Atsuko Sunada

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.

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.

The characteristics of keratomycosis by Beauveria bassiana and its successful treatment with antimycotic agents

Clinical findings and treatment of keratomycosis caused by Beauveria bassiana, an entomopathogenic filamentous fungus, are described for an 80-year-old woman, who was referred to the hospital for ocular pain and redness on the 9th day after an ocular injury caused by the frame of her glasses. She had a long history of recurrent diabetic iritis and continuously used topical antibiotics and corticosteroids. At her first visit, a slit-lamp examination indicated a corneal ulcer confined within the superficial stromal layer, along with a slight infiltration and edema. Only a very few inflammatory cells were seen in the anterior chamber. Direct microscopic examination of corneal scrapings revealed septate fungal hyphae with zig-zag rachis and budding that was subsequently identified as B. bassiana by slide culture. Topical voriconazole with miconazole, pimaricin and oral itraconazole were effective and the lesion disappeared leaving only a mild scar at 2 months. The sensitivity of B. bassiana to various antimycotic agents was confirmed by broth microdilution, agar dilution with the Clinical Laboratory Standard Institute standard, and a disk method using topically applied concentrations. B. bassiana, which exhibits a characteristic appearance in smears and causes superficial keratomycosis, is sensitive to voriconazole with miconazole, pimaricin, and itraconazole.

Utility of Etest in choosing appropriate agents to treat fungal keratitis.

PURPOSE To evaluate the utility of Etest in choosing the appropriate treatment of fungal keratitis. METHODS Etest was used to determine the drug sensitivities of isolates from the eyes of three patients with fungal keratitis, and the clinical outcomes of treatment with selected drugs were evaluated. RESULTS In all cases, drug sensitivity demonstrated by Etest accorded with clinical efficacy of the drugs. CONCLUSION The results in these cases suggest that evaluating drug sensitivities with Etest is an efficient means of selecting optimal pharmacotherapy for fungal keratitis.

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.

In Vitro Combined Effects of Cefozopran/Teicoplanin and Cefozopran/Vancomycin on Methicillin-Resistant Staphylococcus aureus

Abstract We determined the In Vitro effects of teicoplanin (TEIC) or vancomycin (VCM) added to cefozopran (CZOP) on 50 methicillin-resistant Staphylococcus aureus (MRSA) strains, using a modified checkerboard method with serial 1.25-fold dilutions, and assessed the time-kill curve. CZOP + TEIC was synergistic (fractional inhibitory concentration index ≤0.5) against 98% and CZOP + VCM against 20% of strains. Both drug combinations were additive against the remaining strains. A comparison of the fractional bactericidal concentration indices for 32 strains showed synergistic bactericidal effects for CZOP + TEIC in 88%, and in 48% for CZOP +VCM, confirming that CZOP + TEIC is superior to CZOP + VCM. The time-kill curve confirms that the bactericidal potency of these drugs is increased through combined use with CZOP. These results suggest that treatment using TEIC or VCM with CZOP may be useful in treating MRSA infections, including polymicrobial infections and those involving Gram-negative rods.

Accuracy of Commercial Susceptibility Testing Method for Measuring Vancomycin MIC Against Methicillin-Resistant Staphylococcus aureus (MRSA)

Methods: We evaluated the vancomycin (VAN) minimum inhibitory concentrations (MIC) on methicillin-resistant Staphylococcus aureus (MRSA) strains using MicroScan panel (Pos combo 3.1J). We also used the following 2 methods of preparing the bacterial solution: MicroScan Prompt system (P method) and manual preparation with inoculums adjusted to match a 0.5 McFarland turbidity standard (M method). The Clinical and Laboratory Standards Institute (CLSI) method was used as a reference. Results: On comparing the distribution of VAN MICs, the CLSI and M methods showed a good correlation, while the P method results were higher. Colony counts indicated the final inoculum concentrations from the P method were higher than those from the M method, which were within the ideal inoculum concentration. Comparing the results between the M method with the W/A 96 System (18hour incubation period) and the M method with a 24-hour incubation period revealed a discrepancy in the detection of some strains with 2.0 µg/mL of VAN. Conclusion: The P method appears to be less reliable than the M method for measuring VAN MIC against MRSA. Hence, the M method with a 24-hour incubation period should be used instead.

Evaluation of Amphotericin B and Micafungin Combination Against Clinical Isolates of Aspergillus Species

Abstract We aimed to evaluate the efficacy of a combination of amphotericin B (AMB) and micafungin (MCFG) against 25 clinical isolates of Aspergillus species in vitro. We examined fungal growth in the presence of these drugs using a checkerboard method with the tetrazolium salt: 2,3-bis (2- methoxy-4-nitro-5-sulphophenyl)-2H-tetrazolium-5-carboxyanilide inner salt (XTT) to determine the efficacy of an AMB/MCFG combination in inhibition of filamentous fungal growth, evaluated based on 50% reduction of metabolic activity. The fractional inhibitory concentration index showed that the drugs synergistically inhibited 36% of the isolates. Activity was judged as indifferent for 64% isolates; antagonistic interaction was not detected. The AMB/MCFG combination was more effective than AMB alone when sub-inhibitory concentrations of AMB were used. This report demonstrates the efficacy of AMB/MCFG combination for inhibiting the growth of Aspergillus species in vitro, warranting the extension of such studies to animal models.

Utility of Etest in Choosing Appropriate Agents to Treat Fungal Keratitis: Author's Reply

Purpose. To evaluate the utility of Etest in choosing the appropriate treatment of fungal keratitis. Methods. Etest was used to determine the drug sensitivities of isolates from the eyes of three patients with fungal keratitis, and the clinical outcomes of treatment with selected drugs were evaluated. Results. In all cases, drug sensitivity demonstrated by Etest accorded with clinical efficacy of the drugs. Conclusion. The results in these cases suggest that evaluating drug sensitivities with Etest is an efficient means of selecting optimal pharmacotherapy for fungal keratitis.