Evaluation of agar culture plates to efficiently identify small colony variants of methicillin-resistant Staphylococcus aureus.

Background: Small-colony variants of methicillin-resistant Staphylococcus aureus (SCV-MRSA) recently were described as slow-growing, thymidine-dependent strains; typically, SCV-MRSA were isolated from patients receiving sulfamethoxazole-trimethoprim, but detection of these strains frequently was delayed because of their small colony size and slow growth. Bacteremia cases due to SCV-MRSA sometimes become lethal when the initiation of treatment with intravenous anti-methicillin-resistant Staphylococcus aureus (MRSA) drugs starts too late. Methods: Here, we evaluated the use of general MRSA-specific agar plates in Japan, including MS-CFX, X-MRSA, and CHROMagar, for the efficient detection of SCV-MRSA, including the comparative detection efficiencies of these media for stock strains and clinical isolates. Results: Among the three MRSA-specific agar plates that were tested, X-MRSA and CHROMagar showed similar detection efficiencies for both 24 and 48 hrs culturing; in contrast, MS-CFS did not permit the detection of SCV-MRSA in stock strains. For clinical isolates of SCV-MRSA, X-MRSA plates permitted detection of the smallest and slowest-growing colonies of SCV-MRSA at 48 hrs of culturing; in contrast, CHROMagar and MS-CFX sometimes did not identify SCV-MRSA at 24 and 48 hrs. Conclusion: Optimization of media and incubation times will be necessary for efficient identification for SCV-MRSA, which would prevent delays in diagnosis and treatment with anti-MRSA drugs.

Evaluation of blood culture broths with lysis buffer to directly identify specific pathogens by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry methods.

BACKGROUND: Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is now widely used to detect pathogens in clinical settings in Japan. METHOD: Here, we report the effects of adding lysis buffer in the MALDI-TOF MS method to directly detect bacteria from 3 blood culture systems and compare their detection efficiencies for each pathogen. RESULTS: Blood culture broths from BD, bioMérieux, and Oxoid showed similar detection efficiencies without lysis buffer use and Escherichia coli, Klebsiella pneumoniae, and Pseudomonas aeruginosa were efficiently detected in all broths when lysis buffer was used. However, Streptococcus pneumoniae was not detected in BD broth when lysis buffer was added. Furthermore, Haemophilus influenzae and Bacteroides fragilis were not detected in all 3 systems when lysis buffer was used. CONCLUSION: Optimization of blood culture system and lysis buffer is necessary according to each pathogen for direct identification by MALDI-TOF MS methods.

Usefulness of matrix-assisted laser desorption ionization time-of-flight mass spectrometry to identify pathogens, including polymicrobial samples, directly from blood culture broths.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (TOF-MS) is now widely used to detect pathogens in clinical settings in Japan. Here, we report the ability of TOF-MS to detect bacteria from blood culture (BC) broths, and compare the efficacy of TOF-MS to that of conventional culture methods. Bacteria were correctly detected from 63 monomicrobial samples within 80 minutes; results matched those obtained by conventional BC methods, although the conventional methods took 2-3 days. In addition to the 63 monomicrobial samples, another three polymicrobial samples were tested; notably, the infecting bacteria were not correctly identified in two of these three samples. To better assess the TOF-MS detection of polymicrobial samples, we tested various ratios of mixed broth samples, including combinations of the bacteria that we were unable to detect in clinical samples. Combinations of Enterobacter cloacae and Pseudomonas aeruginosa were correctly detected at a culture ratio of 2:1, but not in the 3:1 mixture. These results suggested that TOF-MS is a strong tool for the rapid and correct detection of pathogens from monomicrobial BC samples, though results need to be carefully checked when handling known or suspected polymicrobial samples.

Ability of procalcitonin to diagnose bacterial infection and bacteria types compared with blood culture findings.

Procalcitonin (PCT) and C-reactive protein serve as biomarkers of infection in patients with sepsis/bacteremia. The present study assessed the clinical characteristics of 280 patients with suspected sepsis who were admitted to Tohoku Medical and Pharmaceutical University Hospital between January 2012 and December 2013. Among the patients, 133 and 147 were positive and negative for PCT, respectively. Patients who were PCT positive were older and more frequently male, had reduced levels of platelets and albumin, and increased levels of aspartate aminotransferase, alanine aminotransferase, blood urea nitrogen, creatinine, and C-reactive protein. Patients who were PCT positive had significantly higher blood culture positivity compared with those who were PCT negative, and the sensitivity and specificity of PCT for detecting positive blood cultures were 74.5% and 59.1%, respectively. Escherichia coli was detected in PCT-positive patients, whereas Staphylococcus epidermidis and Staphylococcus lugdunensis were frequently detected in PCT-negative patients. Levels of PCT were higher in the patients infected with gram-negative rods than those with gram-positive cocci. Furthermore, extended-spectrum ß-lactamase (ESBL)-producing bacteria cases showed higher levels of PCT than those of non-ESBL cases. These results suggest that PCT may be a useful biomarker of sepsis, and it might serve as a strong tool to detect patients with severe gram-negative rod bacteremia including ESBL-producing bacteria cases early due to its relative high sensitivity.

Association of influenza with severe pneumonia/empyema in the community, hospital, and healthcare-associated setting.

We presented three cases of influenza-related severe pneumonia/empyema that occurred in one season. CASE 1: A 76-year-old diabetic man, developed empyema as a result of severe community-acquired pneumonia (CAP) secondary to Haemophilus influenzae, as confirmed on sputum culture. Nasal swab was positive for influenza A antigen. After drainage of empyema, intravenous peramivir and piperacillin/tazobactam were administered for 3 days and 2 weeks, respectively, followed by oral levofloxacin for 2 weeks. Eventually, he recovered. In this case, the isolated H. influenzae was non-typeable and negative for beta-lactamase. CASE 2: A 55-year-old man with suspected cerebral infarction and diabetes mellitus (DM) developed severe pneumonia/empyema as result of hospital-acquired pneumonia (HAP). Although influenza A antigen was detected, no bacterium was isolated from the sputum, blood, or pleural effusion. He showed severe hypoxia, but recovered after administration of peramivir and levofloxacin with prednisolone for 5 days and 2 weeks, respectively. CASE 3: A 76-year-old woman with heart failure and DM was followed-up on an outpatient basis and was under nursing home care for four months. Subsequently, she developed pneumonia and was admitted to our hospital; influenza antigen was isolated from nasal swab. Healthcare-associated pneumonia (HCAP)/empyema were diagnosed and were effectively treated with peramivir and levofloxacin for 4 days and 1 week, respectively. In diabetic patients, influenza virus may possibly accelerate pneumonia/empyema due to bacterial coinfection. Although non-typeable H. influenzae is a rare causative pathogen of empyema, it can be expected as a result of "pathogen shift" due to the increased use of the H. influenzae type b vaccine in Japan.