) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
University of Baghdad
College Name
College of sciences
Department
Department of Biology
Full Name as written
in Passport
Hula Younis Fadil Al-Sadi
e-mail
hulayounis@yahoo.com
Career
Assistant Lecturer
Master
Lecturer
Assistant Professor
Professor
PhD
Thesis Title
Genotyping of Human Papillomavirus in
a sample of Iraqi women with cervical abnormalities by using Real-Time PCR
and Macroarray
Year
September-2011
Summary
The present study was conducted to investigate the relationship
Abstract
between Human papillomavirus (HPV) infection and cervical
cytological abnormalities in Iraqi women. One hundred twenty two
women were included in this study with a cervical infection. A sample
of 110 women with abnormal cytology cervical dysplasia / cancer, 45
of which 40.9% had HPV infection and 12 (16.7%) women had
chronic infection with normal cytology during 2010. A control group
with 10 women of normal cytology and no HPV infection was included
in study for comparison.
Pap smear test results showed two types of cervical cytological
abnormalities, low squamous intraepithelial lesion (LSIL) which
comprised of 70% of HPV positive cases, and high squamous
intraepithelial lesion (HSIL) which comprised of 26% of HPV positive
cases. Whereas 4 % out of the total risk group was represented in
women with normal cytology. Koilocytosis is pathognonomic for HPV
) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
infection. Furthermore, Koilocytosis was accompanied with 100% with
genital wart presence and 16.7% was accompanied with flat wart
presence.
HPV-DNA studied using Real-Time PCR and MacroArray assay
(GenoArray) which applied for the first time in Iraq indicates the
presence of HPV-DNA in 35.6% of all tested groups. Results indicate
the presence of 17 different HPV genotypes among 47 infected women
by using macroarray technique. Fourteen of these HPV genotypes
which comprised of 82.35% were high risk (HR-type) and 17.56%
(3/17) were low risk (LR-type).One of the important results in the
present study indicates the presence of ten different genotypes which
may be recorded for first time in Iraqi women (HPV-31, -44, -45, -51, 52, -53, -58, -59, -66 and -68 types).
Out of 17 genotypes HPV-16 was the predominant one, this
genotype together with genotypes 59, 6, 39 and 66 comprised of the
five most common types detected in HPV positive cases. Moreover,
percentage of HPV-16, and -59 were extended to involve women with
normal cytology. Seven different genotypes -6, -11, 16, 59, -39, -66
and -44 have been identified in genital wart specimens.
Results indicated that 32 (68.09%) out of 47 infected women had
HR type alone, whether single or multiple types, while eight women
(17.02%) had HR types in association with LR types and seven women
(14.89%) had LR type only.
Transmission electron microscope (TEM) was used for viral
particles detection from ten HPV DNA positive samples with different
cytological changes. Results indicated the presence of viral particles in
six samples with different grades of disease and four samples showed
no viral particles.
) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
To evaluate the immune status of patients lymphocyte
transformation assay was applied. Results indicated that transformed
lymphocyte were found to be high in women with normal cytology
which may have HPV as a latent infection, while they were decreased
in women with abnormal cytology. Approximately 40% of genital wart
cases have higher mean of proliferative T cell response when they were
compared with control group. However, proliferative T cell responses
were statistically independent on HPV infection type, viral load and
aging group.
Real-time PCR assay was employed for HPV detection and
quantitation (viral load estimation) from the range of HR-HPV DNA
13 genotypes most frequently found in different grades of cervical
intraepithelial neoplasia, cervical tumors and health controls from (132
samples). Results showed that out of 47 positive cases 35 cases can be
detected by real-time PCR which comprised of 74.5%. Failures in
detection by this technique included 12 cases which comprised of
25.5% of the positive samples. Therefore, viral DNA copies number
for the most HR-HPV types in cervical samples was determined from
35 sample with low and high-grade cervical intraepithelial neoplasia
(CIN I, II, III and Squamous Cell Carcinoma), and with normal
cytology. However, the viral load of the 33 samples spread over a wide
range from 1×10 to 9.4×106 copies/ml (mean 4.17×104 copies/ml). In
addition, two samples were around the standard curve values as:
approximately 5×107 and 1×109 copies/ml considered. Viral load was
estimated approximately 46% constituted as high viral load, 34% as
low viral load and 20% as slightly high viral load.
) ‫أنموذج ( أ ) الخاص برسائل الماجستير و اطاريح الدكتوراة ( اخر شهادة‬
Macroarray was applied in 68 samples, 36 of which were positive
and 32 were negative by the real-time PCR test. High-risk genotypes
were found in 35/36 (97%) real-time PCR positive samples, whereas
remaining two samples were negative for HPV DNA (false-positive)
by macroarray assay. The low-risk and probable high-risk (PHR) HPV
genotypes were detected in the 35 HPV-positive samples which are not
detected by real-time PCR included genotypes: HPV-6 (in three
samples), -11, -53 and -66 (in two samples for each).
HR, PHR and LR HPV genotypes were detected by macroarray in
the 11/31 HPV-negative samples in real-time PCR which comprised of
genotypes: -39, -58, -66, -11, -6 and -44. The GenoArray (GA) is
capable of detecting 21 HPV multiple genotypes, including 8 types not
detecting in real-time PCR. These types represented 32.84% of HPV
types frequency and 33.33% of HPV positive cases had multiple types,
this may reflect the important factor potentially favoring the GA for
HPV detection. Therefore, the GA genotyping assay appears to be
more accurate and sensitive technique for the detection and genotyping
of HPV infections than real-time PCR one.