Multidrug-resistant Trichosporon asahii infection of nongranulocytopenic patients in three intensive care units.

Trichosporon asahii (Trichosporon beigelii) infections are rare but have been associated with a wide spectrum of clinical manifestations, ranging from superficial involvement in immunocompetent individuals to severe systemic disease in immunocompromised patients. We report on the recent recovery of T. asahii isolates with reduced susceptibility in vitro to amphotericin B (AMB), flucytosine, and azoles from six nongranulocytopenic patients who exhibited risk factors and who developed either superficial infections (four individuals) or invasive infections (two individuals) while in intensive care units. The latter two patients responded clinically and microbiologically to AMB treatment. All six isolates were closely related according to random amplified polymorphic DNA studies and showed 71% similarity by amplified fragment length polymorphism analysis, suggesting a common nosocomial origin. We also review the literature pertaining to T. asahii infections and discuss the salient characteristics of this fungus and recent taxonomic proposals for the genus.

Differentiation of Candida dubliniensis from Candida albicans on staib agar and caffeic acid-ferric citrate agar.

The methods currently available for the identification of the pathogenic yeast Candida dubliniensis all have disadvantages in that they are time-consuming, expensive, and/or, in some cases, unreliable. In a recent study (P. Staib and J. Morschhäuser, Mycoses 42:521-524; 1999) of 14 C. dubliniensis and 11 C. albicans isolates, it was suggested that the ability of C. dubliniensis to produce rough colonies and chlamydospores (chlamydoconidia) on Staib agar (SA) provided a simple means of differentiating it from its close relative C. albicans. In the present investigation, we examined the colony morphology and chlamydospore production of 130 C. dubliniensis and 166 C. albicans isolates on SA and on the related defined medium caffeic acid-ferric citrate agar (CAF). All of the C. dubliniensis and C. albicans isolates produced chlamydospores on the control medium, i.e., rice-agar-Tween agar. However, while none of the C. albicans isolates produced chlamydospores on either SA or CAF, 85.4 and 83.8% of the C. dubliniensis isolates produced chlamydospores on SA and CAF, respectively. All of the C. albicans isolates grew as smooth, shiny colonies on SA after 48 to 72 h of incubation at 30 degrees C, while 97.7% of the C. dubliniensis isolates grew as rough colonies, many (65%) with a hyphal fringe. In contrast, 87.4% of the C. albicans and 93.8% of the C. dubliniensis isolates yielded rough colonies on CAF. Although the results of this study confirm that SA is a good medium for distinguishing between C. dubliniensis and C. albicans, we believe that discrimination between these two species is best achieved on the basis of colony morphology rather than chlamydospore production.

Recovery of Candida dubliniensis from non-human immunodeficiency virus-infected patients in Israel.

Candida dubliniensis is a recently discovered yeast species principally associated with carriage and disease in the oral cavities of human immunodeficiency virus (HIV)-infected individuals. To date the majority of isolates of this species have been identified in Europe and North America. In this study, five Candida isolates recovered from separate HIV-negative hospitalized patients in Jerusalem, Israel, were presumptively identified as C. dubliniensis on the basis of their dark green coloration when grown on CHROMagar Candida medium. Their identification was confirmed by a variety of techniques, including carbohydrate assimilation profiles, absence of growth at 45 degrees C, positive reaction with C. dubliniensis-specific antibodies as determined by indirect immunofluorescence analysis, and positive amplification with C. dubliniensis-specific PCR primers. All five strains were shown to be susceptible to a range of antifungal agents, including fluconazole. One of the five isolates was recovered from urine specimens, while the remaining four were recovered from upper respiratory tract and oral samples. While none of the patients was HIV positive, all were receiving broad-spectrum antibacterials at the time isolates of C. dubliniensis were obtained from clinical specimens. This study describes the first isolates of C. dubliniensis from the Middle East and confirms that this yeast can be associated with carriage and infection in the absence of HIV infection.

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.

Evaluation of mycology laboratory proficiency testing.

Changes over the last decade in overt proficiency testing (OPT) regulations have been ostensibly directed at improving laboratory performance on patient samples. However, the overt (unblinded) format of the tests and regulatory penalties associated with incorrect values allow and encourage laboratorians to take extra precautions with OPT analytes. As a result OPT may measure optimal laboratory performance instead of the intended target of typical performance attained during routine patient testing. This study addresses this issue by evaluating medical mycology OPT and comparing its fungal specimen identification error rates to those obtained in a covert (blinded) proficiency testing (CPT) program. Identifications from 188 laboratories participating in the New York State mycology OPT from 1982 to 1994 were compared with the identifications of the same fungi recovered from patient specimens in 1989 and 1994 as part of the routine procedures of 88 of these laboratories. The consistency in the identification of OPT specimens was sufficient to make accurate predictions of OPT error rates. However, while the error rates in OPT and CPT were similar for Candida albicans, significantly higher error rates were found in CPT for Candida tropicalis, Candida glabrata, and other common pathogenic fungi. These differences may, in part, be due to OPT's use of ideal organism representatives cultured under optimum growth conditions. This difference, as well as the organism-dependent error rate differences, reflects the limitations of OPT as a means of assessing the quality of routine laboratory performance in medical mycology.

Cryptococcus neoformans var. grubii: separate varietal status for Cryptococcus neoformans serotype A isolates.

Cryptococcus neoformans var. neoformans presently includes isolates which have been determined by the immunologic reactivity of their capsular polysaccharides to be serotype A and those which have been determined to be serotype D. However, recent analyses of the URA5 sequences and DNA fingerprinting patterns suggest significant genetic differences between the two serotypes. Therefore, we propose to recognize these genotypic distinctions, as well as previously reported phenotypic differences, by restricting C. neoformans var. neoformans to isolates which are serotype D and describing a new variety, C. neoformans var. grubii, for serotype A isolates.

Efficacy of API 20C and ID 32C systems for identification of common and rare clinical yeast isolates.

The abilities of the API 20C and ID 32C yeast identification systems to identify 123 common and 120 rare clinical yeast isolates were compared. API 20C facilitated correct identification of 97% common and 88% rare isolates while ID 32C facilitated correct identification of 92% common and 85% rare isolates.

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.

A new medium for the presumptive identification of dermatophytes.

A new medium, Dermatophyte Identification Medium (DIM) (trade mark pending), was specifically developed to eliminate problems of false-positive results associated with commercially marketed media, such as dermatophyte test medium (DTM). Previous investigations had demonstrated that DTM only partially suppressed growth of nondermatophytes and that several of these nondermatophytic fungi that were morphologically similar to dermatophytes caused false-positive results. Presumptive identification of an unknown isolate as a dermatophyte required only the transfer of a portion of the suspected colony recovered from the specimen to DIM. Positive results, evidenced by a change in the color of the medium, were observed within 24 to 48 h. In studies of over 500 isolates of dermatophytes and common nondermatophyte molds, as well as close to 600 yeast isolates, false-positive results were always associated with bacterial contamination of the mold isolates while false negatives were only observed with occasional isolates of Trichophyton verrucosum. DIM culture was an inexpensive, rapid, and accurate method for the presumptive identification of dermatophytes in the clinical mycology laboratory.

Clinical isolates of Candida guilliermondii include Candida fermentati.

Clinical isolates of Candida guilliermondii that were investigated by isoenzyme and randomly amplified polymorphic DNA analyses represented two distinct species. The two species were distinguished on the basis of delayed fermentation of galactose. The larger group of isolates was closely related to the anamorph C. guilliermondii ATCC 6260T (T = type strain) and its teleomorph, Yamadazyma (= Pichia) guilliermondii ATCC 46036T. The remaining group, whose members fermented galactose, was very similar to Candida fermentati CBS 2022, which had for many years been placed in synonymy with C. guilliermondii. Three additional groups were represented by individual strains; these strains included C. guilliermondii var. soya ATCC 20216, which was found to represent Yamadazyma ohmeri. The type strain of Y. guilliermondii is redefined.

Phialophora verrucosa: a new cause of mycetoma.

A 29-year-old Thai woman had draining sinus tracts, tumefaction, and granules on the plantar aspect of the foot. Phialophora verrucosa was isolated from the lesion. P. verrucosa is a major agent of chromoblastomycosis, which is known to rarely cause subcutaneous phaeohyphomycosis. This dematiaceous fungus has not been previously reported to cause mycetoma. This case illustrates the clinical spectrum of disease of this fungus. The salient features of mycetoma and management options are presented.

Ribosomal DNA internal transcribed spacer analysis supports synonomy of Scedosporium inflatum and Lomentospora prolificans.

Scedosporium inflatum is a dematiaceous opportunistic pathogen originally described by D. Malloch and I.F. Salkin (Mycotaxon 21:247-255, 1984). However, E. Gueho and G. S. De Hoog (J. Mycol. Med. 118:3-9, 1991) recently suggested reducing this mold to synonomy with Lomentospora prolificans on the basis of their similar morphological and molecular characteristics. We have investigated the ribosomal DNA internal transcribed spacers (ITS), i.e., ITS I and ITS II, of 18 isolates, including these two fungi and a closely related pathogen, Scedosporium apiospermum, and its telemorph, Pseudallescheria boydii. Identical ITS restriction fragment length polymorphisms were found in eight isolates of S. inflatum and L. prolificans. These results support Gueho and De Hoog's proposal to combine S. inflatum and L. prolificans into the binomial Scedosporium prolificans. However, the ITS I sequence of S. apiospermum and the ITS restriction fragment length polymorphisms of S. apiospermum and P. boydii were found to be significantly different from those of S. inflatum and L. prolificans. The ITS restriction pattern differences may be valuable in clinical settings for distinguishing these fungi.

Cokeromyces recurvatus, a mucoraceous zygomycete rarely isolated in clinical laboratories.

Cokeromyces recurvatus Poitras was isolated from an endocervical specimen obtained from a 37-year-old, insulin-dependent diabetic. The patient's diabetic condition had been well controlled for 10 years, and she had no other known medical problem. This is only the fourth time that this zygomycete has been recovered from a human source. While there was no evidence of tissue invasion in the present patient, the observation of fungus-like structures in two separate Papanicolaou-stained cervical smears prepared 1 year apart suggests that C. recurvatus may be capable of colonizing endocervical tissue.

Disseminated visceral fusariosis treated with amphotericin B-phospholipid complex.

Fusariosis, a rare infectious disease of the immunocompromised host, is relatively resistant to amphotericin B (AmB) or other antifungal agents. We describe a 5-year follow-up of a 40 year old woman with T-type acute lymphoblastic leukemia who following chemotherapy developed prolonged high fever, chills, night sweats, and severe weakness. Liver function tests were impaired and abdominal computerized tomography (CT) showed multiple lesions in the liver and abnormal structure of the spleen. A laparotomy revealed multiple granulomas containing Fusarium sp. in the liver, and the spleen was heavily infiltrated by the same fungus. The patient failed to respond to the conventional AmB dosage form (Fungizone) even after a total dose of 3.0 g was given, and developed significant renal impairment. AmB was complexed (in a mole ratio of 1:16) with a mixture of the phospholipids dimyristoyl phosphatidylcholine and dimyristoyl phosphatidylglycerol (mixed in 7:3 mole ratio). The resulting drug complex, AmB-PLC, was then administered (1-4 mg/kg/day, total dose 4.2 g) and subsequently the patient was cured of all symptoms of fusariosis. There were only mild side effects and no nephrotoxicity was evident. On the contrary, marked improvement of the renal function tests occurred during AmB-PLC treatment. Eight months later, she developed a spinal lesion with dense consistency in L5 and S1, and after receiving another course of AmB-PLC (3.1 g) she recovered completely. In a 2 year follow-up period the patient had no further relapse of the fungal disease. Subsequent chemotherapy given for relapse of the leukemia was followed by a new fungal infection, which was treated with AmB-cholesteryl sulfate complex (Amphocil).(ABSTRACT TRUNCATED AT 250 WORDS)