The Epidemiology of Circulating Respiratory Pathogens during the COVID-19 Pandemic.

Objective To survey the epidemiology of respiratory pathogens during the coronavirus disease 2019 (COVID-19) pandemic using multiplex polymerase chain reaction (PCR). Methods Specimens were assayed using multiplex nested PCR. Materials Specimens were obtained from outpatients who presented with symptoms of upper respiratory tract infection and asymptomatic outpatients who had contact with patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection at Tohoku Medical and Pharmaceutical University Hospital in Sendai, Japan, from November 1, 2020, to May 31, 2023. The analysis included multiple specimens collected from the same patients at different time-points. Data were collected from the electronic records after testing. Results This study included 8,335 patients (4,311 men) with a median age of 59 years old, and 11,741 total specimens were collected. At least 1 positive SARS-CoV-2 result was obtained for 1,710 (14.6%) specimens. Furthermore, 15 pathogens were identified in the positive specimens, and rhinovirus/enterovirus was detected more frequently than other viruses. We identified a larger number of SARS-CoV-2-positive specimens in patients ≥10 years old. In contrast, in patients 0-9 years old, we identified a larger number of specimens positive for rhinovirus/enterovirus than for other viruses. Conclusion In this study, we examined the epidemiology of circulating respiratory pathogens during the COVID-19 pandemic era.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures.

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/µL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Epidemiologic and genomic investigations of an unusual increase in Salmonella enterica serovar Paratyphi A infection among travelers returning from Myanmar in 2015.

OBJECTIVES: An unusual increase in Salmonella enterica serovar Paratyphi A infection rate in Japanese travelers returning from Myanmar was observed in 2015. METHODS: We analyzed epidemiologic data of returned travelers with enteric fever from 2005-2019. We also analyzed 193 Salmonella Paratyphi A isolates, including 121 isolates with published genomes. RESULTS: Annual notification trends showed a rapid increase in Salmonella Paratyphi A infection in travelers returning from Myanmar in 2015: 2-4 cases/100,000 travelers in 2012-2014 and 13 cases/100,000 travelers in 2015 (P <0.001). The genomic analyses revealed that 11 Myanmar-related isolates in 2015 formed a tight cluster in clade 3 with a single nucleotide variant (SNV) distance of 0-11 (primarily 0-7), yielding a wider SNV range than outbreak-associated isolates from Cambodia in 2013 (0-6 SNVs) or China in 2010 (0-5 SNVs). Although all Cambodia-related isolates in 2013 harbored the wild-type gyrA sequence, all Myanmar-related isolates in 2015 had a single, identical mutation (Ser83Phe) in the gyrA gene. CONCLUSION: The epidemiologic and molecular investigations suggested an increase in the infection rate with genetically closely related Salmonella Paratyphi A in travelers returning from Myanmar in 2015. Careful monitoring of the infection in Myanmar as an endemic country is warranted, considering the resumption of cross-border travel during the COVID-19 pandemic.

Clinical evaluation of cell-direct polymerase chain reaction-based nucleic acid lateral flow immunoassay for rapid detection of bacterial pathogens in clinically suspected sepsis: A multi-center study in Japan.

Blood culture, a method for identifying causative agents of bacterial sepsis, requires several days. The combination of cell-direct polymerase chain reaction and nucleic acid lateral flow immunoassay (cdPCR-NALFIA) is a simple and sensitive detection method for identifying pathogenic bacteria. Furthermore, this assay, when applied directly to blood samples yields results within 4.5 h, without requiring culture. This study was performed at five hospitals in Japan between 2013 and 2016. Blood samples from 73 patients with clinically suspected sepsis yielded 18 positive blood cultures, and the isolated bacterial species were detectable using cdPCR-NALFIA in nine samples. Thirteen samples were positive on cdPCR-NALFIA. In total, 17 samples confirmed to have bacterial species were detectable using cdPCR-NALFIA and/or blood culture with a true positive rate of 76.5% and 64.7%, respectively. The combination of blood culture and cdPCR-NALFIA could improve the rate of detection of bacterial sepsis.

Distribution of extraintestinal pathogenic Escherichia coli O-serotypes and antibiotic resistance in blood isolates collected from patients in a surveillance study in Japan.

INTRODUCTION: Invasive extraintestinal pathogenic Escherichia coli (ExPEC) disease (IED), characterised by sepsis and bacteraemia, is a major global healthcare concern worsened by emerging multidrug resistant (MDR) strains. The development of multivalent prophylactic vaccines targeting E. coli strains of IED-associated O-serotypes could address this. A better understanding of O-serotype distribution is required for this purpose. Here, we characterised O-serotype prevalence and drug resistance among ExPEC bacteraemia isolates in Japan. METHODS: E. coli blood isolates from patients aged ≥60 years with bacteraemia were obtained from a retrospective surveillance study in Japan (2015-2017). O-serotyping was performed by agglutination; for isolates non-typeable by agglutination, O-genotyping was performed. Antimicrobial susceptibility was evaluated by broth microdilution using a 21-antibiotic panel. The frequency of drug resistant (DR) isolates was evaluated by antimicrobial susceptibility testing. RESULTS: Of 401 ExPEC bacteraemia isolates evaluated, the most prevalent O-serotype (≥1%) was O25 (28.7% [n = 115]), followed by O1 (14.2% [n = 57]), O2 (8.5% n = 34]), O6 (5.5% [n = 22]), O75, O18, O13, O16, O15, O4, O46/O134, O86, O8 and O83 (each <5% prevalence). These 14 O-serotypes accounted for 81.5% of isolates collected. In total, 19% (n = 77) of isolates were DR ≥ 3, of which 59.7% were O25. Fluoroquinolone-resistance among all and O25 isolates was most prevalent (35.7% and 84.3%, respectively). Almost all (98%) isolates identified as O25 were of subtype O25B. CONCLUSIONS: E. coli serotype O25B showed the highest prevalence and highest multidrug resistance among ExPEC bacteraemia isolates from patients ≥60 years in Japan. Our data may inform development of multivalent glycoconjugate vaccines to prevent IED.

Use of a multiplex polymerase chain reaction assay for the early detection of an outbreak of human parainfluenza virus type 3 infection in a nursery school during the COVID-19 pandemic.

Introduction: Although outbreaks of parainfluenza virus type 3 (PIV-3) have been reported in children, to our knowledge none have been reported in a nursery school. As the symptoms of PIV-3 infection are similar to those of COVID-19 infection, accurate diagnosis of PIV-3 and other respiratory viruses is important during the COVID-19 pandemic. Aims: We experienced an outbreak of upper respiratory symptoms at a nursery school in Miyagi Prefecture, Japan, from 29/5/2021 to 13/6/2021 and aimed to determine the causative organism(s). Methods: A multiplex polymerase chain reaction (PCR) assay which enabled rapid detection of a variety of causative microorganisms of respiratory tract infections was used to analyse 13 nasopharyngeal swabs collected during the outbreak. Infection Prevention and control measures were implemented to prevent further spread of infection. Results: All 13 samples were positive for PIV-3 infection. 2 of the 13 samples were also positive for rhinovirus/enterovirus and 1 sample was also positive rhinovirus/enterovirus and coronavirus NL 63. No samples were positive for SARS-CoV-2. Discussion: Children in school settings are especially vulnerable to respiratory viral infections, including COVID-19. Children under two years are unable to wear masks reliably, and the COVID-19 vaccine was approved only for older children. Multiplex PCR assays can be used for the rapid diagnosis of respiratory infections. Conclusion: We identified an outbreak of PIV-3 in a nursery school during the COVID-19 pandemic. The investigation of the outbreak highlighted that it was important not to overlook other respiratory infections including PIV-3 during the COVID-19 pandemic. The multiplex PCR assay provided rapid and accurate diagnosis of the causative organisms in the outbreak and helped to direct appropriate interventions to control the outbreak.

Disseminated tuberculosis with paradoxical reactions caused by a Mycobacterium tuberculosis strain belonging to the Indo-Oceanic lineage: An imported case in Japan.

Tuberculosis remains a major public health concern. Millions of tuberculosis cases and associated deaths have been reported worldwide. The Indo-Oceanic lineage Mycobacterium tuberculosis is common in Southeast Asia and causes extrapulmonary lesions. Only a few case studies on this lineage with genetic analysis using whole-genome sequencing have been reported in the literature. We present a case of disseminated tuberculosis, characterized by a variety of extrapulmonary lesions and paradoxical reactions, caused by the Indo-Oceanic lineage M. tuberculosis in a woman in Myanmar. A 22-year-old Burmese woman had arthritis in the right knee, with unknown aetiology, and was referred to our hospital. Computed tomography of the trunk revealed multiple nodular shadows in both lungs; swollen mediastinal lymph nodes; and small, low-density areas in the spleen. M. tuberculosis was detected in the sputum sample, joint aspirate, subcutaneous tumor, and exudate. She experienced a variety of paradoxical reactions together with aggressive tuberculosis dissemination in all areas of the body. Whole-genome sequencing of the DNA of MTB obtained from sputum and the right cervical subcutaneous abscess confirmed the Indo-Oceanic lineage of M. tuberculosis, the predominant strain in Myanmar. The Indo-Oceanic lineage M. tuberculosis causes disseminated tuberculosis all over the body including the periungual region. When patients show unusual symptoms, physicians should consider the introduction of new strains from foreign countries. Genetic analyses of the strains are recommended to define and confirm the lineages.

The surveillance of colistin resistance and mobilized colistin resistance genes in multidrug-resistant Enterobacteriaceae isolated in Japan.

BACKGROUND: The plasmid-mediated bacterial colistin-resistant gene, mcr, is of global concern in clinical healthcare. However, there are few reports of surveillance for mcr in Japan. The aim of this study was to assess the prevalence of colistin resistance by identifying nine mcr genes in extended-spectrum beta-lactamase (ESBL)-producing Enterobacteriaceae and carbapenem-resistant Enterobacteriaceae (CRE) isolates in Japan. METHODS: A total of 273 ESBL and CRE clinical isolates were collected from patients in five tertiary hospitals from August 2016 to March 2017. Minimum inhibitory concentration (MIC) of colistin was measured using the microdilution method. Polymerase chain reaction (PCR) was performed to detect mcr-1 to mcr-9 genes in all strains. Whole-genome sequencing (WGS) analysis was conducted for any mcr-genes identified that had not been previously reported in patients from Japan. RESULTS: The rate of colistin resistance was 7.7% in all strains, with a higher rate in the CRE strains than in the ESBL-producing strains (20.4% versus 1.1%). The mcr-5 and mcr-9 gene were detected in one ESBL-producing Escherichia coli strain (1/273, 0.37%) and three CRE strains (3/273, 1.1%), respectively. As the ESBL-producing E. coli strain was the first clinical strain with mcr-5 in Japan, WGS analysis was performed for the strain. The sequence type of the mcr-5-positive strain was ST1642 and it carried two distinct plasmids, ESBL gene-carrying pN-ES-6-1, and mcr-5.1-carrying pN-ES-6-2. CONCLUSIONS: The results of this study showed that the frequency of colistin resistance and mcr-positive strains is not high in Japan. As the MIC for colistin was low in the mcr-5.1 and mcr-9 gene-positive strain, continuous monitoring of mcr genes is necessary.

Utility of a Cell-Direct Polymerase Chain Reaction-Based Nucleic Acid Lateral Flow Immunoassay for Detection of Bacteria in Peripheral Blood Leukocytes of Suspected Sepsis Cases.

BACKGROUND: The detection of the pathogens in the blood is essential for the management of septic patients; however, conventional blood culture takes 2-3 days. Therefore, rapid and convenient methods may be useful to aid clinical decision-making. METHODS: Blood samples with sepsis clinically diagnosed in cases that fulfilled the diagnostic criteria were used and analyzed the utility of a novel bacterial nucleic acid identification test using a cell-direct polymerase chain reaction (cdPCR)-based nucleic acid lateral flow immunoassay (NALFIA) which were named as "DiagnoSep" to detect representative bacteria in peripheral blood leukocytes in patients admitted to our hospital and compared the conventional blood culture results simultaneously taken from the patients. RESULTS: We analyzed the total 42 samples in the terms of this study and found 18 (42.8%) were positive on cdPCR-NALFIA, and 24 (57.1%) were positive on blood cultures. Although the positive rate was higher with blood cultures, 15 samples showed positive results from both blood cultures and cdPCR-NALFIA, and the identified bacteria agreed for 10 samples. Of the 18 cdPCR-NALFIA-positive cases, the results for 8 samples differed from the results of blood cultures; four of them had an implanted pacemaker or prosthetic joint and were positive for Staphylococcus aureus or Staphylococcus epidermidis on cdPCR-NALFIA. CONCLUSION: Blood culture tests are probably the gold standard in identifying causative organisms in sepsis, but the rapid results from cdPCR-NALFIA simultaneously used with blood culture may make it an important auxiliary diagnostic tool for identifying infecting organisms and lead to the improvement of mortality of the septic patients, because these combined results provide the wide information on the possible pathogens in early phase.

Case Report: Successful Treatment of Five Critically Ill Coronavirus Disease 2019 Patients Using Combination Therapy With Etoposide and Corticosteroids.

Acute respiratory distress syndrome (ARDS) is the leading cause of mortality in hospitalized patients with coronavirus disease 2019 (COVID-19) because of limited effective therapies. During infection, the accumulation and activation of macrophages and monocytes in the lungs induce inflammatory mediators and contribute to tissue injury, leading to ARDS. However, therapeutic strategies that directly target activated macrophage and monocytes have not been reported. Combination treatment with etoposide (a cytotoxic agent) and a corticosteroid has been widely used for treating hemophagocytic lymphohistiocytosis characterized by the systemic activation of macrophages with overwhelming inflammation. Herein, we present five cases of COVID-19-associated ARDS treated with etoposide and corticosteroids. Three of the five patients were over 65 years of age and had various underlying diseases, including multiple myeloma. Four patients required invasive mechanical ventilation (MV), and one patient refused to be placed on MV due to underlying diseases. All patients were pre-treated with antiviral and/or other anti-inflammatory agents, but their condition deteriorated and hyperinflammation was noted. All five patients responded well to treatment and had an immediate response, as reflected by improvement in their respiratory condition and inflammatory marker levels and rapid resolution of fever after etoposide administration; however, some patients required a second dose of etoposide and longer course of steroids. All patients recovered, and there were no severe adverse events related to the drugs. Following successful treatment in these five patients, we plan to conduct a clinical trial to evaluate the efficacy and safety of combination therapy with etoposide and corticosteroid for treating COVID-19 patients in Japan.

Azithromycin: A promising treatment option for Mycobacterium avium complex pulmonary disease in case of intolerance to clarithromycin.

Macrolide-based combination chemotherapy is recommended for the treatment of Mycobacterium avium complex (MAC) pulmonary disease (MPD). The susceptibility of the MAC to macrolide antibiotics (MAs) determines the efficacy of treatment and clinical course of MPD. However, MAs cause several adverse effects, resulting in the discontinuation of macrolide-based combination chemotherapy. We encountered two women aged 65 years and 66 years diagnosed with MPD based on bronchoscopic examinations. They were initially treated with clarithromycin-based combination chemotherapy. However, neither patient could continue with chemotherapy owing to adverse events such as rash and edema. We switched clarithromycin with azithromycin, and the patients were able to continue chemotherapy without adverse events. Both patients completed their treatment successfully. Azithromycin, which also belongs to the class of MAs, can be a promising therapeutic option for MPD in case of clarithromycin intolerance.

Proactive Engagement of the Expert Meeting in Managing the Early Phase of the COVID-19 Epidemic, Japan, February-June 2020.

To deal with the risk of emerging diseases with many unknowns, close and timely collaboration and communication between science experts and policymakers are crucial to developing and implementing an effective science-based intervention strategy. The Expert Meeting, an ad hoc medical advisory body, was established in February 2020 to advise Japan's COVID-19 Response Headquarters. The group played an important role in the policymaking process, promoting timely situation awareness and developing science-based proposals on interventions that were promptly reflected in government actions. However, this expert group may have been overly proactive in taking on the government's role in crisis management. For the next stage of managing the coronavirus disease pandemic and future pandemics, the respective roles of the government and its advisory bodies need to be clearly defined. Leadership and strategic risk communication by the government are key.

Pulmonary infection caused by Mycobacterium marinum in a patient with anorexia nervosa.

Mycobacterium marinum can cause pulmonary infection and can grow at ≤32°C. Physicians should consider M. marinum when examining patients with pulmonary infection and low body temperature or anorexia nervosa, and grow the specimen at ≤32°C. https://bit.ly/3jkzBeq.

Nationwide multicenter questionnaire surveys on countermeasures against antimicrobial resistance and infections in hospitals.

BACKGROUND: The goals of the National Action Plan on Antimicrobial Resistance (AMR) of Japan include "implementing appropriate infection prevention and control" and "appropriate use of antimicrobials," which are relevant to healthcare facilities. Specifically, linking efforts between existing infection control teams and antimicrobial stewardship programs was suggested to be important. Previous studies reported that human resources, such as full-time equivalents of infection control practitioners, were related to improvements in antimicrobial stewardship. METHODS: We posted questionnaires to all teaching hospitals (n = 1017) regarding hospital countermeasures against AMR and infections. To evaluate changes over time, surveys were conducted twice (1st survey: Nov 2016, 2nd survey: Feb 2018). A latent transition analysis (LTA) was performed to identify latent statuses, which refer to underlying subgroups of hospitals, and effects of the number of members in infection control teams per bed on being in the better statuses. RESULTS: The number of valid responses was 678 (response rate, 66.7%) for the 1st survey and 559 (55.0%) for the 2nd survey. More than 99% of participating hospitals had infection control teams, with differences in activity among hospitals. Roughly 70% had their own intervention criteria for antibiotics therapies, whereas only about 60 and 50% had criteria established for the use of anti-methicillin-resistant Staphylococcus aureus antibiotics and broad-spectrum antibiotics, respectively. Only 50 and 40% of hospitals conducted surveillance of catheter-associated urinary tract infections and ventilator-associated pneumonia, respectively. Less than 50% of hospitals used maximal barrier precautions for central line catheter insertion. The LTA identified five latent statuses. The membership probability of the most favorable status in the 2nd study period was slightly increased from the 1st study period (23.6 to 25.3%). However, the increase in the least favorable status was higher (26.3 to 31.8%). Results of the LTA did not support a relationship between increasing the number of infection control practitioners per bed, which is reportedly related to improvements in antimicrobial stewardship, and being in more favorable latent statuses. CONCLUSIONS: Our results suggest the need for more comprehensive antimicrobial stewardship programs and increased surveillance activities for healthcare-associated infections to improve antimicrobial stewardship and infection control in hospitals.

Genomic characteristics of listeria monocytogenes causing invasive listeriosis in Japan.

We reviewed 18 listeriosis cases in Japan and performed molecular analysis of causative Listeria monocytogenes (LM) isolates. Strains genetically related to those from other countries caused various types of listeriosis, including vascular listeriosis in immunocompetent elderly people. Our results highlight the importance of integrated clinical and genomic analysis of LM.

A Matched Case-Case-Control Study of the Impact of Clinical Outcomes and Risk Factors of Patients with IMP-Type Carbapenemase-Producing Carbapenem-Resistant Enterobacteriaceae in Japan.

IMP-type carbapenemase, found in various Gram-negative bacteria, has been increasingly detected worldwide. We aimed to study the outcomes and risk factors for acquisition of IMP-type carbapenemase-producing carbapenem-resistant Enterobacteriaceae (IMP-CRE), as this has not been evaluated in detail. We conducted a matched case-case-control study of patients from whom IMP-CRE isolates were obtained. All patients who tested positive for IMP-CRE were included; they were matched with patients with carbapenem-susceptible Enterobacteriaceae (CSE) and with controls at a ratio of 1:1:2. The risk factors for acquisition for the CRE and CSE groups and mortality rates, which were calculated using multivariate logistic regression models with weighting according to the inverse probability of propensity scores, were compared. In total, 192 patients (96 patients each in the CRE and CSE groups, with 130 Enterobacter cloacae isolates and 62 Klebsiella sp. isolates) were included. The IMP-11 type was present in 43 patients, IMP-1 in 33, and IMP-60 and IMP-66 in 1 each; 31 patients with CRE (32.3%) and 34 with CSE (35.4%) developed infections. Multivariate analysis identified the following independent risk factors: gastrostomy, history of intravenous therapy or hemodialysis, and previous exposure to broad-spectrum ß-lactam antibiotics, including penicillin with ß-lactamase inhibitors, cephalosporins, and carbapenems. In propensity score-adjusted analysis, mortality rates for the CRE and CSE groups were similar (15.0% and 19.5%, respectively). We found that IMP-CRE may not contribute to worsened clinical outcomes, compared to CSE, and gastrostomy, previous intravenous therapy, hemodialysis, and broad-spectrum antimicrobial exposure were identified as risk factors for CRE isolation. Fluoroquinolone and aminoglycosides are potentially useful antibiotics for IMP-CRE infections.

Serotype distribution and antimicrobial susceptibility of Streptococcus pneumoniae associated with invasive pneumococcal disease among adults in Japan.

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.

Chromobacterium haemolyticum Pneumonia Associated with Near-Drowning and River Water, Japan.

We report a severe case of Chromobacterium haemolyticum pneumonia associated with near-drowning and detail the investigation of the pathogen and river water. Our genomic and environmental investigation demonstrated that river water in a temperate region can be a source of C. haemolyticum causing human infections.