Volker Rickerts

BACKGROUND: The severe acute respiratory syndrome (SARS) has recently been identified as a new clinical entity. SARS is thought to be caused by an unknown infectious agent. METHODS: Clinical specimens from patients with SARS were searched for unknown viruses with the use of cell cultures and molecular techniques. RESULTS: A novel coronavirus was identified in patients with SARS. The virus was isolated in cell culture, and a sequence 300 nucleotides in length was obtained by a polymerase-chain-reaction (PCR)-based random-amplification procedure. Genetic characterization indicated that the virus is only distantly related to known coronaviruses (identical in 50 to 60 percent of the nucleotide sequence). On the basis of the obtained sequence, conventional and real-time PCR assays for specific and sensitive detection of the novel virus were established. Virus was detected in a variety of clinical specimens from patients with SARS but not in controls. High concentrations of viral RNA of up to 100 million molecules per milliliter were found in sputum. Viral RNA was also detected at extremely low concentrations in plasma during the acute phase and in feces during the late convalescent phase. Infected patients showed seroconversion on the Vero cells in which the virus was isolated. CONCLUSIONS: The novel coronavirus might have a role in causing SARS.

ABSTRACT Cryptococcosis is a potentially lethal disease of humans/animals caused by Cryptococcus neoformans and Cryptococcus gattii . Distinction between the two species is based on phenotypic and genotypic characteristics. Recently, it was proposed that C. neoformans be divided into two species and C. gattii into five species based on a phylogenetic analysis of 115 isolates. While this proposal adds to the knowledge about the genetic diversity and population structure of cryptococcosis agents, the published genotypes of 2,606 strains have already revealed more genetic diversity than is encompassed by seven species. Naming every clade as a separate species at this juncture will lead to continuing nomenclatural instability. In the absence of biological differences between clades and no consensus about how DNA sequence alone can delineate a species, we recommend using “ Cryptococcus neoformans species complex” and “ C. gattii species complex” as a practical intermediate step, rather than creating more species. This strategy recognizes genetic diversity without creating confusion.

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 fumigatus DNA 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 with Absidia corymbifera and A fumigatus in one, and infections with Rhizopus arrhizus and A fumigatus in 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.

BACKGROUND: With the advent of new antifungal agents, the identification of a causative pathogen is crucial to guide the antifungal treatment of invasive mold infection. However, tissue cultures often fail to grow a fungal pathogen in cases of suspected mold infection. METHODS: In a prospective multicenter study, we compared the results of histopathological analysis, culture, and 2 seminested polymerase chain reaction assays identifying Aspergillus species and Zygomycetes as causative agents of invasive mold infections using respiratory tract biopsy samples obtained from 56 immunocompromised patients who had suspected mold infection. RESULTS: Mold hyphae were detected histopathologically in 27 (48%) of the tissue specimens. Hyphae corresponded to either aspergillosis (n=18) or zygomycosis (n=6) or could not be further specified (n=3). A mold was cultured from 14 of 18 samples with aspergillus hyphae, 2 of 6 samples with Zygomycetes hyphae, and 1 of 3 samples with unspecified hyphae. Polymerase chain reaction was superior to culture in detecting the infecting mold (26 of 27 samples vs. 17 of 27 samples, respectively; P=.006) from histopathologically positive samples. Genus or species identification by sequencing of the polymerase chain reaction products were in accordance with culture results in 16 of 18 culture-positive samples. Both polymerase chain reaction assays failed to detect fungal DNA in 1 sample that had unspecified hyphae and negative culture results. CONCLUSION: 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.