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Ministry of Health of the Republic of Kazakhstan
«WEST-KAZAKHSTAN MARAT OSPANOV STATE
MEDICAL UNIVERSITY»
ABSTRACT
Balmagambetova Saule Kabedaevna
The dissertation thesis
" Epidemiological analysis of Human Papillomavirus in Western Kazakhstan in
relation to HPV-attributable cervical pathology"
submitted for the scientific degree of Doctor of Philosophy (PhD) by the specialty
6D110100 - “Medicine”
Scientific advisers:
Prof. A. Tinelli, PhD, University of Salento, Lecce, Italy.
Prof. Bekmukhambetov E.Zh., PhD, Rector of the West Kazakhstan
Marat Ospanov State Medical University.
Time of fulfillment: 2014 - 2017 years.
Topicality
Virtually all cases of cervical cancer are developed on onset a persistent
infection caused by a limited set of human papillomavirus (HPV). The prognosis
published in November 2016 by the American Cancer Society (ACS) and a group of
researchers at the Lancet Center is the following: “for cervical cancer, the number of
diagnoses could rise by at least 25% to over 700,000 by 2030, mainly in low- and
middle-income countries”. Analytical data of Kazakhstani scientists show an increase
in the incidence of cervical cancer from 14.5 per 100,000 female population in 2004
to 20.2 in 2014. It is alarming that "the analysis of the age-specific incidence rates
revealed a significant risk of the disease already at a young age and a marked increase
by 40-44 years. The peak incidence over the past 6 years has shifted to a younger age,
the overall increase in morbidity occurred due to women aged 35 to 55 years".
Despite the existence of a state screening program for the early detection of
cervical cancer in the country since 2008, situation with the incidence and prevention
of new cases remains practically unchanged, and even becomes more tense. And yet,
cervical cancer is a very real object for early detection because of its belonging to the
number of visual forms, and can be largely prevented by both effective screening and
vaccination.
Aim of the research: analysis of situation with human papillomavirus infection
spread amongst female general population in Western Kazakhstan for development
the epidemiological prognosis of further incidence of cervical cancer to determine the
circle of urgent preventive measures.
Objectives of the research
1. determine the overall frequency of human papillomavirus detection among women
in the western region of Kazakhstan;
2. identify the most common genotypes of HPV with the definition of viral load in
different age groups;
3. submit a comparative analysis of diagnostic tools for cervical cancer screening
practiced in the region for development corrective recommendations for the screening
strategy;
4. analyze comprehensively a set of clinical, laboratory and socio-demographic data
for identification of the region-specific risk factors for cervical cancer that determine
the epidemiological prognosis.
The objects of the study are women from the general population who are
permanently residing in the West region of Kazakhstan at the age of 18 to 60 and
over, clinically healthy (a sample for cross-sectional study), as well as with the newly
diagnosed "cervical cancer" (sample for the "case - control" study).
The subject of the study is the clinical and laboratory parameters of the tested
group ( results of the HPV PCR with genotyping and viral load detection for each
case, results of a cytological study conducted by two methods: traditional and liquid
one, results of colposcopic examination), and socio-demographic indicators (results
of the survey for detection risk factors for cervical cancer defined by the WHO).
Personal contribution of the author consists in direct interviewing and
examination of patients according to the developed Protocol, carrying out additional
diagnostic procedures (biopsies from the cervical tissue according to indications),
scientific and statistical analysis of the data obtained towards the development of
practical recommendations.
Scientific novelty
in framework of this study for the first time:
- frequency of detection of the most common HPV types in the female general
population in Western Kazakhstan was determined;
- the most important correlations between age and viral load, viral load and
colposcopic patterns, viral load and cytologic status were revealed;
- risk factors with the largest contribution to the implementation of cervical cancer in
the region were identified; social profiles of infected HPV and those diagnosed with
cervical cancer were compiled;
- a set of epidemiological, socio-demographic and clinical data in the region,
including the HPV prevalence in normal cytology was comprehensively analyzed in
order to complement the existing Kazakhstani guidelines for the prevention of
cervical cancer.
Practical significance of the work
A comprehensive study of the cervical cancer causative factor has not been
carried out yet in Kazakhstan, despite the introduction of a national screening
program for cervical cancer since 2008. The study provides an opportunity to fill the
regional gap in the national and global map of the prevalence of HPV types.
Given the growing incidence of cervical cancer in Kazakhstan, assessment of
screening diagnostic tools to identify the reasons for inadequate effectiveness of
preventive interventions constitutes an urgent task for researchers. This work should
be considered as a start of such a kind of research.
In general, the study provides an opportunity to cover a wide range of causes of
the increased incidence of cervical cancer in the region.
The main points of the research for the thesis argumentation
1) Overall HPV prevalence in the Western region of the Republic of Kazakhstan is
25.0%, with an average viral load 5.53.8. Both indicators are estimated as high for
the general population.
20.3% of the examined female population of total 25.0% is infected with types of HR
group, that is, specific weight of the HR-HPV carriers is 81.2%.
Specific gravity of the so-called "vaccine types" (6, 11, 16, 18), i.e. potentially
eliminated by vaccination, turned to be 43.0% in Western Kazakhstan, and including
31 and 45 types, also potentially preventable by vaccination up to 59.6%.
2) Top-five leading HPV types in the Western region are identified: 16 (26.4%); 31
(10.1%); 51 (9.4%); 52 (9.0%); 6 (7.9%). Of these, only type 6 refers to weakly
carcinogenic, the rest - of the HR-HPV group.
Almost the same identity of the results on viral load determination in the age groups
18/29 - 30/39 - 40/49 years is observed, followed by a decrease in the group 50-60
years.
The greatest amount of emissions (abnormally high values) is recorded in the age
group 30-39 years, when, according to the worldwide data, process of the viral
persistence and the development of CIN begins.
Top-5 HPV leading types in the group of patients with cervical cancer includes the
following types: 16 (71.9%), 31 (14.1%), 58 (7.8%), 18 (7.8%), 33 (6, 2%).
3) Comparative analysis of diagnostic tools for cervical cancer screening which are
practiced in the region showed some advantage of the traditional cytological method
(Romanovsky-Giemsa coloring) in comparison with LBC. The use cytological
screening methods solely for cervical cancer appears to be conceptually obsolete.
4) The leading risk factor for developing cervical cancer in the region is HPV type
16.
Identification of authentic for the region risk factors and prediction of the epidemic
situation in the prevalence of cervical cancer demonstrated that the probability of
cervical cancer occurrence in the examined women in the region:
- decreases with a relatively high level of per capita income (500$ and above);
- decreases with at least irregular (sometimes) participation in screening for cervical
cancer.
The prognosis is justified for 73.9% of infected with HPV, but not affected with
cervical cancer and 70.3% of the CC cases.
The trend of the CC incidence in the female population of the Western region tends to
increase. The expected incidence for cervical cancer in 2017 is 26,9 per 100,000
women standardized by age.
Approbation of the work
The main points of the dissertation were reported on the profile Problem
Commission of the West Kazakhstan Marat Ospanov State Medical University, at the
Approbation meeting of the unified University’s Problem Commission.
Results of the study were reported:
at the World Congress on Cancer "World Congress on Cancer and Infectious
Diseases", May 3-5, 2017, Madrid, Spain;
at a scientific conference in Baku, Azerbaijan, May 10-11, 2017 The 6th Annual
International Scientific-Practical Conference "Medicine Pressing Questions";
at a scientific-practical Conference with international participation on October
5-6, 2017, dedicated to the 60th anniversary of the WKMOSMU, Aktobe.
According to the results of the thesis, 8 scientific publications were issued, 4 of
them were published in journals recommended by the Education and Science Control
Committee of the Ministry of Education and Science of the Republic of Kazakhstan,
4 in foreign periodicals:
1 in the journal «Asian Pacific Journal of Cancer Prevention» indexed in the
Scopus database (SJR for 2016 - 0.743);
1 article – in Compendiums of International scientific Conferences (abroad);
2 abstracts - in the International Conferences Proceeding’s books.
List of publications on the thesis topic
1 Bekmukhambetov Y.Z., Balmagambetova S.K., Jarkenov T.A., Nurtayeva S.M.,
Mukashev T.Z., Koyshybaev A.K. / Distribution of High Risk Human Papillomavirus
Types in Western Kazakhstan – Retrospective Analysis of PCR Data // Asian Pac J
Cancer Prev, - 2016 – vol. 17, no. 5. – P. 2667-72.
2 Е.Ж. Бекмухамбетов, С.К. Балмагамбетова, А.Ж. Жылкайдарова, Ж.Б.
Елеубаева, А.К. Койшыбаев, О.Н. Уразаев, Б.К. Каримсакова, С.Н. Рыжкова,
О.В. Заваленная, С.К. Саханова, Ж.Ж. Уразаева, С.Е. Коктова, К.К. Саркулова,
Л.М. Якупова / Современные тенденции в области скрининга рака шейки
матки // Онкология и радиология Казахстана - 2017 г. - вып. 2 (44) – С. 30-38.
3 Бекмухамбетов ЕЖ, Балмагамбетова СК, Уразаев ОН, Уразаева ЖЖ. /
Применение альфы Кронбаха в контроле согласованности опросника на
предмет выявления факторов риска ВПЧ-инфекции // Серия конференций
ЗКГМУ им. М. Оспанова, Батыс Казахстан медицина журналы, ISSN 1814-5620
- Актобе, 2017 – том I – c. 130-135.
4 Bekmukhambetov Y.Z., Balmagambetova S.K., Jarkenov T.A, Koyshybaev A.K.,
Urazayev O.N., Zavalennaya O.V., Koktova S.I., Sarkulova K.K., Yerimbetova G.G.,
Balmagambetova Zh., Yakupova L.M., Zholdybayeva E.V. / HPV prevalence, type
distribution and vaccination awareness in women of Mangystau province of Western
Kazakhstan // Fundamental and Applied Sciences Today X, Proceedings of the
Conference. North Charleston, 26-27.12.2016, Vol. 3, pp. 36-39 - North Charleston,
SC, USA: CreateSpace, 2017.
5 Е.Ж. Бекмухамбетов, С.К. Балмагамбетова, А.К. Койшыбаев, О.Н. Уразаев,
Г.Г. Еримбетова, Б.К. Каримсакова, С.Н. Рыжкова, О.В. Заваленная, С.К.
Саханова, Ж.Ж. Уразаева, С.Е. Коктова, К.К. Саркулова, Л.М. Якупова, К.А.
Тауекелова / Инфицированность вирусом папилломы человека и
осведомленность о вакцинации против рака шейки матки у женщин г. Уральска
Западно-Казахстанской области // Медицина, 2017 г. - вып. 6 - С. 27-36.
6 Ye.Zh. Bekmukhambetov, S.K. Balmagambetova, A.K. Koyshybaev, O.N.
Urazayev, B.K. Karimsakova, S.N. Ryzhkova, G.G. Yerimbetova, S.K. Sakhanova,
O.V. Zavalennaya, Zh.Zh. Urazayeva / Comparative analysis of diagnostic tools for
cervical cancer screening in Western Kazakhstan // The 6th Annual International
Scientific-Practical Conference “Medicine Pressing Questions”, Proceedings book.
May 10-11, Baku, Azerbaijan. Medical Review, vol. 4, pp. 41-42. ISBN 978-9952-
8176-4-5.
7 Заваленная ОВ, Балмагамбетова СК, Каримсакова БК, Уразаев ОН, Саханова
СК, Коктова СИ, Якупова ЛМ, Саркулова КК, Жолдыбаева ЕВ, Балмагамбетова
Ж. / Результаты экспериментальной работы по экстракции ДНК вируса
папилломы человека и генитальных инфекций из жидкостной цитологической
транспортной среды CELL SCAN // Батыс Казахстан медицина журналы -
Актобе, №1 (53) 2017 - с. 25-36. ISSN 1814-5620.
8 Yerbol Bekmukhambetov, Saule Balmagambetova, Arip Koyshybaev, Olzhas
Urazayev, Oxana Zavalennaya, Zhanna Balmagambetova, Elena Zholdybayeva,
Kogershin Tauekelova / Some peculiarities of HPV types causing cervical cancer in
Western Kazakhstan // World Congress on Cancer and Infectious Diseases,
Proceedings book. May 03-05, 2017, Madrid, Spain. JBR - Translational Clinical and
Experimental Oncology, May, p. 29.
Structure and scope of the dissertation
The thesis is made in the volume of 121 pages and consists of the following
chapters: Introduction; Current state of the problem (literature review); Materials and
methods used in the study; Results and Discussion; Conclusion; List of references;
Applications.
The work is illustrated by 39 tables and 20 figures.
The List of references contains 140 sources, including 24 in Russian and 116 in
English.
The research was carried out within the framework of the scientific Project with
grant financing of the Ministry of Education and Science of the Republic of
Kazakhstan "Epidemiological analysis of human papillomavirus in the Western
region of Kazakhstan in relation to HPV-attributable cervical pathology - social,
clinical and genetic aspects" (grant 2230/GF4, State registration No. 0115RK01224,
Agreement with the Science Committee No. 179 12.02.2015, No. 103, April 25
2016, No. 209 03/03/2017).
Based on the data obtained in present scientific work 2 introductions have been
elaborated: University’s teaching Module program for PhD students on HPV-
infection issues and training lecture for sentinel specialists on the same topic.
Results of the research formed the basis for the scientific and methodological
paper "Management of women with cervical pathology in PHC facilities, considering
uptodate data on Papillomavirus infection: Recommendations for the development of
a Protocol for diagnosis and treatment."
Methods of the research
To achieve the goal and solve the problems posed in this work, a combined
design was used that included 3 components: a survey (N 1107), a cross-sectional
study in the general female population of the Western region of Kazakhstan (N
1107), and a study "cervical cancer case - control" (N 64).
Inclusion criteria:
age 18-60 years +;
presence of pathology of the cervix of any degree of severity, including minimal,
suitable for colposcopy;
resident of Western Kazakhstan of any ethnicity;
absence of vaccination in the anamnesis.
Exclusion criteria:
non-residents;
vaccinated.
Note: HIV, 1 trimester pregnancy till 10-11 weeks are not exclusion criteria.
The design and Protocol of the study were approved by the local Ethical
Committee of the University (Minutes No. 3 of 09.10.2014). The work was carried
out in accordance with the Checklist of mandatory items for observational studies
STROBE. The Informed consent form was developed in accordance with the
recommendations of the WHO and all participants who signed the form were fully
informed of the objectives of this analysis.
Calculation of sample (N) for interview and cross-sectional study
In determining the sample, the following points mattered:
- according to a pilot study of the West Kazakhstan University on HPV as of 2013-
2014, N for HPV typing was 1098, with valid statistical results (p0.043);
- from the republican statistical data as of 01.01.2014 the township female population
(capitals of the provinces, suburbs and small towns) was taken from 4 provinces of
Western Kazakhstan. The following factors were taken into account: the ratio of men
/ women - 48% / 52%; the share of the population aged 15-65 is 71%. The ethnic
composition: Kazakhs - 66.1%; Russians - 21.5%; other ethnic groups - 12.4%. In the
Western region, Kazakhs made up 81% in Aktobe; 91.8% - Atyrau; Mangystau-
89.6%; Uralsk-74.3%; other ethnic groups - respectively. In total, N according to
calculations in the program SAS.9.3 (two side type I error of p0.05, CI 95%) was
1152, of which 417 in Aktobe, 253 in Uralsk, 237 in Atyrau and 245 in Mangystau.
The survey was conducted on the basis of medical institutions (outpatient
clinics). For scope different categories of patients to achieve maximally
representative sample and minimize possible bias, state, insurance and private
medical organizations were included.
In Aktau, data was collected in the state outpatient clinic No. 1 (RPN, affixed
population’ register - 114000) and No. 2 (RPN - 91649), as well as in the regional
oncological dispensary, in the medical insurance company “Intertich”, and in the
private clinic “Cha-Kur”.
In Atyrau, the survey was conducted in the state outpatient clinic No. 1 (RPN -
57186), No. 2 (RPN-46000) and No. 7 (RPN-35912), as well as in the regional
oncological dispensary, in the medical insurance company "Intertich", in the private
clinic "Dostar-med".
In Uralsk - on the basis of outpatient clinic No.1 (RPN - 70412), No. 5 (RPN -
46777), No. 6 (RPN - 55704), as well as in the regional oncological dispensary. In the
city of Aksai - on the basis of insurance clinics "Intertich" and "Medicare".
In Aktobe - on the basis of the clinical and diagnostic department of the
Regional Perinatal Center, in the Medical Center of the University, in the private
clinic "World of Women", in the medical insurance company "Intertich".
Randomization of the sample was not performed because of the goal - to identify the
prevalence of HPV infection in female general population in the Western region.
Enrollment of women in the study was conducted either during their routine visit
to the gynecologist, or by ads placed in the clinics lobby, or by the invitation of
sentinel specialists.
The algorithm for women examining:
1) Compiling the patient's socio-behavioral profile by filling in a specially developed
questionnaire, only after the signing of the Informed consent form (developed in two
languages, optional). The questionnaire was developed in a semi-structured manner,
with questions, mostly closed, to collect data reflecting the role of known risk factors
in the development of cervical cancer. To obtain the most truthful answers during the
interview, the local staff was asked to come out from the office and the interviewees
were guaranteed confidentiality of the information provided. To motivate the veracity
of the answers, the researchers also allowed not to indicate the real name and
conducted relevant consultations (explanations) about filling the most "problematic"
graphs - smoking, the number of sexual partners, the level of income.
2) Collection of material for PCR-typing of HPV and determination of viral load for
each identified type.
Smear taking was performed with urogenital probe "Juno" of Belarusian origin.
Qualitative detection and quantification of human papillomavirus by PCR was
performed with HPV Kvant-21 test systems (DNA-Technology LLC, Russia) using
equipment: DT-prime4-M1 detection amplifier, serial number A5D409 LLC "DNA -
Technology", software version V 7.6. Production of the company "DNA-technology"
is certified (ISO 13485: 2012) and registered in the Republic of Kazakhstan (RK-
MT-7-No. 013267 dated July 23, 2014).
Characteristics of the test systems used in the study:
Kit of HPV reagents Quantum-21 (the same production) is designed to identify,
typing and quantifying the DNA of low-risk human papillomavirus (HPV 6, 11, 44)
and possibly / potentially / high (HPV 16, 18, 26, 31, 33 , 35, 39, 45, 51, 52, 53, 56,
58, 59, 66, 68, 73, 82) carcinogenic risk.
In this work, there was planned to carry out an experiment on isolation the HPV
and vaginal microbiota DNA from the residual fluid of the CellScan transportation
medium, according to the worldwide practice of using cytological transportation
media approved by the FDA. In order to isolate the DNA of the vaginal microbiota
from the residual liquid of the CellScan medium, the material obtained from women
with severe clinical manifestations of infection and the control PCR smear were
tested using the "Femoflor-screen" reagent kit. A set of reagents "Femoflor-screen"
allows for qualitative and quantitative assessment of the vaginal biota: normobiota,
aerobic (facultative-anaerobic) and anaerobic conditionally pathogenic biota, myco-
and ureaplasmas and Candida fungi in absolute and logarithmic indices.
Diagnostic sensitivity of the study with "Femoflor-screen" for the detection of
dysbiotic conditions is 88.7%;
specificity - 89.6%; for bacterial vaginosis up to 95%. Specificity of this set of
reagents is confirmed by a sequence of 16s RNA.
3) Sampling of the smear for cytological examination by the methods of liquid
(CellScan) and traditional cytology (Romanovsky-Giemsa coloring).
The material was taken in accordance with the Instructions for use of liquid cytology
by special cytobrashes in a vial with a transportation medium. CellScan
transportation media are being used widely in the Republic of Kazakhstan since 2013
for screening of cervical cancer. Produced by IMSTAR technology (France) and,
according to the manufacturer, tested on a representative sample of 25,000 women.
Specificity of the test is> 85% and sensitivity> 90%. Data were taken from the site
(www.imstar.fr). Industrial introduction and production are carried out by LTD "Tech
Bio Co.", South Korea.
The sampling of the material according to the traditional method (Azur-eosin staining
by Romanovsky-Giemsa) was carried out on a slide using the "Yunona" (endocervix)
and Iire (ectocervix) spatula.
Interpretation of the material obtained by different methods was carried out according
to the terminology system TBS, 2001 (Bethesda system), according to the
"Guidelines for screening of target groups of the female population ..." in the
Republic of Kazakhstan and corresponding methodical recommendations developed
by leading Kazakhstan experts on the basis of WHO expert evaluations.
4) Colposcopic exam and entering information into the database.
Extended colposcopy was performed after taking smears from the cervix, according
to a standard procedure with using 4% acetic acid solution and 3% aqueous Lugol
solution. In the assessment of colposcopic changes, the qualitative classification of
colposcopic patterns by Coppleson-Pixley (1971) was applied, as well as the point
system by Reid. A modern classification of colposcopic pictures which was adopted
at the XIV World Congress on Colposcopy and Cervical Pathology in Brazil (Rio de
Janeiro) in July 2011 was used. Colposcopic examinations were selectively followed
by a material taking (cervical tissue biopsy) at suspected CIN .
For the "case-control" study, all consonants to participate in the study were
selected amongst women with a newly diagnosed "cervical cancer". Randomisation
was not performed.
Inclusion criteria:
- any age;
- any stage of the process;
- histological verification of the diagnosis.
Exclusion criteria:
non-residents of the Western Kazakhstan;
presence of previous medical intervention - radiotherapy, chemotherapy, surgical
treatment.
The design of the study also suggested conducting a survey to identify the role
of the main risk factors for cervical cancer, a similar taking of the material for HPV
PCR-typing and for cytological testing using LBC and traditional methods
(Romanovsky-Giemsa). Colposcopy was not performed when the advanced stages of
cervical cancer.
Calculation of the sample (N) for the "case of cervical cancer - control"
When calculating sample size for the initial data, the number of adult (18+)
female population of the republic was taken and the incidence of cervical cancer in
Kazakhstan equaled 4.8% (data of the Committee on Statistics of the Republic of
Kazakhstan). According to calculations, the sample size should be 64-67 people in
each group (case-control). Given the possible losses among participants, the
estimated amount can be increased to 80.
Methodological basis of the research, summary
To determine the prevalence of HPV infection with the definition of viral load in
general female population and among the cervical cancer patients, the PCR-Real time
method was used based on applying of Russian test systems. Range of the Russian
test system “Quant-21” is 21 types of the virus.
The HPV prevalence in normal cytology was studied for the first time in clinical
practice of the region.
To study the role of potential risk factors for the implementation of cervical
cancer, a 14-item questionnaire was elaborated, divided into 2 blocks: socio-
demographic and behavioral parameters of the studied population. According to
commonly accepted standards, the questionnaire was validated by calculating
Cronbach's alpha (analysis of internal consistency of the test) using Statistica.10
programs (Dell software, USA); SPSS Modeler.
An in-depth analysis of the effect of potential risk factors for the development of
cervical cancer was performed by the method of matching (comparison of the
"cervical cancer” and “control” cases in the ratio 1: 1). Women from the general
population of the same age who were infected with HPV but did not fall ill with
cervical cancer served as a control group. In framework of the match for the
compilation of a social profile of the patients with cervical cancer, an analysis of the
Pearson χ2 contingency tables with the quantification of quantitative variables by the
Mann-Whitney test were performed. Logistic regression analysis with calculation of
the odds ratio was carried out.
To assess the effectiveness of the screening test used for the detection of
cervical cancer in the country, indicators of the CellScan cytological liquid
technology of the South Korean production were compared with those of the
traditional technology (smear coloring according to Romanovsky-Giemsa), in total
494 pairs. In addition, 94 sets of "histological / fluid / traditional cytological findings"
taken from one subject were used. Statistical processing methods relevant for
comparison of diagnostic tests were applied: ROC analysis with curve construction;
Kappa statistics (Cohen's ) using the program www.medcalc.be.
For a preliminary decision on feasibility of introducing an auxiliary screening
method VIA, an assessment of the effectiveness of colposcopy in detecting signs of
HPV infection was carried out, and the Spearman’s rank correlation was calculated.
By methods of linear regression analysis, the trends of general and age
incidence of cervical cancer in the region for the short-term period (least squares
method) were calculated, and the cervical cancer incidence prognosis for 2017
(moving averages method) was performed.
Results
Overall HPV prevalence in the Western region of the Republic of Kazakhstan is
25.0% (22.3;27.7 CI 95%, p = 0.05), with an average viral load in the group of
infected HPV 5.53.8 (CI 95% 5.1;5.9), Me 5.2 (3.1-8.4 by 25/75 quartiles).
65.8% of the total HPV prevalence, or two-thirds, constitute types of the HR group
(total 13), whereas potential / possible and weak carcinogenic types (8 in all) are
34.2%.
20.3% of the examined female population of total 25.0% is infected with types of HR
group, that is, specific weight of the HR-HPV carriers is 81.2%.
Total number of the HPV types detected in one woman reaches 6. With one type of
the virus are infected 77.3% of the examined; with 2 types - 15.5%; 3% - 3.3%; 4% -
2.5%; 5 - 1.1%; 6 - 0.4%.
The leading type in the Western region of Kazakhstan is HPV 16, with a specific
weight up to 26.4%.
Top-5 leading HPV types in the Western region are identified: 16 (26.4%); 31
(10.1%); 51 (9.4%); 52 (9.0%); 6 (7.9%).
Specific gravity of the so-called "vaccine-targeted types" (6, 11, 16, 18), i.e.
potentially being eliminated by vaccination, is 43.0% in western Kazakhstan, and
including 31 and 45 types, also potentially being eliminated by vaccination - 59.6%.
In the group of 18-29 years, the highest HPV prevalence is observed - 42.9%,
decreasing with age.
The average viral load in the Western region is 5.53.8 (CI 95% 5.1;5.9) with a range
of 0.9 up to 22.3, Me 5.2 (3.1-8.4 by 25/75 quartiles).
Almost complete identity of the viral load results in age groups 18/29 - 30/39 - 40/49
years, with a subsequent decrease in the group 50-60 years is revealed. The greatest
number of emissions (abnormally high values) is recorded in the age group 30-39
years.
Assessment of the internal consistency of the questionnaire on the risk factors for
HPV infection and cervical cancer showed: the Cronbach’s alpha was 0.5 (bad), and
increased to 0, 57 (doubtful) when removing the dissonant question (oral
contraceptives application, r = -0.06).
According to the results of the survey, it has been found that only 62.7% of
respondents (34.7% constantly and 28.0% irregularly) attend the screening program
at the state institution.
Among respondents with higher education, 35.1% of those surveyed do not visit the
state outpatient clinic at all, and 51.3% do not attend state screening activities.
About 69.2% of subjects with higher education are aware of vaccination against
cervical cancer, while in the total sample this figure amounted to 56.5% (33.6%
heard, but can not clarify their attitude - approving or negative, 22.9% know and
approve).
Analysis of the Pierson's 2 contingency tables with the definition of the Cramer's V-
criterion to establish the relationship between HPV infection and potential risk factors
for cervical cancer led to the compilation of a social profile of the risk group for HPV
infection in the general female population of the region. These are women with a
relatively prosperous financial status (monthly income from 36 to 90 thousand tenge
per capita, i.e. 200-500$), occupied with skilled job, who had 1 and up to 5 sexual
partners and 3 or more pregnancies in the anamnesis.
Differences in the prevalence of HPV infection were revealed through the regions,
with a magnitude of 19.3% in Mangystau, 23.9% in Aktobe, 28.0% in Atyrau, and up
to 29.5% in Uralsk.
It has been established that the sample through the regions is not homogeneous with
respect to some basic parameters:
- 29,2% those surveyed at the age 18-29 in Uralsk vs. 43.7% in Aktobe;
- 3.6% the other (Caucasian) ethnicity in Mangystau vs. 0% in Uralsk;
- 19.3% Slavic ethnicity in Uralsk vs. 11,6-12,0% in other areas;
- 46.9% unemployed and housewives in Aktobe vs. 24.2% in Uralsk;
- 42.5% having a per capita income of over 90,000 (500$) per month (secured
category) in Mangystau vs. 14.5% in Aktobe.
In course of the heterogeneity analysis in the HPV prevalence amongst the general
sample, difference in the average number of sexual partners has been found: till
1.92.2 in Mangystau and Aktobe to 2.5±3.2 in Atyrau and up to 2.73.6 in Uralsk.
In the group of infected in Mangystau, a reliable but weak association between
ethnicity and a certain type of HPV has been established (p = .083, Cramer's V 0.13,
contribution to ² = 1.04), which confirms the data of the pilot project in 2014, when
a certain correlation between the Caucasian ethnicity and HPV 33 in Aktau (p = .032,
Cramer's V 0.17) has been found. There are no correlation between the ethnic factor
and infection by a specific type of HPV in other areas.
Results of a comparative analysis of cytological screening tools used in the region on
the material 494 pairs of cytological smears taken by different methods and 94 sets
"histological conclusion - fluid cytology CellScan - traditional cytology by
Romanovsky-Giemsa":
- the specific weight of the results being coincided with the histological conclusion is
61.7% in the LBC group, and 60.6% in the Romanovsky-Giemsa group;
- attributive agreements analysis (kappa statistics) (80 histologic conclusions)
demonstrated that the indicator for the traditional method, equal to 0.62, is larger
than that for the liquid method ( = 0.47) (84 histologic conclusions);
- on constructing the ROC curve for liquid cytology, an AUC value of 0.84 is
obtained, whereas for the traditional method it is 0.92 (84 histologic conclusions).
Accordingly, the diagnostic sensitivity of the liquid method is 89.8% versus 95.9%
for the traditional one, provided a 1-specificity is equal to 1;
- specific weight of non-informative material in the Romanovsky-Giemsa coloring
method is 0.4% (4.8% for cervical cancer) versus 5.9% in CellScan (6.8% for
cervical cancer).
When examining the content of HPV in normal cytology, it has been established the
following:
in both groups (LBC and traditional cytology), high and almost identical results for
the prevalence of HPV in NILM (46.0 / 44.8%) are obtained. But, in the "normal"
cytology (grade "C" according to TBS), 9.0% of the HPV content is obtained by the
traditional method, and 18.6% in the liquid cytology respectively, although both
results are in limits of worldwide data.
The average viral load for cytological findings is:
in the NILM group (N = 52) - 5.273.64*103 GE / per sample (range 1.0-20.8);
in the ASCUS group - 6,244,92*103GE / per sample (range 0,9-19,8);
in the CIN group (LSIL, HSIL) - 6,293,41*103GE / per sample (range 2,2-18,0).
Viral load is featured by constant increase proportionally to the severity of the
cytological status.
The specific gravity of type 16 in smears of the NILM class (no intraepithelial lesions
or malignancy) has been established as 30.6%.
Results of the experiment on extraction DNA HPV and vaginal microbiota from the
transportation medium CellScan by Russian test systems "Femoflor-screen" have
been eventually evaluated as unsatisfactory - there are great differences between
experimental and control samples that do not allow the implementation of the
technique on extraction DNA HPV and vaginal microbiota from the liquid
transportation medium CellScan, even at the level of qualitative analysis (yes / no).
Results of colposcopic data analysis:
The viral load indices do not correlate enough with the visual colposcopic view of
lesions of the cervix. There are a lot of "emissions": for example, at 2 points by Reid
(colposcopic picture does not inspire disquietitude) many values of viral load from 5
(105 GE / per sample) and above, i.e. high, which are pointing on possible HPV
persistence, are observed.
The Spearman’s rank correlation coefficient r = 0.33 is obtained.
Results of the study "case of cervical cancer-control":
Top-5 identified HPV types in patients with newly diagnosed cervical cancer is the
following: 16, 31, 58, 18, 33.
The specific gravity of HPV 16 is 7/10 (71.9%) of the total number of identified
types.
Infection with one type of virus is detected in 68.2%; 2 types - 15.9%; 3% - 9,1%;
4% -2,3%.
The average viral load in the group of patients with cervical cancer was 6.94.0 (CI
95% 6.1;7.7), and exceeds the regional average in the total sample (5.53.8 (CI 95%
5,1;5.9)).
A reliable relationship between the staging of cancer and viral load indices has been
revealed (p = 0.043, CI 95%, n = 64). The largest contribution to 2 has been made by
St IIa.
The viral load in the studied samples increases till the TIS stage to St IIAB,
demonstrating the largest range in the TIS stage, and the maximum number of
outliers and extreme points in the St IIAB stage.
Results of the matching, performed to identify the dominant risk factors for the
implementation of cervical cancer in the Western region:
based on the analysis of Pierson's 2 and Cramer's contingency tables and results of
the Mann-Whitney test, which details the analysis of quantitative variables, a profile
(social portrait) of cervical cancer patients has been compiled in the region:
these are women mostly aged 50-60 + years, infected with HPV, mostly type 16,
poorly educated, unemployed, mostly poor, with more than 20 years of sexual
experience, not participating in screening programs and not aware of cervical cancer
prevention measures (vaccination). In the profile of cervical cancer realization, a
large number of pregnancies and a high level of viral load also matter.
Assessment of the likelihood of the disease onset under the influence of the analyzed
risk factors with calculation of the odds ratio established that the probability of the
disease occurrence by cervical cancer:
- decreases up to 14 times with a per capita income level of 90000 tg (500$) and
higher (negative coefficient, -2.64);
- increases to 0,9 times with the experience of sexual life over 20 years (indicator
close to 50/50);
- decreases up to 3.3 times with at least irregular participation in screening for
cervical cancer (coefficient is negative, -1.08).
The prognosis is justified for 73.9% of infected HPV, but not affected by cervical
cancer and 70.3% of cases.
Trends in the incidence of cervical cancer, obtained in linear regression analysis
(least squares method) based on the dynamics of indicators from regional oncological
Register (Form No. 7) over the past 5 years, have shown increase in the female
population with an annual growth rate of Tpr = 3,2%.
Decrease in morbidity trends has been established in the age groups 30-39 years (Tpr
= -6.2%) and 18-29 years (Tpr = -3.1%). The increase is noted in persons over 40
years, especially in persons 40-49 years (Tp = 5.5%) and 70 + (Tpr = 13.6%), which
resulted in a general increase of the cervical cancer incidence in the entire female
population of the region.
Based on calculated trends, epidemiological prognosis has been submitted: the
expected incidence of cervical cancer in 2017 is 26.9 (21.4; 32.4 95% CI) (i.e. 26.9
per every 100 000 female population standardized by age).
Conclusions based on the obtained results
1) Overall HPV prevalence in the Western Kazakhstan 25.0% (22.3;27.7 CI 95%, p =
0.05) is estimated as high for the general population.
Average viral load in the group of infected HPV 5,53,8 (CI 95% 5.1;5.9) is also
high and may indicate the persistence of HPV infection in the majority of the
examined HPV-carriers.
2) The most carcinogenic vaccine-dependent HPV 16 is the leading type amongst
common population of the Western region, with a specific gravity up to 26.4%.
According to the results of the "CC case - control" study, the specific gravity of HPV
16 is 7/10 (71.9%) of the total number of identified types in the group of patients.
In general, specific gravity of the so-called "vaccine types" (6, 11, 16, 18), i.e.
potentially being eliminated by vaccination, is 43.0% in western Kazakhstan, and
including 31 and 45 types, also potentially being eliminated by vaccination - 59.6%.
This index points on the need to restart the immunization program (vaccination
against cervical cancer).
Research on the HPV content in normal cytology, which revealed on our own
material a high (46,0 / 44,8) percentage of HPV in "normal" smears, i.e. without signs
of atypia, constitutes a serious argument in favor of introducing molecular-biological
testing of women on HPV. An even stronger argument is to determine the high
specific gravity of the most carcinogenic type 16 in smears of the NILM class
(without atypia and malignancy) - 30.6%.
In general, the basis for further evaluative studies on the transition to primary HPV-
oriented screening in which cytology plays the role of a sorting tool, has been
obtained.
3) The largest number of abnormally high viral load values recorded in the age group
30-39 years, also correlates with the data of other researchers who established that the
process of the virus persistence and development of CIN begins in this age.
Despite the fact that the analysis of the relationship between HPV infection and risk
factors in this age group did not yield a value of p≤0.05, this age should be identified
as potentially risky in terms of cervical cancer implementation.
4) Comparative analysis of cytological screening tools for cervical cancer led up to
the finding of a certain advantage of the traditional method comparing with LBC,
despite the proportion of the results coincided with the histological conclusion turned
to be 61.7% in the LBC group vs. 60.6% in the Romanovsky-Giemsa group.
The main advantage of the traditional method is its relative reliability: specific weight
of the non-informative material in the Romanovsky-Giemsa coloring method is
negligible compared to CellScan - 0.4% versus 5.9%.
In the region, some diagnostic advantage of the traditional method is presumably
associated with the defective CellScan membrane filters supplied by the
manufacturer, LTD "Tech Bio Co.", South Korea, which led to a low class of
cellularity and a significant number of non-informative conclusions by CellScan.
There is a progressive decrease in the number of non-informative material in the
CellScan group in dynamics.
Further comparative studies with a great number of histological findings are earnestly
needed.
5) In this study, we evaluated the effectiveness of colposcopy in detecting HPV
infection of the cervix as a predictor of cervical cancer that is why the possibility of
experimental introduction of VIA method (visual inspection with acetoacetic acid)
for sparsely populated sites of the region was assessed.
By the results of colposcopic data analysis, it is established that the viral load indices
do not correlate with the picture of cervical lesions visually determined on the
colposcopy. There is a discrepancy between the relatively calm colposcopic picture
and the values of the viral load from 5 (105) GE / per sample and higher, evidencing a
possible persistence of HPV.
By Spearman’s rank correlation index r=0.33 (moderate, closer to weak) obtained in
the present study, the introduction of the VIA method at present is not justified.
Further research with an involvement of larger amount of specialists is needed.
6) Average viral load in the group of patients with cervical cancer, amounting to
6.94.0 (CI 95% 6.1;7.7), exceeds the regional average in the general sample and has
significantly associated with the stage of the cancer process (p=0.043), increasing
from stage TIS to St IIAB. Presumably, the largest range of viral load demonstrated
in the TIS stage may evidence the need for the break through of the cell basal
membrane, after which the immune mechanisms are not longer able to restrain the
viral replication. Further research is needed.
7) According to the survey results, it can be concluded that attendance the state
screening program is extremely inadequate (only 62.7% of respondents visit the
program in the general sample, besides 28% of them irregularly, whereas amongst
educated respondents attendance is even lower). A similar situation arose with
awareness of the measures on primary prevention of the cervical cancer.
Based on the profile (social portrait) of cervical cancer patients in the region –
“women aged 50-60 + years, infected with HPV, predominantly type 16…” - it has
been established that the presence of vaccine-dependent type 16 is one of the most
important risk factors in the region.
The probability of cervical cancer in women infected with HPV in the region can be
reduced or prevented by increasing the standard of living and mobilizing the
participation of women in a national screening program.
Trends in the incidence of cervical cancer in the Western region revealed a course
towards an increase, with an annual growth rate Tpr = 3.2%.
Observed increase in the incidence of cervical cancer in 70+ group is generally
consistent with literature data on the constant evolution of the human papillomavirus
and enhancing the role of potentially / possibly / probably carcinogenic types in the
implementation of cervical cancer.
In the future, this issue should be thoroughly studied in the researchs on local cases,
including the method of HPV genes sequencing.
Expected incidence of cervical cancer in 2017, defined in the range from 21,4 up to
32,4 is regarded to very high and entails the necessity of urgent measures to reduce it.
Final conclusion
Current screening program is characterized by conceptual backwardness, since it
does not take into account high prevalence of HPV infection, which is the causative
factor of cervical cancer in the general female population of reproductive age, and
does not include the necessary diagnostic tools, namely, molecular-biological tests
for HPV determining.
Given the relatively large stratum of enlightened women in the republic, the
unsatisfactory data on awareness of primary prevention and attendance of the national
screening program suggest that state measures to prevent cervical cancer are
inadequate and do not meet the needs of the population, especially its educated part.
The same refers to the situation on the attendance of state medical institutions.
Efficacy of the basic screening instrument - the CellScan fluid system at
present cannot be considered satisfactory for a number of the above mentioned
reasons. Further evaluative studies are needed.
Large prevalence of high carcinogenic risk HPV in Kazakhstan entails the
urgent need to renew the national program of primary infection prevention, i.e.
vaccination of adolescents.
Overall, throughout the region further increase in morbidity and mortality from
cervical cancer is expected, despite screening activities.
It is necessary to revise the existing screening practices in order to reduce the
growing incidence of cervical cancer.
Recommendations
Based on the urgency of the problem, the experience and resources available to
Kazakhstani experts, together with the leading specialists of KazIOR (Kazakh
Institute of Oncology and Radiology), the following recommendations were
developed, previously partly published in the journal “Oncology and Radiology of
Kazakhstan”:
1) Revise methodological approaches to the screening strategy implemented in the
country. To organize the transition to HPV-oriented screening, which proved to be
effective on a global scale, i.e. use screening tools only in the combined HPV test +
PAP mode, according to the latest European expert assessments of screening
technologies, with a level of evidence IA.
2) Approve a mechanism for validating screening methods and conduct all tests of the
population screening through this mechanism. Develop an appropriate Application to
the Committee of the Ministry of Health on the Assessment of Medical Technologies
(HTA), according to the commonly accepted international valuation procedure.
3) Increase coverage of the female population by moving awareness campaign
towards screening and vaccination against cervical cancer to the Internet. According
to the Committee on Statistics of the republic, the percentage of women aged 15-24
using the Internet (social networks, messengers) is 94.6%, while the proportion of
women aged 15-49 years, at least once a week using mass media (newspapers,
magazines, radio, television) is only 16.1%.
4) Increase the responsibility of specialists in cervical pathology (colposcopic
centers) for the final results of screening in the relevant state facilities. Strengthen the
interaction of the screening stages: screening room - general practitioner -
gynecologist – colposcopic cabinet, i.e. to debug the feedback in rehabilitation of
women with revealed pre-tumor pathology.
5) Increase the level of cytologists training for submitting cytological conclusions
only on a basis of Bethesda terminology (TBS, 2001), and for fluent understanding of
molecular-biological tests.
6) Debug a state system for training laboratory technicians to work in state-owned
PCR laboratories, and improve the qualitative and quantitative training of molecular
biologists.
7) Revise the existing Protocols for the diagnosis and treatment of papillomavirus
infections: introduce the concepts of "viral load", "transient" and "persistent" HPV
infection, for the rational management of patients with PCR-HPV and cytological
status positive results.
8) Renew discontinued national immunization program against HPV by including the
mandatory vaccination of 11-12 year old girls in the National Immunization
Schedule.