Identification of Aspergillus and Mucorales in formalin-fixed, paraffin-embedded tissue samples: Comparison of specific and broad-range fungal qPCR assays.

Establishing the etiology of invasive fungal infections is important to guide therapeutic options and for epidemiologic purposes. Formalin-fixed, paraffin-embedded (FFPE) tissue specimens from patients with proven invasive fungal infections are valuable to determine the etiology of systemic fungal infections. We compared different polymerase chain reaction (PCR) amplification strategies from FFPE tissue blocks to identify agents of invasive fungal infections. We found that specific PCR assays show superior sensitivity in the identification of DNA of Mucorales and Aspergillus and mixed infections caused by both as compared to broad-range PCR assays. Shorter amplicon lengths and less detection of contaminating fungal DNA are potential factors involved. However, detection of fungal DNA by highly sensitive specific PCR assays in the absence of demonstration of fungal elements in tissue suggests that PCR results should be interpreted in the context of the histopathology and clinical findings.

Deciphering the aetiology of a mixed fungal infection by broad-range PCR with sequencing and fluorescence in situ hybridisation.

Simultaneous infections with multiple fungi may be misinterpreted as monomicrobial infections by current diagnostics with ramifications for the choice of antimicrobial agents that may impact patient outcomes. The application of molecular methods on tissue samples may be useful to decipher the aetiology of mixed fungal infections. We present a leukaemic patient who died from sepsis due to candidaemia. The postmortem examination documented fungal elements in lung tissue. Fungal DNA was amplified from the lung sample by broad-range PCR assays targeting the 28S ribosomal RNA gene or the internal transcribed spacer 2 (ITS-2). Fluorescence in situ hybridisation (FISH) using differentially labelled fungal probes was applied on the tissue. Sequencing identified the PCR amplicons as Aspergillus fumigatus (28S assay) and Candida tropicalis (ITS-2 assay). As a chromatogram suggested mixed amplicons, the Isentio ripseq(®) tool for in silico analysis was applied and confirmed the presence of both amplicons in the PCR products of both assays. FISH confirmed the presence of Aspergillus and Candida within the infectious process, a prerequisite for inferring a causal relationship with the infection. The combination of broad-range PCR with sequence analysis and FISH applied on tissue samples is a powerful approach to identify the aetiology of invasive fungal infections, including mixed infections.

Clinical and molecular epidemiology of ciprofloxacin-susceptible MRSA encoding PVL in England and Wales.

We aimed to enhance our case ascertainment of meticillin-resistant Staphylococcus aureus encoding Panton-Valentine leucocidin (PVL-MRSA), determine the patient demographic, risk factor and disease associations, and define the clonal diversity amongst isolates referred to the UK Health Protection Agency's Staphylococcus Reference Unit. PVL-MRSA collected during 2005-6 from community-based and hospitalised patients located across England and Wales were identified by polymerase chain reaction (PCR). Representative geographically and temporally unrelated isolates were characterised via toxin gene profiling, SCCmec, spa and agr typing, multilocus sequence typing (MLST) and minimum inhibitory concentration (MIC) determinations. PVL-MRSA were identified from 275 patients. Affected individuals were <1 to 95 years of age (mean 30, median 27 years). Forty-five isolates were from 18 household or community-based clusters and 23 isolates were from outbreaks in healthcare settings. Overall, 58% (n = 161) had skin and soft tissue infections and 9% (n = 25) presented with or developed more serious disease, including eight patients (3%) with necrotising pneumonia, five of whom subsequently died. PVL-MRSA were genetically diverse and harboured SCCmecIV or V(T)/VII. Representatives of MLST clonal complexes (CCs) 8, 30 and 80 were identified the most often. The 275 PVL-MRSA included internationally disseminated community-associated MRSA (CA-MRSA) strains, as well as other minor lineages, and were associated with typical risk factors and disease presentations.