Detection of Fusarium oxysporum DNA and (1-3)-b-D-glucan in serum specimens of experimentally infected mice
Abstract number: 1733_1471
Khan Z.U., Ahmad S., Theyyathel A.
Objective: Invasive Fusarium infection is an emerging opportunistic mycosis associated with high mortality. Its diagnosis is difficult due to non-specific signs and symptoms and similarity in tissue morphology with Aspergillus species. The aim of this study was to develop a sensitive and specific diagnostic procedure based on amplification of Fusarium oxysporum DNA by nested (n) PCR and to compare the results with (1-3)-b-D-Glucan detection in serum samples obtained from experimentally infected mice.
Methods: Sixty mice immunosuppressed with intraperitoneal injections of cyclophosphamide were infected intravenously with a dose of 1×107F. oxysporum conidia/mouse. Six mice were sacrificed every day post-infection. Blood specimens were collected by cardiac puncture. One hundred mL of blood was used for culture and the rest was processed for separation of serum. The lung homogenate was cultured and was also used for direct microscopic examination. The genomic DNA from the reference strains of F. oxysporum and 9 other fungi including Fusariumsolani, Aspergillus fumigatus, A. flavus and A. terreus was isolated and used as template for nPCR specificity. The species-specific primers were derived from the internally transcribed spacer (ITS)-1 and ITS-2 regions of rDNA and their specificity was confirmed by BLAST searches. The DNA from serum specimens was extracted using standard procedures. The PCR amplicons were detected by agarose gel electrophoresis. The (1-3)-b-D-Glucan was detected by Fungitell (Cape Cod Inc. E. Falmouth, MA, USA).
Results: The nPCR was specific for F. oxysporum and detected nearly 440 fg of Fusarium DNA which is roughly equivalent to 10 genome copies. Of the 60 serum samples tested, 55 (92%) were positive for (1-3)-beta-D-Glucan and 47 (78%) were positive for F. oxysporum DNA. The lung homogenates of all the infected animals yielded F. oxysporum in culture. The fungus was also discernible in KOH-calcofluor mount of 40 (67%) of the animals.
Conclusions: A sensitive and species-specific nPCR assay has been developed for the detection of F. oxysporum DNA in serum samples of experimentally-infected mice. During a follow-up of 14 days post-infection, 92% and 78% of serum samples were positive for (1-3)-b-D-Glucan and F. oxysporum DNA, respectively. The data suggest that detection of F. oxysporum DNA by nPCR combined with (1-3)-b-D-Glucan may help in early diagnosis of invasive fusariosis.
Supported by KURA Grant MI 04/02.
|Session name:||European Society of Clinical Microbiology and Infectious Diseases|
|Location:||ICC, Munich, Germany|
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