Coccidioidomycosis in India: Report of a third imported case.

We describe the third fatal case of imported coccidioidomycosis in India in a 31-year-old mechanical engineer originally from Andhra Pradesh, India, who lived in Gwinner, North Dakota. He had traveled to Arizona in summer of 2006, where he drove tractors in a dusty environment at a tractor production facility near Phoenix, Arizona. He was human immunodeficiency virus (HIV) positive. Initially, he was treated in Fargo, North Dakota, in 2006, with liposomal amphotericin B followed by oral fluconazole. Antiretroviral treatment for HIV infection was started. He moved back to India and was admitted to the intensive care unit of St. John's Medical College and Hospital, Bangalore, India. His blood cultures yielded Coccidioides sp. The identity of the isolate was confirmed using the Gen Probe Accuprobe test at the Centers for Disease Control and Prevention, Atlanta, Georgia. In spite of initiation of treatment with antifungal agents (amphotericin B and fluconazole), his condition deteriorated and he expired three days following his admission to the hospital.

Occurrence of pathogenic fungi in soil of burrows of rats and of other sites in bamboo plantations in India and Nepal.

This study examined 215 samples of soil from burrows of rats, other sites in bamboo plantations in different parts of India and Nepal by dilution plating and mouse passage technique for occurrence of Penicillium marneffei and other pathogenic fungi. None of the samples including 25 collected from the burrows of a bamboo rat (Cannomys badius) known to be a carrier of P. marneffei, was positive for the fungus. Among the pathogenic fungi recovered were four isolates of Pseudallescheria boydii (including one from Nepal), two of Trichosporon asteroides, one of Scytalidium hyalinum, 23 isolates of Trichophyton mentagrophytes var. mentagrophytes (including two from Nepal), and two of Microsporum gypseum. Fourteen of the 23 isolates of T. mentagrophytes var. mentagrophytes when tested with the mating types of Arthroderma vanbreuseghemii were found to be of the '+' mating type. The frequent recovery of this dermatophyte from soils of bamboo plantations in several parts of India is remarkable. The study also demonstrates for the first time the occurrence of P. boydii and T. mentagrophytes var. mentagrophytes in Nepalese soil. Among the other fungi recovered were several isolates of species of Aspergillus, Penicillium, Paecilomyces, Fusarium, Chrysosporium, Acremonium, Rhizopus, Mucor, Geotrichum, Trichosporon and Rhodotorula.

Phaeoacremonium krajdenii, a cause of white grain eumycetoma.

We describe the first case of white grain pedal eumycetoma caused by Phaeoacremonium krajdenii in a 41-year-old man from Goa, India. Based on histological examination of biopsy tissue showing serpentine granules, a culture of the granules yielding phaeoid fungal colonies, and morphological characteristics and sequence comparison of the partial beta-tubulin gene with the ex-type isolate of P. krajdenii, the causal agent was identified as P. krajdenii.

Blastomycosis in a South Indian patient after visiting an endemic area in USA.

We describe a case of blastomycosis in a diabetic patient from South India who had visited Milwaukee, Wisconsin, an endemic area for blastomycosis in the USA. After his return to Bangalore, India, the patient developed intermittent fever of moderate to high grade, cough, loss of weight and appetite, and abscesses in the left cubital fossa and thigh regions. Systemic examination at our hospital revealed that he had dullness to percussion over the chest region and decreased breath sounds. Direct examination of Gram-stained smears of the pus from an abscess showed many broad-based budding yeast cells and culture yielded a dimorphic fungus later identified as Blastomyces dermatitidis. Histologic examination of the curettage tissue slides stained with hematoxylin and eosin, periodic acid Schiff's reagent, and Gomori's methenamine silver stain procedures showed many broad-based budding cells characteristic of B. dermatitidis. The patient was successfully treated, initially with amphotericin B, followed by oral itraconazole for a period of 6 months. Blastomycosis cases in India are reviewed and the likely source of infection in this patient is discussed.

Aspergillus endocarditis presenting as femoral artery embolism.

Fungal valvular endocarditis is an unusual cause of endocarditis, yet very important because of its historically poor prognosis. We report two fatal cases of fungal valvular endocarditis following cardiovascular surgery, presenting as femoral artery embolism. Aspergillus terreus and A. flavus were the causative agents of endocarditis in the two patients. Diagnosis was established very early by culture of the emboli and was confirmed later by isolation of the same Aspergillus species from the resected valve tissue.

Subcutaneous phaeohyphomycotic abscess caused by Pleurophomopsis lignicola.

A 41-year-old man with a past medical history of diabetes mellitus type II, AIDS (diagnosed 2 years earlier; CD4 count < 10), peripheral neuropathy, and pulmonary tuberculosis of 2 years duration was admitted to the hospital with abnormal liver function tests. There was a chronic hepatitis/cholestasis that had worsened while the patient was undergoing directly observed tuberculosis therapy. On admission, the patient complained of a painful swelling on his right arm. In the posterior aspect of the arm, there was a 3-4-cm subcutaneous mass that was fluctuant, mobile and tender. Incision of the mass released yellowish pus mixed with blood. Direct examination of the pus in KOH mounts and Gram-stained smears revealed subhyaline, septate, branched hyphae. When the pus was cultured on Sabouraud dextrose agar containing chloramphenicol, several velvety, olivaceous grey colonies grew after 7 days at 25 degrees C. When grown on oatmeal agar, the fungus produced subglobose, rostrate pycnidia with phialidic conidiogenous cells, and 1-celled cylindrical conidia. It was identified as Pleurophomopsis lignicola Petrak. This report describes the third known case of subcutaneous infection caused by P. lignicola in an immunocompromized patient.

Subcutaneous phaeohyphomycosis caused by Veronaea botryosa in the People's Republic of China.

The second case of phaeohyphomycosis caused by Veronaea botryosa in China, in a 12-year-old boy from Jiangsu Province, is presented. Based on direct examination of the scrapings from crusted lesions; histologic examination of the biopsy tissue showing septate, phaeoid hyphal elements; and the culture exhibiting sympodial, conidiogenous cells producing predominantly two-celled, cylindric conidia, the etiologic agent was identified as V. botryosa.

Coccidioidomycosis in India: report of a second imported case.

We describe a fatal case of imported coccidioidomycosis in India in a 22-year-old male who worked in Tucson, Arizona, approximately four years prior to his illness. The diagnosis was based on the presence of characteristic spherules with endospores in biopsy tissue of lymph nodes, bone and pus from a chronic discharging sinus in the left gluteal region and isolation of Coccidioides immitis in culture. C. immitis is one of the most infectious and virulent fungal pathogens and poses a serious occupational hazard for laboratory personnel, especially in areas where the disease is not endemic. To reduce the role of laboratory-acquired infection, all procedures that involve manipulation of cultures of C. immitis should, whenever possible, be conducted in a biological safety cabinet.

Invasive aspergillosis outbreak on a hematology-oncology ward.

An outbreak of invasive aspergillosis occurred in a community hospital in temporal association with construction activity. Epidemiological investigation showed that patients who are at highest risk comprise a small group and are readily identifiable. Clinicians should strive to protect these patients, following guidelines published by the Centers for Disease Control and Prevention.

White grain mycetoma caused by a Cylindrocarpon sp. in India.

We describe a case of white grain eumycetoma of the foot of an Indian male caused by a slow-growing, poorly sporulating fungus that does not match any known agent of this infection. Histologic examination of a biopsy tissue specimen showed oval, lobular, white granules composed of hyaline, septate hyphae, and thick-walled chlamydospores. Culture of granules from a draining sinus yielded compact, very-slow-growing, poorly sporulating colonies producing a strong reddish brown pigment that diffused into the medium. The fungus was identified as a Cylindrocarpon sp. based on the development of rare cylindrical conidia borne from solitary phialides lacking collarettes, in addition to chlamydospores formed singly or in short chains.

Report of the first human case of lobomycosis in the United States.

We describe the first human case of lobomycosis caused by Lacazia loboi in a 42-year-old white male resident of Georgia. The patient had traveled to Venezuela 7 years earlier, where he had planned to rappel down Angel Falls in Canaima. Although he never actually rappelled the falls, he did walk under the falls at least three times, exposing himself to the high water pressures of the falls. He noticed a small pustule with surrounding erythema developing on the skin of his right chest wall. The lesion gradually increased in size and had an appearance of a keloid. For cosmetic reasons, the patient sought medical treatment to remove the lesion. After an uncomplicated excision of the lesion, the patient recovered completely. The excised tissue was fixed in formalin for pathologic examination. Tissue sections stained by hematoxylin and eosin, periodic acid-Schiff stain, and Gomori methenamine silver stain procedures showed numerous histiocytes, multinucleated giant cells, and numerous globose or subglobose, lemon-shaped cells producing multiple blastoconidia connected by narrow tube-like connectors and catenate chains of various lengths characteristic of L. loboi.

Rapid identification of Candida dubliniensis with commercial yeast identification systems.

Candida dubliniensis is a newly described species that is closely related phylogenetically to Candida albicans and that is commonly associated with oral candidiasis in human immunodeficiency virus-positive patients. Several recent studies have attempted to elucidate phenotypic and genotypic characteristics of use in separating the two species. However, results obtained with simple phenotypic tests were too variable and tests that provided more definitive data were too complex for routine use in the clinical laboratory setting. The objective of this study was to determine if reproducible identification of C. dubliniensis could be obtained with commercial identification kits. The substrate reactivity profiles of 80 C. dubliniensis isolates were obtained by using the API 20C AUX, ID 32 C, RapID Yeast Plus, VITEK YBC, and VITEK 2 ID-YST systems. The percentages of C. dubliniensis isolates capable of assimilating or hydrolyzing each substrate were compared with the percentages from the C. albicans profiles in each kit's database, and the results were expressed as percent C. dubliniensis and percent C. albicans. Any substrate that showed >50% difference in reactivity was considered useful in differentiating the species. In addition, assimilation of methyl-alpha-D-glucoside (MDG), D-trehalose (TRE), and D-xylose (XYL) by the same isolates was investigated by the traditional procedure of Wickerham and Burton (L. J. Wickerham and K. A. Burton, J. Bacteriol. 56:363-371, 1948). At 48 h (the time recommended by the manufacturer for its new database), we found that the assimilation of four carbohydrates in the API 20C AUX system could be used to distinguish the species, i.e., glycerol (GLY; 88 and 14%), XYL (0 and 88%), MDG (0 and 85%), and TRE (15 and 97%). Similarly, results with the ID 32 C system at 48 h showed that XYL (0 and 98%), MDG (0 and 98%), lactate (LAT; 0 and 96%), and TRE (30 and 96%) could be used to separate the two species. Phosphatase (PHS; 9 and 76%) and alpha-D-glucosidase (23 and 94%) proved to be the most useful for separation of the species in the RapID Yeast Plus system. While at 24 h the profiles obtained with the VITEK YBC system showed that MDG (10 and 95%), XYL (0 and 95%), and GLY (26 and 80%) could be used to separate the two species, at 48 h only XYL (6 and 95%) could be used to separate the two species. The most useful substrates in the VITEK 2 ID-YST system were TRE (1 and 89%), MDG (1 and 99%), LAT (4 and 98%), and PHS (83 and 1%). While the latter kit was not yet commercially available at the time of the study, it would appear to be the most valuable for the identification of C. dubliniensis. Although assimilation of MDG, TRE, and XYL proved to be the most useful for species differentiation by the majority of commercial systems, the results with these carbohydrates by the Wickerham and Burton procedure were essentially the same for both species, albeit following protracted incubation. Thus, it is the rapidity of the assimilation achieved with the commercial systems that allows the differentiation of C. dubliniensis from C. albicans.

Indigenous disseminated Penicillium marneffei infection in the state of Manipur, India: report of four autochthonous cases.

We describe four cases of disseminated infection caused by endemic Penicillium marneffei in human immunodeficiency virus (HIV)-infected patients from the Manipur state of India. The most common clinical features observed were fever, anorexia, weight loss, hepatosplenomegaly, and, more importantly, skin lesions resembling molluscum contagiosum. The diagnosis in each of the four cases was achieved by direct examination of smears, observance of intracellular yeast-like cells multiplying by fission in biopsied tissue from skin lesions, and isolation of the dimorphic P. marneffei in pure culture in each case. In one case, fluorescent antibody studies allowed specific diagnosis. This report documents a new area in which P. marneffei is endemic, located in eastern India, and describes the first occurrence in India of P. marneffei in HIV-infected patients as well as the extension of the areas of P. marneffei endemicity westward to the northeastern state of Manipur.

Phaeohyphomycotic ulcer caused by Phialophora verrucosa: successful treatment with itraconazole.

We report the first well documented case of subcutaneous phaeohyphomycotic infection caused by Phialophora verrucosa in India. Examination of the biopsied tissue from an ulcerating lesion on the shin of the left leg of a 45-year-old woman from Bombay, India, showed numerous dematiaceous, septate, branching hyphal elements and thick-walled cells characteristic of phaeohyphomycosis. Cultures of the scrapings from the lesion and of the biopsied tissue yielded a pigmented fungus that was identified as P. verrucosa. Initial treatment with fluconazole followed by oral itraconazole for 30 days and local application of a copper sulphate solution resulted in complete resolution of the lesion. Treatment with itraconazole was continued for an additional 3 months after complete healing. No new lesions developed and the patient did not show jaundice, hepatosplenomegaly or any other signs of toxicity.

Misleading manifestations of Coccidioides immitis in vivo.

We describe a case of coccidioidomycosis in which several unusual morphologic forms of Coccidioides immitis occurred in biopsy tissue from the right lower lung of a patient. To our knowledge, this is the first case where so many diverse morphologic forms were manifested in a single patient in the absence of typical endosporulating spherules. Immature spherules demonstrating segmentation mimicked morula forms of Prototheca spp. Certain elements resembled budding cells of Blastomyces dermatitidis. These consisted of juxtaposed immature spherules without endospores, a germinating endospore, or thick-walled hyphal cells. Branched, septate hyphae and moniliform hyphae consisting of chains of thick-walled arthroconidia or immature spherules were also present. Complement fixation and immunodiffusion tests performed on the patient's serum were negative for C. immitis, B. dermatitidis, and Histoplasma capsulatum antibodies. Fluorescent-antibody studies were carried out with a specific C. immitis conjugate. All of the diverse fungal tissue elements stained positive with a moderate to strong (2 to 3+) intensity.

Phaeohyphomycosis caused by Phaeoacremonium inflatipes.

Phaeoacremonium inflatipes, one of three species previously classified as strains of Phialophora parasitica, was identified as the causal agent of a subcutaneous infection of the left foot of an 83-year-old woman from South Carolina. The patient had a granulomatous growth over the anteromedial aspect of her left foot. It was surgically excised, which led to complete healing without complications. Tissue sections of the excised mass stained with hematoxylin and eosin and Gomori's methenamine silver strains showed many septate hyphal elements of various lengths, some exhibiting brownish pigment in the cell walls of the hyphae. Portions of the tissue, when cultured, yielded many colonies which were initially glabrous, off white becoming velvety, greyish brown on aging. Microscopically, their hyphae were septate, branched, and phaeoid and bore lateral and terminal, erect, septate conidiophores. The conidiogenous cells (phialides) were terminal or lateral, mostly monophialidic, subcylindrical to spinelike in shape, and constricted at their bases and bore funnel-shaped, inconspicuous collarettes at their tips. The conidia were subhyaline, oblong, and ellipsoid to allantoid.

Utility of random amplified polymorphic DNA PCR and TaqMan automated detection in molecular identification of Aspergillus fumigatus.

We developed a method for the identification of Aspergillus fumigatus fungal isolates by using random amplified polymorphic DNA (RAPD) PCR (RAPD-PCR) cloning and the TaqMan LS50B fluorogenic detection system (Perkin-Elmer Corp., Applied Biosystems, Foster City, Calif.). DNA from seven clinically important Aspergillus species was screened by RAPD-PCR to identify section- or species-specific amplicons. With the OPZ19 RAPD primer a 1,264-bp product was amplified from all A. fumigatus strains initially examined but not from other species. A partial DNA sequence of this product was used to design a specific primer pair, which generated a single 864-bp fragment with DNA from 90 of 100 A. fumigatus isolates when a "touchdown" (65-->55 degrees C) annealing protocol was used. The TaqMan system, a fluorogenic assay which uses the 5'-->3' endonuclease activity of Taq DNA polymerase, detected this 864-bp product with DNA from 89 of these 90 A. fumigatus strains; 1 DNA sample generated an indeterminate result. With DNA from three morphologically typical A. fumigatus isolates, six white ("albino") A. fumigatus isolates, and five of six Neosartorya species (non-A. fumigatus members of the section Fumigati), the 864-bp product was amplified differentially at an annealing temperature of 56 degrees C but not with the touchdown annealing format. No amplicon was detected with DNA from 56 isolates of heterologous Aspergillus, Penicillium, and Paecilomyces species or from Neosartorya fennelliae; TaqMan assay results were either negative (51 isolates) or indeterminate (5 isolates) for all isolates. This RAPD-PCR and TaqMan assay offers promise as a nucleic acid-based system that can be used for the identification of filamentous fungal isolates and that requires no postamplification sample manipulations.

Biosafety considerations in handling medically important fungi.

Over 500,000 workers in the USA alone are employed in laboratories that range from small physician offices to large clinical laboratories handling microbes for comprehensive research and/or diagnostic work. These workers are exposed to a variety of potential occupational health risks such as exposure to infectious clinical materials, environmental specimens, cultures, complex and inflammable chemicals, radiation, and electrical and mechanical hazards. As members of the International Society for Human and Animal Mycology, we have no policy statement on biosafety standards for handling medically important fungi. The intent of the symposium is to cover some of the important aspects of biosafety; (1) standards in handling dimorphic fungal pathogens; (2) the principles and criteria of biosafety levels and classification of known medically important fungi, aerobic actinomycetes, environmental fungi according to their biosafety levels; (3) medically important fungal waste and its safe disposal; and (4) biosafety and regulatory considerations in handling and mailing medically important fungi in a culture collection.