MALDI-TOF MS identification of Exophiala species isolated in Japan: Library enrichment and faster sample preparation.

Exophiala species cause chromoblastomycosis, mycetoma, and phaeohyphomycosis, which are occasionally fatally in immunocompromised patients. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides rapid and accurate examination of isolated bacteria and some fungal isolates, but the preparation method for filamentous fungi is complicated. In this study, 31 clinical isolates of Exophiala spp. in Japan were identified by MALDI-TOF MS with a library enriched by adding data. To simplify the sample preparation method, two modified methods were compared with the standard method for filamentous fungi. The agar cultivation sample preparation method reduced the time required for liquid culture and was considered suitable for clinical use. In 30 of 31 clinical isolates of Exophiala spp., the species identified by MALDI-TOF MS with the highest score matched the species identified by sequencing the internal transcribed spacer region. Exophiala dermatitidis, E. lecanii-corni, and E. oligosperma were identified above the genus level, while E. jeanselmei and E. xenobiotica were often not identified at the species level. The identification scores tended to be lower for less-registered strains in the in-house library. It is suggested that library enrichment and the modified preparation method may facilitate early diagnosis of rare fungal infections by Exophiala spp. in clinical laboratories using MALDI-TOF MS.

A case of severe pneumonia caused by Legionella longbeachae with positive results by a Legionella urinary antigen detection kit.

Legionella longbeachae is a Legionella bacteria often detected in soil, and is known as a rare cause of Legionella infections in Japan. In addition, detection of this Legionella species is often overlooked due to negative results from Legionella urinary antigen tests, which could lead to errors in the therapeutic approach. An 80-year-old woman was admitted to our hospital because of fever and dyspnea. Her blood tests showed elevated white blood cells, increased C-reactive protein and transaminases, and hyponatremia. Chest computed tomography showed dense consolidation in the right lung. We diagnosed Legionella pneumonia because the Legionella urinary antigen test was positive on the day after her admission. The patient was intubated and mechanically ventilated on the third day of hospitalization, because of respiratory failure. However, her condition did not improve and she died on the 10th day after admission. After her death, L. longbeachae was detected from sputum culture from her tracheal tube, and was diagnosed as the causative organism of her pneumonia. L. longbeachae infection reportedly rarely produces positive urinary antigen test results. Our experience suggests that the urinary antigen test using Ribotest Legionella might be able to detect Legionella spp. other than L. pneumophila.

First reported isolation of hemin-requiring Proteus vulgaris small-colony variant from urine culture.

A hemin-requiring Proteus vulgaris small-colony variant (SCV) was isolated from a urine culture. This isolate was grown on 5% sheep blood agar but not on modified Drigalski agar. The single nucleotide substitution was found in the SCV of the hemC gene (c.55C > T), and this substitution caused a nonsense mutation (p.Gln19Ter). Porphyrin test results showed that the biosynthesis of δ-aminolevulinic acid stopped up to porphobilinogen and not pre-uroporphyrinogen due to a mutation in the hemC gene. To our knowledge, this is the first report of hemin-requiring P. vulgaris.

[Evaluation of Methicillin Resistance Determination Time for MRSA Using Fully Automated Rapid Identification Susceptibility testing system RAISAS S4].

In this study, we examined the accuracy and rapidity of drug susceptibility determination using clinical isolates of MRSA with the fully automated rapid identification susceptibility testing system RAISUS S4. Ninety eight MRSA strains were used and the time until methicillin resistance was determined was analyzed by both of the standard method (18-hr method) and the rapid method. Five strains (5.1%) were determined to be methicillin-sensitive in MPIPC by rapid method only while all strains were determined to be resistant in CFX. The average methicillin resistance determination time was 7.0/5.0 hr for MPIPC, 6.3/5.0 hr for CFX, and 6.3/5.0 hr for the combination of MPIPC and CFX by the standard/rapid method, with the rapid method being significantly shorter (Wilcoxon's signed rank sum test, p<0.01). Strains determined to be methicillin-sensitive by MPIPC tended to have a longer time to methicillin resistance by the standard method, but this effect was much less pronounced for the rapid method using CFX. Methicillin resistance determination by the rapid method using RAISUS S4 enables rapid detection of MRSA without false-susceptible results, which may lead to early and appropriate treatment.

Development of a novel chromogenic method, Penta-well test, for rapid prediction of ß-lactamase classes produced in clinical Enterobacteriaceae isolates.

We developed a novel chromogenic method, Penta-well test, which enables the rapid detection and classification of ß-lactamases in clinical Enterobacteriaceae isolates. This test is based on a combination of nitrocefin and 3 ß-lactamase inhibitors specific to classes A, B, and/or C, with nitrocefin hydrolysis by ß-lactamases being assessed by optical density measurements at 490 nm. When the cutoff value for each ß-lactamase class was determined (0.09, 0.4, and 0.55 for class A, class B, and class C ß-lactamase producers, respectively), the sensitivity and specificity of classification were 93.5% and 68.8% for class A, 93.8% and 100% for class B, and 86.7% and 100% for class C, respectively. Moreover, this method allowed accurate ß-lactamase classification in 20 of 23 (87.0%) isolates producing plural class of ß-lactamases. Thus, the Penta-well test can provide information that would be useful in the accurate detection and classification of ß-lactamases produced by causative bacteria.