During a European Confederation of Medical Mycology (ECMM) prospective survey of cryptococcosis in Europe (from July 1997 to December 1999) 655 cases were reported from 17 countries; 565 of the completed questionnaires were evaluable. Cryptococcosis was associated with HIV infection in 77% of cases (range 57.5-94%). Assessment of the laboratory data highlighted the lack of defined standard procedures for the diagnosis of cryptococcosis: the antigen test was not usually used for screening, the disease was mainly recognised when meningitis occurred (65% of patients) and, with the exception of a few cases, the extent of the infection was not investigated. Cryptococcus neoformans was the etiological agent in all of the cases except for six caused by C. gattii and four by other Cryptococcus species. A total of 311 C. neoformans strains were serotyped by Crypto Check latex agglutination, genotyped by PCR-fingerprinting using the (GACA)4 oligonucleotide as a single primer, and their mating type was determined by PCR of the STE20 alleles. Serotype A was the most represented (51% of the isolates), followed by serotype D (30%) and serotype AD (19%). PCR-fingerprinting analysis significantly increased the percentage of hybrid strains to 30%, as 6% of the serotype A and 28% of the serotype D isolates were of the VN3 or VN4 hybrid genotype. In addition, the mating type determinations revealed the MATa serotype A allele in one haploid strain and 28 hybrids, and hybrid isolates with a single mating type (four Aalpha and two Dalpha) were also identified. This is the first prospective survey to be carried out in Europe which has attempted to investigate the epidemiology of cryptococcosis and the population structure of C. neoformans, and the results obtained thus far show the widespread involvement of AD hybrid strains in C. neoformans infections.

Prions are pathogens with an unusually high tolerance to inactivation and constitute a complex challenge to the re-processing of surgical instruments. On the other hand, however, they provide an informative paradigm which has been exploited successfully for the development of novel broad-range disinfectants simultaneously active also against bacteria, viruses and fungi. Here we report on the development of a methodological platform that further facilitates the use of scrapie prions as model pathogens for disinfection. We used specifically adapted serial protein misfolding cyclic amplification (PMCA) for the quantitative detection, on steel wires providing model carriers for decontamination, of 263K scrapie seeding activity converting normal protease-sensitive into abnormal protease-resistant prion protein. Reference steel wires carrying defined amounts of scrapie infectivity were used for assay calibration, while scrapie-contaminated test steel wires were subjected to fifteen different procedures for disinfection that yielded scrapie titre reductions of ≤101- to ≥105.5-fold. As confirmed by titration in hamsters the residual scrapie infectivity on test wires could be reliably deduced for all examined disinfection procedures, from our quantitative seeding activity assay. Furthermore, we found that scrapie seeding activity present in 263K hamster brain homogenate or multiplied by PMCA of scrapie-contaminated steel wires both triggered accumulation of protease-resistant prion protein and was further propagated in a novel cell assay for 263K scrapie prions, i.e., cerebral glial cell cultures from hamsters. The findings from our PMCA- and glial cell culture assays revealed scrapie seeding activity as a biochemically and biologically replicative principle in vitro, with the former being quantitatively linked to prion infectivity detected on steel wires in vivo. When combined, our in vitro assays provide an alternative to titrations of biological scrapie infectivity in animals that substantially facilitates the use of prions as potentially highly indicative test agents in the search for novel broad-range disinfectants.

The unusual resistance of agents causing transmissible spongiform encephalopathies (TSEs) to chemical or thermal inactivation requires special decontamination procedures in order to prevent accidental transmission of these pathogens by surgical instruments. In the search for effective, instrument-compatible and routinely applicable decontamination procedures, a previous study [Lemmer, K., Mielke, M., . J Gen Virol 85, 3805-3816] identified promising reagents in an in vitro carrier assay using steel wires contaminated with the diseaseassociated prion protein, PrP Sc . In the follow-up study presented here, these reagents were validated for their decontamination potential in vivo. Steel wires initially loaded with ¢3¾10 5 LD 50 of 263K scrapie infectivity were implanted into the brains of hamsters after treatment for decontamination and subsequently monitored for their potential to trigger clinical disease or subclinical cerebral PrP Sc deposition within an observation period of 500 days. It was found that routinely usable reagents such as a commercially available alkaline cleaner (pH 12.2) applied for 1 h at 23 6C or for 10 min at 55 6C and a mixture of 0.2 % SDS and 0.3 % NaOH (pH 12.8) applied for 5 or 10 min at 23 6C achieved removal of 263K scrapie infectivity below the threshold of detection (titre reduction of ¢5.5 log 10 units). The increasing use during the past few years of similar model systems by different research groups will facilitate comparison and integration of findings on the decontamination of steel surfaces from prions. Methods identified as highly effective in the 263K steel wire model need to be validated for human TSE agents on different types of instrument surfaces.

Prion infectivity and its molecular marker, the pathological prion protein PrPSc, accumulate in the central nervous system and often also in lymphoid tissue of animals or humans affected by transmissible spongiform encephalopathies. Recently, PrPSc was found in tissues previously considered not to be invaded by prions (e.g., skeletal muscles). Here, we address the question of whether prions target the skin and show widespread PrPSc deposition in this organ in hamsters perorally or parenterally challenged with scrapie. In hamsters fed with scrapie, PrPSc was detected before the onset of symptoms, but the bulk of skin-associated PrPSc accumulated in the clinical phase. PrPSc was localized in nerve fibres within the skin but not in keratinocytes, and the deposition of PrPSc in skin showed no dependence from the route of infection and lymphotropic dissemination. The data indicated a neurally mediated centrifugal spread of prions to the skin. Furthermore, in a follow-up study, we examined sheep naturally infected with scrapie and detected PrPSc by Western blotting in skin samples from two out of five animals. Our findings point to the skin as a potential reservoir of prions, which should be further investigated in relation to disease transmission.

Effective reprocessing of surgical instruments ensuring elimination of inadvertent contamination with infectious agents causing transmissible spongiform encephalopathies (TSEs) is essential for the prevention of iatrogenic transmission of Creutzfeldt-Jakob disease (CJD) or its new variant (vCJD) from asymptomatic carriers. In a search for effective yet instrument-friendly and routinely applicable reprocessing procedures, we used an in vitro carrier assay to assess the decontamination activity exerted by different reagents on pathological prion protein (PrP Sc ), the biochemical marker for TSE infectivity, attached to steel surfaces. In this assay, steel wires were contaminated with 263K scrapie brain homogenate and reprocessed for decontamination by exposure to several different test reagents. Residual contamination with PrP Sc and its protease-resistant core PrP27-30, still present after reprocessing on the wire surface or in the cleaning solution, was monitored by sensitive Western blot detection without or after proteinase K digestion. Using this approach, various reagents and processing conditions were screened for both their efficacy of decontamination and their active principles, such as detachment, destabilization or degradation of surface-bound prion protein. This revealed that, under appropriate conditions, relatively mild reagents such as 0?2 % SDS/0?3 % NaOH (pH 12?8), a commercially available alkaline cleaner (pH 11?9-12?2), a disinfectant containing 0?2 % peracetic acid and low concentrations of NaOH (pH 8?9) or 5 % SDS (pH 7?1) exert potent decontaminating activities on PrP Sc /PrP27-30 attached to steel surfaces. For in vivo validation, wires reprocessed in these reagents have been implanted into reporter animals in ongoing experiments.