MAJOR ARTICLE

Oral Human Papillomavirus Infection in Adults Is

Associated with Sexual Behavior and HIV Serostatus
Aimee R. Kreimer,1,a Anthony J. Alberg,1 Richard Daniel,2 Patti E. Gravitt,1 Rapheal Viscidi,3 Elizabeth S. Garrett,4

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Keerti V. Shah,2 and Maura L. Gillison5
Departments of 1Epidemiology and 2Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 3Department
of Pediatrics, Johns Hopkins School of Medicine, and Departments of 4Biostatistics and 5Oncology, Sidney Kimmel Comprehensive Cancer Center
at Johns Hopkins, Baltimore, Maryland

The prevalence and risk factors for oral human papillomavirus (HPV) infection are unknown, despite evidence
for an etiological role for HPV in oral cancers. Oral samples from human immunodeficiency virus (HIV)–
seronegative (n p 396) and HIV-seropositive (n p 190 ) adults were tested for HPV DNA. High-risk HPV
infections were present in 2.1% of tonsil and 6.3% of oral-rinse specimens. The prevalence of oral high-risk
HPV infection was greater in HIV-seropositive individuals (13.7% vs. 4.5%; P ! .001 ). In multiple logistic
regression, odds of oral HPV infection increased with age, male sex, and herpes simplex virus (HSV)–2
seropositivity in HIV-seronegative individuals and with CD4 cell count !200 cells/mL, HSV-2 seropositivity,
oral mucosal abnormalities, and 11 oral sex partner during the previous year (odds ratio, 12.8; 95% confidence
interval, 3.1–52.7) among HIV-seropositive individuals. HPV type 16, which is present in most HPV-associated
tonsillar cancers, was the most prevalent high-risk oral HPV infection.

Human papillomavirus (HPV) is etiologically associ-
ated with a subset of head and neck squamous-cell
carcinomas (HNSCC), particularly those that arise from
the lingual and palatine tonsils [1–6]. However, the
prevalence of oral HPV infection in individuals without
oral cancer has been uncertain, and the risk factors for
and natural history of oral infection are largely unex-
plored. Estimates of oral HPV prevalence are highly
variable [7]. Because prevalence estimates in many
studies have been reported without concomitant analy-
ses of demographic and behavioral data, it is difficult
to discern whether such variability reflects methodo-
Received 25 June 2003; accepted 26 August 2003; electronically published 2
February 2004.
Presented in part: 20th International Papillomavirus Conference, Paris, October
2002, (abstract O007).
Financial support: National Institute of Dental and Craniofacial Research (grant
DE13121); State of Maryland Cigarette Restitution Fund (to M.L.G.); Damon Runyon
Cancer Research Foundation (Damon Runyon-Lilly Clinical Investigator award to
M.L.G.).
a
Present affiliation: National Cancer Institute, National Institutes of Health,
Department of Health and Human Services, Bethesda, Maryland.
Reprints or correspondence: Dr. Maura L. Gillison, Sidney Kimmel Comprehensive
Cancer Center at Johns Hopkins, G91, Cancer Research Bldg., 1650 Orleans St.,
Baltimore, MD 21231 (gillima@jhmi.edu).
The Journal of Infectious Diseases 2004; 189:686–98
 2004 by the Infectious Diseases Society of America. All rights reserved.
0022-1899/2004/18904-0017$15.00
logical or population differences. Prevalence estimates
of 9.2%–18.6% in exfoliated oral cells have been re-
ported in studies with disparate specimen collection
methods, study populations, and sensitivity of poly-
merase chain reaction (PCR) methods used for HPV
detection [1, 8, 9]. HPV infection in the tonsillar ep-
ithelium, the most biologically relevant site with respect
to cancer, has not been specifically evaluated, likely be-
cause the sampling of tonsils in healthy individuals is
technically challenging.
Numerous studies have established that cervical HPV
infection is necessary for the development of cervical
cancer and that it is acquired through sexual contact [10].
Similarly, a population-based study of oral cavity and
oropharyngeal cancers determined that a high number
of sex partners, younger age at first intercourse, and a
history of genital warts were associated with the oral
cancer risk in men [1]. Similar associations of increased
sexual behavior with oral cancer risk have been ob-
served in some [11] but not in other [12–14] studies.
Although oral-genital contact is a likely method of
transmission of HPV to the oral mucosa, such contact
has not been associated with oral cancer risk in case-
control studies. However, in a case-case comparison,
the odds of a tumor being HPV-positive increased in

686 • JID 2004:189 (15 February) • Kreimer et al.

individuals who reported a history of oral-genital sex or 16
lifetime sex partners [15]. In a study of sexually transmitted
disease (STD) clinic attendees, the increased odds of oral HPV
infection in individuals with a history of unprotected oral in-
tercourse was no longer observed after adjustment for other
sexual behaviors [8]. No association between incident oral HPV
infection and oral-penile contact during the preceding 12
months was reported in college-aged women [16]. However,
oral HPV infections were rare in this population.
The aim of the present study was to determine the prevalence
and type distribution of genital-mucosal type HPV infections
in the oral region and tonsillar epithelium of HIV-seronegative
and -seropositive adults without HNSCC and to investigate
associations with demographic and sexual behaviors.
SUBJECTS, MATERIALS, AND METHODS
Individuals in Baltimore, Maryland, were recruited during 2001–
2002 to participate in an oral-cancer screening program and
cross-sectional study. Study sites included a Hispanic community
center, a drug rehabilitation program, a medical clinic for HIV-
infected individuals, and a community health center in Baltimore.
The Institutional Review Board of the Johns Hopkins Bloomberg
School of Public Health approved the research protocol, and
informed consent was obtained from all participants.
All study subjects underwent a visual oral-cancer screening
examination by an otolaryngologist. Oral mucosal abnor-
malities (e.g., leukoplakia, erythroplakia, and papillomas)
were recorded, as were the presence of the tonsils. The oto-
laryngologist collected a transepithelial brush biopsy of the
epithelium overlying the palatine tonsils or tonsillar fossae
(in subjects with absent or atrophic tonsils) by use of an
OralCDx cytobrush (CDx Laboratories), as recommended by
the manufacturer [17]. The cytobrush has 94% sensitivity for
the diagnosis of oral dysplasia or carcinoma, compared with
scalpel biopsy [17]. Each sample was transferred by rotation
of the cytobrush into a collection tube that contained normal
saline. For oral-rinse specimens, 15 mL of sterile saline were
swished in the oral cavity for 15 s, gargled for 15 s, and
expectorated into a specimen cup. A blood sample was ob-
tained, and serum was separated by centrifugation. All sam-
ples were stored at ⫺70C until processing.
Behavioral data were collected by self-administered ques-
tionnaire (at the Hispanic community center) or by interview
(at all other study sites). Subjects were queried about their self-
identified racial category (white, black, Hispanic, Asian, or
other), demographic data, and sexual behaviors that included
age at coitarche, age at first performing oral sex, the number
of recent (the previous 12 months) versus lifetime sex partners
via vaginal or oral contact, and those that were casual (defined
as “a 1-night stand” or “sex with a person you do not know”)
Oral HPV Infection and Sexual Behaviors • JID 2004:189 (15 February) • 687
same-sex and opposite-sex partners. Subjects were queried
about a history of a number of STDs, HIV infection, most
recent CD4 cell count if HIV-positive, and exchange of sex for
money or drugs. Women were queried about abnormal Pa-
panicolaou (Pap) smear results. All subjects were asked about
lifetime use of tobacco and alcohol; current and former users
were questioned on the age at initiation, cessation, and average
amount used per day.
Smokers were defined as individuals with ⭓1 pack-year of
cigarette use, and former smokers were defined as those who
had quit for 11 year. Drinkers were defined as ever drinking ⭓1
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drink/week for a year, and former drinkers were defined as those
who had quit for 11 year. The consumption of alcoholic bev-
erages was quantified as the average number of drinks per week
among current drinkers.
HPV DNA detection. Tonsillar epithelial and oral-rinse
specimens were pelleted by centrifugation and resuspended in
normal saline. The sample was incubated in digestion buffer at
a final concentration of 50 mmol/L Tris-HCl (pH 8.5), 1 mmol/
L EDTA, 200 mg/mL proteinase K, and 1% laureth-12 for 1 h at
55C, followed by heat denaturation at 95C for 10 min. Digested
samples were stored at ⫺70C until further analysis.
HPV DNA was detected by multiplex PCR targeted to the
conserved L1 region of the viral genome by use of PGMY09/
11 L1 primer pools [18]. The b-globin gene was coamplified
[19]. Serial dilutions of digested oral-rinse specimens were
analyzed to optimize PCR conditions, because of the possible
presence of a PCR inhibitor [9, 20]. The quantitation of input
cell number in serially diluted samples, by use of real-time
PCR targeted to the single genomic copy of human endog-
enous retrovirus (ERV)–3 (see below), indicated that a 10-
fold sample dilution (final amount, 1 mL) was optimal for
analysis.
PCR products were denatured in 0.13 N NaOH and hy-
bridized to an HPV probe array for genotyping of 38 HPV
types classified as “high risk or probable high risk” or “low or
unknown risk” and b-globin (Roche Molecular Systems) [19,
21]. Positive controls, which consisted of 10 and 100 HPV-16
(SiHa)– or HPV-18 (C4-2)–positive cells diluted in a back-
ground of HPV-negative cells (K562), and a negative control
(K562 cells) were included in each experiment. Samples that
tested positive for b-globin were considered to be of sufficient
quality for analysis.
Quantitation of human cell number. All specimens from
HPV DNA–positive individuals and those from a random sample
of HPV DNA–negative specimens, frequency matched by HIV
serostatus, were selected to quantify the input number of human
cells tested for HPV DNA by use of a TaqMan real-time PCR
assay for a single-copy gene, ERV-3 [22]. Unknown sample quan-
tities were derived by interpolating threshold cycle number values
from a standard curve generated from logarithmic dilutions
Table 1. Characteristics of the study population, by HIV serostatus.

Total population, % HIV seronegative, % HIV seropositive, %

Characteristic (n p 597) (n p 396)a (n p 190)a
Demographic
Age, median (range), years 40.6 (18–85) 38.4 (18–85) 43.5 (26–66)c
Study site
Hispanic community center 17.4 26.3 0.0c
Drug rehabilitation center 25.3 32.3 8.4
HIV clinic 30.7 2.0 90.5
Community health center 26.6 39.4 1.1
Race
White 23.3 29.8 11.1c

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Black 55.8 41.9 82.6
Hispanic 16.9 24.8 1.6
Other 2.7 2.0 4.2
Unknownb 1.3 1.5 0.5
Education
!9th grade/some high school 45.9 45.7 48.4
High school graduate 32.1 31.6 32.6
College graduate and above 20.3 20.2 19.0
Unknownb 1.7 2.5 0
Income, $
!10,000 61.3 55.6 73.1c
10,000–30,000 23.5 24.2 21.6
130,000 9.2 12.1 3.7
Unknownb 6.0 8.1 1.6
Health behaviors
History of tonsillectomy (self-report)
No 74.4 73.0 76.8
Yes 20.4 21.2 19.0
Unknownb 5.2 5.8 4.2
Abnormal Papanicolaou smear results (women only, self-report)
Never 64.8 71.5 51.2c
Ever 32.4 25.5 46.3
Unknownb 2.8 3.0 2.5
Lifetime cigarette use, pack-years
None 29.0 33.1 20.0c
⭓1 and !20 38.5 37.9 40.0
⭓20 31.5 27.5 40.0
Unknownb 1.0 1.5 0
Alcohol use
Never 23.5 25.8 17.9c
Former 49.4 48.0 52.1
Current 24.0 21.5 30.0
Unknown 3.1 4.7 0.0
HPV-16, -18, or -33 serostatus
Seronegative 58.8 66.2 45.3c
Seropositive 39.4 33.8 52.1
Unknowna 1.8 0.0 2.6
Sexual behaviors
Mean (range) age at first intercourse, years 15.7 (6–45) 15.8 (6–29) 15.5 (7–45)
Lifetime no. of sex partners
0–5 38.5 40.9 34.2
⭓6 59.3 56.3 64.7
Unknownb 2.2 2.8 1.1

NOTE. HPV, human papillomavirus.

a Phlebotomy was unsuccessful in 11 individuals.
b Data unavailable because of subject nonresponse.
c Characteristic is significantly different in HIV-seronegative and -seropositive groups (P ! .005)
 Table 2. Human papillomavirus (HPV) DNA type distribution, stratified by specimen collection method.

Tonsil brush biopsy (n p 583) Oral rinse (n p 590)

No. of infections % of study No. of infections % of study
detected population infected detected population infected
High-risk HPV typesa
16 6 1.0 10 1.7
18 1 0.2 4 0.7
35 1 0.2 4 0.7
39 0 0.0 2 0.3
45 1 0.2 1 0.2

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52 0 0.0 1 0.2
53b 0 0.0 1 0.2
56 0 0.0 3 0.5
58 0 0.0 2 0.3
59 1 0.2 4 0.7
66b 0 0.0 3 0.5
67 0 0.0 2 0.3
69 1 0.2 1 0.2
73 0 0.0 2 0.3
82c 0 0.0 5 0.8
83 2 0.3 6 1.0
Total high-risk 13 2.1d 51 6.4d
Low-to-intermediate risk HPV typese
6 0 0.0 2 0.3
55 1 0.2 3 0.5
61 2 0.3 7 1.2
62 3 0.5 6 1.0
64 0 0.0 1 0.2
71 1 0.2 4 0.7
72 4 0.7 6 1.0
81 2 0.3 2 0.3
84 0 0.0 9 1.5
89 3 0.5 10 1.7
Total low risk 16 1.0d 50 5.8d
Total 29 3.1 101 12.2

a High-risk HPV types assayed for but not detected in oral samples: 26, 31, 33, 51, 68, and 70 [28].
b HPV type classified as probable high risk [28].
c HPV-82 includes the MM4 and IS39 subtypes.
d Value different from the sum of columns because of multiply infected individuals.
e Low-risk HPV types assayed for but not detected in oral samples: 11, 40, 42, 54, and 57 [28].

(from 50,000 to 0.5) of a diploid human cell line, CCD-18LU
(ATCC CCL-205). The cell number was defined as the number
of ERV-3 copies (in the diploid genome) divided by 2.
HPV serological testing. Serum samples were tested for
antibodies to HPV-16, -18, and -33 in a viruslike particle–based
ELISA [23]. Serum samples were dichotomized as positive or
negative for each antibody. The assay cutoff was determined by
comparison with the distribution of values obtained for 74 chil-
dren, ages 2–5 years, after the exclusion of outliers [23]. Sero-
positivity was defined as 3 SD above the mean optical density
obtained for the negative control serum samples (excluding
outliers).
HIV serological testing. The presence of HIV-1 infection
was determined according to Centers for Disease Control rec-
ommendations [24]. Serum samples were tested for HIV-1 an-
tibodies using the Vironostika HIV-1 Microelisa System
(bioMerieux), according to the manufacturer’s protocol. All sam-
ples with reactive ELISA results were subsequently evaluated by
use of the Cambridge Biotech HIV-1 Western Blot Kit (Calypte
Biomedical).

Oral HPV Infection and Sexual Behaviors • JID 2004:189 (15 February) • 689
Table 3. Univariate analysis of associations with tonsillar human papillomavirus (HPV) infection in the
total study population.

Tonsil brush biopsy

Characteristic Na HPV DNA n (%) OR (95% CI)

HIV status
Seronegative 387 4 (1.0) 1.0
Seropositive 186 14 (7.5) 7.8 (2.5–24.0)
CD4+ cell count (HIV seropositive only, self-report)
⭓200 99 4 (4.0) 1.0
!200 40 7 (17.5) 5.0 (1.4–18.3)
Unknown 44 3 (6.4) 1.6 (0.3–7.5)

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Abnormal Papanicolaou smear result (self-report, women only)
Never 159 3 (1.9) 1.0
Ever 80 3 (3.8) 2.0 (1.0–8.3)
Oral mucosal abnormality
Absent 545 14 (2.6) 1.0
Present 38 4 (10.5) 4.5 (1.4–14.3)
Cigarette smoking
Never 168 2 (1.2) 1.0
Former 53 0 0.0 (0.0–6.1)
Current use, packs/day
!1 158 7 (4.4) 3.8 (0.8–18.8)
⭓1 190 9 (4.7) 4.1 (0.9–19.4)
Alcohol use
Never 135 3 (2.2) 1.0
Former 289 9 (3.1) 1.4 (0.4–5.3)
Current, drinks/week
!20 67 4 (6.0) 2.8 (0.6–12.9)
⭓20 73 2 (2.7) 1.2 (0.2–7.6)
Serological evidence of STDs
HPV-16, -18, or -33
Seronegative 343 6 (1.8) 1.0
Seropositive 230 12 (5.2) 3.1 (1.1–8.4)
HSV-1
Seronegative 102 1 (1.0) 1.0
Seropositive 470 16 (3.4) 3.6 (0.5–27.1)
HSV-2
Seronegative 242 3 (1.2) 1.0
Seropositive 329 15 (4.6) 3.8 (1.1–13.3)
History of STDs (self-report)
Never 288 5 (1.7) 1.0
Ever 266 13 (4.9) 2.9 (1.0–8.3)
Age at first oral sex
⭓18 years 293 10 (3.4) 1.0
!18 years 109 5 (4.6) 1.4 (0.5–4.1)
Lifetime no. of oral sex partners
0–5 444 12 (2.7) 1.0
⭓6 124 6 (4.8) 1.8 (0.7–5.0)
Recent no. of oral sex partners
0–1 489 14 (2.9) 1.0
⭓2 80 4 (5.0) 1.8 (0.6–5.6)
Same-sex oral sex
Never 495 12 (2.4) 1.0
Ever 62 6 (9.7) 4.3 (1.6–11.9)

(continued)
 Table 3. (Continued.)
Characteristic
Age at first vaginal sex, years
⭓18
!18
Lifetime no. of vaginal sex partners
0–5
⭓6
Lifetime no. of casual sex partners
0–5
⭓6
NOTE. CI, confidence interval; HSV, herpes simplex virus; OR, odds ratio; STD, sexually transmitted disease.
a Columns are not equal to total population because of subject nonresponse or unsuccessful phlebotomy.
Herpes simplex virus (HSV)–1 and -2 serological testing.
Serum samples were analyzed for the presence of antibodies to
HSV-1 and HSV-2 by use of Focus Technologies HerpeSelect-
1 ELISA IgG and HerpeSelect-2 ELISA IgG, respectively [25].
Statistical analysis. HPV DNA prevalence estimates and
exact 95% confidence intervals (CIs) were calculated. The pres-
ence of HPV DNA in tonsillar epithelial and oral-rinse speci-
mens was analyzed separately and combined to represent HPV
DNA in the “oral region.” the presence of HPV DNA was
stratified by type and high- and low-risk classifications. Indi-
viduals coinfected by high- and low-risk types were placed in
the high-risk category. The k statistic was used to compare
agreement between cytobrush and oral-rinse data. Logistic re-
gression was used to model the relationship between age as a
continuous variable and oral HPV infection. To evaluate the
data from ERV-3 cell quantitation, the median cell number and
interquartile range were determined. Matched pairs of tonsillar
and oral rinse specimens from the same individual were com-
pared by use of a 2-sided sign test. The rank sum test was used
to compare median cell number, stratified by specimen collec-
tion method and HIV serostatus or the presence of HPV-DNA.
Associations observed in data when they were stratified by
study site and questionnaire administration method were sim-
ilar in univariate and multivariate analyses; therefore, data were
pooled for further analysis. The HIV-seronegative and -sero-
positive groups were compared by use of the x2 or Fisher’s
exact test for categorical variables and by Student’s t test (with
unequal variance) for continuous variables. Logistic regression
models were used to estimate odds ratios (ORs) (and 95% CIs)
for associations between demographic and exposure variables
and the presence of HPV DNA. Trend tests were conducted
across ordered groups. Variables that were important in HIV-
stratified univariate analysis and for which P ! .20 were eval-
uated in a multiple logistic-regression model, as were variables
that were considered to be relevant on the basis of the literature.
Oral HPV Infection and Sexual Behaviors • JID 2004:189 (15 February) • 691
Tonsil brush biopsy
Na HPV DNA n (%) OR (95% CI)
127 1 (0.8) 1.0
410 15 (3.7) 4.8 (0.6–36.6)
223 4 (1.8) 1.0
342 14 (4.1) 2.3 (0.8–7.2)
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370 8 (2.2) 1.0
179 10 (5.6) 2.7 (1.0–6.9)
The final model was created by the inclusion of variables with
potential biological significance, as well as those that remained
statistically significant after adjustment. All P values reported
are 2-sided and were considered to be statistically significant
at P ! .05.
RESULTS
The characteristics of the study population, stratified by HIV
serostatus, are shown in table 1. Compared with the HIV-sero-
negative group, the HIV-seropositive group was more likely to
be older, black, current alcohol drinkers, in the lowest income
group, and to have histories of more extensive cigarette smok-
ing and abnormal Pap smear results (table 1). Age at first in-
tercourse and total number of lifetime sex partners did not
differ between HIV-seropositive and -seronegative individuals
(table 1). With respect to history of oral sex, HIV-seropositive
individuals reported fewer lifetime partners, were less likely to
have had 11 recent partner, and had an older mean age at first
performing oral sex (23.0 [HIV-positive] vs. 21.2 years; P p
.02). However, HIV-seropositive individuals were more likely
to be seropositive to HPV-16, -18 or -33. The seroprevalence
of HSV-2 in the study population increased with increasing
years of sexual activity (OR, 1.03; 95% CI, 1.01–1.05), history
of STDs (OR, 2.5; 95% CI, 1.8–3.6), and increased number of
lifetime sex partners (0–5, 6–10, and 110 partners; P p .07 for
trend), as reported elsewhere [26].
Prevalence of HPV DNA by specimen collection method.
All subjects had either (or both) a tonsillar epithelial or oral
rinse specimen positive for b-globin by PCR. Tonsillar epithelial
samples from 2.3% and oral rinse specimens from 1.2% were
negative for b-globin by PCR.
HPV DNA was present in the tonsillar epithelium of 18
(3.1%) of 583 (95% CI, 1.8%–4.8%) evaluable individuals.
HPV-16 was the most common type detected (6 infections)
and was present in the tonsillar epithelium of 1.0% (95% CI,
0.4%–2.2%) of individuals. Coinfection of tonsils by multiple
HPV types was observed in 1.4% (95% CI, 0.6%–2.7%), and
2.2% (95% CI, 1.2%–3.8%) had infections with high-risk types.
The prevalence of HPV DNA did not differ in individuals with
or without tonsils by physician examination (13 [2.9%] of 447
vs. 5 [3.9%] of 128; P p .6).
HPV DNA was detected in the oral rinse of 72 (12.2%) of
590 (95% CI, 9.6%–14.9%) evaluable individuals. This was sig-
nificantly higher than the detection rate in the tonsillar epithe-
lium (P ! .001). Classification of an individual as HPV infected
was therefore highly dependent on specimen collection method
(cytobrush vs. oral rinse), with correlation between these meth-
ods being poor (k p 0.16). HPV DNA was detected in both
specimens obtained from 9 of 81 individuals with oral HPV
DNA and was type concordant among 8 of them.
Positive results from tonsillar epithelial and oral rinse spec-
imens were combined as a measurement of HPV DNA presence
in the oral region (subsequently referred to as an oral HPV
infection). Oral HPV infection was detected in 81 (13.6%) of
597 (95% CI, 10.8%–16.3%) individuals. Overall, HPV16 was
detected in 2.5% (95% CI, 1.4%–4.1%), high-risk HPV types
in 7.0% (95% CI, 5.1%–9.4%), and multiple HPV types co-
infected 2.8% (95% CI, 1.7%–4.5%) of the study population.
The type distribution of the 130 HPVs detected is summarized
in table 2.
The number of cells analyzed for HPV DNA (quantified by
ERV-3 TaqMan assay) was almost 10-fold greater in the oral rinse
than in the tonsillar brush biopsy specimens (median, 9743 [in-
terquartile range {IQR} 4070–19,490] vs. median, 971 [IQR 460–
2882]; P ! .001, sign test). Tonsillar brush biopsy samples that
were positive for HPV DNA had more cells than those that tested
negative (median, 2265 [IQR 1117–7160] vs. median, 831 [IQR
435–2533]; P p .002, rank sum). However, no difference was
observed in HPV-positive and -negative oral rinse specimens (me-
dian, 10,980 [IQR 5303–20,586] vs. median, 9411 [IQR 3768–
18,055]; P p .2, rank sum). The number of cells tested for HPV
DNA in tonsillar epithelial and oral rinse specimens did not
differ by HIV serostatus (data not shown).
HPV in the tonsillar epithelium. Because current data
suggest that the tonsillar epithelium is the most biologically
relevant site of exposure in terms of cancer risk, an exploratory
analysis was performed to investigate associations of biomark-
ers and sexual behaviors with the presence of HPV DNA in
tonsillar epithelial cells (table 3). The low prevalence precluded
an HIV-stratified analysis.
HIV seropositivity, CD4+ cell count !200 cells/mL, HSV-2
seropositivity, and HPV-16, -18, or -33 seropositivity were all
significantly associated with the presence of a tonsillar HPV
infection (table 3). Several sexual behaviors were also associated
692 • JID 2004:189 (15 February) • Kreimer et al.
with a tonsillar HPV infection, including a history of same-sex
oral sex, 15 lifetime casual sex partners, and a history of an
STD. The odds of a tonsillar HPV infection were elevated in
current tobacco users, compared with nonusers, and a signif-
icant trend was noted for increased odds with increased pack-
years of use (P p .02 for trend) and with number of packs per
day (P p .03 for trend) among current cigarette smokers. All
associations found to be significant in this univariate analysis
of HPV DNA in the tonsillar epithelium were also significant
in an analysis of HPV DNA in oral-rinse specimens, but as-
sociations were not as strong (data not shown).
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HPV in the oral region. Data from tonsillar epithelial and
oral-rinse specimens were combined as a measurement of over-
all oral HPV infection, to explore associations with validated
serologic biomarkers of sexual behavior [26, 27] and self-
reported behavioral exposures. These analyses were stratified
by HIV serostatus, because, compared with HIV-seronegative
individuals, HIV-seropositive individuals were more likely to
have an oral HPV infection (25.3 vs. 7.6%; P ! .001), to be
infected by 11 HPV type (5.8 vs. 1.5%; P p .004), and to be
infected by a high-risk HPV type (13.7 vs. 4.5%; P ! .001).
When stratified by HIV serostatus, an increase in prevalence
of HPV infection with age was observed in HIV-seronegative
individuals (P ! .001 for trend; figure 1) but not among HIV-
seropositive individuals (P p .95 for trend). The trend was pres-
ent in HIV-seronegative men (P ! .001 for trend) and women
(P p .03 for trend). The odds of HPV DNA presence was es-
timated to increase 5% with each year of age (OR, 1.05; 95%
CI, 1.02–1.08) in the HIV-seronegative group. The distribution
of high- and low-risk types was similar across all age categories
(data not shown).
Among the HIV-seronegative group, factors associated with
an oral HPV infection included being male, HSV-2 seroposi-
tivity, having ⭐1 oral or vaginal sex partner during the pre-
ceding year, having ⭐1 recent casual sex partners, and being
seronegative for HPV-16, -18, or 33 (table 4). When multiple
regression models were fitted to evaluate associations between
demographic and exposure variables and the presence of an
oral HPV infection, male sex (OR, 3.0; 95% CI, 1.3–7.0), HSV-
2 serostatus (OR, 2.7; 95% CI, 1.2–5.2), and older age were
significantly associated with oral HPV infection among HIV-
seronegative individuals (table 5). There was a 5% increase in
the odds of HPV DNA presence with each year of age (OR, 1.05;
95% CI, 1.02–1.07). Other measurements of sexual behavior were
evaluated but did not significantly affect the associations pre-
sented in table 5. In particular, the number of recent oral sex
partners was not significant (OR, 0.2; 95% CI, 0.0–1.2).
Among the HIV-seropositive individuals, factors associated
with oral HPV infection included a self-reported CD4+ cell
count !200 cells/mL, the presence of an oral mucosal abnor-
mality on examination by an otolaryngologist, 11 oral sex part-
ner during the preceding 12-month period, and 11 casual sex
partner (table 4). The associations that remained robust in
multiple-regression analyses included male sex, HSV-2 sero-
status, reported CD4+ cell count, and the number of oral sex
partners during the preceding year (table 5). A particularly
strong association was observed with oral-genital contact
among HIV-seropositive individuals. Individuals who reported
performing oral sex on 11 sex partner during the preceding
12-month period had an estimated 13-fold (95% CI, 3.1–52.7)
increase in the odds of having an oral HPV infection, after
positive (9/18) than in HIV-seronegative individuals (2/20; P !
.01). Current cigarette smokers were twice as likely to have an
oral mucosal abnormality, compared with former cigarette users
and those who had never smoked (OR, 2.0; 95% CI, 1.0–4.1).
DISCUSSION
In the present cross-sectional study, high-risk HPV types
strongly associated with cancer [28] were found in the oral
region of 4.5% of HIV-seronegative and 13.7% of HIV-sero-
positive individuals. HPV-16 was the type most frequently de-

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adjustment for other variables in the model. The addition of
other measurements of recent or lifetime sexual behaviors, in-
cluding the number of sex partners, did not affect the strength,
direction, or significance of this association.
Oral mucosal abnormalities. A total of 39 oral mucosal
abnormalities were noted in 38 (6.4%) of 597 study subjects
on examination by an otolaryngologist (table 6). Oral mucosal
abnormalities with malignant potential, such as leukoplakia and
erythroplakia, accounted for 23 (59%) of 39 lesions noted. HIV-
seropositive individuals were significantly more likely to have
an oral mucosal abnormality (OR, 2.0; 95% CI, 1.0–3.8). Fur-
thermore, oral mucosal abnormalities were more likely to occur
in individuals with an oral HPV infection who were HIV sero-
tected in our study and in the overwhelming majority (90%–
95%) of HPV-associated tonsillar cancers [2, 29].
The evidence for an etiological role for HPV in HNSCCs is
strongest for tonsillar carcinomas [2, 5, 6, 29–31]; therefore,
the tonsillar epithelium is the most biologically relevant site to
sample for HPV infection. To our knowledge, this is the first
study to estimate the prevalence of tonsillar HPV infection in
adults without HNSCCs by using direct sampling of the ton-
sillar epithelium with a validated sampling method. A tonsillar
epithelial HPV infection was found in 3.1% of the study pop-
ulation. Tonsillar HPV infection was strongly associated with
HIV infection, immunosuppression, and several measurements
of sexual behavior in univariate analysis. However, these sta-

Figure 1. The prevalence of oral human papillomavirus (HPV) infection in the study population, stratified by HIV serostatus and age. The prevalence
of an oral HPV infection did not increase with age in individuals with HIV infection (P p .95 for trend) but did in those without (P ! .001 for trend).
HIV-seronegative individuals are represented in black and HIV-seropositive individuals in gray. Sample size within each age category is indicated below,
stratified by HIV serostatus. +, positive; ⫺, negative.

Oral HPV Infection and Sexual Behaviors • JID 2004:189 (15 February) • 693
Table 4. Univariate analysis of associations with an oral human papillomavirus (HPV) infection, by HIV serostatus.

HIV seronegative HIV seropositive

Characteristic Na HPV DNA positive OR (95% CI) Na HPV DNA positive OR (95% CI)
Sex
Female 165 9 (5.5) 1.0 82 16 (19.5) 1.0
Male 231 23 (10.0) 1.9 (0.9–4.3) 108 32 (30.0) 1.7 (0.9–3.4)
CD4+ cell count, cells/mL (HIV-seropositive only, self-report)
1200 NA NA 101 23 (22.8) 1.0
!200 40 16 (40) 2.3 (1.0–5.0)
Unknown 49 9 (18.4) 0.8 (0.3–1.8)
History of abnormal Papanicolaou smear result (self-report,

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women only)
Never 118 6 (5.1) 1.0 42 7 (16.7) 1.0
Ever 42 3 (7.1) 1.4 (0.3–6.0) 38 8 (21.1) 1.3 (0.4–4.1)
Oral mucosal abnormality
Absent 376 30 (8.0) 1.0 172 39 (22.7) 1.0
Present 20 2 (10.0) 1.3 (0.3– 5.8) 18 9 (50.0) 3.4 (1.3–9.2)
Cigarette smoking
Never 131 9 (6.9) 1.0 38 9 (23.7) 1.0
Former 34 3 (8.8) 1.3 (0.3–5.1) 19 3 (15.8) 0.6 (0.1–2.6)
Current use, packs/day
!1 94 9 (9.6) 1.4 (0.5–3.8) 63 19 (30.2) 1.4 (0.6–3.5)
⭓1 124 11 (8.9) 1.3 (0.5–3.3) 69 17 (24.6) 1.1 (0.4–2.7)
P for trend 0.52 0.70
Alcohol use
Never 102 8 (7.8) 1.0 34 8 (23.5) 1.0
Former 190 12 (6.3) 0.8 (0.3–2.0) 99 25 (25.3) 1.1 (0.4–2.7)
Current, drinks/week
!20b 41 6 (14.6) 2.0 (0.7–6.2) 28 8 (28.6) 1.3 (0.4–4.1)
⭓20 44 5 (11.4) 1.5 (0.5–4.9) 29 7 (24.1) 1.0 (0.3–3.3)
P for trend 0.24 0.88
HPV-16, -18, or -33 status
Seronegative 262 26 (9.9) 1.0 86 20 (23.3) 1.0
Seropositive 134 6 (4.5) 0.4 (0.2–1.1) 99 27 (27.3) 1.2 (0.6–2.4)
HSV-1 status
Seronegative 69 3 (4.4) 1.0 33 6 (18.2) 1.0
Seropositive 323 28 (8.7) 2.1 (0.6–7.1) 152 41 (27.0) 1.7 (0.6–4.3)
HSV-2 status
Seronegative 196 10 (5.1) 1.0 45 8 (17.8) 1.0
Seropositive 195 22 (11.3) 2.4 (1.1–5.1) 139 39 (28.1) 1.8 (0.8–4.2)
History of STDs (self-report)
Never 240 21 (8.8) 1.0 50 12 (24.0) 1.0
Ever 135 11 (8.2) 0.9 (0.4- 2.0) 132 35 (26.5) 1.1 (0.5–2.4)
Age at first oral sex, years
⭓18 194 16 (8.3) 1.0 101 29 (28.7) 1.0
!18 71 6 (8.5) 1.0 (0.4–2.7) 37 8 (21.6) 0.7 (0.3–1.7)
Lifetime no. of oral sex partners
0–5 302 23 (7.6) 1.0 150 39 (26.0) 1.0
⭓6 81 9 (11.1) 1.5 (0.7–3.4) 38 9 (23.7) 0.9 (0.4–2.0)
Recent no. of oral sex partners
0–1 317 31 (9.8) 1.0 176 40 (22.7) 1.0
⭓2 67 1 (1.5) 0.1 (0.0–1.0) 12 8 (66.7) 6.8 (1.9–23.8)

(continued)
 Table 4. (Continued.)

HIV seronegative HIV seropositive

Characteristic Na HPV DNA positive OR (95% CI) Na HPV DNA positive OR (95% CI)
Age at first vaginal sex, years
⭓18 95 9 (9.5) 1.0 33 6 (18.2) 1.0
!18 270 21 (7.8) 0.8 (0.4–1.8) 142 37 (26.1) 1.6 (0.6–4.1)
Lifetime no. of vaginal sex partners
0–5 160 14 (8.8) 1.0 65 16 (24.6) 1.0
⭓6 220 18 (8.2) 0.9 (0.4–1.9) 123 32 (26.0) 1.1 (0.5–2.2)
Recent no. of vaginal sex partners
0–1 251 27 (10.8) 1.0 153 40 (26.1) 1.0

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⭓2 139 5 (3.6) 0.3 (0.1–0.8) 37 8 (21.6) 0.8 (0.3–1.8)
Lifetime no. of casual sex partners
0–5 267 23 (8.6) 1.0 109 23 (21.1) 1.0
⭓6 99 9 (9.1) 1.1 (0.5–2.4) 77 24 (31.2) 1.7 (0.9–3.3)
Recent no. of casual sex partners
0–1 305 30 (9.8) 1.0 168 40 (23.8) 1.0
⭓2 63 2 (3.2) 0.3 (0.1–1.3) 20 8 (40.0) 2.1 (0.8–5.6)

NOTE. Data are no. (%) unless otherwise indicated. CI, confidence interval; NA, not applicable; OR, odds ratio; STD, sexually transmitted disease.
a Columns are not equal to total population because of subject nonresponse or unsuccessful phlebotomy.
b The mean value of drinks per week among current drinkers was chosen as the cutoff.

tistically significant associations may not be independent mark-
ers of risk for tonsillar HPV infection because of the low prev-
alence of tonsillar infection.
Our study builds on evidence that oral HPV infection can
be acquired through sexual behavior, particularly recent oral-
genital contact. Oral HPV infection in individuals with and
without HIV infection was associated with HSV-2 seropositiv-
ity, an accepted surrogate measurement of several sexual be-
haviors [27]. Recently, HSV-2 infection was demonstrated to
increase the risk of an incident HPV infection [32], and it has
been suggested that HSV-2 may act to promote cervical disease
progression [33–35], even though HSV-2 sequences have not
been found in tumors [36]. In the present study, however, oral
HPV infection was significantly associated with seroreactivity
to HSV-2 but not with HSV-1, the herpes virus that more
commonly infects the oral cavity. Therefore, we interpret the
association of oral HPV infection with HSV-2 as evidence of
an association with sexual behavior.
The strongly positive association of oral-genital contact with
an HPV infection in HIV-seropositive but not in HIV-sero-
negative adults would suggest that the same behavior may have
different consequences in the setting of immunosuppression.
Evidence for abnormal oral mucosal immunity is provided by
reports of high prevalence of oral infectious complications and
a rise in prevalence with advancing immunosuppression in
HIV-infected individuals [37, 38]. HIV-seropositive individuals
were more likely to have an oral HPV infection, even though
HIV-seropositive individuals reported fewer recent oral sex
partners. Possible explanations for this finding would include
reactivation of a latent infection, infection persistence, or high-
titer infection more likely to be detected in the setting of im-
munosuppression. A prospective investigation of the factors
associated with an incident oral HPV infection may distinguish
from among these possibilities.
Oral-genital contact was not significantly associated with oral
HPV infection in the HIV-seronegative population. Surpris-
ingly, in univariate analysis, the odds of an oral HPV infection
were significantly reduced in HIV-seronegative individuals who
reported 11 recent oral, vaginal, or casual sex partner (table
4), but this finding was not corroborated in the multivariate
model. This finding could be real and could indicate that the
transmission of HPV to the oral cavity is significantly different
in HIV-seronegative adults, but we think this unlikely. Other
possible explanations would include insufficient sample size
caused by a lower prevalence of infection in this group; a finding
caused by chance, given the multiple comparisons performed
in the study; or bias in selection of the study population. In
particular, racial and ethnic heterogeneity was greater in the
HIV-seronegative group. We were unable to evaluate the effects
of race or ethnicity because of colinearity with several other
exposure variables. For example, the HIV clinic served a pre-
dominantly black community. Because of the low prevalence
of oral HPV infection, a larger study is warranted to clarify the
associations of race and sexual behaviors with infection among
HIV-seronegative individuals.
The association of oral-genital contact with oral HPV infec-
tion in our results does not exclude a role for other methods
of transmission of HPV to the oral mucosa. These may in-
clude mother-to-child vertical transmission [39–42], nonsexual
transmission in children prior to their sexual debut [43], or

Oral HPV Infection and Sexual Behaviors • JID 2004:189 (15 February) • 695
Table 5. Associations of selected variables with an oral human analysis was limited to the 38 most common sexually trans-
papillomavirus (HPV) infection, after adjustment for covariates, mitted genital-mucosal HPV types. Unusual HPV types pre-
stratified by HIV serostatus.
viously associated with oral lesions in HIV-seropositive indi-
OR (95% CI)
viduals, such as HPV-7, -13, and -32 [52, 53], would not have
been detected. In the setting of HIV infection, unusual HPV
HIV seronegative HIV seropositive
Variablea (n p 384) (n p 182) types (e.g., cutaneous HPV-7) and unusual associations of HPV
type with particular lesions (e.g., HPV-13, -18, or -32 papil-
Age, yearsb 1.05 (1.02–1.07) 1.0 (0.95–1.05)
lomas) may be found in the oral cavity [52, 53].
Sex
Although HPV infection was strongly associated with mu-
Female 1.0 1.0
cosal abnormalities in HIV-seropositive individuals, an etiologic
Male 3.0 (1.3–7.0) 1.9 (0.8–4.3)
association cannot be asserted because we did not analyze HPV
HSV-2

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DNA in biopsy specimens of lesions per se. The relationship
Seronegative 1.0 1.0
between HPV infection and oral lesions may be attributed to
Seropositive 2.7 (1.2–5.2) 3.2 (1.1–9.0)
residual confounding because both are related to severity of
Oral mucosal abnormality
immuosuppression [54, 55]. HPV is rarely detected in oral
Absent 1.0 1.0
cavity dysplasia in HIV-negative individuals [30]; however,
Present 1.2 (0.2–5.9) 6.3 (2.0–19.6)
dysplasia and carcinoma in situ have been observed in HPV-
CD4 cell count
positive oral warts in HIV-infected individuals [53, 56]. The
1200 NA 1.0
!200 2.6 (1.0–6.2)
Table 6. Oral mucosal abnormalities in the study population.
Unsure 0.7 (0.3–1.8)
No. of recent oral sex No. of No. HPV
partners Abnormality individualsa DNA positive HPV typeb
0–1 1.0 1.0
Leukoplakia
2⭓ 0.2 (0.0–1.2) 12.8 (3.1–52.7)
Buccal mucosa 7 3 6, 39, 84
NOTE. CI, confidence interval; HSV, herpes simplex virus; NA, not appli- Retromolar
cable; OR, odds ratio. trigone 2 1 62, 82
a Adjusted for covariates in the model.
Tongue 8 3 16, 58, 89
b Age was modeled as a continuous linear variable.
Paraglottal fold 1 0
Site not specified 3 1 59
auto-inoculation. For example, respiratory papillomatosis oc-
Erythroplakia
curs as a consequence of both peripartum and sexual trans-
Hard palate 1 0
mission [44, 45].
Tongue 1 0
The prevalence of oral infection by genital-mucosal HPVs
Masses
was significantly higher among HIV-seropositive individuals, as
Tonsil 1 0
has been reported elsewhere [8]. Furthermore, prevalence was
Uvula 1 0
higher in individuals with severe immunosuppression in the
Base of tongue 1 0
oral as previously reported in the cervical and anal regions [46,
Neck 1 0
47]. Similarly, high-risk and concurrent multiple-type oral, cer-
Floor of mouth 1 0
vical [48, 49], anal HPV infections [47, 50] are increased in
Ulcers
HIV-seropositive individuals.
Buccal mucosa 1 1 6
The observed prevalence of oral HPV infection in HIV-sero-
Tongue 1 0
negative individuals in the present study should not be expected
Palate 2 1 73
to extrapolate to the general US population because of the
Papilloma
demographic composition of the subjects in the present study.
Soft palate 1 0
Furthermore, the study subjects had a higher prevalence of
Tongue 2 0
high-risk sexual behaviors, as indicated by the seroprevalence
Tonsillar
for 2 validated markers of sexual behavior, HPV-16 (20.2% in asymmetry 2 1 16
the present study vs. 13.0% in the US population) and HSV- Papillary atrophy of
2 (50% in the present study vs. 23.3% in the US population) the tongue 2 1 83
[51]. By contrast, we may have underestimated oral HPV in- a More than one mucosal abnormality was detected in 1 individual.
fection prevalence in HIV-seropositive individuals. HPV DNA b Only if HPV was present in oral specimen.

696 • JID 2004:189 (15 February) • Kreimer et al.

relationship between HPV and oral lesions with malignant po-
tential (e.g., leukoplakias, erythroplakias, and dysplastic pap-
illomas) in HIV-positive individuals should be clarified. An
increased incidence of oral papillomas in individuals receiving
highly active antiretroviral therapy, in contrast to a decline in
incidence of EBV-associated oral hairy leukoplakia and Kaposi
sarcoma–associated herpesvirus [57], might predate a similar
rise in high-risk HPV associated disease, particularly tonsillar
carcinoma.
High-risk HPVs, predominantly HPV-16, are consistently de-
tected in 40%–60% of tonsillar squamous-cell carcinomas in
Western cultures [1, 2, 15, 58–60]. According to surveillance,
epidemiology, and end results data, the incidence of tonsillar
cancer increased significantly from 1973 to 1995 in men, whereas
the incidence at all other oral sites remained constant [61]. Al-
though changes in tonsillectomy rates may have influenced these
trends, it is conceivable that an increase in oral HPV infections,
in tandem with increased rates of unprotected oral sex [62, 63],
may have contributed. The HIV epidemic may have contributed
as well, because HIV-seropositive individuals have a 2–6-fold
increased in risk of tonsillar and oropharyngeal cancer [64, 65].
The present study represents a step toward a better under-
standing of the prevalence and natural history of oral HPV
infection. The implications of these findings for oral cancer
screening, however, are unclear. The natural history of an oral
HPV infection and the predictive value of infection for the
development of oral cancer remain unknown.
Acknowledgments
We thank Janet Kornegay (Roche Molecular Systems, Pleas-
anton, CA), for generously providing the reagents for the HPV
line blot hybridization; Drore Eisen (CDx Laboratories, Suffern,
NY), for providing OralCDx brushes; the laboratories of Thom-
as Quinn, for performing HIV serological analysis, and Jona-
than Zenilman, for performing herpes simplex virus–1 and-2
analysis; Nasir Bhatti, Patrick Byrne, Joseph Califano, Robert
Ferris, James Sciubba, and Ralph Tufano, for performing oral
cancer screening examinations and OralCDx biopsies; and Da-
vid E. Symer, for comments on the manuscript.
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