccine (PPV) 23 was 61.0%, while those in the pneumococcal conjugate vaccine (PCV) 7 and PCV13 were 2.8% and 28.2%, respectively. Non-vaccine serotypes accounted for 37.9% of all isolates and 50.8% of isolates from immunosuppressed patients. Serotype 12F was the most common vaccine serotype, followed by serotype 3. Conclusions: The continued disease burden of IPD in adults in Japan warrants improved vaccination rates and development of next-generation vaccines that include serotypes not currently covered. Clinical trial registration: Clinical trial summary registration number 160,822,918,146; JapicCTI-163352.

While clonal heterogeneity has been demonstrated in most cancers, quantitative assessment of individual tumor clones has not been translated to inform clinical practice. A few methods have been developed to investigate the tumor clonality of adult T cell leukemia/lymphoma (ATLL), but currently there is no clinically translatable method available for quantifying individual tumor clones in ATLL patients. Here, we present a methodology to assess the tumor clonality of ATLL and quantify patient-specific tumor clones in a clinical setting. The methodology consists of three steps: (1) selective amplification of restriction fragments containing a human T cell leukemia virus type 1 (HTLV-1) integration site, (2) amplicon deep sequencing to estimate the clonal structure and identify HTLV-1 integration sites of dominant clones, and (3) digital PCR targeting the HTLV-1 integration sites of the dominant clones to quantify specific tumor clones. We successfully tracked individual tumor clones using this approach and demonstrated that each clone had a distinct response to therapies. The procedure is straightforward and clinically feasible, which should facilitate the proper assessment and management of ATLL.

Abstract Background Severe fever with thrombocytopenia syndrome (SFTS) is an emerging infectious disease that commonly has a lethal course caused by the tick-borne Huaiyangshan banyang virus [former SFTS virus (SFTSV)]. The viral load in various body fluids in SFTS patients and the best infection control measure for SFTS patients have not been fully established. Case presentation A 79-year-old man was bitten by a tick while working in the bamboo grove in Nagasaki Prefecture in the southwest part of Japan. Due to the occurrence of impaired consciousness, he was referred to Nagasaki University Hospital for treatment. The serum sample tested positive for SFTSV-RNA in the genome amplification assay, and he was diagnosed with SFTS. Furthermore, SFTSV-RNA was detected from the tick that had bitten the patient. He was treated with multimodal therapy, including platelet transfusion, antimicrobials, antifungals, steroids, and continuous hemodiafiltration. His respiration was assisted with mechanical ventilation. On day 5, taking the day on which he was hospitalized as day 0, serum SFTSV-RNA levels reached a peak and then decreased. However, the cerebrospinal fluid collected on day 13 was positive for SFTSV-RNA. In addition, although serum SFTSV-RNA levels decreased below the detectable level on day 16, he was diagnosed with pneumonia with computed tomography. SFTSV-RNA was detected in the bronchoalveolar lavage fluid on day 21. By day 31, he recovered consciousness completely. The pneumonia improved by day 51, but SFTSV-RNA in the sputum remained positive for approximately 4 months after disease onset. Strict countermeasures against droplet/contact infection were continuously conducted. Conclusions Even when SFTSV genome levels become undetectable in the serum of SFTS patients in the convalescent phase, the virus genome remains in body fluids and tissues. It may be possible that body fluids such as respiratory excretions become a source of infection to others; thus, careful infection control management is needed.

Summary: Background: The genomic relationships among Enterococcus faecium isolates are the subject of ongoing research that seeks to clarify the origins of observed lineages and the extent of horizontal gene transfer between them, and to robustly identify links between genotypes and phenotypes. E faecium is considered to form distinct groups—A and B—corresponding to isolates derived from patients who were hospitalised (A) and isolates from humans in the community (B). The additional separation of A into the so-called clades A1 and A2 remains an area of uncertainty. We aimed to investigate the relationships between A1 and non-A1 groups and explore the potential role of non-A1 isolates in shaping the population structure of hospital E faecium. Methods: We collected short-read sequence data from invited groups that had previously published E faecium genome data. This hospital-based isolate collection could be separated into three groups (or clades, A1, A2, and B) by augmenting the study genomes with published sequences derived from human samples representing the previously defined genomic clusters. We performed phylogenetic analyses, by constructing maximum-likelihood phylogenetic trees, and identified historical recombination events. We assessed the pan-genome, did resistome analysis, and examined the genomic data to identify mobile genetic elements. Each genome underwent chromosome painting by use of ChromoPainter within FineSTRUCTURE software to assess ancestry and identify hybrid groups. We further assessed highly admixed regions to infer recombination directionality. Findings: We assembled a collection of 1095 hospital E faecium sequences from 34 countries, further augmented by 33 published sequences. 997 (88%) of 1128 genomes clustered as A1, 92 (8%) as A2, and 39 (4%) as B. We showed that A1 probably emerged as a clone from within A2 and that, because of ongoing gene flow, hospital isolates currently identified as A2 represent a genetic continuum between A1 and community E faecium. This interchange of genetic material between isolates from different groups results in the emergence of hybrid genomes between clusters. Of the 1128 genomes, 49 (4%) hybrid genomes were identified: 33 previously labelled as A2 and 16 previously labelled as A1. These interactions were fuelled by a directional pattern of recombination mediated by mobile genetic elements. By contrast, the contribution of B group genetic material to A1 was limited to a few small regions of the genome and appeared to be driven by genomic sweep events. Interpretation: A2 and B isolates coming into the hospital form an important reservoir for ongoing A1 adaptation, suggesting that effective long-term control of the effect of E faecium could benefit from strategies to reduce these genomic interactions, such as a focus on reducing the acquisition of hospital A1 strains by patients entering the hospital. Funding: Wellcome Trust.

ABSTRACT Streptococcus pneumoniae, the most common cause of community-acquired pneumonia, causes severe invasive infections, including meningitis and bacteremia. The widespread use of macrolides has been reported to increase the prevalence of macrolide-resistant S. pneumoniae (MRSP), thereby leading to treatment failure in patients with pneumococcal pneumonia. However, previous studies have demonstrated that several macrolides and lincosamides have beneficial effects on MRSP infection since they inhibit the production and release of pneumolysin, a pneumococcal pore-forming toxin released during autolysis. In this regard, we previously demonstrated that the mechanisms underlying the inhibition of pneumolysin release by erythromycin involved both the transcriptional downregulation of the gene encoding pneumolysin and the impairment of autolysis in MRSP. Here, using a cell supernatant of the culture, we have shown that clarithromycin inhibits pneumolysin release in MRSP. However, contrary to previous observations in erythromycin-treated MRSP, clarithromycin upregulated the transcription of the pneumococcal autolysis-related lytA gene and enhanced autolysis, leading to the leakage of pneumococcal DNA. On the other hand, compared to erythromycin, clarithromycin significantly downregulated the gene encoding pneumolysin. In a mouse model of MRSP pneumonia, the administration of both clarithromycin and erythromycin significantly decreased the pneumolysin protein level in bronchoalveolar lavage fluid and improved lung injury and arterial oxygen saturation without affecting bacterial load. Collectively, these in vitro and in vivo data reinforce the benefits of macrolides on the clinical outcomes of patients with pneumococcal pneumonia. IMPORTANCE Pneumolysin is a potent intracellular toxin possessing multiple functions that augment pneumococcal virulence. For over 10 years, sub-MICs of macrolides, including clarithromycin, have been recognized to decrease pneumolysin production and release from pneumococcal cells. However, this study indicates that macrolides significantly slowed pneumococcal growth, which may be related to decreased pneumolysin release recorded by previous studies. In this study, we demonstrated that clarithromycin decreases pneumolysin production through downregulation of ply gene transcription, regardless of its inhibitory activity against bacterial growth. Additionally, administration of clarithromycin resulted in the amelioration of lung injury in a mouse model of pneumonia induced by macrolide-resistant pneumococci. Therefore, therapeutic targeting of pneumolysin offers a good strategy to treat pneumococcal pneumonia.

Introduction The COVID-19 pandemic has emerged worldwide. Although several medications have been approved for treating moderate-to-severe COVID-19, very few treatment strategy has been established for patients with mild COVID-19 who do not require oxygen administration. Clarithromycin is a macrolide antimicrobial agent that has been widely used for bacterial respiratory infectious diseases. Clarithromycin also acts an immunomodulating drug and suppresses cytokine storms in viral respiratory diseases, including influenza. In this study, we aim to evaluate the efficacy of clarithromycin in patients with mild COVID-19.Methods and analysis This is an exploratory, multicentre, open-label, randomised controlled trial. This study was initiated in May 2021 and will end in July 2022. Patients with mild COVID-19 pneumonia who do not require oxygen administration will be enrolled and randomly assigned in a 1:1:1 ratio to group A (administration of clarithromycin 800 mg/day), group B (administration of clarithromycin 400 mg/day) or group C (standard treatment without clarithromycin). The planned number of enrolled patients is 60 (20 patients × three groups). The primary endpoint is the number of days required to improve the clinical symptoms as measured by the severity score. Secondary endpoints include days for recovery of the body temperature, proportion of patients with oxygen administration, inflammatory cytokines, viral load, serum immunoglobulins, peripheral blood lymphocytes, blood biomarkers and pneumonia infiltrations.Ethics and dissemination The study protocol was approved by the Clinical Research Review Board of Nagasaki University in accordance with the Clinical Trials Act in Japan. The study will be conducted in accordance with the Declaration of Helsinki, the Clinical Trials Act and other current legal regulations in Japan. Written informed consent will be obtained from all the participants. The results of this study will be reported as journal publications.Trial registration number jRCTs071210011.

The contributions of the complement system have been elucidated in the process of solid organ transplantation, including kidney transplantation. However, the role of complement in liver transplantation is unknown. We sought to elucidate the time-dependent changes of peritransplantational serum complement levels and the relationships with posttransplant outcomes and other immunological biomarkers. We enrolled 82 patients who underwent living-related donor liver transplantation (LDLT). Nine patients (11%) died within 90 days after LDLT (non-survivors). The following immunomarkers were collected preoperatively and at 1, 2, and 4 week(s) after LDLT: serum C3, C4, immunoglobulin G (IgG), and peripheral blood leukocyte populations characterized by CD3, CD4, CD8, CD16, CD19, CD20, CD22, and CD56. Consequently, C3 and C4 increased time-dependently after LDLT. Preoperatively, C3 was negatively correlated with the MELD score, Child–Pugh score, CD16-positive leukocyte percentage, and the CD56-positive leukocyte percentage. Non-survivors had lower levels of C3 at 2 weeks in comparison to survivors (median [interquartile range]: 56 [49-70] mg/dL vs. 88 [71-116] mg/dL, p=0.0059). When the cutoff value of C3 at 2 weeks to distinguish non-survivors was set to 71 mg/dL, the sensitivity, specificity, and area under the ROC curve were 87.5%, 75.0%, and 0.80, respectively. A principal component analysis showed an inverse relationship between the C3 and C4 levels and the percentage of CD8-, CD16-, and CD56-positive leukocytes at 1 and 2 week(s). All non-survivors were included in the cluster that showed higher percentages of CD8-, CD16-, and CD56-positive leukocytes at 2 weeks. In conclusion, we demonstrated the relationship between complement, outcomes, and other immunomarkers in LDLT and suggested the usefulness of C3 at 2 weeks after LDLT in distinguishing the mortality.

ABSTRACTA 68-year-old Japanese man was admitted to our hospital for an acute febrile illness with shivering and impaired consciousness. He was a previous smoker and had a history of chronic obstructive pulmonary disease, for which he inhaled steroid with a long-acting bronchodilator. He had received a 23-valent pneumococcal polysaccharide vaccination 2 years previously. He was intubated and placed on a ventilator in intensive care unit because of acute respiratory failure and hypercapnia. Streptococcus pneumoniae was grown from his blood, sputum, and urine cultures, and he was diagnosed with invasive pneumococcal disease with acute renal failure. He was treated with intravenous beta-lactam and macrolide with continuous hemodiafiltration and was discharged 3 months later. The pneumococcus was identified as serotype 12F, and his serotype-specific IgG and opsonophagocytic index against serotype 12F indicating a lack of protection from IPD among PPV23 serotypes. This case highlights that some individuals may have a serotype-specific polysaccharide antibody failure that makes them susceptible to serotype 12F invasive pneumococcal disease. This case also illustrates the need for serotype-specific IgG and opsonophagocytic index titre cut-offs for each specific pneumococcal serotype in available vaccines to understand the vaccination protection for individual patients better.

Abstract Background Rapid diagnosis and appropriate treatment of Munchausen syndrome is important not only for the patient but also for health care workers because a delay in diagnosis can worsen patients’ clinical outcomes, and result in a substantial medical cost. Case presentation A young and previously healthy 24-year-old Japanese woman, a nurse, presented with complaints of refractory abscess on her left upper limb for 3 months. A physical examination on admission revealed low-grade fever and a subcutaneous abscess in her left forearm. Laboratory data suggested mild systemic inflammation and liver dysfunction, but no abnormalities of the immune system, including changes in the number of lymphocytes and neutrophils, neutrophil phagocytic capacity, and natural killer (NK) cell activity, were observed. A human immunodeficiency virus test was also negative. Multiple modalities, including positron emission tomography-computed tomography, failed to detect any cause and focus of infection except her left upper limb. Streptococcus mitis and Prevotella buccae were detected from the wound, but no microorganisms were detected in a blood culture. The cellulitis promptly resolved; however, exacerbation of the subcutaneous abscess with polymicrobial bacteremia repeatedly occurred unexpectedly. Because of this puzzling clinical course, the possibility of self-injury was finally suspected. Three syringes with needles, with a turbid liquid, were found in our patient’s bag. Enterobacter cloacae and Enterococcus faecalis were detected in the liquid, and an analysis via repetitive element sequence-based polymerase chain reaction determined that Enterococcus faecalis in the wound and syringe contents were genetically identical. She was diagnosed as having Munchausen syndrome and treated with the collaboration of a psychiatrist. She finally confessed that she had injected her own saliva and toilet water into the drip line and wound. Conclusions This case report is valuable in that it is the first case in which this syndrome was diagnosed by a genetic method. Munchausen syndrome should not be neglected as a possible cause of refractory and recurrent infection.

Severe fever with thrombocytopenia syndrome (SFTS) and Japanese spotted fever (JSF; a spotted fever group rickettsiosis) are tick-borne zoonoses that are becoming a significant public health threat in Japan and East Asia. Strategies for treatment and infection control differ between the two; therefore, initial differential diagnosis is important. We aimed to compare the clinical characteristics of SFTS and JSF based on symptomology, physical examination, laboratory data, and radiography findings at admission. This retrospective study included patients with SFTS and JSF treated at five hospitals in Nagasaki Prefecture, western Japan, between 2013 and 2020. Data from 23 patients with SFTS and 38 patients with JSF were examined for differentiating factors and were divided by 7:3 into a training cohort and a validation cohort. Decision tree analysis revealed leukopenia (white blood cell [WBC] < 4000/μL) and altered mental status as the best differentiating factors (AUC 1.000) with 100% sensitivity and 100% specificity. Using only physical examination factors, absence of skin rash and altered mental status resulted in the best differentiating factors with AUC 0.871, 71.4% sensitivity, and 90.0% specificity. When treating patients with suspected tick-borne infection, WBC < 4000/µL, absence of skin rash, and altered mental status are very useful to differentiate SFTS from JSF.

Objective It remains unclear whether human T-cell leukemia virus type 1 (HTLV-1) infection influences therapeutic responses in patients with rheumatic diseases and whether immunosuppressive treatments increase the risk of HTLV-1-related complications in HTLV-1 carriers with rheumatic diseases. We examined the effects of tocilizumab (TCZ), an interleukin (IL)-6 receptor antagonist, on two HTLV-1-infected T-cell lines (HCT-5 and MT-2) in vitro . Methods We evaluated production of cytokines and chemokines, expression of HTLV-I associated genes, HTLV-1 proviral load (PVL), expression of HTLV-1 structural proteins, and apoptosis. Results There were no significant differences in cytokine and chemokine levels in the culture supernatants of HCT-5 and MT-2 cells treated with phosphate-buffered saline (PBS) or TCZ. No significant differences were detected in mRNA abundance of Tax or HBZ, PVL, expression of the HTLV-1 structural protein GAG, or apoptosis among HCT-5 and MT-2 cells treated with PBS or TCZ. Conclusions TCZ had no effect the cytokine profiles, HTLV-1 gene and protein expression, PVL, or apoptosis in HTLV-1-infected T-cell lines. Thus, TCZ treatment has no effect on HTLV-1 infection in vitro .

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.

ObjectivesA 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.MethodsBlood 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.ResultsSensitivity, 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.ConclusionsThese findings suggest that anti-SARS-CoV-2 antibody testing has lower performance in asymptomatic or mild COVID-19 patients than required in the guidelines.

Allergic bronchopulmonary aspergillosis (ABPA) and chronic pulmonary aspergillosis (CPA) are important fungal infections caused by Aspergillus species. An overlap of ABPA and CPA has been reported; therefore, it is critical to determine whether the main pathology is ABPA or CPA and whether antifungals are required. In this study, we investigated whether the serum cytokine profile is useful for understanding the pathology and for differentiating between these diseases. We compared the various serum cytokine levels among healthy subjects and patients diagnosed with asthma, ABPA, or CPA at Nagasaki University Hospital between January 2003 and December 2018. In total, 14 healthy subjects, 19 patients with asthma, 11 with ABPA, and 10 with CPA were enrolled. Interleukin (IL) -5 levels were significantly higher in patients with ABPA than in those with CPA, and IL-33 and tumor necrosis factor (TNF) levels were significantly higher in patients with CPA than in those with asthma (p < 0.05, Dunn’s multiple comparison test). The sensitivity and specificity of the IL-10/IL-5 ratio (cutoff index 2.47) for diagnosing CPA were 70% and 100%, respectively. The serum cytokine profile is useful in understanding the pathology of ABPA and CPA, and the IL-10/IL-5 ratio may be a novel supplemental biomarker for indicating the pathology of CPA.

Background: Methicillin-resistant Staphylococcus aureus (MRSA) is a common causative agent of pneumonia; however, the detailed mechanism underlying severe MRSA pneumonia, including association with oral hygiene or periodontitis, remains poorly characterized. In this study, we examined the pathogenic effect of Prevotella intermedia, a major periodontopathic pathogen, on MRSA pneumonia.Methods: The pathogenic effect of the supernatant of P. intermedia (Pi Sup) was investigated in a murine MRSA pneumonia model, using several clinical strains; whereas the bactericidal activity of polymorphonuclear leukocytes (PMNs) was investigated in vitro. The effect of Pi Sup on messenger RNA (mRNA) expression of the toxin/quorum sensing system (rnaIII) was investigated by quantitative reverse transcription PCR both in vitro and in vivo.Results: Mice infected by hospital-acquired MRSA (HA-MRSA) with Pi Sup exhibited a significantly lower survival rate, higher bacterial loads in the lungs, and higher α-hemolysin (hla) expression in the lungs, than those without Pi Sup. A similar effect of Pi Sup was not observed with MRSA strains producing Panton-Valentine leucocidin (PVL) or toxic shock syndrome toxin (TSST). In vitro, Pi Sup suppressed bactericidal activity of PMNs against the HA-MRSA strain. HA-MRSA was the clinical strain with the highest ability to proliferate in the lungs and was accompanied by time-dependent up-regulation of rnaIII and hla.Conclusions: Our results provide novel evidence that the product of P. intermedia exerts a pathogenic effect on MRSA pneumonia, in particular with a strain exhibiting strong proliferation in the lower airway tract. Moreover, our results indicate that P. intermedia affects MRSA toxin expression via quorum sensing in a strain-dependent fashion, which might be important for understanding the pathogenesis of severe MRSA pneumonia.

Streptococcus pneumoniae is often isolated from patients with community-acquired pneumonia. Antibiotics are the primary line of treatment for pneumococcal pneumonia; however, rising antimicrobial resistance is becoming more prevalent. Hinokitiol, which is isolated from trees in the cypress family, has been demonstrated to exert antibacterial activity against S. pneumoniae in vitro regardless of antimicrobial resistance. In this study, the efficacy of hinokitiol was investigated in a mouse pneumonia model. Male 8-week-old BALB/c mice were intratracheally infected with S. pneumoniae strains D39 (antimicrobial susceptible) and NU4471 (macrolide resistant). After 1 h, hinokitiol was injected via the tracheal route. Hinokitiol significantly decreased the number of S. pneumoniae in the bronchoalveolar lavage fluid (BALF) and the concentration of pneumococcal DNA in the serum, regardless of whether bacteria were resistant or susceptible to macrolides. In addition, hinokitiol decreased the infiltration of neutrophils in the lungs, as well as the concentration of inflammatory cytokines in the BALF and serum. Repeated hinokitiol injection at 18 h intervals showed downward trend in the number of S. pneumoniae in the BALF and the concentration of S. pneumoniae DNA in the serum with the number of hinokitiol administrations. These findings suggest that hinokitiol reduced bacterial load and suppressed excessive host immune response in the pneumonia mouse model. Accordingly, hinokitiol warrants further exploration as a potential candidate for the treatment of pneumococcal pneumonia.