A 30-month ECMM survey of cryptococcosis in Europe: molecular analysis of 316 Cryptococcus neoformans isolates

During an ECMM prospective survey of cryptococcosis in Europe (July 1997-December 1999) 655 cases were reported from 17 countries and 565 forms were analysed. Cryptococcus neoformans was the etiologic agent of all the cases except five caused by C. gattii serotype B (in Austria, Germany, Greece and France) and serotype C (in UK), and four caused by other Cryptococcus species, namely C. albidus (in Russia), C. laurentii (in UK and Bulgaria) and C. luteolus (in Bulgaria). Cryptococcosis was associated with HIV infection in 77% of cases with the highest prevalence in Italy (94%) and Portugal (90%) and was indicative of AIDS in 57.5%. In 99 cases, cryptococcosis occurred in HIV negative patients, mainly associated with cancer (42), organ transplantation (20), autoimmune disease (8), sarcoidosis and trauma (5 each). Corticosteroid therapy was reported in 25 patients. The fungus was cultured in 542 (96%) cases, mostly from CSF. A total of 316 strains from 15 countries were serotyped by latex agglutination (Crypto Check Iatron Labs, Tokyo, Japan), genotyped by PCR fingerprinting using (GACA)4 oligonucleotide as single primer and their mating type was determined by PCR of STE20 alleles. Serotype A strains were the most represented (51%) followed by serotype D (30%) and serotype AD (19%). PCR-fingerprinting analysis changed significantly the percentage of hybrid strains (30% instead of 19%) as 6% of serotype A and 28% of serotype D isolates were shown to be VN3 or VN4 hybrid genotype. In addition, the determination of the mating type revealed the presence of the MATa serotype A allele either in one haploid strain and in 28 hybrids. Hybrid isolates presenting a single mating type, 4 Aalpha and 2 Dalpha , were also identified. The present study is the first European prospective survey of cryptococcosis and represents an attempt to take a picture of the epidemiology of cryptococcosis in the complex Old Continent mosaic. Furthermore, the molecular analysis of the isolates collected during the study has improved the knowledge about the different subpopulations of C. neoformans revealing the large involvement of AD hybrid strains in C. neoformans infection. The results obtained so far are encouraging for keeping the surveillance ongoing in order to better define the geographical occurrence of cryptococcosis and outline a map of distribution of C. neoformans pheno/genotypes in Europe.