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Short-term Stability of Pathogen Specific Nucleic Acid Targets in Clinical Samples 1

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Mohammad R. Hasan1,2*, Rusung Tan1,2, Ghada N. Al-Rawahi1,2, Eva Thomas1,2, Peter Tilley1,2 3

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1Division of Microbiology, Virology and Infection Control, Department of Pathology, Children's 5

and Women's Health Centre of BC, Vancouver, BC, Canada and 2Department of Pathology and 6

Laboratory Medicine, University of British Columbia, Vancouver, BC, Canada 7

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Running title: Stability of Pathogen Specific Amplification Targets 9

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Keywords: Pathogen, Nucleic acid amplification tests (NAATs), nucleic acid stability, clinical 11

specimens, storage temperature 12

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*Corresponding author: 15

Mohammad R. Hasan, Microbiology Laboratory, BC Children's & Women's Hospitals, Rm 16

2F59A, 4500 Oak Street, Vancouver, BC V6H 3N1, Canada 17

Phone: (604)-875-2345 Ext. 5283 18

FAX: (604)-875-3777 19

E-mail: Rubayet.Hasan@cw.bc.ca 20

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.02659-12 JCM Accepts, published online ahead of print on 10 October 2012

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Abstract 24

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The stability of pathogen specific DNA or RNA amplification targets in clinical samples 26

following short-term storage at room temperature, 4oC and -80oC was assessed by real time PCR. 27

In purified nucleic acid extracts, both DNA and RNA targets were stable for up to 30 days, 28

irrespective of storage temperature. In unextracted samples, temperature-dependent loss of 29

targets (p<0.05) was observed in serum and cerebrospinal fluid specimens while no changes 30

were observed for EDTA blood samples. 31

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Nucleic acid amplification tests (NAATs) to detect infectious pathogens are now widely used in 47

clinical microbiology laboratories. Although these assays provide high sensitivity, specificity, 48

and rapid turn-around time, special laboratory practice and procedures are required to avoid false 49

positive and false negative results (1,2). Optimal application of NAATs depends on a number of 50

pre-analytical factors such as specimen collection, transport and storage conditions (3,4). These 51

factors impact the stability of pathogen-associated nucleic acids (NA) which must be protected 52

from physical, chemical or enzymatic degradation during specimen transport and handling. The 53

most important threats to the stability of NA targets in clinical samples are nucleases, such as 54

DNases and RNases, which may be released from lysed human cells and microorganisms (5). In 55

real life clinical situations, questions arise over whether clinical samples or NA extracts, left at 56

room temperature (RT) for a few days or stored in the refrigerator for a week are still valid for 57

molecular testing. Although there are published guidelines for the collection, transport and 58

storage of specimens for molecular tests, described in CLSI (4), they are mostly based on 59

opinions and limited experimental data. Published data on the stability of pathogen-specific 60

amplification targets are also limited to individual pathogens and/or specimen types (3,4,6-10, 61

12). Therefore, in this study, we undertook a broader study to compare the stability of extracted 62

and unextracted clinical samples over time and at different temperatures. 63

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To determine the short-term storage stability of purified DNA and RNA, NA was extracted from 65

known positive specimens containing the following: Neisseria meningitidis in EDTA blood; 66

cytomegalovirus (CMV) and Epstein-Barr virus (EBV) in serum; and influenza A virus in 67

nasopharyngeal washes (NPW). All samples were extracted using the QIAsymphony 68

virus/bacteria kit in an automated DNA extraction platform QIAsymphony SP (Qiagen) except 69

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for EDTA blood, which was extracted manually using QIAamp DNA Blood Mini Kit (Qiagen). 70

Purified eluates were stored in aliquots under sterile conditions at room temperature (RT) (22-71

25oC), 4oC and -80oC for 0-30 days. 72

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For unextracted samples, the target pathogen and the specimen types were chosen so that they (i) 74

represent the most common specimen type for the respective pathogen and ii) had not previously 75

been studied. EDTA blood, serum, cerebrospinal fluid (CSF) and NPW samples were spiked 76

with a N. meningitidis B strain (ATCC 13090), a CMV patient isolate, a human enterovirus 70 77

strain (ATCC VR-836) and an influenza A virus patient isolate, respectively, within 4 hours of 78

collection (Supplemental methods). NA from spiked samples was extracted in the same way 79

following storage at RT, 4oC and -80oC for 0-16 days. Extracted nucleic acids were analyzed 80

either by in-house (Supplemental method and Table 1) or commercial, probe-based, real time 81

PCR assays. Human enterovirus RNA was detected using a commercial eQ-PCR™ EnV 82

Detection Kit (Trimgen Corporation). For kinetic analysis, CT values for each pathogen were 83

plotted against the number of days the samples were stored at different temperatures. The half-84

lives of pathogen-associated nucleic acid targets were obtained from the linear equation 85

(Supplemental methods). For average analysis, fold changes in the relative quantity of 86

amplification targets were calculated based on CT values using the equation FC=2^(-dCT), where 87

dCT=(CT,day X- CT,day 0) (11). The percent loss of targets was calculated from fold changes from 88

the quantity of targets at day ‘0’. Statistical significance was calculated by paired and 2-tailed, 89

student’s T-test and p<0.05 was considered to be statistically significant. Statistical differences 90

between multiple experimental groups were calculated by ANOVA followed by post-test using 91

Bonferroni correction at 95% confidence interval. 92

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There was no loss of extracted N. meningitidis, CMV and EBV DNA derived from EDTA blood 93

and serum samples after storage at RT, 4oC and -80oC for 0, 3, 7, 14 and 30 days (Fig. 1A-i,C-94

i,D-i). To account for sample to sample variation, we performed further tests on additional 95

samples at days 0 and 30 of storage at different temperature, and found no significant differences 96

in the relative quantity of specific amplification targets (Fig. 1A-ii, C-ii, D-ii). In general, DNA, 97

when purified, and stored in a preferred buffer such as Tris/EDTA (pH 7.2) is considered to be 98

stable for at least one year at 2-8oC, and for longer terms at -70oC or lower (3). However, it is 99

important to note that the DNA purity could be dependent on the specimen type and the 100

extraction methods, which could subsequently influence the stability of purified DNA. Our 101

findings suggest that DNA extracted from EDTA blood or serum samples, using an automated 102

and magnetic bead-based extraction method, may be safely stored at RT or lower without any 103

loss for up to one month. Unlike the case with DNA, it is recommended that purified RNA be 104

stored at -70oC or lower, and at a slightly alkaline pH (7.1-7.5) (3). In our lab setting, we 105

observed a small (~1 CT; p<0.05) loss of influenza A RNA, which was not temperature 106

dependent (between RT to -80oC), in purified NPW extracts following storage for a period of one 107

month (Fig. 1B). These results indicate that temporary storage of extracted nucleic acids from 108

clinical samples at RT or 4oC is unlikely to affect NAAT results. 109

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Unlike extracted samples, where purified DNA and RNA molecules are maintained in a defined 111

buffer, crude clinical samples represent a complex biological environment, where an interplay of 112

degradative proteases and nucleases may determine the fate of pathogen-associated amplification 113

targets. Previously published observations on the stability of microbial DNA in clinical samples 114

include i) Trichomonas vaginalis DNA, which was stable for 1 day at RT and 3 days at 4oC in 115

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urine samples (6) ii) spiked Treponema pallidum DNA in CSF which was detectable after 96h of 116

storage at RT and 4oC (12) iii) HSV DNA in positive clinical specimens remained stable for at 117

least 16 months at -20°C (7), and iv) HBV DNA in positive plasma samples was stable at 25oC 118

and at 5oC for at least 25 days (8), whereas, in serum they were stable only for 5 days at 4oC and 119

-70oC and less stable at RT (10). Our results on the stability of spiked N. meningitidis DNA in 120

EDTA blood, and CMV DNA in serum and EDTA blood samples correlate well and 121

complement these observations. Whereas both N. meningitidis and CMV DNA were highly 122

stable in EDTA blood for two weeks at any storage temperature used, significant loss (p<0.05 by 123

ANOVA and post-test) of CMV DNA was observed in serum samples in a temperature-124

dependent manner (Fig. 2A-B). The half-life of CMV DNA based on PCR assays was <1, 2, and 125

3 days when stored at RT, 4oC and -80oC, respectively (Table 1). 126

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Although a loss of RNA targets in crude samples is expected during storage, unless frozen, we 128

noted no dramatic, logarithmic decrease in human enterovirus and influenza A RNA levels in 129

CSF and NPW samples, respectively after two weeks. A small but statistically significant 130

reduction of enterovirus RNA (65%; p<0.05) at RT, and influenza RNA (>50%; p<0.001) at all 131

temperatures was observed (Fig. 2C-D). The half-life of enterovirus RNA in CSF samples at RT 132

was 9 days (Table 1), which correlates well with that of HIV-1 RNA (7 days) in plasma samples, 133

as previously reported (8). Similarly, at 4oC, enterovirus RNA maintained its integrity for 134

approximately two weeks in this study as seen in a previous HIV-1 study (8). The results of 135

influenza A RNA in NPW samples were somewhat surprising as no temperature-dependent 136

effects were detected. One limitation of this study is that we did not analyze RNA stability in 137

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EDTA blood samples. However, a previous study found HCV RNA stable in EDTA blood for up 138

to 96 hours (9), similar to what we observed for DNA targets. 139

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In conclusion, we observed minimal loss of pathogen NAAT targets for both extracted and 141

unextracted clinical samples within a few days of specimen collection. None of the NAAT 142

targets became undetectable during the study period, which was limited to 1 month and 2 weeks, 143

for pure extracts and crude samples, respectively, to represent most common scenarios in a 144

diagnostic microbiology laboratory. The temperature-dependent loss of targets in serum and CSF 145

samples indicates possible enzymatic degradation. In contrast, EDTA blood samples were found 146

to be the most protective medium for nucleic acid stability. This may be because EDTA 147

sequesters metal ions required for enzymatic activity and thus inhibits nucleases and proteases. 148

Based on these observations, we conclude that, if a processing delay is unavoidable, EDTA 149

blood or plasma samples are preferable to serum samples for real time PCR detection of bacterial 150

or viral pathogens. 151

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8. Jose M, Gajardo R, Jorquera JI. 2005. Stability of HCV, HIV-1 and HBV nucleic acids in 179

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tubes on hepatitis C virus level in whole blood samples. J. Clin. Microbiol.39(5):1788-183

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Figure legends 206

FIG 1: Storage stability of DNA and RNA in post-extracted clinical samples. Nucleic acid 207

extracts from clinical samples known positive for N. meningitidis (A), Influenza A virus (B), 208

CMV (C) and EBV (D) were assessed for respective targets by Taqman PCR at day ‘0’ and 209

following storage at RT, 4oC and -80oC for different time intervals as indicated. A-D(i) Kinetic 210

data based on CT values plotted against number of days stored at different temperature. A-D(ii) 211

Average of fold changes calculated from CT values, with respect to CT values at day ‘0’. Baseline 212

CT values in different samples ranged from 29.9 to 38.5 for N. meningitidis, 28.4 to 39.5 for 213

CMV, 28.2 to 38.5 for EBV and 21.3 to 34.8 for Influenza A virus. The CT value difference 214

because of intra-assay variation between duplicate runs was <1 CT. Error bars = SEM; statistical 215

significance calculated by pair-wise student’s T-test (2-tailed); *p<0.05, **p<0.01, ***p<0.001. 216

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FIG 2: Storage stability of DNA and RNA in pre-extracted clinical samples. Clinical 218

samples were spiked with N. meningitidis (A), CMV (B), Enterovirus (C) and Influenza A virus 219

(D). Nucleic acid extraction and Taqman PCR for the respective targets were performed on 220

aliquots of samples at day ‘0’ and following storage at RT, 4oC and -80oC for different time 221

intervals as indicated. A-D(i) Kinetic data based on CT values plotted against number of days 222

stored at different temperature. A-D(ii) and B(iii) Average of fold changes calculated from CT 223

values, with respect to CT values at day ‘0’. Baseline CT values in different spiked samples 224

ranged from 31.9 to 34.0 for N. meningitidis, 29.6 to 31.7 for CMV, 32.7 to 34.1 for enterovirus, 225

and 33.9 to 37.1 for Influenza A. The CT value difference because of intra-assay variation 226

between duplicate runs was <1 CT. Error bars = SEM; statistical significance compared to data at 227

day 0 calculated by pair-wise student’s T-test (2-tailed); *p<0.05, ***p<0.001. Multiple group 228

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comparisons for CMV DNA levels in serum samples were made by ANOVA and post-test (95% 229

confidence interval) indicating a statistically significant (p<0.05) difference between different 230

groups. 231

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Table 1: Half life and % loss of pathogen specific amplification targets in different types of

clinical specimens.

Pathogen Specimen type Days stored

Half life (days) % loss of target after 2 weeks

RT 4oC -80oC RT 4oC -80oC

N.meningitidis EDTA Blood 16 - - - No loss No loss No loss

CMV Serum 16 <1 2 3 79.7±5 50±10 36.9±12

EDTA Blood 16 - - - No loss No loss No loss

Human Enterovirus CSF 16 9 - - 82.1±9 No loss No loss

Influenza A Nasopharyngeal aspirates

14 6 6 10 74±23 70±25 66±27

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