Prospective study of a panfungal PCR assay followed by sequencing, for the detection of fungal DNA in normally sterile specimens in a clinical setting: a complementary tool in the diagnosis of invasive fungal disease?

Abstract

We prospectively analyzed 34 clinical biopsy samples from 23 patients with a suspected invasive fungal infection by fungal culture, histology and a panfungal PCR followed by sequencing. Results were compared to the composite diagnosis according the European Organization for Research and Treatment of Cancer (EORTC) criteria. In 34 samples, culture, histology and panfungal PCR were positive in 35%, 38% and 62%, respectively. On the sample level the panfungal PCR revealed a sensitivity of 69% and a specificity of 62.5% compared to proven IFI according postoperative EORTC criteria. On patient level, the sensitivity of the PCR approach was 100%, specificity 62.5%.

Development and application of two independent real-time PCR assays to detect clinically relevant Mucorales species

“…According to EORTC/MSG criteria (De Pauw et al, 2008), all were classified as having IFD proven either by positive culture or by histology. Owing to molecular identification using DNA eluates of tissue samples, six patients (eight samples) were affected by Mucorales infections (Babouee et al, 2013).…”

“…AA, Aplastic anaemia; ALL, acute lymphatic leukaemia; AML, acute myeloid leukaemia; COPD, chronic obstructive pulmonary disease; NA, not applicable (positive cryptococcal antigen test from cerebrospinal fluid); Neg., negative; Pos., positive. *Molecular identification by ITS-based panfungal PCR as described by Babouee et al (2013). DSpecies were identified by the panfungal and the 18S Mucorales assay (18S PCR); sequencing revealed the same genus level.…”

“…Panfungal PCR was routinely performed directly from extracted tissue samples as described before (Babouee et al, 2013;Babouee Flury et al, 2014). Primers ITS5 and ITS4, targeting ITS regions 1 and 2, were used for fresh tissue samples, and primers ITS3 and ITS4, amplifying ITS region 2, were applied to FF-PET extracts.…”

“…Seven proven nonMucorales IFD were used to test the specificity of the new assays in a clinical context. A panfungal PCR assay was initially used to detect fungal DNA as part of the standard routine in University Hospital Basel, enabling an identification of fungal species in 88% (15/17) of specimens (Babouee et al, 2013;Babouee Flury et al, 2014). In only two samples could no pathogen be detected despite positive histology showing fungal hyphae (patient 7; Table 2).…”

İnvazi̇v Fungal Enfeksi̇yonlarin Tanisinda i̇n-House Poli̇meraz Zi̇nci̇r Reaksi̇yonunun Değerlendi̇ri̇lmesi̇ / Evaluation of in-House Polymerase Chain Reaction for Diagnosis of Invasive Fungal Infection

“…Moleküler yöntemlerin en önemli avantajlarından biri de, yaklaşık 4-6 saat içinde sonuç verebilmeleridir (6). PCR'nin kullanımını değerlendiren çalışmalarda örnek tipi (serum, BAL, tam kan), ekstraksiyon protokolü (enzimatik, mekanik, otomatize, ticari kit vb), amplifikasyon sistemi (nested, in-house), protokol (gerçek zamanlı, kantitatif ve in house), hedeflenen gen bölgesi (18S rRNA, 28S rRNA vb) ya da pimer seçimi (türe özgül, genusa özgül ya da panfungal) gibi çok sayıda faktörde büyük çeşitlilik gözlenmektedir (6,8 (12). Klinik örneklerde mantarların moleküler tanısı ile ilgili yapılan birçok çalışmada da, PCR temelli yöntemlerin duyarlılık ve özgüllük oranları ile ilgili bildirilen farklı sonuçlara rağmen, geleneksel yöntemlere göre daha yüksek olduğu konusunda görüş birliği vardır (13)(14)(15).…”