Diagnosis of invasive fungal infections by a real-time panfungal PCR assay in immunocompromised pediatric patients.

Invasive fungal disease (IFD) is a life-threatening event in immunocompromised patients, and there is an urgent need for reliable screening methods facilitating rapid and broad detection of pathogenic fungi. We have established a two-reaction real-time PCR assay permitting highly sensitive detection of more than 80 fungal pathogens, covering a large spectrum of moulds, yeasts and Zygomycetes. To assess the clinical potential of the assay, more than 600 consecutive specimens from 125 pediatric patients carrying a high risk of IFD were analyzed. An excellent correlation between PCR positivity and the presence of proven, probable or possible fungal infection according to the European Organization for Research and Treatment of Cancer criteria was demonstrated, as revealed by the sensitivity of the assay of 96% (95% CI: 82-99%). The negative predictive value of the panfungal PCR assay presented was 98% (95% CI: 90-100%), while the specificity and the positive predictive value were 77% (95% CI: 66-85%) and 62% (95% CI: 47-75%), respectively. The results indicate that molecular screening of patients during febrile neutropenic episodes by the assay presented could help prevent unnecessary toxicity resulting from empirical antifungal treatment in individuals who may not be at risk of imminent fungal disease. Our observations raise the possibility that rapid species identification may be required to increase the positive predictive value for impending fungus-related disease.

Monitoring of adenovirus load in stool by real-time PCR permits early detection of impending invasive infection in patients after allogeneic stem cell transplantation.

Invasive adenovirus (AdV) infections are associated with high morbidity and mortality in allogeneic stem cell transplant recipients. We observed that molecular detection of the virus in stool specimens commonly precedes AdV viremia, suggesting that intestinal infections may represent a common source of virus dissemination. To address this notion, we have investigated 153 consecutive allogeneic transplantations in 138 pediatric patients by quantitative monitoring of AdV in stool specimens and peripheral blood by a pan-adenovirus real-time (RQ)-PCR approach. AdV was detectable in serial stool specimens in all cases of AdV viremia during the post-transplant course (P<0.0001). The incidence of AdV viremia in individuals with peak virus levels in stool specimens above 1 x 10E6 copies per gram (n=22) was 73% vs 0% in patients with AdV levels in stool specimens below this threshold (n=29; P<0.0001). Serial measurement of AdV levels in stool specimens by RQ-PCR permitted early diagnosis of impending invasive infection with a sensitivity and specificity of 100% (95% confidence interval (CI) 96-100%) and 83% (95% CI 67-92%), respectively. The median time span between detection of AdV loads in stool specimens above 1 x 10E6 copies per gram and first observation of viremia was 11 days (range 0-192). Quantitative monitoring of the AdV load in stool specimens therefore provides a rationale for early initiation of antiviral treatment with the aim of preventing progression to life-threatening invasive infection.

Species-specific identification of a wide range of clinically relevant fungal pathogens by use of Luminex xMAP technology.

In immunocompromised patients suffering from invasive fungal infection, rapid identification of the fungal species is a prerequisite for selection of the most appropriate antifungal treatment. We present an assay permitting reliable identification of a wide range of clinically relevant fungal pathogens based on the high-throughput Luminex microbead hybridization technology. The internal transcribed spacer (ITS2) region, which is highly variable among genomes of individual fungal species, was used to generate oligonucleotide hybridization probes for specific identification. The spectrum of pathogenic fungi covered by the assay includes the most commonly occurring species of the genera Aspergillus and Candida and a number of important emerging fungi, such as Cryptococcus, Fusarium, Trichosporon, Mucor, Rhizopus, Penicillium, Absidia, and Acremonium. Up to three different probes are employed for the detection of each fungal species. The redundancy in the design of the assay should ensure unambiguous fungus identification even in the presence of mutations in individual target regions. The current set of hybridization oligonucleotides includes 75 species- and genus-specific probes which had been carefully tested for specificity by repeated analysis of multiple reference strains. To provide adequate sensitivity for clinical application, the assay includes amplification of the ITS2 region by a seminested PCR approach prior to hybridization of the amplicons to the probe panel using the Luminex technology. A variety of fungal pathogens were successfully identified in clinical specimens that included peripheral blood, samples from biopsies of pulmonary infiltrations, and bronchotracheal secretions derived from patients with documented invasive fungal infections. Our observations demonstrate that the Luminex-based technology presented permits rapid and reliable identification of fungal species and may therefore be instrumental in routine clinical diagnostics.

Identification of fungal species by fragment length analysis of the internally transcribed spacer 2 region.

The rapid identification of fungal pathogens in clinical specimens is a prerequisite for timely onset of the most appropriate treatment. The aim of the present study was to develop a sensitive and rapid method for the species-specific identification of clinically relevant fungi. We employed fluorescent polymerase chain reaction (PCR)-fragment length analysis of the highly variable internally transcribed spacer 2 (ITS2) region to identify individual fungal species by their specific amplicon sizes. The specificity of the technique was ascertained by the detailed analysis of 96 strains derived from 60 different human-pathogenic fungal species. To achieve adequate sensitivity for species identification in patients with invasive fungal infection, who often display very low pathogen loads in peripheral blood, the ITS2 region was amplified by semi-nested PCR prior to amplicon-length analysis. Serial specimens from 26 patients with documented fungal infections were investigated. The fungal pathogens identified included different Aspergillus and Candida species, Rhizopus oryzae and Fusarium oxysporum. Fragment length analysis of the ITS2 region upon amplification by semi-nested PCR permits the sensitive identification of fungal species. The technique can be readily implemented in routine diagnostics.