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Detection of Human Papillomavirus DNA in biopsies of cervical cancer patients in Salem, Tamilnadu 1
1. INTRODUCTION

Human Papilloma Virus (HPV) is the etiological agent of cervical cancer, the
second-most common malignant tumour in the world. In India, cervical cancer is a
leading cancer among women, with an annual incidence of approximately 132,000
and mortality rates of approximately 74,000. HPV infection is the most common
genital infection of females particularly afflicting adolescents and women in their
early 20s. Most young women are infected within the first few years of becoming
sexually active, and the lifetime risk of HPV infection is 70-79%. Adolescent females
harboring infections with high risk HPV seem to be most vulnerable to develop
histological lesions of the cervix that may become precancerous and potentially
invasive cancer. The major reason for high mortality from cervical cancer is late
stage of diagnosis owing to the lack of a cervical cancer screening program.
Moreover, most genital HPV infections are asymptomatic and unapparent. Since
Adolescents are more vulnerable to HPV infection, early detection in this age group at
the appropriate time is essential (Kari Braaten et al., 2008; Marc Steben et al., 2007;
David Hager, 2009).
Human Papilloma Virus (HPV) contains a circular, double-stranded DNA
genome, and more than 40 HPV types can infect the genital area and result in genital
lesions. As the most common sexually transmitted virus, HPV is commonly
transmitted through direct skin-to-skin contact, most often during penetrative genital
contact, followed by contact with infected cutaneous or mucosal surfaces, including
the anogenital epithelium (Xiang Wang et al., 2013).
The prevalence of HPV infection among promiscuous and sexually active
women has been studied in many countries, and different geographic distributions of
various HPV types have been found. For example, HPV-16 and HPV-18 are the
prevalent types among sexually active women in the United States, Brazil, and Spain
(Del Amo et al., 2009; Koutsky, 1997). Where as HPV-52, followed by HPV-16 and
HPV-58, are the most common types in the Philippines (Miyashita et al., 2009).
Female Sex Workers (FSWs) are at a particularly high risk of HPV infection.
(Xiang Wang et al., 2013). Hpv is the main risk factor for cervical cancer and the
third most common cause of cancer-related death among women. It can be
categorized into low-risk and high-risk types (Parkin et al., 2005) the latter of which
there are 13, including HPV-16, HPV-18, HPV-31, HPV-33, HPV-35, HPV-39, HPV-

45, HPV-51, HPV-52, HPV-56, HPV-58, HPV-59 and HPV-68. Although, most of
the HPV infections are transient and disappear without treatment within 2 years
(Bierman et al., 1998).
Cervical infections caused by the Human Papilloma Virus (HPV) cause
preinvasive and invasive cervical neoplasia. Women infected with HIV are at higher
risk for genital HPV infection (Ferenczy et al., 2003). With advances in our
understanding of HPV biology and with the development of technologies for HPV
detection, there is a growing interest in the potential use of HPV DNA testing as a
screening tool for cervical cancer (Ratnam et al., 2000; Sellors et al., 2000; Clavel et
al., 2001; Kulasingam et al., 2002).
HPV types 16 and 18 account for approximately 70% of cervical cancer cases
worldwide with other high-risk types such as HPV-45, HPV-31, HPV-33 and HPV-52
accounting for the majority of the remaining cervical malignancies (Veldhuijzen et
al., 2010) HPV low-risk types (LR-HPV), mainly HPV-6 and HPV-11, are rarely
detected in high grade cervical lesions but cause the majority of anogenital warts
(Middleton et al., 2003).
An understanding of HPV, risk factors, modes of transmission, and the virus
role in cervical cancer is generally low or nonexistent in many populations. A national
survey in 2000 found that less than one third of Americans had heard of HPV and
only 2% of Americans were able to identify HPV as a sexually transmitted disease
(Friedman and Shepeard, 2007). In June of 2006, The Food and Drug Administration
(FDA) approved the use of Mercks vaccine Gardasil, which protects against HPV
types 6, 11, 16, and 18. Types 6 and 11 are low risk strains that cause 90% of genital
warts cases and types 16 and 18 are high-risk strains that cause 70% of cervical
cancer cases (Merck and Co, 2008).
Although HPV infection is considered a sexually transmitted infection, HPVs
can also be transmitted by non-sexual routes, including casual physical contact and
perinatal vertical transmission (Sinal and Woods, 2005; Syrjanen and Puranen, 2002).
HPV is epitheliotropic, resulting in infection and replication in basal epithelial cells.
The virus is species- and tissue type-specific, infecting the skin or mucous
membranes. (Bartholomew, 2004; Kahn and Hillard, 2004). There have been more
than 100 different types of HPV identified, with 30-40 that are known to infect the
genital tract.


However, most HPV infections spontaneously regress and only in a small
percentage of cases the infection persists, Low-grade Intraepithelial Lesions (LSIL)
progress to High-grade Intraepithelial Lesions (HSIL) and, ultimately, develop into
invasive cervical carcinoma (Woodman et al., 2007). Several studies have reported
that self-obtained samples from the anogenital tract were accurate and suitable for
HPV-DNA testing and have shown a similar correlation to clinically obtain samples
(Petignat et al., 2007; Hobbs et al., 2008).
Most HPV infections are asymptomatic and are efficiently controlled by the
immune system, therefore, the outcome of HPV infection is variable, the infection is
usually transient, and complete resolution is generally common within 12-24 months
(Ho gye et al., 1998; Moscicki et al., 2001). The prevalence of HPV infection, overall
and by age, varies by country, region within the country, and population subgroup. To
compare HPV prevalence between geographic areas or countries, data on age-specific
or age-adjusted prevalence using sensitive HPV detection methods are needed. Age-
specific data on HPV prevalence among women would also be useful (in conjunction
with data on age at first intercourse) to inform future policies to maximize the
potential benefits of HPV prophylactic vaccination (Jennifer et al., 2008).
Studies of adolescence represent a unique opportunity to examine HPV
infected in HIV-infected individuals with a more limited history of sexual activity and
relatively recent HIV and HPV infections (Anna-Barbara Moscicki et al., 2000).
Cervical cancer is one of the leading causes of mortality by cancer with
approximately 495000 women newly diagnosed each year (Ferlay et al., 2004). In
Portugal, cervical cancer is the 4
th
most frequent cancer with 950 new diagnoses and
378 deaths per year, and the second most frequent among women between the age of
15 and 44 (Pinheiro et al., 2004).
The association between HPV infection and urinary tract tumors is still under
investigation. Here we show a case of urethral condyloma acuminatum and
concomitant multiple bladder papillomas, and the existence of HPVs in each of the
tumors was assessed by the PCR. (Natsuko Nakazaki et al., 2012). In the genital tract,
HPV is primarily transmitted by skin-to-skin contact, and certain genotypes are
trophic in both men and women. This can result in subclinical infection of the skin
over the entire genital region, which can be detected by HPV DNA testing. A small
proportion of those infected will manifest either benign or malignant disease. This

chapter will review the epidemiologic aspects of HPV infection and the natural
history of HPV disease, primarily in the female genital tract (Deborah et al., 2007).
To test the quality of DNA obtained from exfoliated cells, a polymerase chain
reaction on the human genomic -globin gene was performed. HPV DNA detection
and genotyping was performed, amplified using the SPF10 primer and HPV
amplimers tested on agaroses gels (Kleter et al., 1999). Human papillomavirus (HPV)
DNA screening seems to be an attractive alternative to cytology screening. This was
first supported by split sample studies in which more high-grade Cervical
Intraepithelial Neoplasia (CIN) was detected with HPV DNA screening than with
cytology screening (Cuzick et al., 2008).
Human papilloma virus is a small non enveloped Icosahedral DNA virus that
replicates in the nucleus of squamous epithelial cells (Parkin et al., 2006). They are
called papilloma virus because certain strains may cause warts or papilloma which are
benign (non-cancerous) tumors (Kumar et al., 2007). Cancer of the cervix is the
second most common cancer among women worldwide (Obure et al., 2009). In India
it is the major cause of cancer in women and a leading cause of deaths due to cancer.
Every year, more than 130,000 new cases and about 70,000 deaths were recorded.
The persistent infection by specific High Risk Human Papilloma Viruses (HR-HPVs)
is essential for the progression of cervical lesion and women who are infected with
HR-HPVs are likely to develop cancer. Cancerous HPV types are associated with
cervical cancer, and non-cancerous HPV types are associated with warts of the genital
areas and low grade disease of the cervix (Marais et al., 2008).
Human Papilloma Virus (HPV) prevalence, age-specific prevalence, and type
of distribution differ substantially between populations (Clifford et al., 2005;
Franceschi et al., 2006) and HPV 18 has a greater role in cervical cancer in Indonesia
than in the rest of the world. HPV 18 was found as frequently as HPV 16 in cervical
cancer (Schellekens et al., 2004) or even more frequently than HPV 16 (Bosch et al.,
1995). In October 1999, we initiated a cluster-randomized, controlled trial to evaluate
the effectiveness of a single round of HPV testing, cytologic testing, or VIA in
reducing the incidence of cervical cancer, as compared with a control group that
received usual care in a previously unscreened, high risk population in the
Osmanabad district in the state of Maharashtra, India.


A highly efficacious prophylactic vaccine against HPV types 6, 11, 16, and 18
was licensed in June 2006 and recommended for routine use in females aged 11 to 12
years in the United States. (Koutsky et al., 2002; Villa et al., 2005). Cancer of the
cervix is the second most common cancer among women worldwide (Obure et al.,
2009). In India it is the major cause of cancer in women and a leading cause of deaths
due to cancer. Every year, more than 130,000 new cases and about 70,000 deaths
were recorded. The persistent infection by specific High Risk Human Papilloma
Viruses (HR-HPVs) is essential for the progression of cervical lesion and women who
are infected with HR-HPVs are likely to develop cancer. Cancerous HPV types are
associated with cervical cancer, and non-cancerous HPV types are associated with
warts of the genital areas and low grade disease of the cervix (Marais et al., 2008).
Globally, it is estimated that of 500,000 women develop cervical cancer and
almost 274,000 of them die from the disease per year (WHO, PATH, and the United
Nations Population Fund, 2009). It is the second most common cancer in women
worldwide and the most common in women in under-developed and developing
countries, which bear more than 80% (WHO, 2010) of the global burden of the
disease. This reveals the lack of effective control measures in these countries. The
sensitivity for high-grade lesions in these studies, which have used urine, vaginal
swab or tampon specimens, has ranged from 66% to 94%. Although these findings
appear promising, the studies have had limitations because of test insensitivity, small
sample sizes and methodological flaws, such as verification bias. The primary
objective of the study reported here was to determine the sensitivity and specificity of
self-sampling for HPV in detecting HSIL (CIN 2 or 3) (John et al., 2000).
Most of these HPV infections are found to be transient and only women who
harbor persistent high risk HPV infection are at risk of developing cervical lesions.
High risk HPV infections (hrHPV) seem to persist longer than infections with low
risk HPVs (Franco et al., 1999; Woodman et al., 2001; Guiliano et al., 2002;
Richardson et al., 2003; Munoz et al., 2004; Brown et al., 2005). The HPV-16/18
fraction was found to account for 78.9 per cent of the ICC, 61.5 per cent with High-
grade Squamous Intraepithelial Lesions (HSIL), 30.8 per cent with Low-grade
Squamous Intraepithelial Lesions (LSIL) and 3.9 per cent in women with normal
cytology/histology. The most prevalent HPV types associated with cervical cancer
were HPV-16, -18, -45, -33, -35, 58, -59 and -31 (Clifford et al., 2003).


It is estimated that over 500,000 new cases and more than 250,000 deaths
occur every year due to this preventable disease. In Turkey, CC is the ninth most
common cancer in women with a mortality risk of more than 50 % and is the cause of
5.3 % of all malignancies affecting women (Ozgul, 2007). Cancer of cervix is
preventable, yet approximately 493,100 new cases and more than 273,000 deaths
occur each year among women worldwide (Ferlay et al., 2002) India, which accounts
for the one-sixth of the worlds population also bears the one-fifth of the worlds
burden of cervical cancer (Sankaranaryanan et al., 2001). Cervical cancer is a leading
form of cancer among women living in low resource regions of the world and often
kills women at a young age when they are still raising families (Walboomers et al.,
1999).
The Human Papilloma Virus (HPV) is a non-enveloped, double-stranded DNA
virus infecting deeper layers of skin and the inner mucosal lining of organs. Of the
over 100 known HPV types, 40 preferentially infects the stratified squamous
epithelium of the mucosa and genital skin of the cervix, vagina, vulva, penis, and
perianal areas. HPV infection is very common and, in most cases, transient or self-
limiting. However, 1020% of HPV-infected women develop a persistent infection
and continues to shed HPV DNA from the genital tract for 24 or more months
(Moscicki et al., 2006).
Two HPV vaccines, a quadrivalent vaccine protecting against HPV types
6/11/16/18 and a bivalent one directed against HPV types 16/18, have become
available for the primary prevention of HPV 16/18-related cervical cancer. Self-
sampling has several advantages compared with physician-collected samples for
detection of HPV genital infection. Self-sampling is a less costly and non-invasive
collection procedure. Self-collected samples can be more easily obtained in settings
with limited resources or in populations difficult to reach. Numerous studies have
reported lately that self-obtained samples of the anogenital tract in women were
accurate and suitable for DNA testing (Petignat et al., 2005; Harper et al., 2002;
Harper et al., 2002; Wright et al., 2000; Coutlee et al., 1997).

The International Association for Research in Cancer (IARC), the most
prevalent reported high-risk HPV types, which infect the uterine cervix, are: HPV-16
(53%), HPV- 18 (15%), HPV-45 (9%), HPV-31 (6%), and HPV-33 (3%) (Munoz,
2000). Lower prevalence of other phylogenetically related types is also found (Meyer

et al., 1998). Since HPV cannot be cultured and the clinical performance of
serological assays is poor, diagnosis of HPV infection is almost entirely based on
molecular tools, including liquid hybridization (e.g. Hybrid Capture 2; Digene
Diagnostics, Silver Springs, USA) (Cox et al., 1995).
The HPV virion has a double-stranded, circular DNA genome of
approximately 7900 bp, with eight overlapping open reading frames, comprising early
(E), and late (L) genes and an untranslated long control region. The L1 and L2 genes
encode the major and minor capsid proteins. The capsid contains 72 pentamers of L1,
and approximately 12 molecules of L2. The early genes regulate viral replication and
some have transformation potential (Steenbergen et al., 2005). HPV 16, which is one
of the more common types among cytologically normal women, is also the most
common type among cervical cancer cases (Franco et al., 1999; Ho et al., 1998; Liaw
et al., 1999; Munoz et al., 2003; Richardson et al., 2003; Schiffman 1992; Woodman
et al., 2001).
High-risk HPV infection is asymptomatic, invisible, and may not be
expressed in cervical tissue dysplasia for years (American Social Health Association,
2002). It will not be detected, except when cervical tissue is examined by microscope
in routine pelvic examination. Employing Papanicolaou (Pap) testing; when viral
DNA is identified in cervical cells (National Cancer Institute, 2004); or when invasive
cancer produces symptoms. More than 100 HPV types are known to occur that are
categorized into three broad categories depending upon their oncogenic potential:
high risk types including HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, -
68, -73 and -82; intermediate types including HPV-26, -53, -66 and low-risk types
including HPV-6, -11, -40, -42, -43, -44, -54, -61, -70, -72, -81 and -CP6108 (Munoz
et al., 2003). Genital Human Papilloma Virus (HPV) is the most common STI in the
United States and, perhaps, the most common STI among sexually active women
(Centers for Disease Control and Prevention. 2002-2007).
Although Human Papilloma Virus (HPV) is known to be strongly associated
with the development of cervical cancer, most HPV infections in women are transient
(Ho et al., 1998; Moscicki et al., 1998). The women with persistent infection appear
to have a higher risk of developing significant precancer even as adolescents
(Moscicki et al., 1998; Koutsky et al., 1992; Schlecht et al., 2001; Konno et al.,
1992). Although factors that influence persistence of HPV are not yet well

understood, several studies suggest that alterations in cell-mediated immune responses
play a large role in persistence of HPV.
Human Papilloma Virus (HPV) is a member of the Papoviridae family of
viruses. There are over 100 distinct types of HPV that all infect
different epithelial surfaces, from the hands and feet in the genital region. Some HPV
types are the causative agent of warts and condyloma acuminata (genital warts), while
others have been shown to be the causative agent of cervical cancer (University of
Bristol, 2004). The HPV DNA test is now accepted as a useful adjunctive method that
increases the sensitivity of the conventional cervical cytology (Prusty et al., 2005;
Jacobson et al., 2000; Vossler et al., 1995) Since 1995 we have been studying HPV
infection in a cohort of approximately 650 HIV-infected women, with the aim of
identifying early markers of cervical disease (Uberti-Foppa et al., 1998).
HPV is a relatively small, non-enveloped, double-stranded circular DNA virus
of Papillomaviridae family with a genome of 8000bp. The genome consists of three
major regions Long Control region, early regions (E1, E2, E4, E5, E6 and E7) and late
region (L1 and L2). These viruses are epitheliotropic in nature, infect the basal cells
of the stratified squamous epithelium and the metaplastic cells of the transformation
zone of the cervix squamocolumnar junction and preferentially localize to the ano
genital tract. HPV is enormously diverse, with over 110 HPV types have been
identified on the basis of variations in genotype homology. Among these,
approximately 3040 HPV types infect the epithelial lining of the ano-genital tract
(genital HPVs), of which 1520 types are oncogenic (David Hager, 2009; Amanda
Tristram et al., 2007).
Based on their potential to lead to cervical cancer, HPV has been broadly
classified into high risk and low-risk types. HPV infection, including infection with
the high-risk types associated with cervical cancer and there are 15 high risk-HPV
types (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 66, 68, 73 and 82). On the other
hand, 11 different HPV types that have been mainly associated with genital warts and
benign cervical lesions such as condylomata accuminata and dysplasias are termed as
low-risk HPV types. These include HPV types 6, 11, 40, 42, 43, 44, 54, 61, 70, 81
and CP6108. Of the 15 high-risk oncogenic types, HPV 16 and 18 account for about
75% of all cervical cancers, adenocarcinoma in-situ of the cervix, cervical
intraepithelial neoplasia- III (severe dysplasia), as well as vulvar and vaginal
intraepithelial neoplasia- II and III (moderate and severe dysplasia). Among low-risk

types, HPV6 and HPV11 cause approximately 90% of genital warts (David Hager,
2009; Elizabeth et al., 2005).
The Centers for Disease Control and Prevention (CDC) estimates that one in
every two people will acquire a genital HPV infection in their lifetimes; 50% of
sexually active women will acquire a cervical HPV infection within 5-7 years of
initiating vaginal intercourse and by the age of 50, this proportion reaches 80%. The
infection may progress from residential to episomal and finally, in an integrated form.
Residential infection typically occurs a minimum of 6 weeks from exposure, can
persist without detection for decades, and can be low risk or high risk. From the
episomal state, vitally active HPV is located in the cell nucleus, separate from the
human DNA, whereas in the integrated form, the HPV DNA circle has opened and
joined the human DNA. HPV is typically found in episomal form in cervical lesions
and integrated form in cervical cancer. During integration E1 and E2 are frequently
disrupted whereas E6 and E7 viral oncogenes are retained (Michael Pichichero,
2007). Progression from initial HPV infection to invasive cervical cancer may take
15-20 years and is typically associated with persistent HPV infection (Melissa
Lawson, 2006). Most HPV infections occur during the teenage years or early
twenties, whereas cervical cancer typically presents in women 35 years and older
(Mohammed Akhtar, 2004).
To initiate progression specific changes in cellular control functions including
genetic mutations should occur in host cells. Inactivation of these cellular control
functions permits deregulated transcription of the early viral genes E6 and E7, and
that triggers cell proliferation, inhibition of apoptosis, reprogramming of
differentiation, and chromosomal instability. These changes could support the
integration of episomal HPV genomes into the chromosomes of the host cell and
contribute to further over expression of the viral genes E6 and E7 (Irene Kraus et al.,
2006; Sigrun Ressler et al., 2007).
The clinical manifestations of HPV infection in women usually appear as
either genital condylomatous lesions (HPV warts) or cytological abnormalities. Viral
proteins expressed during active infection induce pathologic changes, including basal
cell proliferation, nuclear enlargement, koilocytosis, and abnormal mitotic figures.
Each of these changes is a defining feature of Squamous Intraepithelial Lesions (SIL).
As such, the development of both Low-grade Squamous Intraepithelial Lesions
(LSIL) and High-grade Squamous Intraepithelial Lesions (HSIL) can be considered

the pathologic consequences of HPV infection. The majority of infections and
subsequent corresponding cytological abnormalities are transient and of minimal
significance (low-grade squamous intraepithelial lesions) in terms of oncogenic
potential.
In natural history studies of women with newly acquired HPV infection, the
average length of infection of detectable HPV is 13 months. Fortunately HPV
infection in women is generally a transient infection that is cleared by the immune
system and undetectable in most people within 24 months with only 5-10% persisting.
A study of married women showed that approximately 70% of women with HPV
infection became HPV negative within 1 year and as many as 91% of them became
HPV negative within 2 years, with a mean duration of infection of 8 months. This
majority of these infections will be reseeded spontaneously (Kari Braaten et al.,
2008). Most infections are asymptomatic, with cell changes on the Pap smear
abnormalities seen in only about 10% of women who are positive for HPV. This study
confirms the transient nature of HPV infection in women (Melissa Lawson, 2006;
Marcia Shew et al., 2005; Anna-Barbara Moscicki, 2005; Michael Pichichero, 2007).
Although HPV infection is frequently transient in women and the natural
history of HPV infection and cytologic abnormalities is still being defined, this
population has a relatively high incidence of abnormal Papanicolaou (Pap) smears.
Mount and Papillo reported that the incidence of Pap smears with squamous
intraepithelial lesion was higher in women, and other investigators have proposed that
the prevalence of abnormal pap smears and cervical dysplasia is increasing worldwide
in women (Jessica Kahn et al., 2002). Moreover, High- and low-risk type HPV is
reported to be a common genital infection in adolescents, with a prevalence ranging
from 9% to 16% in different countries (Frank Ldicke et al., 2001). A recent study
showed that 1519-year-old women had a rate of Squamous Intraepithelial neoplasia
(SIL), second only to 2024-year-old women and 1014-year-olds, had a higher rate
of SIL than women over 30 years (Frega et al., 2003).
The hosts immune response to HPV infection is initially the innate response,
primarily interferon-based, which then triggers circulating humoral and cell-mediated
responses at local lymph nodes. A cell-mediated immune response is required for
HPV containment and lesion regression. In addition to the immune evasion permitted
by the intracellular and the epithelial location of the virus, HPV can induce a local
immune deficiency by depletion of intraepithelial lymphocytes, Langerhans cells,

and CD4+ cells with down-regulation of cytokine production (Amanda Tristram et
al., 2007; Deborah Money et al., 2007).
Nevertheless, the mere presence of a high-risk form of HPV is not itself
sufficient to trigger the carcinogenic process. It is clear that other risk factors are
associated with the acquisition and retention of the virus and viral associated
oncogenesis. Several factors, including host cell factors are necessary for progression
of HPV-infected precursor lesions to cancer. In particular, women seem to exhibit a
number of factors that increase their susceptibility to HPV infection. The behavioral
and biological risk factors associated with the acquisition of HPV in women include
poor genital hygiene, early age of first sexual intercourse, number of lifetime sexual
partners, alcohol and drug abuse, and lack of condom use, hormonal and dietary
factors and immunosuppression. Chlamydia trachomatis, a common infection and
Cigarette smoking may also enhance persistence and progression of HPV infection
(Michael Pichichero, 2007; Frega et al., 2003; Melissa Lawson, 2006; Hauwers, 2009;
Sara, 2009).
Transmission of HPV primarily occurs through direct genital contact, most
commonly by sexual intercourse. But, other types of sexual and non-sexual contacts
can transmit HPV in self reported virgins. Transmission via inanimate objects such
as bed clothing, toilets is thought to be unlikely, but the true risk is unknown (Kari
Braaten et al., 2008).
Until recently Papanicolaou-stained (Pap) smear, cervical cytology was the
only traditional morphological screening method which detects early HPV infection
and should be done only in asymptomatic women. Cellular changes were classified by
the pathologists according to the Bethesda classification system as negative (normal),
Atypical Squamous Cells of Undetermined Significance (ASCUS), Low-grade
Squamous Intraepithelial Lesion (LSIL), High-grade Squamous Intraepithelial Lesion
(HSIL), or suggestive of cancer (Andrea Fuessel et al., 2004). But Pap testing in
adolescents is particularly not feasible because of the vast majority of cytological
abnormalities in adolescents regress or does not represent cervical intraepithelial
neoplasia (CIN) 2/3 or carcinoma in Situ (Jessica Kahn et al., 2005). Colposcopy
permits visualization of the lesions and is performed for women with abnormal
smears or with signs or symptoms suggestive of cancer. HPV DNA testing recently
has been incorporated into guidelines for primary cervical cancer screening in women
older than 30 years of age (Caitlin Wetzel et al., 2007).

Diagnosis of HPV infection has relied primarily on the detection of the viral
genome due to the inborn difficulties associated with the culturing of this virus.
Several molecular biology techniques have been used in detecting HPV infection, in
particular PCR and Hybrid capture II test etc. In adult women with HPV-DNA-PCR
on cervical scrapes, cervical biopsies, vaginal swab, etc. are used to identify the HPV-
type responsible for the infection. A biopsy is needed for women with high grade
cervical lesions or any cancerous growth. Alternatively, detection of HPV DNA in
urine can function as a secondary screening technique for cervical cancer in triage for
women with atypical squamous cells of undetermined significance (ASC-US). It has
been reported that women with cytological abnormalities are likely to have detectable
HPV DNA in urine as in cervical swabs (Eun-Seop Song et al., 2007). Compare to the
PCR urine screen with conventional molecular assays that requires cervical swab
specimens, researchers found that positive urine and cervical swab results had similar
correlations with abnormal Pap smear results. It appears to be a useful way of
determining which types of HPV are present in the cervix.
Persistent infection with High-Risk Human Papillomavirus (HR-HPV) has
been demonstrated to be the necessary causal factor for developing cervical cancer.
To know the most prevalent HR-HPV in different geographical areas is important to
design diagnostic tests and implementation of vaccines. The prevalence of HRHPV in
a total of 1001 patients, 198 with normal cytology results, 498 with Low-grade
Squamous Intraepithelial Lesion (LSIL), and 205 with High-grade Squamous
Intraepithelial Lesion (HSIL) who attended our gynecology department for
opportunistic screening of HPV infection (Maria Lusia Mateos Lindemann et al.,
2010).
Infection by Human Papilloma Virus (HPV) is one of the primary causes of
mortality from cancer in northern Brazil. Sexually active women from Manaus,
Amazonas, without cytological alterations and women with pre-malignant and
malignant cytological alterations were examined for HPV virus, identified via PCR
and sequencing. The target region for this study was part of the L1 capsid gene of
HPV. Twenty-three samples that were PCR-positive were sequenced. Analysis of 336
bp demonstrated a high incidence of high-risk HPV types in the population of
Manaus, identified as HPVs 16, 33, 58, 66, 68. HPV type 16 was the most prevalent,
presenting two variants similar to the Asian-American (AA) and East-Asian type (As)
variants. A rare, HPV type 13 related to Hecks disease was also detected. This

preliminary provides important information about the HPV circulating in Amazonas
State (Castro et al., 2011).
The prevalence of cervical infection with the HPV vaccine campaign in Viet
Nam, it is important to note that one can be infected with multiple types of HPV.
Vaccination does not protect against all types of high risk HPV types. Future vaccine
campaigns should openly disclose this information to women receiving vaccines.
High prevalence of infection with HPV high risk types was observed in this study.
(Lanth vu et al.,2013). Therefore, in the present study, we proposed to investigate the
prevalence of HPV among the married women using biopsy HPV-DNA detection in
Salem district, Tamil Nadu.


AIM & OBJECTIVES

The present study was designed to meet the following broad objectives:

To study the prevalence of HPV infection in married women in Salem district.
Efficiency of L1 PCR analysis with MY09/MY11 consensus primers and specific
primers HPV16.
To estimate the prevalence of high-risk HPV16, by using a highly sensitive
polymerase chain reaction (PCR).
To assess the socio demographic factors and personal habits associated with HPV
positive study subjects.
To assess the sign and symptoms associated with HPV positive study subjects.
To assess the risk factors associated with HPV positive study subjects.


2. REVIEW OF LITERATURE

Cancer of the uterine cervix is the second most common cancer among women
worldwide with an estimated 510,000 newly diagnosed cervical cancer cases and
288,000 deaths. In India, cervical cancer is a leading cancer among women, with an
annual incidence of approximately 132,000 and mortality rates of approximately
74,000. Epidemiological estimates suggest that the worldwide prevalence of HPV
infection is between 9 and 13%, which equates to approximately 630 million infected
women. The cancers that develop from uterine cervix are of two types: (i) squamous
cell carcinomas, which develop from squamous epithelium, cover most visible part of
the cervix; and (ii) adenocarcinoma, which arise from the glandular lining of the
endocervical canal. About 85% to 90% of cervical cancers are squamous cell
carcinomas, and the rest 1015% are adenocarcinoma's. Squamous Cell Carcinoma
(SCC) is preceded by well-recognized epithelial changes, the precancerous lesions,
which develop through several grades: cervical intraepithelial neoplasia (CIN) I to III;
or Low grade Squamous Intraepithelial Lesions (LSIL) to high grade SIL or a mild,
moderate and severe dysplasia leading to Carcinoma-In-Situ (CIS). These lesions may
progress to malignancy, or persist, or regress (Alok Bharti et al., 2010; Bhudev Das et
al., 2000). High-risk Human Papilloma Viruses (HR-HPVs) are the causative agents
of cervical cancer and prophylactic HPV vaccination has been recommended for
adolescents, but no data are available on the prevalence of HPV infection among
adolescents in India.

2.1 ROLE OF HPV
Human Papilloma Virus (HPV) infection is the cause of cervical cancer in
almost 100% of the cases. The association between HPV infection and cervical
neoplasm was established after the link between genital HPV infections and cervical
cancer was first demonstrated in the early 1980s by Harold zur Hausen and a number
of molecular and epidemiologic studies have since demonstrated a strong co-relation
between human papillomavirus (HPV) infection and this disease (Hoenil Jo et al.,
2005). HPV infection can also lead to genital warts, recurrent respiratory
papillomatosis, and vaginal, vulval, anal and penile cancers. HPV types 16 and 18 are
responsible for more than 70% of HPV-related cervical cancers whilst HPV types 6
and 11 cause approximately 90% of the cases of genital warts. HPV infection is most

common among young, sexually active individuals, and it is so prevalent that
approximately 75% to 80% of sexually active individuals will become infected in
their lifetime. Clinical, sub-clinical and latent HPV infections are the most common
sexually transmitted viral diseases today, with a peak in prevalence among young
women soon after the onset of sexual activity. Latent genital HPV infection can be
detected in 5% to 40% of sexually active women of reproductive age. In most cases,
genital HPV infection is transient or intermittent (Alok Bharti et al., 2010; Jha
Urvashi Prasad et al., 2008; Bethany weaver et al., 2006).
Human Papillomavirus (HPV) is the main cause of cervical cancer.
(Walboomers et al., 1999; Harold zur Hausen et al., 1996) More than 35 HPV types
have been identified in the genital tract, and HPV16 and 18 are responsible for
approximately 70% of cervical cancer. (Munoz et al., 2003; Bosch et al.,2003)Two
HPV vaccines, a quadrivalent vaccine that protects against HPV6, 11, 16, and 18 and
a bivalent vaccine that protects against HPV16 and 18, have been developed and
approved by the United States Food and Drug Administration (FDA) for females 9-25
years and 9-26 years of age, respectively (Ault et al., 2007).
Human Papillomavirus (HPV) 6 and 11 are the aetiological agents responsible
for Recurrent Respiratory Papillomatosis (RRP). There is general consensus that
HPV11 results in more aggressive disease compared to HPV6. There are numerous
sub-types or variants of both HPV6 and HPV11. These sub-types have different
activities at least in-vitro. The numbers of different HPV types within RRP tissue may
be more extensive than initially appeared. This depends specifically upon the HPV
types tested. The clinical differences between HPV6 and HPV11 disease may not be
accurately predictable as these viruses exist in numerous sub-types. Also, RRP tissue
may contain more than one subtype or even be co-infected with other viruses that may
influence outcome. In-vitro studies upon cell lines are a reasonable starting point for
evaluation of these differences (Donne et al., 2010).
The role of inflammation in HPV infection and disease is complex since it
involves responses capable of preventing initial infections, clearing those ongoing as
well as promoting persistence and progression of associated lesions. Avoiding the
immune response has been considered a key aspect of HPV persistence which is the
main factor leading to HPV-related neoplasia. HPVs have evolved different ways of
targeting immune signaling pathways. Moreover, host inflammatory response may
promote lesion progression and affect tumor fate by diverse mechanisms including the

direct participation of inflammatory cells. In this review we discuss the interplay
between HPV oncogenic proteins and an array of inflammatory responses that
ultimately may lead to cancer (http://www.ncbi.nlm.nih.gov/pubmed/20819779,
2013).
Human Papilloma Virus (HPV) is the main cause of cervical cancer, and many
countries now offer vaccination against HPV to girls by way of government-funded
national immunization programs. Monitoring HPV prevalence in adolescents could
offer a near-term biological measure of vaccine impact, and urine sampling may be an
attractive large-scale method that could be used (Espen Enerly et al., 2013).

2.2 BIOLOGY OF HPV
The Papilloma Viruses (PV) belongs to the family Papillomaviridae and is
highly species-specific with a tropism for epithelial cells. The Papillomaviruses are
found among many species, and probably occur in most mammals and birds (Villers,
2004). The first Papillomavirus was identified in 1933, and causes warts in cottontail
rabbits (Shope, 1933). This particular virus, cottontail rabbit papillomavirus (CRPV),
later induces malignant transformation (Rous, 1935). Harald Zur Hausen proposed in
the late seventies that Human Papilloma Virus (HPV) can cause cervical cancer
(Hausen, 1976). Human Papillomavirus is a member of the Papillomaviridae family,
designated by the International Committee on Taxonomy of Viruses (ICTV) that
infects only humans (ICTV Virus Taxonomy 2009). Like all Papilloma Viruses, HPV
establishes productive infections only in the epithelium of the skin or mucosa.
Molecular biology has made it possible to study the interactions of viral gene
expression and replication with the host cell phenotype (IARC Monographs 2007).
Major breakthroughs in the understanding of the viral oncoprotein, most notably in
the E5/6/7 proteins, activities of HR-HPV types to the integrity of cellular DNA and
cell-cycle control were made already in the 1990s.
One hundred eighteen Papillomavirus (PV) types have been completely
described, and a yet higher number of presumed new types have been detected by
preliminary data such as subgenomic amplicons. The classification of this diverse
group of viruses, which include important human pathogens, has been debated for
three decades. This article describes the higher-order PV taxonomy following the
general criteria established by the International Committee on the Taxonomy of
Viruses (ICTV), reviews the literature of the lower order taxa, lists all known PV

types, and interprets their phylogenetic relationship. PVs are a taxonomic family of
their own, Papillomaviridae, unrelated to the polyomaviruses. Higher-order
phylogenetic assemblages of PV types, such as the genital human PVs, Are
considered a genus, the latter group, for example, the genus Alpha-Papillomavirus.
Lower-order assemblages of PV types within each genus are treated as species
because they are phylogenetically closely related, but while they have distinct
genomic sequences, they have identical or very similar biological or pathological
properties. The taxonomic status of PV types, subtypes, and variants remains
unchanged and is based on the traditional criteria that the sequence of their L1 genes
should be at least 10%, 210%, and maximally 2% dissimilar from one another
(Ethel-Michele de Villiers et al., 2004).

Classification of HPV in this database was taxonomic scheme developed by
International Committee on Taxonomy of Viruses.
Classification of Human Papillomavirus Viruses

2.2.1 Human Papilloma Virus (HPV) is a DNA virus from the Papilloma
virus family that is capable of infecting humans. Like all Papilloma viruses, HPVs
establish productive infections only in keratinocytes of the skin or mucous membrane.
While the majority of the known types of HPV cause no symptoms in most people,
some types can cause warts (verrucae), while others can in a minority of cases lead to

cancers of the cervix, vulva, vagina, penis, oropharynx and anus ( Genital HPV
Infection, 2009).

2.3 MORPHOLOGY AND GENOME ORGANISATION
HPV is a relatively small (55nm diameter) non-enveloped virus and contain a
double-stranded closed-circular DNA genome of approximately 7200 bp8000 bp. It
has an icosahedral capsid composed of 72 capsomers, which contain at least two
capsid proteins, L1 and L2 replicating as an episome in the nucleus of host cells. The
HPV genome can be divided into three regions, the noncoding Long Control Region
(LCR), or the upper regulatory region (URR), and the early (E) and late (L) gene
region (protein encoding). The LCR/URR constitutes about 10% of the genome,
varying between 800 bp and 900 bp. URR forms a Non-protein-Coding Region
(NCR) of the viral genome, but it contains many cis-acting elements. Viral gene
expression is generally regulated by several viral and host-cell transcription factors,
which bind to the URR. These factors include nuclear factor-1 (NF-1), activator
protein-1 (AP-1), octamer- binding factor-1 (Oct-1), progesterone receptor, Yin and
Yang factor-1 (YY-1), SP-1, KRF-1 and glucocorticoid receptor, etc.
During HPV DNA integration, the viral genome usually breaks in the E1/E2
region. The break usually leads to the loss of the E1 and E2 regions. The loss of E2,
which encodes proteins including one that inhibits the transcription of the E6 and E7
regions, has been known to result in uncontrolled and increased expression of E6 and
E7 oncogenic proteins.
Increased expression of E6 and E7, meanwhile, has been observed to lead to
the malignant transformation of the host cells and to tumor formation (Bosch et al.,
1992; Doorbar et al., 1991; Phelps et al., 1998). HPV viral integration into the host
genomic DNA is associated with progression from polyclonal to monoclonal status in
CIN, and these events play a fundamental role in the progression from low-grade to
high grade cervical Neoplasia (Ueda et al., 2003)


The functions of the products of HPV early region open reading frames
Early region Protein functions
E1
Unwinds the DNA strands working with E2 protein
Modulate the transcription activity of the E2 protein
E2
Enables E1 protein to bind to the viral origin of replication
located within the LCR
Encodes a LCR-binding protein that regulates transcription of
the early region
E4
Encodes a protein that interacts with cytokeratin
Expressed in later stages of infection, when complete virions
are being assembled
E5
Augment cellular proliferation and DNA synthesis in a context
of cell membrane receptors, such as EGF and PDGF
Induces an increase in mitogen-activated protein kinase activity
E6
Binds to p53 and targets it for rapid degradation via a cellular
ubiquitin ligase
Induces telomerase activation
E7
Binds to the hypophosphorylated Rb proteins and liberate E2F,
which results in S phase entry
Interacts with inhibitors of cyclin dependent kinases
Induces abnormal centrosome duplication resulting in
aneuploidy

The LCR regulates transcription from the early and late regions, and therefore
controls the production of viral proteins and particles. The early region is downstream
of the LCR and contains six open reading frames, E1, E2 and E4E7, and is involved
in viral replication and oncogenesis. These encode all viral proteins except for the
viral capsid proteins, which are encoded in the late region. The L1 and L2 genes in the
late region encode the major and minor capsid proteins, both of which are required
late in the viral life cycle to encapsulate the virus (Amanda Tristram et al., 2007;
Hoenil Jo et al., 2005; Bhudev Das et al., 2000).


2.4 HPV TYPES
Human papilloma virus types are recognized based on their genetic sequence
and are numbered in order of their discovery. Sequence differences showing less than
90% homology to any of the known types on the basis of the E6, E7, L1 regions are
consider as a new HPV type and sequence difference of more than 10% in the L1
region are recognized as another type, whereas variations of less than 2% are
recognized as variants of currently numbered types. HPVs can be grouped as
cutaneous or mucosal types. The cutaneous types are quite numerous and are
associated with common hand and foot warts as well as a rare genetic disorder
characterized by widespread warts and frequent skin cancer, erythrodermoplasia
verruciformis. The cutaneous types are being investigated as risk factors for actinic
keratoses, psoriasis, basal cell carcinomas, and squamous cell carcinomas. The
mucosal types are those most commonly found in the anogenital region, but that may
be found elsewhere, including the nail beds and oropharyngeal epithelium.
More than 80 types of Human Papilloma Viruses (HPVs) are known today,
and they are generally classified according to their potential to induce malignant
transformation. HPVs 16, 18, 31, 33, 35, 39, 45, 50, 51, 53, 55, 56, 58, 59, 64 and 68
are considered high risk types because they are detectable in carcinomas and
dysplasias 8, 23. HPVs 31, 33, 35, 51 and 52 are sometimes regarded as intermediate
risks because they are more common in mild or severe dysplastic lesions than in
carcinomas. Among the high-risk strains, HPV 16 and 18 are the most closely
associated with cervical carcinoma. The HPV16 DNA has been found in more than
50% of squamous cell carcinomas, while the HPV18 DNA has been found in more
than 50% of adenocarcinomas (Milde-Langosch et al., 1993).
To date, more than 110 human and animal papillomavirus genotypes have
been characterized and sequenced. Of the approximately 30 HPVs that infect the ano
genital tract, 15 HPV types (HPV types 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59,
68, 73 and 82) have been found to be associated with high grade lesions and invasive
cervical cancer and hence are classified as high-risk types. HPV types 16 and 18 are
the two most common highly oncogenic HPV types found in invasive cervical cancer
and high-grade Cervical Intraepithelial Neoplastic (CIN) lesions. On the other hand,
11 different HPV types that have been mainly associated with genital warts and
benign cervical lesions such as condylomata accuminata and dysplasias are termed as
low-risk HPV types. These include HPV types 6, 11, 40, 42, 43, 44, 54, 61, 70, 81

and CP6108. Among these, HPV6 and HPV11 cause approximately 90% of genital
warts (Alok Bharti et al., 2010).

Genome organization of Human Papilloma Virus cause cervical cancer. (E1-E7
early genes, L1-L2 late genes: capsid)

2.5 LIFE CYCLE OF HPV
HPV are strictly host-specific and infects its host by penetrating through
mucosal tears in the basal membrane. The nature of the cell surface receptor used for
viral attachment is not known, although heparin sulphate and stabilizing
proteoglycans have been suggested to be epithelial cell receptors for HPV. The target
cells for an initial HPV infection are the immature basal cells of the epithelium, and
HPV is thought to reach these cells through micro abrasions or cracks within the
epithelium. Once in a host cell, the life cycle of HPV can be separated into two stages,
i.e., nonproductive and productive. In the nonproductive stage, the virus maintains its
genome as a low copy number episome by using the hosts DNA replication
machinery to synthesize its DNA in basal layer of the epithelium .The pattern of viral
gene expression in these cells is not well defined, but it is generally believed that the
viral E1 and E2 proteins are expressed in order to maintain the viral DNA as an
episome and to facilitate the correct segregation of genomes during cell division. The
number of copies of the HPV genome, as a circular or episomal form, is thus low in
the basal cells nucleus, and virally encoded proteins are expressed at very low levels.
As a result, HPV-infected basal cells show no specific cytologic or histologic changes
and cannot be distinguished from uninfected cells. This stage of an HPV infection is
referred to as a latent or clinically unapparent infection since the woman is HPV

DNA positive, but no lesions can be detected, even by microscopy (Marc Steben et
al., 2007; Anna-Barbara Moscicki, 2005).
The productive stage of the viral life cycle occurs in the terminally
differentiating suprabasal layers of the epithelium. In these cells, the virus switches to
a rolling-circle mode of DNA replication and amplifies its genome to higher copy
number, expresses late genes encoding capsid proteins, and produces viral progeny
(Hoenil Jo et al., 2005) Human papilloma virus is episomal in productive infections
and in most lesions. Integration of the viral genome into host cellular DNA is found in
essentially all cancers with HPV18 and most of those with HPV16. Viral integration
has not been observed in all cancers, but integration clearly has implications in the
perpetuation of the transformed phenotype as well as in tumor progression. HPV
integration appears to be random with respect to the cellular chromosome, but nearly
always occurs within the E1/E2 region of the HPV genome.
Integration may be an irreversible event as a consequence of host cell
genomic instability, or it may be that integration initiates a chain of events including
the impairment of tumor suppressor genes (e.g., TP53 and RB), which results in
genomic instability and cell immortalization. The papillomavirus life cycle differs
from that of all other virus families: infection requires the availability of epidermal or
mucosal epithelial cells that are still able to proliferate. In these cells, viral gene
expression is largely suppressed, although the limited expression of specific early
viral genes (such as E5, E6, and E7) results in enhanced proliferation of the infected
cells and their lateral expansion.6 Following entry into the suprabasal layers, late
viral gene expression is initiated; the circular viral genome is then replicated, and
structural proteins form. In the upper layers of the epidermis or mucosa, complete
viral particles are assembled and released (Amanda Tristram et al., 2007).

Life cycle of genital Human Papilloma Virus.

2.6 PATHOGENESIS OF CERVICAL CANCER
A significant role for malignant transformation can be assigned to the E6 and
E7 genes and their respective proteins. E6 and E7 proteins are encoded by high-risk
HPV such as 16 and 18 and in addition to having in vitro transforming activity, both
E6 and E7 are always actively transcribed in cervical cancer, suggesting that
unregulated expression of these genes plays a significant role in carcinogenesis and
maintenance of the malignant phenotype. These proteins interact with cellular
proteins such as p53 and the product of retinoblastoma gene (pRb), which is normally
responsible for controlling key steps in the cell cycle. P53 is a key cellular regulatory
gene that acts as a transcriptional activator and has characteristics of a tumor
suppressor gene. In non-infected cells, p53 levels increase in response to cellular or
DNA damage or aberrant cellular proliferation signals. High levels of p53, by
interacting with WAF1/cip1 and cdk-cyclin, induce the cell to undergo growth arrest
in the G1 phase of the cell cycle, which provides an opportunity for the cell to either
repair DNA damage or be eliminated through programmed cell death (apoptosis). In
HPV-infected cells E6 protein from high-risk HPVs binds to p53 thus causing loss of
its crucial role in controlling the cell cycle.15 pRb also plays a crucial role in
controlling the transition from G1 to the S phase of the cell cycle by interacting with
other cell cycle proteins including E2F, c-myc and n-myc. E7 encoded by high-risk
HPVs binds with pRb, blocking its ability to interact with E2F and unregulated
proliferation occurs. On the other hand, E6 and E7 from low-risk HPVs have a lower
affinity for p53 and pRB and therefore is not oncogenic (Mohammed Akhtar, 2004).
The E5 gene product induces an increase in mitogen-activated protein kinase
activity, thereby enhancing cellular responses to growth and differentiation factors.
This results in continuous proliferation and delayed differentiation of the host cell.
The inactivation of p53 and pRb proteins can give rise to an increased proliferation
rate and genomic instability. As a consequence, the host cell accumulates more and
more damage DNA that cannot be repaired, leading to transformed cancerous cells. In
addition to the effects of activated oncogenes and chromosome instability, potential
mechanisms contributing to transformation include methylation of viral and cellular
DNA, telomerase activation, and hormonal and immunogenetic factors (Elizabath et
al., 2005).


2.7 PERSISTENCE AND CLEARENCE
Despite the high rates of anogenital infection, very few result in cellular
changes, genital warts, intraepithelial neoplasia, or cancer. Most infections (70-90%)
are contained by host immune responses and become undetectable within 6 to 10
months. Persistence of the virus (ie, continued detection of the virus for longer
periods of time) is a necessary but not sufficient step for the development of both
Cervical Intraepithelial Neoplasia (CIN) and cervical cancer. Persistence consistently
is associated with high-risk viral types, particularly HPV 16. Persistence of the virus
also has been associated with having multiple sexual partners, cigarette smoking,
immune suppression, and oral contraception. HPV viral persistence appears to be a
complex interaction of sexual behaviors, coexisting genital infections, and viral
characteristics (Marcia Shewet et al., 2005).
The majorities of HPV infections are self-limited and spontaneously clear
within a several-year period as a result of cell-mediated immunity. In one study, two-
thirds of adolescents infected with low risk HPV types spontaneously cleared their
infections by 12 months, as did over half of those infected with high-risk HPV types.
By 23 months, more than 80% had cleared their HPV infections. In another follow-up
study of adolescents and women with LSIL, 91% of HPV-infected individuals cleared
their infections after 36 months of follow-up. However, many women who
spontaneously clear one specific type of HPV become infected with another HPV
type. This is part of the reason that infection with multiple types of HPV is quite
common in sexually active adolescents and women (Thomas Wright, 2009).
Persistence of Human Papilloma Virus (HPV) infection is necessary for the
development of cervical cancer. Additionally, infection with HPV is implicated in the
majority of cases of other genital tract malignancies including vulvar, penile, and
vaginal cancer. HPV testing and vaccination are a routine part of
obstetrical/gynecological clinical practice. With an enhanced public awareness of
HPV infections, many patients turn to their obstetricians/gynecologists with questions
about transmission, testing, and prevention. In this review, we will discuss the biology
of HPV, epidemiology of disease, methods and indications for testing, and
vaccination strategies (Erickson et al., 2013).


2.8 DETERMINANTS OF HPV INFECTION
Although HPV is a necessary factor for the causation of cervical cancer, it
appears to be insufficient. Various other factors including host cell factors are
necessary for progression of HPV-infected precursor lesions to cancer. The primary
risk factors for acquiring HPV are generally associated with sexual activity. HPV is so
common and transmissible that having just one sexual partner often results in
infection. Indeed, cumulative prevalence rates are as high as 82% among adolescent
women in select populations. As such, nearly all sexually active adolescents are at
high risk for acquiring HPV (Anna-Barbara Moscicki, 2005). The main factors
associated with HPV infection are sex, age, race, sociodemographic characteristics,
prior sexually transmitted infections, parity, contraceptive methods, Dietary factors
and smoking (Eliane Duarte-Franco, 2004)
The most significant risk factor for HPV infection is sexual behaviour. The
total number of sexual partners and the number of recent partners appear to be the
most consistent factors, particularly for infections with carcinogenic HPVs. Age at the
time of the first sexual contact is a less constant factor of HPV infection .There are
several studies suggesting that the number of sexual partners is an important
mechanism through which adolescent and women acquire HPV. A longitudinal cohort
study of female university students (N =608) proved that a prior coercive sexual
experience was associated with a higher lifetime number of sexual partners (p
<.0001), which in turn associated with subsequent HPV infection (p< .0001) and SIL
(p < .0001). In logistic regression models, coercive sexual experience was associated
significantly with HPV (odds ratio [OR], 1.84; 95% confidence interval [CI], 1.19
2.84) and at a marginal significance level with SIL (OR, 1.90; 95% CI, .973.70)
(Jessica Kahn et al., 2005). A study conducted among randomly selected women aged
18 to 24 years, living in Sicily (south Italy) found that The only risk factor for HPV
infection was the number of sexual partners (women with 2-3 partners versus women
with 1 partner: odds ratio, 3.86; 95% confidence interval, 2.45-6.09) ( Pietro
Ammatuna et al., 2008) Other factors, such as STI history, hormonal factors (oral
contraceptives or pregnancy), condom use, and smoking are occasionally associated
with HPV infection.


2.9 PROGRESSION OF CERVICAL CANCER
Although more than 90% of HPV infections are spontaneously cleared by the
immune system, some infections will cause LSIL, HSIL, or progress to cervical
cancer (Anna-Barbara Moscicki, 2005). Functionally high-risk HPV infection
contributes to carcinogenesis and tumor progression predominantly through the
actions of two viral oncogenes, E6 and E7. These oncogenes are consistently
expressed in cervical cell lines and in human cancers. Both of these oncogenes
interact with and inhibit the activities of critical components of cell cycle regulatory
systems, in particular E6 with p53 and E7 with Rb. The E7 protein interacts with pRB
and inactivates this cellular protein. As a consequence, E2F transcription factor is
released from pRB-E2F complex, leading to transcriptional activation of several
genes involved in cell proliferation. Binding of the E6 protein to the p53 promotes the
degradation of the latter through a ubiquitin -dependant proteolysis system. Also of
significance is that on completion of the degradation of p53 by the ubiquitin-
dependant proteolysis system, the E6 protein is free to interact again with remaining
p53 molecules, leading to further degradation of the latter (Sreekala Nair et al., 2005).

2.10 EVASION OF IMMUNE RESPONSE BY HPV
There are a number of mechanisms by which HPV and its resulting neoplasia
evade recognition by the host:
HPV infects keratinocytes, which are not good APCs.
The virus is not lytic in its normal life cycle, and intraepithelial lesions are not
necrotic, so no danger signals are produced to stimulate the immune system.
Capsid proteins are produced late in the life cycle, in superficial epithelium distant
from normal immune surveillance and at low levels, compared with other viruses.
HPV proteins have immunosuppressive actions.
There is no viraemic stage of infection and therefore little opportunity for
systemic antigen presentation (Amanda Tristram et al., 2007).

2.11 DIAGNOSIS
Human papillomavirus testing is complicated because of the nature of the
virus. It cannot be cultured, and antibody methods are relatively insensitive; therefore,
HPV detection requires some form of nucleic acid test. The viral life cycle is
restricted to differentiating epithelium; therefore, a cellular sample collected from the

site of infection is required. Further, HPV is not a single virus but a family of more
than 100 closely related viral types. The types are distinguished based on differences
in their DNA sequence. Assays must be designed to handle the complexity introduced
by the large number of HPV types. Human papillomavirus testing has been used to
understand the epidemiology and natural history of the virus. The nature of the sample
and the assay will frame the view of infection. Definitions of occult, persistent, or
recurrent infection are complicated because of these issues.
Methods for HPV testing include commercial tests, such as Hybrid Capture
(HC; Digene Corporation, Silver Spring, USA) and a variety of Polymerase Chain
Reaction (PCR) protocols. Hybrid Capture (HC) is a commercially available solution
hybridization test and is the only HPV detection system that is approved by the US
Food and Drug Administration. It is relatively simple to use and therefore suitable in a
screening setting, but it does not provide information regarding individual HPV types
present in a sample. Hybrid Capture 2 contains a cocktail of probes for 13 HR (16, 18,
31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68) and 5 LR (6, 11, 42, 43 and 44) types
(Amanda Tristram et al., 2007).
Cervical cancer and genital warts are caused by HPV. However, testing for the
virus using standard techniques can sometimes give a negative result -- in these cases,
the condylomas are called 'virus-negative' warts. In a new study published
in Virology, researchers assessed the DNA found in samples taken from 40 patients
with 'virus-negative' genital warts. Through a general DNA sequencing approach, the
researchers showed that several of the negative samples did in fact contain HPV DNA
(Hanna Johansson et al., 2013).
Infection of cervical epithelium with high-risk human papilloma virus
(hrHPV) might result in productive or transforming cervical intraepithelial neoplasia
(CIN) lesions, the morphology of which can overlap. In transforming CIN lesions,
aberrations in host cell genes accumulate over time, which is necessary for the
ultimate progression to cancer. On the basis of (epi) genetic changes, early and
advanced transforming CIN lesions can be distinguished. This paves the way for new
molecular tools for cervical screening, diagnosis and management of cervical cancer
precursor lesions (Steenbergan et al., 2014)


2.11.1 TESTING METHODS FOR HPV DETECTION
Several effective molecular diagnostic techniques are available for HPV
infection. The Southern Blot method, Dot Blot method (ViraPap, Viratype), FISH
method (filter in situ hybridization), In situ hybridization (ISH), Hybrid captureII and
PCR (Polymerase Chain Reaction). A new method for HPV detection and genotyping
is the DNA Chip. This method, still under evaluation, uses PCR amplification
products and is able to identify 22 HPV genotypes. HPV detection is strongly affected
by the quality of the samples, in terms of cell quantity. Only the PCRs include a
control of the cells content of the sample.

2.11.1 (a) THE HYBRID CAPTURE II ASSAY (DIGENE)
The Hybrid Capture II assay (Digene) is commercially available. It is a nucleic
acid hybridization assay with signal amplification that utilizes microplate
chemiluminescent detection. The assay is highly sensitive and can detect DNA for 13
High-Risk (16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59, 68) and five low-risk (6, 11,
42, 43, 44) HPV types (Clavel et al., 1998; Chaudhary et al., 2010). Despite its good
performance as a triage test it is not a sensitive or type-specific assay.

2.11.1 (b) PAP TEST
The Pap test is the gold standard for detecting abnormal cell changes in the
uterine cervix due to HPV was developed by Papanicolaou in the 1920s (Carmichael,
1973). Cells are collected from the outer opening of the cervix and the endocervix
(transformation zone) using a speculum and /or brush and fixed on a glass followed
by examination under a microscope to look for abnormalities. The test aims to detect
potentially pre-cancerous changes, which could be removed. It is the most widely
used test in screening for cervical cancer worldwide. In America, the Pap test has
reduced the incidence of cervical cancer from 40/100,000 to 8/100,000 women. In
Finland, Sweden, British Columbia and Canada, the incidence of cervical cancer has
decreased up to 70-80% (Hristova and Hakama, 1997; Nieminen et al., 1999). It has
been observed that the introduction of the Pap test in a population which has never
been screened for cervical cancer may reduce the risk by 60-90% among women over
25 years of age (IARC 2005). Although the Pap test is a gold standard, if done once it
has a sensitivity ranging from 50-80%. The repeated sampling following an organized
programme is crucial.

2.11.1 (c) MULTIPLEX HPV GENOTYPING
Multiplex HPV genotyping is bead-based quantitative and high-throughput
DNA hybridization method that uses the Luminex suspension array technique to
simultaneously detect and genotype up to 100 HPV types (Schmitt et al., 2006). This
method is based on the amplification of a sequence of HPV DNA (in the L1 gene)
identified by the consensus primers GP5+/6+ and the subsequent detection of the
amplicons with type-specific oligonucleotide probes coupled with fluorescence-
labelled polystyrene beads with a diameter of 5.6 m that are internally dyed with
various ratios of two spectrally distinct fluorophores, creating an array of 100
different bead sets with specific absorption spectra (Louvanto et al., 2010). It has a
comparable principle to that of Multiplex HPV serology.

2.11.2 METHODS OF HPV TYPING
The presently existing methods for identifying HPV types include type-
specific PCR, line blot methods and pyrosequencing. Reverse line blot analysis is a
quick and simple HPV typing procedure, based on GP5+/GP6+ PCR amplification
products. This method can analyze 42 samples per day per membrane, with a
concordance of 96.5% for both single and multiple infections.

2.11.3 SEROLOGY
The detection of HPV antibodies is less used, due to the multitude of HPV
genotypes and inconsistent immunological response. Roughly half of the cervical
cancers are associated with HPV 16 and only half of these HPV 16 infections induce a
seroconversion for unknown reasons (Hpfl, 2001). According to Daling (1996), HPV
seroprevalence is an unreliable measure of HPV exposure, its association with the
presence of viral DNA demonstrating weak sensitivity. Among women with HPV 16
DNA detected, only 42.7-46% was seropositive. The sensitivity of serology for
detecting anti-HPV antibodies is approximately 50% in best case scenarios, maybe up
to 65-75%. Specificity is difficult to measure but is estimated at more than 98%
(Dillner, 2000). HPV seropositivity is associated with the number of sexual partners,
cytological lesions and previous HPV 16 infection.
The only HPV test approved by the U.S. FDA for clinical use is Digenes
Hybrid Capture 2 HPV DNA test (HC2). This test can be applied to cervical cells
collected with the Digene Cervical Sampler (includes brush and media), cervical cells

collected for liquid-based cytology, or with cervical biopsies collected in Digene
Specimen Transport Medium. The current assay format uses liquid hybridization in a
microarray platform. Samples are lysed to release nucleic acids and combined with
the RNA probe mixture. The high risk probe mix includes 13 types (HPV16, 18, 31,
33, 35, 39, 45, 51, 52, 56, 58, 59 and 68). The RNA probes hybridize to the DNA
targets in liquid phase and are bound to the wall (i.e., captured) by antibodies specific
for DNARNA hybrids. The same antibody linked to alkaline phosphatase is used to
generate a signal after addition of chemiluminescent substrate. Cutoff for a positive
result is determined by comparison of the intensity of sample to that of the 1 pg/mL
control. The test has good interlaboratorys comparisons and has been widely used in
trials determining the clinical utility of HPV testing.
Type-specific Polymerase Chain Reaction (PCR) assays for HPV are available
and can be particularly useful in quantitative assessments of HPV. However, the large
numbers of types those are of interest limit their usefulness in most studies. PCR
assays that target sequences that are highly conserved are called consensus assays.
These assays will generate a product for nearly all HPV types. Type-specific
identification requires analysis using sequencing, restriction fragment length
polymorphism assays, or hybridization. The three most widely used consensus PCR
assays are PGMY09/11, GP5+/6+, and SPF. All target the L1 region. They differ in
the primer mix, the size of the amplified product, and the method for type
identification. Results differ slightly in terms of type-specific sensitivity and
specificity. Typing assays using linear arrays or strips allow for efficient detection of
multiple HPV types within a sample.
Other HPV assays include serology and in situ hybridization. Serologic assays
use ELISA-based detection of type-specific antibodies against L1-VLPs (virus-like
particles). A positive reaction indicates past or current infection (Elizabeth et al.,
2006)
In the absence of genital samples, Human Papilloma Virus (HPV) serology
may be useful to assess HPV infection in young men and women. HPV
seroprevalence and determinants of seropositivity were assessed in 817 female and
518 male university students in Busan, South Korea, of whom 74% and 44%,
respectively, reported never having had penetrative sexual intercourse. Type-specific
HPV DNA status, assessed by a short PCR fragment primer set, was available from
genital samples. Seropositivity to L1 proteins of HPV types 16, 18, 31, 33, 45, 52, and

58 were assessed using multiplex HPV serology. Among women, HPV
seroprevalence was significantly higher among sexually active (26.1%) than
nonsexually active students [11.1%, odds ratio (OR) = 2.9; 95% confidence interval
(95% CI), 1.8-4.7], although the association was weaker than that for HPV DNA
prevalence (OR, 14; 95% CI, 4.7-42). Furthermore, HPV seroprevalence was higher
among HPV DNA-positive (24%) than HPV DNA-negative women (13%), and there
was a positive correlation of type-specific seroprevalence with the presence of HPV
DNA of the same type (Gary Clifford, 2013).

2.12 VACCINES
The development of HPV vaccines has taken many years, since there is no
effective culture system to propagate HPV. The problem has been solved by using the
L1 coat protein to produce Virus-Like Particles (VLPs), which contain no viral DNA.
However, their outward close resemblance to the actual virus activates a strong
immune response. There are 15 HPV genotypes which have carcinogenic potential; so
far, the vaccines contain VLPs against only two types. It appears that HPV gains entry
through a micro abrasion of the genital epithelium and enters keratinocytes in the
basal epithelium (Stanley, 2010). HPV does not cause a viraemia and, therefore, there
is no systemic infection to trigger antibody production, facilitating the persistence of
the infection, and then transmission by shed epithelial cells. There may be a local cell-
mediated immune response, which results in clearance of the infection, but does not
always result in the production of serum neutralising antibodies. Only around half of
those infected develop antibodies to the virions, and even then the levels are usually
too low to prevent re-infection, even with the same HPV type (Schwarz, 2008;
Safaeian, 2010).
Merck & Co (Gardasil marketed by Commonwealth Serum Laboratories
[CSL] in New Zealand) and Glaxo Smith Kline (GSK Cervarix) have both
developed vaccines that have been licensed in New Zealand. Gardasil contains VLPs
for HPV types 6, 11, 16 and 18 and Cervarix VLPs for HPV types 16 and 18. Gardasil
is licensed in females aged 9 to 45 years and males aged 9 to 26 years (Sexually
Transmitted Infection Education Foundation 2012).On this basis, two prophylactic
vaccines containing type -16 and-18 specific antigens were recently developed; to
date, they have showed a 79 year clinical efficacy of about 100% against cervical
cytological abnormalities caused by HPV-16 and -18 genotypes, with a variable cross-

protective efficacy against cervical lesions associated with other high-risk oncogenic
types (Malago et al., 2012; Wheeler et al., 2012).
Vaccination of adolescents against sexually transmitted infections (STIs) is an
important prevention strategy that may reduce the global burden of disease. The
World Health Organization, Centers for Disease Control and Prevention's Advisory
Committee on Immunization Practices, and other national health agencies recommend
the use of existing STI vaccines, and many countries have incorporated them into
their routine vaccination schedule. Despite this, however, data indicate that STI
vaccine uptake is suboptimal for a variety of reasons. Health care professionals (HCP)
have been shown to have a strong beneficial effect on STI vaccine uptake, yet studies
demonstrate that many HCPs fail to discuss or recommend them to adolescent
patients. This review article focuses on HCP communication about STI vaccines with
adolescents and their parents. It describes STI vaccine message content and delivery
as well as the context in which HCPs formulate their messaging approach. It also
examines other contextual factors that may shape communication about STI vaccines.
Studies from many countries indicate that HCPs often possess misinformation about
adolescents, including their sexual risk behaviors, as well as STIs, vaccine safety and
efficacy, and STI vaccination recommendations. They also have misconceptions of
parental barriers to STI vaccination. These may impact STI vaccine communication
and have a negative influence on STI vaccine uptake. These findings highlight the
critical need for improved HCP education related to adolescent health, sexuality, and
STI vaccination. This may be particularly important in settings without an existing
infrastructure or expertise in caring for this unique patient population (Hofstetter et
al., 2014).
The arena of vaccination, which has been adversely affecting so many
children, is now a center point for documenting how Big Pharma has taken over so
much of science, resulting in outright fraud thats used to promote their products.
Scientists Lucija Tomljenovic Christopher Shaw, Judy Wilyman, Eva Vanamee, and
Toni Bark use the example of Mercks Gardasil, Human Papilloma Virus (HPV)
vaccine, to demonstrate the point that HPV vaccine activism is not based on science,
but instead on misrepresentations of science.


In a letter signed by all five of these scientists, they point out several flaws in
the claims Mercks Gardasil and Pfizers Cervarix make to sell their vaccine:
HPV vaccines have never been shown to prevent cervical cancer.
The end-points used in the studies are based on infections and lesions that usually
heal without
Help, so how can they demonstrate efficacy in preventing cancer several years
later?
The trials are biased to produce false negatives and therefore cannot accurately
estimate the risk of developing cancer.
Passive methods of recording adverse effects cannot accurately reflect how
prevalent they are.
Accurate estimates of the actual frequency of HPV vaccine adverse effects cannot
be made when such effects are automatically dismissed as unrelated to the
vaccine.
Women are not informed that, in some instances the HPV vaccines may increase
the rate at which existing abnormalities develop, thus causing the cancer from
which theyre supposedly being protected.
When information about HPV vaccine risks and limitations are not provided,
women cannot possibly make informed decisions about whether to have the
vaccine (Heidi Stevenson, Green med information, feb,13).
There are currently two HPV vaccines on the market, but if there was any
regard for sound scientific evidence, neither would be promoted as heavily as they
are. The first, Gardasil, was licensed by the US Food and Drug Administration (FDA)
in 2006. It is now recommended as a routine vaccination for girls and women between
the ages of 9-26 in the US. The second HPV vaccine, Cervarix, was licensed in 2009.
(Mercola, 13). The study pointed out that HPV vaccine uptake among young girls in
the US has been low but concluded that: Within four years of vaccine introduction,
the vaccine-type HPV prevalence decreased among females aged 1419 years despite
low vaccine uptake. The estimated vaccine effectiveness was high.
A new study looking at the prevalence of Human Papilloma Virus (HPV)
infections in girls and women before and after the introduction of the HPV vaccine
shows a significant reduction in vaccine-type HPV in U.S. teens. The study, published
in [the June issue of] The Journal of Infectious Diseases reveals that since the vaccine
was introduced in 2006, vaccine-type HPV prevalence decreased 56 percent among

female teenagers 14-19 years of age. About 79 million Americans, most in their late
teens and early 20s, are infected with HPV. Each year, about 14 million people
become newly infected. This report shows that HPV vaccine works well, and the
report should be a wake-up call to our nation to protect the next generation by
increasing HPV vaccination rates, said CDC Director Tom Frieden. Unfortunately
only one third of girls aged 13-17 have been fully vaccinated with HPV vaccine.
Countries such as Rwanda have vaccinated more than 80 percent of their teen girls.
Our low vaccination rates represent 50,000 preventable tragedies 50,000 girls alive
today will develop cervical cancer over their lifetime that would have been prevented
if we reach 80 percent vaccination rates. For every year we delay in doing so, another
4,400 girls will develop cervical cancer in their lifetimes (Division of News &
Electronic Media, 13).
According to CDC, each year in the United States, about 19,000 cancers
caused by HPV occur in women, and cervical cancer is the most common. About
8,000 cancers caused by HPV occur each year in men in the United States, and
oropharyngeal (throat) cancers are the most common. The study by Dr. Lauri
Markowitz and colleagues at the CDC used the National Health and Nutrition
Examination Survey (NHANES) data to compare prevalence or proportion of girls
and women aged 14-59 years with certain types of HPV before the start of the HPV
vaccination program (2003-2006) with the prevalence after vaccine introduction
(2007-2010).
Through these promising results, public health experts and clinicians look
forward to more people getting vaccinated for HPV. Routine vaccination at age 11-12
for both boys and girls is recommended, but according to recent national
immunization surveys, only about half of all girls in the U.S. and far fewer boys
received the first dose of HPV vaccine. A series of three shots is recommended over
six months. HPV vaccination is also recommended for older teens and young adults
who were not vaccinated when younger (CDC, 2013).
The Food and Drug Administration (FDA) has approved two vaccines that
protect against HPV types 16 and 18, which cause 70% of cervical cancers. One of
the vaccines also protects against types 6 and 11, which cause about 90% of genital
warts. The FDA has approved both vaccines for use in girls and women 9 to 26 years
old, and the second vaccine for prevention of genital warts in boys and men 9 through
26 years old. The vaccines are given in three doses over a period of six months. The

American Academy of Pediatrics recommends that both girls and boys receive HPV
vaccines between ages 11 and 12 and that those ages 13 to 21 that haven't had the
vaccine be immunized.
The FDA says the vaccines are considered safe but are only effective if given
before an initial exposure to the virus. AAP recommends that young people who are
sexually active still receive the vaccination as those already infected with one type of
HPV infection may benefit from the protection against other types included in the
vaccine.

2.15.1 ABOUT GARDASIL
GARDASIL is approved for use in more than 125 countries. As of July 2012,
more than 95 million doses of GARDASIL have been distributed worldwide;
however, it is not known how many doses have been administered. Merck is pursuing
a systematic and thoughtful approach to improve access to GARDASIL in the
developing world through four key pillars: innovation, partnerships, pricing and
implementation. The initiative in Uganda follows the launch in April 2011 of a
comprehensive cervical cancer prevention program in Rwanda incorporating both
HPV vaccination and HPV testing, the first program of its kind in Africa. In its initial
year, an estimated 93 percent of eligible girls 12 to 15 years of age in Rwanda were
vaccinated with three doses of GARDASIL. Also, in 2010 Merck partnered with the
Royal Government of Bhutan and Australian Cervical Cancer Foundation to launch a
six-year national vaccination program with GARDASIL for appropriate girls and
young women between the ages of 12 and 18 in Bhutan (Heidi Stevenson, 2013).


3. MATERIALS AND METHODS

3.1 STUDY POPULATION
The study population consists of 119 married women from various parts of
Salem District. All the subjects were recruited between November 2013 to June 2014.
All the participants were volunteers who gave informed written consent. The
exclusion criteria include those who had prior malignancy, mental illness, and the
participants those who had received antibiotics in the previous month.

3.2 SAMPLE COLLECTION
A total of 119 paraffin fixed cervical cancerous patient biopsy were collected
from a Salem biopsy center. Subjects were entered the study providing signed
informed consent form. They were, family interviewed with a structured questionnaire
to obtain information on their age, sex, residence income, and medical history. So, a
total of 119 married women were included in this study. The data and samples were
unlinked and anonymous.
3.3 GENOMIC DNA EXTRACTION METHODS
Two different methods were used to identify the DNA.
Protocol 1: DNA extraction technique for biopsy tissue (Baker & Conway, 1999)
A small section (~20mg) of the sample was removed and washed with DD
H
2
O. Cut into small pieces on glass plate covered with parafilm
Place diced sample in 2.0 ml Microcentrifuge tube containing 500l lysis
buffer. Bring total volume to 900 l with lysis buffer
100l proteinase K (10mg/ml) was added and incubated overnight at 55
o
C with
gentle rotation
Equal volume of equilibrated phenol was added and vortexes briefly. Rotated or
rocked at room temperature for 10-15 mins. Spin down 15-20 min at 13,000
rpms. Carefully drawn off supernatant and placed in new 2.0 ml tube

Added equal volume of chloroform-isoamyl alcohol (24:1). Vortex briefly.
Rotate or rock at room temperature for 10 min. Spin for 15 min at 13,000
rpms. Drew off supernatant and placed in new 2.0 ml tube
Then again repeated the protocol, add equal volume of chloroform-isoamyl
alcohol (24:1). Vortex briefly. Rotated or rocked at room temperature for 10
min. Spin for 15 min at 13,000 rpms. Drawed off supernatant and placed in new
2.0 ml tube
. Split into 2 tubes. Added volume ammonium acetate (7.5 M)
Added 2 volumes of cold 95% EtOH. Place in freezer
DNA may spool into a white cottony ball (high quality DNA) after about an hour
or less at -20
o
C. Remove spooled DNA with sterile 1000 l Pipettor. Place in a
new tube with 200 l 70% EtOH to wash. Spin down for 10 min at 7,000 rpms to
get a pellet. Poured off or pipette off EtOH. Placed tube in block or incubated for
10 min at ~55
o
C to dry off remaining EtOH
Dissolved in 150 l TE at 55
o
C for 1 hr. Label with H (for high quality)
Remaining DNA in 2 tubes can be collected by spinning down into pellets at 7000
rpm's for 20 min. Poured off 95% EtOH. Add ~150 l 70% EtOH to wash
pellet. Spin again at 7000 rpms for 10 min. Poured off EtOH. Dry for 10 min at
~55
o
C. Dissolved in 25 l TE at 55
o
C for 1 hour
Quantify DNA concentration in each tube. Standardize concentrations of samples
to desired levels as much as possible. Some samples may be highly
concentrated. Dilute these further with TE
All instruments and reagents should be sterile. Scalpels, forceps and other
instruments should be cleaned with 70% EtOH and flamed over a burner between
samples


Protocol 2: DNA extraction technique for biopsy tissue
Day 1
Cut 10-20X (30m) sections of formalin fixed paraffin sample into
Eppendrof tubes
1ml xylene was added and incubated at RT for 15 mins.
Spin down for 5 min at 13000 RPM and discarded supernatant.
Added 1ml of 100% ethanol and incubated at RT for 15 min.
Spin down for 5 mins at 13000 RPM and discarded the supernatant.
500l of Proteinase K buffer was added (50mM Tris pH 8, 1mM EDTA, 0.5%
Tween20).
Incubated overnight at 55 C in a shaker.
Day 2
Again added 20l Proteinase K (stock solution 20mg /ml in water, stored at -20 C.
Final conc=0. 4 mg/ml.
Incubated overnight at 55 C in a shaker.
Day 3
Added 500 l phenol chloroform into the tube and wait for 5min at RT.
Spin down for 5 mins at 13000 RPM
Get off the upper layer.
Mixed it with 500l of PCI in eppendrof tubes (Phenol chloroform
Isoamyl alcohol extraction 1:1 v/v).
Shaked gently and incubated for 10 min at RT.
Spin down for 5 min at 13000 RPM.
Supernatant was collected into new eppendrof tubes.
Added 300l of 7.5M ammonium acetate, 1ml cold 100% ethanol and 5l
Glycogen (stock = 20g/ml).
Shaked gently and incubated at -20 C for 2 hours or overnight.

Spin down for 5 min at 13000 RPM and discarded the supernatant.
The pellet was air dried and dissolved in 25l in water or TE buffer.
Proteinase K buffer
Tris, pH 8 0.5 ml
0.5M EDTA 40l
10% Tween 1ml
Todd H
2
O 18.5ml
Total 20ml

Proteinases K dissolve solution
Tris HCL pH 7.5 0.1ml
CaCl
2
0.2ml
Glycerol 10ml
Water 10ml

3.4 DETECTION AND QUANTIFICATION OF DNA
The DNA isolated by these two methods were detected on 0.8% agarose gel
and the purity of DNA extracted by two method were compared based on readings at
260/280nm absorbance ratio to analyze the quality and quantity of DNA extracted.

3.4.1 HPV DNA AMPLIFICATION AND DETECTION
Initially, all samples were subjected to -globin primer amplification to
determine the presence of cellular DNA. Samples negative for -globin were excluded
from the study. All -globulin positive DNA samples were then screened by
MY09/MY11 consensus primer spanning the L1 region of the HPV genome. Then
further analysis of PCR using type-specific primer for HPV-16 was performed. The

primers used, their sequence and amplicon size were given in table 1. PCR was
performed in a 25 l reaction mix containing 6l of template, 2.5mM MgCl
2
,
10mMdNTP, 10M of each primer, 2.5U of Taq polymerase. The thermal cycling
consisted of an initial denaturation step at 94
o
C for 4mins, followed by 40 cycles of
denaturation at 94
o
C for 1min, annealing at 55
o
C for 1min and extension 72
o
C for
1min respectively and terminated by final extension 72
o
C for 7mins.

Reaction setup for PCR
Components Stock Concentration
Working
Concentration
Taq Buffer 10X 1X
dNTPs 10mM 200M
MgCl2 25mM 2mM
Primer 100M 10M
Taq polymerase 5U/1l 5U

The thermal cycling consisted of an initial denaturation step at 94
o
C for 4
mins, followed by 40 cycles of denaturation at 94
o
C for 1min, annealing at 55
o
C for
1min and extension 72
o
C for 1min respectively and terminated by final extension
72
o
C for 7mins. A second round of amplification with the general primer positioned
inside of the MY09 and MY11 primer was performed on 1l of amplified DNA from
the first round of PCR added to 49 l of the PCR master mix. After an initial
denaturation step of 94C for 5 min, the samples were subjected to 40 reaction cycles
of denaturation at 95 C for 1min, primer-template annealing at 40C for 2min and
primer extension at 72C for 2 min. At the end of the cycles a final extension step at
72C for 10 min was performed.


Table 1 Primers used for detection of HPV

3.4.2 DETECTION OF AMPLIFIED PRODUCTS
After PCR amplification, 8l of each single-step PCR product were mixed
with 2l of loading dye and were electrophoresized through 2% agarose gel at 100
volts. Subsequently 5l of the nested PCR product was electrophoresed through 2%
agarose gel. After electrophoresis the gels were stained with ethidium bromide and
examined and photographed under ultraviolet illuminator using Gel documentation
system. The bands of appropriate size were identified by comparison with known size
ladder (100 -1000bp ladder, Bio-Rad Lab.,).
3.5 METHODS OF GEL ELECTROPHORESIS

The edges of a clean, dry glass plate were sealed with tape to form a mold and the
mold was set on a horizontal section of the bench
Sufficient electrophoresis buffer (1x TBE) was prepared to fill the electrophoresis
tank to cast the gel
1.5 gm of agarose powder was added to 100ml of 1x TBE buffer solution in an
Erlenmeyer flask
The slurry was heated in a boiling water bath until the agarose dissolved completely
S.NO: PRIMER PRIMER SEQUENCE SIZE
1.
globin-F
globin-R
GAA GAG CCC AAG GAC AGG TAC
CAA CTT CAT CCA CGT TAG ACC

264bp
2.
MY 09-F
MY 09-R
CGT CCM ARR RGG AWA CTG ATC
GCM CAM GGM CAT AAY AAA TGG

450bp
3.
HPV-16-F
HPV-16-R
AAG GCC AAC TAA ATG TCA C
CTG CTT TTA TAC TAA CCG G

217bp

When the gel had cooled to 55c, ethidium bromide to a final concentration of 20l of
10mg/ml was added
The gel solution was mixed thoroughly with gentle swirling
The comb was positioned 0.5-1.0mm above the plate to form the sample slots in the
gel
The warm agarose solution was poured into the mold and the gel allowed to set
completely (30-45 minutes at room temperature), Then poured a small amount of
electrophoresis buffer on the top of the gel, the comp was removed carefully
Again electrophoresis buffer was poured off and the tape was removed carefully. The
gel was mounted in the electrophoresis tank
Added just enough electrophoresis buffers to cover the gel to a depth of 1mm
10l of PCR product was mixed with 2l 6 x gel-loading buffers and slowly loaded
into the slots of the submerged gel using a micropipette, a 100bp DNA ladder was
used to detect the number of base pairs
The lid of the tank was closed and electrical leads were attached, so that the DNA
migrates towards the positive anode
Apply a voltage of 1-5v/cm. The gel was run until the bromophenol blue had migrated
an appropriate distance thoroughly the gel
Then the electrical current was turned off, the leads and the lid removed from the gel
tank.
The gel was examined carefully by ultraviolet (UV) transilluminator and the results
were noted and photographed the gel

3.6 STATISTICAL ANALYSIS
The t-test analysis was calculated to evaluate the purity of DNA extracted by
protocol1 (Baker & Conway, 1999) and protocol 2 based on the absorbance ratio at
260/280nm. The odds ratio and 95% Confidence Interval were calculated in
determining the prevalence of HPV infection in rural and urban areas of married
women included in this study.


CERVICAL CANCER
QUESTIONNAIRE FOR PRIMARY DATA COLLECTION
Hospital Code:
Lab ID: GVN/CC/
PERSONAL DETAILS
Name
Age
Marital Status Age at Marriage:
Address
Nationality
Occupation, Annual
Income

PERSONAL HABITS
Food Habits Veg Non Veg
Smoking Yes No
Alcohol Yes No
Narcotic Yes No

MEDICAL HISTORY
Stomach Ache Irritation
Fungal Infection Arthritis
Itching Inflammation
Vaginal Discharge Viral Infection
Loss of Body Weight Back Ache
Urinary Tract Infection Lower abdominal pain
Other Complaints

OTHER DETAILS REMARKS
Number of Children, Age at Delivery:
Method of Contraception used:
Type of Delivery Normal Cessarian
Consanguineous Marriage Yes No
Abortion Yes No
Intercourse Often Rare
Irregular Menstruation Prolonged Shortened
Exposure to STD, if yes,
type, treatment undergone
Yes No
HRT (If yes, what HRT
taken and when)
Yes No
Hysterectomy Yes No
Menopause attained, If yes
at what age
Yes No

Undergone any Screening
test for Cervical Cancer? If
yes, type of test and how
many times, time interval
Yes No
History of Abnormal cells Yes No
Anorexia Yes No

OTHER COMPLAINTS:
ONSET OF SYMPTOMS:
Age:
CANCER STAGE:
TYPE OF CERVICAL CANCER: Squammous / Glandular
MODE OF TREATMENT: a) Surgery b) Chemotherapy c) Radiation d) Other
PATIENT CONSENT:
I hereby declare that I am aware of the nature and purpose of the above tests and the
datas provided by me is given with my own willingness.
Date:
Place:
Signature

STD - Sexually Transmitted Disease, HRT - Hormone Replacement Therapy


4. RESULTS

This chapter deals with analysis and results of data collected from the married
women, Salem, Tamilnadu.
The study was based on the sample of 119 subjects in the study population.
The data regarding the HPV and other background variables such as age, occupation,
annual income, Height, Weight, food habits, stomach ache, UTI, Inflammation, Viral
infection, Fungal infection, irritation, itching, loss of body weight, backache, arthritis,
lower abdominal pain, vaginal discharge, Irregular menstruation, Exposure to STD,
HPV 16, screening test for cervical cancer, History of abnormal cell, Anorexia,
Vaccination etc., were elicited from the randomly selected subjects over the
population. The informations were carefully screened and recorded from the
collected samples. The data was put into several statistical analyses to extensively
explicate the prevalence of HPV over the study areas and to explore the results with
various socio-demographic variables.

Analysis of the data focused on the following objectives,
4.1 General Objective
The Prevalence of HPV over the study populations of Salem District.
4.2 Sub Objective
To examine the prevalence status of HPV Positive, High-Risk HPV-16 in the
study subjects.
To determine the sign and symptoms of HPV Positive, High-Risk HPV-16
To determine the risk factors associated with HPV Positive, High-Risk HPV-
16.


4.3 Polymerase Chain Reaction
4.3.1 Single rounds PCR
To check the presence of HPV-DNA, all samples were initially screened with -
globin primer and none of the samples were found negative. Only the HPV-DNA
positive for -globin was further analyzed by PCR for MY09/MY11 and type-specific
primer HPV-16. Out of 119 biopsy samples, 19 were positive for MY09/MY11
consensus primer, and 10 positive for HPV-16. PCR amplification of the L1 region of
HPV was shown in the Plate 4 and the amplified products of HPV-16 were shown in
Plate 5.

4.3.2 DNA Extraction
The purity of DNA extracted by protocol 1 (Baker and Conway, 1999) and
protocol 2 was analyzed using student-t-test. The t-test analysis of purity of DNA
isolated by above mentioned two methods were shown in table 1. The P value was
found to be < 0.0001 indicating there was a statistical significant difference between
two methods employed. The quantity of DNA obtained per biopsy sample was about
6 to 10ng which was sufficient for PCR. This indicates that protocol 1 (Baker and
Conway, 1999) was an effective method for extraction of HPV-DNA from the biopsy
sample.


Table 1: Absorbance ratio of DNA isolation using different protocols

S.NO.
Absorbance at 260/280nm (n-25)
Protocol 1 Protocol 2
1 1.54 0.81
2 1.16 0.73
3 1.80 0.69
4 1.33 0.77
5 1.30 0.82
6 1.32 0.56
7 1.83 1.16
8 2.00 1.58
9 1.67 0.89
10 1.94 0.93
11 2.00 1.49
12 1.95 1.03
13 1.98 1.04
14 1.69 0.86
15 1.49 0.54
16 1.72 0.77
17 2.00 1.05
18 1.78 0.94
19 1.85 1.15
20 1.94 1.17
21 1.99 1.03
22 1.05 1.01
23 1.89 1.16
24 1.75 1.01
25 1.26 0.98

Table-2 Prevalence of HPV over the study population

Figure: 1 Prevalence of HPV over the study population

The above table 2 and figure 1 showed the prevalence of HPV among the
study populations. A total 119 study subjects 19 were positive for HPV infection,
remaining 100 cases were negative for HPV infection. Among 19 positive cases, 10
were positive for HPV 16 DNA, the remaining cases were infected with other HPV
types.

0
20
40
60
80
100
120
No % No %
HPV Positive HPV 16
Positive
Negative
S. No Results
HPV Positive

HPV 16
No % No %
1. Positive 19 15.97 10 8.40
2. Negative 100 84.03 109 91.60
Total 119 100 119 100

Table: 3 Prevalence of HPV over the age distribution of study
Subjects

Figure: 2 Prevalence of HPV over the age distribution of study
Subjects

The age distribution of study subjects over HPV positive and HPV16 were
shown in table 3 and figure 2. A total of 119 study subjects were tested for the present
study, the prevalence of HPV positive 10 and 6 positive subjects were found in the
age groups 30-40 and 41-50 respectively. The maximum numbers of positive subjects
were found in the age group between 30-40. In the case of HPV16 - 6 positive
subjects were found in the age group between 30-40.
0
10
20
30
40
50
60
70
Positive Negative Positive Negative
HPV Positive HPV 16
10
60
6
64
9
40
4
45
30-40 41-50
S. No Age
HPV Positive HPV 16
Positive Negative Positive Negative
1. 30-40 10 60 6 64
2. 41-50 9 40 4 45
Total 19 100 10 109

Table 4: Association of socio demographic variables with HPV positive results

Socio
demographic
variables
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV positive
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative Upper
limit
Lower
limit
No. % No. % No. %
Age of the
study subject
30-40 10 14.3 60 85.7 70 100
0.5506 0.7407 0.2765 1.9844
41-50 9 18.4 40 81.3 49 100
Occupation
Rural 12 15.6 65 84.4 77 100
0.8776 0.9231 0.3333 2.5566
Urban 7 16.7 35 83.3 42 100
Annual
Income
<25,000 5 14.3 30 85.7 35 100
0.7469 0.833 0.2754 2.5213
>25,000 14 16.7 70 83.3 84 100

* P value <0.05 is statistically significant.
The association of socio demographic variables with HPV positive result was
shown in table 4. Prevalence (OR for women by age group 30-40 vs. 41-50 =0.
7407; 95% CI: 0.2765-1.9844). 30-40 years was slightly increased level with HPV
positivity. Occupation (OR for women with rural vs. Urban = 0.9231; 95% CI=
0.3333-2.5566). High level HPV positivity was found to be rural women. Annual
income (OR for their income 25,000 vs. 25,000 = 0.833; 95% CI: 0.2754-2.5213)
higher level HPV infection was more distributed to above 25,000 study subjects.


Table 5: Association of socio demographic variables with HPV - 16 results
Socio
demographic
variables
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV 16
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
limit
Lower
limit
No. % No. % No. %
Age of the
study
subject
30-40 6 8.6 64 91.4 70 100
0.9371 1.0547 0.2814 3.9536
41-50 4 8.16 45 91.8 49 100
Occupation
Rural 7 9.0 70 90.9 77 100
0.7150 1.3000 0.3180 5.1346
Urban 3 7.14 39 92.9 42 100
Annual
Income
<25,000 1 2.9 34 97.1 35 100
0.1905 0.2451 0.0290 2.0123
>25,000 9 10.7 75 89.3 84 100

The association of socio demographic variables with HPV -16 positive results
was shown in above Table 5. Age group (30-40 vs. 41-50 = 1.0547; 95% CI; 0.2814-
3.9536) the prevalence of age group 30-40 years was increased level with HPV16
positivity. Occupation, the HPV16 positivity (9.0%) was found to be in rural women.
Annual income (25,000 vs. 25,000 = 0.2451; 95% CI = 0.0290-2.0123).


Table 6: Association of personal habits with HPV positive results
Personal
habits
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV Positive
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative
Upper
limit
Lower
limit
No. % No. % No. %
Food
habit
Veg 12 13.8 75 86.2 87 100
0.2898 0.514 0.2027 1.6106
Non
veg
7 21.9 25 78.1 32 100

The prevalence of HPV over the personal habitat was studied and the results
were shown in table 5. Positive subjects were found to be only in food habits. (OR for
women, their food habits = 0.514; 95% CI = 0.2027-1.6106) the food habits (Non-
veg) was found to be positive for HPV. The above mentioned personal habits were
not much associated with prevalence of HPV infection in the study subjects.


Table 7: Association of personal habits with HPV 16 results

Personal
habits
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV 16
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
limit
Lower
limit No. % No. % No. %
Food
habit
Veg 7 8.0 80 92 87 100
0.8169 0.8458 0.2049 3.4912
Non
veg
3 9.4 29 90.6 32 100

The prevalence of HPV over the personal habits was studied and the results
were shown in table 7. Positive subjects were found to be only in food habits. (OR for
women, their food habits = 0.8458; 95% CI = 0.2049-3.4912) the food habits (Non-
veg) was found to be positive for HPV. The above mentioned personal habits were
not much associated with prevalence of HPV infection in the study subjects.


Table 8: Association of sign and symptoms with HPV positive results
Sign and
symptoms
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV Positive
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative Lower
limit
Upper
Stomach ache
Yes 15 17.6 70 82.4 85 100
0.4318 1.6071 0.4924 5.2458
No 4 11.8 30 88.2 34 100
Fungal
infection
Yes 2 2.6 74 97.4 76 100
0.0001* 0.0413 0.0089 0.1912
No 17 39.5 26 60.5 43 100
Itching
Yes 15 14.6 8.8 85.4 103 100
0.0171* 5.6250 1.3594 23.2749
No 4 25 12 75 16 100
Vaginal
discharge
Yes 8 9.9 73 90.1 81 100
0.0110* 0.2690 0.0978 0.7401
No 11 28.9 27 71.0 38 100
Loss of body
weight
Yes 1 2.4 40 100 41 100
0.0177* 0.0833 0.01701 0.6493
No 18 23.0 60 77 78 100
Urinary tract
infection
Yes 8 117.2 50 86.2 58 100
0.5290 0.7273 0.2698 1.9602
No 11 18.0 50 82 61 100
Irritation
Yes 6 13.0 40 87 46 100
0.4911 0.6923 0.2430 1.9721
No 13 17.9 60 82.2 73 100
Arthritis
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100
Inflammation
Yes - - - - - -
NA NA NA NA
No 19 35.2 35 65 54 100
Viral
infection
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100
Back ache
Yes 1 2.4 40 97.6 41 100
0.0177* 0.0833 0.0107 0.6493
No 18 23.1 60 77 78 100
Abdominal
pain
Yes 6 13.0 40 87 46 100
0.491 0.6923 0.2430 1.9721
No 13 17.9 60 82.2 73 100


The association of base line characters with HPV positive result was shown in
below Table 8. None of the subjects positive were found to be Arthritis, Inflammation
and Viral infection. Stomach ache (OR for women had stomach ache = 1.6071 95%
CI: 0.4924-5.2458). Fungal Infection (OR for women had Fungal Infection = 0.0413;
95% CI: 0.0089-0.1912: p-value 0.0001) highly sensitive significant associated with
Fungal Infection. Itching (OR for women had itching = 5.6250; 95% CI: 1.3594-
23.2749 p-value 0.0171) highly sensitive significant associated with itching. Vaginal
discharge (OR for women had vaginal discharge = 0.2690; 95% CI: 0.0978-0.7401: p-
value 0.0110) highly sensitive significant associated with vaginal discharge. Loss of
body weight (OR for women had loss of body weight = 0.0833; 95% CI: 0.01701-
0.6493: p-value 0.0177) highly sensitive significant associated with loss of body
weight. UTI (OR for women had UTI = 0.7273; 95% CI: 0.2698-1.9602). Irritation
(OR for women had irritation =0. 6923; 95% CI: 0.2430-1.9721). Back ache (OR for
women had back ache = 0.0833; 95%CI: 0.0107-0.6493: p-value 0.0177) highly
sensitive significant associated with back ache. Abdominal pain (OR for women had
abdominal pain = 0.6923; 95%CI: 0.2430-1.9721). The symptoms such as viral
infection, arthritis and Inflammation were not associated with HPV positive infection.


Table 9: Association of Sign and symptoms with HPV - 16 results
Sign and
symptoms
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV 16
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative
Lower
limit
Upper
Stomach
aches
Yes 8 9.4 77 90.6 85 100
0.5344 1.6623 0.3345 8.2618
No 2 5.9 32 94.1 34 100
Fungal
infection
Yes - - - - - -
NA NA NA NA
No 10 23.3 33 76.7 43 100
Itching
Yes 9 8.7 94 91.3 103 100
0.7399 1.4362 0.1695 12.1662
No 1 6.7 15 93.8 16 100
Vaginal
discharge
Yes 3 3.7 78 96.3 81 100
0.0142* 0.1703 0.0414 0.7012
No 7 18.4 31 81.6 38 100
Loss of body
weight
Yes - - - - - -
NA NA NA NA
No 10 12.8 68 87.2 78 100
Urinary tract
infection
Yes 4 6.9 54 93.1 58 100
0.5653 0.6790 0.1814 2.5411
No 6 9.8 55 90.2 61 100
Irritation
Yes 2 4.3 44 95.7 46 100
0.2212 0.3693 0.0749 1.8221
No 8 11 65 89.0 73 100
Arthritis
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100
Inflammation
Yes - - - - - -
NA NA NA NA
No 10 18.5 44 81.5 54 100
Viral
infection
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100
Back ache
Yes - - - - - -
NA NA NA NA
No 10 12.8 68 87.2 78 100
Abdominal
pain
Yes 2 4.3 44 95.7 46 100
0.2212 0.3693 0.0749 1.8221
No 8 11 65 89.0 73 100


The association of base line characters with HPV16 positive result was
showed in below Table 9. None of the subjects positive were found to be Fungal
Infection, Back ache, Arthritis, Inflammation and Viral infection. Stomach ache (OR
for women had stomach ache = 1.6623; 95% CI: 0.3345-8.2618). Itching (OR for
women had itching = 1.4362; 95% CI: 0.1695-12.1662). Vaginal discharge (OR for
women had vaginal discharge = 0.1703; 95% CI: 0.0414-0.7012: p-value 0.0142)
highly sensitive significant associated with vaginal discharge because, p-vaiue < in
0.05. UTI (OR for women had UTI = 0.6790; 95% CI: 0.1814-2.5411). Irritation (OR
for women had irritation =0.3693; 95% CI: 0.0749-1.8221). Abdominal pain (OR for
women had abdominal pain = 0.3693; 95%CI: 0.0749-1.8221). The symptoms such as
viral infection, arthritis, Fungal Infection, Back ache and Inflammation were not
associated with HPV16 positive infection.


Table 10: Association of risk factors with HPV Positive results
Risk factors
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV Positive
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative Lowe
r
limit
Upper
limit
No. % No. % No. %
Irregular
menstruatio
n
Yes 2 2.6 74 94.4 76 100
0.0001
*
0.041
3
0.008
9
0.1912
No 17 39.5 26 60.5 43 100
Exp to
STD
HPV16
Yes - - - - - -
NA NA NA NA
No 19 35.2 35 65 54 100
Screening
test for
cervical
cancer
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100
History Of
abnormal
cell
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100
Anorexia
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100
Vaccination
Yes - - - - - -
NA NA NA NA
No 19 32.2 40 68 59 100

The prevalence of HPV positive over the associated risk factors was presented
in above table 10. None of positive subjects were found in exposure to STD HPV 16,
screening test for cervical cancer, history of abnormal cell, anorexia and
vaccination).Irregular menstruation for positive subjects (OR= 0.0413; 95% CI:
0.0089-0.1912, p <0.0001) were significantly associated with irregular menstruation.


Table 11: Association of risk factors with HPV 16 results
Risk factors
C
l
a
s
s
i
f
i
c
a
t
i
o
n
s

HPV prevalence
(HPV 16)
Total
P

v
a
l
u
e

Odds
ratio
95%
Confidence
Interval
Positive Negative Lowe
r
limit
Uppe
r
Irregular
menstruatio
n
Yes - - - - - -
0.079 1.0547
0.281
4
3.953
6
No 10 23.3 33 76.7 43 100
Exp to
STD
HPV16
Yes - - - - - -
NA NA NA NA
No 10 18.5 44 81.5 54 100
Screening
test for
cervical
cancer
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100
History of
abnormal
cell
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100
Anorexia
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100
Vaccination
Yes - - - - - -
NA NA NA NA
No 10 17 49 83.0 59 100

The prevalence of HPV16 over the associated risk factors was presented in the
above table-11. Positive subjects were found in exposure to STD HPV16, screening
test for cervical cancer, history of abnormal cell, anorexia, vaccination and irregular
menstruation for positive subjects (OR= 1.0547; 95% CI: 0.2814-3.9536). None of
the risk factors were not significantly associated with HPV 16 infection.


6. DISCUSSION

Human Papilloma Virus infections (HPV) remain the most common STI of
females particularly afflicting adolescents and women in their early 20s. The greatest
risk factors for infection are young age and number of sexual partners, with higher
rates of infection in women less than 25 years of age (Manop Kanato et al., 2006;
Jessica Kahn et al., 2005; Moscicki et al., 2001). It is accepted that in young, sexually
active women the likelihood of being exposed to HPV and subsequent infection is
extremely high. In adolescents, the majority of infections and subsequent
corresponding cytological abnormalities are transient and of minimal significance in
terms of oncogenic potential. Only a smaller percentage of adolescents will have
persistent infections with higher risk subtypes and manifest high grade cytological
abnormalities. In this small percentage of women who fail to clear these infections,
persistent infection can predispose to the development of high-grade cancer precursor
lesions and cervical cancer. The Prevalence of HPV infection in pre-pubertal girls,
college girls and married women was also well documented in previous study (Gallo
et al., 2003; Powell et al., 2003). So, early detection of HPV infection and cervical
cancer in this age group is required.
In this study, biopsy samples from sexually active married women for the
presence of HPV infection in married women in Salem, Tamilnadu. A total of 119
women was initially recruited in this study. Two different DNA extraction protocol
was evaluated to find a suitable method for HPV- DNA extraction from the biopsy
and of that DNA isolated by (Baker & Conway, 1999) method was found to be of
good quality and presented in table 1. Among the 119 study subjects HPV DNA was
detected in 19 (15.97%) after a single round amplification with L1 consensus primer,
MY09/MY11 (Plate 4). On further analysis of type-specific HPV primer, only 10
were positive for HPV-16 (Plate 5).
Out of 119 study subjects, 19 (15.97%) were positive for HPV infection by L1
consensus primer and HR-HPV, particularly, HPV-16 was detected only in 10
(8.40%) were recorded in table 2 and figure1. HPV16 are more commonly detected
than non-oncogenic HPV types as these women were sexually active and relatively of
little higher age group.


Pavani et al. (2005) made similar observations; the most prevalent HPV types
found in the invasive cervical cancer in Andra Pradesh were HPV-16 (66.7%). A
study from Chennai reported 99.5% presence of high risk in invasive. Cervical cancer
and 23 different types were found, although HPV-16 was most common (Franceschi
et al., 2003). In comparison, in our study to previous study performed by other
countries like Srilanka 100% (De Silva et al., 2006), Brazil 96% (Eleuterio et al.,
2006), India 95% (Peedicayil et al., 2006), Norway 92% (Kraus et al., 2006),
Lithuania 92% (Gudleviciene et al., 2005), Australia 90% (Liu et al., 2004), Korea
85% (An et al., 2005), Japan 87% (Asato et al., 2004), Italy 85% (Tornesello et al.,
2006), Grace 74% (Konidaris et al., 2007) and China 75-83% (Qiu et al., 2007) have
shown higher HPV prevalence. This may be explained by the difference in the
number of samples the type of case group (high-risk or low-risk group) or cultural
limitations.
In the present study, the age distributions of study subjects over the
HPVL1and HPV16 prevalence were presented in table 3 and figure 2. A total of 119
study subjects were tested for the present study, the prevalence of HPVL1 10 and 9
positive subjects were found in the age groups 30-40 and 41-50 respectively. The
maximum numbers of positive subjects were found in the age group 30-40. In the
case of HPV16 six and 4 positive subjects were found in the age group 30-40 and 41-
50 respectively. The result revealed that the sexually active age group was more prone
to HPV infection. Similar results are also obtained by Franceschi et al., (2005).
In our present study the associations of socio demographic variables with
HPV and HPV16 positive results were illustrated in table 4 and table 5. Age group
associated with HPV prevalence (OR for women by age group 30-40 vs. 41-50 =
0.7407; 95%CI: 0.2765-1.9849) 30-40 years was slightly increased level with HPV
positivity. Occupation (OR for women working in rural vs. urban=0. 9231; 95% CI:
0.3333-2.5565) high level HPV positivity was found to be rural women. Annual
income (OR for women, their income <25000vs>25000=0.833; 95%CI: 0.2754-
2.5213) higher level HPV infection was seen only higher income group. Age group
associated with HPV16 prevalence (OR for women by age group 30-40 vs. 41-50 =1.
0547; 95%CI: 0.2814-3.936). The HPV16 positivity (9.0%) was found to be in rural
women. Annual income (OR for women, their income <25000vs>25000=0.2451;
95%CI; 0.0299-2.0123) higher level HPV16 infection was seen only higher income
group. Our study was very close to the results observed by Aggarwal et al., (2006).

The prevalence of HPV positive and HPV16 over the personal habits was
recorded table 6 and table 7. Food habitat associated with HPV prevalence (OR:
0.514; 95%CI: 0.2027-1.6106). And the positive subjects were found to be habit of
vegetarian. None of the positive subjects were found to be habit of vegetarian (OR:
0.8458; 95% CI: 0.2049-3.4912) in HPV16 respectively. The above mentioned
personal habits were not much associated with prevalence of HPV infection in the
study subjects.
The associations of symptoms with HPV positive and HPV16 positive study
subjects were presented in table 8 and table 9. The stomach ache associated with
HPV positive prevalence (OR for women had symptom of stomach ache vs. Without
a stomach ache =1. 6071; 95%CI:0.4924-5.2458). Fungal infection (OR for women
had fungal infection vs. Without fungal infection =0. 0413; 95%CI: 0.0089-0.1912).
Urinary tract infection (OR for women had UTI vs. Without UTI=0. 7273; 95%CI:
0.2698-1.9602). Vaginal discharge (OR for women had vaginal discharge vs. Without
vaginal discharge = 0.2690; 95%CI: 0.9878-0.7401). Loss of body weight (OR for
women had loss of body weight vs. without loss of body weight =0.0833; 95%CI:
0.01701-0.6493). Abdominal pain (OR for women had abdominal pain vs. without
abdominal pain = 0.6923; 95%CI: 0.2430-1.9721). The back ache (OR for women had
back ache vs. without back ache = 0.0833; 95%CI: 0.0107-0.6493). The itching (OR
for women had itching vs. Without itching =5. 6290; 95% CI: 1.3594-23.2749) the
irritation (OR for women had irritation vs. without irritation = 0.6923 95% CI:
0.2430-1.9721). The symptoms such as viral infection, Inflammation and arthritis
were not associated with HPV positive infection. The prevalence of HPV16 positive
results were equally distributed in symptoms such as stomach ache (OR=1. 6623;
95%CI: 0.3345-8.2618), UTI (OR=0. 6790; 95%CI: 0.1814-2.5411), Vaginal
discharge (OR=0. 1703, 95%CI: 0.0414-0.7012), Itching (OR=1. 4362, 95%CI:
0.1695-12.1662), Irritation (OR=0. 3693; 95%CI: 0.0749-1.8221), Abdominal pain
(OR=0. 3693; 95%CI: 0.0749-1.8221). The symptoms such as fungal infection, loss
of body weight, inflammation, arthritis and viral infection were not associated with
HPV 16 positive infection. In the previous study the prevalence of HPV DNA higher
in poor hygienic condition. It is collaborated by the observation that significant
number i.e. 58% of positive women had vaginitis (Aggarwal et al., 2006). Poor
hygiene was noted to be associated with higher prevalence of HPV in women the
control group by (Franceschi et al., 2005) women who did not toilet their genitals

after intercourse or during menstruation have been found to be greater risk (Chaouki
et al., 1998). The symptoms like pain in lower abdomen, history of discharge purities
vulvae were commonly correlated with presence of genital after intercourse or during
menstruation have been found to be at greater risk (Cooke et al., 1998). Women using
homemade pads during menstruation have been shown to have a 3 to 4 fold increased
risk of cervical cancer (Duttagupta et al., 2004).
The prevalence of HPV positive and HPV16 over the associated risk factors
were studied and presented in table 10 and table11. None of positive subjects were
found in EXP in STD HPV16, Screening test for cervical cancer, history of abnormal
cell, anorexia and vaccination. Irregular menstruation (OR: 0.0413; 95% CI: 0.0089-
0.1912). The prevalence of HPV16, none of positive subjects were found in EXP in
STD, Screening test for cervical cancer, History of abnormal cell, Vaccination,
anorexia. Irregular menstruation (OR: 1.0547; 95% CI: 0.2814-3.9536). The previous
study showed that HPV infections were more pronounced in women ever used
hormonal or contraceptives (p <0.05for each) (Howayda et al., 2007).
The results of three hundred and eighty (380) of 1,275 (29.8%) women were
followed up for a median time of 18.5 months (inter-quartile range 9.7-26.6). Sixty-
nine (69) women had incident HPV infections during 226 person-years of follow-up
reflecting an incidence rate of 30.5 per 100 person-years. Incident HPV infections
were marginally associated with HIV positivity (RR = 2.8, 95% CI: 0.9 - 8.3).
Clearance for HPV type-specific infections were frequent ranging between 42.3%and
100.0% for high- and 50% and 100% for low-risk types. Only 31.2% of women
cleared all their infections. Clearance was associated with HIV negativity (Adjusted
clearance = 0.2, 95% CI: 0.1 - 0.7) but not with age at study entry, lifetime number of
sexual partners and multiplicity of infections. The prevalence of low-grade squamous
intraepithelial lesions (LSILs) was 53/365 (14.5%). None of the women had a high-
grade cervical lesion (HSIL) or cancer. Twenty-two (22) of150 (14.7%) HPV
negative women at baseline developed incident LSIL during follow-up. The risk for
LSIL appeared to be elevated among women with HPV 18-related types compared to
women not infected with those types (RR = 3.5, 95% CI: 1.0 - 11.8) (Elisabete
wilderness et al., 2010).
The results of 12.38 million adolescent girls aged 12 to 17 years, 3.69 million
(29.76%) was recommended to receive the HPV vaccine by their health care provider.
The majority who received the HPV vaccine recommendation were 13 to 17 years of

age (83%), were white (71%), and had one or more preventive visits (94%). Among
those for whom the HPV vaccine was recommended, 48.75% (95% CI 45.37-52.13)
received the vaccine. Multivariate logistic regression analysis of those who were
recommended revealed that enabling and predisposing factors were significantly
associated with the HPV vaccination. Children living at 101% to 200% of the Federal
Poverty Level (odds ratio 0.54; 95% CI 0.30-0.98) and children in households with
two or more adults (0.51; 0.33-0.80) were negatively associated with HPV
vaccination, whereas children with any preventive medical care visit(s) (2.28 [1.36
3.84]) in the previous year were positively associated with HPV vaccination (Swetha
Rao Palli et al., 2012).
Type-specific HPV-DNA was detected in 4.4% (n = 79) of the 1800 girls:
2.7% (n = 49) concerned a high risk HPV type (hrHPV-DNA). The three most
common types were HPV type 16, 18 and 51 (40%). Out of the hrHPV-DNA positive
girls, 32% were seropositive vs. 12% in HPV-DNA negative girls (p < 0.001). Risk
factors independently associated with hrHPV-DNA infection among the sexually
active girls were age >15 years vs. 1415 years (OR = 2.6 (1.25.9)), age of sexual
debut <14 vs. Above 14 years (OR = 3.0 (1.18.2)), total number of lifetime partners
above two vs. Less than two partners (OR = 3.2 (1.38.0)) and age of partner >17 vs.
Under 17 years (OR = 4.2 (1.513.0)). Low hrHPV- DNA prevalence was found in
the adolescent girls. The observed vs. Expected age-related increase in HPV-DNA
prevalence in this cohort in the coming years (with increased sexual activity) will
provide an understanding of the effect of HPV vaccination. Furthermore, this cohort
study will offer the opportunity to improve knowledge of antibody responses
following natural infection and vaccination (Mollers et al., 2012).
The prevalence of cervical infection with the HPV vaccine campaign in Viet
Nam, it is important to note that one can be infected with multiple types of HPV.
Vaccination does not protect against all types of high risk HPV types. Future vaccine
campaigns should openly disclose this information to women receiving vaccines.
High prevalence of infection with HPV high risk types was observed in this study. As
the HPV infection has a high correlation with cervical cancer, this study emphasizes
the need for both primary prevention of cervical cancer with HPV vaccines as well as
secondary prevention with screening (Lanth vu et al., 2013).
Two hundred and forty five women between 20 and 73 years old, were
enrolled in the study (mean age: 38.1 years; SD 10.7 years). Two hundred and thirty

nine of the 245 cervical samples (97.6%) were positive by human -globin
amplification and 208 of the 226 urine samples (92.4%). Fifty one woman were not
included in the statistical analysis due to their samples' low DNA quality (negative
result for -globin) or a lack of either of the samples (cervical or urine) (Marina
Munoz et al., 2013)
Our findings confirm that HPV is shed into the biopsy and that PCR
performed biopsy on specimens is capable of detecting HPV DNA in the vast
majority of HPV infected individuals. The result suggests that biopsy sample can be
used as an important tool for HPV-DNA detection in large populations unwilling to
undergo a pelvic exam or unwilling to provide invasive specimens and screening of
symptom-free normal healthy women who may harbor oncogenic high-risk HPVs or
other HPV-related infections. This will help in early detection and monitoring of
women at high risk for developing cervical cancer. Further research on HPV infection
in married women should focus on identification of behavioral and biological risk
factors for HPV infection, persistence and progression to cervical cancer.
In conclusion, the study found a prevalence of 15.97% for HPV in the study
population. Cervical cancer screening practices are inconsistent in India. The
prevalence is higher in rural, low social, economical, poor hygienic and sexually
active women. It can be controlled or prevented by regular screening of cervical
cytology by similar Pap testing in every woman started at the age above 23. In another
way health education, promotion of condom usage and need to follow healthy,
hygienic practices in during menstruation is the most cost effective approach in
reducing the incidence of cervical cancer.
Vaccination also available to high risk types 16 and 18, but not in other HPV
types. Vaccination does not prevent the entire life. Still (Viral like particle) is
developed to prevent the all types of HPV and easily available to all the women (less
cost effective). The government will take the responsibility for the screening
programmed and vaccination to the women and control the cervical cancer death in
India.


6. SUMMARY

In the present study, a total of 119 biopsy samples were collected from
married women. Two different DNA extraction protocol was evaluated to find
suitable methods for HPV-DNA extraction from the biopsy sample. By comparing the
quality and quantity of DNA extracted by two methods it was found that Baker and
Conway, 1999 protocol was the best method. DNA isolated by this method was taken
for further analysis. All DNA were screened with -globin to assess the presence of
cellular DNA and only -globin positive samples were taken for PCR analysis with
consensus and type-specific primers.

Out of 119 biopsy samples 19 were positive for L1 consensus primer, 19 for MY
nested, and 10 for HPV-16
The overall prevalence of HPV positive and HPV 16 was found to be 19 (15.97%)
and 10 (8.40%). In age wise distribution, 10 positive subjects were found in 30-40 age
group and 9 subjects found to be below 41-50 age group
The prevalence of HPV positive over the socio-demographic variables of study
subjects were analyzed statistically and recorded. HPV prevalence in variables among
married women were also compared and statistically analyzed to calculate OR and
95% CI. Though there was no statistical significant association, HPV prevalence was
higher in subjects from rural areas, age group 30-40, annual income above 25, 000
The prevalence of HPV positive and HPV16 over the personal habits of study subjects
were analysed statistically and recorded. None of the positive subjects were found in
the variables of both HPV 16 and positive subjects were found in HPV positive
Out of 12 symptoms, some symptoms were positively associate with HPV positive
and some subject was positively associated with HPV16, the positive subjects were
statistically significant
Risk factors of positive and negative cases were assessed statistically, but they are not
statistically significant
The study revealed that the following points are the most probable mode of
HPV transmission among the study subjects.
Poor hygiene condition
Premarital and extramarital sexual habits
Unsafe sexual practices

Lack of medical facilities
Lack of awareness on health care
Suggestions:
A large scale study may be conducted by Governments or some other sponsoring
agencies to assess the prevalence of HPV among the study subjects.
A red alert should be rounded powerfully to promote the knowledge on health care.
Health care personnel to be activated to promote the knowledge on signs, symptoms
and risk of HPV.
Frequent pap screening should be implemented.
HPV typing is an important factor for cervical cancer diagnosis and PCR & RFLP
methods are used for detection of HPV types.
The prophylactic trial HPV vaccination (GARDASIL) should be insisted, among the
study population.
Awareness programmes should be conducted by the Government through media,
stages.., etc.
Government should take the responsibilities to provide the screening programme and
vaccination to the women and to control the cervical cancer death in India.


7. CONCLUSION

In conclusion, the present study showed that even in married women HPV
infection of the uterine cervix is common. HPV infection is risk for cervical cancer as
there are studies indicating that oncogenic HPVs particularly HPV-16 contribute to
the development of cervical cancer. The married women with healthy cervix had the
advantage of enabling prevalence of HPV infection using non-invasive biopsy HPV-
DNA detection. These findings confirm that HPV is shed into the biopsy and that
PCR performed on biopsy specimen is capable of detecting HPV DNA in the vast
majority of HPV infected individuals. The result suggests that biopsy sample can be
used as an important tool for HPV-DNA detection in population and screening of
healthy women who may harbor oncogenic high-risk HPVs or other HPV-related
infections. This will help in early detection and monitoring of married women at high
risk for developing cervical cancer. Further research on HPV infection in married
women should focused on identification of behavioral and biological risk factors for
HPV infection, persistence to cervical cancer.

Detection of Human Papillomavirus DNA in biopsies of cervical cancer patients in Salem, Tamilnadu i

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Detection of Human Papillomavirus DNA in biopsies of cervical cancer patients in Salem, Tamilnadu xx

9. APPENDIX

9.1 REAGENTS FOR DNA EXTRACTION
Lysis Buffer (pH 8.0)
50Mm Tris HCL (pH 8.0)
200Mm EDTA (pH 8.0)
1% SDS

PROTEINASE K BUFFER
Tris pH 8.0
0.5M EDTA
10% Tween

TE BUFFER
10mM Tris-HCL
1mM EDTA (pH 8.0)
Autoclave it for 121C for 15mins at 15lbs and store at 4C.
9.2 ELECTROPHORESIS REAGENTS
Tris Acetic acid EDTA (50XTAE) Buffer
Tris base 24.2g
Glacial acetic acid 5.7ml
EDTA 3.72g
Milli-Q 100ml
pH 8.5
Detection of Human Papillomavirus DNA in biopsies of cervical cancer patients in Salem, Tamilnadu xxi

Tracking dye
Bromophenol Blue 2.5mg
Xylene cyanol 2.5mg
Glycerol 0.2ml
Distilled water 1ml.

Detection of Human Papillomavirus DNA in biopsies of cervical cancer patients in Salem, Tamilnadu xxii

PUBLICATIONS
A PAPER PUBLISHED OTHER THAN RELATED TO THESIS
Divya N, Thenmozhi S, Sureshkumar BT, and Selvan M. Antibacterial activity
of medicinal plant against wound infected pathogens. International Journal of
Pharmaceutical Sciences and Research 2014; 5 (11): 1000-09.

Divya et al, IJPSR, 2014; Vol. 5(11): 1000-09.
E-ISSN: 0975-8232; P-ISSN: 2320-5148

International Journal of Pharmaceutical Sciences and Research
1000
IJPSR (2014), Vol. 5, Issue 11 (Research Article)
Received on 09 April, 2014; received in revised form, 27 May, 2014;
accepted, 13 July, 2014; published 01 November, 2014

ANTIBACTERIAL ACTIVITY OF MEDICINAL PLANT AGAINST WOUND
INFECTED PATHOGENS
N.Divya
1*
, S.Thenmozhi
1
, B.T.Sureshkumar
1
, M.Selvan
2
.
1
Department of Microbiology, Vivekanandha College of Arts and Sciences for
Women (Autonomous), Elayampalayam- 637205, Tiruchengode, Namakkal District,
Tamilnadu (India)
2
Departments of Microbiology, Muthayammal College of Arts and Science,
Rasipuram- 637408, Namakkal District, Tamilnadu (India).
ABSTRACT
Medicinal plants contribute in the human health care system. Most of the plants
utilized by village peoples as a folk medicine. The effects of plant extracts on bacteria
have been studied by a very large number of researchers in different part of the world.
In this study antimicrobial activity of Acalypha indica were investigated against two
strains of human pathogenic bacteria. A total of 15 pus samples were collected from
Namakkal private hospital, among that two strains were isolated as Staphylococcus
aureus and Pseudomonas aeruginosa. Isolation and identification of bacterial isolates
by using standard biochemical tests. The above isolated organisms were tested for
their sensitivity towards the Acalypha indica medicinal plant leaves extract by disc
diffusion and Agar well methods. In this study the highest antibacterial activity was
observed in water extracts of Acalypha indica than compared with acetone extract.
The inhibitory effect of medicinal plant against Staphylococcus aureus in well
diffusion is (17mm), and Pseudomonas aeruginosa is (20mm) in water extract. In
acetone extract 7mm and 9mm inhibitory zone showed by Staphylococcus aureus and
Pseudomonas aeruginosa, respectively.
Keywords: Pseudomonas aeruginosa, Staphylococcus aureus, Acalypha indica,
Acetone and water extract, Antibacterial Activity.

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