Clinical Significance of Low CMV DNA Levels in...

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Clinical Significance of Low CMV DNA Levels in Human Plasma 1

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Running Title: Significance of Low CMV DNA Levels in Human Plasma 3

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Jesse Waggoner1, Dora Y. Ho1, Paolo Libiran2, and Benjamin A. Pinsky1, 2,* 5

1Department of Medicine, Division of Infectious Diseases and Geographic Medicine, Stanford 6

University School of Medicine, Stanford, CA, USA. 7

2Department of Pathology, Stanford University School of Medicine, Stanford, CA, USA. 8

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*To whom correspondence should be addressed. 10

3375 Hillview Ave, Room 2913 11

Palo Alto, CA 94304 12

Phone: 650-721-1896 13

Fax: 650-723-6918 14

E-mail: [email protected] 15

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Copyright © 2012, American Society for Microbiology. All Rights Reserved.J. Clin. Microbiol. doi:10.1128/JCM.06800-11 JCM Accepts, published online ahead of print on 18 April 2012

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ABSTRACT 17

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The clinical significance of low copy numbers of CMV DNA detected in immune suppressed 19

patients remains unclear. In this study, we compare the artus CMV Rotor-Gene PCR, utilizing 20

automated nucleic acid extraction and assay setup (the artus CMV protocol), with COBAS 21

Amplicor CMV Monitor (our reference protocol). We then analyzed the results of all CMV PCR 22

tests ordered following the implementation of the artus CMV protocol at our institution and 23

followed 91 adult patients with a positive test result. The artus CMV protocol had a linear range 24

extending from 2.0 to 7.0 log10 copies/mL and had a lower limit of 95% detection of 57 25

copies/mL. Using archived plasma samples, it demonstrated 100% sensitivity and 94% 26

specificity for the detection of CMV DNA. Following implementation of the artus CMV 27

protocol, 320 of 1403 (22.8%) plasma samples tested positive (compared with 323/3579 (9.0%) 28

samples in the preceding six months), and 227 (16.2%) samples had copy numbers <400 29

copies/mL. Ninety-one adult patients had at least one positive test. The data was analyzed using 30

a threshold of 200 copies/mL, and in 22 episodes, the viral load increased from <200 copies/mL 31

to ≥200 copies/mL on sequential tests. In 21 of these 22 episodes, the viral load either continued 32

to increase or antiviral treatment was initiated in response to the repeat value. In summary, we 33

evaluate the performance characteristics of a protocol utilizing the artus CMV PCR and identify 34

clinically meaningful changes in CMV DNA copy number even when initially detected at a low 35

level. 36


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INTRODUCTION 37

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Cytomegalovirus (CMV) remains a major cause of morbidity following solid organ 39

transplantation (SOT) and hematopoietic stem cell transplantation (HSCT), and even with 40

antiviral treatment, mortality from certain forms of CMV end-organ disease, such as CMV 41

pneumonitis, remains high (14, 17, 18). Viremia has been associated with end-organ disease, 42

and an increase in CMV DNA titer in serial blood samples, typically detected using a 43

quantitative PCR assay, can predict disease development (10, 20). When used in a pre-emptive 44

treatment protocol, quantitative PCR helps decrease the incidence of CMV disease and the use of 45

antiviral therapy compared to using viral culture (9). However, despite the widespread use of 46

quantitative CMV PCR assays, thresholds for the diagnosis of disease or the initiation of pre-47

emptive therapy have not been established (9, 14, 17). This situation has resulted from the use of 48

different clinical diagnostic tests for CMV (both commercially available and lab-developed), the 49

broad range of values over which CMV disease can occur, and until recently, the lack of a 50

universal standard for CMV DNA quantitation (8, 13, 24). 51

52

The COBAS Amplicor CMV Monitor was the first commercially available quantitative CMV 53

PCR and remains commonly used in clinical virology laboratories (7). It has a lower limit of 54

detection of 400 copies/mL of plasma and a linear range from 2.78log10 to 5.0 log10 copies/mL 55

(600 to 100,000 copies/mL) (4). The limits of detection and quantitation of this assay result in a 56

large number of patients with ongoing yet unquantifiable or even undetectable viremia. 57

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The artus CMV Rotor-Gene (RG) PCR is a commercially-available, quantitative CMV PCR 59

protocol. Initial reports have demonstrated a broad linear range and a lower limit of detection 60

below that of the COBAS CMV Monitor (3). While such low copy numbers of CMV DNA as 61

those detected with the artus protocol have been reported, the clinical significance of these “low 62

positives” in a population of immune suppressed patients has not been well described (2, 11, 15). 63

64

In this study, we report the analytical performance of a protocol utilizing the artus CMV PCR 65

combined with automated sample preparation and assay set-up (SP/AS) on the QIAsymphony 66

SP/AS device (hereinafter referred to as the artus CMV protocol). Using archived human 67

plasma samples, we determined the sensitivity and specificity of the artus CMV protocol for the 68

detection of CMV DNA compared to the COBAS CMV Monitor assay following extraction on 69

the MagNA Pure LC (hereinafter referred to as the reference protocol). We document an 70

increased rate of detection of CMV in patient plasma samples following implementation of the 71

artus CMV protocol at our institution. We then followed 91 adult patients with positive tests, 72

and using a group of transplant recipients with serial testing, we identified clinically relevant 73

increases in CMV DNA copy number, even when quantified at low levels, previously 74

undetectable with the reference protocol. 75

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MATERIALS AND METHODS 77

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Samples and Control Material 79

82 archived plasma samples that had been previously tested by the reference protocol were tested 80

using the artus CMV protocol. Basematrix 53 defibrinated human plasma (DHP; SeraCare, 81

Milford, MA) was used as a negative control in all PCR runs. Stock CMV AD-169 (ATCC, 82

Manassas, VA) diluted to 5.0 and 3.0 log10 copies/mL (100,000 and 1,000 copies/mL) in DHP 83

was used as high and low positive controls, respectively. The concentration of the original stock 84

was determined using the reference protocol. 85

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DNA Extraction and Assay Set-up 87

DNA extraction for the artus CMV protocol was performed on 1.0mL of acid citrate dextrose 88

(ACD) plasma (1.2mL of input volume required to account for instrument dead volume) using 89

the Qiagen virus/bacteria Midi Kit on the QIAsymphony SP device (both from Qiagen, Valencia, 90

CA). DNA was eluted in a final volume of 95μL. Following DNA extraction, the artus CMV 91

PCR was set-up using the QIAsymphony AS. 10µL of extracted nucleic acids, 12.5µL CMV RG 92

Master Mix, and 2.5µL of CMV magnesium solution were used, for a final reaction volume of 93

25µL. 94

95

For the reference protocol, 100µL of each specimen was extracted on the MagNA Pure LC using 96

the MagNA Pure LC DNA isolation kit with the DNA I Blood Cell High Performance protocol 97

(Roche, Indianapolis, IN). Reactions were manually set-up by pipetting 50µL out of the 100µL 98

elution into COBAS Amplicor amplification tubes (A-tubes) containing 50 µL of the COBAS 99

CMV Monitor Master Mix with Magnesium. 100


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Quantitative PCR Assays 102

The artus CMV PCR (Qiagen, Valencia, CA) is a real-time, fluorescein-labeled, hydrolysis-103

probe-based PCR targeting the CMV major immediate early gene. Reactions were carried out on 104

the Rotor-Gene Q (RGQ) and underwent an initial 10 min at 95°C, then 10 cycles of touchdown 105

PCR; 95°C for 15 sec, 65°C for 30 sec, and 72°C for 20 sec, with the annealing step decreasing 106

by 1°C each cycle. This was followed by 35 cycles of 95°C for 15 sec, 56°C for 30 sec, and 107

72°C for 20 sec. Data was collected on the green and yellow channels during annealing. An 108

internal control is added to each primary sample prior to extraction, and amplification is 109

performed with specific primers and JOE-labeled, hydrolysis probes contained in the artus CMV 110

Master Mix to ensure adequate extraction efficiency and the absence of inhibitors. Positive 111

samples produced a signal above a threshold value of 0.1 in the green channel. The number of 112

CMV copies/mL was calculated by comparing the cycle number at which the fluorescent signal 113

crossed this threshold (Ct) with the four-point standard curve included on each run. The standard 114

curve was generated using four quantitation standards (Qiagen, Valencia, CA) with 115

concentrations of 10,000; 1,000; 100; and 10 copies/µL. Results were reported in copies/mL of 116

the original plasma sample. Samples with detectable virus quantified at <150 copies/mL were 117

reported as “Detected, <150 copies/mL”. 118

119

The artus CMV protocol was subsequently calibrated to the 1st WHO International Standard for 120

human cytomegalovirus for NAT based assays (NIBSC code 09/162) obtained from the National 121

Institute for Biological Standards and Controls (NIBSC; Hertfordshire, United Kingdom). The 122

WHO International Standard was diluted to 5.0, 4.7, 4.0, 3.7 and 3.0 log10 IU/mL and 6 123


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replicates at each concentration were run on 4 separate days. The data collected in copies/mL 124

was compared to the expected IU/mL. A conversion factor was calculated by taking the mean 125

ratio of IU/mL to copies/mL for all data points. 126

127

The COBAS Amplicor CMV Monitor (Roche, Indianapolis, IN) utilized in the reference 128

protocol is a non-saturating, end-point PCR targeting the CMV polymerase UL54 gene followed 129

by quantitation with an automated enzyme-linked oligosorbent assay (ELOSA). A separate 130

internal quantitation standard is added to the primary specimen prior to extraction. 131

Amplification and detection were performed as described previously (23). 132

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Samples that yielded discordant results by the artus CMV and reference protocols were tested 134

using the CMV R-gene real-time PCR assay (hereinafter referred to as the CMV R-gene assay; 135

Argene, Sherley, NY), which targets the pp65 UL83 gene. This assay has a reported lower limit 136

of detection (LLOD) of 50 copies/mL and a linear range extending to 7.0log10 copies/mL (6). 137

10μL of DNA extracted using the Qiagen SP device was added to 15μL of amplification premix. 138

Reaction conditions on the RGQ were 95°C for 15 min, followed by 45 cycles at 95°C for 10 sec 139

and 60°C for 40 sec. Positive samples produced a signal above a threshold value of 0.025 in the 140

green channel. The number of CMV copies/mL was calculated by comparing the cycle number 141

at which the fluorescent signal crossed this threshold (Ct) with the four-point standard curve 142

included on each run. The standard curve was generated using four quantitation standards 143

(Argene, Sherley, NY) with concentrations of 5,000; 500; 50; and 5 copies/µL. 144

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Linearity, Lower Limit of Detection, and Accuracy 146


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Linearity of the artus CMV protocol was determined using serial 10-fold dilutions of CMV AD-147

169 in DHP. Replicates of each dilution were taken through the entire assay, from extraction to 148

quantitation. To determine the LLOD, Optiquant CMV Panel samples (AcroMetrix, Benicia, 149

CA) were diluted to 100, 50, and 25 copies/mL. Twenty replicates were run at each 150

concentration, and probit analysis was performed to determine the lower limit of 95% detection. 151

The quantitative results obtained with the artus CMV and reference protocols were compared 152

using 82 previously tested clinical samples. Samples that yielded discordant results were re-153

tested using the CMV R-gene assay. 154

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Clinical Study Design 156

This research was approved by the Stanford University Institutional Review Board. We 157

analyzed the general test characteristics for plasma CMV PCR assays sent at our institution 158

during the study period, which extended from June 20, 2011, the date on which the artus CMV 159

protocol was implemented, to September 12, 2011. Adult patients with a single positive result 160

using the artus CMV protocol during this interval were then identified. We collected relevant 161

clinical data, including patient age, sex, and significant past medical history. For transplant 162

recipients, we documented the type of transplant, indication for transplantation, CMV status at 163

transplant, and antiviral prophylaxis. We also documented any treatment administered during 164

the study. The death summary was used for the documentation of cause of death where 165

applicable. 166

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Definitions used for CMV infection and end-organ disease were consistent with previously 168

published recommendations (14, 17, 19). CMV infection was defined by the detection of CMV 169


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DNA in patient plasma. The confirmation of CMV disease required a biopsy with consistent 170

histopathology as well as virus isolation or positive immunohistochemistry (IHC). The diagnosis 171

of “CMV syndrome” was considered only for SOT recipients. Recurrent infection was defined 172

as a rising CMV viral load following the completion of a course of antiviral treatment, with the 173

documented absence of detectable CMV DNA or CMV DNA that was detectable but <200 174

copies/mL at the completion of treatment. 175

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Immune suppression regimens varied by transplant organ, time from transplantation, and 177

evidence of graft rejection or graft-versus-host disease. Transplant patients received antiviral 178

prophylaxis according to protocols employed by the different transplant services at our 179

institution. Two HSCT patients remained on treatment for CMV infection throughout the study 180

period and were not included in the tally of patients receiving prophylaxis. Pre-emptive therapy 181

and treatment for CMV infection or disease was initiated and the duration of therapy was 182

determined by the attending physician caring for the patient. Repeat CMV testing during therapy 183

and follow-up were performed by the clinical laboratory at the discretion of the treatment team. 184

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Statistical Analysis 186

Agreement between the artus CMV and reference protocols was assessed using a Bland-Altman 187

plot (1). Standard statistical analyses were performed using Excel (Microsoft, Redmond, WA). 188

Probit analysis was performed using SPSS (IBM, Armonk, NY). Two-tailed Fisher’s exact tests 189

and unpaired t-tests were performed using GraphPad software (GraphPad, La Jolla, CA). 190

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RESULTS 192

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Analytical Characteristics of the artus CMV Protocol 194

The linear range of the artus CMV protocol extended from 2.0log10 to 7.0log10 copies/mL (100 195

to 10,000,000 copies/mL). Using probit analysis at 95% detection, the LLOD was found to be 196

57 copies/mL. Assay precision was further evaluated using samples with concentrations of 197

5.0log10, 3.0log10, and 2.30log10 copies/mL (100,000, 1,000, and 200 copies/mL). The mean 198

values were 5.08log10, 2.97log10, and 2.27log10 copies/mL (120,000, 933, and 195 copies/mL) 199

and the inter-run percent coefficients of variation (%COV) were 1.4, 3.7, and 5.8, respectively 200

(calculated for logarithmic values). A checkerboard experiment using 24 CMV-negative plasma 201

samples and 24 CMV AD-169 spiked plasma samples [concentration 6.0log10 copies/mL 202

(1,000,000 copies/mL)] was performed to evaluate the QIAsymphony SP/AS device; no 203

evidence of cross-contamination was observed (data not shown). 204

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The artus CMV protocol was calibrated to the 1st WHO International Standard for human 206

cytomegalovirus for NAT based assays resulting in a conversion factor of 0.76 IU/copy of CMV 207

DNA. 208

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Comparison of the artus CMV and Reference Protocols 210

A total of 82 archived clinical samples (42 positive, 40 negative) that had previously been tested 211

using the reference protocol were re-tested using the artus CMV protocol on the QIAsymphony 212

SP/AS/RGQ system. All 42 samples with detectable CMV DNA by the reference protocol were 213

positive by the artus CMV protocol (sensitivity, 100%; Table 1). There was good agreement in 214

the quantitative results between the artus CMV and reference protocols (Figure 1). Except for 215


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two samples (discussed below) all samples yielded results that differed by <2 SD of the mean 216

difference. 217

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Of 40 samples with undetectable CMV DNA using the reference protocol, 10 were positive 219

using the artus CMV protocol (specificity, 75%; Table 1). The mean concentration in the 10 220

discordant samples using the artus CMV protocol was 255 copies/mL (range 13 - 986), and 8 221

samples had copy numbers <400. Seven patients had a positive CMV PCR by the reference 222

protocol prior to (4 patients) or subsequent to (3 patients) the discordant sample that was used in 223

this validation. On retesting using the CMV R-gene assay, eight of 10 discordant samples (80%) 224

had detectable CMV DNA and two remained undetectable. The revised sensitivity and 225

specificity for the artus CMV protocol, using any two positive tests as the “gold standard,” were 226

100% and 94%, respectively. 227

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Two samples, collected from a single patient at different time points, were detected but under-229

quantitated by the artus CMV protocol compared to the reference protocol (2.82 vs. 3.73log10 230

copies/mL, and 2.44 vs. 3.22log10 copies/mL for the artus CMV and reference protocols, 231

respectively). The results differed by an amount ≥2 SD from the mean difference (Figure 2). 232

For these discordant samples, the CMV R-gene assay results were consistent with the reference 233

protocol and remained elevated compared to the artus CMV protocol. 234

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Clinical Results 236

During the study period, 1403 plasma CMV assays were performed at our institution using the 237

artus CMV protocol. Of these, 320 had detectable CMV DNA (22.8%), with 93 quantitated at > 238


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400 copies/mL (6.6%) and 227 (16.2%) detected at <400 copies/mL. In the six months prior to 239

this change, using the reference protocol, 323 out of 3579 tests had detectable CMV DNA (9.0%, 240

p<0.0001). All of these samples had copy numbers ≥400 copies/mL. The number of patients 241

with at least one positive test also increased with the use of the artus CMV protocol over the 242

same time interval (17.8% vs. 6.9% of patients per month, p<0.0001). The average number of 243

tests did not vary significantly throughout this period (593 vs. 539 tests per month, respectively, 244

p=0.052). 245

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We identified and collected clinical information on the 91 adult patients who had at least one 247

plasma sample with detectable CMV DNA using the artus CMV protocol. The mean age for 248

patients was 51.2 (standard deviation 14.8), and 53 patients were male. The majority of patients 249

had received a transplant (n = 82, 90.1%), of which 55 (60.4%) had an HSCT and 27 (29.7%) 250

had an SOT (lung, 9; renal, 6; heart, 5; liver 3; combined transplants, 4). Table 2 shows 251

characteristics of the transplant recipients, including CMV status at the time of transplantation 252

and antiviral prophylaxis following transplantation. 253

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The number of tests per patient during the study period (June 20, 2011 through September 12, 255

2011) varied significantly based on their underlying condition. HSCT recipients had a mean of 256

7.0 tests performed per patient (standard deviation 3.8), whereas non-HSCT patients had a mean 257

of 2.7 tests per patient (standard deviation 2.5; p<0.0001). 258

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Treatment Outcomes and Recurrence 260


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Forty-five patients (49.5%) received antiviral treatment for CMV infection or disease. There 261

was no difference in the proportion of patients treated for CMV based on the type of transplant 262

(58.2% for HSCT vs. 40.7% for SOT, p=0.163). All patients received valganciclovir or 263

ganciclovir as treatment. Four patients also received foscarnet during the study (8.9%). Twenty-264

eight treatment episodes were started and completed during the study. The average duration of 265

antiviral therapy for these episodes was 30.5 days (standard deviation 15.7, range 10-78). 266

Twenty-one patients had at least two weeks of follow-up off therapy, and eight patients had 267

recurrent CMV infection. The rate of recurrence was not significantly different if CMV DNA 268

remained detectable but <200 copies/mL at the end of treatment (50%, n = 10) or if it had 269

become undetectable (27%, n = 11; p=0.387). 270

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Two patients had confirmed end-organ disease. The first patient, without a transplant, presented 272

with DKA and odynophagia and was found to have esophageal ulcerations positive for CMV by 273

IHC. The serum viral load was 3.78log10 copies/mL (6,020 copies/mL). The second patient 274

received a renal transplant (R-/D+) 17 months before presenting with abdominal pain and 275

diarrhea. Serum CMV showed 5.41log10 copies/mL (259,000 copies/mL), and colon biopsies 276

revealed viral inclusions positive for CMV stain by IHC. Three other patients received treatment 277

for possible CMV disease [colitis (2) and CMV syndrome (1)]. One patient with possible colitis 278

did not undergo a colonoscopy until after treatment had started. The second patient had a 279

colonoscopy at an outside facility, the results of which were not available to us. The patient with 280

possible CMV syndrome improved with antiviral treatment; a limited work-up did not reveal 281

other possible causes for his illness. 282

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Eight patients died during the study; causes of death included intra-cerebral hemorrhage, end-284

stage liver disease (2), graft versus host disease (2), pulmonary embolism and pneumonia, 285

congestive heart failure, and AML with blast crisis. None of these patients had confirmed CMV 286

disease. Three patients were receiving treatment for CMV infection at the end of life. 287

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Patients with Detectable CMV DNA at Low Levels 289

To determine the clinical significance of low positive CMV DNA levels we evaluated our data 290

based on a threshold of 200 copies/mL. This threshold was chosen after initial review of our 291

data for 91 patients with a positive test. 80% (4/5) of patients with quantified viral loads 292

between 150 and 200 copies/mL had a spontaneous decline in the viral load below 150 293

copies/mL, whereas 21/22 patients with viral loads that increased to ≥200 copies/mL had viral 294

loads that continued to increase or received treatment (p=0.0014). 295

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As shown in Figure 2, 86 (94.5%) patients in this study had at least one CMV level detected at 297

<200 copies/mL. Five (5.5%) of the patients always had elevated CMV DNA levels >1000 298

copies/mL during the study period and were not further evaluated. Sixteen (17.6%) patients had 299

samples with viral loads >200 copies/mL at the beginning of the study period (median viral load 300

1348 copies/mL, range 354-9320 copies/mL). Fifteen of these patients received antiviral 301

treatment. A single patient had an increased viral load in the setting of a disseminated varicella-302

zoster infection, and the CMV copy number declined with high dose acyclovir treatment. The 303

transition from a viral load of <200 to >200 copies/mL was not captured during the study period 304

for these 16 patients. 305

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Forty-nine (53.8%) patients only had samples with CMV DNA detected but <200 copies/mL. Of 307

these 49 patients, 16 had a single test performed during the study period. Of the 33 patients with 308

multiple tests, the rate of positive tests per patient varied from 8.3% to 81.8% (1 out of 12 to 9 309

out of 11 samples). None of these patients received treatment during the study period, though 15 310

had been treated for CMV infection or disease previously. 311

312

Twenty-one patients developed 22 clinical episodes during which the CMV DNA copy number 313

increased from a level <200 copies/mL (either undetected or detected but <200 copies/mL) to a 314

level ≥200 copies/mL on sequential tests (Table 3). All 21 patients had received an HSCT 315

(R+/D+, n=10; R+/D-, n=9; R-/D+, n=2). The median time between sequential tests was 7 days 316

(range 1-27 days). 317

318

We further categorized these 22 episodes based on quantification of the subsequent tests. In 319

45.5% (10/22) of episodes the subsequent viral load was ≥600 copies/mL, levels quantifiable by 320

the reference assay. In five of these episodes antiviral treatment was initiated in response to this 321

viral load (median viral load 999 copies/mL, range 937-8390 copies/mL). In the other five 322

episodes, the CMV level continued to increase, and all five were eventually treated (median viral 323

load at the start of treatment 946 copies/mL, range 763-2570 copies/mL). In 13.6% (3/22) of 324

episodes the subsequent viral load was 400-600 copies/mL, a range detectable but not 325

quantifiable by the reference method. A CMV level of 541 copies/mL prompted antiviral 326

treatment during one episode. In two episodes, the viral load continued to increase and both 327

episodes were subsequently treated (viral load at the start of treatment 1160 and 761 copies/mL, 328

respectively). 329


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330

In 40.9% (9/22) of episodes the subsequent viral load ranged from 200-400 copies/mL, a range 331

that was not detectable by the reference method. In 8 of these episodes, the viral load continued 332

to increase. Seven of these episodes were ultimately treated with antivirals and one was treated 333

with reduced immune suppression (median viral load at the start of treatment 888 copies/mL, 334

range 323-7950 copies/mL). There was a single episode in which the viral load increased from 335

<200 to >200 copies/mL on sequential tests (262 copies/mL) and then decreased without 336

intervention. 337

338

In 72.2% (16/22) of episodes, patients did not immediately receive treatment in response to their 339

subsequent CMV viral load. In fifteen of these clinical episodes CMV levels continued to 340

increase and ultimately required antiviral therapy or reduced immune suppression. 341

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DISCUSSION 343

344

In this study, we evaluated a CMV testing method that involves automated sample preparation, 345

assay set-up, and real-time, quantitative PCR using the artus CMV PCR. This protocol showed 346

good numerical agreement when compared with the reference protocol, and the artus CMV 347

protocol has a broader linear range and lower LLOD. In addition, we also report the finding of a 348

significant increase in the rate of positive tests following the implementation of the artus CMV 349

protocol for the detection and monitoring of CMV in patient plasma samples at our institution. 350

This increase appears to be accounted for by the detection of patients with viral loads 351

undetectable by the reference method, though this cannot be confirmed without testing all 352

samples by both methods. 353

354

The artus CMV PCR has been evaluated in a number of previous studies (3, 5, 7, 12, 22). Our 355

results agree with the findings of the only previous report to compare artus CMV PCR reagents 356

to the COBAS Amplicor CMV Monitor (3). However, we demonstrate a lower LLOD and 357

extend the previous results to include the use of a different DNA extraction protocol and smaller 358

reaction volumes. 359

360

We did identify two specimens that amplified poorly in the artus CMV protocol. These samples 361

were drawn at separate times from a single patient, and repeat testing using the reference 362

protocol and CMV R-gene assay showed consistent and elevated quantitative results. The three 363

assays used in this study target different regions of the CMV genome. One potential explanation 364

for the discrepant quantitation is that the CMV isolate from this patient contains a mutation in the 365


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proprietary major immediate early gene sequences targeted by the artus CMV PCR. Such 366

mutations have been reported to cause decreased amplification in other assays that target this 367

gene, but they have not previously been reported for the artus CMV PCR (5, 16). 368

369

Recently, the WHO approved an international quantitation standard for CMV in an effort to 370

address concerns regarding the agreement of quantitative results for CMV PCR testing between 371

centers (14, 21, 24). Our results were generated using a commercially available set of 372

quantitation standards, and a conversion factor of 0.76 IU/copy of CMV DNA was subsequently 373

calculated by calibrating the artus CMV protocol to the 1st WHO International Standard for 374

human cytomegalovirus. To our knowledge, this is one of the first reports to provide such a 375

conversion factor. Reporting clinical results in IU/mL should allow for more accurate 376

comparisons of quantitative results generated using different assays. 377

378

Previous studies have documented the ability of PCR assays to detect CMV DNA copy numbers 379

below 100 copies/mL, though the clinical significance of such values remained unclear (2, 15). 380

Boeckh et al. designed a sensitive real-time PCR assay and reported on two patients diagnosed 381

with CMV disease who had viral loads detectable in stored samples from up to seven weeks prior 382

to diagnosis (2). Kaiser et al. modified the COBAS Amplicor CMV Monitor such that they 383

could detect CMV DNA down to 20 copies/mL of patient plasma (15). They then identified 16 384

HSCT recipients with detectable CMV DNA by their new protocol. Three patients in that study 385

had low detectable viral loads that resolved without therapy. However, most patients received 386

treatment following the detection of CMV DNA, making it difficult to draw conclusions 387

regarding changes in the viral load from this report. 388


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In HIV-positive patients with low CD4 counts, Erice et al. studied the use of a CMV antigen 389

assay (limit of detection reported as 200 copies/mL), and the reference protocol used in our study 390

(11). In univariate analysis after adjusting for HIV viral load and CD4 count, an increase in the 391

CMV copy number to >200 copies/mL during the study was significantly associated with 392

decreased survival. While the numerical cut-off used in their study cannot be generalized to our 393

population given the significant differences in testing modalities, these results support the 394

conclusion that increases in CMV viral load are significant even when initially detected at a very 395

low level. 396

397

During our study, 94.5% (n = 86) of patients had a CMV viral load that was detectable but <200 398

copies/mL, and many patients had repeatedly detectable viral loads at <200 copies/mL. The 399

finding of detectable virus at this level is difficult to interpret in isolation. However, the CMV 400

viral load continued to rise or antiviral treatment was initiated in 21 out of 22 clinical episodes 401

during which the viral load increased from <200 to >200 copies/mL. Twenty of these patients 402

eventually received antiviral treatment (Table 3), and in only a single episode did the viral load 403

decline without any intervention (from 262 to <200 copies/mL). All 21 patients in this sub-404

group had received an HSCT and were being followed using a pre-emptive treatment protocol; 405

none of them developed confirmed CMV disease. 406

407

Recommendations for the duration of treatment for CMV infection and disease are available, but 408

these have not been studied in a randomized setting (14, 17). Detectable CMV viral loads at the 409

end of treatment have been shown to predict recurrence in SOT patients, though this report 410

utilized a less analytically sensitive assay than the test used here (23). In our patients, the rates 411


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of recurrent infection in patients with a detectable viral load at the end of therapy did not differ 412

significantly from rates in patients with undetectable CMV at the end of therapy. The number of 413

patients in this analysis was small (n = 21), however, and this merits further study in a larger 414

population. 415

416

Limitations of our study include the retrospective design and the reliance on treating physicians 417

to determine when to initiate antiviral therapy during periods of viremia. Despite these 418

limitations, we were able to demonstrate that episodes of CMV viremia eventually treated with 419

antiviral therapy can be detected earlier using a more sensitive testing method. A prospective 420

study would better define clinical benefits that may result from using the CMV threshold 421

identified here (i.e. shorter duration of antiviral therapy or decreased toxicity). While the 422

incidence of end-organ disease would provide a more rigorous end-point, its occurrence is likely 423

too rare for it to be used in this fashion. 424

425

In summary, we report the performance characteristics of an automated protocol using the artus 426

CMV PCR in comparison with a widely used reference protocol and report our findings from the 427

study of a cohort of immune compromised patients monitored with this test. In a subgroup of 428

HSCT recipients, we observed clinically meaningful increases in the viral load, even when 429

quantified at levels previously undetectable in our reference protocol. These findings support the 430

use of a low threshold for the initiation of pre-emptive therapy in transplant patients. 431

432


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ACKNOWLEDGEMENTS 433

We thank the staff of the Stanford Clinical Virology Laboratory for their hard work, support, and 434

technical expertise. Qiagen provided the artus CMV RG PCR reagents and QIAsymphony 435

SP/AS extraction kits and consumables used for assay evaluation. 436

437


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22. Raggam, R. B., M. Bozic, H. J. F. Salzer, S. Hammerschmidt, C. Homberg, K. Ruzicka, and 506

H. H. Kessler. 2010. Rapid quantitation of cytomegalovirus DNA in whole blood by a new 507

molecular assay based on automated sample preparation and real-time PCR. Med Microbiol 508

Immunol. 199:311-316. 509

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transplantation. J Infect Dis. 181:717-720. 512

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Multi-site PCR-based CMV viral load assessment - assays demonstrate linearity and precision, 514

but lack numeric standardization. J Mol Diagn. 11:87-92. 515

516


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Reference Protocol Total artus C

MV

P

roto

col Pos Neg

Pos 42 10 52 Neg 0 30 30

Total 42 40 82

517

Table 1. Comparison of the artus CMV protocol to the reference protocol using 82 clinical 518

samples. The 10 samples positive by the artus CMV protocol and negative by the reference 519

protocol were retested using the CMV R-gene assay. Eight of 10 samples were positive by the 520

CMV R-gene assay. 521

522


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Transplant Recipients Patients (%)

Total 82 (100) SOT 27 (32.9) CMV status at transplant R+/D+ 15

R+/D- 5 R-/D+ 6 R+/D unknown 1 Prophylaxis

Acyclovir 3 Valganciclovir 15 None 9 HSCT 55 (67.1) Allogenic 53 Autologous 2 CMV status at transplant R+/D+ 29 R+/D- 22 R-/D+ 4 Prophylaxis 53 Acyclovir 51

Valganciclovir 0 None 2

523

Table 2. Characteristics of 82 transplant recipients with a positive plasma CMV PCR during the 524

study period. The two autologous HSCT transplants were CMV positive (R+) at transplant. 525

526


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Subsequent Viral Load N Continued to

Increase Decreased,

No Intervention Treatment

200-400 copies/mL 9 8 1 8

400-600 copies/mL 3 2 0 3

>600 copies/mL 10 5 0 10

Total 22 15 1 21 527

Table 3: Outcome for 22 clinical episodes during which the CMV viral load increased from 528

<200 to >200 copies/mL on sequential tests. The quantifiable level to which the viral load 529

increased on the second test is expressed as the subsequent viral load. 530

531


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FIGURE LEGENDS 532

533

Figure 1. Bland-Altman plot for the comparison of the artus CMV and reference protocols 534

using 42 clinical samples. The mean difference was zero (labeled, bold-faced line); dashed lines 535

indicate mean +/- 2 standard deviations. 536

537

Figure 2. Flowchart of the 91 adult patients with at least one positive plasma CMV PCR during 538

the study period. 539

540


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Figure 1 541

542

543

-1.20

-0.90

-0.60

-0.30

0.00

0.30

0.60

0.90

1.20

0.00 1.00 2.00 3.00 4.00 5.00 6.00

artusC

MV

-re

fere

nce

pro

toco

l (lo

g 10

cop

ies/

mL

)

Mean (log10 copies/mL)

Mean Difference


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Figure 2544

545

Patients with positive CMV PCR during study period

n=91

≥1 CMV viral load detected at <200 copies/mL

n=86

Viral load always <200 copies/mL

n=49

Viral load increased from <200 to >200 copies/mL

n=21

Viral load initially >200 copies/mL

n=16

Viral load always >1000 copies/mL

n=5


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