The performance of a Fully Automated Urine Particle Analyzer, Sysmex UF-5000, in detecting fastidious bacteria in urine samples.

Several types of fastidious bacteria can cause tract infections. We evaluated the performance of counting fastidious bacteria using a Fully Automated Urine Particle Analyzer UF-5000. The results showed that UF-5000 counts fastidious bacteria in urine without the need for culture using measurement principles based on flow cytometry.

Multi-drug resistance pattern and genome-wide SNP detection in levofloxacin-resistant uropathogenic Escherichia coli strains.

OBJECTIVES: Antibiotic treatment is extremely stressful for bacteria and has profound effects on their viability. Such administration induces physiological changes in bacterial cells, with considerable impact on their genome structure that induces mutations throughout the entire genome. This study investigated drug resistance profiles and structural changes in the entire genome of uropathogenic Escherichia coli (UPEC) strains isolated from six adapted clones that had evolved under laboratory conditions. METHODS: Eight UPEC strains, including two parental strains and six adapted clones, with different fluoroquinolone resistance levels originally isolated from two patients were used. The minimum inhibitory concentration (MIC) of 28 different antibiotics including levofloxacin was determined for each of the eight strains. In addition, the effects of mutations acquired with increased drug resistance in the levofloxacin-resistant strains on expression of genes implicated to be involved in drug resistance were examined. RESULTS: Of the eight UPEC strains used to test the MIC of 28 different antibiotics, two highly fluoroquinolone-resistant strains showed increased MIC in association with many of the antibiotics. As drug resistance increased, some genes acquired mutations, including the transcriptional regulator acrR and DNA-binding transcriptional repressor marR. Two strain groups with genetically different backgrounds (GUC9 and GFCS1) commonly acquired mutations in acrR and marR. Notably, acquired mutations related to efflux pump upregulation also contributed to increases in MIC for various antibiotics other than fluoroquinolone. CONCLUSIONS: The present results obtained using strains with artificially acquired drug resistance clarify the underlying mechanism of resistance to fluoroquinolones and other types of antibiotics.

Prediction of presence of fastidious bacteria by the Fully Automated Urine Particle Analyzer UF-1000i in the case of ineffective antimicrobial therapy for urinary tract infection.

INTRODUCTION: Recent studies have reported associations between fastidious bacteria that are difficult to grow and isolate in conventional urine culture conditions and urinary tract infections (UTIs). Because the Fully Automated Urine Particle Analyzer UF-1000i (hereinafter referred to as "UF-1000i") detects fastidious bacteria without being affected by culture conditions, owing to its flow cytometry-based principle, we evaluated the robustness of UF-1000i detection using clinical urine samples from patients with UTIs following ineffective antimicrobial therapy. METHODS: A total of 150 patients diagnosed with UTIs were enrolled, and their laboratory findings were analyzed, focusing on the discrepancy in bacterial numbers between UF-1000i and conventional culture at each antimicrobial therapy effectiveness classification. In addition, gene identification was conducted by molecular analysis using 16S ribosomal RNA gene sequencing and next-generation sequencing (NGS) to elucidate the reason for the presence of fastidious bacteria in these samples. RESULTS: The ineffective therapy cases showed more than 100-fold discrepancy in bacterial counts, with a higher proportion (30.8%) than effective therapy cases without secondary administration (5.7%) between the bacterial counts in UF-1000i and conventional culture methods. The presence rates of fastidious bacteria were 100% and 66.7% in discrepant cases of ineffective and effective without secondary administrations, respectively. CONCLUSION: This study suggests that discrepancies in bacterial numbers between the conventional culture method and UF-1000i measurement at the primary visit can predict the presence of fastidious bacteria, especially in cases of ineffective antimicrobial therapy.

Nationwide external quality assessment of SARS-CoV-2 nucleic acid amplification tests in Japan.

OBJECTIVES: We conducted a nationwide external quality assessment (EQA) study of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) nucleic acid amplification testing in Japan. METHODS: A total of 563 public health and private sector laboratories participated. The EQA samples comprised 6 RNA and full-process controls. RESULTS: The overall agreements were 99.3% and 97.9% for the RNA and full-process controls, respectively. A total of 530/563 (94.1%) laboratories reported correct results; public health laboratories had the highest accuracy. Thirty-three laboratories reported at least one incorrect result (26 laboratories of medical facilities, 5 commercial laboratories, 1 public health laboratory, and 1 other). Sixteen laboratories of medical facilities that used a fully automated assay system failed to detect the presence of the full-process control, due to inherent insufficiency in the limit of detection (LOD). Other causes of incorrect results included failure to ensure the LOD (n = 13), error in result judging or reporting (n = 3), and error in sample handling (n = 1). CONCLUSIONS: Performance was mostly dependent on the laboratory category and assay evaluation, particularly the LOD. Guidance should be developed based on these results, particularly in the phase of new entry into laboratory services for SARS-CoV-2.

Genomic Sequences of Uropathogenic Escherichia coli Strains with Various Fluoroquinolone Resistance Profiles.

The emergence of drug-resistant uropathogenic Escherichia coli (UPEC) has hampered antibiotic therapy for urinary tract infections. To elucidate the resistance mechanisms of UPEC, we performed whole-genome sequencing of eight UPEC strains with different fluoroquinolone resistance levels. Here, we report our sequencing data, providing a valuable resource for understanding such mechanisms.

Combination analysis of a whole lymph node by one-step nucleic acid amplification and histology for intraoperative detection of micrometastasis.

OBJECTIVE: The aim was to develop a more efficient molecular detection system than histological examination (HE) for lymph node (LN) metastasis. METHODS: Cytokeratin (CK) 19 mRNA copy numbers of 5 colon carcinoma cell lines (Lovo, DLD1, WiDr, Colo201 and Colo320) were calculated and compared by one-step nucleic acid amplification (OSNA) and conventional real-time reverse-transcription polymerase chain reaction (RT-PCR). Then, 91 LN submitted for HE from 6 patients with advanced colorectal adenocarcinoma and 64 LN submitted for frozen diagnosis from 47 patients with different malignancies were examined by OSNA and HE. RESULTS: CK19 mRNA copy numbers of all but Colo320 cells detected by OSNA were within double of those detected by RT-PCR. The least cell count of Lovo cells detected at one reaction (2 microl) by OSNA was calculated as 0.8 cells. Carcinoma metastasis showing either HE+ or OSNA+ was detected in 7.9% of the LN from advanced colorectal adenocarcinomas and in 30.0% of the LN for frozen diagnosis from different malignancies; HE-/OSNA+ metastasis was detected in 4.8 and 4.0%, respectively. OSNA analysis of 1 LN could be completed within 40 min. CONCLUSION: A combined analysis of LN by HE and OSNA could increase the sensitivity for detecting micrometastasis during surgery.

The use of flow cytometry as a diagnostic test for malaria parasites.

A total of 453 clinical blood samples were determined for malaria parasites by flow cytometric assay (FCM) and reagents from Sysmex Corporation, Japan. In this study, the FCM greatly simplified and accelerated parasite detection, with sensitivity of 91.26%, specificity 86.28% and accuracy 87.42%. Overall, the parasite counts by flow cytometric measurement correlated well with the parasitemia measured by microscopic assay (regression coefficient = 0.9409). The detection limit was 0.05-0.1% parasitemia. No evidence of malaria parasites in either blood donor volunteers or other disease patients groups was determined by FCM. However, 48 samples who had been treated with antimalarial drugs and whose parasite microscopic counts were negative, showed false-positive results. When the data of these 48 samples were analyzed, they were found to have high levels of reticulocytes, ranging from 2.0-18.9%. This finding suggested that a high reticulocyte concentration in the blood may interfere with the performance of the FCM. Further improvement, by eliminating this interference, will make the FCM one of the most promising tests for malaria diagnosis.