Molecular diagnostics in cancer patients with suspected respiratory mold infections

Abstract

Invasive fungal infections (IFIs), including aspergillosis and mucormycosis, are serious complications in cancer patients. Reliable detection of etiologic agents is challenging but important for optimal patient management. In a prospective multicenter study, we compared fungal detection by culture, specific and unspecific qPCRs, and fluorescence in situ hybridization (FISH) targeting Aspergillus and Mucorales in bronchoalveolar lavage fluid (BALF) and serum of adult cancer patients with suspected fungal pneumonia. In subgroups of patients, fungal DNA was amplified from serum. We evaluated 210 IFI episodes (proven: 3, probable: 72, possible: 107, unclassifiable: 28). Broad-range fungal PCR from BALF was terminated early due to frequent amplification of colonizing yeasts. Specific qPCR assays targeting molds (Aspergillus, Mucorales) were superior for mold detection (26%) compared to FISH (15%) and culture (8%). Detection of fungi by FISH predicted subsequent fungal culture positivity. In proven/probable IFI episodes, detection of Aspergillus DNA was more likely in BALF than in serum (35/75 [47%] vs 11/66 [17%]; P = 0.0002) in contrast to Mucorales DNA (7/75 [9%] vs 5/66 [8%]; P = 0.73). Mucorales DNA was detected together with Aspergillus DNA in 6 of 7 BALF samples but not in serum. Specific qPCRs targeting Aspergillus and Mucorales from BALF are the most sensitive means to identify molds in adult cancer patients with suspected fungal pneumonia. BALF qPCRs suggest frequent fungal coinfection, while viruses were rarely detected. Fungal detection by FISH may predict subsequent positive fungal cultures.IMPORTANCEMold pneumonia is a serious complication in cancer patients. Reliable detection of etiologic agents is critical for patient care, but optimal testing strategies are undefined. We performed a prospective study comparing molecular tests (specific and unspecific qPCR and FISH) and culture on bronchoalveolar lavage fluid (BALF) and serum, focusing on aspergillosis and mucormycosis to gain insights into mold detection in high-risk patients. We demonstrate that FISH visualizes fungal elements in BALF, predominantly conidia, suggesting recent fungal exposure or failure to clear inhaled conidia at the time of diagnostic testing. We find that specific qPCRs from BALF are the most sensitive method to detect Aspergillus and Mucorales, superior to broad-range qPCR, culture, and testing of serum. Importantly, Mucorales DNA is mostly co-detected with Aspergillus, suggesting co-infections. Although DNA detection in serum is more likely in mucormycosis than in aspergillosis, sole serum testing may miss mucormycosis, precluding optimal patient care.

Keywords: Aspergillosis; BALF; CMV; FISH; PCR; cancer; mucormycosis.

Fig 1

Classification of enrolled patients and respective episodes of suspected respiratory mold infections according to EORTC/MSG 2008 criteria.

Fig 2

Comparison of PCR, culture, and FISH positivity for detection of Aspergillus and Mucorales in BALF samples from proven (n = 3) or probable (n = 72) episodes. Abbreviations: FISH, fluorescence in situ hybridization; Asp, Aspergillus; Mucor, Mucorales; +, positive.

Fig 3

Fluorescence microscopy of BALF samples (each row represents one sample) using DAPI staining (targeting DNA) and FISH targeting ribosomal RNA by an unspecific probe (EUK) and specific probes targeting Aspergillus using the Cy3 dye and Mucorales using the Cy5 dye. First row: conidia, hybridizing with Aspergillus probe with leuco- and erythrocytes (autofluorescence Cy3 and FITC). Second row: hyphae, hybridizing with Aspergillus probe. Third row: germinating conidia hybridizing with Mucorales probe. Fourth row: yeast cells hybridizing with unspecific probe (EUK) only. Fifth row: hyphal fragment without hybridization of Aspergillus and Mucorales probes. Sixth row: yeasts hybridizing with unspecific probe and swollen conidia hybridizing with Aspergillus probe. Bar represents 5 µm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindol; EUK, eukaryotic probe; Cy, cyanine; BALF, bronchoalveolar lavage fluid; FISH, fluorescence in situ hybridization.