Vol 7, No 2 (2017)

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Full Issue

REVIEWS

LIVE ATTENUATED VACCINES FOR THE IMMUNOPROPHYLAXIS

Shamsutdinova O.A.

Abstract

The review focuses on the history of the production of live antiviral vaccines and their use for the prevention of infectious diseases. It was noted that before the beginning of the 20th century, only three live vaccines were developed and put into practice — against smallpox, rabies, plague. The discovery of D. Enders, T.H. Weller and F.Ch. Robins of the ability of the polio virus, and then of a number of other viruses, to reproduce in vitro in cell cultures of various types, greatly expanded the studies on the production of attenuated strains of viruses for live vaccines. The historical stages of obtaining and introducing live vaccines for the prevention of smallpox, poliomyelitis, measles, rubella, and mumps are highlighted. Arguments in favor of the use of associated vaccine preparations for the prevention of viral infections are presented. Various variants of the strategy and tactics of using live vaccines, which are used for specific prevention of viral infections in different countries, are described. The review provides information on technological methods for obtaining antiviral vaccines. The publications testifying to the development of specific reactions in immunized vaccine strains of measles, mumps, poliomyelitis and rubella viruses, such as aseptic meningitis (vaccine strains of mumps virus), acute arthritis (vaccine rubella virus strains), temperature reactions, rash (vaccine strains of the virus Measles), vaccine-associated paralytic poliomyelitis (VAPP) vaccine vaccine poliovirus. It is particularly noted that the long experience of vaccine prevention both in Russia and abroad convincingly shows that the risk of developing post-vaccination complications is incommensurably lower than the risk of causing harm to health from the corresponding infections. It is concluded that despite introduction of new third and fourth generation vaccines into practice, live attenuated vaccines do not lose their significance and are used in vaccine-preventable infections as the most effective immunobiological drugs.
Russian Journal of Infection and Immunity. 2017;7(2):107-116
pages 107-116 views

ORIGINAL ARTICLES

PRODUCTION OF HYBRID RECOMBINANT PROTEIN Flu-Chim, CONTAINING INFLUENZA VIRUSES A AND B MAJOR EPITOPES

Dukhovlinov I.V., Dobrovolskaia O.A., Orlov A.I.

Abstract

The influenza virus is highly contagious diseases of people, birds and mammals. Approximately 250 000– 500 000 deaths are caused by influenza epidemics worldwide yearly, and the death number may be up to millions in a possible influenza pandemic. Vaccination is the most cost-effective way to reduce the considerable disease burden of seasonal influenza. Although seasonal influenza vaccines are effective, their performance in the elderly and immunocompromised individuals would benefit from improvement. Major problems related to the development and production of pandemic influenza vaccines are response time and production capacity as well as vaccine efficacy and safety. Reverse genetics techniques can speed up the generation of seed viruses and new mathematical modelling methods improve vaccine strain selection. Using vaccines based on recombinant proteins, we avoid the risks associated with the introduction of the virus into the body, even inactivated. In this paper, we have got a highly purified recombinant fusion protein composed of fragments of the hemagglutinin of influenza viruses A and B. As adjuvant we used components of flagellin. We used the most immunogenic and conserved areas of hemagglutinin H1, H3, H5 and B, which cause the formation of specific antibodies which can cross-react with homologous epitopes among the various strains of influenza A and B. Vaccine efficacy is increased by using multiple epitopes of various proteins. The aim of this study was to clone and express the hybrid recombinant protein Flu-Chim, containing immunogenic epitopes of influenza A/H1N1, A/H3N2, A/H5N1 and B fused with fragments of flagellin in Escherichia coli expression system and its subsequent purification. During the study was created high-yield E. coli strain, which produces the recombinant protein Flu-Chim, selected the optimal protocol of induction of the gene encoding the protein. The protein was purified using metal affinity chromatography. The purity of the final preparation reached 98%. In the future, we are going to study the immunogenic properties of the protein and use it as a component of the candidate vaccine against influenza.
Russian Journal of Infection and Immunity. 2017;7(2):117-122
pages 117-122 views

POLYLACTIC ACID NANOPARTICLES INFLUENCE ON IMMUNOGENICITY OF THE PROTEIN BOUND WITH THEM

Polyakov D.S., Antimonova O.I., Sakhabeev R.G., Grudinina N.A., Khodova A.E., Sinitsyna E.S., Korzhikov-Vlakh V.A., Tennikova T.B., Shavlovsky M.M.

Abstract

We investigated immunogenic properties of proteins bound with nanoparticles. A process for producing spherical nanoparticles having size of 20 microns by polymerization of lactic acid and an optimal method of nanoparticle surface activation were described. Activated nanoparticles were used for covalent binding of model fusion protein comprising sequences of human beta-2 microglobulin and green fluorescent protein. It is shown that the nanoparticles were able to bind 3 micrograms of the protein per 1 mg of the polymer. According to the results of confocal microscopy and electrophoresis the protein is firmly adsorbed on the surface of the granules. F1 (CBA x C57BL) mice were subjected to intraperitoneal immunization with fusion protein modified nanoparticles and equivalent mixture of unmodified nanoparticles and unbound fusion protein. Blood was taken at 2 weeks after three-time intraperitoneal immunization. Antibody level to model protein was determined in mouse sera using enzyme-linked immunosorbent assay. Each of experimental and control groups comprised 39 animals. The validity of the results was evaluated using the Mann–Whitney test. It is shown that the average antibody level in the control group was 1.8 times greater than that in the experimental group. The diffe rence was significant (p < 0.004). We discuss the significance of the results in terms of development traps capable to bind virus particles in blood and to provide immune response.
Russian Journal of Infection and Immunity. 2017;7(2):123-129
pages 123-129 views

POLYMORPHISMS WITHIN INNATE IMMUNE RESPONSE, CALCIUM METABOLISM AND LIPID METABOLISM ARE PREDICTORS OF INFECTIVE ENDOCARDITIS

Ponasenko A.V., Kutikhin A.G., Khutornaya M.V., Rutkovskaya N.V., Kondyukova N.V., Odarenko Y.N., Kazachek Y.V., Tsepokina A.V., Yuzhalin A.E., Barbarash L.S., Barbarash O.L.

Abstract

Infective endocarditis (IE) is a septic inflammation of the endocardium generally caused by bacteria. Despite certain advances in treatment, case fatality rate and mortality of IE are still relatively high, particularly in high-risk groups. This requires the development of novel efficient preventive approaches; one of them is a personalized medicine. Recognition of microbial patterns, cytokine and acute phase responses, hemostasis features and alterations in plasma lipid and calcium profile all have been reported to affect pathogenesis and clinical course of IE. We hypothesized that inherited genomic variation in the abovementioned pathways may determine individual susceptibility to IE. Having recruited 124 patients with IE and 300 age-, sex-, and ethnicity-matched healthy controls, we profiled their genomic DNA for 35 functionally significant polymorphisms within the 22 selected genes involved in pathways mentioned above, with the further genetic association analysis. Genotyping was performed using TaqMan allelic discrimination assay while statistical analysis was carried out utilizing SNPStats, a web tool for genetic association analysis. We found that the G/A genotype of the rs1143634 polymorphism within the IL1B gene, the G/T genotype of the rs3212227 polymorphism within the IL12B gene, the A/G genotype of the rs1130864 polymorphism within the CRP gene, and the G allele of the rs1801197 polymorphism within the CALCR gene are associated with a decreased risk of IE whereas the T/T genotype of the rs1205 polymorphism within the CRP gene is associated with a higher risk of IE. Furthermore, heterozygous genotypes of the rs1143634 and rs3212227 polymorphisms were associated with the higher plasma levels of IL-1β and IL-12, respectively, suggesting their possible importance for IE development. Our results indicate that inherited variation in the cytokine, acute phase response, and calcium metabolism pathways may be linked to IE. However, further molecular epidemiology studies are needed to thoroughly uncover the genetic basis of IE.
Russian Journal of Infection and Immunity. 2017;7(2):130-140
pages 130-140 views

ROLE OF CD95 AND DR3 RECEPTORS IN NA VE T-LYMPHOCYTES APOPTOSIS IN CHILDREN WITH INFECTIOUS MONONUCLEOSIS DURING CONVALESCENCE

Filatova E.N., Anisenkova E.V., Presnyakova N.B., Kulova E.A., Utkin O.V.

Abstract

Infectious mononucleosis is a widespread disease caused by certain members of Herpesviridae family. Acute infectious mononucleosis develops predominantly in children and is accompanied by an increase of the number of circulating naive CD4+ and naive CD8+ T-lymphocytes in the peripheral blood. The normalization of immunological parameters is achieved within 4–6 months after recovery and that is an indicator of a proper functioning of the immune system. CD95 and DR3 death receptors are involved in the initiation of apoptosis of naive T-lymphocytes in healthy people and in patients with infectious mononucleosis. The aim of the study was to evaluate the ability of CD95 and DR3 receptors to initiate apoptosis of naive CD4+ and naive CD8+ T-lymphocytes in children with infectious mononucleosis during convalescence. The material for the study was the samples of the peripheral blood of children who previously had infectious mononucleosis. The blood sampling was carried out again after 4–6 months after the disease. At the time of the study, children did not display clinical and laboratory signs of infectious mononucleosis. Same children who were examined earlier in the period of the development of acute infectious mononucleosis, as well as relatively healthy children were used as the comparison groups. Isolation of naive CD4+ and naive CD8+ T-lymphocytes was performed by negative magnetic immunoseparation. For specific stimulation of CD95 and DR3 receptors monoclonal antibodies were used. The level of apoptosis and expression of death receptors were evaluated by flow cytometry. Freshly isolated cells were analyzed, as well as cells cultured with the addition of appropriate monoclonal antibodies. It was shown that the recovery period was accompanied by increased apoptosis of freshly isolated naive CD4+ and naive CD8+ T-lymphocytes compared with the acute phase of infectious mononucleosis. Thus in both populations of naive T-cells showed an increase of CD95+ cells’ susceptibility to apoptosis. CD95 stimulation in the cell culture did not lead to the change in the level of apoptosis of naive CD4+ and naive CD8+ T-lymphocytes. The freshly isolated naive CD4+ and naive CD8+ T-lymphocytes DR3+ cells were resistant to apoptosis, and in the process of cultivating their sensitivity varied depending on the subpopulation belonging. Thus in the culture of naive CD4+ T-lymphocytes DR3 was not involved in the transfer of pro-apoptotic signal. In the culture of naive CD8+ T-lymphocytes DR3+ cells were possible to increase the apoptosis of DR3-negative cells. At the same time the DR3 activation by monoclonal antibodies in the culture caused the death of DR3+ naive CD8+ T-lymphocytes that naturally associated with decreased proapoptotic activity of these cells and resulted in inhibition of apoptosis of total pool of naive CD8+ T-lymphocytes. Thus, the functional ability of CD95 and DR3 receptors to trigger an apoptosis of naive T-lymphocytes in children during convalescence of infectious mononucleosis varied and depended on their belonging to naive CD4+ or naive CD8+ T-lymphocytes.
Russian Journal of Infection and Immunity. 2017;7(2):141-150
pages 141-150 views

IMPACT OF HIV INFECTION AND TUBERCULOSIS ON THE PERIPHERAL BLOOD T-CELL DIFFERENTIATION

Vasileva E.V., Kudryavtsev I.V., Maximov G.V., Verbov V.N., Serebriakova M.K., Tkachuk A.P., Totolian A.A.

Abstract

Tuberculosis is the leading cause of death among HIV infected individuals. In this regard, an important task is the timely detection of tuberculosis in HIV infected patients. Previously, we have shown that the diagnostic value of in vitro test, QuantiFERON-TB Gold In-Tube is not decreased in patients with HIV infection against the background of tuberculosis. However, it remains unclear what kind of cell populations produce IFNγ in response to specific Mycobacterium tuberculosis antigens stimulation in vitro, because the immunodeficiency, caused by HIV, makes primarily for a decrease the abundance and attenuation functions of CD4 T-lymphocytes. The aim of thшы work was to compare the degree of differentiation of T-lymphocytes CD4 (Th) and CD8 (Tcyt) in patients with pulmonary tuberculosis and healthy donors against the background of HIV infection. The study data were obtained during the examination of 28 patients with pulmonary tuberculosis without HIV infection (HIV–TB+), 23 patients with HIV infection (TB–HIV+) and 30 patients coinfected with HIV and tuberculosis (TB+HIV+). The comparison group consisted of 37 healthy individuals (TB–HIV–). Аbsolute and abundance (relative content) of major subpopulations of T-lymphocytes (based on the expression of CD27 marker, CD28, CD45RA and CD62L) in the peripheral blood for all patients included in the study (n = 118) were evaluated by flow cytometry approach. For patients with pulmonary tuberculosis (n = 58) QuantiFERON-TB Gold In Tube (Qiagen, QFT) test was performed. Th/Tcyt ratio was not significantly different among the groups of TB–HIV– and TB+HIV– (1.76 [1.51; 2.30] against 1.86 [1.22; 2.79], p = 0.960). At that time, the size of both subpopulations “terminally differentiated” Tcyt (Tcyt Eff, CD27–CD28– CD62L–CD45RA–) Th lymphocytes and effector memory lymphocytes (Th EM, CD27–CD28+CD62L–CD45RA–), was significantly different in all four study groups. Multidirectional changes of the absolute and abundance (relative content) in these cell populations in comparison with healthy donors for tuberculosis and HIV infection was noticed. Absolute content of Tcyt Eff, compared with healthy donors (76.1 [20.7; 143.5]), 4-fold increases in the group of HIV+TB+ and 2 times in groups TB+HIV– and TB–HIV+. Th EM content increases only at TB+HIV– group compare to healthy donors. In groups of patients with HIV infection (TB–HIV+ and TB+HIV+) a decrease in the content of these cells was observed. Thus, our work shows that the population of Th EM and Tcyt Eff could potentially be viewed as universal biomarkers for two socially significant infectious diseases: tuberculosis and HIV infection. In future experiments, it is necessary to validate these results to ensure specificity and determine the number of Th EM and Tcyt Eff specificly induced by Mtb antigens.
Russian Journal of Infection and Immunity. 2017;7(2):151-161
pages 151-161 views

EFFECTIVENESS OF MOLECULAR-GENETIC DIAGNOSTICS DURING PERTUSSIS INFECTION FOCI EXAMINATION

Pimenova A.S., Borisova O.Y., Tsvircun O.V., Basov A.S., Aleshkin V.A., Afanasiev S.S., Donskich E.E., Pikina A.P., Kafarskaya L.I., Afanasiev M.S., Karaulov A.V.

Abstract

Purpose: whooping cough diagnostics by molecular-genetic methods.

Materials and methods. Under observation there were 4930 people during the period from 2012 to 2015. All samples were received in 8 schools of Moscow and the Moscow region: in 3 schools had been found children with whooping cough, in other 5 schools – only children with prolonged cough. Whooping cough diagnosis had been given earlier by bacteriological and serological methods. 430 clinical samples were studied by 2 methods: PCR with fluorescent hybridized detection of amplified products and isothermal amplification.

Results. In three of eight schools whooping cough cases at 7 children at the age of 7, 9, 11 and 15 years were revealed earlier. The diagnosis of whooping cough at them was confirmed by means of bacteriological and serological methods. 33 positive DNA samples (7.7%) are revealed. DNA-positive samples are allocated from 18 pupils and 15 employees of schools. In two schools where earlier infection sources were established, 15 DNA-positive samples from which in three cases clinical manifestations were observed are revealed. In those schools where it wasn’t earlier established a source of an infection and examinations conducted it is long the coughing children, 18 DNA-positive samples are revealed, and in two cases clinical manifestations in the form of cough were observed.

Conclusion. Performed research confirmed high efectiveness of molecular-genetic methods during pertussis infection foci examination in schools for infection source identification also amongst long coughing children. 

Russian Journal of Infection and Immunity. 2017;7(2):162-170
pages 162-170 views

DYNAMICS OF TICKS’ INFESTATION WITH BORRELIA GENUS BACTERIA AND TICK-BORNE ENCEPHALITIS VIRUS IN KIROV REGION

Bessolitsyna E.A., Volkov S.A., Stolbova F.S.

Abstract

Tick-borne encephalitis and tick-borne borreliosis (or Lyme disease) are one of the most known and the most spread transmissible diseases of human. Nowadays it was proved that these diseases are diagnosed among domestic animals as well. Ixodida ticks are often serve as vectors for these diseases. It is common that only ticks gathered from human after bite are studied for presence of infection. But such approach does not give a full distribution picture of infection among the ticks. Thus, it is necessary to study ticks gathered not only from human but from other sources as well. The aim of this study is to determine the presence of TBEV and Borrelia burgdoferi sensu lato genetic material in ticks depending on its species, sexual identity and object it was gathered from in 2007–2016. The main tick vectors were determined. It was shown that percentage of infected with different diseases ticks fluctuates separately during the period of the study. It was also shown that percentage of infected ticks tends to increase. It is often that only tick female are studied as it stay for a long time after the bite. But it was proved that male ticks can also be a vector for diseases. It was proved that percentage of infected male ticks is increasing. The percentage of infected female ticks is also increasing along with general percentage of infected ticks. Fluctuations of percentage of infected ticks among different species was shown. Such fluctuation can be a sign tick’s areal spreading at unusual territories. It can be related to climate change. The difference of percentage of infected ticks of different species can be related to the study of ticks usual for the area and ticks which migrate to the Kirov region. The percentage of infected ticks was also studied. It was also proved that the maximum percentage of infected ticks had the ones gathered from human and minimal percentage had the ones gathered from plants.
Russian Journal of Infection and Immunity. 2017;7(2):171-180
pages 171-180 views

СARBAPENEMASE-PRODUCING GRAM-NEGATIVE BACTERIA IN A SPECIALIZED HOSPITAL OF ST. PETERSBURG

Polischouk A.G., Jakubovich E.I., Poluhina O.V., Osovskich V.V., Evtushenko V.I.

Abstract

Species composition, susceptibility to antimicrobial agents, incidence and type of carbapenemase of carbapenemase-producing gram-negative bacteria, isolated in the Russian Research Centre of Radiology and Surgical Technologies from February 2014 to April 2016 were described. Screening of carbapenem-resistant bacteria was conducted by plating biomaterial on the chromogenic medium (“CHROMagar KPC”, DRG, France), species identification by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) on the analyzer VITEK MS (bioMé- rieux, France). The sensitivity of gram-negative bacteria to antimicrobial drugs was determined on the analyzer VITEK-2 (bioMérieux), and the obtained results were interpreted in accordance with EUCAST criteria v.6.0, 2016. Genes encoding carbapenemase of KPC, OXA-48/162, VIM, IMP, NDM, OXA-51, OXA40/24, OXA-23 and OXA-58 groups were detected by multiplex real-time PCR (AmpliSens, Russia). 813 clinically relevant bacteria (602 patients) were obtained, including 405 gram-negative, among which 5.2% (21 strain from 16 patients) non-susceptible to meropenem and/or imipenem: Klebsiella pneumoniae (n = 5), Enterobacter cloacae (n = 2), Serratia marcescens (n = 1), Pseudomonas aeruginosa (n = 3), Acinetobacter baumannii (n = 10). Clinical material from 4 patients was infected by up to 3 carbapenem-non-susceptible strains of different species of gram-negative bacteria at the same time. The genes encoding acquired carbapenemases were detected in 84% of the nonsusceptible to carbapenems isolates: in A. baumannii — OXA40/24 (n = 8); in K. pneumoniae — OXA-48 (n = 1), KPC (n = 1) and NDM (n = 2); in P. aeruginosa — VIM (n = 1) and KPC (n = 1); in E. cloacae — KPC (n = 1). OXA-48 carbapenemase was also detected in one carbapenem-sensitive strain of K. pneumonia. All carbapenemase-producing strains had a phenotype of multidrug resistance. The results of the study showed that over the analyzed period, the prevalence of the carbapenem-nonsusceptible strains of gram-negative bacteria were relatively low, but all these strains were multidrug resistant and most of them possessed acquired carbapenemase genes. The species carbapenemnonsusceptible gram-negative bacteria and the types of the detected acquired carbapenemase were typical for Russia. For K. pneumoniae and P. aeruginosa no predominance of any one type of carbapenemase was revealed, while in all strains of A. baumannii the gene of OXA40/24 carbapenemase was found.
Russian Journal of Infection and Immunity. 2017;7(2):181-192
pages 181-192 views

IMMUNOGENICITY OF ADJUVANT INFLUENZA VACCINE FOR PREGNANT WOMEN

Kostinov M.P., Cherdantsev A.P., Shmitko A.D., Praulova D.A., Protasov A.D., Dagil U.A., Kostinova T.A., Akhmatova N.K., Chromova E.A., Ryzhov A.A., Blagovidov D.A., Polishchuk V.B., Magarshak O.O., Korovkina E.S.

Abstract

Recent epidemiological events showed that pregnant women are the most vulnerable part of population if there is the flu in the country and they die much more often than the rest part of people. That is why influenza vaccination of population including pregnant women is one of the priorities of public health service in our state. Worldwide experience of influenza vaccination of either adults or children by new adjuvant vaccine has caused our research of its efficiency among pregnant women. The aim of the study was to investigate the level of antibodies to influenza virus strain A/H1N1/v, A/H3N2 and B in pregnant women vaccinated adjuvant trivalent subunit vaccine. Our research is randomized and comparative on parallel groups. It was carried out within the demands of Russian Federation and International ethic norms adapted to such kind of researches. Evaluation of the immunogenicity of the vaccine was conducted in 27 pregnant women in the II trimester of gestation, and in 23 pregnant women in the III trimester of gestation, 19 non-pregnant women was in the control group. The level of antibodies in the serum was determined using a reaction of hemagglutination inhibition before and 1, 3, 6, 9 and 12 months after the vaccination. Revealed that influenza vaccination of pregnant women in the II and III trimester, causes the increase in titers of antibodies to vaccine influenza strains A and B, to fully meet the required criteria CPMP, and does not differ from the nonpregnant group. In a month after vaccination the level of seroprotective against A/H1N1/v was 77.0%, A/H3N2 — 88.9%, B — 85.2% after vaccination in II trimester, and 87.0; 87.0; 91.35% in III trimester of gestation. The factor of seroconversion after vaccination in II trimester for A/H1N1/v was equal to 6.5, A/H3N2 — 7.2, B — 6.5, after vaccination in III trimester of pregnancy: 7.1, 6.5 and 5.1 correspondingly. At the same time revealed accelerated decline in antibody titer against influenza in pregnant women compared to nonpregnant women in a year after immunization The set of results of the study can be concluded that adjuvant trivalent subunit vaccine effective in vaccination of pregnant women in II and III trimester of pregnancy, and it is able to effectively provide immune protection against viruses of the flu A and B within all pregnancy long.
Russian Journal of Infection and Immunity. 2017;7(2):193-202
pages 193-202 views

SHORT COMMUNICATIONS

THE ANTIGEN-SPECIFIC CELL IN VITRO TESTS FOR POST-VACCINATION ANTIPLAGUE IMMUNITY FORMATION

Kulichenko A.N., Abzaeva N.V., Gostischeva S.E., Rakitina E.L., Ponomarenko D.G., Kostuchenko M.V.

Abstract

The possibility of post-vaccination anti-plague immunity evaluation was researched using antigen-stimulated cells tests in vitro and cytometry analysis. The object of study — the blood samples of 17 people immunised by the live plague vaccine (Yersinia pestis EV) epicutaneously. Blood taking was carried out before vaccination and after immunisation on 7 and on 21 days, in 3 and in 6 months. Intensity antigen reactivity of lymphocytes was detected by cell tests in vitro, analysing markers of early (CD45+CD3+CD25+) and late (CD45+CD3+HLA-DR+) lymphocyte activation using flow cytometry. The complex of water-soluble Y. pestis antigens and allergen — pestin PP was tested as antigen. The high stimulating potential was defined of the water-soluble antigens Y. pestis complex. It is shown that coefficient of stimulation of relative level T- lymphocytes which express receptors for IL-2 was positive for all observation times after immunisation. The coefficient of stimulation had maximum values at 21 days (56.37%) and at 3 (47.41%) months. In identifying HLADR-positive lymphocytes before vaccination, the negative coefficient of stimulation was indicated on 7 and 21 days and the positive coefficient of stimulation was indicated at 3 and at 6 months. Analysis of intensity expression of early and late lymphocyte activation markers dynamics showed the possibility and prospect of application of cellular in vitro tests for the laboratory evaluation of specific reactivity of cellular immunity in both the early (7 days) and late (6 months) periods after vaccination. The results can be the basis for developing a new algorithm for assessment of immunological effectiveness of vaccination people against plague. It is the algorithm based on the identification of lymphocyte activation markers by antigen stimulation in conditions in vitro.
Russian Journal of Infection and Immunity. 2017;7(2):203-208
pages 203-208 views


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