Clinical evaluation of anterior nasal cavity swab specimens by a rapid antigen test using a GLINE-2019-nCoV Ag Kit to diagnose COVID-19.

The promising diagnostic performance of rapid antigen tests (RATs) using non-invasive anterior nasal (AN) swab specimens to diagnose COVID-19 has been reported. A large number of RATs are commercially available; however, the careful assessment of RATs is essential prior to their implementation in clinical practice. We evaluated the clinical performance of the GLINE-2019-nCoV Ag Kit as a RAT using AN swabs in a prospective, blinded study. Adult patients who visited outpatient departments and received SARS-CoV-2 tests between August 16 and September 8, 2022, were eligible for this study. Patients who were aged under 18 years and patients without appropriate specimens were excluded. Two sets of AN and nasopharyngeal (NP) swabs were collected from all patients. Each set of specimens was tested by the RAT and quantitative reverse-transcription polymerase chain reaction (RT-qPCR). Of the 138 recruited patients, 84 were positive and 54 were negative by RT-qPCR using NP swabs. The positive agreement rate between RT-qPCR using NP swabs and RAT using AN swabs was 78.6% (95% confidence interval [CI], 68.3%-86.8%), the negative agreement rate was 98.1% (95% CI, 90.1%-99.9%), and the overall agreement rate was 86.2% (95% CI, 79.3%-91.5%), with a κ coefficient of 0.73. The positive agreement rate in the early phase (≤3 days from symptom onset) was >80%, but this fell to 50% in the late phase (≥4 days). This study demonstrates that the GLINE-2019-nCoV Ag Kit using AN swabs has good clinical performance and might be a reliable alternative method for diagnosing COVID-19.

Evaluation of an immunochromatography-based rapid antigen test, Inspecter Kowa® SARS-CoV-2, using saliva specimens for the detection of SARS-CoV-2.

BACKGROUND: In the context of the coronavirus disease 2019 (COVID-19) pandemic, a rapid and reliable point-of-care test is an essential tool for controlling the spread of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). In particular, an immunochromatography test (ICT) that uses saliva specimens for rapid antigen detection not only reduces the risk of secondary infections but also reduces the burden on medical personnel. METHODS: The newly developed salivary antigen test kit "Inspecter Kowa® SARS-CoV-2" is an ICT to which saliva specimens can be directly applied. We evaluated its usefulness in comparison with reverse transcription quantitative PCR (RT-qPCR) and the Espline® SARS-CoV-2 Kit for the detection of SARS-CoV-2 using nasopharyngeal swab specimens. In this study, 140 patients with suspected symptomatic COVID-19 who visited our hospital were enrolled, and nasopharyngeal swab and saliva specimens were collected after they consented to participate in the study. RESULTS: Inspector Kowa SARS-CoV-2 was positive in 45 of 61 (73.8%) saliva that were positive by RT-qPCR and the Espline® SARS-CoV-2 Kit was also positive in 56 of 60 (93.3%) Np swabs that were positive by RT-qPCR. Good antigen detection was achieved by ICT with saliva and nasopharyngeal swab specimens when viral load was ≥105 copies/mL, whereas detection sensitivity was low when viral load was <105 copies/mL, especially in saliva specimens. CONCLUSION: This ICT for the detection of SARS-CoV-2 salivary antigen is an attractive tool that does not require specialized equipment and allows patients to perform the entire process from sample collection to self-diagnose and to reduce the burden on medical care during a pandemic.

Evaluation of the correlation between the access SARS-CoV-2 IgM and IgG II antibody tests with the SARS-CoV-2 surrogate virus neutralization test.

Fully automated immunoassays for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) antibodies that are strongly correlated with neutralization antibodies (nAbs) are clinically important because they enable the assessment of humoral immunity after infection and vaccination. Access SARS-CoV-2 immunoglobulin M (IgM) and immunoglobulin G (IgG) II antibody tests are semi-quantitative, fully automated immunoassays that detect anti-receptor-binding domain (RBD) antibodies and might reflect nAb levels in coronavirus disease 2019 (COVID-19). However, no studies have investigated the clinical utility of these tests in association with nAbs to date. To evaluate the clinical utility of Access SARS-CoV-2 IgM and IgG II antibody tests and their correlation with the SARS-CoV-2 surrogate virus neutralization test (sVNT) that measures nAbs in patients with COVID-19, we analyzed 54 convalescent serum samples from COVID-19 patients and 89 serum samples from non-COVID-19 patients. The presence of anti-RBD antibodies was detected using Access SARS-CoV-2 IgM and IgG II antibody tests, while nAbs were measured by sVNT. The sensitivity and specificity of sVNT were 94.4% and 98.9%, respectively. There were strong positive correlations between the inhibition values of sVNT and the results of the Access SARS-CoV-2 IgM (R = 0.95, R2 = 0.90, p < 0.001) and IgG II antibody tests (R = 0.96, R2 = 0.92, p < 0.001). In terms of the presence of nAbs, the sensitivity and specificity were 98.1% and 98.9% in the IgM assay and 100.0% and 100.0% in the IgG II assay, respectively. The Access SARS-CoV-2 IgM and IgG II antibody tests showed high sensitivity and specificity for the detection of nAbs in COVID-19 patients and might be alternatives for measuring nAbs.

Point Prevalence Surveys of Antimicrobial Prescribing in a Non-Acute Care Hospital in Saitama Prefecture, Japan.

Background: The global point prevalence survey (Global-PPS) is the standard for the surveillance of prescribed antimicrobials among inpatients and provides data for the development of hospital antimicrobial stewardship programs. Aim: To evaluate the prevalence and quality of antimicrobial prescriptions using the universally standardized Global-PPS protocol in a non-acute care hospital in Saitama Prefecture, Japan. Methods: Antimicrobial prescriptions for inpatients, staying at the hospital overnight, were surveyed on three separate week days in November 2018, January 2019, and May 2019. Information on the prescribed antimicrobials on the survey target day was obtained from the in-hospital pharmacy. Survey data were collected by physicians, based on the extracted information. Patient information was anonymized and entered in the Global-PPS Web application by physicians. We report the antimicrobial use prevalence, the indication for prescription, diagnosis, the most prescribed antimicrobials, and a set of quality indicators related to antimicrobial prescribing. Results: In total, 6.7% of the surveyed inpatients (120/1796) were prescribed antimicrobials on the survey day. Sulfamethoxazole/trimethoprim was the most commonly prescribed, with 20.0% of systemic antibiotic prescriptions (ATC J01). Of all antibiotics for systemic use, up to 58.4% were Watch antibiotics, as defined by the World Health Organization AWaRe classification. The most prescribed group of systemic antibiotics was non-penicillin beta-lactam antibiotics (34.4%), followed by penicillin antibiotics in combination with beta-lactamase inhibitors (25.6%), and sulfonamides with trimethoprim (20.8%). Healthcare-associated infections and medical prophylaxis were the most common indications reported in 69.3% and 26.3% of prescriptions, respectively. The most common diagnosis for systemic antibiotic prescriptions was pneumonia (49.6%). Reasons for antimicrobial prescriptions were indicated in the medical records for 67.1% of prescriptions, and the stop/review date was documented to be 50.3%. Compliance with local guidelines reached 66.7%. Conclusions: This study highlights important challenges related to antimicrobial prescription in a highly specific, non-acute care patient population.

Evaluation of a chemiluminescent enzyme immunoassay-based high-throughput SARS-CoV-2 antigen assay for the diagnosis of COVID-19: The VITROS® SARS-CoV-2 Antigen Test.

A high-throughput, fully automated antigen detection test for SARS-CoV-2 is a viable alternative to reverse-transcription polymerase chain reaction (RT-qPCR) for mass screening during outbreaks. In this study, we compared RT-qPCR for viral load and the VITROS® SARS-CoV-2 Antigen Test with reference to the results of the LUMIPULSE® SARS-CoV-2 Ag Test. Of 128 nasopharyngeal swab specimens taken from patients suspected of being infected with SARS-CoV-2, 49 were positive and 79 were negative according to RT-qPCR. Consistent dose-dependent detection with VITROS® assay was successfully achieved when using nasopharyngeal swab specimens with Ct values of 32.0 or lesser, whereas the CLEIA-based LUMIPULSE® assay was able to detect lower viral loads compared with the VITROS® assay. Our results show that the performance of the VITROS® assay was satisfactory for the diagnosis of contagious COVID-19 patients in the clinical setting. Highlights The performance of the VITROS® SARS-CoV-2 Antigen Test was sufficient for the diagnosis of contagious COVID-19. This test showed high sensitivity and specificity in the detection of SARS-CoV-2 in samples with a Ct value of 32 or less.

Antibody response patterns in COVID-19 patients with different levels of disease severity in Japan.

We analyzed antibody response patterns according to the level of disease severity in patients with novel coronavirus disease 2019 (COVID-19) in Japan. We analyzed 611 serum specimens from 231 patients with COVID-19 (mild, 170; severe, 31; critical, 30). Immunoglobulin M (IgM) and IgG antibodies against nucleocapsid protein (N) and spike 1 protein (S1) were detected by enzyme-linked immunosorbent assays. The peaks of fitting curves for the optical density (OD) values of IgM and IgG antibodies against N appeared simultaneously, while those against S1 were delayed compared with N. The OD values of IgM against N and IgG against both N and S1 were significantly higher in the severe and critical cases than in the mild cases at 11 days after symptom onset. The seroconversion rates of IgG were higher than those of IgM against both N and S1 during the clinical course based on the optimal cut-off values defined in this study. The seroconversion rates of IgG and IgM against N and S1 were higher in the severe and critical cases than in the mild cases. Our findings show that a stronger antibody response occurred in COVID-19 patients with greater disease severity and there were low seroconversion rates of antibodies against N and S1 in the mild cases.

Bloodstream infection by multidrug-resistant Streptococcus oralis in a leukemic patient with febrile neutropenia after hematopoietic stem cell transplantation.

We reported the case of a patient with leukemia who developed febrile neutropenia after hematopoietic stem cell transplantation. Blood culture results revealed the presence of Streptococcus oralis, while antimicrobial susceptibility testing showed the resistance to penicillin and cephem. Furthermore, isolates were not susceptible to either meropenem or daptomycin but not to vancomycin. S oralis is known to belong to Streptococcus mitis group and be a causative agent of bacteremia in the neutropenic patients, but multidrug resistance of S oralis is rare. Our findings suggest that we might pay attention to the emergence of the microorganisms acquiring multidrug resistance in neutropenic patients.

Thoracic aortic mycotic aneurysm due to Staphylococcus argenteus: A case report.

Staphylococcus argenteus was subdivided as a novel species from Staphylococcus aureus in 2014. We herein report a case of mycotic aneurysm caused by S. argenteus. A 59-year-old woman with diabetes and schizophrenia visited at the emergency room because of falling. Chest computed tomography revealed a left humerus fracture and a thoracic aortic aneurysm. With her elevated WBC count and CRP level, she was suspected to have a mycotic aneurysm. After being transferred to our hospital, vascular graft replacement surgery was performed. Isolates of blood cultures and surgical specimens were identified as S. argenteus by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MAS MALDI Biotyper Ver. 8.0). Although S. argenteus lacks staphyloxanthin, a carotenoid pigment, it is coagulase positive. In addition to traditional and automated biochemical identification systems, even MALDI-TOF MAS may misidentify the organism as S. aureus depending on its version. S. argenteus should be considered when coagulase-negative Staphylococcus like colonies are obtained from samples of S. aureus infection. To our knowledge, this is the first case of aortic mycotic aneurysm caused by S. argenteus in Japan. Although S. argenteus is considered less virulent than Staphylococcus aureus, we should closely monitor the prevalence and the clinical impact of this pathogen on community-acquired infections and health care-associated infections.

Endogenous endophthalmitis secondary to septic arthritis caused by group A Streptococcus infection: A case report and literature review.

Streptococcus pyogenes is a rare pathogen that causes endogenous endophthalmitis (EE). A healthy 58-year-old woman was diagnosed with EE secondary to septic arthritis caused by S. pyogenes. She underwent enucleation after hospitalization for 14 days with appropriate antibiotic cover. A literature search for outcomes of this condition revealed reports on only 10 eyes among 8 cases identified: 8 eyes (80%) developed poor visual outcome and 5 eyes (50%) underwent enucleation. There were no cases with immunocompromise. Our case report and literature review suggest the importance of awareness of the occurrence of S. pyogenes infection in immunocompetent hosts, and thus early diagnosis and aggressive treatment may be required to improve visual outcome.

Successful treatment of severe acute respiratory distress syndrome due to Group A streptococcus induced toxic shock syndrome in the third trimester of pregnancy-effectiveness of venoarterial extracorporeal membrane oxygenation: A case report.

We report a case of Group A streptococcal infection-induced toxic shock syndrome (GAS-TSS) with severe acute respiratory distress syndrome (ARDS), successfully treated with venoarterial extracorporeal membrane oxygenation (V-A ECMO). A 31-year-old woman was transferred due to high fever, continuous uterine contractions and fetal bradycardia at 31 weeks of gestation. She was in a shock status on arrival, and as fetal heart beat disappeared, we canceled the cesarean section and took priority in maternal rescue. At 21 h after the admission, pulseless ventricular tachycardia occurred, and V-A ECMO was introduced after defibrillation, which dramatically improved her respiratory and circulatory conditions. On the 3rd day, GAS was isolated from blood culture. The patient was freed from V-A ECMO on the 5th day and was discharged on the 25th day without permanent impairment. V-A ECMO should be considered as an effective therapeutic option against ARDS and circulation failure in GAS-TSS during pregnancy.

Antimicrobial susceptibility and prevalence of resistance genes in Bacteroides fragilis isolated from blood culture bottles in two tertiary care hospitals in Japan.

The in vitro susceptibilities of Bacteroides fragilis to antimicrobial agents, especially to carbapenem, are a major concern in the treatment of patients with bloodstream infections. In this study, 50 isolates of B. fragilis were obtained from positive blood bottles from 2014 to 2019 in Saitama, Japan. Their susceptibility to ampicillin/sulbactam was reduced to 70.0% compared with a previous report, whereas they were still sufficiently susceptible to piperacillin/tazobactam (94.0%). Five cfiA-positive isolates (5/50, 10.0%) were identified that were resistant to doripenem and meropenem, and two of them carried an insertion sequence located upstream of the cfiA-coding region. In particular, imipenem should be considered as a first-line carbapenem for the empirical treatment of B. fragilis infection because only insertion sequence and cfiA double-positive strains showed resistance to imipenem. Thirty-six percent of the isolates had a reduced minimum inhibitory concentration for moxifloxacin. In addition, metronidazole should still be considered as an active agent for B. fragilis because all isolates were susceptible to this antibiotic and the prevalence of the nim gene was low in Japan.

Clinical characteristics in blood stream infections caused by Klebsiella pneumoniae, Klebsiella variicola, and Klebsiella quasipneumoniae: a comparative study, Japan, 2014-2017.

BACKGROUND: Klebsiella variicola and K. quasipneumoniae are new species distinguishable from K. pneumoniae but they are often misidentified as K. pneumoniae in clinical settings. Several reports have demonstrated the possibility that the virulence factors and clinical features differ among these three phylogroups. In this study, we aimed to clarify whether there were differences in clinical and bacterial features between the three phylogroups isolated from patients with bloodstream infections (BSIs) in Japan. METHODS: Isolates from all patients with BSIs caused by K. pneumoniae admitted to two hospitals between 2014 and 2017 (n = 119) were included in the study. Bacterial species were identified via sequence analysis, and their virulence factors and serotypes were analyzed via multiplex PCR results. Clinical data were retrieved from medical records. RESULTS: Of the 119 isolates, 21 (17.7%) were identified as K. variicola and 11 (9.2%) as K. quasipneumoniae; K1 serotype was found in 16 (13.4%), and K2 serotype in 13 (10.9%). Significant differences in the prevalence of rmpA, iutA, ybtS, entB and kfu (p < 0.001), and allS genes (p < 0.05) were found between the three phylogroups. However, there were no significant differences in clinical features, including the 30-day mortality rate, between the three organisms, although K. variicola was more frequently detected in patients over 80 years old compared with other Klebsiella species (p < 0.005), and K. quasipneumoniae more frequently occurred in patients with malignancy (p < 0.05). CONCLUSIONS: Our findings demonstrated the differences in bacterial pathogenicity and clinical features among these three phylogroups. Further epidemiological studies into BSI caused by Klebsiella species are warranted.

An innovative diagnostic technology for the codon mutation C580Y in kelch13 of Plasmodium falciparum with MinION nanopore sequencer.

BACKGROUND: The recent spread of artemisinin (ART)-resistant Plasmodium falciparum represents an emerging global threat to public health. In Southeast Asia, the C580Y mutation of kelch13 (k13) is the dominant mutation of ART-resistant P. falciparum. Therefore, a simple method for the detection of C580Y mutation is urgently needed to enable widespread routine surveillance in the field. The aim of this study is to develop a new diagnostic procedure for the C580Y mutation using loop-mediated isothermal amplification (LAMP) combined with the MinION nanopore sequencer. RESULTS: A LAMP assay for the k13 gene of P. falciparum to detect the C580Y mutation was successfully developed. The detection limit of this procedure was 10 copies of the reference plasmid harboring the k13 gene within 60 min. Thereafter, amplicon sequencing of the LAMP products using the MinION nanopore sequencer was performed to clarify the nucleotide sequences of the gene. The C580Y mutation was identified based on the sequence data collected from MinION reads 30 min after the start of sequencing. Further, clinical evaluation of the LAMP assay in 34 human blood samples collected from patients with P. falciparum malaria in Indonesia revealed a positive detection rate of 100%. All LAMP amplicons of up to 12 specimens were simultaneously sequenced using MinION. The results of sequencing were consistent with those of the conventional PCR and Sanger sequencing protocol. All procedures from DNA extraction to variant calling were completed within 3 h. The C580Y mutation was not found among these 34 P. falciparum isolates in Indonesia. CONCLUSIONS: An innovative method combining LAMP and MinION will enable simple, rapid, and high-sensitivity detection of the C580Y mutation of P. falciparum, even in resource-limited situations in developing countries.

First report of the isolation of blaIMI-1-producing colistin-heteroresistant Enterobacter cloacae in Japan, September 2016.

The isolation of a carbapenem-resistant Enterobacter cloacae strain harboring the IMI-1 variant of blaIMI-1 carbapenemase points to the worldwide emergence of multidrug resistant bacteria as a potential source of health care infections. In this report, we describe the first isolation of E. cloacae with blaIMI-1 carbapenemase isolated from a Japanese patient in September 2016. The isolate was resistant to carbapenems, levofloxacin, and aminoglycosides, and heteroresistant to colistin but sensitive to fourth-generation cephalosporins. All microbiology laboratories worldwide should be made aware of these blaIMI-1-producing subtypes with unusual antibiotic susceptibility profiles.

A novel diagnostic method for malaria using loop-mediated isothermal amplification (LAMP) and MinION™ nanopore sequencer.

BACKGROUND: A simple and accurate molecular diagnostic method for malaria is urgently needed due to the limitations of conventional microscopic examination. In this study, we demonstrate a new diagnostic procedure for human malaria using loop mediated isothermal amplification (LAMP) and the MinION™ nanopore sequencer. METHODS: We generated specific LAMP primers targeting the 18S-rRNA gene of all five human Plasmodium species including two P. ovale subspecies (P. falciparum, P. vivax, P. ovale wallikeri, P. ovale curtisi, P. knowlesi and P. malariae) and examined human blood samples collected from 63 malaria patients in Indonesia. Additionally, we performed amplicon sequencing of our LAMP products using MinION™ nanopore sequencer to identify each Plasmodium species. RESULTS: Our LAMP method allowed amplification of all targeted 18S-rRNA genes of the reference plasmids with detection limits of 10-100 copies per reaction. Among the 63 clinical samples, 54 and 55 samples were positive by nested PCR and our LAMP method, respectively. Identification of the Plasmodium species by LAMP amplicon sequencing analysis using the MinION™ was consistent with the reference plasmid sequences and the results of nested PCR. CONCLUSIONS: Our diagnostic method combined with LAMP and MinION™ could become a simple and accurate tool for the identification of human Plasmodium species, even in resource-limited situations.

Utility of the MALDI-TOF MS method to identify nontuberculous mycobacteria.

In comparison to the conventional real-time polymerase chain reaction method (PCR method) or the DNA-DNA hybridization method (DDH method), the utility of NTM identification by the matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) method has seldom been reported. In this study, 75 clinical NTM isolates from our hospital between April 2013 and July 2014 were identified and analyzed using PCR, DDH, and MALDI-TOF MS methods, and the results for the MALDI-TOF MS method were compared with the others. Identification at the species level was in agreement for 71 (94.5%) of the 75 isolates. For further details, identification was possible for 23 (95.8%) of 24 Mycobacterium avium, 11 (100%) of 11 Mycobacterium intracellulare, and 1 (50%) of 2 isolates mixed with M. avium and M. intracellulare. Mycobacterium ksansasii, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium gordonae, and Mycobacterium chelonae identified by DDH method were same result by MALDI-TOF MS. Additionally, Mycobacterium mucogenicum, which could not be identified by the DDH method, was identified by the MALDI-TOF MS method. However, two isolates identified as Mycobacterium terrae by DDH method could not be identified by the MALDI-TOF MS method and were determined to be Mycobacterium arupense by 16S ribosomal RNA (rRNA) sequence analysis. The present findings show that, for rare bacterial species, identification is sometimes not possible, but, in most cases, the results of identification by the MALDI-TOF MS method have a high concordance rate with the results of the PCR and DDH methods.

Exacerbation of invasive Candida albicans infection by commensal bacteria or a glycolipid through IFN-γ produced in part by iNKT cells.

BACKGROUND: The commensal yeast Candida albicans is a major cause of invasive fungal infections. Despite treatment with antifungal agents, the mortality rate attributed to these types of infection is high. Although numerous cases have been reported regarding a poor outcome for patients with bacterial and C. albicans coinfection, the mechanisms by which the coinfecting bacteria exacerbate the C. albicans infection remain elusive. METHODS AND RESULTS: We evaluated how glycolipid-mediated activation of invariant natural killer T (iNKT) cells affects the clearance of C. albicans. Surprisingly, C. albicans-infected, glycolipid-treated mice exhibited significantly lower survival rates, increased fungal burden, and higher interleukin (IL)-6 production in the kidneys compared with control mice. Glycolipid-induced exacerbation of C. albicans infection was not observed in interferon-gamma knockout (IFN-γKO) mice. In the C. albicans-infected, glycolipid-treated mice, the number of neutrophils in the blood and bone marrow dramatically decreased in an IFN-γ-dependent manner. Furthermore, mice that were coinfected with C. albicans and nonfermentative gram-negative commensal bacteria exhibited increased fungal burden and inflammatory cytokine production in the kidneys that were dependent on IFN-γ and iNKT cells. CONCLUSIONS: Our results indicate that coinfecting commensal bacteria exacerbate C. albicans infection through IFN-γ produced, in part, by iNKT cells.

Unique interplay between sugar and lipid in determining the antigenic potency of bacterial antigens for NKT cells.

Invariant natural killer T (iNKT) cells are an evolutionary conserved T cell population characterized by features of both the innate and adaptive immune response. Studies have shown that iNKT cells are required for protective responses to Gram-positive pathogens such as Streptococcus pneumoniae, and that these cells recognize bacterial diacylglycerol antigens presented by CD1d, a non-classical antigen-presenting molecule. The combination of a lipid backbone containing an unusual fatty acid, vaccenic acid, as well as a glucose sugar that is weaker or not stimulatory when linked to other lipids, is required for iNKT cell stimulation by these antigens. Here we have carried out structural and biophysical studies that illuminate the reasons for the stringent requirement for this unique combination. The data indicate that vaccenic acid bound to the CD1d groove orients the protruding glucose sugar for TCR recognition, and it allows for an additional hydrogen bond of the glucose with CD1d when in complex with the TCR. Furthermore, TCR binding causes an induced fit in both the sugar and CD1d, and we have identified the CD1d amino acids important for iNKT TCR recognition and the stability of the ternary complex. The studies show also how hydrogen bonds formed by the glucose sugar can account for the distinct binding kinetics of the TCR for this CD1d-glycolipid complex. Therefore, our studies illuminate the mechanism of glycolipid recognition for antigens from important pathogens.