Fluconazole, with or without dexamethasone for experimental cryptococcosis: impact of treatment timing.

The time of initiation of fluconazole treatment with or without dexamethasone, and the impact on mycological outcome and drug pharmacokinetics were assessed in a murine model of disseminated cryptococcosis. Non-infected mice and mice with disseminated cryptococcosis were given saline, dexamethasone, or fluconazole +/- dexamethasone, 1 or 8 days after infection. Cfus were counted in tissues, and fluconazole concentrations were determined in plasma and tissues by HPLC and a bioassay. Despite fluconazole tissue and plasma concentrations which were above the minimal inhibitory concentration, the numbers of cfus in brain and lung tissues were reduced after early (P = 0.002 and 0.04, respectively), but not after late fluconazole treatment. The administration of dexamethasone did not have a deleterious effect on the number of cfus, fluconazole pharmacokinetics or antifungal activity. In conclusion, the size of the fungal burden influences the effective level of fluconazole activity in lung and brain. These results strongly suggest that potential antifungal agents should be studied following both early and late administration in experimental cryptococcosis.

DNA typing suggests pigeon droppings as a source of pathogenic Cryptococcus neoformans serotype D.

The unusual proportion of serotype D strains of Cryptococcus neoformans infecting patients diagnosed with cryptococcosis in some parts of France prompted the analysis of DNA fingerprints obtained with 26 clinical and 29 environmental isolates from the same area. Our results suggest that pigeon droppings are a potential source of pathogenic strains of C. neoformans serotype D, as previously demonstrated for serotype A.

Individual and environmental factors associated with infection due to Cryptococcus neoformans serotype D. French Cryptococcosis Study Group.

Differences between the two varieties of Cryptococcus neoformans with regard to geographic distribution, epidemiology, and even pathogenicity have been described. None between C. neoformans variety neoformans serotypes A and D have been reported. We reviewed 452 cases of cryptococcosis diagnosed in France, where serotype D is responsible for 21% of the infections. Univariate analysis showed that the frequency of serotype D infections was significantly higher among patients > 60 years of age, those born in Europe, those receiving corticosteroid therapy, and those who had skin lesions. In the multivariate analysis, the risk of serotype D infection was significantly higher for patients with skin lesions, those receiving corticosteroid therapy, and those living in certain regions in France. It was significantly lower for patients with meningitis those coming from Africa, and females. Among the 350 human immunodeficiency virus-infected patients, the risk was significantly higher for those > 60 years old, those who were intravenous drug abusers, and those with skin lesions. The risk was significantly lower for patients from Africa and in cases of meningitis. These results suggest that individual and environmental factors can be associated with the serotype of the infecting strain of C. neoformans.

Molecular typing of Cryptococcus neoformans serotype D clinical isolates.

Cryptococcus neoformans serotype A is responsible for the majority of cryptococcal infections in AIDS patients. In France, approximately 17% of the patients are infected with serotype D, regardless of their human immunodeficiency virus status. In a retrospective study of 273 patients, we found that serotype D was unevenly distributed in France. We wondered if this was related to the yeast's genetic background. We used karyotyping and DNA fingerprints generated by UT-4p to analyze 40 serotype D clinical isolates. We found an extensive polymorphism, with only two conserved karyotypes from drug-addicted patients living in the same area. Although highly variable, the DNA fingerprints were classified into 10 groups. Four pairs of isolates were identical; three of these pairs were from patients living in the same area, but there was no other correlation with the geographical area. The two isolates with identical karyotypes belonged to the same fingerprint group. Five of the six isolates that made up fingerprint group I were recovered from drug-addicted patients (P < 0.002; chi-square), and all five isolates found in fingerprint group III were from male homosexual patients (P < 0.02). Finally, five of the seven isolates from patients with cryptococcal pneumonia were classified as fingerprint group V (P < 0.04). These results suggest that there are possible relationships between characteristics of the isolates and body localization or even risk factors. Results of the present study warrant other studies on isolates of all serotypes and on isolates from clinical and environmental sources.

Serotyping of Cryptococcus neoformans by using a monoclonal antibody specific for capsular polysaccharide.

The importance of epidemiological studies of cryptococcosis has increased since the beginning of the AIDS epidemic. Cryptococcus neoformans exists in two varieties defining four serotypes, Cryptococcus neoformans var. neoformans (serotypes A and D) and Cryptococcus neoformans var. gattii (serotypes B and C). The varieties are easy to distinguish by their differences of growth on diagnostic media. We propose here an easy serotyping method combining diagnostic media and a direct immunofluorescence assay with one monoclonal antibody (E1) specific for cryptococcal polysaccharide. The method was validated by the blinded testing of four to five reference strains of each serotype. Immunofluorescence patterns were characteristic of a given serotype provided that the variety of the strain had been defined before. For C. neoformans var. neoformans, a bright, homogeneous staining with several cell aggregates was characteristics of serotype A, whereas only a few serotype D cells were positive. For C. neoformans var. gattii, a completely negative isolate was serotype C, whereas the population of serotype B included a majority of negative cells but also included positive cells with a speckled pattern. The method was then used to serotype 156 clinical isolates from France and isolates from areas where C. neoformans var. gattii was endemic before the AIDS epidemic (13 strains from Rwanda and Zaire and 5 strains from Australia). The specificity of E1 was defined by its reactivity with various Cryptococcus spp. and analyzed according to the described cryptococcal antigenic factors. We conclude from this study that E1 provides a rapid and reliable means to serotype multiple isolates of C. neoformans.

Evaluation of a monoclonal antibody-based latex agglutination test for diagnosis of cryptococcosis: comparison with two tests using polyclonal antibodies.

Cryptococcal antigen detection has become a routine biological test performed for patients with AIDS. The poor prognosis of cryptococcosis explains the need for reliable tests. We evaluated the performances of a newly commercialized agglutination test that uses a monoclonal antibody specific for cryptococcal capsular polysaccharide (Pastorex Cryptococcus; Sanofi-Diagnostics Pasteur, Marnes-la-Coquette, France) and compared them with those of tests that use polyclonal immune sera (Cryptococcal Antigen Latex Agglutination System, Meridian Diagnostics, Inc., Cincinnati, Ohio; and Crypto-LA, International Biological Labs Inc., Cranbury, N.J.). The sensitivities and specificities of the tests were compared by using purified polysaccharides and yeast suspensions. Clinical specimens (131 serum samples, 41 cerebrospinal fluid samples, 34 urine samples, and 19 bronchoalveolar lavage samples) from 87 human immunodeficiency virus-positive subjects with (40 patients) and without (47 patients) culture-proven cryptococcosis were retrospectively tested during a blinded study. The effect of pronase treatment of samples was assessed for Pastorex Cryptococcus and the Cryptococcal Antigen Latex Agglutination System, and the antigen titers were compared. Our results show that (i) during the screening, concordance among the three tests was 97%; (ii) the use of pronase enhanced both the sensitivities and specificities of the Pastorex Cryptococcus test; (iii) titers agreed for 67% of the cerebrospinal fluid samples and 60% of the serum samples; and (iv) cryptococcosis was detected equally well with Pastorex Cryptococcus and with the other tests, whatever the infecting serotype (A, B, or D). The meaning of in vitro sensitivity and the relationship between titers and sensitivity are discussed. The results show that Pastorex Cryptococcus is a rapid and reliable test for the detection of cryptococcal antigen in body fluids and suggest that kits cannot be used interchangeably to monitor antigen titers in patients.

Isolation of Cryptococcus neoformans var. gattii from an Asian patient in France: evidence for dormant infection in healthy subjects.

We report the first case of cryptococcosis due to Cryptococcus neoformans var. gattii in France. This Cambodian, non-AIDS patient had been living in France for 23 years when he developed cryptococcosis due to C. neoformans serotype B in 1985. It is possible that he was infected back in Asia or several months before, in Zaire, where he lived between 1981 and 1984. To our knowledge, this is the second case providing clinical evidence for reactivation of latent infection with C. neoformans. The mechanism is probably similar to that for C. neoformans var. neoformans.