Treatments for invasive fungal infections remain unsatisfactory. We evaluated the efficacy, tolerability, and safety of voriconazole as salvage treatment for 273 patients with refractory and intolerant-to-treatment fungal infections and as primary treatment for 28 patients with infections for which there is no approved therapy. Voriconazole was associated with satisfactory global responses in 50% of the overall cohort; specifically, successful outcomes were observed in 47% of patients whose infections failed to respond to previous antifungal therapy and in 68% of patients whose infections have no approved antifungal therapy. In this population at high risk for treatment failure, the efficacy rates for voriconazole were 43.7% for aspergillosis, 57.5% for candidiasis, 38.9% for cryptococcosis, 45.5% for fusariosis, and 30% for scedosporiosis. Voriconazole was well tolerated, and treatment-related discontinuations of therapy or dose reductions occurred for <10% of patients. Voriconazole is an effective and well-tolerated treatment for refractory or less-common invasive fungal infections.

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.

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.

Aspergillus fumigatus is often found in the respiratory tract secretions of patients with cystic fibrosis (CF), although the role of the fungus for progression of pulmonary disease remains unclear. This study aimed to investigate the frequency of A. fumigatus and other fungi in sputum of adult CF patients using different methods for culture and microscopy. Results from the analysis of 369 samples from 94 patients showed that A. fumigatus could be isolated in 45.7% of patients. Other moulds were rare, but the yeast Candida albicans was another frequent isolate, detected in 75.5% of patients. A comparison of different culture media showed no difference between a selective medium developed to specifically inhibit Pseudomonas aeruginosa and a standard fungal culture medium for growth of A. fumigatus, although both were more efficient for detection of fungi than other bacterial culture media. Fluorescent microscopy with calcofluor white was more sensitive for detection of fungal hyphae in undiluted sputum than standard methylene blue staining. This study shows that A. fumigatus and C. albicans have a high frequency in adult CF patients. Microbiological analysis should routinely include methods for specific identification of fungi to monitor for potential complications arising from fungal disease in these patients.

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.

Scedosporium prolificans is one of the most life-threatening fungal opportunistic pathogens due to its high resistance to common systemic antifungal agents. While a close relative of Pseudallescheria boydii, S. prolificans has a more limited geographic range being primarily found in Australia, USA and Spain. Infections have also been reported from several other European countries and from Chile. Twenty patients with Scedosporium prolificans infection or colonization from August 1993 to May 2007 were retrospectively reviewed in Germany. They had all been identified at or reported to the Reference Laboratory for Pseudallescheria/Scedosporium spp. in Berlin. Twelve of 13 patients with haematological disorders and/or on immunosuppressive therapy developed a fatal invasive scedosporiosis. Colonization of the respiratory tract was reported for one patient after heart-lung-transplantation, all six patients with cystic fibrosis and one with chronic sinusitis. Molecular studies of the S. prolificans isolates confirmed that parts of the 18S, the Internal Transcribed Spacer (ITS) regions and the D1/D2 domain of the 28S region of rDNA are monomorphic. However, sequencing of parts of the translation elongation factor EF1-alpha (EF-1alpha) and the chitin synthase (CHS-1) genes revealed the presence of three and two distinct genotypes, respectively. Two informative mutations were found in EF-1alpha and a single nucleotide exchange in the CHS-1 gene.

Cunninghamella bertholletiae is a rare cause of pulmonary mucormycosis. We describe a cluster of invasive pulmonary infections caused by C. bertholletiae in 4 immunocompromised patients that occurred during a 2-year period at 1 center. Three of the patients were receiving antifungal prophylaxis with itraconazole. Presenting symptoms were fever unresponsive to antibacterial chemotherapy, hemoptysis, and infiltrates on chest radiograms. Three patients were treated with liposomal amphotericin B. Only 1 patient survived.Invasive fungal infections are serious and often fatal complications in immunocompromised patients. Candida species, Aspergillus species, and Cryptococcus neoformans are the most frequent pathogens of these infections [1,2]. Similar conditions caused by other fungal pathogens such as Trichosporon beigelii, Fusarium species, and Penicillium marneffei have been described, and these pathogens are being isolated with increasing frequency [3,4].Cunninghamella bertholletiae (class Zygomycetes, order Mucorales) is a saprophytic, ubiquitous fungus that is found in soil. It is rarely isolated as an agent of zygomycoses in immunocompromised patients [5]. We reviewed the medical records of 4 immunocompromised patients who developed serious pulmonary infections caused by C. bertholletiae during a 2-year period at the University Hospital in Frankfurt, Germany; we describe this cluster of infections here (table 1). Case ReportsCase 1. A 60-year-old woman in first complete remission of acute lymphoblastic leukemia was admitted to our hospital in November 1998 for consolidation therapy with cytarabine and teniposide (German ALL [acute lymphoblastic leukemia] Study Group protocol) [6]. Eight days after the start of chemotherapy, she developed neutropenia (absolute neutrophil count, !500/mL). She developed fever and abdominal pain 1 day later. CT of the abdomen revealed evidence of appendicitis and cholecystitis. Cholecystectomy and appendectomy were performed the same day. Both diagnoses were confirmed his- topathologically. There was no evidence of invasive fungal infection of the gallbladder or appendix. The bone marrow regenerated after 14 days of neutropenia, and the patient developed mild hemoptysis. The platelet count, prothrombin time, and partial thromboplastin time were normal. A CT scan of the chest showed a homogenous alveolar opacification in the left upper and right lower lobes. Treatment was not started.Follow-up CT performed 7 days later showed decreasing infiltrates, and the patient was discharged from the hospital 4 weeks thereafter in stable condition. Four days later, the patient developed massive hemoptysis and was readmitted. A CT scan of the chest revealed multiple cavities in both lower lobes and left middle and upper lobes. Antifungal therapy with amphotericin B (1 mg/kg/day) was started on the same day. Bronchoscopy was done; this procedure revealed hemorrhage of the right lower lobe and a mass obstructing the left upper bronchus. Analysis of bronchial aspirates (BAs) revealed broad, nonsepta...