Panfungal PCR assay for detection of fungal infection in human blood specimens

Abstract

A novel panfungal PCR assay which detects the small-subunit rRNA gene sequence of the two major fungal organism groups was used to test whole-blood specimens obtained from a series of blood or bone marrow transplant recipients. The 580-bp PCR product was identified after amplification by panfungal primers and hybridization to a 245-bp digoxigenin-labeled probe. The lower limit of detection of the assay was approximately four organisms per milliliter of blood. Multiple whole-blood specimens from five patients without fungal infection or colonization had negative PCR results. Specimens from 11 infected patients had positive PCR results. Blood from three patients with pulmonary aspergillosis had positive PCR results: one patient's blood specimen obtained in the week prior to the diagnosis of infection by a positive bronchoalveolar lavage fluid culture result was positive by PCR, and blood specimens obtained from two patients 1 to 2 days after lung biopsy and which were sterile by culture were positive by PCR. The blood of four patients with candidemia, three patients with mixed fungal infections, and one patient with fusariosis also had positive PCR signals. The panfungal PCR assay can detect multiple fungal genera and may be used as an adjunct to conventional methods for the detection of fungal infection or for describing the natural history of fungal infection. Further studies are needed to define the sensitivity and specificity of this assay for the diagnosis of fungal infection prior to the existence of other clinical or laboratory indications of invasive fungal infection.

FIG. 1

Results for candidemic patients 10022 (C. parapsilosis), 11958 (C. parapsilosis), 10701 (C. glabrata), and 10490 (C. tropicalis, C. albicans, and a Candida strain whose species was not determined). Solid symbols indicate positive results; ▴, Hickman catheter tip culture; •, blood culture; and ▪, whole-blood PCR assay. Open symbols indicate negative results; ○, blood culture; □, whole-blood PCR assay.

FIG. 2

Results for patients with pulmonary aspergillosis. Infection in patient 9282 was diagnosed by bronchoscopy, 3 days after a CT scan demonstrated pulmonary nodules. A. fumigatus cultured from open lung biopsy specimens from patients 9886 and 10850. ▴, CT scan with nodules; ▵, CT scan without nodules; +, bronchoalveolar fluid culture positive; ×, sputum culture positive; ⧫, lung biopsy specimen positive; •, blood culture positive; ○, blood culture negative; ▪, PCR-positive blood; □, PCR-negative blood.

FIG. 3

Clinical course for patients with mixed infections. Patient 10916 had multiorgan failure in the setting of difficult-to-control graft-versus-host disease. Infection with neither C. albicans nor A. fumigatus was suspected, and no antifungal therapy had been initiated prior to death. Patient 10149 had pulmonary infection with P. carinii followed by infection with A. fumigatus. Patient 7872 died of fusariosis. Patient 9937 died of disseminated yeast and mold infection after struggling for months with severe chronic graft-versus-host disease. (A to D) ▪, positive PCR result for blood; □, negative PCR result for blood; and ○, culture-negative blood specimen. (A) ×, lung sample culture positive for Aspergillus at autopsy; •, blood specimen culture positive for Torulopsis (Candida) glabrata. (B) ▿, normal chest radiograph; ▾, chest radiograph with infiltrates; ×, lung specimen culture positive for Aspergillus at autopsy; ∗, bronchoalveolar lavage fluid positive for P. carinii by silver stain. (C) +, sputum specimen culture positive for Fusarium; ×, skin biopsy specimen positive for hyphae; •, blood specimen culture positive for Fusarium. (D) +, blood specimen culture positive for a bacterial organism; ∗, cultures of organ specimens obtained at autopsy grew Torulopsis and sterile mycelia; ▴, blood culture grew a zygomycete; ▾, blood culture grew Prototheca; ⧫, blood culture grew Penicillium.