HPV Detection and genotyping in males from the city of Córdoba

184
Revista Argentina de Microbiología (2010)
42:0325-7541
184-188
ISSN
Revista Argentina de Microbiología (2010) 42: 184-188
INFORME BREVE
HPV Detection and genotyping in males from the city of
Córdoba, Argentina
F. VENEZUELA1*, L .E. KREMER2, X. KIGUEN1, C. CUFFINI1
1
Instituto de Virología “Dr.J. M. Vanella” U.N.C. Calle Enfermera Gordillo Gómez s/n (entre avenidas Enrique Barros y
Valparaiso) Ciudad Universitaria, Córdoba (5016); 2Hospital Nacional de Clínicas-Córdoba.
Calle Santa Rosa 1500, B° Alberdi, Córdoba (5000)
*Correspondence. Email: venezuelafernando@hotmail.com
ABSTRACT
A wide range of human papillomavirus (HPV) types can infect the anogenital region of males. Although there is a vast
knowledge on HPV infections in women as well as on their association with cervical cancer, the study of HPV infections
in males is scarce and controversial. The aim of the present work was to detect and typify HPV infections of the
anogenital region in males and analyze the associated risk factors in the population studied. Anogenital samples from
37 patients (30 of whom were HIV carriers) attending the Infectology Service at the Hospital Nacional de Clínicas in
Córdoba, Argentina, were studied. Nine of these patients tested HPV-positive and five out of these nine were found to
have mixed infections, being 18 and 61 the most frequent genotypes. There was a significant correlation between the
HPV-positive patients and those having an HPV-compatible lesion or AIDS. The present work is the first study in the
city of Cordoba which contributes relevant results to the knowledge of HPV infection and to the possible implementation
of measures for its prevention.
Key words: Male, genotyping, HPV, AIDS, anogenital, Córdoba
RESUMEN
Detección y genotipificación de VPH en varones de la ciudad de Córdoba, Argentina. Un amplio espectro de
tipos de virus papiloma humano (VPH) puede infectar la zona anogenital de los varones. Si bien existe un vasto
conocimiento de la infección por VPH en las mujeres y su asociación con el cáncer de cérvix, el estudio de la infección
por VPH en los varones ha sido escaso y sus resultados controvertidos. El presente trabajo tuvo como objetivo
detectar y tipificar infecciones por VPH en la región anogenital de varones y analizar los factores de riesgo asociados
en la población estudiada. Se estudiaron muestras anogenitales de 37 pacientes (30 portadores del VIH) que asistieron al Servicio de Infectología del Hospital Nacional de Clínicas de la ciudad de Córdoba. Nueve resultaron positivas
para VPH, de las que 5 correspondían a infecciones mixtas. Los genotipos de mayor frecuencia fueron el 18 y el 61.
Hubo una correlación significativa entre los resultados VPH positivos en aquellos pacientes con lesión compatible o
con sida. Nuestro trabajo es el primero de su tipo realizado en la ciudad de Córdoba y aporta resultados relevantes
para el conocimiento de la infección por VPH y la implementación de medidas de prevención.
Palabras clave: Varón, genotipificación, HPV, sida, anogenital
Human papillomavirus (HPV) infection has become
one of the most important sexually transmitted diseases
(13, 14). HPV causes not only benign lesions (like
condylomata acuminata), but also a wide range of
preneoplastic lesions and carcinoma. Although the relationship between HPV and the etiopathogeny of cervical
cancer is well known, there are few studies on HPV infection in male anogenital lesions. In addition, it has been
proposed that males play an important role as reservoirs
and transmitting agents of HPV infection (4, 11).
During March-June 2009, in order to detect and typify
HPV infections in the anogenital region of males, we studied 40 samples from 37 patients attending the Infectology
Service at the Hospital Nacional de Clínicas in Córdoba,
Argentina, who presented at least one of the following
conditions: a) a lesion compatible with HPV in the
anogenital region, b) their last sexual partner presented
a lesion compatible with HPV in the anogenital region,
and c) more than three sexual partners in the previous
year. The average age of patients was 36.9 years (range
21-57 years).
The Infectology Service provides care to patients with
different types of lesions. However, it is interesting to point
out that over 80% of the patients included in this study
were carriers of the human immunodeficiency virus
(twenty-nine of HIV and one of HIV-HCV). In accordance
with the Center for Disease Control and Prevention (CDC),
16 of the 29 HIV carriers were classified as HIV-AIDS
(A3: 3; C1: 2; C2: 6 and C3: 5).
An epidemiological chart was performed for each patient and, according to the data collected, either a urethral
swab/penile brushing or a brushing of the anal/perianal
HPV Detection and genotyping in males from the city of Córdoba, Argentina
region was obtained, since these samples are considered optimal to evaluate the infection in the anogenital
region (5). A total of 28 urethral swabs/penile brushing
and 12 anal/perianal were obtained. In three of the patients reporting both passive and active sexual intercourse, both types of samples were collected. The reasons why each patient attended the Infectology Service
are detailed in Table 1.
Of the 29 patients that attended the Infectology
Service for reasons other than an HPV-compatible lesion,
six presented this kind of lesion in the anogenital region
and 12 stated that they had had previous HPV lesions
(Table1).
Of the 14 patients who presented an HPV-compatible
lesion, two presented perianal condyloma acuminata and
12 presented flat warts (four in the perianal region, three
in the glans penis and five on the surface of the penis).
The samples were collected in 500 µl phosphate-buffered
saline (PBS). DNA was then extracted using the commercial AccuPrep Genomic DNA Extraction Kit (Bioneer
Inc., CA, USA), according to the manufacturer’s instructions. A 450-bp segment, corresponding to the L1 region
of the viral genome, was amplified by PCR, using the
degenerate primers MY09 and MY11 (1). The product was
detected by electrophoresis in agarose gel at 1.5% using
an U.V. transilluminator.
The Beta globin gene was used as a DNA preservation marker. Viral genotypification was carried out by
analysis of restriction fragment length polymorphism
(RFLP), following the technique described by Bernard et
al. (1). A statistical analysis was carried out with Epi Info™,
Version 3.5.1, 2008 (http://www.cdc.gov/epiinfo/).
Of all the samples analyzed, nine were positive for
HPV and belonged to patients that presented an HPVcompatible lesion (Table 3). In addition, five patients that
presented an HPV-compatible lesion were HPV-negative.
Table 1. Distribution according to the inclusion conditions (n = 37)
Reason for consultation
HPV-compatible lesions
Clinical check up and in search of medication
Other (traumatisms, allergies, pneumonia, etc.)
8
22
7
Reason for enrollment in the study
HPV-compatible lesion (A)
Sexual partner with HPV-compatible lesion (B)
More than three sexual partners in the last year (C)
A+B
A+C
B+C
A+B+C
6
3
17
2
5
3
1
185
This could be due to the fact that the primers used in this
study detected the mucosal HPV types (1). This may indicate that the lesions were caused by non-mucosal HPV
types, since the β-globin gene was the one amplified in
all the samples. Another hypothesis is that the lesions
could have been caused by another etiologic agent, since
they were classified as HPV-compatible, always under a
clinical criterion.
The largest number of patients presenting lesions was
observed in the compatible range of 21 to 30 years of
age (57%), which correlates with the positive results for
HPV (28.6%).
Several articles have reported the detection of HPV
in men, whose infection rates range between 1.0% and
82.9% (3) depending on the population studied, the
anatomic site of the sample, the collection method and
the sensitivity of the technique for the detection of HPV
(6, 7, 9).
In the present work, the percentage of patients who
attended the Infectology Service presenting an HPV infection was 24.4%, a value comparable to the 33% found
in the males attending a private hospital specialized in
sexually transmitted diseases in the USA (15). It is worth
pointing out that the methodology used, the population
included and the type of sample obtained were similar in
both studies.
Table 2 shows that HPV-positive patients, with more
than three sexual partners in the year previous to our
study, reported not having used a condom (never / sometimes) in any of their sexual encounters. If we point out
that all of them had at least a high school degree, we
could conclude that the educational background does not
guarantee the knowledge and application of the measures to prevent a sexually-transmitted infection.
This table also shows that three out of the six patients
presenting an HPV-compatible lesion who had attended
the Infectology Service for other reasons tested positive.
It is interesting to point out that one of them (patient No.
4) presented a mixed infection, whose genotypes belonged to the high risk group. This patient had had sexual
intercourse with the opposite sex, which indicates the lack
of knowledge of the population of the risks of this infection, not only for themselves, but also for their sexual
partner(s). This result is very important for the purposes
of determining the guidelines for the prevention of HPV
infection.
Table 2 also indicates that of all the genotypes identified (6, 11, 16, 18, 26, 44, 45, 59, 61 and 68), the most
frequent ones were 18 and 61, and shows that in patients with condylomata acuminata, only the low-risk HPV
genotypes 61 and 11 were detected.
Of the three patients from whom both kinds of samples were taken, only one (patient n° 6) was positive
(genotype 18-68) and corresponded to the sample taken
from the perianal region.
Reason for
Finished
high school
Educational
background
16-45
[C2]
Yes
high school
Finished
Never
>5
Heterosexual
2 months
Penis surface
61
Yes
education
tertiary level
Finished
Sometimes
>5
Homosexual
6 months
18-68
[C3]
Yes
university
Finished
Always
1
Homosexual
1 month
Perianus
Flat wart
2
lesion
Compatible
A
52
6
18
[C2]
Yes
education
tertiary level
Finished
Never
>10
Homo sexual
2 months
Perianus
Flat wart
2
lesion
Compatible
A+B+C
30
7
26-59
[C2]
Yes
high school
Finished
Always
1
Homosexual
0.5 months
Penis surface
Flat wart
1
check up
Clinical
A
37
8
11
[C3]
Yes
university
Did not finish
Sometimes
1
Homosexual
12months
Perianus
acuminata
Condyloma
2
lesion
Compatible
A
47
9
Reason for enrollment in the study: (A) Lesion compatible with HPV, (B) sexual partner with lesion compatible with HPV, (C) more than three sexual partners. (2)Type of sample: (1) urethral swab/penile brushing,
(2) anal/perianal brushing
(1)
61-18
6-61
44
Genotype
Yes
education
tertiary level
Finished
Always
1
Homosexual
1 month
[A3]
—
high school
Finished
Never
>3
Heterosexual
2 months
HIV carrier (CDC)
—
Always
Use of condom
Classification of
1
Heterosexual
Sexual preference
No. of patners
2 months
Time of the lesion
Penis surface
Condyloma
2
lesión
Compatible
A+C
45
5
Perianus
Glans
Flat wart
1
Other
A+C
30
4
Lesion location
Flat wart
1
check up
Clinical
A
25
3
acuminata
Flat wart
1
lesion
Compatible
A+B
31
2
the lesion
Glans
Flat wart
Characteristics of
(2)
1
lesion
Type of sample
Compatible
Reason for
A+B
23
1
consultation
study
enrolment in the
(1)
Age
patients
HPV-positive
Table 2. Characteristics of HPV-positive patients
186
Revista Argentina de Microbiología (2010) 42: 184-188
HPV Detection and genotyping in males from the city of Córdoba, Argentina
Regarding the genotype frequency, no significant differences between the low-risk and the high-risk HPV types
were observed. It can be noticed that every two HPVpositive patients with compatible lesions, one could have
at least one infection caused by a high-risk genotype.
Likewise, 80% of the patients with an AIDS diagnosis by
the presence of the disease marker (C2 and C3) were
positive for at least one high-risk genotype, although for
HIV-carriers any genotype should be considered high risk.
It should also be noted that, since the implementation of
antiretroviral therapy has failed to reduce the incidence
of some cancers caused by HPV (10), all the necessary
187
precautions had been taken in the Infectology Service
where this study was carried out.
The RFLP analysis of three (HIV positive) out of the
seven HPV positive-patients, showed DNA bands that
were not attributed to other genotypes present. Since a
significant association with the presence of multiple HPV
infections in HIV patients has been reported (8), this
result suggests that some of them may have had co-infections
with other HPV genotypes. Importantly, the two HCVpositive patients were negative for HPV detection.
HPV detection was significantly associated with two
factors: AIDS and the HPV-compatible lesion, which is in
Table 3. Frequency of detection of HPV vs risk factors
Total
Total N° of patients
HIV: YES
AIDS
Yes
No
HIV: NO
Sexual preference
Homosexual
Heterosexual
Bisexual
LESION
Yes
No
Partner with lesion
Yes
No
Use of condom
Always
Never
Sometimes
N° of partners
1
>3
>5
>10
>20
Educational background
Did not finish elementary school
Finished elementary school
Did not finish high school
Finished high school
Did not finish tertiary level education
Finished tertiary level education
Did not finish university
Finished university
HPV - (%)
HPV + (%)
37
30
28
23
(75.6)
(76.7)
9 (24.4)
7 (23.3)
16
14
7
10
13
5
(62.5)
(92.9)
(71.4)
6 (37.5)
1 (7.1)
2 (28.6)
24
10
3
19
7
2
(79.0)
(70.0)
(66.7)
5 (21.0)
3 (30.0)
1 (33.3)
14
23
5 (35.7)
23 (100.0)
9 (64.3)
0 (0.0)
9
28
6
22
(66.7)
(78.6)
3 (33.3)
6 (21.4)
17
6
14
13
3
12
(76.5)
(50.0)
(85.7)
4 (23.5)
3 (50.0)
2 (14.3)
p
0.77110
0.04980
0.56580
0.00001
0.46890
0.23210
0.28000
11
10
4
8
4
6 (54.5)
9 (90.0)
3 (75.0)
6 (75.0)
4 (100.0)
5
1
1
2
0
(45.5)
(10.0)
(25.0)
(25.0)
(0.0)
1
3
2
13
1
6
5
6
1
3
2
9
1
3
4
5
0
0
0
4
0
3
1
1
(0.0)
(0.0)
(0.0)
(30.8)
(0.0)
(50.0)
(20.0)
(16.7)
0.66800
(100.0)
(100.0)
(100.0)
(69.2)
(100.0)
(50.0)
(80.0)
(83.3)
188
Revista Argentina de Microbiología (2010) 42: 184-188
agreement with the results reported by other authors (2,
12). The presence of HPV in patients with an AIDS diagnosis could be due to the immunosupression caused by
HIV, which may lead to a higher degree of HPV replication, a higher viral load and, consequently, an increase in
epithelial mucosal lesions (12).
In the present work, we found no significant association between HIV and HPV infections, which may be
mainly due to a difference in the number of HIV carriers
(n = 30) and non-carriers (n = 7) included in this study
(Table 3).
The understanding of HPV infection in males is important in order to reduce transmission to women as well as
for its possible association with penis and anus cancer.
According to the results obtained in this study, the preventive measures in this age group (21-30 years old)
should be emphasized.
Our work is the first of its kind in the city of Córdoba
and contributes relevant data to the knowledge of HPV
infection and to the possible implementation of measures
to prevent HPV in the male population.
Giuliano A. Prevalence of HPV infection among men: a
systematic review of the literature. J Infect Dis 2006; 194:
1044-57.
Franceschi S, Castellsagué X, Dal Maso L, Smith JS,
Plummer M, Ngelangel C, et al. Prevalence and determinants of human papillomavirus genital infection in men. Br
J Cancer 2002; 86: 705-11.
Giovannelli L, Migliore MC, Capra G, Caleca MP, Bellavia
C, Perino A, et al. Penile, urethral, and seminal sampling
for diagnosis of human papillomavirus infection in men. J
Clin Microbiol 2007; 45: 248-51.
Grupo de estudio de VPH en hombres de Brasil-Estados
Unidos-México. Human Papillomavirus infection in men
residing in Brazil, Mexico, and the USA. Salud Pública Mex
2008; 50: 408-18.
Lajous M, Mueller N, Cruz-Valdéz A, Aguilar LV, Franceschi
S, Lazcano-Ponce, et al. Determinants of prevalence, acquisition, and persistence of human papillomavirus in healthy
mexican military men. Cancer Epidemiol Biomarkers Prev
2005; 14: 1710-6.
Müller EE, Chirwa TF, Lewis DA. Human papillomavirus
(HPV) infection in heterosexual South African men attending sexual health services: associations between HPV and
HIV serostatus. Sex Transm Infect. 2010; 86: 175-80.
Nielson CM, Flores R, Harris RB, Abrahamsen M,
Papenfuss MR, Dunne EF, et al. Human papillomavirus
prevalence and type distribution in male anogenital sites
and semen. Cancer Epidemiol Biomarkers Prev 2007; 16:
1107-14.
Palefsky J. Human papillomavirus-related disease in people with HIV. Curr Opin HIV AIDS 2009; 4: 52-6.
Partridge JM, and Koutsky LA. Genital human papillomavirus in men. Lancet Infect Dis 2006; 6: 21-31.
Sirera G, Videla S, Herranz P, Corzo-Delgado J. Human
papilloma virus and HIV/AIDS. Enferm Infecc Microbiol Clin
2006; 2: 40-6.
Watson R. Human Papillomavirus: Confronting the epidemic. A urologist’s perspective. NJ Rev Urol 2005; 7:
135-44.
Weaver BA. Epidemiology and natural history of genital human papillomavirus infection. J Am Osteopath Assoc 2006;
106: 2-8.
Weaver BA, Feng Q, Holmes KK, Kiviat N, Lee SK, Meyer
C, et al. Evaluation of genital sites and sampling techniques
for detection of human papillomavirus DNA in men. J Infect
Dis 2004; 15: 677-85.
Acknowledgements: We thank all the patients enrolled in
this study and Drs Fanny N. Cohen, Carlos M. Quinteros and
Luis Allende, from the Infectology Service at the Hospital Nacional de Clínicas in Córdoba, Argentina, for their invaluable
contribution to this study.
4.
5.
6.
7.
8.
9.
10.
11.
12.
REFERENCES
1. Bernard H, Chan SY, Manos MM, Ong CK, Villa LL, Delius
H, et al. Identification and assessment of known and novel
human papillomaviruses by polymerase chain reaction
amplification, restriction fragment length polymorphisms,
nucleotide sequence, and phylogenetic algorithms. J Inf Dis
1994; 170: 1077-85.
2. Chan PK, Luk AC, Luk TN, Lee KF, Cheung JL, Ho KM, et
al. Distribution of human papillomavirus types in anogenital
warts of men. J Clin Virol 2009; 44: 111-4.
3. Dunne EF, Nielson CM, Stone KM, Markowitz LE, and
13.
14.
15.
Recibido: 06/10/09 – Aceptado: 05/05/10