A consensus on fungal polymerase chain reaction diagnosis?: a United Kingdom-Ireland evaluation of polymerase chain reaction methods for detection of systemic fungal infections

Abstract

The limitations of classical diagnostic methods for invasive fungal infections (IFIs) have led to the development of molecular techniques to aid in the detection of IFIs. Despite good published performance, interlaboratory reproduction of these assays is variable, and no consensus has been reached for an optimal method. This publication describes the first multicenter study of polymerase chain reaction methods, for the detection of Aspergillus and Candida species, currently used in the UK and Ireland by distribution and analysis of multiple specimen control panels. All three Candida methods were comparable, achieving a satisfactory level of detection (10 cfu), and the method of preference was dependent on the requirements of the particular laboratory. The results for the five Aspergillus assays were more variable, but two methods (2Asp and 4Asp) were superior (10(1) conidia). Formally, the overall performances of the two Aspergillus assays were comparable (kappa statistic = 0.77). However, on the Roche LightCycler, there was a clear sample-type effect that greatly reduced the detection limit of the 4Asp method when testing whole blood samples. Therefore, the preferred Aspergillus method relied on the amplification platform available to the user. This study represents the initial process to achieve a consensus method for the diagnosis of IFIs.

Figure 1

Sensitivities of the Aspergillus PCR assays using different PCR platforms with different fungal loads and sample types. a: Roche LightCycler, b: Corbett Rotor–Gene, and c: Applied Biosystems TaqMan.

Figure 2

Detection limits for the Aspergillus assays using the Roche LightCycler platform and Corbett Rotor–Gene with DNA extracted from EDTA whole blood spiked with Aspergillus conidia. a: 2Asp assay and b: 4Asp assay.

Figure 3

Sequence similarity between a: 4Asp forward primer; b: 4Asp probe; c: A. fumigatus 18S rRNA gene partial sequence (accession no. AF548063, bp 1212 to 1367); d: Homo sapiens 18S rRNA gene partial sequence (accession no. X03205, bp 1346 to 1508); e–h: 4Asp LC products; and i: 4Asp reverse primer.

Figure 4

Schematic proposing a structure for a pan-fungal PCR using the current methods as described and assessed.