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Clin Infect Dis. 2017 Nov 29;65(12):2035-2041. doi: 10.1093/cid/cix728.

Performance of Targeted Fungal Sequencing for Culture-Independent Diagnosis of Invasive Fungal Disease.

Author information

1
Department of Pathology.
2
Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford.
3
Clinical Microbiology Laboratory, Stanford University Medical Center, Palo Alto, California.

Abstract

Background:

Identification of fungi causing invasive fungal disease (IFD) is critical for guiding antifungal therapy. We describe the performance and clinical impact of a targeted panfungal polymerase chain reaction (PCR) amplicon sequencing assay for culture-independent diagnosis of IFD.

Methods:

Between January 2009 and September 2016, 233 specimens, consisting of fresh and formalin-fixed, paraffin-embedded (FFPE) tissues and sterile body fluids with known diagnosis of IFD based on reference method results (n = 117), and specimens with negative fungal culture, but with microscopic and ancillary findings indicative of IFD (n = 116), were included. PCR amplicons from the internal transcribed spacer 2 and the D2 region of 28S ribosomal RNA gene were sequenced and fungi identified.

Results:

Sensitivity and specificity of fungal sequencing in specimens with known diagnosis were 96.6% (95% confidence interval [CI], 87.4%-99.4%; 58/60) and 98.2% (95% CI, 89.4%-99.9%; 56/57). In patients with suspected IFD, the diagnostic yield of fungal sequencing was 62.9% (73/116) overall and 71.3% (57/80) in patients classified with proven IFD based on the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and Mycoses Study Group (EORTC/MSG) criteria. Samples obtained by open biopsy had a significantly higher diagnostic yield (71.5% [40/56]) compared with core-needle biopsy (50% [17/34] P = .04) and fine needle aspiration (0% [0/2]; P = .009). Additionally, D2 sequencing diagnosed 5 cases of invasive protozoal infections due to Toxoplasma gondii (n = 3), Trypanosoma cruzi, and Leishmania species. Sequencing results altered patient management in the majority of suspected cases.

Conclusions:

The targeted fungal sequencing assay allowed accurate identification of fungi causing IFD and additionally provided partial-protozoal coverage. The diagnostic yield was dependent on the amount of tissue available for testing.

KEYWORDS:

invasive fungal disease; invasive fungal infection; panfungal PCR; ribosomal RNA; sequencing

PMID:
29020284
DOI:
10.1093/cid/cix728
[Indexed for MEDLINE]

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