Short-Term Stability of Pathogen-Specific Nucleic Acid Targets in Clinical Samples

Abstract

bThe stability of pathogen-specific DNA or RNA amplification targets in clinical samples following short-term storage at room temperature, 4°C, and ؊80°C was assessed by real-time PCR. In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, irrespective of storage temperature. In unextracted samples, temperature-dependent loss of targets (P < 0.05) was observed in serum and cerebrospinal fluid specimens, while no changes were observed for EDTA blood samples. N ucleic acid amplification tests (NAATs) to detect infectious pathogens are now widely used in clinical microbiology laboratories. Although these assays provide high sensitivity, high specificity, and rapid turnaround time, special laboratory practice and procedures are required to avoid false-positive and false-negative results (1, 2). Optimal application of NAATs depends on a number of preanalytical factors, such as specimen collection, transport, and storage conditions (3, 4). These factors impact the stability of pathogen-associated nucleic acids (NA), which must be protected from physical, chemical, or enzymatic degradation during specimen transport and handling. The most important threats to the stability of NA targets in clinical samples are nucleases, such as DNases and RNases, which may be released from lysed human cells and microorganisms (5). In real-life clinical situations, questions arise over whether clinical samples or NA extracts, left at room temperature (RT) for a few days or stored in the refrigerator for a week, are still valid for molecular testing. Although there are published guidelines for the collection, transport, and storage of specimens for molecular tests, described by CLSI (4), they are based mostly on opinions and limited experimental data. Published data on the stability of pathogen-specific amplification targets are also limited to individual pathogens and/or specimen types (3,4,(6)(7)(8)(9)(10)12). Therefore, in this study, we undertook a broader study to compare the stability of extracted and unextracted clinical samples over time and at different temperatures.To determine the short-term storage stability of purified DNA and RNA, NA was extracted from known positive specimens containing the following: Neisseria meningitidis in EDTA blood, cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in serum, and influenza A virus in nasopharyngeal washes (NPW). All samples were extracted using the QIAsymphony virus/bacteria kit in an automated DNA extraction platform QIAsymphony SP (Qiagen) except for EDTA blood, which was extracted manually using the QIAamp DNA blood minikit (Qiagen). Purified eluates were stored in aliquots under sterile conditions at room temperature (RT) (22 to 25°C), 4°C, and Ϫ80°C for 0 to 30 days.For unextracted samples, the target pathogen and the specimen types were chosen so that they (i) represent the most common specimen type for the respective pathogen and (ii) had not previously been studied. EDTA blood, serum, cerebrospinal fluid (CSF), and NPW samples were spiked w...

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