Aspergillus DNA contamination in blood collection tubes

Abstract

Fungal PCR-based diagnostic methods are at risk of contamination. Sample collection containers were investigated for fungal DNA contamination using real-time PCR assays. Up to 18% of blood collection tubes were contaminated with fungal DNA, probably A. fumigatus. Lower proportions of contamination in other vessels were observed. The incidence of invasive aspergillosis (IA) is increasing amongst immunocompromised individuals with an estimated one million deaths worldwide (Maschmeyer and Haas 2008).The need for a universally applicable rapid and sensitive diagnostic method is pressing (Denning 1998). PCR based detection of Aspergillus nucleic acids provides a well explored diagnostic technique that could fulfil these criteria (Chen et al 2002, Perlin andZhao 2009). However, Aspergillus is ubiquitous in the environment and DNA contamination in a diagnostic assay can arise from dead or fragmented fungi. Given the costs and side effects of the preferred antifungal therapeutics (Menzin et al 2009), even very low levels of false positive results or contamination have serious consequences. The most convenient source of material for diagnosis is blood. An 8% contamination rate in over 3,000 fungal PCR assays performed on whole blood over 2 years has been reported (Loeffler et al 1999). Contaminating DNA from 12 different Aspergillus species caused a false positive rate of 19% with sources of contamination including DNA extraction and PCR reagents (Palmer et al 2001). Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. We investigated the prevalence of fungal DNA contamination originating from blood collection tubes and a variety of other clinically relevant vessels from a range of manufacturers using a DNA extraction kit that has been proven free from fungal DNA (data not shown) (MycXtra ™ , Myconostica, Manchester, UK). Extracted DNA was tested using a quality controlled commercial molecular beacon (MB) real-time PCR assay that detects Aspergillus and Penicillium spp. (FXG:Resp (Asp+), Myconostica). Positive samples were screened using a second PCR assay with a TaqMan (TM) probe specific for Aspergillus fumigatus only. NIH Public AccessCollection vessels tested and rates of contamination detected are listed in table 1. To test for microbiological contamination, sterile PBS-Tween 80 (1mL) was added to each vessel and agitated on a vortex mixer for 1 minute. An aliquot (100μL) was then spread onto Sabouraud dextrose agar plates and incubated at 30°C for 2 weeks. To test for fungal DNA contamination, molecular grade sterile water (1mL) was added to each vessel and agitated on a ...

Molecular and Nonmolecular Diagnostic Methods for Invasive Fungal Infections

“…Another problem associated with fungal PCR is the potential for contamination. Fungi are ubiquitous in the environment and can easily contaminate surfaces and materials used in all steps of fungal PCR, including commercially available reagents (123) and collection tubes (124). Therefore, careful precautions and highly experienced personnel are necessary to avoid false-positive findings associated with contaminants.…”

Fungal DNA Detected in Blood Samples of Patients Who Received Contaminated Methylprednisolone Injections Reveals Increased Complexity of Causative Agents

“…However, the contamination of the methylprednisolone vials with non-Exserohilum fungi provides a foundation for concern that these signals in patients with localized symptoms reflect a more complex pattern than heretofore appreciated. Second, due to its broad coverage, the panfungal real-time PCR is vulnerable to trace contamination at any step from sample collection to PCR amplification (17,18). However, bearing in mind the possibility of contamination amid blood testing, we carefully applied extreme precautions to prevent airborne and carryover contaminations, used sufficient negative controls, and applied a rigorous C T cutoff to the analysis of panfungal real-time PCR results.…”

Broad-Range PCR Coupled with Electrospray Ionization Time of Flight Mass Spectrometry for Detection of Bacteremia and Fungemia in Patients with Neutropenic Fever

“…A. fumigatus detection in these two patients was in concordance with the diagnosis of probable invasive fungal infection. On the other hand, lowering the cutoff could have the unintended consequence of detection of reagent contamination, as described previously for A. fumigatus (17), or increased detection of potential contaminants, such as P. acnes. The protocol evaluated here was selective to avoid environmental organisms from being overreported.…”