Comparison of quantitative real time PCR with Sequencing and ribosomal RNA-FISH for the identification of fungi in formalin fixed, paraffin-embedded tissue specimens.

BACKGROUND: Identification of the causative agents of invasive fungal infections (IFI) is critical for guiding antifungal therapy. Cultures remain negative in a substantial number of IFI cases. Accordingly, species identification from formalin fixed, paraffin embedded (FFPE) tissue specimens by molecular methods such as fluorescence in situ hybridisation (FISH) and PCR provides an appealing approach to improve management of patients. METHODS: We designed FISH probes targeting the 28S rRNA of Aspergillus and Candida and evaluated them with type strains. Fluorescence microscopy (FM), using FISH probes and quantitative broad-range fungal PCR targeting the rRNA gene were applied to FFPE tissue specimens from patients with proven IFI in order to explore benefits and limitations of each approach. RESULTS: PCR followed by sequencing identified a broad spectrum of pathogenic fungi in 28 of 40 evaluable samples (70%). Hybridisation of FISH probes to fungal rRNA was documented in 19 of 40 tissue samples (47.5%), including 3 PCR negative samples with low fungal burden. The use of FISH was highly sensitive in invasive yeast infections, but less sensitive for moulds. In samples with hyphal elements, the evaluation of hybridisation was impaired due to autofluorescence of hyphae and necrotic tissue background. CONCLUSIONS: While PCR appears to be more sensitive in identifying the causative agents of IFI, some PCR negative and FISH positive samples suggest that FISH has some potential in the rapid identification of fungi from FFPE tissue samples.

Haemorrhagic cystitis and polyomavirus JC infection in ataxia telangiectasia.

We report the case of a young adolescent with ataxia telangiectasia (AT) and life-threatening haemorrhage from the bladder due to a combination of bladder wall telangiectasis, immunosuppressive therapy and an infection with polyomavirus JC. BK and JC are both members of the polyomavirus family. BK virus is a known cause of haemorrhagic cystitis in bone-marrow and nephropathy in kidney transplant patients, whereas JC virus is mainly associated with progressive multifocal leukoencephalopathy and only rarely found in haemorrhagic cystitis. Although opportunistic infections are uncommon in AT and virus replication was described as being down-regulated in ATM (AT mutated protein)-deficient cells, clinicians should be aware that severe haematuria in a patient with AT and undergoing immunosuppressive therapy is suggestive for polyomavirus JC-induced haemorrhagic cystitis.

Comparison of histopathological analysis, culture, and polymerase chain reaction assays to detect invasive mold infections from biopsy specimens.

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.

Rhinocerebral zygomycosis in a young girl undergoing allogeneic stem cell transplantation for severe aplastic anaemia.

We report on a 10-year-old girl with severe aplastic anaemia who developed rhinocerebral infection caused by Absidia corymbifera and a possible co-infection caused by Alternaria alternata. Despite prolonged neutropenia, therapy with liposomal amphotericin B and posaconazole improved the clinical condition. Subsequently, the girl underwent allogeneic haematopoietic stem cell transplantation (HSCT) for the underlying disease, but the fungal infection remained under control with the antifungal treatment. No severe side effect of the antifungal drugs was noted. Unfortunately, the girl died 5 months after HSCT due to disseminated adenovirus infection.