Genotypic variation and antifungal susceptibilities of Candida pelliculosa clinical isolates.

At the Istituto Ricovero Cura Carattere Scientifico, Ospedale Maggiore di Milano, Italy, Candida pelliculosa accounted for 3.3 and 4.4 % of all Candida species other than Candida albicans collected during 1996 and 1998, respectively. Genetic variability was investigated by electrophoretic karyotyping and inter-repeat PCR, and the susceptibility to five antifungal agents of 46 strains isolated from 37 patients during these 2 years was determined. Combination of the two typing methods yielded 14 different DNA types. Although the majority of DNA types were randomly distributed among different units, one DNA type was significantly more common in patients hospitalized in a given unit compared with those from other wards (P=0.034), whereas another DNA type was more frequently isolated in patients hospitalized during 1996 than in those hospitalized during 1998 (P=0.002). Fluconazole, itraconazole and posaconazole MIC90 values were 16, 1 and 4 microg ml-1, respectively. All isolates but three were susceptible in vitro to flucytosine. All isolates were susceptible in vitro to amphotericin B. These data suggest that there are possible relationships among strains of C. pelliculosa, wards and time of isolation. Amphotericin B seems to be the optimal drug therapy in infections due to this yeast species.

Comparative analysis of pathogenicity of Cryptococcus neoformans serotypes A, D and AD in murine cryptococcosis.

OBJECTIVES: To characterize the pathogenicity of 15 strains of Cryptococcus neoformans belonging to several serotype/mating type allele patterns (Dalpha, Da, A(alpha), A(a), A(alpha)/D(a) and D(alpha)/A(a)) in experimental models of murine cryptococcosis. METHODS: CD1-infected mice were examined for survival and fungal loads in either brain or lung during the course of infection. RESULTS: All strains, with the exception of one Da strain, produced melanin in vitro. Similarly, all strains were encapsulated and produced phospholipase. When CD1 mice were challenged intravenously (i.v.) with 5x10(5)CFU/mouse and observed for 60 days post-infection, a significant variation of mortality rate was observed among mice infected with different strains. A(alpha) and A(alpha)/D(a) strains all produced 100% mortality within the study period with mean survivals significantly shorter than those of mice infected with strains belonging to any other allele type (P<0.0001). A wide range of pathogenicity was shown by haploid and diploid strains presenting D(alpha) allele. This finding was confirmed by an intranasal model of challenge. To investigate the progression of infection, the mice were challenged i.v. with 5x10(4)CFU/mouse and tissue burden experiments (brain and lung) were performed on days 6 and 12 post-infection. Only the mice infected with A(alpha) and A(alpha)/D(a) strains showed a >1 log(10) increase of CFU/g in both tissues throughout the study period. CONCLUSIONS: Our results suggest that the presence of the A(alpha) mating type allele in either haploid or diploid strains is correlated with virulence, while the presence of the A(a) or D(a) allele in haploid strains is associated with moderate or no virulence. Finally, either haploid or diploid strains presenting D(alpha) allele vary in virulence.

Multilocus enzyme electrophoresis analysis of Aspergillus fumigatus strains isolated from the first clinical sample from patients with invasive aspergillosis. EBGA Network. European Research Group on Biotype and Genotype of Aspergillus.

The genotypes of 50 isolates of Aspergillus fumigatus from 11 patients with invasive aspergillosis, obtained from three hospitals in different geographical areas, were determined by multilocus enzyme electrophoresis (MLEE). The study analysed the genetic polymorphism of multiple isolates from the first sample. Seven of the 14 enzymic loci studied were polymorphic, giving rise to eight different electrophoretic types. For nine of 11 patients studied, no polymorphism was observed in isolates within the first clinical sample. Analysis of genetic distance between electrophoretic types demonstrated a genetic heterogeneity within each geographical site. Moreover, some genotypes were preferentially found in a given area and this revealed a population structure within these geographical sites. Therefore, the epidemiology of A. fumigatus should be considered separately for each of these areas. The multiple discriminatory markers of MLEE seem to provide a powerful tool for increasing the understanding of the biology of this fungus.

Combination of three typing methods for the molecular epidemiology of Aspergillus fumigatus infections. European Research Group on Biotype and Genotype of Aspergillus.

This study investigated the source of infection and strain relatedness of Aspergillus fumigatus isolates from bronchial colonisation and invasive aspergillosis (IA) in four transplant patients. Environmental isolates from the patient's home and from the hospital and infecting isolates were obtained for patient A who developed IA. Clinic environmental and colonising isolates were obtained for patient B. Sequential isolates were obtained from various organs from patient C who developed IA and also from patient D who had a bronchitic aspergillosis that developed into IA. Ninety-one A. fumigatus isolates were analysed by three typing methods: multi-locus enzyme electrophoresis (MLEE), random amplified polymorphic DNA (RAPD) and sequence-specific DNA primers (SSDP). The three combined typing methods demonstrated a greater differentiation of isolates than the typing methods used separately or in pairs. This demonstrated the genotypic variability of A. fumigatus and facilitated better epidemiological analysis. Large polymorphisms were demonstrated for each patient isolate between and colonies within various samples. The relatedness of the isolates suggested nosocomially acquired aspergillosis for patient B, but the source of infection for patient A remained unclear. The results suggested at least three multiple infections among the four patients. This study enabled the identification of the source of infection and strain relatedness, which in turn facilitates the development of preventive measures for patient management in the future.

Determination of Cryptococcus neoformans var. neoformans mating type by multiplex PCR.

Mating type plays an important role in the epidemiology and virulence of Cryptococcus neoformans. The present study designed a multiplex PCR method to distinguish the six mating type patterns (Aa, Da, Aalpha, Dalpha, Aa/Dalpha, and Aalpha/Da) of C. neoformans var. neoformans. PCR amplification identified one fragment for Aa (860 bp), Dalpha (413 bp) and Da (645 bp) strains, two fragments for Aalpha (320 and 400 bp) and Aa/Dalpha (860 and 413 bp) strains, and three fragments (645, 400, 320 bp) for an Aalpha/Da strain. The method appears to be a valid, simple and relatively inexpensive tool for epidemiological and virulence studies.

Molecular typing of Aspergillus fumigatus strains by sequence-specific DNA primer (SSDP) analysis.

A PCR typing method has been developed and tested to investigate the polymorphism of clinical strains of Aspergillus fumigatus. Firstly, the DNA fragments from random amplified polymorphic DNA (RAPD) patterns of nine epidemiologically and geographically non-related monosporal strains of A. fumigatus were cloned and sequenced. The pairs of five sequence-specific DNA primers (SSDP), characteristic of the 5' and 3' extremities of the RAPD products, were then used in high stringency PCR to type 43 clinical strains of A. fumigatus from 13 patients, according to the presence or absence of a single amplified band. This original approach, which uses the advantages of PCR, has made it possible to overcome the difficulties resulting from the low stringency amplification. The SSDP analysis of 51 A. fumigatus strains (9 unrelated monosporal strains and 43 clinical strains from 13 patients) can be classed into 22 different types with a high reproducibility and a high level of discrimination (D = 0.96). The results suggest that seven lung transplant patients with necrotizing aspergillosis, bronchitis aspergillosis and bronchial colonization were infected by multiple strain genotypes, whereas three patients with invasive aspergillosis seem to have been infected by a single strain.

Cryptococcal meningitis: diagnosis and treatment.

In the last decade, largely due to the increasing number of immunocompromised patients, particularly those with AIDS, there has been a dramatic increase in the incidence of cryptococcosis. The majority of human cryptoccoccal infections are caused by C. neoformans. Pulmonary cryptococcosis is the commonest form of infection and meningitis is the most severe, being fatal in most cases. Diagnosis is usually by culture and/or serology. Combined therapy with amphotericin B and flucytosine remains the initial treatment of choice, although a short course of amphotericin B alone followed by high dose fluconazole or itraconazole and the combination of flucytosine with fluconazole or with itraconazole may be effective alternatives. Both azoles, given as single agent maintenance therapy, have been shown to be beneficial as they are more effective and less toxic than the weekly infusion of amphotericin B. Primary prophylaxis with fluconazole has been advocated but problems of resistance are a concern. To date, itraconazole has not been associated with problems of resistance.

Candidosis in the intensive care unit: a 20-year survey.

Deep-seated candidosis is a major problem in critically ill patients. Colonization with candida has been identified as an important independent risk factor for the development of candidaemia. Since the 1980s routine surveillance cultures have been performed on patients admitted for six or more days to the 'E. Vecla' intensive care unit (ICU) of the IRCCS Ospedale Maggiore di Milano. Colonization was observed on admission to the ICU in 59 of 117 (50%) patients in 2000 and 10 others developed colonization during their stay on the unit. A similar colonization rate was found in a survey performed 16 years earlier. The incidence of non-albicans Candida species, however, increased in 2000. In particular, 24 patients were culture positive for Candida glabrata at some point during their hospital stay, whereas this species was isolated from only one patient in 1983-1984. Antifungal susceptibility testing performed by Sensititre Yeast One revealed no resistance among 19 C. albicans strains tested. In contrast, fluconazole resistance was observed in two of 39 (5%) C. glabrata isolates from 23 patients. In the period 1983-2002, 28 candida bloodstream infections were identified and 12 were considered to be ICU-acquired (2.6/1000 hospitalized patients; 0.33/1000 patient days). The low rate of ICU-acquired candidaemia despite the inclusion of severely compromised patients in this study confirms the usefulness of routine mycological surveillance in preventing deep-seated candidosis.

Hospital-acquired Aspergillus fumigatus infection: can molecular typing methods identify an environmental source?

Aspergillus fumigatus infection in hospitalized immunocompromised patients often raises suspicion regarding the potential for hospital acquisition. Hospital staff have an important responsibility in implementing preventive measures, especially since the advent of current legislation concerning hospital-acquired infections. There have been high expectations that molecular typing methods might determine the source of Aspergillus fumigatus, a ubiquitous mould. The aim of the present epidemiological study, was therefore, to identify the origin(s) of Aspergillus infection in six well-documented patients. All the clinical strains (N=33), and those from hospital (N=14) and home environments (N=34) were isolated according to a standardized protocol and typed by sequence-specific DNA primer analysis. The results confirmed the huge biodiversity of the A. fumigatus population, and consequently the difficulty in ascertaining a hospital source of the infection, as opposed to infections due to other Aspergillus species less frequently encountered.

Infection due to Absidia corymbifera in a patient with a massive crush trauma of the foot.

We report the case of a patient with a massive crushing trauma of the right foot who developed a local infection due to Absidia corymbifera. Systemic and local antifungal therapy with ketoconazole associated with hyperbaric oxygen therapy (HBO) yielded a rapid clinical and microbiological resolution. Controlled clinical studies are warranted to further elucidate the potential utility of HBO/antifungal combination therapy.

Experience with itraconazole in cryptococcosis and aspergillosis.

Cryptococcosis and aspergillosis in immunocompromised patients are extremely difficult clinical conditions to manage and treatment with available antifungal drugs often fails. Itraconazole, R-51211, Janssen Pharmaceutica, a new orally absorbed triazole, is a possible alternative drug which is potentially effective and nontoxic. Preliminary experience with 28 patients, eight with cryptococcosis and 20 with aspergillosis, is reported. Of these patients, 16 were immunocompromised (seven with the acquired immune-deficiency syndrome (AIDS), five heart transplant recipients and four with leukaemia or lymphoma). Overall, results of treatment were good (18 in remission, four markedly improved, four moderately improved and two failed). Prevention of relapses of cryptococcosis was obtained in all patients with AIDS on long-term itraconazole monotherapy (3 mg/kg). Treatment of invasive aspergillosis required a higher dosage (about 5 mg/kg) and prolonged administration. Besides its efficacy this antifungal agent allowed outpatient management.

Flucytosine and cryptococcosis: which in vitro test is the best predictor of outcome?

The combination of flucytosine and amphotericin B is first choice treatment for active cryptococcosis. Because of innate or acquired resistance of Cryptococcus neoformans to flucytosine, in vitro testing is mandatory. Yeast nitrogen base (YNB) at pH 7.0 is the recommended medium for the broth microdilution test (NCCLS M27-A) and for the E-test. In order to verify if minimum inhibitory concentrations (MICs) were able to predict treatment outcome, the susceptibility of 24 isolates from 21 patients treated with flucytosine alone or in combination was tested by the broth microdilution, agar dilution and E-test using YNB either at pH 7.0 or at pH 5.4. Only those MICs obtained on YNB pH 5.4 proved to correlate with treatment outcome. The present study suggests that in vitro susceptibility to flucytosine of C. neoformans isolates should be evaluated on YNB pH 5.4 and the test should be standardized accordingly.

Effect of medium composition on static and cidal activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against Aspergillus fumigatus: a multicenter study.

The effect of the medium composition on the fungistatic (MIC) and fungicidal (MLC) activity of amphotericin B, itraconazole, voriconazole, posaconazole and terbinafine against four Aspergillus fumigatus strains has been investigated by four European laboratories. MICs were determined by broth microdilution, using RPMI 1640 and Antibiotic Medium 3 (AM3), three times in three independent determinations by the four laboratories. MLCs were determined for the three independent determinations by the four laboratories, subculturing 100 microl from each well showing no visible growth after 48 hours. Except for a 2-dilution difference observed in three cases, no differences were observed between MICs determined on the two media. In contrast, a 3- to 6-dilution discrepancy between the MLCs was observed for the azoles. Endpoints on RPMI were higher than those on AM3. A 1-2 dilution difference was noted between both the endpoints of amphotericin B and of terbinafine. The highest inter- and intra-laboratory agreements were reached on AM3. The azoles showed a medium-dependent fungicidal activity.

[Mycoses as opportunistic infections in AIDS patients]. / Mykosen als opportunistische Infektionen bei AIDS-Patienten.

Since January 1985 more than 100 patients with deep fungal infections have been treated with itraconazole (200 to 400 mg/day) in Northern Italy. Evaluation of the drug efficacy and tolerance was possible in one patient with sporotrichosis, in 34 with aspergillosis, and in 36 with cryptococcosis (mainly patients positive for human immunodeficiency virus). Response to itraconazole alone was obtained in the case of sporotrichosis and in 24 of 34 patients with different forms of aspergillosis (of the 18 patients with invasive pulmonary aspergillosis, 15 were cured). Patients with cryptococcosis received itraconazole for active infection and/or for prevention of relapse. Active infection was treated successfully with itraconazole alone in nine of twelve patients and with itraconazole plus flucytosine in eight of ten patients. Of the 31 patients who received itraconazole maintenance therapy for up to 27 months, four (13%) had relapses; 14 (45%) did not have relapses, and decline of serum antigen was detected in twelve of them; and 13 (42%) were completely cured (serum antigen titer dropped to zero). With the exception of hypokalemia in one patient, itraconazole was well tolerated even in patients who received the drug for several months or years.

[Disseminated histoplasmosis in patients with AIDS. 2 case reports]. / Istoplasmosi disseminata in pazienti affetti da AIDS. Descrizione di due casi.

Histoplasmosis is endemic in some areas of United States and in South America, and generally causes an acute self-limiting respiratory infection. In elderly and immunosuppressed patients the infection can spread through the blood, causing a severe systemic illness. Here we describe two cases of disseminated histoplasmosis in AIDS patients. The first was observed in an Italian woman who had never visited endemic countries, and was recognized only at autopsy; the second was observed in a trans-sexual patient, arrived in Italy from Brazil. Clinical suspicion of histoplasmosis is important in immunocompromised patients of non-endemic areas as symptoms are often aspecific and misdiagnosis is frequent.

[Ketoconazole treatment of candidiasis in children: clinico-pharmacokinetic study]. / Il trattamento delle candidosi in età pediatrica con ketoconazolo: dati clinici e farmacocinetica.

Twenty-one children, age range 5 mo - 14 yrs, affected by candidosis, were treated with ketoconazole (tablets or suspension). Patients had alimentary tract involvement (12), oesophagitis (1), urinary tract candidosis (3), vaginitis (2), septicaemia (1), endophtalmitis (1) and chronic pulmonary illness with persistence of Candida albicans in sputum (3). Daily drug doses ranged from 3 to 13 mg/Kg and treatment period from 7 days to 14 months. Pharmacokinetic study in 15 children showed large individual variability of drug serum levels. Pharmacokinetic parametres, related to different schedules of the two ketoconazole formulations (tablets and suspension) are reported; drug levels after chronic administration are also evaluated. A daily dose of 3 mg/Kg of ketoconazole suspension, given in 3 administrations, did not result in sufficiently high levels, which indeed were obtained with a daily dose of 10 mg/Kg (3.3 X 3). The effect of treatment is proven by negativization of cultures in 90% of patients, by disappearance of clinical signs in 67% by improvement in 9%. The therapeutic effect on the remaining 24% (5 patients) is not evaluable. Adverse effects were only nausea and pyrosis in four cases; no laboratory abnormalities were found. A daily dosage from 7 to 10 mg/Kg, in two or three administrations, is suggested to obtain therapeutic levels in children.