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.