Impact of Genomic Sequence Variability on Quantitative PCR Assays for Diagnosis of Polyomavirus BK Infection Parmjeet Randhawa, Jeffrey Kant, Ron Shapiro, Henkie Tan, Antik Basu and Chunming Luo J. Clin. Microbiol. 2011, 49(12):4072. DOI: 10.1128/JCM.01230-11. Published Ahead of Print 28 September 2011. Updated information and services can be found at: http://jcm.asm.org/content/49/12/4072

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JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 2011, p. 4072–4076 Vol. 49, No. 12 0095-1137/11/$12.00 doi:10.1128/JCM.01230-11 Copyright © 2011, American Society for Microbiology. All Rights Reserved.

Impact of Genomic Sequence Variability on Quantitative PCR Assays for

Diagnosis of Polyomavirus BK Infection

Parmjeet Randhawa,1* Jeffrey Kant,

1,2 Ron Shapiro,

3 Henkie Tan,

3 Antik Basu,

3 and

Chunming Luo1

Departments of Pathology,1 Human Genetics,

2 and Surgery,

3 University of Pittsburgh Medical Center, Pittsburgh, Pennsylvania

Received 20 June 2011/Returned for modification 6 September 2011/Accepted 22 September 2011

Knowledge of polyomavirus BK (BKV) genomic diversity has greatly expanded. The implications of BKV

DNA sequence variation for the performance of molecular diagnostic assays is not well studied. We analyzed

184 publically available VP-1 sequences encompassing the BKV genomic region targeted by an in-house quantitative hydrolysis probe-based PCR assay. A perfect match with the PCR primers and probe was seen in 81 sequences. One Dun and 13 variant prototype oligonucleotides were synthesized as artificial targets to

determine how they affected the performance of PCR. The sensitivity of detection of BKV in the PCR assay was a function of the viral genotype. Prototype 1 (BKV Dun) could be reliably detected at concentrations as low as 10 copies/ l. However, consistent detection of all BKV variants was possible only at concentrations of

10,000 copies/ l or higher. For BKV prototypes with 2 or more mismatches (representing genotype IV, genotype II, and genotype 1c strains), the calculated viral loads were 0.57 to 3.26% of the expected values. In

conclusion, variant BKV strains lower the sensitivity of detection and may have a substantial effect on quantitation of the viral load. Physicians need to be cognizant of these effects when interpreting the results of quantitative PCR testing in transplant recipients, particularly if there is a discrepancy between the clinical

impression and the measured viral load.

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Polyomavirus BK (BKV) has become an important patho-gen

in kidney transplant patients. Immunosuppression given to prevent

acute rejection triggers BK viruria in 10 to 60%, viremia in 5 to

30%, and biopsy-proven viral nephropathy in 1 to 10% of patients

(8, 10, 11). Initial graft loss rates associated with BKV

nephropathy were very high but have now dropped to 25%. This

success has been attributed to intensive viral monitoring followed

by preemptive reduction in immunosuppression (1, 14). In a

recent survey that had an overall response rate of 55.5%, 173 of

200 (86.5%) kidney transplant centers reported screening for

BKV in blood by quantitative PCR, while 111 of 202 (55.5%)

performed viral screening in urine (2). In the latter category, 90%

of respon-dents preferred PCR screening to urine cytology. While

cytology is a useful modality to screen for viral nephropathy in

low-resource settings, it is less sensitive than quantitative PCR for

detecting viral replication prior to the onset of clinical

nephropathy. In addition, it cannot differentiate BKV infection

from infection by the related polyomavirus JC, which causes

significant graft dysfunction at a substan-tially lower frequency.

Current quantitative PCR assays were developed several years

ago using BKV Dun or similar genotype I strains as reference

sequences for the design of primers and probes. However, our

knowledge of BKV genomic diversity has ex-panded enormously

(6, 7, 12, 16). PCR-based diagnostic and treatment algorithms

must be reevaluated to take into account newly discovered BKV

single-nucleotide polymorphisms. One

* Corresponding author. Mailing address: Division of Transplant

Pathology, University of Pittsburgh, Department of Pathology, E737 UPMC-Montefiore Hospital, 3459 Fifth Ave., Pittsburgh, PA 15213. Phone: (412) 647-7646. Fax: (412) 647-5237. E-mail: randhawapa @upmc.edu.

Published ahead of print on 28 September 2011.

approach to define the potential extent of this problem is to assay the same sample using a panel of different PCR assays (5). This is a labor-intensive method that is not practical for

routine application by clinical laboratories. We employed an

alternate approach that consists of aligning PCR primer and probe

sequences with large data sets of BKV sequences (3). This

bioinformatic evaluation was followed by experimental

amplification of 14 custom oligonucleotides of extended length

designed to comprehensively represent genetic variability in the

targeted area of the (VP-1) gene. Our results show that variant

BKV strains significantly lower the sensitivity of detect-ing viral

DNA and have a substantial effect on quantitation of the viral

load. We recommend that molecular diagnostic lab-oratories

offering BKV testing regularly reevaluate their cur-rent assays for

the ability to accurately identify and quantitate the majority of

viral strains circulating in the communities they serve. This

recommendation is particularly applicable to geo-graphic

locations with a high incidence of genotypes other than type 1. It

is worth recalling that BKV genotype IV has a reported

prevalence of 54% in Mongolia, while genotype III accounts for

9% of BKV isolates reported from Africa (17).

MATERIALS AND METHODS

Retrieval of public sequences. A total of 184 BKV VP-1 sequences matching

primer and probe sequences of the PCR assay used in our laboratory were retrieved

from GenBank. These sequences were aligned by Clustal X with de-fault

multialignment parameters (13). The alignments were manually adjusted using

BioEdit (T. Hall, Department of Microbiology, North Carolina State University;

available at http://www.mbio.ncsu.edu/BioEdit/BioEdit.html). Phylogenetic analysis. When not already known, genotype assignment of the

sequences was based on phylogenetic clustering using known reference se-quences,

as previously described (7). Neighbor-joining trees were constructed in Mega 4.1

using Kimura’s two-parameter method and the complete deletion option for gaps and

missing data. Trees were viewed using the Tree Explorer program. A bootstrap test

with 1,000 replicates was used to estimate the confi-dence of branching patterns in

the trees.

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VOL. 49, 2011 BK VIRUS ASSAYS 4073

TABLE 1. GenBank accession numbers of 184 publicly available BKV sequences classified into 14 prototypes

Prototype (no.) GenBank sequence

1 (81) .............................. Ia_CAF-15; Ia_CAF-5; Ia_CAF-9; Ia_Dunlop; Ia_KEN-1; Ia_KEN-4; Ia_MM; Ia_PittNP4; Ia_PittNP5; Ia_PittVR4; Ia_PittVR9; Ia_UT; Ia_Z19534; Ia_ZAF-1; Ib1_CAP-h2; Ib1_CAP-h22; Ib1_CAP-h5; Ib1_CAP-h8; Ib1_CAP- m13; Ib1_CAP-m18; Ib1_CAP-m5; Ib1_CAP-m9; Ib1_Dik; Ib1_GBR-6; Ib1_HI-u5; Ib1_HI-u6; Ib1_HI-u8; Ib1_J2B-2; Ib1_KEN-3; Ib1_KOM-1; Ib1_KOM-5; Ib1_LAB-18; Ib1_LAB-27; Ib1_MMR-6; Ib1_NER-1; Ib1_OKN-18; Ib1_PHL-6; Ib1_PHL-7; Ib1_PittNP1; Ib1_PittVM2; Ib1_PittVR8; Ib1_VNM-9; Ib1_WW; Ib2_ESP-2; Ib2_ETH-4; Ib2_FIN-10; Ib2_FIN-11; Ib2_FIN-13; Ib2_FIN-14; Ib2_FIN-23; Ib2_FNL-12; Ib2_FNL- 22; Ib2_GBR-4; Ib2_GBR-8; Ib2_GBR-9; Ib2_HC-u2; Ib2_HC-u5; Ib2_HC-u9; Ib2_J2B-13; Ib2_J2B-9; Ib2_JL; Ib2_LAB-14; Ib2_LAB-20; Ib2_LAB-22; Ib2_LAB-25; Ib2_LAB-29; Ib2_LAB-7; Ib2_PittNP2; Ib2_PittVM4; Ib2_PittVM5; Ib2_PittVR10; Ib2_PittVR2; Ib2_PittVR3; Ib2_PittVR5; Ib2_PittVR6; Ib2_SWE-2; Ib2_TUR-5; Ic_RYU-2; Ic_TW-8; Ic_TW-8a, II_ETH-3

2 (1) ................................ Ib1_CAP-m2 3 (48) .............................. II_GBR-12; IVa1_MMR-24; IVa1_PHL-8; IVa1_SEC-3; IVa1_VNM-7; IVb1_JPN-32; IVb1_JPN-33; IVb1_JPN-36;

IVb1_THK-8; IVb1_TW-3; IVb1_TW-3a; IVb2_JPN-15; IVb2_JPN-34; IVb2_JPN-35; IVb2_KOM-2; IVb2_KOM-7; IVb2_MON-8; IVc1_FUJ-18; IVc1_FUJ-32; IVc1_MMR-28; IVc1_MMR-29; IVc1_MON-1; IVc1_MON-6; IVc1_NEC-14; IVc1_NEC-24; IVc1_NEC-4; IVc1_NWC-14; IVc1_NWC-15; IVc1_NWC-8; IVc1_SEC-6; IVc1_SWC-1; IVc1_SWC-2; IVc1_SWC-4; IVc1_VNM-1; IVc2_FIN-2; IVc2_FIN-9; IVc2_FNL-17; IVc2_Fin-4; IVc2_GRC-3; IVc2_GRC-4; IVc2_GRC-5; IVc2_ITA-3; IVc2_LAB-33; IVc2_MON-2; IVc2_MON- 3; IVc2_MON-5; IVc2_NWC-7; IVc2_SWE-4

4 (6) ................................ IVa2_FUJ-13;IVa2_MMR-1;IVa2_MMR-43;IVa2_RYU-3;IVa2_SEC-21;IVa2_VNM-2 5 (5) ................................ III_AS; III_KOM-3; III_Z19536; II_EF376992; II_J2B-11 6 (22) .............................. Ic_J2B-1; Ic_KOM-6; Ic_MT; Ic_NAR-12; Ic_NAR-13; Ic_NEA-25; Ic_NEB-6; Ic_NEC-12; Ic_NEC-8; Ic_NGY-38;

Ic_NGY-5; Ic_OKN-14; Ic_RYU-1; Ic_THK-6; Ic_THK-9; Ic_THK-9a; Ic_TW-1; Ic_TW-1a; Ic_TW-1b; Ic_TW-4; Ic_TW-5; Ic_TW-7

7 (7) ................................ Ib2_DQ366598; Ib2_LAB-21; Ib2_LAB-8; Ib2_PittNP3; Ib2_PittVM1; Ib2_PittVM3; Ib2_PittVR7 8 (1) ................................ Ib2_V_ITA-5 9 (1) ................................ Ib2_V_PittVR1

10 (4) .............................. Ic_AB181542; Ic_AB181555; Ic_ab268370; Ic_ab276279 11 (3) .............................. II_AB268401;II_AB276173;II_AB276304 12 (2) .............................. Ib2_DQ533639;Ib2_DQ533641 13 (1) .............................. Ia_AB276228 14 (2) .............................. Ic_AB245326;Ia_AB268405

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Synthesis of oligonucleotides. Fourteen chromatographically purified synthetic

oligonucleotides, each 135 nucleotides in length and representing all known BKV

genetic variation in the VP-1 gene region targeted by the PCR assay, were purchased

for use as artificial targets to compare amplification efficiencies (In-tegrated DNA

Technologies, Coralville, IA). Nucleotides AGGG were incorpo-rated at one end of

these synthetic nucleotides, and AAAT at the other end. Oligonucleotide solutions in

EB buffer (Qiagen) were standardized by measuring the absorbance at 260 nm and

represented target sequence concentrations rang-ing from 1E8 to 1E0 copies/ l.

Real-time PCR. The assay targeted the BKV VP-1 gene as follows: forward

primer, 5 -GCAGCTCCCAAAAAGCCAAA-3 (1600 to 1619; Dun number-ing);

reverse primer, 5 -CTGGGTTTAGGAAGCATTCTA-3 (1726 to 1706; Dun

numbering); probe, 5 -ACCCGTGCAAGTGCCAAAACTACTAATAAA AGG-3

(1623 to 1655; Dun numbering). The real-time PCR was performed in a total volume of 20 l and contained the

following components: 10 l TaqMan Fast Universal PCR Master Mix (2 ; catalogue

number 4352042), 1.5 l of each primer (prepared at 1 M), 1 l of probe (prepared at 10

M), and 6 l oligonucleotides at specified concentra-tions. The PCR cycling program

consisted of the following steps: 95°C for 4 min and then 95°C for 10 s, 60°C for 30

s, and 72°C for 10 s for a total of 40 cycles. Thermal cycling was performed using an

Applied Biosystems 7500 apparatus. Standard precautions were employed to prevent

PCR contamination. Pre- and postamplification steps were done in separate

laboratories. The quantitation of target copy numbers used a standard curve with the

pBKV(34-2) plasmid, which contains the BKV Dun genome (ATCC 45025).

Data analysis. Analysis of the real-time PCR assay was performed using SDS

software (Applied Biosystems). Unknown target concentrations were deter-mined by

linear regression using threshold cycles (CT) plotted against the log10 copy number

of the standard BKV plasmid. Corrections for sample dilution and descriptive

statistics were performed in Microsoft Excel 2007.

RESULTS

The breakdown of genotypes for the 184 sequences retrieved

for the study was as follows: 16 Ia, 30 Ib1, 45 Ib2, 30 Ic, 53 IV,

7 II, and 3 type III. An alignment of these sequences indicated a

perfect match with the PCR primers and probe for 81 se-quences

(13 genotype Ia, 29 genotype Ib1, 35 genotype Ib2, 3 genotype 1c,

and 1 genotype II). All genomic variability in the BKV VP-1

region targeted by our quantitative PCR assay could be

represented by 14 unique prototype sequences (Table 1).

Prototype 1 matches the BKV Dun reference sequence, as well as

several other sequences, all except one of which are genotype 1.

Prototypes 2 thru 14 correspond to a broad spec-trum of

sequences that includes genotypes II, III, and IV at coverage

frequencies that are summarized in Table 2. It is apparent that

prototypes 1, 2, 6, 7, 8, 9, 10, 12, 13, and 14 cover primarily

genotype I strains. Genotype II is represented by prototypes 5 and

11, genotype III by prototype 5, and genotype IV by prototypes 3

and 4. The locations of nucleotide mis-matches between the viral

prototype and PCR primer/probe sequences are depicted in Fig. 1

and enumerated in Table 3, which shows the relative amplification

efficiencies of the dif-ferent prototype sequences.

The results indicate that the sensitivity of detection of BKV is a

function of the viral genotype. Thus, prototype 1, which

represents BKV Dun and similar strains, could be consistently

detected at concentrations as low as 10 copies/ l (10,000 cop-

ies/ml). This is clinically relevant, since plasma BKV loads of this

magnitude have been used as a trigger to lower immuno-

suppression and initiate antiviral therapy. Notably, at lower

concentrations, namely, 1 copy/ l, detection of prototype 1 was

not possible in 1 of 3 replicates (Table 3). Prototype 3, which

covered the majority of genotype IV strains, behaved essen-

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4074 RANDHAWA ET AL.

TABLE 2. Frequency distribution of BKV genotypes I to IV as

represented by prototype sequences 1 to 14a

No. of sequences of BKV genotype:

Prototype

I II III IV Totalb (%)

(n 121) (n 7) (n 3) (n 53)

1 80 1 0 0 81 (44)

2 1 0 0 0 1 (0.5)

3 0 1 0 47 48 (26.1)

4 0 0 0 6 6 (3.3)

5 0 2 3 0 5 (2.7)

6 22 0 0 0 22 (12)

7 7 0 0 0 7 (3.8)

8 1 0 0 0 1 (0.5)

9 1 0 0 0 1 (0.5)

10 4 0 0 0 4 (2.2)

11 0 3 0 0 3 (1.6)

12 2 0 0 0 2 (1.1)

13 1 0 0 0 1 (0.5)

14 2 0 0 0 2 (1.1)

a Reported frequencies for the major BKV genotypes were 46 to 82% for genotype

I, 0 to 6% for genotype II, 0 to 9% for genotype III, and 12 to 54% for genotype IV.

Genotype IV has the highest reported incidence in China and Mongolia, while

genotype I is the predominant strain in most other parts of the world. Subgroups of

genotype I may also have a predilection for specific geo-graphic regions. Thus,

Zheng et al. report incidence figures of 79% for genotype 1a in Africa, 64% for

genotype 1b in West Asia, and 100% for genotype 1c in northeast Asia (17).

b Out of 184 sequences.

tially similarly to prototype 1 in terms of the sensitivity of

detection. However, prototype 4, which corresponds to 6/53

(11%) known genotype IV strains, could not be detected in 2 of 3

replicates set up at concentrations of 1E2, i.e., 100 cop-ies/ l and

1E1, i.e., 10 copies/ l. Consistent detection (defined as detection

in 3 of 3 replicates) of all 14 BKV prototypes was possible only at

concentrations of 1E4, i.e., 10,000 copies/ l or higher. At

concentrations of 1E1, i.e., 10 copies/ l, the assay was able to

detect only 6 of 14 prototype sequences in all three replicates,

while lowering of the prototype concentration to 1E0, i.e., 1 copy/

l, reduced the performance of the assay to reproducible detection

of only 3 prototypes. Another observation of interest is that, compared to the

reference prototype (number 1), the measured viral load is

variably underestimated for all other prototypes (numbers 2 to

14). This effect is most pronounced at low concentrations. In

general, quantitation of the BKV load by real-time PCR de-pends

principally on two factors: (i) the linearity of the stan-dard curve

(prepared using the BKV Dun strain plasmid in our

J. CLIN. MICROBIOL. assay) and (ii) the amplification efficiency of the target se-quence.

Both these factors appear to contribute to the proto-type-specific

variations seen in our experimental system. The linear part of a

real-time PCR standard curve is characterized by a CT difference

of approximately 3.3 between serial 10-fold dilutions of the

plasmid standard. Using this yardstick, CT measurements were in

the linear range for BKV Dun proto-type 1 at all concentrations

up to 1E2, i.e., 100 copies/ l. All values outside the linear range

are indicated in boldface in Table 3. For 13 variant BKV strains

(prototypes 2 through 14, taken together), linearity was observed

only at concentrations up to 1E5, i.e., 100,000 copies/ l. The linear

range of the PCR assay for variant strains fell sharply at lower

concentrations: linear measurements were obtained for only 6/13

prototypes at concentrations 1E3, i.e., 1,000 copies/ l; 3/13

prototypes at concentrations 1E2, i.e., 100 copies/ l; and 0/13

prototypes at concentrations 1E1, i.e., 10 copies/ l. Amplification

effi-ciency was also significantly reduced for BKV variants

(proto-types 2 to 14) expressed as a percentage of the BKV Dun

reference (prototype 1), which was assumed to represent 100%

efficiency (Table 3). Thus, for prototype 2, the viral-DNA yield

was 13.31% of the expected value at 1E7copies/ l and 15.51% of

the expected value at 1E6, i.e., 1,000,000 copies/ l. For

measurements in the lower nonlinear part of the standard curve

(boldface in Table 3), the calculated yields were quite variable

and inaccurate, which explains why the mean of sev-eral

calculated results was 100% of input DNA.

Finally, the experiments conducted illustrate that the ampli-

fication efficiency is a function of the number of sequence

mismatches between the viral target and PCR assay primer/ probe

sequences. A sequence alignment of all prototype se-quences is

shown in Fig. 1, including the locations of all variant nucleotides

in relation to the PCR primers and probe. Table 3 enumerates the

mismatches between the target sequence and the forward primer

(F), reverse primer (R), and probe (P) sequences. Prototypes 3, 4,

10, and 11 had 2 or more mis-matches. Prototypes 2, 5, 12, 13,

and 14 had 1 mismatch with the primer sequences, while

prototypes 6, 7, 8, and 9 had 1 mismatch with the probe. For 4/13

BKV variants with 2 or more primer/probe mismatches, the

calculated viral loads were 0.57 to 3.26% of the expected values

(i.e., approximately 100-fold lower).Considering 5/13 BKV

variant prototypes with only 1 primer mismatch, 2/5 and 3/5,

respectively, yielded calculated target copy numbers

approximately 10- and 20-fold lower than the expected values.

Finally, for 4/13 BKV variants with only 1

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FIG. 1. Alignment of 14 prototype BKV sequences (lightface) with PCR primer and probe sequences (boldface and underlined). Identical nucleotides at

the same position are represented by dots.

VOL. 49, 2011 BK VIRUS ASSAYS 4075

TABLE 3. Comparative amplification efficiencies of prototypes 1 to 14

Prototype Calculated copy no. of prototypes 2–14

a No. of mismatchesb

1E7 1E6 1E5 1E4 1E3 1E2 1E1 1E0

F R P

1 100.00 100.00 100.00 100.00 100.00 100.00 100.00 100.00

2 13.31 15.51 17.07 16.89 39.59 63.4 308.45 120.28 1

3 1.06 0.71 0.63 1.22 12.55 57.61 190.14 48.89 2

4 2.47 1.10 1.00 3.26 7.09 23.07 77.32 136.94 2 1

5 14.74 10.53 9.70 9.36 21.94 47.48 87.32 44.07 1

6 76.66 91.13 59.54 90.81 130.73 116.85 291.55 64.72 1

7 84.51 88.87 83.89 162.56 212.90 139.92 136.48 ND 1

8 54.32 30.68 15.72 24.38 38.89 423.53 342.25 175.00 1

9 18.10 13.74 20.16 39.09 121.29 190.76 270.86 374.07 1

10 0.57 0.68 0.57 2.29 52.9 71.01 453.52 86.11 1 1

11 0.72 1.49 0.84 2.04 16.89 68.95 254.79 48.61 2

12 7.37 4.91 3.69 3.92 26.92 49.54 155.07 72.22 1

13 6.12 3.93 2.96 2.82 17.13 46.22 194.37 90.28 1

14 8.21 5.55 3.53 3.48 21.64 77.73 155.21 46.11 1

a Each BKV prototype sequence was set up at 8 different concentrations ranging from 1E0 to 1E7 target copies per l. The calculated copy numbers (means of three

independent determinations) for each prototype are expressed as percentages of prototype 1. The values in boldface are outside the linear range of the standard curve. Calculated values for prototypes set up at these low concentrations were extremely variable, and the mean often appeared to be greater than the expected values ( 100%). Additionally, detection of the target at these low copy numbers was not reproducible and frequently failed in 1 of 3 replicates (cells shaded in gray) or in 2 of 3 replicates (cells shaded in gray and underlined). ND, not determined.

b The number of mismatches between the target sequence and the forward primer (F), reverse primer (R), or probe (P) sequence.

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probe mismatch, underestimation of the target copy number was

less pronounced and observed deviation was within 5-fold of the

expected value.

DISCUSSION

Quantitative PCR is now widely used to monitor BKV in-

fection after kidney transplantation. Different laboratories em-

ploy different viral targets and primer sequences for amplifying

viral DNA. Assays are typically based on the BKV Dun strain or

similar reference sequences of genotype I. In the assay evaluated

here, primers and probes designed in this manner showed a

perfect match with only 87/184 (47.3%) and one or more

mismatches with 97/184 (52.7%) publicly available se-quences.

Mismatches were seen most often with genotype IV, followed by

genotypes 1c, 1b, II, and III. This rank order is consistent with the

known phylogenetic distances between dif-ferent viral strains.

To study the impact of genetic variability on BKV PCR, 14

prototypesequencesincorporatingrepresentativesingle-nucleo-tide

polymorphisms in 184 viral strains were synthesized. These

prototypes generally corresponded to specific genotypes, but with

occasional exceptions, which may represent more recent

mutational events occurring after the divergence of major ge-

notypes in the course of viral evolution. The ability of the

quantitative real-time PCR assay to accurately detect virus was

maximal for BKV genotypes 1a, 1b1, and 1b2. Sequences rep-

resented by prototype I could be consistently detected at all

concentrations up to 1E1, i.e., 10 copies/ l (i.e., 1E4, or 10,000

copies/ml), which is a threshold that has been frequently used for

reducing immunosuppression in patients with BK viremia or viral

nephropathy (4). For genotype IV, comparable detec-tion was

observed for sequences corresponding to prototype 3, but those

represented by prototype 4 were consistently ampli-fied only at a

concentration that was 2 log units higher. Geno-type II/III

sequences corresponding to prototype 5 could be detected in all

replicates only if the concentration was 1E2, i.e.,

100 copies/ l or higher. For genotype III sequences (prototype

sequence 11), the threshold for consistent detection was 1E4, i.e.,

10,000 copies/ l. The thresholds described are specific for the

assay used and could be altered by modifying the amplifi-cation

conditions. Nonetheless, the comparative data dramat-ically

illustrate the effect of the viral genotype on detection of viral

DNA. Clinical management of BKV infection depends not only on the

detection of virus, but also on estimation of the viral load in body

fluids. By definition, the amplification efficiency was 100% for

prototype 1, which represents 81 genotype 1a, 1b1, and 1b2

sequences showing a perfect match with PCR primer/ probe

sequences. However, as shown in one prior investigation (5),

quantitation of the viral load was markedly compromised for

variant BKV strains. Thus, at a DNA input of 1E7, or 10,000,000

copies/ l, the calculated viral load for genotype IV sequences was

1.06% of the expected value for sequences represented by

prototype 3 and 2.47% for prototype 4 (Table 3). For genotype II

and III sequences represented by prototype 5, amplification

efficiencies as low as 9.36% were observed. Quantitation of

genotype 1c sequences (prototype 6) was gen-erally not as

severely affected, with the exception of one se-quence from East

Asia (AB245326, prototype 14). Two geno-type 1a sequences,

AB276228 from South Africa (prototype 13) and AB268405 from

Japan (prototype 14), also showed poor amplification. In clinical

practice, changes in the viral load of 10-fold or higher are often

considered to be meaning-ful. Using this criterion, only the

variants corresponding to prototypes 6, 7, 8, and 9 gave

acceptable results, since the calculated copy numbers for other

prototypes were less than 10% of the expected values.

Underestimation of the target copy number was not markedly

dependent on the target con-centration per se, but marked assay

variability was observed for samples tested at the lower end of the

standard curve. It is notable that in several instances a single-nucleotide primer

mismatch with the target sequence substantially com-promised

detection by PCR. Mismatches at the level of the

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4076 RANDHAWA ET AL. probe sequence were less critical. Analogous findings have been

reported in the literature. Thus, single-nucleotide poly-morphisms

have been reported to not always be detrimental to assay

performance (5), while 2 mismatches at the 3 end of a PCR primer

can result in up to 2-log-unit differences in the calculated

cytomegalovirus load (9). Large numbers of mis-matches result in

complete lack of amplification of the viral target sequence (a

false-negative result) (15). Two previously published studies on the effect of BKV ge-netic

variation on assay performance deserve mention. Hoff-man et al.

used seven different primer/probe sets to perform PCR on urine

specimens and found substantial interassay dis-agreements that

were most striking for genotype III and IV strains (5). Expected

and observed DNA copy numbers varied by as much as 4.2 log10

templates per reaction. Notably, sig-nificant assay variation was

seen with primers directed at the BKV large T antigen, as well as

VP-1, while the agnogene was not evaluated. Dumoulin and

Hirsch found gene polymor-phisms in the target sequence of their

assay in 32%, 23%, and 82% of sequences corresponding to the

forward primer, re-verse primer, and probe, respectively (3). The

effects of these polymorphisms on amplification of genotype-

specific se-quences were not evaluated. Modification of the PCR

assay using primer or probe sequences with degeneracy at 4

nucle-otide positions was able to correct for the majority of the

observed sequence mismatches. However, no attempt was made to

provide coverage for more divergent variant BKV strains, which

constituted approximately 10% of published se-quences. The

modified PCR assay was found to be comparable to the original

PCR assay, but its performance with respect to detection and

accurate quantitation or uncommon BKV strains other than

genotype I was not evaluated.

In conclusion, our studies indicate that BKV quantitative PCR

assays designed using genotype 1 reference sequences, such as

BKV Dun, do not perform satisfactorily for mutant viral strains.

Detection sensitivity and amplification efficiencies are

particularly compromised for genotypes Ic, II, III, and IV. While

these genotypes are relatively uncommon in the United States,

genotype IV strains occur more frequently in the Far East.

Transplant physicians should suspect variant viral strains when

unexplained graft dysfunction occurs in the setting of low-level

BK viruria and absence of viremia. A high index of suspicion for

the presence of a variant strain should also arise in patients with

rejection-like infiltrates at biopsy that do not respond to steroid

treatment. Sensitive virus detection and accurate quantitation of

the viral load in such patients may be achieved by using alternate

PCR assays targeting a different

J. CLIN. MICROBIOL. viral gene, employing degenerate primers to account for vari-ant

sequences (3), or by using a multiplex approach that allows

simultaneous amplification of common variant strains (5).

ACKNOWLEDGMENTS

This study was supported by NIH grant RO1 AI 51227 to P.R. The

content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the National Institute of Allergy and Infectious Disease.

Jill March provided excellent secretarial assistance. We have no conflicts of interest.

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