Journal of Medical Virology 85:288–294 (2013)
Infection With High-Risk HPV Types Among Female
Sex Workers in Northern Vietnam
Huyen Thi Thanh Hoang,1,2,3Azumi Ishizaki,1Cuong Hung Nguyen,1,2Vuong Thi Tran,1,2
Kaori Matsushita,1Kunikazu Saikawa,4Norimitsu Hosaka,1,5Hung Viet Pham,1Xiuqiong Bi,1
Van Thanh Ta,3Thuc Van Pham,1and Hiroshi Ichimura1*
1Department of Viral Infection and International Health, Graduate School of Medical Science, Kanazawa University,
2Hai Phong Medical University, Hai Phong, Vietnam
3Hanoi Medical University, Hanoi, Vietnam
4Department of Human Pathology, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
5Eiken Chemical Co., Ohtawara, Tochigi, Japan
Vaccines against two high-risk human papillo-
mavirus (HPV) types, HPV-16, and HPV-18,
are in use currently, with high efficacy for pre-
venting infections with these HPV types and
consequent cervical cancers. However, circulat-
ing HPV types can vary with geography and
ethnicity. The aim of this study was to investi-
gate the prevalence of HPV types and the asso-
cervical cytology among female sex workers in
Northern Vietnam. Cervical swabs and plasma
samples were collected from 281 female sex
workers at two health centers in Hanoi and Hai
Phong in 2009. The HPV L1 gene was amplified
by PCR using original and modified GP5þ/6þ
primers. Amplified PCR products were geno-
typed by the microarray system GeneSquare
(KURABO) and/or clonal sequencing. Of the 281
women, 139 (49.5%) were positive for HPV
DNA. Among the HPV-positive samples, 339
strains and 29 different types were identified.
Multiple-type and high risk-type HPV infections
were found in 85 (61.2%) and 124 (89.2%) wom-
en, respectively. The most common genotype
was HPV-52, followed by HPV-16, HPV-18, and
detected in 3.2% (9/281) of the women, and all
of these samples were positive for HPV-DNA.
Age ?25 years and infection with human
immunodeficiency virus were associated posi-
tively with HPV infection among the women
while ever smoking was associated negatively.
These results show that HPV-52 is most preva-
lent among female sex workers in Northern
Vietnam, most of whom had normal cervical
cytology. This information may be important
for designing vaccination strategies in Vietnam.
J. Med. Virol. 85:288–294, 2013.
? 2012 Wiley Periodicals, Inc.
HPV prevalence; vaccine; cervi-
cal cancer; Vietnam
Genital human papillomavirus (HPV) infection is
the most common infection transmitted sexually
among women and the main cause of cervical cancer
worldwide, especially in developing countries, where
85% of cervical cancer cases occur [Ferlay et al.,
2010]. There were an estimated 529,000 new cases
and 275,000 cervical cancer—related deaths globally
in 2008, including 312,000 (59%) new cases in Asia,
and HPV infections are prevalent particularly in
South and Southeast Asia [Ferlay et al., 2010; WHO,
2010]. Cervical cancer screening using cytological
testing and HPV vaccination are of paramount impor-
tance for preventing cervical cancer in young women.
HPV belongs to the family papillomaviridae. More
than 100 distinct HPV genotypes have been charac-
terized molecularly, and about 40 HPV types have
been identified in the mucosal epithelia of the hu-
man genital tract [Munoz et al., 2006]. Cervical can-
cer is caused by HPV types that belong to a few
‘‘high-risk’’ species of the mucosotropic alpha genus,
such as alpha-5, -6, -7, -9, and -11 [Bouvard et al.,
2009; Schiffman et al., 2010]. Eight HPV types
(HPV-16, -18, -31, -33, -35, -45, -52, and -58) are
*Correspondence to: Hiroshi Ichimura, MD, PhD, Department
of Viral Infection and International Health, Graduate school of
Medical Science, Kanazawa University, 13-1 Takaramachi,
Kanazawa 920-8640, Japan.
Accepted 12 September 2012
Published online 14 November 2012 in Wiley Online Library
? 2012 WILEY PERIODICALS, INC.
observed most frequently and are responsible for
about 90% of all cases of cervical cancer worldwide
[Munoz et al., 2006]. In particular, HPV-16 and
HPV-18 are observed in 70% of cervical cancer
et al., 2006].
HPV-16 and HPV-18 hold great promise for reducing
the global burden of cervical cancer [Harper et al.,
2006; Wheeler, 2007]. However, circulating HPV
types can vary by geography and ethnicity, and the
current vaccine formulary for these two high-risk
types is less effective against some other oncogenic
HPV types, although a recent study has shown
cross-protective efficacy of the HPV-16/18 vaccine
against oncogenic HPV types such as HPV-31, HPV-
33, HPV-45, and HPV-51 [Wheeler et al., 2012]. In
Europe and America, HPV-16 and HPV-18 are the
most common HPV types [Clifford et al., 2005]
whereas in Asia, in addition to HPV-16, HPV-52,
and HPV-58 are most common [Bao et al., 2008].
Particularly in Japan, the Philippines, Taiwan, and
the Zhejiang province of southeast China, HPV-52 is
reported to be the HPV type identified most fre-
quently [Lin et al., 2006; de Sanjose ´ et al., 2007;
Miyashita et al., 2009; Ye et al., 2010]. Thus, an un-
derstanding of the geographical distribution of HPV
types is necessary to estimate vaccine efficacy accu-
rately and prevent HPV infection and the subse-
quent development of cervical cancer.
In Vietnam, more than 6,000 new cases of cervical
cancer (incidence rate: 11.7 per 100,000 women per
year) and 3,000 cervical cancer—related deaths are
estimated to occur each year. Cervical cancer ranks as
the second most common cancer in women ages 15–45
years [Domingo et al., 2008]. Screening for cervical
cancer with the Pap smear test and HPV DNA detec-
tion are not available widely in Vietnam [WHO,
2002]. Previous studies identified HPV-16 and HPV-
58 as the most common high-risk HPV types in a gen-
eral population of Vietnamese women [Pham et al.,
2003; Domingo et al., 2008] while another study
reported that HPV-52 was the most common type
among female sex workers in Southern Vietnam
[Hernandez et al., 2008]. However, population-based
information on the distribution of HPV types among
Vietnamese women is limited still [Bao et al., 2008].
In the current study, the prevalence of HPV infection,
the distribution of HPV types, and risk factors for
HPV infection among female sex workers in Northern
Vietnam were determined. The association between
HPV types and abnormal cervical cytology was also
et al.,2003; Clifford
SUBJECTS AND METHODS
Subjects and Sample Collection
A cross-sectional survey of HPV infection and geno-
type distribution among female sex workers in North-
ern Vietnam was conducted from June to November
27.6 ? 8.0 years) had been commercial sex workers
previously and were concentrated in two rehabilita-
tion centers in Hanoi and Hai Phong, the largest cit-
ies in Northern Vietnam. They were recruited after
giving written informed consent. A gynecological ex-
amination was performed, and two cervical-swab sam-
ples were collected using a cervical brush (Honest
Uterine Cervical Brushes; Honest Medical, Tokyo,
Japan). The cervical swabs were smeared onto a slide,
fixed with alcohol solution (Rapid Fix; Muto, Tokyo,
Japan), and stained according to standard procedures
for the Pap smear test. The remainder of each sample
was suspended in 1 ml of lysis buffer (TBE buffer,
50 mM Tris–HCl, 5 mM EDTA, 2% SDS) and stored
at ?808C until use. Sociodemographic information
was collected using questionnaires. Blood samples
were collected and plasma samples stored at ?808C
until use. The study protocol was reviewed and ap-
proved by the ethics committees of Hanoi Medical
The281participants(meanage ? SD:
Genomic DNA was extracted from cervical cells in
lysis buffer using a DNA extraction kit (SMI Test;
Genome Science Laboratories, Fukushima, Japan)
according to the manufacturer’s instructions. The
quality of the extracted DNA was evaluated by ampli-
fying the glyceraldehyde-3-phosphate dehydrogenase
et al., 2002]. All extracted DNA samples were con-
firmed as adequate for HPV, Chlamydia (C.) tracho-
matis, and Neisseria (N.) gonorrhoeae testing.
HPV DNA was detected by PCR using three pairs
of modified GP5þ/6þprimers: GP5þM1-2 (50-TTTRTT-
ACTGTTGTWGATACTAC-30) and GP5þM2-2 (50-TG-
TWACTGTTGTWGATACCAC-30); GP5þM3-2 (50-GT-
WACTGTTGTRGACACCAC-30) and GP6þM1-2 (50-AA
[Yamada et al., 2008; Miyashita et al., 2009]. These
modified GP5þ/6þprimers were designed to minimize
mismatches between primer sequences and comple-
ment target HPV L1 genes and to amplify a 140-bp
fragment of the HPV L1 gene. Amplification was per-
formed as follows: one cycle at 958C for 10 min, fol-
lowed by 45 cycles at 958C for 30 sec, 458C for 30 sec,
and 748C for 30 sec, with a final extension at 748C for
10 min. The presence of HPV DNA was confirmed by
ethidium bromide staining of the PCR products fol-
lowing agarose gel electrophoresis. PCR was repeated
using the original GP5þ/GP6þprimers for the HPV
DNA-negative samples [de Roda Husman et al.,
HPV Infection in Female Sex Workers in Vietnam 289
J. Med. Virol. DOI 10.1002/jmv
HPV genotyping was performed with a DNA micro-
(KURABO, Okayama, Japan), which uses multiplex
PCR targeting different genes from type to type [Ermel
et al., 2010]. The sensitivity and specificity of the
GeneSquare is equal reportedly to that of the Roche
Linear Array HPV Genotyping Assay. The GeneSquare
microarray contains 23 type-specific probes: HPV-6,
-11, -16, -18, -30, -31, -33, -34, -35, -39, -40, -42, -45,
-51, -52, -53, -54, -56, -58, -59, -61, -66, and -68.
The original and/or modified GP5þ/6þPCR products
of the samples that were not genotyped by the GeneS-
quare microarray were cloned using the TOPO TA
Cloning Kit (Invitrogen, Carlsbad, CA) and sequenced
according to the manufacturer’s instructions. The sim-
ilarity between the L1 sequences obtained from the
PCR products and those of various HPV genotypes
registered in the GenBank database was determined
HPV types that belong to the mucosotropic alpha
genus were classified as high-risk types (HPV-16, -18,
-31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -68) or
possibly high-risk types (HPV-26, -53, -66, -67, -69, -
70, -73, -82, -85, and -97) according to the classifica-
tion of the International Agency for Research on Can-
cer [Bouvard et al., 2009], or as low-risk types (HPV-
6, -11, -40, -42, -43, -44, -54, -55, -61, -62, -72, -81, -83,
-84, and -89) [Munoz et al., 2003] or unknown-risk
types (HPV-2, -3, -7, -10, -13, -27, -28, -29, -30, -32, -
34, -55, -57, -62 -71, -74, -77, -78, -86, -87, -90, -91, -
94, -102, -106, and JBE2) [Schiffman et al., 2010].
Detection of Infections Transmitted
Sexually or Blood Borne
Anti-human immunodeficiency virus (HIV) anti-
body, anti-hepatitis C virus (HCV) antibody, and hep-
atitis B virus surface antigen (HBsAg) were tested
using serological test kits (Abbott, Tokyo, Japan).
C. trachomatis and N. gonorrhoeae were detected
method [Hong et al., 2004; Poon et al., 2005].
Classification of Cervical Cytology
The Bethesda Reporting System 2001 was used to
classify cervical cytology [Solomon et al., 2002] as nor-
mal (negative for intraepithelial lesion or malignan-
cy), atypical glandular cells/atypical squamous cells of
undetermined significance, low-grade squamous intra-
epithelial lesion, high-grade squamous intraepithelial
lesion, or adenocarcinoma in situ.
Statistical analysis was performed using SPSS Ver-
sion 19.0 for Windows. The chi-square test and/or
Fisher’s exact test were used for comparisons between
HPV DNA-positive and -negative groups, and univari-
ate analysis was performed to assess the association
between HPV DNA-positive results and risk factors or
other infections. A multivariate analysis was per-
formed by using a stepwise binary logistic regression
model to confirm the association. P-values of ?0.05
were considered to indicate statistical significance.
Profile of Cervical HPV Infection
Of the 281 women, 139 (49.5%) were positive for
HPV DNA. Of these, 54 (38.8%) were infected with a
single HPV type, and 85 (61.2%) were infected with
multiple types. Multiple-type HPV infection was more
common in women ?25 years than in those >25 years
old (OR: 2.1; 95% CI: 1.3–2.6), and also more common
in women infected with HIV than in women without
HIV infection (OR: 2.2; 95% CI: 1.1–4.4). From the 139
HPV DNA-positive samples, 339 HPV strains and 29
different genotypes were isolated (Fig. 1). Of the 339
Fig. 1. Prevalence of HPV genotypes among female sex workers in Northern Vietnam.
290Hoang et al.
J. Med. Virol. DOI 10.1002/jmv
strains, 228 (67.3%) were high-risk, 27 (8.0%) possible
high-risk, 54 (15.9%) low-risk, and 30 (8.8%) unknown-
risk HPV types. Infection with high-risk HPV types
was found in 89.2% (124/139) of the women infected
with HPV. Among the high-risk HPV types, HPV-52
was the most common type (28.1%; 39/139), followed by
HPV-16 (18.7%; 26/139), HPV-18 (16.5%; 23/139),
HPV-51 (16.5%; 23/139), and HPV-58 (16.5%; 23/139).
Prevalence of Infections Transmitted
Sexually or Blood Borne
The 281 women were tested for infections transmit-
ted sexually or blood borne, such as infections with
HIV, HBV, HCV, N. gonorrhoeae, and C. trachomatis.
Of the 281 women, 177 (63.0%) had at least one infec-
tion. The prevalence of these infections was 12.8%
(36/281) for HIV-1, 6.8% (19/281) for HBV, 18.5% (52/
281) for HCV, 1.4% (4/281) for N. gonorrhoeae, and
6.8% (19/281) for C. trachomatis.
Risk Factors for Cervical
To determine the risk factors associated with cervi-
cal HPV infection, a univariate analysis was per-
formed. Age ?25 years (OR: 2.2; 95% CI: 1.4–3.6),
being single (OR: 1.9; 95% CI: 1.2–3.1), smoking (OR:
0.6; 95% CI: 0.3–0.9), and HIV infection (OR: 4.2; 95%
CI: 1.2–3.1) were associated significantly with HPV
infection (Table I). When all of the variables were ad-
justed for multivariate analysis, age ?25 years (OR:
2.3; 95% CI: 1.4–3.9), smoking (OR: 0.5; 95% CI: 0.3–
0.8), and HIV infection (OR: 7.9; 95% CI: 3.1–20.2)
were confirmed as independent factors predicting
high-risk HPV infection.
Association Between High-Risk HPV Types
and Abnormal Cervical Cytology
Of the 281 women, 272 (96.8%) had normal cervical
cytology, and nine (3.2%) had abnormal cervical cytol-
ogy (four with atypical glandular cells/atypical squa-
mous cells of undetermined significance, four with a
low-grade squamous intraepithelial lesion, and one
with a high-grade squamous intraepithelial lesion)
with the Pap smear test. HPV DNA was detected in
the four women with atypical glandular cells/atypical
squamous cells of undetermined significance, as well
as in the five women with abnormal cervical cytology.
All of them were infected with high-risk HPV types
The sequences described in this report have been
deposited in GenBank/EMBL/DDBJ under accession
In the current study, the prevalence of cervical
HPV infection among female sex workers in Northern
Vietnam was 49.5%. This value is lower than the
prevalence reported for a similar population in South-
ern Vietnam (85%) in 2008 [Hernandez et al., 2008].
The difference may reflect the previous finding that
HPV prevalence among the general population of
women in Southern Vietnam is fivefold higher than in
Northern Vietnam [Pham et al., 2003]. Nevertheless,
the HPV prevalence in this study is similar to that
among female sex workers in other Asian countries,
such as the Philippines (57.2%) [Miyashita et al.,
2009], Japan (52.6%) [Matsushita et al., 2011], and
Korea (47%) [Choi et al., 2003]. A similar HPV preva-
lence among female sex workers was reported in
Kenya (55.6%) [Luchters et al., 2010], Tunisia (44.1%)
[Znazen et al., 2010], Peru (50.6%) [Montano et al.,
2011], and Mexico (48.9%) [Jua ´rez-Figueroa et al.,
2001]. A lower HPV prevalence among female sex
workers was reported in Spain (39%) [del Amo et al.,
2005] and Australia (32%) [Tideman et al., 2003].
HPV-52 was found to be the most prevalent HPV
type among female sex workers in Northern Vietnam,
most of whom had normal cervical cytology. This re-
sult is consistent with those of a previous study of fe-
male sex workers in Southern Vietnam [Hernandez
et al., 2008], as well as with results from other Asian
regions, such as South Taiwan [Lin et al., 2006], the
Philippines [Miyashita et al., 2009], and Japan [Mat-
sushita et al., 2011]. These findings suggest that
HPV-52 is common in Asian countries in general, al-
though HPV-16 has been reported to be the most
prevalent type in Asia except for Japan and Taiwan
[de Sanjose ´ et al., 2007; Bruni et al., 2010].
It was reported previously that HPV-16 is the most
common HPV type among the general population of
Vietnamese women; those results were obtained using
the original GP5þ/GP6þprimers and an enzyme im-
munoassay for genotyping [Pham et al., 2003]. The
difference between these previous results and those
reported here might result from the primer set used
for HPV PCR. Although the original GP5þ/6þprimer
set has been used in many epidemiological studies, it
does not amplify HPV-52 as effectively as HPV-16 and
HPV-18 because of sequence mismatches between the
target gene and the primers [Yamada et al., 2008,
Miyashita et al., 2009]. Thus, studies using only the
GP5þ/6þprimer set could have underestimated HPV-
52 prevalence. In this study, modified GP5þ/6þprimer
sets were used together with the original set to broad-
en the spectrum of detectable HPV types [Miyashita
et al., 2009].
In the current study, the risk of HPV infection was
significantly higher in women ?25 years than in wom-
en >25 years of age (OR: 2.1, 95% CI: 1.4–3.9). This
finding is consistent with previous reports that the
prevalence of HPV is age dependent, with a peak in
young women after the onset of sexual activity
[Molano et al., 2003; Miyashita et al., 2009]; one ex-
planation might be that in young women, there is a
higher probability of exposure to HPV and less ac-
quired immunity to HPV from past exposure. Thus,
HPV Infection in Female Sex Workers in Vietnam291
J. Med. Virol. DOI 10.1002/jmv
younger women would benefit more from HPV vacci-
Smoking increases the risk of squamous-cell carci-
noma of the cervix. However, previous studies have
found a negative [Ho et al., 1998], positive [Minkoff
et al., 2004; Pista et al., 2012], or null [Vaccarella
et al., 2008] association between smoking and HPV
infection over time. In this study, ever smoking was
associated with a lower risk of HPV infection,
against HPV infection and whether it is a biologic or a
confounding effect are unknown.
Among the female sex workers in Northern Viet-
nam, HPV infection was the most prevalent (49.5%),
followed by HCV (18.5%), HIV (12.8%), HBV (6.8%),
C. trachomatis (6.8%), and N. gonorrhoeae (1.4%)
TABLE I. Risk Factors Associated With HPV Infection
HPV DNA (þ)
1.4–3.60.001 2.3 1.4–3.90.001
Age at first sexual
Education level 0.7–2.0
Pregnancy history0.4–1.2 0.183
Contraception use 114
Condom use 0.5–1.30.476
Smoking 0.3–0.90.025 0.50.3–0.8 0.006
N. gonorrhoeae43 0.3–30.30.367
FSW, female sex workers; HBV, hepatitis B virus; HCV, hepatitis C virus; HIV, human immunodeficiency virus; HPV, human
aOdds ratio adjusted for all variables.
TABLE II. Relationship Between HPV Genotypes and Cervical Cytology in Female Sex Workers in Northern Vietnam
Cervical cytology (n)Sample ID AgeSmokingInfection
Low riskHigh risk
cells of undetermined
HIV, C. trachomatis
16, 33, 51, 52, 53, 58
6, 42, 54 35, 51, 68
C. trachomatis 40
HIV, human immunodeficiency virus; HPV, human papillomavirus; HCV, hepatitis C virus; N. gonorrhoeae, Neisseria gonorrhoeae; C. tra-
chomatis, Chlamydia trachomatis.
292Hoang et al.
J. Med. Virol. DOI 10.1002/jmv
infections. Of these infections, only HIV infection had
a significantly higher association with HPV infection
in the women in the current study (OR: 7.9; 95% CI:
3.1–20.2), which is consistent with previous reports
that HIV-related immunosuppression increases the
risk of genital HPV infection and affects HPV replica-
tion [Ho et al., 1994]. Humoral immune responses
are altered within a few months after HIV infection
[Marais et al., 2009], which may reduce the ability
of women infected with HIV to produce HPV-specific
secretory IgA antibodies, resulting in an increase in
the HPV infection rate. It was also reported that HPV
infections are more likely to persist in women infected
with HIV compared to women not infected with HIV
[Clifford et al., 2006; Luchters et al., 2010].
In this study, the Pap smear test revealed that only
nine women (3.2%) had ‘‘abnormal’’ cervical cytology
(four with atypical glandular cells/atypical squamous
cells of undetermined significance, four with a low-
grade squamous intraepithelial lesion, and one with a
high-grade squamous intraepithelial lesion). The fre-
quency of abnormal cervical cytology in this study is
much lower than that among female sex workers in
the Philippines (15.2%) [Miyashita et al., 2009] and
Japan (12.8%) [Matsushita et al., 2011]. The differ-
ence might be due to whether those study subjects
were active female sex workers (in the studies of the
Philippines and Japan) or not (in this study). Howev-
er, considering that the HPV prevalence in this study
is similar to that among female sex workers in those
previous studies [Miyashita et al., 2009; Matsushita
et al., 2011], the possibility that collection and fixation
of cervical swab samples were done under limited con-
ditions, which affected the Pap smear analysis, could
not be excluded completely in this study. All nine of
the women with abnormal cervical cytology were
infected with one or more high-risk HPV types. Thus,
the correlation between infection with high-risk HPV
types and abnormal cervical cytology was confirmed.
Although population-based Pap smear screening has
been introduced in Vietnam, only 4.9% of the general
population of women ages 18–69 years have under-
gone the screening [WHO, 2010], and cervical cancer
has become progressively a leading cause of cancer-re-
lated death among women in Vietnam [Domingo
et al., 2008]. Considering these data, an active pro-
gram is needed to control cervical cancer effectively
using not only the Pap smear test but also the cervical
HPV DNA test in Vietnam.
In conclusion, HPV-52 was the most prevalent high-
risk HPV type among female sex workers in Northern
Vietnam, most of whom had normal cervical cytology.
Age ?25 years and HIV infection were associated pos-
itively with HPV infection among the women and ever
smoking was associated negatively. These findings
suggest that the current HPV vaccines targeting
HPV-16 and HPV-18 may not be sufficient to prevent
infection with high-risk HPV types in this region.
Thus, second-generation HPV prophylactic vaccines
that include HPV-52 might be necessary to prevent
HPV infection in Northern Vietnam. However, the
clinical relevance of the use of new vaccines that in-
clude other high-risk types such as HPV-52 was not
demonstrated in this study. Even though HPV-52 was
the most prevalent type, it was not found to be related
to a high-grade squamous intraepithelial lesion, as
was HPV-16. It is, therefore, important to investigate
the prevalence of HPV-52 among patients with cervi-
cal cancer in this geographic area, which is ongoing.
We are grateful to all of the participants in this
study; to Dr. Lihana of Kanazawa University; and
to the staff (Ms. Thuy, Ms. Thanh, Ms. Xuan,
Ms. Huong V.T., Ms. Ngoc, Ms Binh, Mr. Huy) of Hai
Phong Medical University.
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