Sabouraud Dextrose Agar Slants Research Articles

Conjunctival Fungal Flora in a Tertiary Eye Hospital in Nigeria.

The aim of this study is to determine the hospital incidence and pattern of conjunctival fungal flora in adult patients at the Guinness Eye Center Onitsha, Nigeria. New adult patients, without anterior segment disease, were randomly recruited. Using a sterile swab stick, specimen was taken from the inferior conjunctival fornix of each participant's right eye and inoculated into Sabouraud dextrose agar slant in a test tube and incubated at 27°C. The specimens were examined for fungal growth every 48 h for 4 weeks. Specimens with fungal growth were further examined under a high power microscope for fungal identification and characterization. A total of 225 patients (105 males, 120 females) were examined. The age range was 18-75 years; mean age was 41 ± 17.1 years; 62 (27.6%) were culture-positive: 25 (40.3%) were males and 37 (59.7%) were females (P >0.05). Both moulds and yeasts were isolated with moulds constituting 44 (74.2%). Aspergillus [26 (41.9%)] and Candida [16 (25.8%)] were the commonest organisms. Participants >60 years had the greatest burden. Pensioners (61.5%), traders (44.0%), farmers (30.1%), and artisans (27.3%) were occupational groups with significantly higher proportions of culture-positive specimens (P < 0.05). Over a quarter of new adult patients without anterior segment disease harbour fungi, some of which are pathogenic, in their conjunctival fungal organisms. While Aspergillus and Candida were the commonest isolates, older participants, pensioners, traders, farmers, and artisans had significantly higher proportion of culture-positive specimens. These findings should be considered when formulating pre-operative guidelines for ocular surgery in our environment.

Cutaneous Sporotrichosis Presenting as Clinical Feature of Facial Cellulitis in an Adult.

Dear Editor: Sporotrichosis is subcutaneous mycosis due to the dimorphic fungus Sporothrix schenckii complex1,2,3. Fixed cutaneous sporotrichosis may present as acneiform, nodular, ulcerated, or verrucous. Less often the patients may present with facial cellulitis. Clinically initial cutaneous lesion is most frequently on an exposed area, commonly the face in children and the arms in adults4. We report the case of cutaneous sporotrichosis of the upper eyelid in adult. The patient was a 57-year-old female who showed with localized erythematous plaques with swelling on the right upper eyelid for 1 month (Fig. 1A). She has done persimmon tree farming without any recongnizable injury and took blepharoplasty 3 months before the lesion appears. Both of them were suspicious as a route of the infection. Cultures from biopsy specimen were performed in Sabouraud's dextrose agar slants. As a result, black, moist and wrinkled colonies of S. schenckii were observed in two slants (Fig. 2A). Septate branched mycelia and clustered conidia were observed in slide culture (Fig. 2B). Histopathologically, pseudoepitheliomatous hyperplasia and chronic granulomatous inflammation were observed on H&E stain. Many round spores were found around dermis in periodic acid Schiff stain (Fig. 2C). The nucleotide sequence of internal transcribed spacer for clinical isolate was identical to that of S. schenckii strain KMU 3360 (GenBank accession number {type:entrez-nucleotide,attrs:{text:AB122043.1,term_id:40714495,term_text:AB122043.1}}AB122043.1). Fig. 1 (A) Localized erythematous plaques with swelling on the right upper eyelid. (B) Skin lesions showing completely cured after six months of treatment. Fig. 2 (A) Dark brown to black, moist and wrinkled colonies on Sabouraud's dextrose agar slants at 25℃ for 2 weeks. (B) Septate branched mycelia and clustered conidia were observed in slide culture (lactophenol cotton blue stain, ×1,000), (C) ... Itraconazole is the drug of choice for treatment of sporotrichosis5. The patient was treated with oral itraconazole 200 mg/day for six months. The skin lesions were completely cured and no recurrence is observed during 1 year of follow up (Fig. 1B). Although cutaneous sporotrichosis is not unusual, the clinical presentation and the location of the lesion might have led dermatologist into misdiagnosis as facial cellulitis. Therefore, we report this case to emphasize the fungal culture of the skin lesions presenting as clinical feature of faical cellulitis.

Clinicopathologic assessment of Candida colonization of oral leukoplakia.

Leukoplakia is the most common premalignant lesion of the oral mucosa. We studied the colonization of Candida in oral leukoplakia using direct microscopy, culture and histopathology to determine if there is a statistical correlation between Candida invasion and the clinical appearance and presence of epithelial dysplasia in leukoplakia. Samples were collected from 40 patients with oral leukoplakia and 21 controls. The swabs collected were used to inoculate Sabouraud's dextrose agar slant and for direct microscopy with Gram's stain. Culture growths were subjected to germ tube and corn meal agar tests to differentiate between Candida albicans and non-albicans groups. Biopsies were also done in all patients for histopathological confirmation; Gomori's methanamine silver stain was used to identify fungal invasion of lesional epithelium. Nineteen cases of leukoplakia showed Candida on direct smears, compared to 3 controls. Eighteen cases and one control showed growth of Candida on culture. Non-homogenous leukoplakia showed a higher positivity rate on microscopy and culture than homogenous lesions. All these correlations were statistically significant. Forty percent of leukoplakia cases were simultaneously positive for Candida on direct microscopy, culture and histopathologic evaluation. No significant difference was found between non-dysplastic and distinctly dysplastic lesions with respect to Candida detection on microscopy or culture.

English

&nbsp; &nbsp;&nbsp; &nbsp; &nbsp;Earthworm plays a major role in the proper functioning of the soil ecosystem. It acts as scavenger and helps in recycling of dead and decayed plant material by feeding on them. Earthworm increases the soil fertility and is often referred to as a farmer&rsquo;s friend. Earthworms have been used in medicine for various remedies. The paste prepared from earthworm, Eudrilus eugeniae was tested for antibacterial, antifungal activities. For the antimicrobial screening, four species of bacterial isolate and two species of fungal isolates were selected. The bacterial cultures were used for antimicrobial testing maintained on nutrient agar slant and the fungal strains were maintained on Sabouraud dextrose agar slant at 4&deg;C. Minimum inhibitory concentration (MIC) was determined using micro dilution broth method. Earthworm paste at a dose of 100 &micro;l was able to inhibit the growth of bacteria of S. aureus at a maximum level as compared to other bacteria; the growth of fungal Candida albicans was much inhibited. The MIC results indicated that earthworm paste at a dose of 200 &micro;l inhibited the bacterial growth. These studies may lead to the formulation of new antimicrobial drug. The antimicrobial activity of the paste was determined by an agar diffusion method using well and disc, the study clearly indicates that the paste contain a good antibacterial potential and the bioactive compounds to inhibit the growth of bacteria and fungi. Hence earthworm paste (EP) has a good potential to develop a new drug. &nbsp; Key words: Eudrilus eugeniae, antimicrobial activity, fungal and bacteria strains

Dermatophytes from skin lesions of domestic animals in Nsukka, Enugu State, Nigeria

Dermatophytes are well-recognized cutaneous fungi with public health implications. In Nigeria, several studies have been carried out on dermatophytosis in humans; however, data on dermatophytes in animals are lacking. This study was conducted to determine the occurrence and species of dermatophytes in skin lesions in domestic animals in Nsukka Agricultural Zone of Enugu State, Nigeria. Forty-six domestic animals (dogs, goats, sheep and pigs) presented for sale in the local markets in the study area and with suspected lesions of dermatophytosis were used for the study. Plucked hairs and epidermal scales from the skin lesions of affected animals were inoculated on Sabouraud dextrose agar slants containing 0.05 mg/mL of chloramphenicol and 0.5 mg/mL of cycloheximide. Inoculated slants were incubated at room temperature (27°C) for up to 4 weeks and examined at 2-3 day intervals for fungal growth. Laboratory identification of the fungal isolates was based on their colonial, microscopic and biochemical characteristics. Of the 46 animals with suspected lesions of dermatophytosis, six (13.0%) were positive for a dermatophyte, and the following dermatophytes were identified: Microsporum gypseum, two of 12 sheep; Microsporum audouinii, one of 16 dogs; Trichophyton mentagrophytes, one of 16 dogs and one of 12 sheep; and Trichophyton schoenleinii, one of 13 goats. Anthropophilic dermatophytes are among the fungal agents associated with dermatophytosis in animals in Nsukka Agricultural Zone. These dermatophytes could constitute health risks to humans in contact with the animals.

Sporotrichosis Caused By Sporothrix globosa in Rio De Janeiro, Brazil: Case Report

This report describes the first isolation of Sporothrix globosa from a Brazilian patient. A 77-year-old woman was examined for sporotrichosis infection. Histopathological examination of skin biopsy revealed chronic granulomatous infiltrate with microabcess. Furthermore, S. schenckii-like yeasts were evident as demonstrated by PAS and Grocott stains. The fungus was identified based on colony morphology on Sabouraud Dextrose Agar slants, Potato Dextrose Agar, and Corn Meal Agar, microscopic morphology on slides cultures, and assimilation of different carbon sources. The species confirmation was made by molecular methodology.

Cutaneous phaeohyphomycosis caused by Veronaea botryosa observed as sclerotic cells in tissue

A 65‐year‐old Japanese female farmer had an erythematous, slightly scaly, indurated plaque on the dorsum of the left wrist of more than 3 years’ duration. She had a history of hepatitis C. Her medical history and family history were otherwise unremarkable. The lesion had begun as a small region of erythema on the dorsum of the left wrist 3 years previously and had gradually enlarged. The patient had seen a local physician and had been treated with topical steroids, topical antifungals, and topical vitamin D3 agents without improvement.Initial laboratory studies revealed slightly decreased numbers of white blood cells and thrombocytes, decreased albumin levels, and elevated liver enzymes, reflecting chronic hepatitis C. Clinical examination of the lesion revealed a well‐demarcated, erythematous, slightly scaly, indurated plaque of 22 mm × 28 mm in size on the dorsum of the left wrist (Fig. 1). Superficial lymph nodes were nonpalpable. Direct microscopy revealed aggregates of thick‐walled spores, considered to be sclerotic cells, from which budding brown hyphae emanated (Fig. 2a).A well‐demarcated, erythematous, slightly scaly, indurated plaque of 22 mm × 28 mm in size on the dorsum of the left wristimage(a) Microphotograph of scales in KOH reveals aggregates of thick‐walled, brown sclerotic cells and budding brown hyphae (×400). (b) Conidia are bicellular, gourd‐shaped at maturity and pale brown. They are sympodially produced by darker conidiogenous cells (slide culture, ×400)imageCutaneous biopsy specimens revealed a chronic granulomatous inflammation infiltrated with lymphocytes, histiocytes, neutrophils, and multinucleated giant cells. Within the microabscess and within one of the giant cells were large, brown, thick‐walled cells, which were identified as sclerotic cells, and brown septate hyphae.Culture on a Sabouraud dextrose agar slant at 26 °C yielded villous, green‐tinged, black–gray colonies, with a central button‐like protuberance. By 28 days, these colonies had reached 4.8 cm in diameter. A slide culture showed pale brown septate hyphae bearing lateral and terminal conidiophores. Conidiophores were erect, straight, or flexuous, unbranched or occasionally loosely branched, smooth‐walled, septate, and brown. Conidiogenous cells were terminal or lateral and cylindrical in the apical part with numerous dark brown scars fading away to the apex. Conidia were sympodial and bicellular, with rounded apices and truncated bases (Fig. 2b). On the basis of these morphologic features, we identified the mold as Veronaea botryosa. Surgical excision of the lesion was performed, resulting in complete healing.

A Nail Drilling Method Suitable for the Diagnosis of Onychomycosis

We describe a nail drilling method suitable for the diagnosis of onychomycosis. Thirty-three patients with onychomycosis in which the big toenail had a white band or spike were enrolled in this study. We drilled a hole about 3-mm-wide in the most proximal part of the white band or spike using a ball-shaped metal file and then, through the hole, sampled the underlying nail material softened by fungi after removing the superficial hard nail plate. Fungi in 32 (97.0%) of the nail samples were detected by direct KOH examination. When incubated on Sabouraud's dextrose agar slant with chloramphenicol and cycloheximide, fungal cultures were obtained from 27 (81.8%) of the 33 nail samples. Fourteen of the fungal isolates were identified as T. rubrum, 11 as T. mentagrophytes, and 2 as Acremonium sp. The nail drilling method is suitable for diagnosing onychomycosis with a white band or spike, because it gives a high isolation rate and leaves the patients' nail relatively more intact compared with other methods.

Frequency and risk factors of dermatophytosis in students living in rural areas in Eskişehir, Turkey.

Our study included 2384 students from five villages around Eskişehir, Turkey. We asked every student for their personal identification and also for their sanitation in order to get an idea about dermatophytosis. Samples taken from suspicious lesion were collected and inoculated onto Sabouraud dextrose agar slants. For identification of fungi which were grown, macroscopic appearance of colonies, microscopic examination and biochemical tests were used. We found suspicious lesions in 245 (10.3%) and diagnosed dermatophytosis in 86 (3.6%) of the students. The dermatophyte species were Trichophyton rubrum 37 (43%) at first, Trichophyton mentagrophytes 17 (19.8%), Microsporum canis 11 (12.8%), Microsporum gypseum 8(9.3%), Epidermophyton floccosum 6 (7%), Trichophyton verrucosum 6 (7%) and Trichophyton violaceum 1 (1.1%). Tinea pedis (59.3%) was the most frequent clinic form of dermatophytosis, followed by tinea corporis (22.1%), tinea capitis (9.3%), tinea manum (7.0%) and tinea unguium (2.3%). Older age, male gender, poor hygiene, living in dormitory, low level mother education, history of dermatophytosis within family and sanitary conditions were computed as independently variables associated with dermatophytosis infection. For prevention and control of dermatophyte infection in children living rural areas, field studies should be done and sanitary conditions should be improved.

Are giant cells conidia in Sporothrix schenckii? -Freeze-fracture electron microscopic observation-.

Sporothrix schenckii is a well-known pathogenic dimorphic fungus. In this study, we focused on the plasma membrane ultrastructures of giant cells of S. schenckii seen mainly on Sabouraud's dextroseagar slant medium. In the organisms grown for 1, 2, 3, 4, 6 and 8 weeks at 27 and 37 degrees C on brain heart infusion and Sabouraud's dextrose agar slant media, the number of conidia, hyphae, brownish and non-brownish giant cells were counted in ten separated areas under light microscope (x100) to determine the culture conditions under which giant cells were generated. The results showed that brownish giant cells were predominantly seen after longer cultivation periods. Using freeze-fracture electron microscopy, larger oval- or round shaped cells can be identified as conidia by their plasma membrane ultrastructure characteristics, i.e, trench-like invaginations seen in ordinary mature conidia (Maeda M et al.; Can J Microbiol 33: 40, 1987). From these structural features seen by freeze-fracture electron microscopy, giant cells appeared possibly be conidia and were suggested to be starved because of their predominant existence under longer cultivation conditions.

Antigenic relations between pathogenic Paecilomyces lilacinus and P. variotii and other hyalohyphomycotic agents

Objective: The antigenic relations between Paecilomyces variotii and Paecilomyces lilacinus were studied by comparison of their exoantigens. Methods: Forty-one isolates of hyalohyphomycosis-causing fungi including P. lilacinus (n = 15) and P. variotii (n = 12), one isolate each of Paecilomyces inflatus, Paecilomyces marquandii, and Paecilomyces carneus, Fusarium species (n = 3), Neosartorya pseudofischeri (n = 5), and Penicillium species (n = 3), were studied using the exo-antigen test. Ten-day-old extracts derived from Sabouraud's dextrose agar slants were concentrated (25x), and reacted against rabbit anti- P. lilacinus and anti- P. variotii sera in the presence of partially purified homologous antigens (20x) prepared from 5-week-old shaken cultures. Extracts of isolates of P. lilacinus and P. variotii produced one to three lines of identity against the reference homologous antisera. No cross-reactivity or bands of non-identity were observed with other Paecilomyces isolates, N. pseudofischeri, Fusarium species, and Penicillium species isolates. Conclusion: The results of this investigation indicate that P. lilacinus and P. variotii are antigenically distinct and that they can be identified rapidly and accurately by their exoantigens. Exoantigen extracts of morphologically similar fungi and other species of Paecilomyces isolates studied did not possess antigens common to P. lilacinus and P. variotii.

Safety considerations in handling exoantigen extracts from pathogenic fungi.

Currently, exoantigen test procedures for identifying mycelial form cultures of pathogenic molds require that the fungi being extracted be treated with thimerosal to render them safe for handling. Recent studies have demonstrated that thimerosal may not be fungicidal. In view of these reports, we investigated the effects of thimerosal and formaldehyde on a variety of exoantigen preparations. Mature mycelial form fungal cultures, including cultures of Blastomyces dermatitidis, Coccidioides immitis, and Histoplasma capsulatum and morphologically similar fungi, were grown on Sabouraud dextrose agar slants and treated with 0.02, 0.04, and 0.08% thimerosal for 24 and 48 h and with 0.2 and 0.5% formaldehyde for 24 and 48 h. We found that 0.5% formaldehyde killed all of the fungi studied, whereas 0.2% formaldehyde permitted the growth of only one fungus; 0.02, 0.04, and 0.08% thimerosal were fungistatic. Furthermore, 0.2 and 0.5% formaldehyde and 0.08% thimerosal affected certain antigens adversely. For those investigators who prefer to use 0.02% thimerosal and to work with sterile extracts, we recommend that the procedure be modified, and we advocate sterilization of extracts by passage through membrane filters.

Freeze-etching observations of Trichophyton Violaceum.

Trichophyton violaceum was cultured on a Sabouraud's dextrose agar slants for freeze-fracture replication. It was cryoprotected with glycerol and sucrose. The surface structure of the cell wall showed a rodlet pattern. Invagination-like craters were observed in the plasmalemma. The nuclear envelope with pores, mitochondria, and vacuoles were clearly shown. Three-dimensional views of the fine structure were presented.

Accidental Isolation of Trichophyton Mentagrophytes from the Floor of a Schoolhouse

A proven case of cryptococcosis was seen in the Department of Dermatology, University of Michigan Hospital, and attempts were made to trace the possible source of infection. At the onset of his illness, the patient was working as a janitor in the Foster School of Ypsilanti, Michigan. This is a rambling, one-story schoolhouse built during the Second World War. In view of the recent reports of the isolation of Cryptococcus neoformans in nature (1, 4, 5), samples of soil and floor dust from the schoolhouse were obtained: Sample 1 and Sample 2 were soils from under drains, Sample 3 was from the sweepings gathered in the bag of a vacuum cleaner used to clean the floors, and Sample 4 was from dirt accumulated on a motor-driven rotary floor brush. Each sample was suspended in physiological saline solution in a 100 ml graduate with vigorous agitation and left to settle at room temperature. After 3 hours, portions of the top layers of the suspensions were inoculated on duplicate Sabouraud's-dextrose-agar slants, both with and without penicillin and streptomycin. One set of the cultures was left at room temperature and the duplicate set, at incubator temperature. These were examined periodically for growth and discarded after six weeks. For animal inoculation, one ml of the supernatant layer of each suspension was injected intraperitoneally into mice. Each suspension was used to inoculate five mice. Pieces of the livers, spleens and lungs of animals that died were planted on Sabouraud's dextrose agar slants. The remaining animals were sacrified after three weeks and the organs similarly planted. The cultures directly planted with the samples did not show any growth of pathogenic fungi, and most eventually became overgrown with contaminants. However, Trichophyton mentagrophytes was isolated from the livers or spleens of two animals inoculated with portions of Sample 3, composed of vacuum cleaner sweepings. The macroscopic and microscopic features of these isolates were characteristic (FIGS. 1,