Multicenter prospective observational study of fungal keratitis in Japan: analyses of culture-positive cases.

PURPOSE: To investigate the clinical characteristics and causative fungi in patients with fungal keratitis in Japan, and to determine factors related to the prognosis. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eligible patients were enrolled from November 2011 to October 2013 at the 1st stage and from April 2015 to March 2016 at the 2nd stage. The corneal foci were scraped, and the scrapings were cultured in potato dextrose agar. The isolated fungi were identified by gene analyses. Data were collected from the clinical records and statistically analyzed by Cox and logistic regression analyses. RESULTS: Ninety-four fungal strains were isolated from 93 cases, including 42 yeast-like fungi and 52 filamentous fungi. The fungi affected the deep layers of the cornea in 23 cases (24.7%) and the peripheral cornea in 29 cases (31.2%). The incidences of lid swelling/redness, ciliary injection, anterior chamber cells/flare, anterior chamber fibrin, and hyphate ulcer in cases of filamentous fungi were significantly higher than in yeast-like fungi. No history of topical steroids, absence of a main lesion in the peripheral cornea, and best-corrected visual acuity (BCVA) of more than 0.04 at the first visit were related to a shorter healing time. No history of ocular surgery, absence of lesion at one-third deep stromal layer and BCVA of more than 0.04 at the first visit were correlated with BCVA at 3 months after the initial examination. CONCLUSION: Fungal keratitis is caused by various species of fungi and can become refractory due to poor prognosis factors.

Multicenter prospective observational study of fungal keratitis in Japan: analyses of in vitro susceptibility tests for combinations of drugs.

PURPOSE: To determine the effects of a combination of two antifungal drugs against causative fungi of fungal keratitis in Japan. STUDY DESIGN: Multicenter prospective observational study. METHODS: Eighteen isolates of yeast-like fungi and 22 isolates of filamentous fungi collected by the Multicenter Prospective Observational Study of Fungal Keratitis in Japan were studied. Specially manufactured minimum inhibitory concentration (MIC) measurement plates were used to test the effectiveness of 10 combinations of two antifungal drugs against the isolates. The combinations were pimaricin (PMR) + voriconazole (VRCZ), PMR + fluconazole (FLCZ), PMR + miconazole (MCZ), PMR + micafungin (MCFG), VRCZ + FLCZ, VRCZ + MCZ, VRCZ + MCFG, VRCZ + amphotericin-B (AMPH-B), MCZ + FLCZ, and MCZ + MCFG. The checkerboard microdilution method was used, and the fractional inhibitory concentration (FIC) index was calculated based on the guidelines of The Clinical & Laboratory Standards Institute (CLSI). RESULTS: In yeast-like fungi, additive effects were observed between PMR and MCFG in 77.8% of the isolates, and they were also observed between the azoles. Synergistic effects were observed on 11.1% of the isolates for MCZ and FLCZ. On the other hand, antagonistic effects were present between PMR and azoles with 88.9% between PMR and VRCZ, 72.2% between PMR and FLCZ, and 94.4% between PMR and MCZ. In filamentous fungi, additive effects were observed between PMR and MCFG in 40.9% of the isolates, and between VRCZ and MCZ in 40.9% of the isolates. Antagonistic effects were observed for PMR and the azoles. CONCLUSIONS: The combination of drugs prescribed for fungal keratitis incurs a possibility of synergistic, additive, indifferent, or antagonistic effects, depending on drug combinations and fungal strains.

Successful Conservative Treatment of Mycotic Pulmonary Artery Aneurysms Caused by MRSA Bacteremia.

Mycotic pulmonary artery aneurysms (MPAAs) are rare and life-threatening with currently no recommended treatment strategies. In this report, we describe a successfully treated case of ventricular septal defect in an 11-month-old girl who developed bacteremia, infective endocarditis, and MPAA caused by methicillin-resistant Staphylococcus aureus (MRSA). We first started vancomycin, gentamycin, and panipenem-betamipron for infective endocarditis but switched to teicoplanin and arbekacin on day 3 after initiating treatment because bacteremia persisted, and vancomycin minimum inhibitory concentration was relatively high at 2 mg/L. Although we added clindamycin on day 5 and fosfomycin on day 7, MRSA bacteremia persisted, and we finally added daptomycin at 10 mg/kg per day on day 8, whereupon the bacteremia subsided within a day. Although the bacteremia subsided, the patient developed septic pulmonary embolisms and septic arthritis on her left knee. We continued daptomycin but switched the concomitant drug to linezolid, trimethoprim-sulfamethoxazole, and rifampicin on day 11. After several repeats of puncture and lavage of her knee joint, she became afebrile on day 16. Computed tomography scans taken on day 32 revealed right pulmonary artery MPAAs. She was treated with long-term multidrug therapy, and MPAAs were absent on subsequent computed tomography scans on day 184. Multidrug therapy mainly based on daptomycin could be a possible salvage therapy for refractory MRSA bacteremia with high vancomycin minimum inhibitory concentration. Conservative treatment should be selectively considered as a treatment option for clinically stable MPAA instead of surgical and endovascular treatment.

Roussoella solani causing keratomycosis, with an observed both sexual and asexual morphs.

We describe an 82-year-old male farmer who had diabetes mellitus with no history of ocular trauma by soil or plants and who developed a corneal infection due to a fungus. The organism was identified as Roussoella solani based on both the morphological characteristics and phylogenetic analysis using LSU and ITS nrDNA sequences. The sexual stage of R. solani is described and illustrated for the first time. The patient was treated successfully with a combination of topical and systemic voriconazole and micafungin. This case is the first report of keratomycosis caused by R. solani.

[Multicenter Prospective Observational Study of Fungal Keratitis--Identification and Susceptibility Test of Fungi].

PURPOSE: To investigate the causative fungi of fungal keratitis in Japan and their drug susceptibility. METHODS: Identification and antifungal susceptibility test for 8 drugs (micafungin, amphotericin B, flucytosine, fluconazole, itraconazole, voriconazole, miconazole and pimaricin) were performed using isolated fungi from patients with fungal keratitis treated at 27 facilities in Japan between November 1, 2011 and October 31, 2013. RESULTS: Fungal strains were detected in 72 (50.7%) out of 142 samples. The major isolates were Fusarium spp. (18), Candida parapsilosis (12), C. albicans (11) and Alternaria spp. (6), in all, fungi of 31 species were identified by gene analysis. In the yeast-like fungi, susceptibility rates were evident for more than 80% in voriconazole, pimaricin, flucytosine, micafungin, amphotericin B and fluconazole. In filamentous fungi, the susceptibility rate was less than 50% except for PMR (90%). Fusarium spp., which were susceptible to amphotericin B and pimaricin, showed lower susceptibility rates compared with other genera. CONCLUSIONS: Although various genera and species of fungi cause fungal keratitis, the obtained drug susceptibility data in this study demonstrates the different susceptibility patterns among the major isolates (Fusarium spp., C. parapsilosis, C. albicans and other groups). This is important evidence useful for fungal keratitis treatment.

[Multicenter Prospective Observational Study of Fungal Keratitis--Current Status of Patients' Background, Clinical Findings, Treatment and Prognosis].

PURPOSE: To investigate the current status of fungal keratitis in Japan. METHODS: The patients with fungal keratitis were examined at 27 facilities in Japan from November 1st 2011 to October 31st 2013, concerning isolates, patient background, clinical findings, treatment and prognosis. RESULTS: Out of 139 cases, 133 were diagnosed as fungal keratitis, of which fungi were isolated from 72 samples of 71 cases (yeast-like fungi 32 strains and filamentous fungi 40 strains). The corrected visual acuity at the first visit of 88 cases (66.2%) was less than 20/200 and 42 cases (31.6%) were involved with deep stromal lesions, indicating high proportion of severe cases in this study. Three months later, 56 cases (42.1%) were still under treatment, and corrected visual acuity of 57 cases (42.9%) was less than 20/200. In cases with yeast-like fungi, there were significantly more cases with past history of corneal diseases, ocular surgery including keratoplasty, and eye drops' use such as steroids than those with filamentous fungi. On the other hand, there were significantly more cases of filamentous fungi, with trauma on the onset and with intervention of previously attending doctors than those with yeast-like fungi. Logistic regression analyses revealed that contact lens wearing was a significant factor of good prognosis, and yeast-like fungi as one of poor outcome compared with no fungal isolation. CONCLUSION: Although the choice of antifungal drugs has been increasing, fungal keratitis is still severe, refractory and vision-threatening disease.

Performance evaluation of four dominant anti-hepatitis B core antigen (HBcAb) kits in Japan for preventing de novo hepatitis B virus (HBV) infection.

BACKGROUND: The determination of antibody to hepatitis B core antigen (HBcAb) has become an important means of evaluating the risk factors of de novo hepatitis B virus (HBV) infection before starting intensive immunosuppressive drug therapies. Four dominant HBcAb determination reagents used in Japan were evaluated with HBcIgM, HBsAg, HBsAb, HBeAb, and HBV DNA reagents in order to study their clinical utility. METHODS: Four kinds of HBcAb reagent kits (HBcAb Total and HBcAb-IgG reagent) were evaluated with 526 clinical specimens, including 344 negative specimens, at Osaka University Hospital. The dynamic range of each kit was evaluated by testing serially diluted serum from pooled sera with high HBcAb concentration. RESULTS: The reagent that showed the largest dynamic range was the Lumipulse HBcAb-N (HBcAb-IgG reagent). Regarding clinical sensitivity and specificity, Centaur HBcAb (HBcAb Total reagent) gave several "doubtful negative" results and ARCHITECT HBcII (HBcAb Total reagent) had the most discrepant positive results. By comparing the cut-off-index distribution of negative specimens using a parameter of "distance from the mean to the cut-off divided by the SD", Centaur was determined to be the best (distance/SD = 12.65), with Lumipulse and Elecsys Anti-HBc (HBcAb Total reagent) in the second group (8.13 and 7.00, respectively), and ARCHITECT rated as the worst (3.25). CONCLUSIONS: In this evaluation, Elecsys and Lumipulse HBcAb kits showed good clinical sensitivity and specificity and were considered to be suitable for evaluating the risk factors of de novo HBV infection.

Fungal keratitis caused by Beauveria bassiana: drug and temperature sensitivity profiles: a case report.

BACKGROUND: Beauveria bassiana is an entomopathogenic fungus and is a rare cause of keratitis. We present a case of fungal keratitis caused by B. bassiana that was diagnosed by in vivo confocal microscopy and in vitro corneal cultures. In addition, we determined the temperature- and drug-sensitivities of the isolated strain of B. bassiana. CASE PRESENTATION: A 59-year-old Japanese man with a 2-month history of keratitis was examined by slit-lamp biomicroscopy, in vivo confocal microscopy, and histology and cultures of corneal scrapings. The corneal scrapings were used to determine the minimal inhibitory concentrations of different antifungal drugs and also to determine the temperature-sensitivity. In vivo confocal microscopy and histological examinations showed filamentous fungal keratitis. The characteristics of the fungal growth indicated that the keratitis was caused by B. bassiana. The keratitis responded poorly to systemic and topical voriconazole and to natamycin ointment. However, it was resolved after changing the natamycin to micafungin combined with surgical debridement. The isolated strain was sensitive to itraconazole, miconazole, micafungin, voriconazole, and resistant to flucytosine and fluconazole. It was moderately sensitive to amphotericin B, and natamycin. After 7 days in culture, the isolate grew small white colonies at 25 °C, very small colonies at 35 °C and 37 °C. CONCLUSION: The drug-sensitivity and temperature-sensitivity profiles of B. bassiana should be helpful in the treatment of B. bassiana keratitis. Therapeutic surgery may be helpful for mycotic keratitis poorly responsive to medical therapy alone.

In vitro evaluations of topical agents to treat Acanthamoeba keratitis.

PURPOSE: To evaluate the effectiveness of topical agents for the treatment of Acanthamoeba keratitis (AK). DESIGN: Laboratory research. PARTICIPANTS: Fifty-six Acanthamoeba isolates from 56 patients with clinically proven AK were studied. METHODS: The effectiveness of 7 agents against Acanthamoeba cysts was determined in vitro. The agents were 1.0% povidone-iodine, 0.05% benzalkonium chloride (BZC), 0.02% chlorhexidine gluconate (CHG), 0.1% propamidine isethionate, 0.02% polyhexamethylene biguanide (PHMB), 5.0% natamycin, and 1.0% voriconazole (VRCZ). These concentrations are those recommended for patients. In addition, 10-fold dilutions of each of the agents were tested. After exposing the cysts to each agent at 35°C for 1 hour or 24 hours, the agents were removed by centrifugal washing. The exposed cysts were observed by optical microscopy for 7 days. In addition, the fine structures of the exposed isolates were examined by transmission electron microscopy (TEM). The genotype of the isolates was determined by 18S rDNA fragment sequencing. MAIN OUTCOME MEASURES: The in vitro susceptibility was determined by complete growth inhibition, and the morphologic appearance was determined by TEM. The genotypes of the 56 isolates were determined by 18S rDNA fragment sequencing. RESULTS: The Acanthamoeba cysts were most susceptible to natamycin, followed by povidone-iodine, BZC, PHMB, propamidine, and CHG. None of the strains was susceptible to VRCZ. The susceptibilities to PHMB and CHG may be time dependent and to propamidine may be concentration dependent. Transmission electron microscopy showed changes in the inner structure of the cysts exposed to natamycin and povidone-iodine. The Acanthamoeba genotype was T4 in 52 isolates, and cysts with the same genotype had different agent susceptibilities. CONCLUSIONS: Natamycin and povidone-iodine had excellent cysti-static (or cystcidal) effects, and PHMB and propamidine did not. There was no correlation between agent effectiveness and Acanthamoeba genotype. Therefore, susceptibility tests of isolates are needed to choose the most appropriate agent, and our results can be a guideline for choosing the most appropriate agent for immediate empirical treatment of AK.

First case of fungal keratitis caused by Pestalotiopsis clavispora.

PURPOSE: To report the isolation of Pestalotiopsis clavispora from the cornea of a patient with recurrent keratitis. CASE REPORT: A 73-year-old male gardener presented with conjunctival injection and an oval infiltrate with feathery margins in the temporal half of the cornea in the right eye. His ocular history in the right eye included cataract surgery, five episodes of herpes simplex keratitis, three glaucoma surgeries, and bullous keratopathy. He had been treated with corticosteroids for years. Light microscopy of corneal scrapings revealed a filamentous fungus, and fungal keratitis was diagnosed. Treatment with topical voriconazole and pimaricin ointment was commenced. One month later, the infiltrate resolved. The antifungal agents were discontinued 7 months later, and keratitis relapsed 4 days after the discontinuation. The fungus was isolated and identified by molecular techniques as P. clavispora. Based on the results of antifungal susceptibility testing, treatment with topical and intravenous micafungin was initiated. The corneal infiltrate resolved 1 month after the relapse. CONCLUSION: Molecular identification of the pathogen, and antifungal susceptibility testing, are useful in treating patients with fungal keratitis caused by a rare human pathogen.

Reliable and reproducible method for rapid identification of Nocardia species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenged for the identification of Nocardia species. However, the standard ethanol-formic acid extraction alone is insufficient in allowing the membrane proteins of Nocardia species to be ionized by the matrix. We therefore aimed to establish our new extraction method for the MALDI-TOF MS-based identification of Nocardia species isolates. Our modified extraction procedure is through dissociation in 0.5% Tween-20 followed by bacterial heat-inactivation, mechanical breaking of the cell wall by acid-washed glass beads and protein extraction with formic acid and acetonitrile. As reference methods for species identification, full-length 16S rRNA gene sequencing and some phenotypical tests were used. In a first step, we made our own Nocardia database by analyzing 13 strains (13 different species including N. elegans, N. otitidiscaviarum, N. asiatica, N. abscessus, N. brasiliensis, N. thailandica, N. farcinica, N. nova, N. mikamii, N. cyriacigeorgica, N. asteroids, Nocardiopsis alba, and Micromonospora sp.) and registered to the MALDI BioTyper database. Then we established our database. The analysis of 12 challenge strains using the our database gave a 100% correct identification, including 8 strains identified to the species level and 4 strains to the genus level (N. elegans, N. nova, N. farcinica, Micromonospora sp.) according to the manufacture's log score specifications. In the estimation of reproducibility of our method intended for 4 strains, both within-run and between-run reproducibility were excellent. These data indicates that our method for rapid identification of Nocardia species is with reliability, reproducibility and cost effective.

[Antimicrobial susceptibilityof clinical isolates of aerobic gram-negative bacteria in 2008].

We determined MICs of antibacterial agents against 1145 clinical strains of aerobic Gram-negative bacteria (22 species) isolated at 16 Japanese facilities in 2008. MICs were determined using mostly broth microdilution method and antibacterial activity was assessed. Strains producing extended-spectrum beta-lactamases (ESBL) accounted for 3.8% of Escherichia coli, 2.6% of Klebsiella pneumoniae, 6.8% of Klebsiella oxytoca, 5.5% of Proteus mirabilis and 1.8% of Proteus vulgaris. ESBL produced strains were 6.8% at K. oxytoca that increased compared with 3.2% and 5.5% at P. mirabilis that decreased compared with 18.8% in 2006. Among Haemophilus influenzae, 61.7% that decreased compared with 67.7% in 2006, equaled 58.7% in 2004, were strains when classified by penicillin-binding protein 3 mutation. Against Pseudomonas aeruginosa, the activity of most antibacterial agents was similar to that in 2006. Although two antibacterial agents that tobramycin showed an MIC90 of 1 microg/mL and doripenem showed an MIC90 of 4 microg/mL against P. aeruginosa have potent activity. Of all P. aeruginosa strains, 4.3% were resistant to six agents of nine antipseudomonal agents, that decreased compared to 12.2% in 2004 and 5.7% in 2006. Against other glucose-non-fermentative Gram-negative rods, the activity of most antibacterial agents was similar to that in 2006.

[Antimicrobial susceptibility of clinical isolates of aerobic gram-positive cocci and anaerobic bacteria in 2008].

The activity of antibacterial agents against aerobic Gram-positive cocci (25 genus or species, 1029 strains) and anaerobic bacteria (21 genus or species, 187 strains) isolated from clinical specimens in 2008 at 16 clinical facilities in Japan were studied using either broth microdilution or agar dilution method. The ratio of methicillin-resistant strains among Staphylococcus aureus and Staphylococcus epidermidis was 59.6% and 81.2%, suggesting that resistant strains were isolated at high frequency. Vancomycin (VCM), linezolid (LZD) and quinupristin/dalfopristin (QPR/DPR) had good antibacterial activity against methicillin-resistant S. aureus and methicillin-resistant S. epidermidis, with MIC90s of < or = 2 microg/mL. The ratio of penicillin (PC) intermediate and resistant strains classified by mutations of PC-binding proteins among Streptococcus pneumoniae was 92.0% that was highest among our previous reports. Cefpirome, carbapenems, VCM, teicoplanin (TEIC), LZD and QPR/DPR had MIC90s of < or = 1 microg/mL against PC-intermediate and resistant S. pneumoniae strains. Against all strains of Enterococcus faecalis and Enterococcus faecium, the MICs of VCM and TEIC were under 2 microg/mL, and no resistant strain was detected, suggesting that these agents had excellent activities against these species. 15.9% of E. faecalis strains and 1.2% of E. faecium strains showed intermediate to LZD. 17.1% of E. faecium strains showed intermediate or resistant to QPR/DPR. Against all strains of Clostridium difficile, the MIC of VCM was under 1 microg/mL, suggesting that VCM had excellent activity. Carbapenems showed good activity against Clostridiales, Bacteroides spp., and Prevotella spp., but one strain of Bacteroides fragilis showed resistant to carbapenems. And so, the susceptibility of this species should be well-focused in the future at detecting continuously.

Correlation between the consumption of meropenem or doripenem and meropenem susceptibility of Pseudomonas aeruginosa in a university hospital in Japan.

The appropriate use of carbapenems is essential in order to prevent resistance in Pseudomonas aeruginosa. We investigated the correlation between the consumption of meropenem or doripenem and the susceptibility of P. aeruginosa to meropenem in a Japanese university hospital from 2004 to 2009. The susceptibility data of P. aeruginosa and the annual consumption of meropenem or doripenem were analyzed. The consumption of meropenem or doripenem was calculated using the Anatomic Therapeutic Chemical classification and defined daily doses methodology. Meropenem consumption decreased and doripenem consumption increased and throughout the entire investigation period, total consumption of meropenem plus doripenem was stable. Although the annual number of isolated P. aeruginosa has not changed, the annual number of isolated multidrug resistant P. aeruginosa decreased by measures against nosocomial infection. The rate of meropenem resistant P. aeruginosa decreased in a time-dependent manner. Meropenem consumption was positively correlated with the meropenem resistance rate among P. aeruginosa (r = 0.9455, p<0.01). The total consumption of meropenem and doripenem was not correlated with the meropenem resistance rate (r = -0.6601, p>0.1). These results suggested that even if the total consumption of meropenem plus doripenem was not changed, meropenem susceptibility among P. aeruginosa improved by the decrease of meropenem consumption. Although meropenem and doripenem have been suggested to show cross-resistance with each other, the reduction of meropenem consumption might be effective for preventing an increase of meropenem-resistant P. aeruginosa.

Evaluation of human immunodeficiency virus-1/2 antigen/antibody immunochromatographic assay.

BACKGROUND: In Japan, an immunochromatographic HIV-1/2 assay has been used for local Health Care Centers to prevent HIV spread in early stages and for hospitals at night or on holidays, where automated instruments are not available. In 2009, a new immunochromatographic HIV-1/2 assay became available which detects both antigen (Ag) and antibody (Ab) simultaneously and on separate bars. This study was conducted to evaluate the clinical performance of this new assay against three HIV-1/2 assays. METHODS: One immunochromatographic assay (ICA) for HIV Ab detection, one chemiluminescent enzyme immunoassay (CLEIA) for Ab detection and one ELISA for Ag/Ab combination assay were evaluated with the ICA for Ag/Ab detection. Serum samples from 955 patients were used at Osaka University Hospital, who had been tested initially for HIV infection status. The 900 were negative and 55 were positive. In addition, the samples in 10 commercially available panels [2 containing relatively rare HIV subtypes/genotypes and 8 containing seroconversion samples] were tested using all HIV assays. RESULTS: The HIV Ag/Ab ICA showed 100% (900/900, 95% confidence interval (95 CI) 99.59 - 100%) clinical specificity and was better than 99.8% (898/900, 95 CI 99.20 - 99.97%) of the existing ICA. The CLEIA and ELISA showed 100% (600/600, 95 CI 99.39 - 100%) and 99.8% (598/600, 95 CI 98.80 - 99.96%) specificity, respectively. The HIV Ag/Ab ICA showed 100% (55/55, 95 CI 93.51 - 100%) clinical sensitivity and detected all the relatively rare HIV subtypes/genotype panels; these results were the same as the other three assays. The HIV Ag/Ab ICA detected 5 seroconversion panels earlier than antibody-only-detection assays but detected later with 2 panels than ELISA for Ag/Ab combination assay. CONCLUSIONS: The HIV Ag/Ab ICA demonstrated good performances in clinical specificity and sensitivity as a rapid assay and is a suitable assay for qualitative judgement of HIV Ag and Ab simultaneously in human serum and plasma.

Clinical characteristics of keratitis due to Colletotrichum gloeosporioides.

PURPOSE: To determine the characteristics of the keratitis due to Colletotrichum gloeosporioides. METHODS: The medical records of 3 cases of fungal keratitis caused by C. gloeosporioides were reviewed to determine the clinical characteristics. The minimal inhibitory concentrations of different antifungal drugs for all 3 isolates were determined. All 3 isolates were grown on Sabouraud dextrose agar at 25°C, 35°C, and 37°C to determine the temperature-sensitive growth. RESULTS: All 3 patients lived in the southwestern part of Japan and had an ocular trauma involving organic materials. The infectious foci were localized in the anterior stroma, and they did not extend deep into the stroma in all cases. The keratitis was treated with antifungal medications including topical voriconazole and natamycin eye ointment, and was resolved in 2-3 weeks. All of the isolated strains grew well at 25°C but poorly at 35°C and 37°C. All isolated strains had similar drug-sensitivity profiles; they were sensitive to amphotericin B, itraconazole, miconazole, micafungin, and voriconazole, and relatively resistant to flucytosine, fluconazole, and natamycin. CONCLUSIONS: All 3 cases of C. gloeosporioides keratitis had similar clinical features. The similarities in the drug-sensitivity profiles should be helpful in treating C. gloeosporioides keratitis.

Evaluation of pathogen detection from clinical samples by real-time polymerase chain reaction using a sepsis pathogen DNA detection kit.

INTRODUCTION: Sepsis is a serious medical condition that requires rapidly administered, appropriate antibiotic treatment. Conventional methods take three or more days for final pathogen identification and antimicrobial susceptibility testing. We organized a prospective observational multicenter study in three study sites to evaluate the diagnostic accuracy and potential clinical utility of the SeptiFast system, a multiplex pathogen detection system used in the clinical setting to support early diagnosis of bloodstream infections. METHODS: A total of 212 patients, suspected of having systemic inflammatory response syndrome (SIRS) caused by bacterial or fungal infection, were enrolled in the study. From these patients, 407 blood samples were taken and blood culture analysis was performed to identify pathogens. Whole blood was also collected for DNA Detection Kit analysis immediately after its collection for blood culture. The results of the DNA Detection Kit, blood culture and other culture tests were compared. The chosen antimicrobial treatment in patients whose samples tested positive in the DNA Detection Kit and/or blood culture analysis was examined to evaluate the effect of concomitant antibiotic exposure on the results of these analyses. RESULTS: SeptiFast analysis gave a positive result for 55 samples, while 43 samples were positive in blood culture analysis. The DNA Detection Kit identified a pathogen in 11.3% (45/400) of the samples, compared to 8.0% (32/400) by blood culture analysis. Twenty-three pathogens were detected by SeptiFast only; conversely, this system missed five episodes of clinically significant bacteremia (Methicillin-resistant Staphylococcus aureus (MRSA), 2; Pseudomonas aeruginosa, 1; Klebsiella spp, 1; Enterococcus faecium, 1). The number of samples that tested positive was significantly increased by combining the result of the blood culture analysis with those of the DNA Detection Kit analysis (P = 0.01). Among antibiotic pre-treated patients (prevalence, 72%), SeptiFast analysis detected more bacteria/fungi, and was less influenced by antibiotic exposure, compared with blood culture analysis (P = 0.02). CONCLUSIONS: This rapid multiplex pathogen detection system complemented traditional culture-based methods and offered some added diagnostic value for the timely detection of causative pathogens, particularly in antibiotic pre-treated patients. Adequately designed intervention studies are needed to prove its clinical effectiveness in improving appropriate antibiotic selection and patient outcomes.

[The new method for detecting the outbreak sign of MRSA].

Conventional outbreak detection laboratory-based made one unit from the beginning of the month to the end of the month, totaled and analyzed, cannot correctly detect outbreaks continued during two months. The real-time analysis (RTA) we devised adapts to methicillin-resistant Staphylococcus aureus (MRSA) and avoids the problems of conventional detection. RTA analyzes all data for the last 30 days when MRSA is newly isolated 48 hours or more after hospital admission. In the three years from April 2006 to March 2009, we compared the day and number of MRSA outbreaks newly isolated 48 hours or more after hospital admission in 572 subjects using the conventional method and RTA. We also calculated the RTA infection prevention effect. The number of outbreaks detected conventionally numbered 68 cases and those detected by RTA numbered 106 cases. The number of outbreaks newly detected by RTA numbered 38 cases in three years, averaging 4.3 days earlier than conventional detection using conventional method A an average of 15.7 days earlier than conventionally which totals for every end of the month using conventional method B. The effect of infection prevention in the change of RTA from conventional method A presumably decreases MRSA infection to 14-18 persons and it in the change of RTA from conventional method B decreases MRSA infection to 18-25 persons in one year. These results suggested that outbreak detection by RTA could help prevent MRSA outbreak and decrease MRSA infection frequency.