A case of infective endocarditis caused by "Neisseria skkuensis".

"Neisseria skkuensis" is a gram-negative coccus that is endemic in the human oral cavity, with only few reports of infection in humans. Herein, we report a case of a male patient in his sixties presenting with infective endocarditis (IE) caused by "N. skkuensis". To our knowledge, this is the second case of IE confirmed using 16S rRNA gene to have been caused by "N. skkuensis". The accurate diagnosis of rare or difficult-to-identify pathogens is a major challenge for clinical microbiological laboratories. Although Neisseria spp. are common in the oral cavity and are often seen in routine tests, identification of their biochemical properties and mass spectrometric analysis are difficult. In this case report, we describe the accurate identification of "N. skkuensis" by 16S rRNA gene sequencing analysis compared to other identification methods. Further cases of "N. skkuensis" are needed to fully evaluate the clinical approach of this detection method.

Establishing an internal quality control method for the stable extraction of nucleic acids of severe acute respiratory syndrome coronavirus 2 and RT-PCR-based detection.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19), is detected using real-time RT-PCR. However, there are limitations pertaining to quality control, particularly with respect to establishing quality control measures for extraction of viral nucleic acids. Here, we investigated the quality control measures for the various processes using an extrinsic quality control substance and quality control charts. METHODS: An extrinsic quality control substance was added to the sample, and then, real-time RT-PCR was performed. Samples with negative test results and the corresponding data were analyzed; a quality control chart was created and examined. RESULTS: Data analysis and the quality control charts indicated that SARS-CoV-2 could be reliably detected using real-time RT-PCR, even when different nucleic acid extraction methods were used or when different technicians were employed. CONCLUSION: With the use of quality control substances, it is possible to achieve quality control throughout the process-from nucleic acid extraction to nucleic acid detection-even upon using varying extraction methods. Further, generating quality control charts would guarantee the stable detection of SARS-CoV-2.

A case of pediatric patient with acute enteritis due to CTX-M-1 5 extended-spectrum ß-lactamase-producing Salmonella Blockley.

This clinical case report concerns a pediatric patient with acute enteritis caused by multi-drug resistant Salmonella enterica serovar Blockley (Salmonella Blockley). A 3-year-old boy presented to our emergency room with a 5-day history of fever, abdominal pain, and bloody diarrhea. Stool culture tested positive for a Salmonella species, while the blood culture was negative. The patient was successfully treated with an oral antibiotic regimen of fosfomycin. The stool isolate was found to be resistant to multiple drugs, including cefpodoxime, cefotaxime, ceftazidime, and aztreonam, and was confirmed to be a CTX-M-15 extended-spectrum ß-lactamase (ESBL)-producing strain of Salmonella Blockley. This is the first report of a pediatric patient in Japan with acute enteritis caused by a CTX-M-15 ESBL- producing strain of Salmonella Blockley.

Breakthrough invasive Candida glabrata in patients on micafungin: a novel FKS gene conversion correlated with sequential elevation of MIC.

Candida glabrata strains sequentially isolated from blood developed resistance to micafungin (MICs from <0.015 to 4 µg/ml). A novel mutation identified in micafungin-resistant strains at bp 262 of FKS2 (containing a deletion of F659 [F659del]) was inserted into the homologous region in FKS1.

A psoas abscess caused by Propionibacterium propionicum.

Pelvic actinomycosis-like disease due to Propionibacterium propionicum has been very rare and only a few cases have been reported in the literature. We herein report a probable first case of a psoas abscess caused by P. propionicum. Since P. propionicum is indistinguishable from Actinomyces israelii by morphological features or routine biochemical tests, 16S rRNA gene sequencing was useful to discriminate these two species in this case.

A case of multiple abscesses caused by Nocardia farcinica rapidly diagnosed by MALDI-TOF MS.

Identifying Nocardia species in routine tests is often difficult owing to its culture time. We report a case of nocardiosis characterized by multiple abscesses of disseminated disease. A man in his 50 's presented with inflammation on the left buttock and right lower leg. Nocardia farcinica was isolated from the abscess on his buttock and confirmed using matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS). Radiological assessment revealed multiple abscesses in his brain, left lung, and right thigh. He was successfully treated with antibiotics. Accurate species identification by MALDI-TOF MS aids in the optimal treatment of nocardiosis.

Background of patients with Aerococcus spp. detected in blood cultures, and drug sensitivity of Aerococcus spp.

This study aimed to review the clinical characteristics of patients with Aerococcus spp. detected by blood culture, and drug susceptibility of Aerococcus spp. All cases of Aerococcus spp. determined using blood culture between June 2013 and May 2020 in a single institution were included; patient information (age, sex, comorbidities, outcome, diagnosis, antimicrobial agents) was analyzed. The cohort comprised 25 patients (18 [72%] men and 7 [28%] women; median age, 84.5 [range, 75-87] years). Thirteen (52%) patients had urinary tract infections(UTI) caused by Aerococcus spp. All patients had a favorable prognosis, except 1 who died owing to infective endocarditis. Drug susceptibility testing showed that most isolates were susceptible to ß-lactams except 1. However, 24 (96%) cases were resistant to trimethoprim-sulfamethoxazole and 10 (40%) to quinolones. Aerococcus spp. are important causative agents of bacteremia and UTI. The increasing reports of Aerococcus spp. infections could lead to better treatment schemes and facilitate diagnosis.

Clinical Surveillance of Candidemia at Our Hospital.

Treatment of Candidemia has become increasingly complicated as more and more non-albicans Candida species are being isolated in recent years.We launched an investigation of the species, the MIC value, and the state of administration of antifungal drugs for all the cases with Candida spp. confirmed by blood cultures for the 7-year period from 2012 to 2018 at our hospital. In total, 192 cases were found and 206 strains of Candida species were isolated. Overall, 49.5% of the 206 isolated strains were Candida albicans (102 strains), followed by Candida glabrata (40 strains, 19.4%), and Candida parapsilosis (38 strains, 18.4%). The most frequently used antifungal drug for the initial dose was MCFG (120 cases, 59.2%), while the most frequently switched antifungal agent was L-AMB. Cases with an inappropriate end-of-treatment time represented 58.7% of all the cases.We investigated the Candidemia situation at our hospital for a period of seven years. We believe that it is important for medical institutions to gather detailed data on candidemia at their own hospitals. Likewise, the hospital's Infection Control Team/Antimicrobial Stewardship Team should inform the physicians-in-charge about the appropriate diagnosis and treatment based on the data obtained.

The significance of bacterial engulfment in Gram-stained sputum in patients with respiratory infections.

In general, physicians believe that the presence of bacterial engulfment in white blood cells (WBCs) on Gram-stained sputum is a hallmark of lower respiratory infection. However, no studies have described the significance or diagnostic accuracy of engulfment in lower respiratory tract infections.We prospectively studied sputum samples by Gram staining (Favor method) for their quality and engulfment score in WBCs obtained from patients with respiratory symptoms at inpatient and outpatient settings at Kyorin University Hospital between December 2012 and April 2015.A total of 163 patients were enrolled. The patients were classified into an infection (n = 93) or non-infection (n = 70) group based on clinical or radiological findings prior to the evaluation of sputum samples. The proportion of engulfment-positive cases was equal in the infection and non-infection groups (49.5% vs 35.7%, P = 0.11). In the infection group, the engulfment score (%) for Streptococcus pneumoniae was significantly lower (median 3%, interquartile range [IQR]: 2% to 5%, P = 0.005) than that of the non-S. pneumoniae bacteria (H. influenzae, M. catarrhalis, and methicillin-susceptible Staphylococcus aureus (MSSA))(median 22.5%, IQR: 17% to 35.5%). The engulfment score of S. pneumoniae in the WBC was low in the infection group, and no cases were recognized in the non-infection group. Using a cut-off value of 3%, the diagnostic accuracy for infection was as follows: sensitivity: 50%, specificity: 65.7%, and area under the curve (AUC): 0.579 (95% CI 0.464 to 0.694). For the non-S. pneumoniae bacteria (H. influenzae, M. catarrhalis, and MSSA), the engulfment score was significantly higher in the infection group (median 22.5%, IQR 17 to 35.5%) than in the non-infection group (median 6.0%, IQR: 3 to 13%, P = 0.011), and the diagnostic accuracy for infection was as follows: sensitivity: 75%, specificity: 85.7%, and AUC: 0.902 (95% CI 0.75 to 1.00) when the threshold for the engulfment score was defined as 18%.This study provides the first evidence that the engulfment of bacteria in WBCs is not always indicative of infection and that the engulfment score can fluctuate according to the pathogen.

Direct identification of microorganisms from positive blood cultures by MALDI-TOF MS using an in-house saponin method.

OBJECTIVES: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a fast and reliable method for the identification of bacteria. A MALDI Sepsityper kit is generally used to prepare samples obtained directly from culture bottles. However, the relatively high cost of this kit is a major obstacle to introducing this method into routine clinical use. In this study, the accuracies of three different preparation methods for rapid direct identification of bacteria from positive blood culture bottles by MALDI-TOF MS analysis were compared. METHODS: In total, 195 positive bottles were included in this study. RESULTS: Overall, 78.5%, 68.7%, and 76.4% of bacteria were correctly identified to the genus level (score ≥1.7) directly from positive blood cultures using the Sepsityper, centrifugation, and saponin methods, respectively. The identification rates using the Sepsityper and saponin methods were significantly higher than that using the centrifugation method (Sepsityper vs. centrifugation, p<0.001; saponin vs. centrifugation, p=0.003). CONCLUSIONS: These results suggest that the saponin method is superior to the centrifugation method and comparable to the Sepsityper method in the accuracy of rapid bacterial identification directly from blood culture bottles, and could be a less expensive alternative to the Sepsityper method.

Methicillin-resistant Staphylococcus aureus enterocolitis sequentially complicated with septic arthritis: a case report and review of the literature.

BACKGROUND: Although most reports describing patients infected with methicillin-resistant Staphylococcus aureus enterocolitis have been published in Japan, this concept remains a matter of debate and diagnostic criteria have not yet been defined. CASE PRESENTATION: The general status of a 74-year-old Japanese man referred to our hospital (day 1) with severe community-acquired pneumococcal pneumonia gradually improved with antibiotic therapy. Thereafter, up to 4 L/day of acute watery diarrhea that started on day 19 was refractory to metronidazole but responded immediately to oral vancomycin. Gram staining stool samples was positive for abundant fecal leukocytes from which dominant methicillin-resistant Staphylococcus aureus (10(4) CFU/mL) were isolated, suggesting methicillin-resistant Staphylococcus aureus enterocolitis. High fever with methicillin-resistant Staphylococcus aureus bacteremia was evident at day 30, and suppurative right hip arthritis developed around day 71. All methicillin-resistant Staphylococcus aureus strains isolated from stools, blood and aspirated synovial fluid separated in the same manner on pulsed-field gel electrophoresis, as well as two other strains isolated from sputum, belonged to the same clone as sequence type (ST) 764 (complex clonal 5), and carried SCCmec type II. CONCLUSION: The clinical, microbiological and molecular biological findings of this patient indicated methicillin-resistant Staphylococcus aureus enterocolitis that led to septic methicillin-resistant Staphylococcus aureus arthritis.

Whole-Genome Sequence of Mycobacterium kyorinense.

We report here the first draft genome sequence of Mycobacterium kyorinense, which was described in 2009 and exhibits significant pathogenicity to humans.

Direct inoculation method using BacT/ALERT 3D and BD Phoenix System allows rapid and accurate identification and susceptibility testing for both Gram-positive cocci and Gram-negative rods in aerobic blood cultures.

This study describes a direct inoculation method using the automated BacT/ALERT 3D and the BD Phoenix System in combination for identification and susceptibility testing of isolates from positive blood cultures. Organism identification and susceptibility results were compared with the conventional method for 211 positive aerobic blood cultures. Of 110 Gram-positive cocci (GPCs), 98 (89.1%) isolates were correctly identified to the species level. Of 101 Gram-negative rods (GNRs), 98 (97.0%) isolates were correctly identified to the species level. The overall categorical agreement in antimicrobial susceptibility testing among the 110 GPCs was 92.7%, with 0.04% very major and 0.7% major error rates. The overall categorical agreement among 78 isolates of enterobacteria and 23 isolates of nonfermenters in GNRs was 99.5% and 91.1%, respectively, with no major errors identified. We conclude that, compared with previously reported direct inoculation methods, our method is superior in identification and susceptibility testing of GPCs.

High-resolution CT findings of patients with pulmonary nocardiosis.

BACKGROUND: Opportunistic pulmonary infection with Nocardia species is rare in humans, and only a few studies have radiologically analyzed patients with pulmonary nocardiosis using high-resolution computed tomography (HRCT). METHODS: We retrospectively reviewed the medical records of patients with pulmonary nocardiosis at our hospital between April 2006 and December 2011 to assess HRCT and clinical findings. We also searched the medical literature for pulmonary nocardiosis reported in Japan between 2002 and 2011 for comparison. RESULTS: We identified seven patients at our institution and 33 reported infections in Japan. Four of our patients were immunocompetent, whereas the other three had impaired cellular immunity due to type 2 diabetes mellitus or having been inappropriately treated with steroid. Thoracic HRCT revealed no zonal predominance, but tropism for distribution from the middle to the peripheral area, and radiological findings of nodules, cavitation, mass, consolidations, bronchial wall thickening, septal line thickening and ground glass opacity (GGO) were evident. The main HRCT finding in our study comprised nodules (n=5, 71.4%) <30 mm and four patients had multiple nodules as described in other reports. Furthermore, we discovered a crazy paving appearance (CPA) around nodules, cavities, masses or consolidations in five patients (71.4%). CONCLUSIONS: Multiple nodules distributed from the middle to the peripheral area on HRCT might reflect pulmonary nocardiosis, and CPA seemed to be a worth paying attention to the diagnosis.

Mycobacterium kyorinense sp. nov., a novel, slow-growing species, related to Mycobacterium celatum, isolated from human clinical specimens.

A novel, non-pigmented, slow-growing mycobacterium was identified on the basis of biochemical and nucleic acid analyses, as well as growth characteristics. Three isolates were cultured from clinical samples (two from sputum and one from pus in lymph nodes) obtained from three immunocompetent patients with infections. Bacterial growth occurred at 28-42 degrees C on Middlebrook 7H11-OADC agar. The isolates showed negative results for Tween hydrolysis, nitrate reductase, semiquantitative catalase, urease activity, 3 day arylsulfatase activity, pyrazinamidase, tellurite reduction and niacin accumulation tests, but positive results for 14 day arylsulfatase activity and heat-stable catalase tests. The isolates contained alpha-, keto-, and dicarboxymycolates in their cell walls. Sequence analysis revealed that all isolates had identical, unique 16S rRNA sequences. Phylogenetic analysis of the 16S rRNA, rpoB, hsp65 and sodA gene sequences confirmed that these isolates are unique but closely related to Mycobacterium celatum. DNA-DNA hybridization of the isolates demonstrated less than 50 % reassociation with M. celatum and Mycobacterium branderi. On the basis of these findings, a novel species designated Mycobacterium kyorinense sp. nov. is proposed. The type strain is KUM 060204(T) (=JCM 15038(T)=DSM 45166(T)).