Prevalence and genotypes of human papillomavirus among Thai women.

Abstract

One of the most common cancers in women worldwide is cervical cancer, with death rates highest in less developed countries, including Thailand. This study was conducted to explore the prevalence of human papillomavirus (HPV) and its related cytological abnormalities among women attending cervical screening clinics in Thailand using the polymerase chain reaction (PCR). LBC specimens (ThinPrep, Hologic, West Sussex, UK) were subjected to PCR of the E1 region to identify the most prevalent HPV types. Information on age and cytology grade was also collected. Among a total of 1,662 women, 29 different HPV types were found and the overall HPV prevalence was 8.7%. HPV prevalence among the general population amounted to 7.8%. The following HPV types were identified: HPV16 (17.9%), HPV90 (16.6%) and HPV71 (10.3%). The rates of other types were as follows; HPV66 (6.9%), HPV52 (6.2%), HPV34 (5.5%), HPV31 (5.3%), HPV42 (4.8%) and HPV39 (3.4%). HPV infection peaked in women aged around 20-39 years and thereafter gradually declined. As expected, HPV DNA can be found in normal cytology specimens. These results which elucidate HPV distribution in Thailand could be useful for vaccine development and the national cervical cancer prevention program.

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Asian Pacific Journal of Cancer Prevention, Vol 11, 2010 117
Prevalence and Genotypes of Human Papillomavirus among Thai Women
Asian Pacific J Cancer Prev, 11, 117-122
Introduction
More than 400,000 women throughout the world are
affected by cervical cancer every year, the second most
malignant cancer after breast cancer (Gheit et al., 2006).
Results obtained from epidemiological studies associate
high-risk genotypes of human papillomavirus (HPV) with
cervical carcinoma and malignant transformation of
cervical epithelial cells. Recent research has suggested
that HPV DNA can be detected in more than 90% of
cervical cancers, while some studies have demonstrated
HPV in all cases of cervical cancer (Hadzisejdic et al.,
2006). HPV is classified as either cutaneous or mucosal
depending on the type of epithelium host cells. The
mucosal types are sub-divided depending on their
oncogenic potential. Low-risk types such as HPV6 and
HPV11 cause benign warts, whereas the high-risk types
HPV16 and HPV18 are associated with female malignant
disease of the lower genital tract (Crosbie and Kitchener,
2006).
The second most common cancer in women
worldwide, cervical cancer contains DNA sequences from
a high-risk oncogenic genital HPV. HPV16 and HPV18
are the most prevalent types found in 50–70% and 7–20%
of cases, respectively (Parkin and Bray, 2006). Cervical
1Center of Excellence in Clinical Virology, Department of Paediatrics, 2Department of Obstetrics and Gynecology, 3Department of
Pathology and Clinical Epidemiology Unit, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
*For correspondence: Yong.P@chula.ac.th
Abstract
One of the most common cancers in women worldwide is cervical cancer, with death rates highest in less
developed countries, including Thailand. This study was conducted to explore the prevalence of human
papillomavirus (HPV) and its related cytological abnormalities among women attending cervical screening
clinics in Thailand using the polymerase chain reaction (PCR). LBC specimens (ThinPrep®, Hologic, West
Sussex, UK) were subjected to PCR of the E1 region to identify the most prevalent HPV types. Information on
age and cytology grade was also collected. Among a total of 1,662 women, 29 different HPV types were found
and the overall HPV prevalence was 8.7%. HPV prevalence among the general population amounted to 7.8%.
The following HPV types were identified: HPV16 (17.9%), HPV90 (16.6%) and HPV71 (10.3%). The rates of
other types were as follows; HPV66 (6.9%), HPV52 (6.2%), HPV34 (5.5%), HPV31 (5.3%), HPV42 (4.8%) and
HPV39 (3.4%). HPV infection peaked in women aged around 20-39 years and thereafter gradually declined. As
expected, HPV DNA can be found in normal cytology specimens. These results which elucidate HPV distribution
in Thailand could be useful for vaccine development and the national cervical cancer prevention program.
Key Words: Human papillomavirus subtypes - LBC specimens - relative prevalence - Thailand
cancers are differentiated based on the spectrum of intra-
epithelial abnormalities. The spectrum in the cervix ranges
from CIN 1 (low-grade cervical intra-epithelial neoplasia)
over moderate CIN2 to high-grade CIN3. High-grade
CIN3 lesions are the necessary precursor lesions for
cervical cancer and approximately 90% of high-grade CIN
harbor high-risk HPV, with HPV16 most frequently
detected (Clifford et al., 2006). Normally, the risk of HPV
infection is age related and increases with promiscuity.
Thus, the highest incidence is observed in the 15- to 25-
year age group while it declines in women above the age
of 65. Over the last 50 years, both incidence and mortality
due to invasive cervical cancer in the United States and
in other developed countries have decreased to 70% as a
consequence of more frequent cytological screening (Pap
smear) which can facilitate detection of abnormal cervical
cytology and intraepithelial neoplasia at an early stage
and thus, result in efficient treatment. During previous
years, various organizations have provided screening
recommendations on which screening guidelines of most
primary care practices have been based (Ogunmodede et
al., 2007).
Recently, along with the advances in molecular
biology techniques, highly sensitive and effective HPV
detection tests have been developed. Among these are
RESEARCH COMMUNICATION
Prevalence and Genotypes of Human Papillomavirus among
Thai Women
Jira Chansaenroj1, Woradee Lurchachaiwong1, Wichai Termrungruanglert2,
Damrong Tresukosol2, Somchai Niruthisard2, Prasert Trivijitsilp2, Pichet
Sampatanukul3, Yong Poovorawan1*

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Jira Chansaenroj et al
hybrid capture (HC), filter in situ hybridization (FISH),
southern hybridization (SH), and polymerase chain
reaction (PCR) (Khair et al., 2009). More recent data have
demonstrated that the risk for acute infection to proceed
to malignant transformation is substantially different for
individual HR-HPV types. Several clinical and
epidemiological studies have indicated that women
without cervical abnormalities but detectable HPV16 and
18 infections tend to develop more severe preneoplastic
lesions within a shorter period of time than women
infected with other types (Vinokurova et al., 2008).
Identification of individual HPV types is essential to
investigate epidemiology and clinical characteristics of
particular types. At present, several consensus PCR
systems have been modified for use in large-scale
epidemiological studies (Jiang et al., 2006). Recently,
clinical trials have been performed to test the efficacy of
prophylactic vaccines which target the two oncogenic
types, HPV-16 and HPV-18 (Gravitt et al., 2007).
Detection of high-risk HPV types in genital specimens
has been approved in several countries for the triage of
women with a cytological diagnosis of atypical squamous
cells of undetermined significance (ASCUS) and also for
primary cervical cancer screening in women aged 30 years
and above as an adjunct to cytology (Coutlée et al., 2007).
In the management of women with ASCUS, HPV
characterization helps improve the understanding of
prevalence, individual risk stratification, persistence, re-
infection, co-infection and development of the most
effective vaccine (Gillio-Tos et al., 2006). At present, the
molecular detection and typing techniques have been
proposed to be applied instead of the current cytological
screening. The conventional methods for DNA or RNA
extraction from clinical samples present the most labor-
intensive and critical part in current diagnostic assays
(Broccolo and Cocuzza, 2008). Type-specific HR-HPV
prevalence in the population and in cervical cancer is
essential to predict the burden (De Vuyst et al., 2009).
The 8-kilo base pair genome comprises the genes E1,
E2, E4, E5, E6 and E7 which are essential for viral DNA
replication, viral gene transcription and cellular
transformation. In addition, two late (L) genes encode the
viral capsid protein. DNA replication is one of the viral
maintenance processes and thus, details of the mechanism
involved would facilitate antiviral therapy design. The
most interesting gene is E1 which is involved in all steps
of replication initiation such as origin recognition, ATP-
dependent DNA melting, and unwinding of DNA (Auster
and Joshua-Tor, 2004).
In general, there is strong evidence of variation in HPV
prevalence among the female population. Our study has
aimed at elucidating prevalence and genome distribution
of HPV DNA in hospital-based specimens in Bangkok,
Thailand by using PCR for E1 gene amplification. The
amplification assay detects target HPV-DNA from
different carcinogenic HPV types (high risk genotypes:
16, 18, 30, 31, 33, 35, 39, 42, 45, 51, 52, 56, 58, 59, 68,
73 and 82, probably high-risk genotypes: 66, low risk
genotypes: 6 and 11) These studies have improved our
knowledge and can be used for preventive and control
measures of cervical cancer in Thailand.
Asian Pacific Journal of Cancer Prevention, Vol 11, 2010
118
Subjects and Methods
Study population
This study protocol has been approved by the Ethics
Committee of the hospital and faculty of Medicine,
Chulalongkorn University. The cervical cells were
randomly obtained in the course of the patients’ routine
check up or investigation and treatment between January
2008 and December 2009. These specimens were sent as
anonymous with a coding number. The available data were
based on cytology and age at sampling. All specimens
were exclusively used for academic research.
Sample collection and preparation
All cervical cells were collected for cytology by LBC
(ThinPrep®, Hologic, West Sussex, UK). These 848
samples were obtained during hospital-based routine
check up or investigation and treatment at King
Chulalongkorn Memorial hospital, Bangkok and 852 from
Bangkok 9 International hospital, Bangkok. After
centrifugation at 4,000 rpm for 10 min the cell pellets
were separated from the supernatant and stored at -70°C
until further tested.
Cytological diagnoses
All cervical smears were analyzed by a qualified
pathologist using The Bathesda System 2001 (TBS 2001).
To prevent bias the participating patients’ personal details
were not disclosed.
DNA extraction and house keeping gene detection
DNA was extracted by organic extraction (phenol–
chloroform) of the samples. Briefly, cellular pellets were
re-suspended in 400 µl of lysis buffer. Samples were
incubated at 95˚C for 30 min, mixed for 2 min, and
digested with 50 µl of proteinase K (20µg/l). After
overnight incubation at 50∞C, samples were heated to
95˚C for 10 min to inactivate the proteinase K. Phenol–
chloroform extraction followed by high-salt isopropanol
precipitation was performed as described previously
(Broccolo et al., 2005) and purified material was re-
suspended in a final volume of 30 µl deionized water,
respectively. The γ-globin gene was selected to serve as
an internal control for DNA extraction, using conventional
PCR as a detection method. Primer sequences for the _-
globin gene have been previously described (Shadrina et
al., 2007). The reaction mixture consisted of 2 µl DNA,
0.5 µM γ-globin forward primer and γ-globin reverse
primer, 10 µl 2.5X Eppendorf masterMix (Eppendorf,
Hamburg, Germany), and nuclease-free water to a final
volume of 25 µl. The amplification reaction was performed
in a thermal cycler (Eppendorf, Hamburg, Germany) under
the following conditions: Denaturation at 94°C for 3 min,
followed by 40 amplification cycles consisting of
denaturation at 94°C for 30 sec, primer annealing at 60°C
for 30 sec, and extension at 72°C for 30 sec, and concluded
by a final extension at 72°C for 7 min.
HPV detection by PCR and direct sequencing
To detect HPV DNA the primers chosen were part of
the E1 gene sequence. Based on a previous study

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Asian Pacific Journal of Cancer Prevention, Vol 11, 2010 119
Prevalence and Genotypes of Human Papillomavirus among Thai Women
(Lurchachaiwong et al., 2009), the E1 gene can be used
for HPV genotype detection. The amplification reaction
was performed based on this previous study. To identify
HPV DNA positive samples, the PCR products were
subjected to electrophoresis in 2% agarose gel (FMC
Bioproducts, Rockland, ME). The DNA bands were
stained with ethidium bromide and visualized by UV
transillumination (Gel Doc 1000, BIO-RAD, CA). These
were further purified using the HiYield Gel/PCR DNA
Fragments Extraction kit (Bioscience) according to the
manufacturer’s specifications. The purified DNA served
as templates for DNA sequencing performed by
FirstBASE Laboratories SDNBHD (Selangor Darul
Ehsan, Malaysia).The nucleotide sequences were analyzed
in both directions using forward and reverse primers to
confirm the consistency of the sequencing result and
ensure that variations of nucleotide sequences were not
due to sequencing errors.
Sequence analysis
The resulting sequences were analyzed by BLAST and
genotyping was analyzed by comparing with the
sequences stored at
(www.ncbi.nlm.nih.gov).
GENBANK database
Statistical analysis
HPV prevalence was determined as individual
genotypes. The proportion of positive samples was
analyzed in relation to age-groups.
Results
Of a total of 1698 ThinPrep specimens collected from
hospital-based routine check-up or investigation and
treatment in Thailand, 36 had inadequate cytology results
and age. In total, 1662 specimens were included. The
average age of the participants was 43.4 years. In this
study, we used E1 primers designed for semi-nested PCR.
The PCR primers were type-specific for high-risk HPV
genotypes. Data were classified according to cytological
type, with 1,622 (97.6%) showing normal, and 40 (2.4%)
abnormal cytology.
In the single infection group, the prevalence analysis
was based on 1,662 specimens. According to the results
obtained, of those 1662, there were 145 specimens (8.7%)
were positive for HPV DNA, with HPV16 most frequently
detected (26;17.9%), followed by candHPV90
(24;16.6%), HPV71 (15;10.3%), HPV66 (10;6.9%),
HPV52 (9;6.2%), HPV34 (8;5.5%), HPV31 (7;4.8%),
HPV42 (7;4.8%) and HPV39 (5;3.4%) whereas each of
the remaining HPV types was found in less than 3.0%
(Figure 1). Of all HPV DNA positive samples 65 (44.8%)
were high-risk HPV. Twelve different high-risk HPV types
except for genotypes 33, 45, 73 were identified among
the women. Age specific prevalence increased from 22.8%
at the age of 20-29 years to 37.2% at 30-39 and decreased
from 17.2% to 15.9 and 4.8% at the age of 40-49, 50-59
and 60-69, respectively. As shown in Table 1, HPV
infection is common among the female population below
the age of 30, with HR-HPV particularly prevalent in
women between 30 and 39 and gradually decreasing with
advancing age. All the sequences were submitted to the
Genbank database under the accession number GQ161664
Figure 1. Distribution of Positive HPV Specific Types
by PCR-sequencing Assay
0
2
4
6
8
10
12
14
16
18
6
111618
3031 3234 3539
42
51 525355 5659
66 68
707174
81 82
cand90
91
35H
68a
cand85
Genotype
Table 1. Distribution of Type-specific Human Papillomavirus among 1662 Women in 10 Year Age Groups
Age group <20 20-29 30-39 40-49 50-59 60-69 70-79 ≥80
Total No.
HPV DNA-ve No (%)
HPV DNA +ve No (%) 0 (0.0)
LR-HPV No.
PHR-HPV No.
HR-HPV No.
Any type No.
6
6 (100)
208 455 506
481 (95.1)
25 (4.9)
2
1
10
12
325
302 (92.9)
23 (7.1)
131
124 (94.7)
7 (5.3)
25 6
5 (83.3)
1 (16.7)
175 (84.1) 401 (88.1)
33 (15.9) 54 (11.9)
4
5
14
10
23 (92.0)
2 (8.0)
6
3
24
21
3
11
9
1
1 4
3
1
Table 2. Prevalence of Human Papillomavirus Infection in Cervical Lesions among 1,662 women in Thailand
Cytology Normal ASC-US ASC-H AGC LSIL HSIL CA All Total No (%)
Total No.
HPV DNA negative
HPV DNA positive
LR-HPV No.
PHR-HPV No.
HR-HPV No.
Any types No.
1,622 (97.6) 13
1,495 (90.0) 9 (69.2) 2 (100)
127 (7.6) 4 (30.8)
8 2
10
55 2
54
23
2 (66.7) 6 (50)
1 (33.3) 6 (50)
128
3 (37.5)
5 (62.5)
1
1
3
240 (2.4)
22 (1.3)
18 (1.1)
4
3
10
1
1,662 (100)
1517 (91.3)
145 (8.7)
12
13
65
55
2 (100)
1
2
2
1
12

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Jira Chansaenroj et al
- GQ161751 and GU447239 - GU447289.
We compared the results of cytology with HPV
genotype. Abnormal Pap smear indicated ASCUS 32.5%
(13/40), ASC-H 5.0% (2/40), AGC 7.5% (3/40), LSIL
30.0% (12/40), HSIL 20.0% (8/40) and carcinoma 5.0%
(2/40). HPV DNA positive samples with normal cytology
amounted to 7.8% (127/1622).
The number of specific HPV types in relation to
cytology is shown in table 2. A high percentage of HR-
HPV is associated with abnormal cytology. As for
subtypes, the carcinogenic HPV type 9 species (e.g.
HPV16, HPV31) were highly prevalent in samples of both
normal and abnormal cytology. Second in prevalence are
non-carcinogenic HPV type 15 species (e.g. HPV71,
HPV90) which are only found in samples with normal
cytology. Third are 6 and 7 species.
Asian Pacific Journal of Cancer Prevention, Vol 11, 2010
120
Discussion
Information on HPV cervical infection in Thailand is
scarce. Our aim was to determine the prevalence of HR-
HPV types causing cervical infections by amplifying the
E1 gene of HR-HPV by semi-nested PCR performed on
1662 hospital-based specimens. Altogether, 8.7% were
HPV DNA positive. HPV DNA positive samples with
normal cytology amounted to 7.8% (127/1622). HR-HPV
prevalence was 44.8%
There are various HPV types involved in cervical
cancer depending on geographic distribution. HPV types
rare in cervical cancer are commonly found in the general
population. In future, HPV testing will focus on primary
screening and cervical cancer prevention. HPV genotype
distribution will provide basic knowledge for HPV-based
cervical cancer screening, cost-effective prophylactic HPV
vaccine and assessment of vaccination against specific
HPV infection in each geographic area (Lai et al., 2007).
In routine set-up or high-throughput, HPV typing is
feasible.
HPV-DNA detection in the Cervicovaginal tract has a
higher sensitivity than sampling from urine specimens,
although obtaining samples from the population is more
labor intensive. Despite differences in collection methods
and techniques, it has become apparent that prevalence
differs notably between geographical regions but still,
HPV16 is typically most prevalent (Manhart et al., 2006).
This corresponds with the literature which suggests that
the prevalence of HPV in women with normal cytology
does not exceed 10% in the North-Western part of Europe
(Baay et al., 2005). For example, of a population of 3,305
women between 15 and 69 years of age with normal
cytology examined in the Netherlands, 4.6% were HPV
positive and subsequently subjected to GP5+/bioGP6+
polymerase chain reaction-enzyme immunoassay (PCR-
EIA) (Jacobs et al., 2000). Research conducted on samples
obtained from a cervical screening program of 6,123
women between 32 and 38 years old from 5 different
regions in Sweden detected HR-HPV in 6.8% by using
HPV primer (GP5+/GP6+) PCR-EIA (Forslund et al.,
2002). According to a previous report, prevalence in Asia
and Australia amounts to between 5% and 11%. For
example, based on GP5+/GP6+ PCR-EIA performed on
randomly, selected married women aged between 15 and
69 years, 2.0% were HPV positive in Hanoi and 10.9% in
Ho Chi Minh City (Pham et al., 2003). In Indonesia, of
2,686 women aged 15-70 years, 11.4% were HPV positive
(Vet et al., 2008).
According to a study, HPV16 and/or HPV18 can be
detected in more than 50% high-grade squamous
intraepithelial lesions, 70% invasive cervical cancer and
81.5% adenocarcinomas. We implied that HPV16 and
HPV18 are more prone to persist and progress to cervical
lesion than other high-risk types (De Sanjosé et al., 2007).
Among the HR-HPV types in many regions, type 16 is
dominant. HPV prevalence varies depending on many
factors such as age and region. As a result, most cervical
carcinomas the worldwide contain HR HPV DNA. A
decrease in HR-HPV infection with advanced age is the
main cause for the decline in HPV prevalence observed
upon screening this population group (Jacobs et al., 2000).
Therefore, prevalence tendencies observed in this study
may be used as baseline profiles to monitor the efficacy
of HPV vaccination strategies in Thailand. Previous study
of infection by multiple HPV genotypes showed that single
infection of HPV genotype have more significant effect
on increasing risk of high-grade cervical lesions than
multiple infection of HPV genotypes. Moreover, the
multiple HPV infections are less frequent in high-grade
than low-grade cervical neoplasia and less common in
cervical adenocarcinoma (ADC) (Zielinski et al., 2003;
Cuschieri et al., 2004; Hadzisejdi et al., 2006).
This study has been in agreement with previous reports
in that screening of a population aged between 30 and 60
years revealed a significant decline in HR-HPV
prevalence. Also, de novo HR-HPV infections will
decrease with advancing age. HPV prevalence peaks at
the age of 40 and thereafter declines for all HR-HPV types.
Pap smear is not replaced by HPV test but they can
complement each other to reduce negative results in
samples with normal cytology or LSIL. A recommendation
by the American Cancer Society suggests that HPV testing
should be used in women after the age of 30, because
infection is mainly asymptomatic in populations under
30 years of age and often transient (Wright et al., 2004).
In Southeast Asia, the age-standardized incidence rate
(ASR) is 18.3. In Thailand, 6,300 new cases of cervical
cancer are diagnosed per year with ASR=19.5 per 100,000
with variations according to geographical region. In the
north ASR=25.6, in the center ASR=20.7, in the south
ASR=16.1 and in the north-east ASR=15.0 (Sriplung et
al., 2005). Epidemiological studies on HPV genotype
distribution are limited. As reported by the World Health
Organization, a large percentage of women living in cities
had their first sexual experience once they had reached
the age of 24. Hence, most studies reported a high
incidence of HPV infection in healthy populations below
30 years of age (Suwannarurk et al., 2009). For example,
the study conducted in Lampang and Songkla provinces
in 1997-2000 reported only 11% HPV infection rate in
the age group of 25 or less. HPV DNA was found in 82-
91% of cervical carcinomas and in 9-20% of normal
cervical smears (Sukvirach et al., 1994).
HPV9 (e.g. HPV16, HPV31) species are high-risk-

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Prevalence and Genotypes of Human Papillomavirus among Thai Women
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Acknowledgements
We would like to express our gratitude to the
Commission on Higher Education, Ministry of Education,
the Center of Excellence in Clinical Virology,
Chulalongkorn University, CU Centenary Academic
Development Project and King Chulalongkorn Memorial
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