Karin Lemmer

Follow-up of epidemiological data of cryptococcosis in Austria, Germany and Switzerland with special focus on the characterization of clinical isolates.

The present survey in Austria, Germany and Switzerland continued the survey of cryptococcosis set up by the European Confederation of Medical Mycology (ECMM) in 1997. From 2000 to 2003 77 cases have been reported. An HIV infection is still the most important risk factor (68%). Young HIV+ women from ASIA contributed to the increase of cryptococcosis in females. A total of 129 clinical isolates of both surveys were genotyped by PCR fingerprinting to study the prevalence of different genotypes. The prevalence of Cryptococcus neoformans var. grubii (serotype A) with the genotypes VNA1 and VNA2 was higher in Germany and Austria (74.5%) than in Switzerland (52%), while in Switzerland the Cr. neoformans hybrids AD (26%) and Cr. neoformans var. neoformans (serotype D) (22%) were more prevalent compared with Germany and Austria (8 and 17.5% respectively). Cryptococcus gattii isolates were studied by FT‐IR spectroscopy. DNA in the ITS region was sequenced to get further information about Cr. neoformans serotype AD strains and about the geographical origin of the Cr. gattii isolates. The ITS sequence of the serotype AD isolates of the genotypes VNAD1, VNAD2 and VNAD4 is usually identical to serotype A or serotype D respectively. In the three isolates of the genotype VNAD3 a genotype‐specific sequence pattern was detected. Two autochthonous infections due to Cr. gattii could indicate that the genotype VGIV with the ITS type ‘Asia 2’ might be endemic in Europe.

Accumulation of pathological prion protein PrPSc in the skin of animals with experimental and natural scrapie.

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.

Quantitative detection and biological propagation of scrapie seeding activity in vitro facilitate use of prions as model pathogens for disinfection.

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.

Fast, broad-range disinfection of bacteria, fungi, viruses and prions

Effective disinfectants are of key importance for the safe handling and reprocessing of surgical instruments. This study tested whether new formulations containing SDS, NaOH and 1-propanol (n-propanol) are simultaneously active against a broad range of pathogens including bacteria, fungi, non-enveloped viruses and prions. Inactivation and disinfection were examined in suspension and on carriers, using coagulated blood or brain homogenate as an organic contaminant. Coomassie blue staining was used to assess whether the formulations undesirably fixed proteins to rough surfaces. A mixture of 0.2 % SDS and 0.3 % NaOH in 20 % n-propanol achieved potent decontamination of steel carriers contaminated with PrP(TSE), the biochemical marker for prion infectivity, from 263K scrapie hamsters or from patients with sporadic or variant Creutzfeldt-Jakob disease. 263K scrapie infectivity on carriers was decreased by > or =5.5 logs. Furthermore, the formulation effectively inactivated poliovirus, hepatitis A virus and caliciviruses (including murine norovirus) in suspension tests. It also yielded significant titre reductions of bacteria (Enterococcus faecium, Mycobacterium avium; >6 logs), fungi (spores of Aspergillus niger; > or =5 logs) and poliovirus (>4 logs) embedded in coagulated blood on carriers. The formulation was not found to fix proteins more than was observed with water as the cleaning reagent. In conclusion, SDS, NaOH and n-propanol can synergistically achieve fast, broad-range disinfection.

Quality control assessment for the serological diagnosis of dengue virus infections

BACKGROUND A major drawback of modern societys rapidly increasing mobility is the ease with which dangerous infections can be imported into Europe. Often these infections are not diagnosed because physicians are not familiar with the symptoms and laboratory tests are not always available in local diagnostic centres. Improving diagnostics is the most important step in detecting and dealing with these pathogens and quality control measures are, therefore, essential tools. OBJECTIVES To assess the diagnosis of imported dengue virus infections in Europe by (1) running a pre-evaluation panel (four serum samples, sent out in 1999) and optimising sample preparation and shipping procedures and (2) initiating an External Quality Assurance (EQA) program (20 serum samples, sent out in 2002). STUDY DESIGN All serum samples sent out were to be tested for the presence of dengue virus-specific IgM and IgG. For the pre-evaluation panel, four samples were distributed (one sample IgM+/IgG+, one sample IgM-/IgG+, two samples IgM-/IgG-) and for the EQA 20 samples (12 samples IgM+/IgG+, five samples IgM-/lgG+, one sample lgM+/IgG- two samples IgM-/IgG-). 13 laboratories took part in the pre-evaluation panel and 18 laboratories participated in the first EQA run. RESULTS For the pre-evaluation panel, the participants reported concurrent and correct results for 88% of the IgG-positive samples and for 100% of the IgG-negative samples. The results for the IgM-positive sample were correct in 91% of the reported tests and in 97% of the IgM-negative samples. For the EQA, the participants reported concurrent and correct results for 71% of the IgG-positive samples and 89% of the IgG-negative samples. 58% concurrent and correct results were reported for the IgM-positive samples and 97% for the IgM-negative samples. CONCLUSIONS The results presented here demonstrate the importance of quality measures for imported viral pathogens like dengue viruses and clearly indicate the need for improving the existing test systems.

Molecular typing of Cryptococcus neoformans by PCR fingerprinting, in comparison with serotyping and Fourier transform infrared-spectroscopy-based phenotyping

Molecular typing by PCR fingerprinting using the single primer (GACA)4 was performed with 110 isolates of Cryptococcus neoformans. Seventy clinical isolates of C. neoformans var. neoformans from Germany (n = 52) and Africa (n = 18) were included. Of these, serotype A (C. neoformans var. grubii) accounted for 47 isolates, serotype D for 12 and serotype AD for 11. Fourier transform infrared (FT-IR) spectroscopy was evaluated for its discriminatory power in phenotyping. Molecular types, defined by different PCR fingerprinting patterns, were compared to serotypes, and both sets of results were compared with the results of analysis by FT-IR spectroscopy. PCR fingerprinting revealed genotypic diversity within each serotype; it showed three different genotypes (designated VNA1-VNA3) within serotype A, two within serotype D (VND1 and VND2), and three within serotype AD (VNAD1-VNAD3). The nomenclature of molecular types within C. n. var. neoformans, as seen in publications to date, is not uniform. In this study, the name assigned to each genotype was based on the 98.6% concordance of genotypes with serotypes, a correspondence that facilitates interlaboratory comparison. This nomenclature is tentatively recommended as a standard. FT-IR spectroscopy combined with hierarchical cluster analysis successfully distinguished C n. var. neoformans from C. n. var. gattii. For C. n. var. neoformans, FT-IR confirmed three distinct genotypes within serotype A and was able to distinguish isolates derived from particular patients as well as isolates differing at the sub-genotype level. Within C. n. var. gattii, the serotypes B and C did not correlate with the four genotypes VGI-VGIV. However, these serotypes could clearly be separated by FT-IR spectroscopy. The molecular profiles were reproducible, and were more stable and more discriminating than serotyping. In connection with a standardized nomenclature, PCR fingerprinting can be a beneficial tool for global epidemiological studies. FT-IR spectroscopy adds an additional level of resolution.

Quality control measures for the serological diagnosis of hantavirus infections

BACKGROUND With societys rapidly increasing mobility, patients infected with severe viral infections can become seriously ill at any place in Europe and elsewhere. Improving the diagnostics of these infections is the most important step in detecting the pathogens and dealing with them, and for this purpose, quality control measures are essential tools. OBJECTIVES To assess the diagnostic reality for rare hantavirus infections in Europe by (1) running a pre-evaluation panel (four samples, sent out in 1999) to optimise sample preparation and shipping procedure and afterwards (2) starting an External Quality Assurance (EQA) program (20 samples, sent out in 2001). STUDY DESIGN All samples sent out had to be tested for the presence of specific IgG and IgM antibodies against hantavirus. For the pre-evaluation panel, four samples were distributed (two samples IgG+/IgM-, one sample IgG-borderline/IgM-, one sample IgG-/IgM-), for the EQA 20 samples (six samples IgG+/IgM+, eight samples IgG+/IgM-, one sample IgG-borderline/IgM-, five samples IgG-/IgM-). Thirteen laboratories took part in the pre-evaluation panel, 18 laboratories participated in the first EQA run. RESULTS For the pre-evaluation panel, the participants reported correct results for 64% of the IgG-positive samples (85% excluding borderline-positive sample), and 92% for the IgG-negative sample. IgM testing was correctly negative in all laboratories. For the EQA, the participants reported correct results for 76% of the IgG-positive samples, and 97% correct results for the IgG-negative samples. For the IgM-positive samples, 53% correct results were reported, and 98% correct results for the IgM-negative samples. CONCLUSIONS The results presented here prove the importance of quality measures also for viruses only rarely suspected, like hantavirus, and they clearly demonstrate the need for improvement of the existing test systems.

Inaktivierung und Entfernung von Prionen bei der Aufbereitung von Medizinprodukten

Zu den Aufgaben des Robert Koch-Institutes (RKI) gehört es, gesundheitliche Risiken zu identifizieren, zu analysieren und Maßnahmen bzw. Empfehlungen zu ihrer Minimierung zu erarbeiten und zu kommunizieren. Dementsprechend wurde in Reaktion auf die BSE-Problematik1in einer umfangreichen Arbeitsgruppe beim RKI der Bericht der „Task Force vCJK“ zur Minimierung des Risikos einer iatrogenen Übertragung von Prionen durch chirurgische Instrumente erarbeitet und im April 2002 auf der Basis der damaligen Kenntnis veröffentlicht [1]. Danach stellt eine geeignete alkalische Reinigung mit anschließender Dampfsterilisation bei 134°C ein routinefähiges Verfahren zur Minimierung des Übertragungsrisikos von Prionen durch chirurgische Instrumente dar. Vorliegende Ergebnisse zur inaktivierenden Wirkung anderer Verfahren wurden bisher mit unterschiedlichen Prüfmethoden gewonnen und nach individuellen Kriterien bewertet. Wir teilen deshalb die Meinung von Experten, dass eine Vereinheitlichung der Prüfmethoden erforderlich ist [2], nicht zuletzt um die Entwicklung weiterer für die Routine und spezielle Anforderungen geeigneter Verfahren zur Dekontamination von Prionen zu fördern. Wir möchten hier Anforderungen an die Prüfung und Deklaration entsprechender Verfahren zur Diskussion stellen.Ausdrücklich wird darauf hingewiesen, dass in allen Fällen eines erkennbaren Risikos für das Vorliegen einer transmissiblen spongiformen Enzephalopathie (TSE) den bestehenden Empfehlungen des RKI [1, 3] zu folgen ist.1 BSE ist die bovine, spongiforme Enzephalopathie, deren Erreger, auf den Menschen übertragen, die Variante der Creutzfeldt-Jakob-Krankheit (vCJK) auslösen können. Als Verursacher von transmissiblen spongiformen Enzephalopathien (TSE) gelten Prionen (proteinaceous infectious particles).

Molekularbiologische Identifizierung von Cunninghamella spec.

Zusammenfassung. Mit den klassischen mikrobiologischen Verfahren ist eine eindeutige Identifizierung der bislang einzigen klinisch relevanten Cunninghamella‐Art, C. bertholletiae, nicht möglich. Mittels PCR und Sequenzierung der Internal‐transcribed‐spacer‐(ITS)‐DNA‐Region konnten von neun klinischen Isolaten der Gattung Cunninghamella sieben als C. bertholletiae und zwei als C. echinulata identifiziert werden. Ein Umgebungsisolat eines mit C. echinulata infizierten Patienten konnte ebenfalls als C. echinulata identifiziert werden. Alle Isolate der Spezies C. bertholletiae zeigten eine hohe Sequenzhomologie der ITS‐Region. Innerhalb der Spezies C. echinulata und C. elegans konnte aufgrund unterschiedlicher Restriktionsfragmentlängen‐Profile nach Inkubation der ITS‐Amplifikate mit TaqI und HinfI eine Subspeziesdifferenzierung erreicht werden. Ähnliche Ergebnisse wurden auch mittels PCR fingerprinting der Gesamt‐DNA mit den Microsatellit‐DNA‐Primern (GTG)5 and (GAC)5 erhalten. Erstmalig konnte C. echinulata als Erreger einer Zygomykose beim Menschen identifiziert werden.

Test methods for estimating the efficacy of the fast‐acting disinfectant peracetic acid on surfaces of personal protective equipment

The work aimed at developing and evaluating practically relevant methods for testing of disinfectants on contaminated personal protective equipment (PPE).