Seishi Asari

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

IntroductionSepsis 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.MethodsA 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.ResultsSeptiFast 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).ConclusionsThis 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.

Acanthamoeba Keratitis With Symbiosis of Hartmannella Ameba

PURPOSE To report a case of severe amebic keratitis in which both Hartmannella and Acanthamoeba were isolated simultaneously from the same lesion. METHOD Case report. The deep corneal lesion was scraped for cytopathology and isolation of the pathogens. We tested the in vitro sensitivities of the pathogens to several drugs. RESULTS Cultures of the corneal scrapings and of the solution in the patients contact lens storage case were positive for Acanthamoeba E9 cysts and trophozoites. Hartmannella ameba coexisted with Acanthamoeba in the cornea. When tested in vitro, Acanthamoeba trophozoites were sensitive to both miconazole nitrate and natamycin, while cysts were sensitive only to natamycin. However, the patient did not respond to these antiamebic drugs. CONCLUSIONS This case suggests that Acanthamoeba is not the only origin of amebic keratitis. Hartmannella may also cause severe drug-resistant keratitis.

Effects of various isolation methods for human peripheral lymphocytes on T cell subsets determined in a fluorescence activated cell sorter (FACS), and demonstration of a sex difference of suppressor/cytotoxic T cells

The effects of various methods for removal of monocytes and residual erythrocytes from human peripheral blood mononuclear cells (PBMC) on T cell subsets were examined. T cell subsets were determined in a fluorescence activated cell sorter (FACS) using fluorescein-conjugated monoclonal antibodies (anti-Leu-1, anti-Leu-2a and anti-Leu-3a antibodies). Removal of monocytes by methods based on adherence or phagocytosis decreased yields of lymphocytes and caused changes in the percentage and/or the fluorescence intensity of T cell subsets. Exclusion of monocytes from the FACS analysis by setting of the scatter gates was incomplete (about 80%). Removal of residual erythrocytes after Ficoll separation by ammonium chloride treatment also changed the percentage of T cell subsets. A method using PBMC without removal of monocytes and erythrocytes was chosen as best and simplest for routine use in FACS analysis of lymphocytes. Erythrocytes could be excluded from the FACS analysis by setting the scatter gates and the percentage of T cell subsets was corrected after measurement of monocytes, identified by peroxidase staining. The reproducibility of measurements of T cell subsets made at different times was examined using PBMC obtained from the same healthy man during 12 weeks. For standardization of the assay, the peak positions of scatter and fluorescence intensity of each PBMC labeled with anti-Leu-3a were adjusted to standard values in each FACS analysis. Under these conditions, variations of other parameters of this standard PBMC were very small in 12 different assays. Using this standard PBMC, satisfactory, reproducible results were also observed on PBMC obtained from another normal subject. Therefore, this standard PBMC labeled with anti-Leu-3a was used as a standard in FACS analysis. Under these accurately standardized conditions, it was demonstrated that the peak position of fluorescence intensity of Leu-2a (suppressor/cytotoxic T) cells was significantly lower (P less than 0.01) in women than in men.

In vitro antifungal combination effects of micafungin with fluconazole, voriconazole, amphotericin B, and flucytosine against clinical isolates of Candida species

Micafungin (MCFG) is an echinocandin antifungal agent that exhibits potent activity against most species of Candida and Aspergillus. We investigated the in vitro antifungal combination effects of MCFG with four other antifungal agents — fluconazole (FLCZ), voriconazole (VRCZ), amphotericin B, and flucytosine — against clinical isolates of 54 Candida spp. by checkerboard analysis. The synergistic antifungal effects of MCFG-FLCZ and MCFG-VRCZ were 11% and 15%, respectively, and the latter displayed a synergistic activity of 63% against Candida glabrata. Antagonism was not observed in any of the combinations tested.

Characteristics of Pseudomonas corneal infection related to orthokeratology.

Purpose: To describe a Pseudomonas aeruginosa corneal infection resulting from orthokeratology. Methods: Case report. Results: A 17-year-old boy wearing orthokeratology (OK) lenses was referred to our clinic because of redness in his right eye in spite of his usage of ofloxacin (OFLX) eye drops. An excavated paracentral corneal ulcer with an immune ring and hypopyon was observed. It was positioned under the paracentral steeper portion of the optic of the OK lens. Culture of the lens solution revealed P. aeruginosa. The patient was treated with topical OFLX and cefmenoxime (CMX) plus intravenous and subconjunctival injections of cefozopran (CZOP), successfully. The antibiotic susceptibility of P. aeruginosa by the disk diffusion susceptibility test was reduced under moderately hypoxic conditions. Glycocalyx slime was formed on the OK lens in vitro by P. aeruginosa isolated from the case. Conclusions: Changes in P. aeruginosa susceptibility to antibiotics under moderately hypoxic conditions and glycocalyx slime formation might affect the features of OK lens-associated infections.

Antimicrobial Susceptibility of Respiratory Tract Pathogens in Japan During PROTEKT Years 1–5 (1999–2004)

Susceptibility to a range of antimicrobial agents was determined among isolates of Streptococcus pneumoniae, Streptococcus pyogenes, and Haemophilus influenzae collected in 12 centers throughout Japan during years 1-5 (the respiratory seasons of 1999-2004) of the longitudinal Prospective Resistant Organism Tracking and Epidemiology for the Ketolide Telithromycin study. The most frequent source of isolates of S. pneumoniae was from patients with community-acquired pneumonia (CAP) (25.3%). Reduced susceptibility to penicillin or erythromycin resistance was common among S. pneumoniae isolates (30.9-44.5% and 77.2-81.9%, respectively). The macrolide MIC(50) for S. pneumoniae was >or=128 microg/ml (azithromycin and erythromycin) and >or=64 microg/ml (clarithromycin). The erm(B) genotype accounted for the most erythromycin-resistant isolates in each study year. H. influenzae isolates were most commonly derived from patients with CAP (26.2%). The proportion of H. influenzae isolates that were beta-lactamase positive ranged between 4.3% and 9.7%. The prevalence of beta-lactamase-negative ampicillin-resistant isolates increased from 0.4% to 2.6% between years 1 and 4 then to 19.7% in year 5. S. pyogenes isolates were highly susceptible to most antimicrobial agents except macrolides and tetracycline. Telithromycin was highly active against all three pathogens examined throughout the study.

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.

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.

Peripheral K cells in normal human pregnancy: decrease during pregnancy and increase after delivery

Peripheral blood levels of K cells were measured in normal pregnant and post-partum women by a plaque-forming cell technique that detects K cells on the basis of their activity of antibody-dependent cell-mediated cytotoxicity (ADCC). Peripheral K cells decreased throughout normal pregnancy (n = 46; 7.8 +/- 3.4%, P less than 0.01; 0.13 +/- 0.08 X 10(9)/l, P less than 0.001) and increased in the post-partum period (n = 17; 14.2 +/- 6.1%, P less than 0.05; 0.32 +/- 0.20 X 10(9)/l, P less than 0.01) in comparison with those in normal non-pregnant controls (n = 29; 10.5 +/- 4.2%; 0.20 +/- 0.09 X 10(9)/l). These findings suggest that a decrease of K cells during pregnancy may contribute in part to maternal acceptance of the fetal allograft and that the post-partum increase of K cells may represent a post-partum increase of cell-mediated cytotoxicity, which may contribute to natural defense against puerperal infection.

Changes in drug susceptibility and the quinolone-resistance determining region of Staphylococcus epidermidis after administration of fluoroquinolones.

PURPOSE: To evaluate the correlation between changes in the susceptibility of bacteria and mutations in the quinolone‐resistance determining region (QRDR) after 3 weeks of continuous fluoroquinolone instillation. SETTING: Miyata Eye Hospital, Miyazaki, Japan. METHODS: In this prospective randomized study, gatifloxacin 0.3% eyedrops or levofloxacin 0.5% eyedrops were administered for 1 week before cataract surgery and for 2 weeks after surgery. Samples were collected from the conjunctival sac before instillation of the antibiotic agent and 14 days after surgery. Susceptibility to the fluoroquinolones and gene mutations in the QRDR of the isolated Staphylococcus epidermidis were analyzed. RESULTS: The detection rate of S epidermidis was 27% in the gatifloxacin group (n=79 eyes) and 21% in the levofloxacin group (n=73 eyes) before instillation of the antibiotic and 6% and 19%, respectively, 14 days postoperatively. The susceptibility rates of S epidermidis strains to levofloxacin were statistically significantly lower after instillation than before antibiotic instillation, and the number of gene mutations in the QRDR was statistically significantly higher after instillation. There was no difference in the gatifloxacin group between before and after antibiotic instillation. In the 9 eyes in which S epidermidis was detected in samples taken before and after antibiotic instillation, most strains were genetically different from each other between the 2 time points. CONCLUSIONS: Three‐week continuous instillation of levofloxacin affected the indigenous bacterial flora in the conjunctival sac, suggesting possible induction of microbial substitution to fluoroquinolone‐resistant S epidermidis. However, there was no change with gatifloxacin.