Background Invasive fungal diseases (IFDs) remain important causes of morbidity and mortality. The consensus definitions of the Infectious Diseases Group of the European Organization for Research and Treatment of Cancer and the Mycoses Study Group have been of immense value to researchers who conduct clinical trials of antifungals, assess diagnostic tests, and undertake epidemiologic studies. However, their utility has not extended beyond patients with cancer or recipients of stem cell or solid organ transplants. With newer diagnostic techniques available, it was clear that an update of these definitions was essential. Methods To achieve this, 10 working groups looked closely at imaging, laboratory diagnosis, and special populations at risk of IFD. A final version of the manuscript was agreed upon after the groups’ findings were presented at a scientific symposium and after a 3-month period for public comment. There were several rounds of discussion before a final version of the manuscript was approved. Results There is no change in the classifications of “proven,” “probable,” and “possible” IFD, although the definition of “probable” has been expanded and the scope of the category “possible” has been diminished. The category of proven IFD can apply to any patient, regardless of whether the patient is immunocompromised. The probable and possible categories are proposed for immunocompromised patients only, except for endemic mycoses. Conclusions These updated definitions of IFDs should prove applicable in clinical, diagnostic, and epidemiologic research of a broader range of patients at high-risk.

Background:Invasive fungal infections are often diagnosed by histopathology without identification of the causative fungi, which show significantly different antifungal susceptibilities.Aims:To establish and evaluate a system of two seminested polymerase chain reaction (PCR) assays to identify and discriminate between agents of aspergillosis and mucormycosis in paraffin wax embedded tissue samples.Methods:DNA of 52 blinded samples from five different centres was extracted and used as a template in two PCR assays targeting the mitochondrial aspergillosis DNA and the 18S ribosomal DNA of zygomycetes.Results:Specific fungal DNA was identified in 27 of 44 samples in accordance with a histopathological diagnosis of zygomycosis or aspergillosis, respectively.Aspergillus fumigatusDNA was amplified from one specimen of zygomycosis (diagnosed by histopathology). In four of 16 PCR negative samples no human DNA was amplified, possibly as a result of the destruction of DNA before paraffin wax embedding. In addition, eight samples from clinically suspected fungal infections (without histopathological proof) were examined. The two PCR assays detected a concomitant infection withAbsidia corymbiferaandA fumigatusin one, and infections withRhizopus arrhizusandA fumigatusin another two cases.Conclusions:The two seminested PCR assays described here can support a histopathological diagnosis of mucormycosis or aspergillosis, and can identify the infective agent, thereby optimising antifungal treatment.

We assessed Calcofluor white staining, Aspergillus polymerase chain reaction, and a galactomannan enzyme immunoassay for diagnosis of fungal infection with use of computed tomography-guided percutaneous lung biopsy specimens obtained from 61 patients. The sensitivity and specificity of computerized tomography, Aspergillus polymerase chain reaction, and galactomannan enzyme immunoassay were 100% and 50%, 100% and 86%, and 88% and 94%, respectively.Invasive aspergillosis (IA) is a major cause of morbidity and mortality among immunosuppressed patients. Case-fatality rates range from 30% to 80% among neutropenic patients, and death results, at least in part, from difficulties in obtaining a reliable diagnosis in the early stage of disease [1]. No method has proven to be sufficiently sensitive and specific to allow adequate diagnosis.The use of CT allows diagnosis early in the course of pulmonary IA and helps to improve the overall survival rate among febrile, neutropenic patients [2]. Previous studies reported that the CT finding of a halo sign is indicative of pulmonary aspergillosis in neutropenic patients [3]. Thus far, it is impossible to distinguish Aspergillus species from other fungi on the basis of clinical signs [4]. However, identification of the causative organisms is highly warranted in the clinical context to deter- mine adequate therapy. Zygomycetes have in vivo and in vitro resistance to the newer antifungals, such as voriconazole and caspofungin [5]. The number of infections due to zygomycetes have increased in our hospital and in other health care centers, indicating the need for a powerful means of diagnosis [6].We evaluated the utility of Calcofluor white staining (CFWS), galactomannan EIA (GM EIA), and Aspergillus PCR of CTguided lung biopsy specimens for diagnosis of rapidly invasive fungal infection in immunosuppressed patients.Methods. A prospective study conducted from October 2003 through September 2006 evaluated 61 patients who had hematologic malignancies (46 patients) or who had undergone solid-organ transplantation (15 patients) and who had CT findings highly suggestive of an invasive fungal infection. The specimens were obtained by CT-guided percutaneous biopsy and were investigated for the presence of fungal elements. All CTguided percutaneous biopsies were performed with an automated biopsy gun that contained a detachable coaxial cutting needle system, as described by Lucidarme et al. [7]. In our study, an outer coaxial needle with a 17-gauge diameter and an inner biopsy needle with an 18-gauge diameter were chosen. Interventions were performed only for patients with platelet counts of 50,000 platelets/mL and with prothrombin and partial thromboplastin times within the normal limits. Lesions that had a diameter 11 cm and that were most easily assessable were chosen for biopsy.Biopsy specimens were transferred to 2 mL of NaCl, minced, and homogenized aseptically. Samples were then vortexed, stored at room temperature for 30 min, and centrifuged. Supernatants and homogenized tissues wer...

In order to evaluate the diagnostic relevance of two nested PCR assays for diagnosis of histoplasmosis in clinical specimens, 100 paraffin-embedded biopsy specimens were examined. Upon microscopy of tissue, 50 biopsy specimens were histoplasma positive and 50 were negative. Due to destruction by formalin fixation, successful extraction of amplifiable human DNA was limited to 29 and 33 samples, respectively. A product of the Histoplasma capsulatum nested PCR assay targeting the gene encoding the unique fungal 100-kDa-like protein was detected in 20 histopathologically positive biopsy specimens but in none of the microscopically negative samples. Sequencing revealed that all 20 products of 210 bp were identical to the sequence of H. capsulatum in the GenBank database. In contrast, the nested PCR assay targeting the fungal 18S rRNA genes amplified products in 26 histopathologically positive but also in 18 microscopically negative biopsy specimens. However, sequencing revealed that only 20 of these 44 PCR products (231 bp) were identical to the sequence of H. capsulatum. The remaining 24 sequences were homologous to those of several Euascomycetes. These PCR products were detected only in tissues possibly colonized by nonpathogenic fungi, possibly causing these nonspecific amplifications. The detection limit of both H. capsulatum nested PCR assays was 1 to 5 fungal cells per sample. The two assays were similarly sensitive in identifying H. capsulatum. In this preliminary study, the novel 100-kDa-like-protein gene nested PCR revealed a specificity of 100% without requiring sequencing, which was necessary for identification of the 18S ribosomal DNA nested PCR products in order to avoid a high rate of false-positive results.

The PCR assays offer a reliable etiologic diagnosis that is superior to culture in patients with proven invasive mold infection. This may improve patient management through tailored antifungal therapy when cultures fail to grow a pathogen.

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Aspergillosis and mucormycosis are the most common mold infections in patients with hematological malignancies. Infections caused by species of the genus Aspergillus and the order Mucorales require different antifungal treatments depending on the in vitro susceptibility of the causative strain. Cultures from biopsy specimens frequently do not grow fungal pathogens, even from histopathologically proven cases of invasive fungal infection. Two seminested PCR assays were evaluated by amplifying DNA of zygomycetes and Aspergillus spp. from organ biopsies of 21 immunocompromised patients. The PCR assays correctly identified five cases of invasive aspergillosis and six cases of mucormycosis. They showed evidence of double mold infection in two cases. Both assays were negative in five negative controls and in two patients with yeast infections. Sequencing of the PCR products was in accordance with culture results in all culture-positive cases. In six patients without positive cultures but with positive histopathology, sequencing suggested a causative organism. Detection of fungal DNA from biopsy specimens allows rapid identification of the causative organism of invasive aspergillosis and mucormycosis. The use of these PCR assays may allow guided antifungal treatment in patients with invasive mold infections.

SummaryScabies has been diagnosed surprisingly frequently in Germany in recent years, and the use of acaricides has risen markedly. Present figures indicate an increase in the prevalence/incidence of scabies, but do not prove or quantify it for the following reasons: (a) scabies is not a notifiable disease in Germany; (b) the diagnosis is not always confirmed lege artis by means of light microscopy or dermatoscopy (which may lead to a comparatively high proportion of false‐positive diagnoses due to the low overall prevalence of scabies); (c) repeated treatments of the same patient and treatment of contact persons are included in the total number of prescriptions. Therefore, there are no valid data on disease occurrence, either in the current situation or from previous periods.Observations of ineffective treatment with permethrin have led to speculations that Sarcoptes mites are developing resistance to this drug. However, there is little evidence for this assumption.We discuss risk groups (children, elderly people in need of care, migrant health personnel in nursing institutions, refugees, sexually active young adults) and evaluate their possible contribution, albeit in the absence of evidence. None of the groups would be solely responsible for an increased frequency.We have compiled recommendations on how the management of scabies could be improved, and present a way of differentiating permethrin resistance from application errors and/or lack of compliance. The goal is to solve the epidemiological and parasitological questions mentioned above.