Inteferon gamma and granulocyte-macrophage colony-stimulating factor augment the antifungal activity of human polymorphonuclear leukocytes against Scedosporium spp.: comparison with Aspergillus spp.

While Aspergillus spp. have been the most frequent filamentous fungi causing infections in immunocompromised patients, Scedosporium spp. are emerging as life-threatening pathogens. We studied the effects of interferon gamma (IFN-gamma) and granulocyte-macrophage colony-stimulating factor (GM-CSF) alone or combined on the antifungal activities of human polymorphonuclear leukocytes (PMN) against Scedosporium apiospermum and Scedosporium prolificans. We paralleled these activities to those against Aspergillus fumigatus and Aspergillus flavus. Incubation of PMN with IFN-gamma and GM-CSF for 22 h enhanced PMN-induced hyphal damage of both Aspergillus spp. and S. prolificans (p < 0.05) but not of S. apiospermum. However, hyphae of S. apiospermum were damaged significantly more after incubation with PMN that had been treated with IFN-gamma and GM-CSF for 2 h. In addition, incubation of PMN with GM-CSF for 2 h enhanced PMN oxidative burst measured as superoxide anion (O2-) production in response to nonopsonized hyphae of A. flavus and Scedosporium spp. (p < 0.05). In contrast, after 2 h, IFN-gamma and GM-CSF alone did not enhance PMN O2- in response to opsonized hyphae of A. flavus and Scedosporium spp.; however, the combination of IFN-gamma and GM-CSF showed significant enhancement against these species. Thus, IFN-gamma and GM-CSF, particularly in combination, demonstrate a species- and time-dependent augmentation of PMN responses to Scedosporium spp.     

Susceptibility of Pseudallescheria boydii and Scedosporium apiospermum to new antifungal agents.

Hyalohyphomycoses caused by Pseudallescheria boydii and Scedosporium apiospermum have recently been on the increase. To find the appropriate treatment for this emerging disease, we examined the antifungal susceptibility of 10 isolates of P. boydii and 17 isolates of S. apiospermum, most of which were isolated from clinical specimens. When the NCCLS M38-P microdilution method was used, itraconazole showed strong antifungal activities, while amphotericin B had little efficacy. A new triazole agent, voriconazole showed a strong effect against isolates of P. boydii and S. apiospermum (MIC50 0.06 micro g/ml), whereas micafungin, a newly developed echinocandin, had little effect (MIC50 >16 micro g/ml). There was no significant difference in the susceptibilities between P. boydii and S. apiospermum isolates against any antifungal agents. Our study suggests that voriconazole is a promising new drug in these infections, and that the same antifungal strategy can be employed in the infections by P. boydii and S. apiospermum.     

Human phagocytic cell responses to Scedosporium prolificans.

Scedosporium prolificans (SP) is an emerging opportunistic dematiaceous mould that causes serious infections in immunocompromised patients. Antifungal activities of human polymorphonuclear (PMN) and mononuclear (MNC) leukocytes against five SP isolates and Aspergillus fumigatus (AF) were evaluated. While monocyte-derived macrophages (MDM) phagocytosed conidia of both organisms comparably, they inhibited the germination of S. prolificans conidia less efficiently than those of A. fumigatus. Unopsonized hyphae of SP strains decreased the superoxide anion (O2-) produced by both PMN and MNC, whereas opsonized hyphae significantly stimulated it. In comparison to AF, phagocytes generally exhibited equal oxidative burst in response to SP. While PMN- and MNC-induced hyphal damage was similar among SP strains, phagocytes tended to damage SP hyphae to an equal or higher degree than AF hyphae. The susceptibility of SP to phagocytes contrasts with its high resistance to antifungal agents and may be related with the very low pathogenicity of the mould.     

Mechanisms of fungal pathogenicity: correlation of virulence in vivo, susceptibility to killing by polymorphonuclear neutrophils in vitro, and neutrophil superoxide anion induction among Blastomyces dermatitidis isolates.

Seven Blastomyces dermatitidis isolates varying in virulence for mice were compared for susceptibility to polymorphonuclear neutrophil (PMN) killing and the ability to induce superoxide anion (O2-) production by PMNs in vitro. In vitro killing of six B. dermatitidis isolates by murine peripheral blood PMNs or by PMNs elicited from the peritoneal cavity by a local immune reaction (B. dermatitidis-immune mice given killed B. dermatitidis intraperitoneally 24 h earlier) inversely correlated with in vivo virulence (most to least virulent) isolates: VV, V, V40, KL-1, A2, and GA-1). The capacity of isolates to induce O2- production by PMNs also inversely correlated with in vivo virulence. Isolate A, of intermediate in vivo virulence, was a good inducer of O2- production in vitro but was no more susceptible to in vitro killing by PMNs than isolate V, VV, or V40. Fungal intracellular superoxide dismutase or catalase content did not correlate with in vivo virulence or in vitro killing by PMNs. Isolate A, however, had two to four times the intracellular catalase activity as did other B. dermatitidis isolates, suggesting a possible mechanism for its enhanced resistance to in vitro killing by PMNs. Therefore, while in vitro killing by PMNs and the capacity to induce O2- production by PMNs inversely correlated with virulence for six B. dermatitidis isolates, isolate A was an exception: its resistance to killing by PMN-generated oxygen metabolites in vitro but its susceptibility to killing in vivo suggest that its in vivo killing occurs by other, perhaps nonoxidative, mechanisms.     

Prevention of corticosteroid-induced suppression of human polymorphonuclear leukocyte-induced damage of Aspergillus fumigatus hyphae by granulocyte colony-stimulating factor and gamma interferon.

Neutrophils (PMNs) are a critical line of defense against Aspergillus fumigatus infection. Increased frequency of invasive aspergillosis has been observed in patients receiving corticosteroids, suggesting a deleterious effect of these compounds on PMN antifungal function. To investigate this hypothesis and to determine the potential preventive utility of granulocyte colony-stimulating factor (G-CSF) and gamma interferon (IFN-gamma), the effects of hydrocortisone (HCS) and dexamethasone (DXS) on PMN-induced damage of Aspergillus fumigatus hyphae were studied with or without pretreatment of PMNs with G-CSF and IFN-gamma. PMNs treated with HCS (> or = 3,000 microM) or DXS (> or = 10 microM) during a 2-h colorimetric tetrazolium metabolic assay (using methylthiotetrazolium) showed suppressed percentage of hyphal damage (P < 0.02). In addition, both HCS (> or = 30 microM) and DXS (> or = 1 microM) significantly suppressed oxidative burst measured as superoxide anion release by PMNs in response to opsonized and nonopsonized hyphae as well as to N-formylmethionyl leucyl phenylalanine. Pretreatment of PMNs with G-CSF (4,000 U/ml) and/or IFN-gamma (100 and 1,000 U/ml) for 90 min prevented the suppression of hyphal damage that occurred in the presence of HCS (3,000 microM; P < 0.01) or DXS (10 microM; P < or = 0.001). G-CSF (4,000 U/ml) and IFN-gamma (100 U/ml) combined had an additive effect on increasing the antifungal activity of HCS-treated but not of DXS-treated PMNs compared with IFN-gamma alone (P = 0.015). Thus, these findings reveal that corticosteroids impair PMN function in response to A. fumigatus and that G-CSF and IFN-gamma prevent this impairment.     

Tumor Necrosis Factor Alpha Enhances Antifungal Activities of Polymorphonuclear and Mononuclear Phagocytes against Aspergillus fumigatus

Invasive aspergillosis is a serious complication in immunocompromised patients. The effects of recombinant human tumor necrosis factor alpha (TNF-α) on antifungal activities of human neutrophils (polymorphonuclear leukocytes [PMNs]), human monocytes (MNCs), and rabbit pulmonary alveolar macrophages (PAMs) against Aspergillus fumigatus were studied. The percentage of PMN-induced hyphal damage was increased after 30 min of incubation of PMNs with 0.1 ng of TNF-α per ml at 37°C (P = 0.043). At 0.1 to 10 ng/ml, TNF-α also increased superoxide anion (O₂⁻) produced by PMNs in response to phorbol myristate acetate, N-formylmethionyl leucyl phenylalanine, and unopsonized hyphae (P < 0.01) but did not exert any effect on PMN phagocytosis of conidia in the presence of serum. By comparison, TNF-α induced only a slight increase in O₂⁻ production by MNCs in response to phorbol myristate acetate (P = 0.05) and no concomitant increase in the percentage of MNC-induced hyphal damage. Incubation of MNCs with TNF-α at 0.001 to 10 ng/ml for 2 days had no effect on phagocytosis or conidiocidal activity. By contrast, incubation of PAMs with TNF-α at 0.1 to 10 ng/ml for 2 days increased phagocytosis of conidia (P = 0.03). Thus, TNF-α augments the capacity of PMNs to damage Aspergillus hyphae, possibly through enhanced oxidative mechanisms, and increases PAM phagocytic activity against conidia. As such, TNF-α may have an important role in host defense against aspergillosis, and neutralization of its activity may be complicated by increased susceptibility to aspergillosis.     

Comparison between Aspergillus fumigatus conidia and hyphae susceptibilities to amphotericin B, itraconazole, and voriconazole by use of the mold rapid susceptibility assay.

Although hyphae are the morphological form observed in tissue during invasive Aspergillus fumigatus infections, antifungal susceptibility testing for A. fumigatus utilizes conidial inocula. Previous studies have yielded conflicting results as to whether conidia adequately reflect antifungal susceptibility of hyphae, but the ease of handling and quantification of conidia have prompted their use in such assays. The mold rapid susceptibility assay, which utilizes a conidial inoculum (cRSA), was adapted as a novel method to assess the utility of conidial versus hyphal inocula (hRSA) to further evaluate the susceptibility of A. fumigatus conidia and hyphae to amphotericin B (AMB), itraconazole (ITC), and voriconazole (VRC). Conidial inocula were prepared as previously described for the cRSA and minimum inhibitory values (MIC) were determined. For the hRSA, microtiter test wells lacking antifungal drug were inoculated with a standardized conidial inoculum and incubated for 12 h at 35-37 degrees C to allow formation of hyphae. Following addition of antifungal drug and 48 h incubation at 35-37 degrees C, hRSA antifungal minimum inhibitory concentration (MIC) values were determined by analysing the pattern of residual glucose levels in hRSA test wells. hRSA MIC values of each strain were influenced by hyphal inoculum size, with increasing hyphal inoculum size corresponding to increased AMB, ITC and VRC MIC values. Comparisons between the hRSA and cRSA MIC values demonstrated insignificant differences in conidial and hyphal susceptibility to drug, thus justifying the use of either fungal form in RSA-based susceptibility testing of A. fumigatus isolates. The RSA may be adapted for use of similar testing of other invasive molds that predominate as hyphal forms in tissue.     

Standardization of a hyphal inoculum of aspergilli for amphotericin B susceptibility testing.

Standardized, evenly dispersed hyphal suspensions served as the inoculum in a microtiter technique for amphotericin B antifungal susceptibility testing. Preliminary testing with six strains of Aspergillus fumigatus and A. flavus produced consistent and reproducible results at 30 degrees C over 24 h. The observed amphotericin B MICs required for hyphae (0.3 to 0.6 microgram/ml) were comparable to MICs required for conidia (0.16 to 0.6 microgram/ml). The results were evaluated and compared with previously published information.     

Tumor cell lysis by activated human neutrophils: Analysis of neutrophil-delivered oxidative attack and role of leukocyte function-associated antigen 1

The lysis of tumor cells, and other nucleated mammalian cells, by neutrophilic polymorphonuclear leukocytes (PMNs) triggered by phorbol myristate acetate (PMA) represents a widely used model system to dissect the PMN cytolytic armamentarium, potentially responsible for the cell damage at tissue sites of PMN activation. Although oxidants are generally considered to be instrumental in the target lysis by PMNs, the mediators actually involved remain a matter of controversy. Moreover, other factors potentially crucial to the lysis have not been clearly identified. In order to reexamine the determinants of the cytolytic process, we studied the events underlying the PMA-triggered PMN-delivered attack against two different targets, selected on the basis of preliminary experiments (B lymphoblastoid Daudi cells and erythroleukemic K 562 cells). The results suggest that the lysis is promoted by hypochlorous acid (HOCl) or a compound with characteristics very similar to HOCl itself. No evidence was obtained for the intervention or contribution of hydrogen peroxide (H₂O₂), hydroxyl (OH ·) radicals, and the major HOCl-derived chloramines. PMNs appeared to use 35% of the generated H₂O₂ to produce HOCl, while the remainder appears to be consumed by PMNs themselves and target cells as well. Moreover, PMNs and target cells coaggregated at an early step of the cytolytic reaction, through a process efficiently prevented by a monoclonal antibody (MoAb J-90) directed against leukocyte function-associated antigen-1 (LFA-1). The inhibition of the PMN-target aggregation by the MoAb J-90 resulted in the impairment of the lysis, despite a normal generation of HOCl. Thus, the data demonstrate that the PMA-triggered lysis of tumor target cells by PMNs requires at least two events, occurring simultaneously: the LFA-1-mediated effector-target adherence and the PMN production of HOCl. The intervention of the LFA-1-mediated PMN-target adherence in the PMA-triggered lysis is likely to allow PMNs to focus HOCl on the target cell surface and suggests that the process requires a sort of molecule to molecule recognition at the effector-target surface level.Supported by grants from Italian C.N.R. and M.P.I. Dr. Luciano Ottonello is a recepient of a Fellowship from C.N.R.     

Scedosporium apiospermum keratitis treated with itraconazole.

Mycotic keratitis usually occurs in conjunction with trauma to the cornea. Scedosporium apiospermum, a dematiaceous fungus linked to the teleomorph Pseudallescheria boydii is not a common agent of mycotic keratitis. A 22-year old male patient with mycotic keratitis due to S. apiospermum is presented. In in vitro susceptibility testing, the isolate showed resistance against amphotericin B (minimum inhibitory concentration [MIC] 16 microg ml(-1)) but was susceptible to itraconazole (ITC) and fluconazole with MICs of 0.125 microg ml(-1) and 4 microg ml(-1), respectively. The patient was cured clinically after ITC treatment and surgical intervention. Azoles may be superior for eliminating S. apiospermum from infected ocular sites.     

Invasive Nattrassia mangiferae infections: case report, literature review, and therapeutic and taxonomic appraisal.

We report on a case of subcutaneous infection of the arm caused by the coelomycetous fungus Nattrassia mangiferae (formerly Hendersonula toruloidea) in a steroid-dependent diabetic man with chronic obstructive lung disease. The man was a resident of Arizona, where the fungus is known to be endemic on Eucalyptus camaldulensis and on citrus trees. Diagnosis of fungal infection was made by observation of narrow hyphal filaments by histopathology of biopsy specimens and isolation of a fast-growing black mold which demonstrated hyphae and arthroconidia of varying widths typical of the Scytalidium synanamorph (S. dimidiatum). The formation of pycnidia, which at maturity expressed conidia with a central median dark band, allowed for the confirmation of the isolate as N. mangiferae. Remission of the lesions occurred following intravenous therapy with amphotericin B, followed by topical clotrimazole treatment. We use this patient's case report as an opportunity to review the literature on cases of deep infection caused by Scytalidium species, to evaluate the antifungal susceptibilities of a spectrum of Scytalidium isolates, and to review the taxonomy of Scytalidium species isolated from human infections.     

In vitro susceptibility of chromoblastomycosis and phaeohyphomycosis agents to antifungal drugs.

The in vitro susceptibility of chromoblastomycosis and phaeohyphomycosis agents to antifungal drugs was appraised using the reference macrodilution method proposed by the National Committee for Clinical Laboratory Standards (NCCLS) for yeasts modified for filamentous fungi. The antifungal drugs amphotericin B, 5-fluorocytosine, itraconazole and fluconazole were tested against one environmental and 18 clinical isolates. This work amended the macrodilution methods proposed by NCCLS and suggests that a conidial suspension free of hyphae leads to a more reliable assay and provides for better reproducibility. The macrodilution method was performed with 10(4) conidia ml-1. The MIC values ranged from 1.0 to 16.0 micrograms ml-1 for amphotericin B and 3.12 to 25.0 micrograms ml-1 for 5-fluorocytosine. A MIC range of 0.06 to 1.95 micrograms ml-1 was determined for itraconazole while 2.0 to 64.0 micrograms ml-1 was detected for fluconazole.     

Comparison of E-test and broth microdilution methods for antifungal drug susceptibility testing of molds

We compared the E test with a broth microdilution method, performed according to National Committee for Clinical Laboratory Standards document M27-A guidelines, for determining the in vitro susceptibilities of 90 isolates of pathogenic molds (10 Absidia corymbifera, 10 Aspergillus flavus, 10 Aspergillus fumigatus, 10 Aspergillus niger, 10 Aspergillus terreus, 10 Exophiala dermatitidis, 10 Fusarium solani, 10 Scedosporium apiospermum, 5 Scedosporium prolificans, and 5 Scopulariopsis brevicaulis). Overall, there was 71% agreement between the results of the two methods for amphotericin B (E-test MICs within +/-2 log2 dilutions of broth microdilution MICs) and 88% agreement with the results for itraconazole. The overall levels of agreement (within +/-2 log2 dilutions) were >/=80% for 5 of the 10 species tested against amphotericin B and 8 of the 10 species tested against itraconazole. The best agreement between the results was seen with A. fumigatus and A. terreus (100% of results for both agents within +/-2 log2 dilutions). The poorest agreement was seen with S. apiospermum, S. prolificans, and S. brevicaulis tested against amphotericin B (20% of results within +/-2 log2 dilutions). In every instance, this low level of agreement was due to isolates for which the broth microdilution MICs were low but for which the E-test MICs were much higher. The E test appears to be a suitable alternative procedure for testing the susceptibility of Aspergillus spp. and some other molds to amphotericin B or itraconazole.     

In vitro activities of voriconazole and five licensed antifungal agents against Candida dubliniensis: comparison of CLSI M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods

We compared the in vitro activity of six antifungal agents against 62 isolates of Candida dubliniensis by the Clinical Laboratory Standards Institute (CLSI [formerly National Committee for the Clinical Laboratory Standards]) M27-A2, Sensititre YeastOne, disk diffusion, and Etest methods and we studied the effect of the time of reading. For the azoles, voriconazole was the most potent in vitro followed by fluconazole, ketoconazole, and itraconazole. All the isolates were susceptible to amphotericin B and flucytosine. The highest rate of resistance was obtained against itraconazole with a high number of isolates defined as susceptible dose-dependent. At 24 hr, 100% of the isolates were susceptible to ketoconazole, amphotericin B, and flucytosine, 98% susceptible to voriconazole and fluconazole, and 95% for itraconazole. At 48 hr, 100% of the isolates remained susceptible for flucytosine and amphotericin B, 95% for voriconazole, 93% for fluconazole, 90% for ketoconazole, and 82% for itraconazole. The agreement between the CLSI and the other methods was better at 24 than 48 hr.     

Global trends in the antifungal susceptibility of Cryptococcus neoformans (1990 to 2004)

The antifungal susceptibilities of 1,811 clinical isolates of Cryptococcus neoformans obtained from 100 laboratories in 5 geographic regions worldwide between 1990 and 2004 were determined. The MICs of amphotericin B, flucytosine, fluconazole, voriconazole, posaconazole, and ravuconazole were determined by the National Committee for Clinical Laboratory Standards broth microdilution method. Isolates were submitted to a central reference laboratory (University of Iowa) from study centers in Africa (5 centers, 395 isolates), Europe (14 centers, 102 isolates), Latin America (14 centers, 82 isolates), the Pacific region (7 centers, 50 isolates), and North America (60 centers, 1,182 isolates). Resistance to amphotericin B, flucytosine, and fluconazole was < or = 1% overall. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) ranged from 35% in North America to 68% in Latin America. Similarly, only 75% of isolates from North America were susceptible to fluconazole (MIC, < or = 8 microg/ml) compared to 94 to 100% in the other regions. Isolates remained highly susceptible to amphotericin B (99% susceptibility at a MIC of < or = 1 microg/ml) over the entire 15-year period. Susceptibility to flucytosine (MIC, < or = 4 microg/ml) increased from 34% in 1990 to 1994 to 66% in 2000 to 2004. Susceptibility to fluconazole (MIC, < or = 8 microg/ml) increased from 72% in 1990 to 1994 to 96% in 2000 to 2004. Voriconazole, posaconazole, and ravuconazole all were very active (99% of isolates susceptible at MIC of < or = 1 microg/ml) against this geographically diverse collection of isolates. We conclude that in vitro resistance to antifungal agents used in the treatment of cryptococcosis remains uncommon among isolates of C. neoformans from five broad geographic regions and has not increased over a 15-year period.     

Influence of amphotericin B and amphotericin B colloidal dispersion on the functions of human phagocytes in defence against Aspergillus species

This study investigated the in vitro effects of amphotericin B and amphotericin B colloidal dispersion (ABCD) on phagocytosis and inhibition of germination of clinical isolates of Aspergillus spp. by monocyte-derived macrophages (MDMs).Both amphotericin B and ABCD caused significant reductions in uptake of conidia of Aspergillus spp. by MDMs (p < 0.01). The inhibition of germination was superior with conidia of Aspergillus fumigatus, Aspergillus niger and Aspergillus terreus isolates. Aspergillus flavus growth was significantly less inhibited of either antimycotic as compared to A. fumigatus and A. terreus (p < 0.01).We demonstrate that amphotericin B or ABCD acts as a potent inhibitor of Aspergillus germination. By contrast, treatment of MDMs with these antimycotics diminished phagocytosis of conidia in vitro.     

Clinical, microbiological, and experimental animal studies of Candida lipolytica.

Candida lipolytica was recovered from six patients in three different clinical centers. The index isolate caused a persistent fungemia with catheter-associated Candida thrombophlebitis, the second isolate was from a polymicrobial sinusitis, and the remaining four isolates were involved in tissue colonization. These and 20 other isolates were consistent in their morphological and physiological characteristics. All formed true hyphae and blastoconidia on cornmeal-Tween 80 agar and all assimilated glucose, glycerol, and erythritol. In a murine model of disseminated candidiasis, the index isolate that caused clinical fungemia caused no mortality and produced only two lesions on a kidney, as determined at necropsy. The nine isolates selected for in vitro antifungal susceptibility studies had intermediate susceptibilities to amphotericin B but were susceptible to ketoconazole. We conclude that C. lipolytica is a weakly virulent pathogen which may require an intravascular foreign body to cause fungemia.     

In vitro activity of amphotericin B,fluconazole and voriconazole against 162 <Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> isolates from Africa and Cambodia

In order to determine the potential role that various antifungal agents might have in the management of cryptococcosis in tropical areas, the in vitro susceptibility of Cryptococcus neoformans isolates from Africa (n=52) and Cambodia (n=110) to three antifungal agents (amphotericin B, fluconazole and voriconazole) were compared using the E-test method. The results of this study (i) confirm the value of the E-test for testing the in vitro susceptibility of C. neoformans towards voriconazole; (ii) provide the first evidence demonstrating good activity of amphotericin B, fluconazole and voriconazole against Cambodian isolates; and (iii) show there are differences in susceptibility between African and Asian C. neoformans isolates, with Cambodian isolates appearing less susceptible to the agents tested but with amphotericin B maintaining good activity.     

Ocular infections caused by Candida species: Type of species,in vitro susceptibility and treatment outcome

Purpose: To report clinical and microbiological profile of patients with ocular candidiasis. Materials and Methods: Patients with ocular candidiasis were retrospectively identified from microbiology records. Significant isolates of Candida species were identified by Vitek 2 compact system. Minimum inhibitory concentration (MIC) of antifungal agents such as amphotericin B, itraconazole, voriconazole, fluconazole and caspofungin was determined by E test and of natamycin by microbroth dilution assay. Data on treatment and outcome were collected from medical records. Results: A total of 42 isolates of Candida were isolated from patients with keratitis-29, endophthalmitis-12 and orbital cellulitis-1. The most common species isolated was Candida albicans (12-keratitis, 4-endophthalmitis, 1-orbital cellulitis). All except one isolate were susceptible to amphotericin B. MIC of caspofungin was in the susceptible range in 28 (96.5%) corneal isolates while 12 out of 29 (41.3%) corneal isolates were sensitive to fluconazole. Resistance to voriconazole was seen in four corneal isolates. All isolates were susceptible to natamycin and all except two isolates were resistant or susceptible dose-dependent to itraconazole. Outcome of healed ulcer was achieved in 12/18 (66.6%) patients treated medically, while surgical intervention was required in 11 patients. Among the isolates from endophthalmitis patients, 11/12 were susceptible to amphotericin B, 6/12 to voriconazole and all to natamycin. Ten out of 11 patients (one patient required evisceration) with endophthalmitis were given intravitreal amphotericin B injection with variable outcome. Conclusions: Ocular candidiasis needs early and specific treatment for optimal results. Candida species continue to be susceptible to most commonly available antifungals including amphotericin B, voriconazole and natamycin.     

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates

Purpose: To standardize in-vitro antifungal susceptibility testing by agar dilution method to find out the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole and ketoconazole on ocular fungal isolates. Methods: A total of 180 ocular fungal isolates (130 filamentous fungi and 50 yeasts) were included. The antifungal drugs such as amphotericin B (0.0625-8 μg/mL), fluconazole (0.2-819.6 μg/mL) and ketoconazole (0.025-6.4 μg/mL) were incorporated in doubling dilutions in the yeast nitrogen base medium. The MIC was determined as the lowest concentration of the antifungal drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug - free control plates. Results: All 50 ocular isolates of yeast were susceptible to amphotericin B, while two (4%) and five (10%) strains were resistant to fluconazole and ketoconazole respectively. Of the 130 filamentous fungi tested, six (4.6%) were resistant to amphotericin B, 49 (37.7%) and 10 (7.6%) were resistant to fluconazole and ketoconazole respectively. Percentile 50 (MIC 50) and Percentile 90 (MIC 90) for all the three antifungal agents were calculated. Aspergillus niger, Aspergillus terreus and Candida krusei were found to be resistant to fluconazole and ketoconazole. Conclusion: This technique was found to be reliable, cost effective and easy to perform with consistent results.