JOURNAL OF CLINICAL MICROBIOLOGY, Nov. 2005, p. 55265535 0095-1137/05/$08.00 0 doi:10.1128/JCM.43.11.55265535.2005 Copyright 2005, American Society for Microbiology.

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Vol. 43, No. 11

Analysis of the Effect of DNA Purication on Detection of Human Papillomavirus in Oral Rinse Samples by PCR
Gypsyamber DSouza,1 Elizabeth Sugar,2 William Ruby,3 Patti Gravitt,1 and Maura Gillison2*
Department of Epidemiology, Johns Hopkins University, Baltimore, Maryland1; Department of Oncology, Johns Hopkins University, Baltimore, Maryland2; and Department of Medicine, Johns Hopkins University, Baltimore, Maryland3
Received 14 June 2005/Returned for modication 2 August 2005/Accepted 11 August 2005

Human papillomavirus (HPV) has recently been associated with oral cancers. To prepare for a study of the natural history of oral HPV infection, the effect of the DNA purication method on HPV genomic DNA detection in Scope mouthwash oral rinse samples and the reproducibility of HPV detection in rinse samples collected 7 days apart were investigated. The study was conducted with a population at high risk for oral HPV infection: human immunodeciency virus-infected men with CD4-cell counts <200. Five DNA purication methods were compared among equal aliquots of oral rinse samples collected from a subset of individuals. The purication methods included (i) proteinase K digestion (PKD) and heat inactivation; (ii) PKD and ethanol precipitation (EP); (iii) PKD, phenol-chloroform extraction, and EP; (iv) use of the Puregene DNA purication kit; and (v) use of the QIAamp DNA Blood Midi kit. HPV was detected by PCR amplication with PGMY09 and PGMY11 L1 primer pools and by use of a Roche linear array. Puregene-puried samples had higher human DNA yields and purities, and Puregene purication detected the greatest number of HPV-positive subjects and total HPV infections in comparison to the numbers detected by all other methods. The total number of HPV infections and HPV prevalence estimates were also higher for Puregene-processed oral rinse samples when a xed volume (10 l) rather than a xed cell number ( 50,000 cells) was used for PCR amplication. A good concordance was observed for oral HPV infection status (agreement, 80%; kappa value, 0.60) and type-specic infection (agreement, 98%; kappa value, 0.57) in matched oral rinse samples. The method of DNA purication signicantly affects the detection of HPV genomic DNA from oral rinse samples and may result in exposure misclassication that could contribute to the inconsistent associations reported in the literature.

Human papillomavirus (HPV), like alcohol and tobacco, has been established as an etiologic agent for a subset of head and neck squamous cell carcinomas (HNSCCs). These HPV-associated HNSCCs, which arise predominantly from the oropharynx, have distinctive clinical, histopathologic, molecular, and prognostic features compared to the features of HPV-negative tumors (6). High-risk oral HPV infection has been associated with risk of HNSCCs in case-control studies (27, 28). Furthermore, a temporal relationship between HPV exposure and the risk for HNSCCs is supported by the 14-fold increased risk for subsequent oropharyngeal cancer observed among HPV type 16 (HPV-16) L1-seropositive individuals (19). It is therefore logical to infer that HPV-associated HNSCCs are the consequence of oral HPV infection. However, little is know about the prevalence, risk factors, or natural history of oral HPV infection. This information is needed to evaluate whether there is the potential for the incorporation of HPV DNA testing into oral cancer screening programs. Clarication of the risks associated with oral HPV infection is hampered by the absence of a gold standard method for oral HPV detection. Prevalence estimates for oral HPV infection in individuals without oral cancer vary from 9.2% in a population-based study (26) to 4.8 to 18.3% (7, 14, 27, 28) in

* Corresponding author. Mailing address: Department of Oncology, Johns Hopkins University, Cancer Research Building, Room G91, 1650 Orleans St., Baltimore, MD 21231. Phone: (410) 502-7412. Fax: (410) 614-9334. E-mail: gillima@jhmi.edu. 5526

hospital-based controls. Differences in study populations and sampling, processing, and HPV detection methodologies likely contribute to this variability. Sampling by the use of oral rinses resulted in the highest prevalence estimate in a study that compared oral HPV detection from matched oral biopsy, rinse, and cytobrush samples collected from healthy individuals (17). Oral rinse sample collection and storage methods that improve DNA quality and yield have been evaluated for use in studies of the genetic factors associated with human disease (5, 13). Oral rinse collection with Scope mouthwash is preferred by study subjects and provides a high DNA yield (13). After collection, the method of DNA purication from oral rinse samples can further inuence the DNA yield and quality (5, 13). However, the inuence of DNA purication methods on HPV detection has not been evaluated. It is important to recognize that moderate levels of inhibition or total DNA yield may only subtly affect the ability to perform analyses targeting the human genome, as the total copy number of each human target will remain sufcient for accurate analysis. In contrast, viral nucleic acid concentrations are variable, depending on the extent of infection and shedding; and therefore, recognition of even modest compromises to PCR sensitivity become increasingly relevant. It is possible that misclassication of infected individuals as uninfected due to the sample processing methodology could bias associations toward the null and may explain why some studies have found a signicant association between oral HPV infection and HNSCC risk (27, 28), while others have not (14, 26). Clarication of the impact of sample

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FIG. 1. Flowchart of study and number of participants. Abbreviations: Prot. K, proteinase K; ETOH, ethanol.

processing on HPV detection is important for the accurate estimation of the prevalence of infection and the risks associated with oral HPV infection, as well as for the reproducible detection of oral HPV in longitudinal studies designed to evaluate the natural history of oral HPV infection. The principal objectives of this study were (i) to compare the effects of oral rinse processing methods on HPV detection and (ii) to assess the agreement of oral HPV infection status between two oral rinse samples collected 7 days apart.
MATERIALS AND METHODS Study population. Human immunodeciency virus (HIV)-infected individuals are at increased risk for both tonsillar cancer (4) and tonsillar HPV infection (16). In previous work, we determined that HIV-infected men with CD4-cell counts 200 were at the greatest risk for oral HPV infection (16), so this high-prevalence population was targeted. Eligibility criteria included male gender, CD4-cell count 200, attendance at the Johns Hopkins HIV (Moore) Clinic, age 18 years, willingness to return for repeat sample collection, and ability to provide informed consent. This protocol was approved by the Johns Hopkins Hospital Institutional Review Board. At the enrollment visit, information about demographics and lifetime and recent sexual behaviors was collected by use of a self-administered questionnaire. An oral rinse sample was collected by use of a 30-s rinse and gargle with 10 ml of Scope mouthwash and was stored at 4C until further processing (maximum, 48 h). The subjects were instructed to return for a second visit 7 days after enrollment. At the follow-up visit, a second oral rinse sample was collected, and a questionnaire about interval sexual behaviors was completed. Data for the most recent CD4-cell count, HIV viral load, and current use of antiretroviral medications was extracted from the medical record. As a control for specimen collection and processing, a 10-ml sample of the stock Scope mouthwash bottle was aliquoted after every 20th sample collected, sent to the laboratory, and processed with all other oral rinse samples. Study schema. The study schema is presented in Fig. 1. Equal aliquots from 20 randomly chosen oral rinse samples from visit 1 were used to compare HPV detection results obtained with ve different DNA purication methods (Fig. 1, left). Garcia-Closas and colleagues (5) previously compared the quantity and quality of human genomic DNA extracted from Scope oral rinse samples by use

of phenol-chloroform and the Puregene and QiaAmp kits. We therefore chose to evaluate the effects of these DNA extraction methods on HPV DNA detection from Scope oral rinse samples. Two additional methods, proteinase K digestion with and without ethanol precipitation, were also chosen for comparison because these methods are commonly used to extract DNA from cervical vaginal lavage specimens prior to HPV detection (15). To ensure comparability between the methods, equal aliquots of the oral rinse sample (1.5 ml) were processed by the different methods, described in detail below, and resuspended in a nal volume of 100 l. After each purication method, sample DNA quantity and purity were determined by spectrophotometry (Nanodrop Technologies Inc., Wilmington, DE), and the sample was stored at 80C until further analysis. A xed volume (10 l) of the processed oral rinse sample was used for PCR detection of HPV DNA. To evaluate the concordance between the results for matched oral rinse samples collected from the same individual 7 days apart, the oral rinse samples were prepared in two ways prior to PCR detection of HPV DNA (Fig. 1, right). One-tenth of the nal volume (10 of 100 l) of Puregene-puried oral rinse sample DNA was used for volume-standardized PCR detection. Based on the work of Smith and colleagues (27, 28), in which the oral epithelial cell number was quantied and standardized to 30,000 cells by hemocytometry, the number of human cells per microliter in Puregene-puried samples, as measured by a human endogenous retrovirus 3 (ERV-3)-specic real-time PCR, was used to standardize the concentration to 5,000 cells per microliter (see below). Ten microliters ( 50,000 cells per PCR) was then used for PCR detection of HPV DNA (referred to subsequently as cell number-standardized samples; Fig. 1). Oral rinse sample processing. The oral rinse specimens were transferred into a 15-ml tube and centrifuged at 3,000 g for 10 min at 4C. The supernatant was decanted, the pellet was resuspended in 10 ml phosphate-buffered saline (PBS), and centrifugation was repeated. PBS was chosen because of its compatibility with all subsequent DNA purication methods. The pellet was resuspended in 6 ml PBS with repeat pipetting and vortexing to ensure even sample distribution, immediately divided into four equal volume aliquots (1.5 ml each), and stored at 80C until further processing. DNA purication methods. The DNA purication methods are summarized in Table 1. Proteinase K digestion with and without ethanol precipitation. A crude proteinase K digestion was performed as recommended for cervical vaginal lavage specimens (15). Briey, a 1.5-ml aliquot of the oral rinse sample was centrifuged, resuspended in 100 l of 1 digestion buffer (50 mM Tris-HCl, pH 8.5, 1 mM

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TABLE 1. DNA purication schematic: summary of the steps in each of the ve DNA purication methods
Methoda Purication step(s) Puregeneb Phenol/ chloroform extraction QIAGENc Proteinase K digestion Proteinase K digestion with ethanol Purpose

Centrifugation Cell lysis RNAase Proteinase K digestion and heat inactivation Additional DNA purication Ethanol precipitation with glycogen Washing with 70% ethanol
a b

X X X X X X X
d

X X X X X X X
e

X X X X X
f

X X X

X X X X X

Pellets cells, separates from collection media Disrupts cellular membranes Degrades RNA Degrades cellular protein Removes protein/lipid component Precipitates DNA Washes DNA precipitate

X, purication step was carried out. The DNA purication from buccal cells in mouthwash kit (Puregene DNA purication kit; Gentra Systems) was used. c The QIAamp DNA Blood Midi kit (Qiagen Inc.) was used. d Additional DNA purication was done with Puregene precipitation solution. e Additional DNA purication was done by phenol/chloroform/isoamyl extraction for phase separation of nucleic acids. f Additional DNA purication was done with the QIAamp midi column.

EDTA, proteinase K at 0.4 mg/ml, 2% Laureth-12), and incubated for 60 min at 55C, followed by heat inactivation for 10 min at 95C. For proteinase K digestion with ethanol precipitation, half of the sample described above (50 l) was further processed by ethanol precipitation with glycogen (0.2 mg/ml; Roche Molecular Systems, Inc., Alameda, CA). The pellet was washed with 70% ethanol, dried, resuspended in 50 l of LoTE (3 mM Tris, 0.2 mM EDTA), and incubated for 1 h at 65C. Puregene. A 1.5-ml aliquot of the oral rinse sample was pelleted by centrifugation at 4,000 g for 10 min. The supernatant was decanted, and the pellet was resuspended in 1 ml Puregene cell lysis solution and incubated at 37C for 15 min. The sample was digested with DNase-free RNase A (5 g/ml) for 30 min at 37C. Proteinase K (20 mg/ml in diethyl pyrocarbonate-treated water; Sigma) was added to a nal concentration of 0.5 mg/ml; and digestion was performed overnight at 55C, followed by heat inactivation at 95C for 10 min. The sample was cooled to room temperature (RT). Protein precipitation solution (340 l) was added to each sample, and the sample was further processed as indicated by the manufacturers protocol for DNA purication from buccal cells in mouthwash (Puregene DNA purication kit; Gentra Systems, Minneapolis, MN). After the sample was air dried, 100 l of DNA hydration solution was added, followed by a 1-h incubation at 65C and an overnight incubation at RT. Phenol-chloroform extraction. A 1.5-ml aliquot of an oral rinse sample was thawed and centrifuged for 10 min at 4,000 g, and the supernatant was discarded. The pellet was resuspended in 250 l of digestion buffer (50 mM Tris-HCl, EDTA, pH 8.5) and incubated for 10 min at RT, followed by RNase A (5 g/ml) digestion for 30 min at 37C. Proteinase K (0.5 mg/ml) digestion was performed overnight at 55C, and heat inactivation was performed for 10 min at 95C. Extraction with phenol-chloroform and isoamyl alcohol (Invitrogen, Carlsbad, CA) was performed until the protein precipitate was clear, followed by ethanol precipitation with glycogen (0.2 mg/ml; Roche Molecular Systems, Inc.). The pellet was washed with 70% ethanol, dried, resuspended in 100 l of LoTE (3 mM Tris, 0.2 mM EDTA), and incubated at 65C for 1 h. Use of QIAamp DNA Blood Midi kit. DNA purication was performed with the fourth aliquot by use of the QIAamp DNA Blood Midi kit (QIAGEN Inc., Valencia, CA). In brief, a 1.5-ml aliquot of the oral rinse sample was centrifuged at 4,000 g for 10 min. The pellet was resuspended in 2.0 ml of PBS, transferred to a 15-ml screw-cap tube, and equilibrated to RT. RNase A (5 g/ml) digestion was performed at 37C for 30 min, followed by proteinase K digestion (0.5 mg/ml) in Qiagen Buffer AL at 56C overnight. The sample was loaded into a QIAamp Midi column, washed, and eluted as recommended by the manufacturer, followed by ethanol precipitation in the presence of glycogen (0.2 mg/ml). The pellet was washed with 70% ethanol, dried at 37C, resuspended in 100 l of LoTE, and then incubated at 65C for 1 h and overnight at RT. ERV-3 (human cell number) quantitation. The total DNA concentration cannot be used to measure the amount of human DNA in oral rinse samples because of intersample variability in the sources of nonhuman DNA (e.g., bacteria) in specimens. Therefore, human diploid cell genome equivalents (referred to subsequently as human cell number) in oral rinse specimens were quantied by

use of a TaqMan real-time PCR targeting a single copy human gene on chromosome 7, ERV-3 (30). The ERV-3-specic primer and probe sequences used were those published previously (30). The protocol was optimized for hot-start real-time PCR, as described previously (16). The number of human cells, as measured by ERV-3 diploid genome equivalents per l, was used to (i) screen the oral rinse samples for PCR inhibition in a series of 10-fold sample dilutions, (ii) estimate the human cell number per l in the sample, (iii) standardize the oral rinse samples to a concentration to 3,000 to 5,000 cells per l prior to HPV detection (subsequently referred to as cell number standardized), and (iv) normalize the HPV-16 and HPV-18 viral loads to the human cell number. HPV detection: line blot assay. HPV DNA was detected in oral rinse samples by multiplex PCR targeted to the conserved L1 region of the viral genome by use of PGMY09 and PGMY11 L1 primer pools (11). The PCR products were denatured in 0.13 N NaOH and hybridized to an HPV probe array for genotyping of 38 HPV types classied as high risk or probable high risk or low or unknown risk and -globin (Roche Molecular Systems, Inc.) (12, 24). Positive controls, consisting of 10 and 100 cells positive for HPV-16 (SiHa cells) or HPV-18 (C4-2 cells) diluted in a background of HPV-negative cells (K562 cells), and a negative control (K562 cells) were included in each experiment. Samples positive for -globin were considered for analysis. Samples were reported as negative or positive for HPV DNA, and the HPV type specication was reported for positive samples. HPV-16 and HPV-18 viral load quantitation. The viral load in 10 l of the Puregene-puried oral rinse sample was determined as described previously by use of real-time TaqMan PCR methods (10). The following modications have been made to the published protocol: (i) a standard curve was created by vefold serial dilution (5 104 to 1.6 101) of a plasmid (pGEM containing HPV-16 or HPV-18) containing the HPV genome in a background of 50 ng/ l human placental DNA. (ii) We found that a standard curve generated by use of supercoiled plasmid results in a 10-fold underestimation of the HPV viral load, necessitating linearization of the plasmid control (e.g., by BamHI restriction for HPV-16 and EcoRI digestion for HPV-18). Therefore, DNA was added to a 50- l reaction mixture containing 1 U of restriction enzyme, and a 30-min incubation at 37C was performed prior to AmpliTaq activation in the amplication cycle. The samples were amplied in an ABI 5700 apparatus, an ABI 7300 apparatus, or a Bio-Rad iCycler. The cycle threshold (CT) of unknown samples was determined from an equation derived from a linear regression through the log CT of the standard curve. Samples with one viral copy or more were considered positive. The viral load quantity was reported for positive samples. Statistical analysis. The HPV DNA detection results reported are stratied by standardization method (cell number versus volume), visit (rinse samples 1 and 2), and HPV type classication (high and low risk) (21). Individuals coinfected by high- and low-risk types were included in each category. HPV DNA prevalence estimates and exact 95% condence intervals (95% CIs) were calculated for visits 1 and 2 for both volume- and cell number-standardized samples. For nonnormally distributed, continuous variables or for samples of small size, medians and interquartile ranges (IQRs) were determined. Median measures of DNA quality and quantity for the different methods of DNA purication were compared by

VOL. 43, 2005 TABLE 2. Descriptive characteristics of study populationa

Characteristic No. (%) of individuals

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Male gender................................................................................109 (100) Currently on HAART ............................................................... 82 (75) Race African American................................................................... 94 (87) Caucasian ................................................................................ 14 (13) Lifetime no. of female sexual partners 0................................................................................................ 15............................................................................................ 5 ............................................................................................ Unknown ................................................................................. Lifetime no. of male sexual partners 0................................................................................................ 15............................................................................................ 5 ............................................................................................ Unknown ................................................................................. 7 (6) 24 (22) 77 (71) 1 (1) 73 (68) 9 (8) 25 (23) 2 (2)

cordant HPV results between visit 1 and visit 2 were compared to those of individuals with concordant positive and negative results by use of the Fishers exact test for categorical variables and the Wilcoxon rank-sum test for continuous variables. All P values reported are two sided and considered statistically signicant at a P value 0.05. Intercooled Stata, version 8.2 (Stata, College Station, TX), was used for all statistical analyses.

RESULTS A total of 109 subjects were enrolled in the study, and 100 subjects returned for the second visit a median of 7 days (range, 3 to 34 days) later. The characteristics of all 109 enrolled subjects previously demonstrated to affect the risk of oral HPV infection (16) are displayed in Table 2. The median numbers of days between the most recent CD4-cell count determination and enrollment and HIV viral load determination and enrollment were 42 days (IQR, 23 to 77 days) and 56 days (IQR, 24 to 113 days), respectively. Oral rinse digestion. Twenty of the 109 oral rinse samples collected at enrollment were randomly selected for a study of the effect of the sample processing method on the prevalence of oral HPV in samples (Table 1). In previous studies by our group (16) and others (25, 29), oral rinse samples were found to contain PCR inhibitors. When an aliquot of the sample was processed by crude digestion with proteinase K (see Materials and Methods), as commonly performed in studies of cervical HPV infection (8), 50% (9 of 18) of the samples were -globin negative (Table 3). Quantication of the cell number by use of an ERV-3-specic TaqMan PCR in a 10-fold dilution series of proteinase K-digested samples conrmed complete PCR inhibition with an input volume of 10 l of sample (Fig. 2). Stepwise modications to the oral rinse digestion procedure were performed in an attempt to remove PCR inhibitors from the oral rinse samples and were evaluated by use of ERV-3 quantication in a 10-fold dilution series. There was substantial intersample variability in the cause of inhibition, including the protein, RNA, and total nucleic acid contents (data not shown). The following protocol was determined to be optimal

Sexually active in the past 30 days........................................... 38 (35) Performed oral-anal or oral-genital sex in prior 30 days...... 18 (17)
a The study population comprised 109 individuals. The median age was 46 years (IQR, 42 to 50 years), the median CD4-cell count was 86 (IQR, 25 to 152), and the median HIV viral load was 19,265 (IQR, 224 to 198,800). IQR reports the 25th and 75th percentiles and is a measure of data variability.

use of the Wilcoxon signed-rank test, and HPV DNA detection results were compared by use of McNemars test. The kappa statistic was used to compare the agreement of HPV infection status (infected or uninfected), HPV type classication (high- and low-risk types), and HPV type-specic infection between visits. Agreement for type-specic infections was calculated by creating binary variables of each possible infection and including the nonoccurrence in each individual as (0, 0). This allowed calculation of the number of infections gained based on the set of possible infections and prevented the unequal weighting of individuals based upon the number of infections. Inclusion of each type-specic nonoccurring infection results in high agreement, but the kappa statistic accounts for the low rate of type-specic occurrence. The characteristics of individuals with dis-

TABLE 3. DNA quality, quantity, and HPV detection results obtained from matched oral rinse samples treated with ve different DNA purication methods
Spectrophotometry Method of DNA purication No. of samples testeda DNA purity (A260 A280) Medianb IQRi Total nucleic acid (ng) per PCRc Medianb IQR ERV-3 j TaqMan PCR No. of human genome copies per PCRc Medianb (103) IQR HPV detection by line blotc

No. globin positived

No. HPV positived

Total no. of HPV infections detected

Puregene Phenol-chloroform QIAGEN Proteinase K-ethanol precipitation Proteinase K digestion

a b c

20 20 20 18 18

1.91 1.58 e 1.81 h 1.62 e 1.86 h

1.871.96 1.521.62 1.732.01 1.441.70 1.632.22

407 547 h 117 e 596 f 2,002 e

312900 447752 8176 3651,110 1,5113,658

199 76.3 e 35.2 e 94.2 f 10.6 e

91462 50211 1476 28191 253

20 of 20 13 of 20 20 of 20 h 7 of 20 g 20 of 20 h 5 of 20 e 17 of 18 h 7 of 18 g 9 of 18 f 2 of 18 e

19 12 9 10 3

Only 18 of 20 samples were available for proteinase K digestion and proteinase K digestion followed by ethanol precipitation. P values were determined by Wilcoxons sign-rank test and are for comparison to Puregene data. A xed volume of 10 l of each sample was added to each PCR mixture. d P values were determined by McNemars matched-pair test and are for comparison to Puregene data. e P 0.001. f 0.001 P 0.01. g 0.01 P 0.05. h P 0.05. i IQR reports the 25th and 75th percentiles and is a measure of data variability. j ERV-3 is a single-copy human gene used to measure the number of human diploid genome equivalents.

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FIG. 2. Tenfold serial dilution series comparing the number of human genome diploid equivalents (measured by human endogenous retrovirus 3, ERV-3, copies) in paired proteinase K-digested (solid lines, squares) and Puregene-processed (dotted lines, triangles) samples. The results for 8 representative samples (of 20) are shown.

for the removal of inhibitors prior to further DNA purication (data not shown): (i) cell lysis in digestion buffer (50 mM Tris-HCl, EDTA, pH 8.5) for 10 min at 37C; (ii) RNase A (5 g/ml) digestion (DNase-free RNase; Roche Molecular Systems, Inc.) for 30 min at 37C; (iii) proteinase K digestion (0.5 mg/ml; Sigma) overnight at 55C; and (iv) heat inactivation for 10 min at 95C. This digestion procedure was therefore incorporated into the Puregene, phenol-chloroform, and QIAGEN procedures described above in the Materials and Methods section. Comparison of methods of purication of DNA from digested oral rinse specimens. The median values for total nucleic acid (ng) and human cell number (per ERV-3 TaqMan PCR) input into the amplication reaction (in a xed volume of 10 l) for matched samples prepared by ve DNA purication methods are compared in Table 3. DNA purity, as measured by spectrophotometry, is also presented. DNA purity and both total nucleic acid and human DNA yields varied signicantly as a function of the DNA purication method. Puregene provided signicantly higher human cell yields than any of the other methods. Although proteinase K digestion was the only method with signicantly fewer -globin-positive samples compared to the number obtained by the Puregene method, the number of individuals who were found to have an oral HPV infection was signicantly higher by Puregene purication than by any of the other methods (Table 3). We therefore prepared a 10-fold dilution series of Puregene-puried samples and compared them to the matched dilution series of crudely digested samples (Fig. 2). DNA purication by use of Puregene resulted in the expected linear relationship between each serial dilution and human cell copy number in the sample, indicating the successful removal of PCR inhibitors. Therefore, for the second component of this study, all oral rinse samples were puried by use of the Puregene method prior to HPV detection. Line blot detection of oral HPV in samples. All oral rinse samples from visit 1 and visit 2 that were cell number standardized were positive for -globin (n 100 pairs). All Scope mouthwash stock bottle control samples were -globin and

HPV negative. Three samples from visit 1 were exhausted prior to volume-standardized analysis (due to their use for purication analysis detailed above), six samples were -globin negative, and two samples were weakly -globin positive. Therefore, analysis of HPV detection results and concordance between rinses 1 and 2 is restricted to 89 pairs (Fig. 1). The HPV type distribution detected in 89 matched pairs of oral rinse samples is displayed in Table 4, stratied by visit and standardization method. The majority of infections were classied as high-risk types (68 to 74%). HPV-16, the most relevant type with regard to oral cancer pathogenesis, was the most common HPV type detected in the oral rinse, regardless of the visit or the standardization method. The number of HPV infections detected in volume-standardized samples was greater than the number detected in cell number-standardized samples (rinse 1, 74 and 62, respectively; rinse 2, 97, and 73, respectively). The oral HPV prevalence in the study population ranged from 44 to 52%, depending on the visit, and the estimates tended to be higher in volume-standardized samples (Table 5). Concurrent infection by more than one HPV type was common in the study population. For volume-standardized samples, 18 of 44 (41%; 95% CI, 26 to 57) and 25 of 46 (54%; 95% CI, 39 to 69) HPV-positive individuals had multiple infections (two to eight) detected on visit 1 and visit 2, respectively. For cell number-standardized samples, multiple infections (two to seven) were detected in 17 of 39 (44%; 95% CI, 28 to 60) individuals with an oral HPV infection detected on visit 1 and 20 of 40 (50%; 95% CI, 34 to 66) individuals on visit 2. Concordance of oral HPV measures between visits 1 week apart. Measures of agreement between HPV detection results at visit 1 and visit 2 are displayed in Table 5. This analysis is restricted to the 89 pairs of both the volume- and the cell number-standardized samples that were evaluable. Agreement for both overall HPV status (with or without at least one oral HPV infection) and type-specic HPV detection were considered. In the analysis of volume-standardized samples, 36 of the 89 individuals had an oral HPV infection detected at both visits, 35 were consistently negative at both visits, and 18 were

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TABLE 4. HPV type distribution in matched oral rinse sample pairs from visit 1 and visit 2 stratied by standardization methoda
No. of samples HPV type Visit 1 Cell no. standardized Visit 2 Visit 1 Vol standardized Visit 2

High-risk HPV 16 18 26 31 33 35 39 45 51 52 53 56 58 59 66 67 68 69 73 82 83 No. high-risk HPVs (% of total, 95% CI) Low-risk HPV 6 11 54 55 61 62 64 70 71 72 81 84 89 No. low-risk HPVs (% of total, 95% CI) Total no. of HPV infections
a

9 4 1 0 1 7 0 4 2 0 0 4 1 2 0 1 2 1 2 0 4 45 (73, 6083)

8 4 2 0 1 6 1 4 2 0 1 3 2 2 1 2 1 2 2 1 5 50 (68, 5779)

10 6 1 0 1 8 0 5 2 0 0 6 2 2 0 2 2 1 2 0 5 55 (74, 6384)

11 5 3 1 1 8 2 6 2 3 2 5 2 3 1 3 1 3 2 1 5 70 (72, 6281)

1 0 0 5 1 1 0 0 1 2 5 1 0 17 (27, 1740) 62

3 2 1 3 5 1 0 0 2 2 3 0 1 23 (32, 2143) 73

2 1 1 5 3 1 0 0 0 2 3 1 0 19 (26, 1637) 74

3 2 1 3 5 1 2 1 2 2 3 2 0 27 (28, 1938) 97

Data are presented for the 89 individuals with complete cell number- and volume-standardized data.

categorized differently at each visit (8 were positive only at the rst visit; 10 were positive only at the second visit). Men who had an oral HPV infection at the rst visit were also likely to have an infection at the second visit (agreement, 80%; kappa value, 0.60). Agreement between visits was good for individuals with a high-risk HPV infection (agreement, 80%; kappa value, 0.58) or a low-risk HPV infection (agreement, 84%; kappa value, 0.53). Measures of agreement between visit 1 and visit 2 were similar for cell number-standardized samples (Table 5). There were a total of 171 type-specic infections detected in the 89 evaluated volume-standardized oral rinse samples from visits 1 and 2 (Table 5). This included 51 concordant typespecic HPV infections detected at both visits, 23 type-specic infections detected only at visit 1, 46 infections detected only at visit 2, and 40 concordant negative oral rinse sample pairs. The percent agreement was very high for both cell number- and

volume-standardized samples, largely because of a high number of concordant negative samples. Type-specic concordance was good for high-risk infections (kappa value, 0.59) and lowrisk infections (kappa value, 0.51) in volume-standardized samples, with similar concordances in cell number-standardized samples (Table 5). There was very strong agreement for both HPV status and type-specic infection when the results for volume-standardized and cell number-standardized samples were compared (Table 5). HPV-16 and HPV-18 viral load quantitation in samples standardized to human cell number. Quantitative PCR for HPV types 16 and 18 was performed only with Puregenepuried samples standardized to human cell number. One or more copies of HPV-16 and HPV-18 were detected in 9.6% (20 of 209) and 9.1% (19 of 209) of the samples, respectively. The viral loads for HPV-16-infected samples ranged from 3 to

5532 DSOUZA ET AL.

TABLE 5. Measures of agreement in HPV detection results between oral rinse samples collected at visit 1 and visit 2, stratied by standardization method
Vol standardized (89 pairs) Measures of agreement Visit 1 Visit 2 Kappa (95% CI)
d d

Cell no. standardized (89 pairs) Measures of agreement Visit 2 Kappa (95% CI)
d

Cell no. versus vol standardized measures of agreement Visit 1 (89 pairs) % Agreement (95% CI) Visit 2 (89 pairs) % Agreement (95% CI) Kappa (95% CI)

Visit 1

% Agreement (95% CI)

% Agreement (95% CI)

Kappa (95% CI)

Oral HPV infection status by individual HPV negative 50 (56, 4567) 48 (54, 4365) 80 (6988) 0.59 (0.410.77) 39 (44, 4355) 41 (46, 3557) HPV positivea High-risk HPV 31 (35, 2546) 32 (36, 2647) 85 (7692) 0.68 (0.520.84) Low-risk HPV 15 (17, 926) 19 (21, 1331) 84 (7592) 0.49 (0.250.73) 45 (51, 3961) 44 (49, 3960) 33 (37, 2748) 18 (20, 1230) 80 (6988) 84 (7592) 43 (48, 3759) 46 (52, 4162) 37 (42, 3153) 20 (22, 1433) 80 (6988)

0.60 (0.420.78) 90 (8196) 0.58 (0.400.76) 93 (8698) 0.53 (0.310.75) 92 (8497)

0.80 (0.680.92) 94 (8799) 0.85 (0.730.97) 94 (8799) 0.74 (0.560.92) 94 (8799)

0.89 (0.810.97) 0.88 (0.780.98) 0.83 (0.690.97)

Type-specic infections detected (no.) High-risk HPV Low-risk HPV Total HPVs detected 50 23 73 98 (9799) 0.64 (0.520.76) 98 (9799) 0.59 (0.310.67) 98 (9799) 0.62 (0.520.72) 55 19 74 70 27 97 97 (9698) 98 (9799) 98 (9799)

45 17 62

0.59 (0.490.69) 99 (98100) 0.83 (0.750.91) 99 (98100) 0.79 (0.710.87) 0.51 (0.330.69) 99 (98100) 0.77 (0.610.93) 99 (98100) 0.80 (0.680.92) 0.57 (0.470.67) 99 (98100) 0.82 (0.740.90) 99 (98100) 0.79 (0.730.85)

Human cell no. per PCR Medianb Interquartile range c 127,523 216,500 58,400251,000 67,000485,000

48,100 47,580 37,99250,265 22,73550,400

0.81; for rinse 1 and rinse 2 for volume-standardized samples, P 0.88 in cell number-standardized samples and P

0.64; for rinse 1, cell number versus volume standardized, 0.0008 in volume-standardized samples.

By McNemars test, for rinse 1 and rinse 2 for cell number-standardized samples, P 0.18; for rinse 2, cell number versus volume standardized, P 0.03. b Wilcoxon signed-rank test comparing ERV-3 in rinse 1 versus ERV-3 in rinse 2, P c IQR reports the 25th and 75th percentiles and is a measure of data variability. d Unless otherwise indicated, values are numbers of samples (percent, 95% CI).

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1,430,000 copies per 10 l of sample, and those for HPV-18infected samples ranged from 1.4 to 138,384 copies per 10 l of sample. Three samples positive for HPV-16 by quantitation were negative by the line blot assay, and each had 80 viral copies (range, 3 to 79 viral copies). Nine samples positive for HPV-18 by quantitation were negative by the line blot assay. Six of the nine samples had viral loads 50 copies; three samples had between 780 and 1,819 copies, but all had two or more concurrent HPV infections detected on line blotting (the presence of infections with multiple types is known to decrease the sensitivity of the multiplex assay). Seven of 20 HPV-16infected individuals and 2 of 19 HPV-18-infected individuals had more than one viral copy per human cell analyzed. The characteristics of the individuals who were either consistently positive or consistently negative for oral HPV infection were compared to those of individuals with discordant results to explore the results for possible explanations for the discordance. Discordant results could not be accounted for by age, interval sexual behaviors, alcohol or tobacco use, time interval between visits, or differences in human cell numbers analyzed for HPV genomic DNA. However, the HPV viral loads in oral samples from individuals with concordant positive HPV-16 and HPV-18 results (median HPV viral load, 4,384 copies per 10 l) were signicantly higher than those in oral samples from individuals with discordant results (median 965 copies per 10 l; Wilcoxon rank-sum test, P 0.06). The systemic HIV viral load also tended to be higher among individuals with concordant results (median 35,866 copies) than among individuals with discordant results (median 400 copies; Wilcoxon rank-sum test, P 0.03). DISCUSSION Our data suggest that the method of DNA purication from oral rinse samples has a potentially large impact upon the ability to detect HPV genomic DNA in these samples by PCR amplication. Our data further suggest that amplication of a human gene present in high copy number, such as -globin, may not be appropriate for categorization of the sample as adequate for detection of small-copy-number nonhuman sequences. This conclusion is supported by our observation that samples processed by some of the methods were uniformly -globin positive, and yet up to half of the oral HPV infections still failed to be detected (Table 3). Puregene purication preceded by thorough RNA and protein digestion resulted in the detection of the greatest numbers of HPV-positive subjects and HPV infections in matched samples compared to the numbers detected by all other methods evaluated. The inability to detect HPV genomic DNA in proteinase K-digested samples could be attributed to the presence of PCR inhibitors in the oral rinse samples, as previously observed by our group (16) and others (25, 29). Stepwise modications to the digestion procedure revealed that the cause of PCR inhibition from sample to sample was quite heterogeneous and could include total nucleic acid overload, protein inhibition, and specic inhibition by RNA. We recognize that ERV-3 assay-based estimates may be inaccurate in the presence of PCR inhibitors because this is also a PCR-based assay. However, the use of serial 10-fold dilutions overcame this potential problem and allowed evaluation of the effects of

DNA digestion and the purication procedures on DNA amplication. It would appear that PCR inhibition is a larger problem in oral samples than samples from other anatomic sites prone to HPV infection. However, some studies report an inability to amplify -globin from more than 5% of cervical swab samples (20, 23) and more than 15% of anal swab samples (3, 22), suggesting that PCR inhibition may play a role in the amplication of HPV DNA from samples from other anatomic sites. We have shown than DNA purication can eliminate this PCR inhibition. The relative loss of human DNA during purication (e.g., purication with the QIAGEN kit and phenol-chloroform) also appeared to affect the ability to detect HPV genomic DNA in oral samples. Puregene provided high DNA purity while preserving human DNA yield, factors which likely affected our improved results with Puregene. Our data indicated that a signicant relative loss of human DNA (QIAGEN kit or phenol-chloroform versus Puregene) did not affect the detection of a high-copy-number human gene ( -globin) but did signicantly affect HPV detection (Table 3). We acknowledge that other DNA purication methods not evaluated as part of this study design may be equally successful in reducing PCR inhibition and in detecting oral HPV infection. Furthermore, we did not evaluate the effects of the DNA purication methods on the detection of HPV in samples from other anatomic sites, such as cervical swab samples. The variability in oral HPV detection observed with different DNA purication methods in this study suggests that evaluation of the effect of the DNA purication method on HPV DNA detection in samples taken from other anatomic sites may be warranted. This research has direct relevance to the interpretation of existing and future studies, suggesting that HPV prevalence may be underestimated in studies that report results for unpuried oral exfoliate samples. According to our data, oral HPV studies that performed no further DNA purication after protein removal (1, 7, 14, 18) may have underestimated the oral HPV prevalence by as much as sixfold, while studies that used only ethanol precipitation or phenol-chloroform extraction (17, 2628) may have underestimated the prevalence by 40 to 75%. Our data underscore the importance of DNA purication to avoid the misclassication of HPV status (false-negative results) in oral exfoliate samples and suggest that misclassication is highly dependent on the purication method used. While this research focused on the effect of optimization of laboratory assays for HPV DNA detection, misclassication may also occur due to factors that affect sampling in the oral region; potential sources of sampling variation include behavioral factors, such as tooth brushing; clinical factors, such as treatment for cancer; procedural differences, such as use of a swab compared to use of a rinse and whether a gargle is done; and variation in viral shedding (which may differ in cases and controls). This study was intentionally performed with HIV-positive men with CD4-cell counts 200 because the high oral HPV infection prevalence in this population increased the power of the study to detect differences in HPV detection between methods. We acknowledge that this study did not include women or immunocompetent subjects. However, the effect of DNA purication method may be equally important and potentially more important in these populations. In this

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study, the HPV-16 and the HPV-18 viral loads were associated with the ability to detect infection at both visits. The improved sensitivity of the assay may therefore be more important in immunocompetent individuals who may have lower oral HPV viral loads. Misclassication of oral HPV exposure (false-negative results) due to nonoptimal DNA processing may also contribute to underestimates of the cancer risk associated with oral HPV infection. Two of the three studies of oral HPV infection predicted to have the least DNA extraction-related misclassication based on the data explored above reported increased odds of HNSCCs in individuals with oral HPV (27, 27), while the study that did not nd a relationship used posttreatment cases (26) (see below). The other widely cited study that did not report an increased oral cancer risk used purication methods subject to more inhibition (14). Whether DNA puricationrelated misclassication of oral HPV exposure in cases and controls is nondifferential has not been evaluated but would bias the estimation of cancer risk toward the null. There are currently no data comparing HPV viral loads in oral exfoliates of cases and controls, so the direction of any possible differential misclassication is unknown. Two widely cited studies of oral HPV infection (18, 26) used posttreatment cases, the results for which may be more likely than those for controls to be misclassied as false negative due to lower viral loads as a result of therapy; this could underestimate the risk of oral HPV infection. We chose to amplify target HPV genomic DNA with PGMY primer pools because of their relatively equal amplication sensitivities across HPV types, an increased type-specic HPV prevalence (11), and an improved ability to detect multiple concurrent infections (11) commonly observed in the oral cavity. We recognize, however, that we may have underestimated the frequency of oral HPV infection in the study population by limiting detection to the HPV types present on the Roche line blot assay. Although there is substantial heterogeneity in primer choice for PCR amplication, most molecular epidemiological studies of HPV infection rely on PCR amplication of HPV genomic DNA. Therefore, we believe that our results could extend to any HPV detection method that relies on PCR amplication of the target. There were some samples that were positive for HPV-16 and HPV-18 by quantitative PCR that were negative for HPV-16 or HPV-18 by line blot analysis. The majority of these samples had viral loads less than 50 copies per PCR mixture, consistent with the expected lower sensitivity of consensus compared to that of type-specic PCR. However, there were some samples with viral loads as high as 1,800 copies that were negative by line blot assays. In every case, the sample was also positive for two or more other HPV types. This is likely due to outcompetition for primers by other HPV types with higher viral loads in the sample: the presence of infections with multiple types is known to decrease multiplex assay sensitivity. In our study design, cell number-standardized samples were created from dilutions of the stock Puregene samples that were used for both ERV-3 quantitation and volume standardization. Analysis of volume-standardized samples was performed last. Eight of 197 ( 4%) samples available for volume-standardized analysis were -globin negative on line blotting, whereas all of these samples were previously -globin positive on cell num-

ber-standardized analyses, and all performed well in the ERV-3 amplication. Because of the nature of the study design, the samples had undergone repeated manipulation, and it is likely that repeated freezing-thawing of the samples may have caused some DNA degradation. We observed very high rates of agreement between HPV detection results using volume-standardized versus cell number-standardized samples that were comparable to the 91% overall agreement in HPV infection status observed with cervical samples collected by different methods on the same day (9) as well as the same cervical samples tested on different days (2). The strong agreement with repeat testing of the same samples provides evidence that the variability observed between visits is not due to limitations in the reproducibility of the HPV detection assay. By contrast, the lower concordance observed for oral exfoliate samples collected 1 week apart either may be ascribed to sampling differences on the 2 days (e.g., the time since the subject last ate or brushed his teeth) or may indicate that oral HPV infection is quite dynamic in this immunocompromised patient population. HPV DNA detection at all sites is subject to variability in sampling and viral load, and no method that reproducibly detect HPV infection all of the time exists. We conclude that the optimized oral rinse method described above can reproducibly categorize an individual as HPV infected and is capable of reproducibly detecting type-specic oral HPV infections. Longitudinal studies sampling the oral cavity at short intervals are now needed to determine the incidence and clearance of oral HPV infection and how frequently sampling is required to monitor the natural history of these infections. Our data show an increased consistency in HPV detection in patients with higher HPV viral loads because of problems with the reproducibility of the assay at the lower limit of detection (21). Individuals with discordant HPV status were also more likely to have low HIV viral loads, suggesting that high systemic HIV viral loads may result in higher oral HPV viral loads in those who are infected. This association was independent of the CD4-cell count, suggesting that a high systemic HIV viral load may either directly or indirectly effect local oral mucosal immunity.
ACKNOWLEDGMENTS We thank Janet Kornegay (Roche Molecular Systems, Pleasanton, CA) for generously providing the line blot reagents and strips. This work was supported by a grant to M.G. from the Damon Runyon Cancer Research Foundation.
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