Successful treatment with voriconazole of prolonged Paecilomyces lilacinus fungemia in a chronic hemodialyzed patient.

Paecilomyces lilacinus is a saprophytic mould which rarely causes infection in humans. We report a case of Paecilomyces lilacinus catheter-related fungemia in a chronic hemodialyzed patient. Blood cultures remained positive for 8 weeks. The infection was cured after eventual acceptance by the patient of oral voriconazole treatment for 6 weeks and removal of the tunneled catheter. The literature on Paecilomyces fungemia in humans is reviewed.

Bloodstream yeast infections: a 15-month survey.

A 15-month survey of 412 bloodstream yeast isolates from 54 Belgian hospitals was undertaken. Candida albicans was the most common species (47.3%) followed by C. glabrata (25.7%), C. parapsilosis (8.0%), C. tropicalis (6.8%) and Saccharomyces cerevisiae (5.1%). Common predisposing factors were antibacterial therapy (45%), hospitalization in intensive care units (34%), presence of in-dwelling catheters (32%), underlying cancer (23%) and major surgery (11%). Most patients had more than one predisposing factor. Fluconazole alone or in combination with another antifungal agent was the treatment of choice for 86.6% of the cases. Susceptibility testing revealed that 93.5% were susceptible to amphotericin B, 39.6% to itraconazole, 42.8% to fluconazole and 87% to voriconazole. Resistance to azoles was more common among C. glabrata isolates.

Isolation of Cryptococcus neoformans in Antwerp Zoo's nocturnal house.

Cryptococcosis was diagnosed postmortem in a striped grass mouse (Lemniscomys barbarus) housed in the nocturnal department of Antwerp Zoo. Eight of the remaining mice in the cage were captured. Cryptococcus neoformans was isolated from the lung of one animal. Two mice had an elevated serum cryptococcal antigen titre. On examination of the pooled faecal samples collected from 17 animal species housed in 23 cages of the nocturnal department, the pathogenic yeast was isolated from the faeces of the striped grass mice and a degu (Octodon degus). Numerous Cr. neoformans colonies were isolated from a tree-trunk, tree-stumps, and decaying wood collected from a hollow tree used to decorate the animals cage. Subsequent examination in four other cages of the nocturnal department revealed that all the sampled tree-trunks were colonized by Cr. neoformans. The fungus was isolated from the air sampled in the cage of the degu. Air samples collected in the public and service corridors remained negative. All the isolated strains were identified as Cr. neoformans var. neoformans serotype A.

In vitro activities of voriconazole (UK-109, 496), fluconazole, itraconazole and amphotericin B against 132 non-albicans bloodstream yeast isolates (CANARI study).

The aim was to evaluate the in vitro activity of voriconazole compared with those of amphotericin B, itraconazole and fluconazole against 132 bloodstream isolates of Candida non-albicans and Saccharomyces cerevisiae species. The minimal inhibitory concentrations (MICs) were determined by an adapted National Committee for Clinical Laboratory Standards (NCCLS) M27-A method using RPMI 1640 as test medium supplemented with 2% glucose. MIC end-points were determined with a spectrophotometer after incubation for 48 h at 35 degrees C. Optical density data were used for the calculation of the MIC end-points. For amphotericin B, the end-point was defined as the minimal antifungal concentration that exerts 90% inhibition compared with the control well growth. For the azoles, the end-points were determined at 50% inhibition of growth. Amphotericin B is highly active with 97% of isolates inhibited by < or =1 microg ml(-1). Decreased susceptibility or resistance to fluconazole was the rule among C. krusei, which is intrinsically resistant to fluconazole. For C. glabrata isolates, resistance to fluconazole and itraconazole was measured in 13% and 17% of the isolates respectively. Voriconazole was quite active in vitro against all the isolates with a MIC90% of < or =1 microg ml(-1) and we conclude that it may be useful in the treatment of non-albicans bloodstream infections.

In vitro activity of amphotericin B, fluconazole and voriconazole against 162 Cryptococcus neoformans 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.

A one-year survey of candidemia in Belgium in 2002.

A total of 211 episodes of bloodstream yeast infections in 207 patients, hospitalized in 28 Belgian hospitals participating in a National Surveillance Program, were evaluated. A total of 81% of the patients were more than 50 years of age. Candida albicans was the cause of infections in 55% of patients, 22% were due to C. glabrata and 13% to C. parapsilosis. The most common predisposing factors were antibacterial therapy (42%), residence in an intensive care unit (32.9%) and presence of an intravascular catheter (29.7%). Most patients had more than one predisposing factor. Fluconazole alone or in association with another antifungal agent was the treatment of choice for 89.7% of the cases. In vitro susceptibility testing of the isolates revealed that 99% were susceptible to amphotericin B, 95% to 5-fluorocytosine, 82% to fluconazole and 69% to itraconazole. Resistance to azoles was more common among C. glabrata isolates in the elderly. We conclude that the frequency of C. albicans infection is decreasing in Belgium and this is associated with the emergence of other species, most notably, C. glabrata.

Isolation of Onychocola canadensis from four cases of onychomycosis in Belgium.

Onychocola canadensis can cause onychomycosis of the toenails. Thirty-two cases have been described up to now. We report on the isolation of Onychocola canadensis from four patients with onychomycosis who acquired their infection in Belgium. Direct examination was positive. Onychocola canadensis was isolated in pure culture. According to the previously published cases, the patients affected were elderly and the preferential site of infection was the big toenail. In contrast to previous reports, we found a predominance in males. Treatment was started in all patients. Two out of the three patients about whom information was available, did not improve after treatment.

Detection of single cells of Cryptococcus neoformans in clinical samples by solid-phase cytometry.

A method based on solid-phase cytometry for the detection and enumeration of single cells of Cryptococcus neoformans in serum and cerebrospinal fluid is described. Both viable and nonviable cells are detected by using fluorescence viability labeling and immunofluorescence. This 30-min procedure has a detection limit of 3 to 6 cells per ml.

Prevalence of Candida dubliniensis in the BCCM/IHEM Biomedical Fungi/Yeasts culture collection (isolates before 1990).

The BCCM/IHEM Biomedical Fungi/Yeasts collection hosts 1200 Candida albicans strains of the Vanbreuseghem mycotheque isolated between 1951 and 1997. From this collection, 469 freeze-dried C. albicans strains, producing chlamydospores, germ tubes and forming green colonies on CHROMagar, all isolated before 1990, were screened to identify the Candida dubliniensis isolates. Screening was performed in different steps using the growth at 45 degrees C, the assimilation of xylose, the intracellular beta-glucosidase activity test and C. dubliniensis-specific polymerase chain reaction (PCR) with primers from ACT1 intron sequence. Five isolates (1%) were identified as C. dubliniensis: one isolate was not documented, the others were of oropharyngeal origin of which two (1987 and 1990) were from proven human immunodeficiency virus patients.

Repeated isolation of Cryptococcus laurentii from the oropharynx of an immunocompromized patient.

Cryptococcus laurentii is one of the non-neoformans cryptococci that have rarely been isolated from humans. We report a case of repeated colonization of the oropharynx by Cr. laurentii in a patient with erythroleukaemia. The isolate was identified by phenotypic and genotypic tests and showed resistance to fluconazole.

AIDS-associated cryptococcal meningitis in Rwanda (1983-1992): epidemiologic and diagnostic features.

OBJECTIVES: to document the trend of AIDS-associated Cryptococcus neoformans meningitis (CM) in Kigali, Rwanda, during 1983-1992, and to highlight some diagnostic and epidemiological features of the disease. METHODS: during the study period, 3476 cerebrospinal fluid (CSF) specimens from 2824 adults (1578 men, 1246 women) were analysed in the Laboratory of Microbiology at the Centre Hospitalier de Kigali, Rwanda, Central Africa, using direct examination, culture and detection of the cryptococcal antigen (CrAg) in the CSF. RESULTS: CM was diagnosed among 549 (19%) patients (347 men, 202 women) and was by far the leading cause of meningitis before Neisseria meningitidis (n=115), Streptococcus pneumoniae (n=68), Mycobacterium tuberculosis (n=26). E. coli, Klebsiella pneumoniae, non-typhoid Salmonella (n=l5) and streptococci (n=4). The number of CM increased from one case in 1983 to 130 new cases in 1992. All 293 tested CM patients had HIV-1 antibodies. The male/female ratio declined from 3.31 during 1983-1987 to 1.58 during 1988-1992. CM showed a seasonal fluctuation, the highest number of infections being observed during the long rainy season. The sensitivity and specificity of the latex test for diagnosing CM was 98% and 99%, respectively. Cryptococcus neoformans var. gattii was cultured from eight (1.6%) of the 499 culture positive patients. CONCLUSION: CM is an important opportunistic infection among AIDS patients in Central Africa. It remains a problematic diagnosis in areas with limited diagnostic facilities.

Comparative evaluation of Fungitest-, Neo-Sensitabs- and M27T-NCCLS broth microdilution methods for antifungal drug susceptibility testing of Candida species and Cryptococcus neoformans.

Two commercial antifungal susceptibility testing systems (Fungitest and Neo-Sensitabs) were compared with the M27T-NCCLS reference broth microdilution method using one hundred isolates of Candida sp. and Crptococcus neoformans. Six different antifungal drugs were tested: amphotericin B, 5-fluorocytosine, fluconazole, itraconazole, ketoconazole and miconazole. The overall agreement between the Fungitest and the reference methods was much better than between the Neo-Sensitabs and the reference methods: the agreement for the Fungitest ranged from 100% for amphotericin B to 76.7% for itraconazole whereas for the Neo-Sensitabs, it ranged from 90.4% for amphotericin B to 36% for ketoconazole. For the total number of tests performed with Neo-Sensitabs, there were 37.8% of discrepancies with the reference method whereas for the tests performed with Fungitest, there was only 16.5% of discrepancies. Major discrepancies, defined as results that classified an isolate as susceptible by one method and resistant by another, occurred in 21 cases for the Neo-Sensitabs test and only in four cases with the Fungitest, namely 0.6% of the cases. We conclude that the Fungitest method constitutes a simple and reliable procedure for antifungal drug susceptibility testing.

Isolation of Basidiobolus ranarum from ectotherms in Antwerp zoo with special reference to characterization of the isolated strains.

Ten Basidiobolus ranarum (= Basidiobolus haptosporus) strains, isolated from faeces of 102 different lower vertebrates (ectotherms) exhibited in Antwerp Zoo, or from their environment were studied for their temperature requirements, haemolysis and other enzyme activities in vitro. All isolates grew well at 25 and 37 degrees C. Three strains that produced undulated zygospore walls were haemolytic and positive for hyaluronidase. All the isolates produced urease, N-acetyl-beta-glucosaminidase, trypsin, lipase, lecithinase, gelatinase, collagenase and elastase, but failed to produce amylase, keratinase and beta-glucosidase. Three isolates failed to produce phosphatase. Only one strain failed to produce DNase. Aesculin was not hydrolysed. Chitinase activity was inconclusive. The results of this study illustrate the importance of exotic animals kept in temperate regions as carriers of potentially pathogenic organisms. In addition to the morphological characteristics, the identification can be based on enzymatic profiles. Enzymatic activity detection may help to explain the pathogenic mechanism of the fungus.

Genetic multilocus studies of different strains of Cryptococcus neoformans: taxonomy and genetic structure.

The genotypes of 107 strains of Cryptococcus isolated from the environment or from patients from various geographical areas were determined by multilocus enzyme electrophoresis (MLEE). We analyzed the relationships between genotype structure and serotype and between genotype structure and strain origin. Twelve of the 14 enzyme-encoding loci studied were polymorphic, giving rise to 48 electrophoretic types. The genotypes of C. neoformans and C. laurentii were very similar. MLEE could not distinguish between these two pathogenic species. A correlation between the genetic multilocus structure and the origin of the sample (from the environment or patients) existed. A second analysis detected a correlation between genotype distribution and serotype. The second analysis considered three serotype groups (B, C, and A plus D plus A/D), proving that serotypes A, D, and A/D are closely related. MLEE is a useful epidemiological tool for improving our understanding of the biology of this fungus.

[Cryptococcus neoformans var. gattii isolates from two Peruvian patients.]. / Aislamiento de Cryptococcus neoformans variedad gattii en dos pacientes peruanos.

The ecology of Cryptococcus neoformans var. gatti is restricted to tropical and subtropical areas whereas C. neoformans var. neoformans is cosmopolitan. Perú is a country with three different well defined geographic areas, some of them have the conditions for the presence of the variety gattii. In order to determine the presence of the two varieties of C. neoformans in Perú, we made the C. neoformans differentiation from the clinical isolates. C. neoformans var. gattii was identified by their ability to assimilate D-proline. We tested 68 strains; only two of them were recognized as the variety gattii and were recovered from two patients without any predisposing factor. We described the clinical spectrum of these two patients, who were diagnosed with disseminated cryptococcosis and cryptococcal meningitis. Neither the presence of Eucalyptus camaldulensis nor Eucalyptus tereticornis has been reported in Perú. So there should exist other ecologic niches for the presence of C. neoformans var. gattii in our country, different from those mentioned.

Molecular typing of Cryptococcus neoformans: taxonomic and epidemiological aspects.

Pulsed-field gel electrophoresis (PFGE), randomly amplified polymorphic DNA (RAPD) analysis, serotype, and killer toxin sensitivity patterns of a wide range of saprobic, clinical, and veterinary isolates of both varieties of Cryptococcus neoformans were examined. C. neoformans var. neoformans and C. neoformans var. gattii differed in chromosomal makeup, RAPD patterns, and killer sensitivity patterns. These results suggest that there are two separate species rather than two varieties. No clear genetic or phenotypic differences were observed among the clinical, saprobic, and veterinary isolates within each taxon. The serotypes differed substantially in their RAPD characteristics. Geographical clustering was observed among the isolates of C. neoformans var. gattii, but not among the isolates of C. neoformans var. neoformans. The isolates of each taxon that originated from restricted geographical areas often had identical or similar karyotypes and RAPD patterns, suggesting that clonal reproduction had occurred. The combination of PFGE and RAPD analysis allowed us to distinguish almost all isolates. This combination of techniques is recommended for further research on epidemiological, ecological, and population issues.

Isolation of yeasts from bovine milk in Belgium.

A total of 436 milk samples from non-infected and 80 from infected quarters were investigated: 24.5% of the samples collected from non-infected and 55% of those collected from infected quarters were positive. Normal milk yielded not less than 16 different species and among them many potentially pathogenic yeast species such as C. parapsilosis, C. guilliermondii, C. tropicalis, C. glabrata and T. asahii, all five able to grow at 40 degrees C. In contrast, the yeasts isolated from infected quarters were from 3 species: C. kefyr, C. catenulata and C. lambica, which were also among the yeasts species recovered from normal milk. Among the three species, only one i.e. Candida kefyr is able to grow above 40 degrees C and from there can be considered as potentially pathogenic, even if bacterial association is necessary to cause mastitis.

Diversity of DNA fingerprints in Cryptococcus neoformans.

DNA fingerprint patterns of 156 Cryptococcus neoformans isolates (26 AIDS patients, 46 non-AIDS patients, and 40 environmental sources) from both varieties (126 C. neoformans var. neoformans and 30 C. neoformans var. gattii isolates) and from seven countries were analyzed by using the DNA probe UT-4p. Nine and twelve distinct DNA fingerprint patterns were observed for isolates of the C. neoformans var. neoformans and var. gattii, respectively. No pattern was unique to AIDS patients, non-AIDS patients, or the environment. Pattern II was observed more often in non-AIDS patients (8 of 23) than in AIDS patients (0 of 25). Pattern V was the most prevalent pattern (42 of 82) in clinical and environmental isolates. Isolates from three AIDS patients in Burundi and Zaire exhibited patterns identical to each other but different from those of isolates collected from their houses (i.e., dust of floors, walls, etc.) or a nearby pigeon coop. DNA fingerprint stability was determined for 53 isolates from nine non-AIDS patients at different time intervals during 5 to 128 weeks of antifungal therapy. For eight patients, the fingerprint pattern was stable while the ninth may have had a mixed infection. Pattern II was observed in 4 of 9 patients, which is similar to 4 of 14 in other non-AIDS patients as reported here. In spite of the extensive pattern heterogeneity among 15 C. neoformans var. gattii isolates in Australia, the patterns observed in seven California isolates were quite different from those in Australia. Among isolates of C. neoformans var. gattii, one fingerprint pattern (designated b) was observed in several countries of the Far East. The fingerprint patterns of two of three environmental isolates from Eucalyptus camaldulensis trees in Australia were identical to those of 2 of the 12 clinical isolates from the country.

[Tests for an early detection of pulmonary cryptococcosis by sputum culture]. / Essai de dépistage précoce de la cryptococcose pulmonaire par mise en culture d'expectorations.

Cryptococcosis is a serious opportunistic infection occurring in acquired immunodeficiency syndrome (AIDS) patients. As the number of infected patients with the human immunodeficiency virus (HIV) in Central Africa and especially in Rwanda increases, the prevalence of cryptococcosis can also be expected to rise. An earlier diagnosis and treatment will improve the prognosis of cryptococcosis. As it is widely accepted that the lungs are the portal of entry for the yeast, 270 sputum samples coming from 230 patients attending the Centre Hospitalier de Kigali (CHK)--Rwanda for lung diseases, were investigated. Cr. neoformans var neoformans was cultured from 8 samples coming from 5 out of 230 patients. A retrospective review showed that 4 out of 5 patients were infected with HIV, a predisposing factor for cryptococcosis.