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Journal of Clinical Virology 56 (2013) 354– 359

Contents lists available at SciVerse ScienceDirect

Journal of Clinical Virology

jo u r n al hom epage: www.elsev ier .com/ locate / j cv

omparison of three real-time PCR for the quantification of polyomavirus BK

.A. Stellrechta,b,∗, A.A. Espinoa, S.M. Nattanmaia, W.F. Jacksona, D.J. Conti c

Department of Pathology and Laboratory Medicine, Albany Medical Center Hospital, Albany, New York, United StatesDepartment of Pathology and Laboratory Medicine, Albany Medical College, Albany, New York, United StatesDepartment of Surgery, Albany Medical College, Albany, New York, United States

r t i c l e i n f o

rticle history:eceived 28 August 2012eceived in revised form 4 December 2012ccepted 5 December 2012

eywords:K virus quantificationK viral load, Real-time PCR

a b s t r a c t

Background: Reactivation of latent polyomavirus BK is associated with nephropathy (PVAN) after renaltransplantation. BK viral load determinations are a highly sensitive and specific method for predictingrisk for PVAN.Objectives and study design: The performance of three real-time PCR for BKV DNA quantification(MultiCode®-RTx BK virus ASR [MC-RTx], MGB-Alert BKV ASR [MGB] and a laboratory developed assay[LDA]) were evaluated against a conventional PCR (test of record, TOR) in terms of linearity, dynamicrange, and accuracy.Results: The LOD (log10 copies/ml) were 2.0, 2.0 and 3.0 for MC-RTx, MGB and LDA, respectively with acommercial plasma panel and 2.0, 2.6 and 3.5 with a urine panel. These assays demonstrated excellentlinearity (r2 = 1.0) and reproducibility (CV range = 0.7–20.4%, 0.9–13.2%, and 0.5–13%, respectively). In ananalysis of 100 clinical specimens, all 76 samples defined as true positive for BKV DNA (positive by two ormore methods or a recent history of positivity) were detected with MC-RTx, while only 64 were detectedwith MGB and 55 were detected with LDA. BKV DNA was not detected by any method in the true negative

specimens. Based on these results, the sensitivities were 100% for MC-RTx, 84% for MGB and 72% for LDA.The greatest linear correlation with the mean concentration was observed with MC-RTx (r2 = 0.96) withtwo samples (3%) with greater than 0.5 log10 variance in quantification versus seven (11%) with MGB andten (18%) with LDA.Conclusions: These real-time assays for BKV load demonstrated excellent performance characteristics,

tratin

with the MC-RTx demons

. Background

BK virus, a member of the family Polyomaviridae, is ubiqui-ous and primary infection is usually acquired early in life witheroprevalence rates in the general adult population ranging from0% to 90%.1,2 During initial infection virions infect uroepithe-

ial cells and establish a latent infection. Viral reactivation occursuring states of host immune suppression. Infection with poly-mavirus BK has emerged as a significant complication afterenal transplantation,3–5 particularly with the introduction of moreotent immunosuppressive agents.6,7 Typically, viral particles arerst detectable in the urine with an incidence of BK viruria of0–60% by 90 days post transplantation.8,9 This may be followed

y viremia and subsequently nephropathy.3 The incidence of BKiremia has been found to be as high as 10–28% with subsequentVAN developing in 5–10% in the first year post-transplant.8–11

∗ Corresponding author at: Albany Medical Center, Department of Clinical Micro-iology, MC-22, 43 New Scotland Avenue, Albany, New York 12208, United States.el.: +1 518 262 3587; fax: +1 518 262 4337.

E-mail address: stellrk@mail.amc.edu (K.A. Stellrecht).

386-6532/$ – see front matter © 2012 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.jcv.2012.12.003

g the greatest sensitivity.© 2012 Elsevier B.V. All rights reserved.

Graft loss occurs in as many as one-third of patients with PVAN andgrafts survival is often associated with significant deterioration inrenal function and decreased long-term survival.4,8,10,12

Successful strategies of intensive monitoring of renal transplantrecipients for BK viremia with preemptive reduction of immuno-suppression have been reported.8,11 Accurate quantification of BKVDNA is essential for a successful transplant program. Real-timequantitative PCR is the state-of-the-art technology for the quan-tification of nucleic acids. However, standardized PCR primers,probes, and calibrants are unavailable for BKV. Further compound-ing the problem is the genetic variability among BKV DNA in clinicalspecimens. BKV isolates have been grouped into subtypes by phy-logenetic analysis of full-genome viral DNA sequences.13,14 Thedistribution of subtypes among human populations has a stronggeographic component.15,16 Indeed, variability in the measurementof BKV loads by real-time PCR assays has been shown to be substan-tially influenced by the BKV subtype.17,18

2. Objectives

The goal of the study was to evaluate the performance of threereal-time PCR assays for BKV DNA quantification by assessing the

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imit of detection, the dynamic range, linearity, precision and accu-acy with BKV dilution panels, as well as with clinical samples.

. Study design

.1. Clinical samples

In a retrospective study, 100 EDTA anticoagulated plasma spec-mens, collected from 69 renal transplant patients, sent to theaboratory for BK viral load determinations by our TOR, were ana-yzed with the three real-time PCR assays. Samples were storedt −80 ◦C until testing. Specimens were defined as true positivef BKV DNA was detected by two or more assays or if the patientad a recent history of being positive for BKV DNA. Studies wereerformed in accordance with IRB requirements at Albany Medicalenter.

.2. LOD, LOQ, linearity, dynamic range, efficiency ofmplification and precision

The AcroMetrix® BKV Panel (Life Technologies, Grand Island,Y), with additional 10-fold serial dilutions in normal humanlasma (NHP), as well as serial dilutions of a viral stock (VR837,TCC, Manassas, VA) in a BKV negative urine sample pool werextracted in triplicate and each extract was amplified in duplicate.he concentration of the BK stock was determined by PCR withC-RTx using the BK Human polyomavirus quantitated viral DNA

Advanced Biotechnologies, Columbia, MD) as the quantificationtandard. The urine sample pool was derived from six specimensent to the laboratory for routine bacterial culture. Results fromesting controls were also used for precision studies.

.3. Nucleic acid extraction

DNA was extracted from 0.1 ml of sample with the NucliSensasyMAG System (bioMérieux, Durham, NC) with an elution vol-me of 50 �l. DNA for three internal controls (5 �l DNA samplerocessing control [Luminex, Austin, TX], 20 �l of IC2, 103 copies/�lEpoch Biosciences, Bothell, WA], 10 �l plasmid DNA pHCVis con-aining bacteriophage phiX-174 sequences, 102 copies/�l) weredded after sample lysis.

.4. Polyomavirus PCR-ELOSA LDA (TOR)

Polyomavirus PCR was performed as previously described11

rom 5 �l of eluate with primers and probe targeting the large Tntigen19 (Integrated DNA Technologies [IDT], Coralville, IA). Prod-cts were detected by enzyme labeled oligo sorbent assay.20,21

uantification of viral DNA was determined by standard curvenalysis generated by amplification of serial dilutions of plasmiderived from the target region.

.5. Real-time PCR

Viral and internal control DNA were amplified on a LightCycler80 (Roche Applied Science, Indianapolis, IN) from 5 �l of eluate as

ndicated below. Quantification of viral DNA for all three real-timessays was determined by standard curve analysis generated bymplification of serial dilutions of BK Human polyomavirus quan-itated viral DNA. AcroMetrix® BKV low and high controls (Lifeechnologies) and NHP were included on each run.

MC-RTx: BK virus sequences from the VP2/VP3 region were

mplified with the BK Virus Primers ASR, Control Primers, 5XSOlution (Luminex Corp. [EraGen]) and Titanium Taq (Clontechaboratories, Inc., Mountain View, CA) in 25 �l reactions using theollowing amplification profile: 1 cycle of 2 min at 95 ◦C and 45

al Virology 56 (2013) 354– 359 355

cycles of 95 ◦C for 5 s, 60 ◦C for 30 s and 72 ◦C for 20 s, followed bya melt curve analysis of 95 ◦C for 10 s, 60 ◦C for 30 s and ramping to95 ◦C with continuous acquisitions of 2.5 per ◦C.

MGB: BK virus sequences from the VP1 region were amplifiedwith the MGB Alert BK Virus Primers & Probe ASRs, MGB Alert N2Primers & Probe, 2X MGB Alert Hot Start Mix and PCR Enhancer(Epoch Biosciences) in 20 �l reactions using the following ampli-fication profile: 1 cycle of 2 min at 50 ◦C and 95 ◦C followed by 50cycles of 95 ◦C for 15 s, 56 ◦C for 30 s and 72 ◦C for 30 s. Melt curveanalysis was performed at 95 ◦C for 15 s, 40 ◦C for 30 s and rampingto 80 ◦C with continuous acquisitions of 1 per ◦C.

LDA: BK virus sequences from two regions in large T antigengene22,23 were co-amplified with dual FAM and CY5-labeled probes(IDT), along with internal control comprised of a plasmid containinga fragment of phiX-174 DNA (CY3-labeled probe), using SsoFastTM

Probes Supermix (Bio-Rad Life Science, Hercules, CA) in 20 �l reac-tions. Amplification were performed with 1 cycle of 2 min at 95 ◦C,followed by 50, two-step cycles of 95 ◦C for 5 s, 60 ◦C for 15 s.

3.6. Statistical analysis

Probit analyses for the limit of detection with a 95% probabilityof detection were performed using SPSS version 8.0 (IBM, Armonk,NY). The accuracy represents the inverse of the percent deviation ofthe mean observed value log10 copies/ml from the expected value.The lower and upper limits of quantification (LOQ) were defined asthe concentrations which maintained a CV ≤ 25% and an accuracyof >90%. Accuracy, coefficient of variance, linear regression, andBland–Altman test of agreement were performed using MicrosoftExcel 2003 (Redmond, WA). Melt temperature predictions weredetermined using OligoAnalyzer 3.1 (IDT24).

4. Results

4.1. LOD, LOQ, dynamic range and linearity

Analysis of a commercial panel with additional dilutions(0.7–6.7 log10 copies/ml) demonstrated that all real-time assay pro-duced results on average of 0.23 logs lower than the manufacturer’sexpected concentration, but results were similar between assays.The LOD by probit analysis from these studies were 2.0, 2.0 and 3.0log10 copies/ml, for MC-RTx, MGB and LDA, respectively. To betterassess the dynamic ranges, BK virus was diluted serially ten-fold ina urine sample pool, as well as two- and three-fold dilutions nearthe LOD (1.9–9.9 log10 copies/ml). The LOD determined from theviral isolate dilution series in a urine matrix was 2.0, 2.6 and 3.6log10 copies/ml for the three assays (Table 1).

The lower and upper LOQ (log10 copies/ml) were also assessed.The lower LOQ for MC-RTx and the LDA were equivalent to theirLOD values of 2.0 and 3.5, respectively, with coefficients of variance(CV) and accuracies of 20% and 96% for MC-RTx and 13.2% and 98%for LDA at the LOD/LOQ (Table 1). On the other hand, the LOQ forMGB was only 2.86 log10 copies/ml, as the precision at 2.6 log10copies/ml was 38%. Furthermore, it was evident that the accuracybegan to show deviation at 2.86 log10 copies/ml. The upper LOQwas at least 10 billion copies/ml, with excellent CVs and accuraciesfor all three assays. Lastly, all three assays demonstrated exquisitelinearity with r2 values of 1.0 for either plasma (Fig. 1A) and urinesamples (Fig. 1B).

4.2. Precision and accuracy

From the studies above, the precision across the dynamic rangeranged from 0.7% to 20.4% for MC-RTx, 0.9–13.2% for MGB and0.5–13% for LDA (Table 1). The real-time PCR assays demonstratedinter-assay precision with controls, with a coefficient of variance

356 K.A. Stellrecht et al. / Journal of Clinical Virology 56 (2013) 354– 359

Table 1Limit of detection, precision and accuracy of the real-time PCR assays in urine.

Exp. No. tested MC-RTx MGB LDA

No. pos Obs CV Accuracy No. pos Obs CV Accuracy No. pos Obs CV Accuracy

9.86 6 6 9.80 0.7% 99.3% 6 9.67 1.2% 98.0% 6 9.86 0.5% 99.9%8.86 6 6 8.89 0.7% 99.7% 6 8.78 1.4% 99.1% 6 8.93 2.9% 99.3%7.86 6 6 8.03 1.7% 97.9% 6 7.84 1.1% 99.7% 6 8.04 1.0% 97.8%6.86 6 6 6.92 1.0% 99.2% 6 6.85 1.0% 99.8% 6 7.10 0.8% 96.5%5.86 6 6 5.90 1.3% 99.4% 6 5.91 0.9% 99.2% 6 6.06 1.3% 96.7%4.86 6 6 4.95 4.1% 98.2% 6 5.01 3.2% 97.0% 6 5.17 4.9% 93.8%3.86 6 6 3.88 1.8% 99.6% 6 4.06 2.3% 95.0% 6 3.74 10.0% 96.8%2.86 6 6 2.67 7.4% 94.3% 6 3.14 13.2% 90.2% 2 2.82 13.0% 98.4%2.56 6 6 2.67 10.5% 96.0% 6 2.43 38.4% 94.8% 0 – – –2.39 6 6 2.43 17.4% 98.4% 6 2.50 33.6% 95.4% 0 – – –2.09 6 6 2.00 20.4% 95.9% 3 2.26 29.1% 91.7% 0 – – –

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xp = expected concentration (log10 copies/ml); Obs = observed concentration (log1

or log10 values of 4.2 and 2.8%, 4.0 and 4.2%, and 7.5 and 4.7%or the BK low and high controls with MC-RTx, MGB and LDA,espectively (data not shown). MC-RTx demonstrated the great-st accuracy across the dynamic range, with values ranging from4% to 100% (Table 1). MGB also showed very good accuracy downo 3.86 log10 copies/ml (95–100%), but began to show more devi-tion at lower concentrations (90.2%). The LDA showed very goodccuracy though the limited dynamic range (94–100%).

.3. Performance with clinical samples

One hundred clinical specimens from 69 patients sent to the lab-ratory for routine polyomavirus measurements by the TOR werenalyzed with all three real-time PCR assays. Of these samples, noamples were inhibitory for PCR and 76 were defined as true posi-ive for BKV DNA. The viral load range for these samples was from.71 to 7.55 log10 copies/ml (median 3.01). MC-RTx was positiveor all 76 specimens whereas MGB was positive for 64 and LDA wasositive for 55 (Fig. 2). All but two true positive sample not detectedy MGB or LDA had levels of BKV DNA lower than the assays’ LOD.ne of these samples had a viral load of 2.96 log10 copies/ml andas detected by all assays except MGB, while the viral load for other

ample was 2.63 log10 copies/ml and was detected by MC-RTx andOR. There were two samples with BKV detected by MC-RTx onlyith viral loads near the LOD for this assay. Both of these sam-les were from patients with detectable virus by multiple methods

ithin the previous one to two months. BKV was not detected by

ny method from the 24 true negative specimens. Based on theseesults, the sensitivities were 100% for MC-RTx, 84% for MGB and2% for LDA and specificities were 100% for all three.

Fig. 1. Linear regression analysis of real-time PCR assay with dilution

1.65 73.9% 91.0% 0 – – –

s/ml).

Excellent correlation was seen between real-time assays for thespecimens with detectable virus, with r2 values of 0.96 for MC-RTx, 0.93 for MGB and 0.89 for LDA (Fig. 3). The mean differencein viral load values for MC-RTx from the mean values was 0.03log10 copies/ml and the 95% limit of agreement (LOA) was between−0.52 and 0.46, suggesting little bias. For MGB the mean bias was−0.13 with a 95% LOA between −0.7 and 0.45 and for LDA themean bias was 0.18 with a 95% LOA between −0.56 and 0.92. Fur-thermore, the viral load values by MC-RTx were in agreement towithin 0.5 log10 copies/ml for all but 2 (3%) samples, both of whichwere under quantified by MC-RTx. On the other hand, deviationsof greater than 0.5 log10 copies/ml were seen with 7 (11%) and 10(18%) samples with MGB and LDA, respectively. There were no sam-ples with greater than 1.0 log10 copies/ml variance from the meanvalue by any assay, with the greatest variance seen being 0.95 log10copies/ml for one sample with LDA.

4.4. Performance with genotype II, III or IV samples

Within the MC-RTx target region, between primer binding sites,a single base pair polymorphism occurs in genotypes II, III or IVvirus, (data not shown) which results in a predicted 0.8 ◦C increasein melting temperature of the amplicon (Table 2). Of the 76 sam-ples with detectable BKV with MC-RTx, 67 had a mean ampliconmelt temperature of 76.9 ◦C and 9 had a mean melt tempera-ture of 77.8 ◦C, suggesting 12% of the samples were genotype II,

III or IV. BKV was detected by all three assays in these samples.However, MGB appeared to under quantify the viral DNA witha mean bias of 0.49 log10 copies/ml and LOA of 0.23 and 0.75as compared with MC-RTx. Furthermore, a deviation of greater

panels. (A) Correlation in plasma and (B) correlation in urine.

K.A. Stellrecht et al. / Journal of Clinical Virology 56 (2013) 354– 359 357

LDA + 50MGB + 59

LDA - 9TOR+ 68

LDA + 3MGB - 9

LDA - 6TP 76 MC-RTx + 76

LDA + 1MGB + 5

LDA - 4TOR- 8

LDA + 1MGB - 3

Specimens 100 LDA - 2

TN 24 MC-RTx - 24 TOR- 24 MGB - 24 LDA - 24

Fig. 2. Assay performance with clinical samples. Results from four PCR tests were compared in 76 true positive (TP) and 24 true negative (TN) plasma samples.

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ig. 3. Correlation of real-time PCR assay with clinical specimens. (A) linear regresshe observed values for each assay. Solid lines depict 0.5 log10 copies/ml from the m

han 0.5 log10 copies/ml was seen with three (33%) samples withGB.

. Discussion

Accurate quantification of BKV DNA is essential for a successfulenal transplant program. However, variability in the performancef primer and probes has been reported.17,18 The main purpose ofhis study was to evaluate the performance of different real-time

CR assays with different gene targets with regards to the detectionnd measurement of BK viral loads. Specifically, MC-RTx targets a3 bp region in VP2/3, while MGB targets a 72 bp region in VP1Fig. 4). In an attempt to amplify more strains of BK with sequence

able 2redicted melting temperatures of MC-RTx amplicons.

Strain Genebank accession Genotype Tm (◦C)

Dunlop V01108 Ia 76.9TW-1 AB211381 Ic 76.9J2B-11 AB301101 II 77.7KOM-3 AB211386 III 77.7TW-3 AB211391 IV 77.7THK-9 AB211379 V 76.9ETH-4 AB263917 VI 76.9

alysis, (B) Bland–Altman plot of the mean viral load (log10 copies/ml) compared todashed line).

divergence, we designed the LDA as multiplex targeting two regionsin the large T antigen, a 133 bp region22,23 and a 176 bp region.23

The precision for all three assays was excellent across thedynamic range. As expected, these assays demonstrated superiorprecision over the TOR as quantitative measurements occurredearly, in the exponential phase of amplification rather, than at anendpoint. Albeit, the TOR is a probe based method with a kineticread for substrate which increases the dynamic range for a con-ventional PCR, the dynamic range is still limited. However, thesensitivity of the TOR is very good and interestingly, only MC-RTxwas more sensitive.

Factors which can reduce the sensitivity of real-time PCR includeprimer and probe design, in particular, relative melting tem-peratures of the probe to the primers.25 These factors can befurther compounded with sequence divergence. Indeed, it hasbeen reported that the VP2/3 regions of the BK genome is highlystable,26 whereas numerous reports of sequence divergence hasbeen reported for both the VP-1 and large T antigen.17,27,28 Fur-thermore, increased mutations of the VP-1 gene in patients withBK nephropathy have been reported.27,29,30

Since the LDA is designed as a two target assay, targeting boththe VP-1 and large T antigen, the assay can be used to assess theperformance of each target individually. Interestingly, both targetswere amplified with similar efficiency for 37 of the 55 positive

358 K.A. Stellrecht et al. / Journal of Clinical Virology 56 (2013) 354– 359

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amples with a mean bias in quantification for the individualarget of 0.16 log10 copies/ml. However, there were ten samplesor which only the VP-1 target was detected and eight samplesor which the only the T-ag target was detected (data not shown).lthough most of these discrepant samples had viral loads near

he limit of detection, there were two samples for which only theP-1 target was detected which had elevated viral loads (3.83 and.62 log10 copies/ml). Neither of these samples appeared to beenotype II, III or IV by melt curve analysis. In fact of the 9 sampleshich appeared to be genotype II, III or IV, seven were amplified

fficiently by both targets, and two were amplified by only target,ne with VP-1 and one with T-ag.

Not surprisingly, MGB, which targets the VP-1 gene, under quan-ified samples which appeared to be genotype II, III or IV. Thisnding supports the role of sequence divergence in reduced sen-itivity of the assay. In our patient population 12% of the samplesontained these more divergent strains of BK. Similar findings waseported by Rennert et al. from the metropolitan New York area31

n which 9% samples were genotypes II, III or IV. It is well knowhat the distribution of BK subtypes among human populations has

strong geographic component world wide.15 Furthermore, geo-raphic variations have been seen within the United States with aeported frequency for genotypes II, III or IV of 20% in the Seattlerea.17

MC-RTx demonstrated the greatest analytical and clinical sen-itivity. However the need for such low level sensitivity could beuestioned, particularly since the recommendations from an inter-ational panel are to refraining from altering immunosuppressionntil the viral loads reaches 4 log10 copies/ml.32 At our institu-ion, all renal transplant patients are monitored monthly for BKiremia beginning 1 month post transplant and continuing through4 months. After 24 months of monthly monitoring for BK viremia,onitoring is reduced to quarterly analysis. We have found the

ower analytical detection useful particularly for these patients, asow level detection prompts for more frequent monitoring.

Initially, 50% reduction in mycophenolate mofetil (MMF) dos-ng is initiated after viremia reaches approximately 3.0 log10opies/ml and if clearance of viremia is not obtained within

months, MMF is discontinued completely. This program ofrompt reduction of immunosuppression has not only preventedhe development of PVAN in our patient population but haslso decreased the development of CMV and/or EBV disease,s BK viremia has been shown to be a useful marker for overmmunosuppression.33

In conclusion, all three real-time PCR assay are reliable assay foreasuring BKV loads for the identification of patients at risk for

KVAN. These assays demonstrated superiority over the TOR withegards to precision. MC-RTx appears to have enhanced sensitivitynd better performance among divergent strains of BK.

unding

None.

1

r AB464953) with PCR target regions notated.

Competing interests

The authors declare no competing interests.

Ethical approval

Obtained from Albany Medical Center.

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