Detection and identification of fungal pathogens in blood by using molecular probes

Abstract

A PCR assay was developed for the detection and identification of Candida and Aspergillus species. The design of the oligonucleotide primer pair as well as the species-specific probes used for species identification was derived from a comparison of the sequences of the 18S rRNA genes of various fungal pathogens. The primers targeted a consensus sequence for a variety of fungal pathogens. The assay was tested for sensitivity and specificity with 134 fungal and 85 nonfungal isolates. To assess clinical applicability, 601 blood samples from four defined groups were tested: group A (n ‫؍‬ 35), controls; groups B to D (n ‫؍‬ 86), patients with febrile neutropenia, without fungal colonization (group B; n ‫؍‬ 29) and with fungal colonization (group C; n ‫؍‬ 36); and patients with documented invasive fungal infection (IFI) (group D; n ‫؍‬ 21). The assay detected and, by species-specific hybridization, identified most of the clinically relevant Candida and Aspergillus species at 1 CFU/ml of blood. Amplification was 100% sensitive for all molds and yeasts tested, with Histoplasma capsulatum being the only non-Aspergillus species hybridizing with the Aspergillus spp. probe. None of 35 group A patients and only 3 of 65 group B and C patients were PCR positive. The sensitivity of the assay for specimens from patients with IFI (21 patients in group D) was 100% if two specimens were tested. For specificity, 3 of 189 specimens from patients at risk but with negative cultures were positive by the assay, for a specificity of 98%. PCR preceded radiological signs by a median of 4 days (range, 4 to 7 days) for 12 of 17 patients with hepatosplenic candidiasis or pulmonary aspergillosis. For the 10 patients with IFI responding to antifungal therapy, PCR assays became persistently negative after 14 days of treatment, in contrast to the case for 11 patients, who remained PCR positive while not responding to antifungal therapy. Thus, the described PCR assay allows for the highly sensitive and specific detection and identification of fungal pathogens in vitro and in vivo. Preliminary data from the screening of a selected group of patients revealed some value in the early diagnosis and monitoring of antifungal therapy.

Advances in the molecular and serological diagnosis of invasive fungal infection in haemato‐oncology patients

“…The sensitivity of the assay for invasive candidiasis, including hepatosplenic candidiasis, and aspergillosis was 100% if two specimens were tested, with positive PCR results preceding radiological evidence of infection in the majority of cases. None of the controls were positive and, significantly, only three specimens from three of 36 neutropenic, colonized patients with negative fungal cultures were positive (Einsele et al, 1997).…”

“…Other assays have been developed which utilize serum, plasma or whole blood specimens. Experimental models and clinical studies have shown PCR to be more sensitive than culture for detection of candidaemia (Chryssanthou et al, 1994;van Deventer et al, 1995;Burnie et al, 1997;Einsele et al, 1997;Morace et al, 1999;Wahyuningsih et al, 2000). Einsele used a panfungal PCR assay to retrospectively analyse 601 blood specimens from patients with documented IFI, those with febrile neutropenia with and without colonization, and from controls.…”

Screening for Aspergillus spp. using polymerase chain reaction of whole blood samples from patients with haematological malignancies

“…PCR assay. DNA extraction of 10 ml of EDTA-anticoagulated blood was performed using recombinant lyticase (Sigma, Wien, Austria) and the QIAmp Tissue Kit (Qiagen, Wien, Austria), as previously described (Einsele et al, 1997). Genes of the 18S rRNA of various fungal pathogens were amplified by PCR using specific primers.…”

Mucormycosis and entomophthoramycosis: a review of the clinical manifestations, diagnosis and treatment

“…Polymerase chain reaction may become a diagnostic modality to identify these organisms [139]. Rickerts et al diagnosed disseminated C. bertholletiae infection in a leukaemic patient using a polymerase chain reaction assay targeting 18S ribosomal DNA in blood.…”