Isolation and identification of Wickerhamiella tropicalis from blood culture by MALDI-MS.

Wickerhamiella is a genus of budding yeast that is mainly isolated from environmental samples, and 40 species have been detected. The yeast isolated from human clinical samples usually only contain three species: W. infanticola, W. pararugosa and W. sorbophila. In this study, we isolated W. tropicalis from a blood sample of a six-year-old female with a history of B-cell precursor lymphoblastic leukemia in Japan in 2022. Though the strain was morphologically identified as Candida species by routine microbiological examinations, it was subsequently identified as W. tropicalis by sequencing the internal transcribed spacer (ITS) of ribosomal DNA (rDNA). The isolate had amino acid substitutions in ERG11 and FKS1 associated with azole and echinocandin resistance, respectively, in Candida species and showed intermediate-resistant to fluconazole and micafungin. The patient was successfully treated with micafungin. Furthermore, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) detected three novel peaks that are specific for W. tropicalis, indicating that MALDI-MS analysis is useful for rapid detection of Wickerhamiella species in routine microbiological examinations.

Impact of the COVID-19 Pandemic on Initiation of Antibiotic Treatment After Performing a Blood Culture and Intervention by the Antimicrobial Stewardship Team.

Purpose: Whether the coronavirus disease 2019 (COVID-19) pandemic had any effect on the time between blood culture collection and administration of antibiotics in the outpatient Department of Emergency Medicine in a single university hospital in Japan was investigated, and the intervention carried out by the antimicrobial stewardship team (AST) to promote the appropriate use of antibiotics was examined. Patients and Methods: The monthly percentage of patients who visited the outpatient Department of Emergency Medicine between January 2019 and December 2021 and received an intravenous antibiotic within 3 hours of blood culture collection was calculated. The AST calculated a quality indicator (QI) based on the results of the investigation and started QI monitoring and hospital feedback. Results: From January 2020 to March 2021 (the third COVID-19 wave), the implementation rate of antibiotic administration within 3 hours after blood culture collection decreased as the COVID-19 pandemic spread, and the implementation rate tended to increase as the number of COVID-19-positive patients decreased. However, when the AST started monitoring and feedback from April 2021, although there was a temporary decline in the early stages of the fifth wave when the scale of infection was large, the implementation rate rose and was maintained by AST intervention. (the fourth and the fifth COVID-19 waves) (P<0.01). Also, the implementation rate was significantly lower during the COVID-19 pandemic than during the non- pandemic (P<0.05). Conclusion: The early COVID-19 pandemic may have affected the delay in time from blood culture collection to antibiotic administration. Later, in recurring COVID-19 pandemics, AST intervention eliminated this problem. When a bacterial infection such as sepsis is suspected, delayed treatment can be prevented by promptly collecting a blood culture, irrespective of concerns about COVID-19 infection. Calculating the QI may promote AST activities and the appropriate use of antibiotics.

Successful Treatment of a Case of Metallo-Beta-Lactamase-Producing Raoultella ornithinolytica Bacteremia by Antimicrobial Stewardship Team Intervention and Therapeutic Drug Monitoring-Based Amikacin Treatment.

An 80-year-old woman underwent pancreatoduodenectomy. Post-operation, she experienced a fever, and a culture of blood revealed metallo-beta-lactamase-producing Raoultella ornithinolytica. For treatments with aminoglycoside antimicrobial agents, a therapeutic drug monitoring-based dosing design can lower the risk of adverse events and enable appropriate treatment. Key Clinical Message. When aminoglycoside antimicrobial agents are administered for MBL-producing bacteremia, prescription suggestions based on TDM by antimicrobial stewardship team can reduce the occurrence of adverse events and enable appropriate treatment.

Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva.

BACKGROUND: Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using saliva samples has emerged as a preferred technique since sample collection is easy and noninvasive. In addition, several commercial high-throughput PCR kits that do not require RNA extraction/purification have been developed and are now available for testing saliva samples. However, an optimal protocol for SARS-CoV-2 RT-PCR testing of saliva samples using the RNA extraction/purification-free kits has not yet been established. The aim of this study was to establish optimal preanalytical conditions, including saliva sample collection, storage, and dilution for RNA extraction/purification-free RT-PCR (direct RT-PCR). METHODS: Patients suspected with COVID-19 from March 02 to August 31, 2020, were enrolled in this study. A total of 248 samples, including 43 nasopharyngeal swabs and 205 saliva samples, were collected from 66 patients (37 outpatients and 29 inpatients) and tested using the 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan). RESULTS: The detection results obtained using nasopharyngeal swabs and saliva samples matched 100%. The sampling time, i.e., either awakening time or post-breakfast, had no significant effect on the viral load of the saliva samples. Although saliva samples are routinely diluted to reduce viscosity, we observed that dilution negatively affected PCR sensitivity. Saliva samples could be stored at room temperature (25°C) for 24 hours or at 4°C for up to 48 hours. CONCLUSIONS: This study demonstrated the appropriate conditions of saliva sample collection, processing, and storage, and indicated that the nCoV-DK is applicable to saliva samples, making the diagnosis method simple and safe.

The 24-h reporting of Gram stains from positive blood cultures contributes to physician's use of appropriate antimicrobials: Experience at a university hospital.

Although recent technological advances for the diagnosis of bloodstream infection (BSI) provide rapid and accurate results, blood culture maintains a key role in the diagnosis of BSI. The objective of this study was to determine whether 24-h reporting by telephone to disclose the suspected microorganism based on the Gram stain morphology from positive blood cultures (first laboratory report) affects a physician's use of appropriate antimicrobials. A total of 627 (14%) out of 4413 blood samples, excluding duplicate samples from the same patient on the same day, were positive for blood cultures between January and December 2016. The contamination rate of blood cultures during the study period was 2.3%. Among 627 patients with positive blood cultures, 538 (86%) were receiving antibiotics at the time of the first laboratory report, of which 502 (80%) thereafter continued the same antimicrobials, and the remaining 36 (6%) were changed to appropriate antimicrobials after the first laboratory report. An additional 25 (4%) were newly administered appropriate antimicrobials after the first laboratory report, whereas an additional 21 (3%) were newly administered appropriate antimicrobials after infection control team (ICT)-intervention. The median time lag (interquartile ranges) from flagging culture bottles as positive to a physician's use of appropriate antimicrobials after the first laboratory report (4 h, 2-7) was significantly (p < 0.001) shorter than that after ICT-intervention (12 h, 10-17). During the study period, no cases of discrepancy between the Gram stain morphology in the first laboratory report and definitive identification of microorganisms in the final laboratory report were observed. Because the timing of flagging culture bottles as positive tends to fall outside normal working hours, immediate 24-h reporting by telephone to disclose the suspected microorganism based on the Gram stain morphology from positive blood cultures may contribute to an early recognition of bacteremia and the physician's use of appropriate antimicrobials.

Performance and usefulness of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit for the diagnosis of COVID-19.

Here, we aimed to evaluate the clinical performance of a novel automated immunoassay HISCL SARS-CoV-2 Antigen assay kit designed to detect the nucleocapsid (N) protein of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). This kit comprises automated chemiluminescence detection systems. Western blot analysis confirmed that anti-SARS-CoV antibodies detected SARS-CoV-2N proteins. The best cut-off index was determined, and clinical performance was tested using 115 serum samples obtained from 46 patients with coronavirus disease 2019 (COVID-19) and 69 individuals who tested negative for COVID-19 through reverse transcription quantitative polymerase chain reaction (RT-qPCR). The HISCL Antigen assay kit showed a sensitivity of 95.4% and 16.6% in samples with copy numbers > 100 and < 99, respectively. The kit did not cross-react with human coronaviruses causing seasonal common cold and influenza, and none of the 69 individuals without COVID-19 were diagnosed with positive results. Importantly, 81.8% of the samples with low virus load (< 50 copy numbers) were diagnosed as negative. Thus, using HISCL antigen assay kits may reduce overdiagnosis compared with RT-qPCR tests. The rapid and high-throughput HISCL SARS-CoV-2 Antigen assay kit developed here proved suitable for screening infectious COVID-19 and may help control the pandemic.

The synergetic effect of Imipenem-clarithromycin combination in the Mycobacteroides abscessus complex.

BACKGROUND: Nontuberculous mycobacteria (NTM) are ubiquitous organisms and the incidence of NTM infections has been increasing in recent years. Mycobacteroides abscessus (M. abscessus) is one of the most antimicrobial-resistant NTM; however, no reliable antibiotic regimen can be officially advocated. We evaluated the efficacy of clarithromycin in combination with various antimicrobial agents against the M. abscessus complex. RESULTS: Twenty-nine clinical strains of M. abscessus were isolated from various clinical samples. Of the isolates, 10 (34.5%) were of M. abscessus subsp. abscessus, 18 (62.1%) of M. abscessus subsp. massiliense, and 1 (3.4%) of M. abscessus subsp. bolletii. MICs of three antimicrobial agents (amikacin, imipenem, and moxifloxacin) were measured with or without clarithromycin. The imipenem-clarithromycin combination significantly reduced MICs compared to clarithromycin and imipenem monotherapies, including against resistant strains. The association between susceptibility of the M. abscessus complex and each combination of agents was significant (p = 0.001). Adjusted residuals indicated that the imipenem-clarithromycin combination had the synergistic effect (adjusted residual = 3.1) and suppressed the antagonistic effect (adjusted residual = - 3.1). In subspecies of M. abscessus complex, the association with susceptibility of M. abscessus subsp. massiliense was similarly statistically significant (p = 0.036: adjusted residuals of synergistic and antagonistic effect respectively: 2.6 and - 2.6). The association with susceptibility of M. abscessus subsp. abscessus also showed a similar trend but did not reach statistical significance. CONCLUSION: Our data suggest that the imipenem-clarithromycin combination could be the recommended therapeutic choice for the treatment of M. abscessus complex owing to its ability to restore antimicrobial susceptibility.

Efficacy of PCR-based open reading frame typing assay for outbreak investigation of metallo-ß-lactamase-producing Pseudomonas aeruginosa in hematology unit.

We investigated the efficacy of the PCR-based open reading frame typing (POT) assay for outbreak investigation of metallo-ß-lactamase (MBL)-producing Pseudomonas aeruginosa (MBL-PA). A total of 53 P. aeruginosa isolates were detected between January 2010 and December 2012 on a hematology ward, of which 6 were identified as MBL-PA with the blaIMP-1 gene. The POT assay revealed the same genotype (207-41) in 3 of 6 MBL-PA, suggesting an outbreak caused by a single strain. Environmental investigation of bathroom samples revealed the same POT genotype (207-41) as those of the clinical isolates and no other MBL-PA strains. Genetic relatedness of the MBL-PA isolates was confirmed by the DiversiLab repetitive-sequence-based PCR typing system, suggesting the POT type 207-41 as a genetically identical clone. The POT assay can be successfully applied to MBL-PA genotyping.