Comparative efficacy of amphotericin B,clotrimazole and itraconazole againstAspergillus spp.

The susceptibilities of two isolates ofAspergillus flavus, one from a human case of recalcitrant mycotic keratitis, and an environmental isolate ofA. fumigatus, to itraconazole, clotrimazole and amphotericin B were measured. Observations of macroscopic growth and microscopic evaluations of conidia germination both indicated that the two isolates ofA. flavus were markedly more resistant to amphotericin B than to itraconazole and clotrimazole. Itraconazole was more effective than clotrimazole for all isolates. Ourin vitro susceptibility results suggest the use of itraconazole should be a primary consideration in the treatment ofAspergillus keratitis.     

Mycotic keratitis caused by Curvularia lunata var. aeria

A mycotic keratitis case which was caused by Curvularia lunata var. aeria in a patient with an injury in the eye is described. The diagnosis was based on the mycologic analysis of several samples taken from the ulcer of cornea. In vitro tests of the sensitivity of the isolated species to several antifungal drugs were made. The results were related to the response in vivo to the treatment.     

Clinical and experimental mycotic corneal ulcer caused by Aspergillus fumigatus and the effect of oral ketoconazole in the treatment

Aspergillus fumigatus was isolated from a case of keratomycosis. The patient, a 12-year-old boy presented with large corneal ulcer with hypopyon. The direct microscopic examination of scrapings revealed hyaline, septate mycelium. In vitro some antimycotics (amphotericin B,5-fluorocytosine, oxiconazole, amorolfine and ketoconazole) were tested against A. fumigatus by agar dilution method. Ketoconazole with minimum inhibitory concentration of 30 g/ml after 11 days of incubation was most effective against A. fumigatus. Experimental corneal ulcer was produced by injecting intralamellary spore suspension (2.5×10⁶ c.f.u.) into the right eyes of previously immunosuppressed albino and black wild types of rabbits. The extent of ocular infection was graded up to 32 days. Histopathologic examination showed infiltration and large destruction of corneal stroma. Oral ketoconazole therapy exhibited partial response followed by relapse. The black type of rabbit appeared more suitable as an animal model for mycotic keratitis.This paper was presented at the X^th congress of the International Society for Human and Animal Mycology at Barcelona, Spain from June 27 to July 1, 1988.     

Mitogenic lectins from Cephalosporium curvulum (CSL) and Aspergillus oryzae (AOL) mediate host–pathogen interactions leading to mycotic keratitis

A core-fucose-specific lectin, CSL from Cephalosporium curvulum, has been reported earlier. Here we assign the role for CSL and another lectin AOL, from pathogenic fungus Aspergillus oryzae, in causing mycotic keratitis. CSL and AOL show strong binding to immortalized and primary human corneal epithelial cells (HCECs) which are inhibited by asialofetuin, confirming their glycan-mediated binding. CSL and AOL showed increase in viability at lower concentrations (0.07 µg/ml) whereas at higher concentrations (0.15 µg/ml and 0.30 µg/ml), have inhibitory effect on immortalized HCECs. Lectin-mediated effect was comparable with the effect induced by the Colony Forming Units (CFUs) of C. curvulum and A. oryzae. CFUs induced more than 1.5-fold increase in HCECs proliferation. Both lectins and fungal CFUs induce secretion of proinflammatory cytokines IL6 and IL8 implicated in ocular diseases. This was supported by upregulation of TLR2 and 4 by lectins as revealed by flow cytometry and RT-PCR. CSL and AOL mediate host–pathogen interactions leading to mycotic keratitis. The mechanism of pathogenesis is possibly initiated through surface binding of mycelia through the lectins to TLR2/4 followed by upregulation of proinflammatory cytokines IL6, IL8 and TLR2 and 4. Understanding the mechanism of pathogenesis is of clinical significance in designing and developing therapeutic strategy to control the infection.

     

Role of opportunistic fungi in ocular infections in Nigeria

The importance of opportunistic fungal pathogens in causing ocular infections is emphasized. A study was conducted over a period of 4 years (1974–1977) to investigate the role of opportunistic fungi in causing mycotic keratitis and to elucidate certain aspects of epidemiology of this disease in Nigeria. Fifty-nine cases of corneal ulcers of suspected mycotic etiology were investigated. Fungal etiology was confirmed in 42 of these cases. The predominant etiological agent was Fusarium solani in 14 cases (33.33%) followed by Penicillium citrinum in 8 cases (19.04%) and Aspergillus fumigatus in 5 cases (11.90%). The yeasts were responsible for only 3 cases (7.14%) i.e. one each caused by Candida albicans, C. parapsilosis and C. guilliermondii. Among the remaining 12 cases, one was caused by F. moniliforme, 3 by A. flavus, 2 each by A. niger, Penicillium expansum and Penicillium sp., and one each by Cladosporium cladosporioides and Cladosporium sp. The clinical features of the cases are briefly described.The incidence of mycotic keratitis in relation to sex, age, occupation, trauma and other factors has been analysed. Corneal trauma appeared to be an important predisposing factor as 27 (67.28%) of the patients gave a history of injuries to the eye. Notably, a large number of patients were farmers and trauma was most often from palm tree leaf, thorn, kernel or other plant objects. Topical application of corticosteroids or broad spectrum antibiotics did not seem to play an important role in the etiology of keratomycosis. Cases were recorded throughout the year although the number of cases was higher in the months of March –May, and November–December than that during the rest of the year.The isolates of the causative agents were studied in detail for their morphological and cultural characters. The isolates of F. solani grew well at 37°C and survived at 40°C for more than 3 weeks. In vitro drug sensitivity tests indicated good antifungal activity of pimaricin and econazole for F. solani, clotrimazole and econazole for Aspergillus fumigatus, A. flavus and Penicillium citrinum, and 5-fluorocytosine for Candida spp.Investigations on the incidence of fungi in normal healthy eyes of 450 persons comprising 204 adults and 246 children yielded 204 isolates belonging to 21 genera of fungi. Cladosporium was most frequent (12.88%) followed by Penicillium (10.22%) and Aspergillus (6.66%). Another important fungus was Fusarium represented by 10 isolates, viz. 4 of F. solani, 2 of F. moniliforme, 1 of F. exysporum, and 3 of Fusarium sp. The yeasts were represented by two isolates each of Candida tropicalis, C. pseudotropicalis, C. krusei, Trichosporon sp and Cryptococcus albidus, and one of Candida guilliermondii. Successive culturing of fungi from normal eyes in a small group indicated that fungi occur in the outer eye generally as transients.The epidemiology of mycotic keratitis has been discussed in relation to the present findings and in comparison with observations of other investigators.     

Characterisation and inhibition effect of cetrimide on collagenase produced byAspergillus flavus, isolated from mycotic ulcers

Collagenase was found to be the most important enzyme, produced by mycotic keratitis fungi. Therefore,Aspergillus flavus collagenase enzyme has been purified by ammonium sulphate precipitation, Sephadex G25 and DEAE-cellulose chromatography. Electrophoretic analysis for the purified enzyme indicated one subunit of molecular weight of 70–90 KDa when examining on SDS-PAGE. Cetrimide (cetyl trimethyl ammonium bromide) has been tested against the purified collagenase enzyme and indicated reversible competitive inhibitor (kis=0.15 mg/ml) with high promising activity. Cetrimide might be used to inhibit mycotic keratitis fungi.     

Studies on a strain of Fusarium solani (Mart.) Sacc. Isolated from a case of mycotic keratitis

A strain of Fusarium solani (Mart.) Sacc. (IMI-216517), isolated from a patient of mycotic keratitis, produced experimental keratomycosis in albino rabbit cornea and survived in internal tissues of albino mice for varying periods. Alantolactone, isolated from the plant — Inula racemosa Hook. f. exhibited marked in vitro fungistatic activity against this strain of F. solani at 100–200 g/ml concentrations. The strain was less sensitive to amphotericin B and showed more acid than alkaline proteinase and phosphatase activities.Communication No. 2526.     

<Emphasis Type="Italic">Aspergillus flavus</Emphasis> Keratitis: Experience of a Tertiary Eye Clinic in Turkey

We investigated the clinical and mycological characteristics of four cases of mycotic keratitis caused by Aspergillus flavus that occurred from July 2014 to May 2015 at Çukurova University Hospital, Adana, Turkey. In a 10-month period, a total of 64 corneal smear/scrapings were examined from patients with suspected mycotic keratitis. Fungal cultures were positive in six of these patients, indicating a 9.4% incidence of mycotic keratitis in this region, including four cases of A. flavus and two cases of Fusarium spp. The predisposing factors, clinical presentation, and success of the therapeutic approaches were further evaluated. For all cases, topical voriconazole was the first choice of treatment. Surgical procedures were required to control infection in 3 of the 4 cases, including intrastromal voriconazole injection for two cases and keratoplasty for one case. Predisposing factors included trauma (two cases, 50%), contact lens use (one case, 25%), and previous ocular surgery (one case, 25%). The clinical presentations also differed, including a well-limited ulcer (one case), an ulcer with an irregular feathery margin (one case), and ulcers with satellite lesions (two cases). The mean duration between the time of presentation and definitive diagnosis by culture was 14 days (8–25 days). We observed that A. flavus keratitis can present with different underlying factors and clinical conditions. A combination of antifungal therapy and supportive surgical intervention may resolve infections caused by A. flavus in the cornea.     

Biological characterization of a clinical and an environmental isolate of <Emphasis Type="Italic">Acanthamoeba polyphaga</Emphasis>: analysis of relevant parameters to decode pathogenicity

Acanthamoeba spp. consists of free-living amoebae, widespread in nature, which occasionally can cause human infections including granulomatous amoebic encephalitis and amoebic keratitis. Acanthamoeba pathogenesis is not entirely known and correlations between pathogenic potential and taxonomy are complex issues. In order to decipher the definition of a pathogenic amoeba, the objective of this work was to decipher the definition of pathogenic amoeba by characterizing two isolates of Acanthamoeba polyphaga obtained from different origins (a keratitis patient and freshwater), looking for differences among them. The clinical isolate grew faster in Peptone-yeast extract-glucose (PYG) medium, transformed more rapidly from a trophozoite to cyst and exhibited increased cytopathic effect on cultured cells. Morphological differences were also noted, since freshwater amoebae presented more acanthopodia than the clinical isolate. Moreover, actin labeling demonstrated that microfilament organization varies between isolates, with the presence of locomotory structures as lobopodia and lamellipodia in the keratitis isolate, which were less adherent on plastic. Zymography demonstrated that the keratitis isolates presented higher proteolytic activity and also were more able to invade collagen matrices. Altogether, we conclude that a group of stable physiological characteristics exist in Acanthamoeba that can be related to pathogenicity.     

Mycotic granulomatosis found in two species of ornamental fishes imported from Singapore

Golden gourami,Trichogaster trichopterus, and snakehead,Channa pleurophthalmus, were newly discovered to be hosts of mycotic granulomatosis caused byAphanomyces sp., the first such cases to be reported in Japan.     

Evaluation of in vitro activities of extracellular enzymes from Aspergillus species isolated from corneal ulcer/keratitis

Mycotic/fungal keratitis is a suppurative, generally ulcerative infection of the cornea. The filamentous fungi, Aspergillus spp. are the second leading cause of mycotic keratitis, particularly in India. Aspergillus spp. produce a range of extracellular enzymes that are used to break down complex molecules and used for growth and reproduction, also for survival on/in host organism. The current study was designed with an objective to screen in vitro extracellular enzyme activity of Fusarium and Aspergillus isolates from mycotic keratitis patients and to correlate the same as a putative virulence factor. Extracellular enzymes viz., deoxyribonuclease (DNase), protease, lipase, elastase, keratinase, etc., produced by Aspergillus have key role in keratomycosis and hence their (n = 85) in vitro activities were investigated. It was found that, the majority of the Aspergillus isolates produced protease (n = 75; 88% of 85) followed by lipase (n = 59; 69% of 85), DNase (n = 35; 41% of 85), elastase (n = 26; 31% of 85) and keratinase (n = 13; 15% of 85). The enzyme activity indices (EAI) for DNase, elastase, protease and lipase ranged between 1.01 and 1.98, whereas elastase EAI varied between 1.26 and 1.92. DNase, protease and lipase showed a maximum EAI of 1.98 and lowest EAI value of 1.01, respectively. Extracellular enzymes of Aspergillus spp. may have potential role in the onset and progression of keratitis.     

Recent advances on mycotic keratitis caused by dematiaceous hyphomycetes

Dematiaceous hyphomycetes (DH) are darkly pigmented fungi ubiquitously found all over the world as plant pathogens and saprophytes, and many of the members of this group have emerged as opportunistic pathogens. These fungi are responsible for a wide variety of infections including mycotic keratitis, which is considered as one of the major causes of corneal blindness, particularly in tropical and subtropical countries with an annual global burden of about 1 000 000 patients. The infection is more common in workers working in an outdoor environment. Moreover, trauma is found to be the most important predisposing cause of mycotic keratitis. Considerable delay in diagnosis and scarcity of effective pharmacological drugs are the major factors responsible for increased morbidity and visual impairment. Considering the crucial role of DH in mycotic keratitis, in the present review, we have focused on major DH with special emphasis on their pathogenicity, diagnosis and treatment strategies.     

Mycotic Keratitis Caused by <Emphasis Type="Italic">Fusarium solani sensu stricto</Emphasis> (FSSC5): A Case Series

Owing to a lack of appropriate diagnostic and therapeutic approaches for mycotic keratitis, approximately one million cases of preventable corneal blindness are reported each year. The number of keratitis cases due to infection with Fusarium is increasing significantly worldwide, many of which are not treated adequately and in a timely manner due to frequent misdiagnosis. In the current report, we describe three cases of keratitis caused by Fusarium solani sensu stricto (FSSC5) from Turkey and The Netherlands, following ocular trauma. The etiological agent of keratitis, FSSC5, identified by sequencing of the partial tef1-α gene, exhibited low minimum inhibitory concentrations (MICs) of 1 µg/mL for amphotericin B and high MICs above the published epidemiological cutoff values for voriconazole (8 µg/mL). Patients were successfully treated with topical amphotericin B and voriconazole with complete recovery.     

Fusariosis in the shrimp Penaeus semisulcatus cultured in Israel

The first case of a mycotic infection in shrimp in Israel is reported. Fusarium solani produced a large melanized lesion in a specimen of Penaeus semisulcatus cultured at Eilat, on the Red Sea. Fungal hyphae elicited a strong hemocytic response in cuticular and connective tissues. In the underlying muscle, the inflammatory reaction appeared weaker, suggesting a gradual failure by the host to resist mycelial invasion. Three cases of human keratomycosis by F. solani have been reported in recent years in Israel, suggesting that handling infected shrimp may represent a hazard to aquaculturists.     

Dermatophytes and other keratinophilic fungi from coypus (Myocastor coypus) and brown rats (Rattus norvegicus)

The occurrence of mycotic agents was investigated by hair-brush technique on the coat of 162 naturalized coypus (Myocastor coypus) and 64 indigenous brown rats (Rattus norvegicus) originating from the same protected area in Central Italy. The isolates from positive coypus (29.6%) were identified as Microsporum gypseum (14.8%), Trichophyton terrestre (9.8%), Alternaria sp. (3.7%), Trichophyton mentagrophytes (2.5%), Cladosporium sp. (1.8%), Scopulariopsis sp. (1.2%), and Chrysosporium keratinophilum (0.6%), whereas the isolates from positive rats (46.7%) were identified as M. gypseum (28.1%), T. mentagrophytes (12.5%), Chrysosporium tropicum and T. terrestre (3.1% each), and Chrysosporium inops (1.6%). Statistically, brown rats were more likely to harbor fungal agents (P < 0.05) on their coat than coypus, especially T. mentagrophytes (P < 0.01) and M. gypseum (P < 0.05). No positive animal showed dermatological lesions. The present results are the first providing basic information on the coat’s mycoflora of a wild coypu population. It is concluded that the coypu, an allochthonous rodent in Italy, can play a role as natural animal reservoir of dermatophytes and as carrier of keratinophilic and saprophytic fungi within its new habitat, though to a lesser extent than indigenous rats. Therefore, people who are exposed to the risk of contact with the coat of rats and coypus should take all the cautions needed to protect themselves from mycotic agents potentially transmissible to humans.     

Cellulose biosynthesis pathway is a potential target in the improved treatment of <Emphasis Type="Italic">Acanthamoeba</Emphasis> keratitis

Acanthamoeba is an opportunistic protozoan pathogen that can cause blinding keratitis as well as fatal granulomatous encephalitis. One of the distressing aspects in combating Acanthamoeba infections is the prolonged and problematic treatment. For example, current treatment against Acanthamoeba keratitis requires early diagnosis followed by hourly topical application of a mixture of drugs that can last up to a year. The aggressive and prolonged management is due to the ability of Acanthamoeba to rapidly adapt to harsh conditions and switch phenotypes into a resistant cyst form. One possibility of improving the treatment of Acanthamoeba infections is to inhibit the ability of these parasites to switch into the cyst form. The cyst wall is partially made of cellulose. Here, we tested whether a cellulose synthesis inhibitor, 2,6-dichlorobenzonitrile (DCB), can enhance the effects of the antiamoebic drug pentamidine isethionate (PMD). Our findings revealed that DCB can block Acanthamoeba encystment and may improve the antiamoebic effects of PMD. Using in vitro assays, the findings revealed that DCB enhanced the inhibitory effects of PMD on Acanthamoeba binding to and cytotoxicity of the host cells, suggesting the cellulose biosynthesis pathway as a novel target for the improved treatment of Acanthamoeba infections.     

Tinea Nigra: Report of Twelve Cases in Venezuela

Tinea nigra is a superficial mycotic infection produced by Hortaea werneckii, formerly known as Phaeoannelomyces werneckii, formerly known as Exophiala werneckii, and Stenella araguata, the latter autochthonous in Venezuela, it was first described and named as Cladosporium castellanii in 1973. The present report describes 12 cases in the period of 1972–2002, diagnosed at the Medical Mycology Section at the Tropical Institute of the Universidad Central de Venezuela, in Caracas. This mycosis is more prevalent among young people, with fair skin, from 3 to 28 years of age, who visited beaches and in whom the lesions are more evident. Out of these 12 patients, 8 (66.66%) had Phaeoannelomyces werneckii as the causal agent and 2 (16.67%) Stenella araguata was isolated. Two patients had more than one macule, and curiously in these cases, each lesion was caused by a different species of the aforementioned fungus.     

Successful treatment of antifungal- and cryotherapy-resistant subcutaneous hyalohyphomycosis in an immunocompetent case with topical 5% imiquimod cream

Hyalohyphomycosis is an unusual opportunistic mycotic infection where the tissue morphology of the causative organism is mycelial. Etiological agents, which are not responsible for the otherwise-named infections like aspergillosis, are the species of non-dematiaceous hyaline hyphomycetes including Penicillium, Paecilomyces, Acremonium (formerly known Cephalosporium), Beauveria, Fusarium, and Scopulariopsis. Several cases of Acremonium infection have been described in immunocompromised patients; however it can cause invasive disease in an immunocompetent person very rarely. Optimum therapy of Acremonium infection is unclear because of the limited number of reported cases and conflicting results of therapies. Imiquimod, an imidazoquinoline with potent antiviral, antitumor and immunoregulatory properties, is currently approved for the topical treatment of external anogenital warts and actinic keratosis. Imiquimod has also been found to be effective for other virus-associated dermatologic lesions, including common and flat warts, molluscum contagiosum, and herpes simplex virus type-2 as well as for some cases of cutaneous leishmaniasis. We report herein, for the first time, a case of unusually recalcitrant hyalohyphomycosis of the face due to Acremonium strictum successfully treated with topical 5% imiquimod in an immunocompetent patient, who had failed to respond to various antifungals, including itraconazole, and cryotherapy.     

Keratitis Due to the Wood Saprobic Ascomycete, Auerswaldia lignicola (Family Botryosphaeriaceae), in a Carpenter in India

Keratitis due to Auerswaldia lignicola in a 32-year-old Indian male carpenter is described. At presentation, the patient reported persistent pain and tearing (left eye) in spite of topical antimicrobial therapy for more than 3 weeks. Clinically, mycotic keratitis was suspected, and direct microscopy of corneal scrapings stained by lactophenol cotton blue and Gram stains revealed broad septate hyphae. Intensive topical antifungal therapy was then given for 15 days. The keratitis continued to progress, necessitating therapeutic penetrating keratoplasty. Following the keratoplasty, there was rapid reduction in inflammation and gradual quietening of the eye. Brown–black fungal colonies resembling Lasiodiplodia theobromae were isolated from corneal scrape and corneal button (post-surgery) material on Sabouraud glucose–neopeptone agar; however, sporulation did not occur, so the morphological identification could not be confirmed. Sequence analysis of the 18S rRNA region of extracted fungal genomic DNA yielded an identification of A. lignicola Ariyawansa, J.K. Liu & K.D. Hyde; the sequence data have been deposited in GenBank (A. lignicola strain DK/V4, accession number KC866317.1). Medical management of keratitis due to such rarely reported fungal species may be difficult, necessitating surgical procedures.     

Epidemio-Clinico-Microbiological Study of Mycotic Keratitis in North-West Region of Rajasthan

Aim and Objectives

Mycotic keratitis, with its diverse clinical presentation and difficulties in treatment, makes it a challenging task for clinicians and an important object of study. The aim of present study was to determine the frequency of occurrence and epidemiological association with identification of fungal isolates from mycotic keratitis cases.

Materials and Methods

This was a prospective and observational study conducted in Mycology Laboratory, Department of Microbiology, S.P. Medical College, Bikaner, on corneal scrapings and swabs of a total of 480 patients attending the Ophthalmology OPD, P.B.M. Hospital, Bikaner, during July 2005 to June 2012.

Results

Out of 480 suspected cases, 180 were found to be positive for fungus by smear/culture examination. Increased incidence was seen in the months of May to September with Aspergillus fumigatus being the most common isolate.

Conclusion

Mycotic keratitis, though an age-old disease, still presented with challenging aspects of diagnosis and treatment. The study showed fungal keratitis is prevalent in rural parts of north-west Rajasthan, mainly found in males (age group 21–40 years) with low socio-economic status (farm or factory workers). The most common cause of fungal keratitis was found to be species of Aspergillus.