[Serologic study of experimental cholera polymer antigen diagnosticums].

AIM: Evaluation of quality indicators of constructed cholera antigen polymer diagnosticums by using a complex of specific anti-cholera sera. MATERIALS AND METHODS. Cell lysates of cholera vibrio strains Vibrio cholerae cholerae 1395, V. eltor Ogawa 2044, V. eltor Inaba 13020, V. cholerae O139 16064 were sensitins for experimental preparations. 3 sera from cholera patients, normal human sera, cholera O1 (Ogawa, Inaba) commercial horse, cholera O139 commercial rabbit and heterologic sera against shigella, salmonella, escherichia and yersinia as well as experimental cholera rabbit sera against O1 and O139 were used as control. RESULTS: The study established that diagnosticums based on V. cholerae cholerae 1395 and V. cholerae O139 16064 strain sensitins by quality indicators may be used in the future for construction of these diagnosticums. CONCLUSION: Antibody containing preparations--commercial horse O1 sera, rabbit experimental and commercial sera and MCA O139 demonstrating titers not lower than 1/5120-1/10240 may serve as a control of experimental diagnosticums in the absence of human sera from cholera patients.

[Cholera vibrios lectins as main pathogenicity and persistence factors (biotechnological aspects of use)].

Literature data and results of our studies of lectins are analyzed in the review. All the leading pathogenicity factors of cholera vibrios that possess enzymatic activity--cholera toxin, hemolysin, neuraminidase, chitinase have several lectin domains, that determine not only their pathogenetic role but also open perspectives for their use in medical practice. At the same time the variable receptor profile of cholera vibrios cells of various biovars and epidemical significance established with hemagglutination inhibition reaction by carbohydrates could be used to develop new principles of testing and typing of cholera vibrios.

[Design of a polymer drug for serological diagnosis of hepatitis C].

A new immunobiological polymer drug has been designed for the serological identification of hepatitis C. The drug is able to reveal specific antibodies in the sera of patients with hepatitis C, meets the current requirements of diagnostic test systems, and shows a high sensitivity and specificity. It is based on polyacroleinic microspheres; the concentrated cell culture biomass of hepatitis C virus (HCV), which contains an adequate set of viral antigens, is used as sensitin. A new diagnosticum is proposed to be used during primary (screening) laboratory studies based on the serological detection of total antibodies to HCV antigens in the volume agglomeration test. The latter is both one of the alternative methods during serological studies and an additional procedure when a set of diagnostic techniques is used.

[Comparative evaluation of protein hydrolysates in designing their based universal culture medium for the diagnosis of plague and cholera].

Various protein hydrolysates made in Russia and foreign countries were comparatively evaluated to use them to design a universal agarized culture medium for the diagnosis of plague and cholera. Pancreatic baker's yeast broth was found to be most effective among the test media.

[Evaluation of toxin producing abilities of non-O1/non-O139 serogroup Vibrio cholerae isolated from humans].

AIM: Determination of non-O1/non-O139 Vibrio cholerae toxin (CT) gene expression by using EIA, and biological effect of non-O1/non-O139 V. cholerae supernatant on cell cultures evaluation. MATERIALS AND METHODS: 39 V. cholerae strains from various serological groups were studied. Hemolytic activity of strains was determined by using Greig test, and cholera toxin production--in GM1-EIA and in continuous cell lines by registering cytotonic, cytotoxic and proteolitic effect. RESULTS: GM1-EIA method does not detect CT production in 29 museum strains of non-O1/non-O139 V. cholerae in vitro. CT was detected only in 1 non-O1/non-O139 V. cholerae strain supernatant with OD = 0.577 that is substantially lower than in O1 V. cholerae strains (OD = 2.176). In cell cultures non-O1/non-O139 V. cholerae supernatants diluted to 1:100 caused elongation only in single cells. CONCLUSION: Cytological model is a more sensitive technique to evaluate toxin producing abilities of non-O1/non-O139 V. cholerae strains and is appropriate for use.

[Serotyping and genotypic characteristic of Vibrio cholerae non-O1/non-O139 serogroups isolated from water of surface basins and sewages of Rostov-on-Don city in 2003 - 2008].

AIM: Determination of serogroup and PCR-genotyping of Vibrio cholerae non-O1/non-O139 strains isolated from surface basins and sewages of Rostov-on-Don city in 2003 - 2008. MATERIALS AND METHODS: Seven hundred strains of V. cholerae non-O1/non-O139 serogroups were studied in reaction of slide-agglutination with array of 80 diagnostic sera for non-O1/non-O139 serogroups. Selective screening of strains representing dominating serogroups was performed for extended number of genetic determinants of pathogenicity factors. RESULTS: It was established that V. cholerae belonging to serogroups O53, O67, O75, and O76 are dominating in water ecosystems of Rostov-on-Don city at this time. All studied strains were characterized by lack of cholera toxin genes and toxin-coregulated pili but had different combinations of genes of additional virulence factors. There was no correlation between genotypic characteristics and serogroup. CONCLUSION: The study showed that change of serologic landscape of V. cholerae non-O1/non-O139 occurred in water objects in studied area during last decades. Necessity of dynamic surveillance for circulation of V. cholerae non-O1/non-O139 in aquatic environment with widening of studied spectrum of their biological features was demonstrated.

[Comparative assessment of dot-immunoassay and immunochromatography methods for detection of 01 serogroup of vibrio cholerae].

AIM: Comparative study of sensitivity and specificity of immunochromatographic (IC) assay kit and dot-immunoanalysis for assessment of feasibility of their use for laboratory diagnostics of cholera. MATERIALS AND METHODS: Experimental lots of IC assay kit and dot-immunoassay (DIA) for detection of Vibrio cholerae serogroup 01 serovars Ogava and Inaba were constructed on the basis of species-specific monoclonal antibodies (MCA) conjugated with colloid gold (IC) and peroxidase (DIA). Hybridoma-producer of MCAwas obtained and stored in liquid nitrogen in Rostov-on-Don Research Institute for Plague Control. It was deposited in specialized collection of cell cultures of vertebrates in Institute of Cytology (Saint Petersburg). RESULTS: Strong specificity of IC assay kit and DIA relative to cholera vibrios 01 and absence of crossreactivity with closely related and heterologous microorganisms were shown. Minimal quantity of vibrios, which could be detected using IC assay kit and DIA, was 107 and 105-106 microbial cells respectively. CONCLUSION: Performance of IC assay takes 5-15 min, DIA--1.5 hour, they allow to visually assess the reaction, do not require instrumentation and in perspective both methods could be used on defined stages of scheme for laboratory analysis of cholera.

[Identification of Vibrio cholerae serogroups O1 and O139 by monoclonal antibodies in the slide agglutination test].

The agglutinating properties of MCA-O1 of the IgG class and MCA-O139 of the IgM class towards epitopes of O-antigen of Vibrio cholerae O1 and accordingly Vibrio cholerae O139 were studied. The ascitic and cultural fluids by hybridomas F8G12 and D11 deposited in the specialized Collection of Cell Cultures of Vertebrates (Saint Petersburg) under RKKK (II) 386 D and RKKK (II) 674 D were the sources of monoclonal immunoglobulins. The advantage of diagnostic monoclonal immunoglobulins is that they are distinguished for strict specificity and their use in practical health care contributes to the higher specificity of a laboratory test for cholera and to its shorter performance.

[Laboratory diagnosis of cholera: analysis and prospects for improvement].

Microbiological monitoring of the circulation of Vibro cholerae remains one of the key factors contributing to optimization of epidemiological surveillance in a specific area and the laboratory diagnosis of cholera is a basic applied tool for the detection and characterization of isolated cultures. The quality of etiological identification of the pathogen, the competent use of procedures, and the observance of a laboratory diagnosis scheme increases the likelihood of the cholera pathogen being detected in the samples taken from human beings and environmental objects, which allows to timely notify the disease and to prevent it. The current goals of investigations include the development of more accessible and rapid methods that would further find their place in the scheme for the laboratory diagnosis of cholera.

[Novel diagnostic kit on the basis of stained polymeric carriers for epidemiologic surveillance in natural foci of Crimean-Congo hemorrhagic fever].

AIM: To study diagnostic value of developed antigenic polymeric diagnostic kit for epidemiologic surveillance in natural foci of Crimean-Congo hemorrhagic fever (CCHF) in South federal district. MATERIALS AND METHODS: Novel antigenic diagnostic kit on the basis of polymeric microspheres for reaction of volume agglomeration was developed. The kit is designed for detection of virus-specific antibodies in human serum and in serum of agricultural animals. RESULTS: Laboratory and field trials of the kit showed its high diagnostic potency, it was included in methodical recommendations "Organization and accomplishment of measures against Crimean-Congo hemorrhagic fever on the territory of its natural foci in Russia". Use of antigenic polymeric kit for epidemiological surveillance allows for more complete and systemic understanding of CCHF epidemic process. CONCLUSION: At present, the diagnostic kit is successfully used, alongside with ELISA and PCR, on different levels of epidemiologic surveillance for CCHF in Rostov region.

[Characterization of the adhesive activity of cholera vibrions in mammalian red blood cells as an additional test for assessment of their epidemic significance].

The in vitro study of the adhesive properties of V. cholerae eltor and V. cholerae O139 on a model of mammalian red blood cells revealed a correlation of their adhesive properties, the presence of the ctx AB, tcpA genes, and their hemolytic activity when blood group A (II) red blood cells were used. In the latter case, the strains having the characteristics of ctx(+) tcp(+) Hly(-) were ascertained to have a mean adhesive value (MAV) of > 1.5, a red blood cell involvement coefficient (RBCIC) of > 50%, while those with the characteristics of ctx(-) tcp(-) Hly(+) had a MAV of < 1.5 and a RBCIC of < 50%. In terms of the adhesive activity, the cultures with the genotype ctx tcp(+) Hly(+) is a heterogeneous group and may be low (MAV < 1.5) and high (MAV > 1.5) adhesive. According to the data of our studies, their adhesiveness is associated with the region of identification and independent on its object.

[Vibrio cholerae serogroups O1 and O139: susceptibility to antibiotics during 7th cholera pandemic].

The review presents data on circulation of antibiotic resistant and susceptible strains of Vibrio cholerae serogroups O1 and O139 isolated from cholera patients and healthy persons as well as from the environment, in Asia, Africa, Australia, and Europe (including New Independent States) during 7th cholera pandemic.

[Vibrio cholerae hemolytic activity].

Mechanisms of realization of Vibrio cholerae hemolytic activitywere analyzed using summarized own results and data from the literature. It has been shown that lectin receptor, which coded by hlyA gene, participates in lysis of sheep erythrocytes, but not of rabbit erythrocytes, as well as interact with D-galactose with selectivity to 3 anomers. Lectin nature of HlyA can determine formation of its complexes with lypopolysaccharides (LPS) and enzymes, which promote realization of hemolysis (by lipase, lecitinase, neuraminidase). It has been determined that lipase activity correlates with hemolytic activity of nonepidemic variants of V. cholerae. Lipase is considered as the enzyme marker of sheep erythrocytes hemolysis. It is assumed that LPS and lipase play shaperon-like role during interaction of HlyA with lipids, which promote denaturation of hemolytic active monomer in hemagglutinating oligomer.

[Cholera caused by Vibrio cholerae O1 ctxAB- tcpA+].

Results of analysis of cholera outbreak during which V. cholerae O1 biovar El-Tor ctxAB- tcpA+ was isolated from 2 patients and 30 carriers are presented. Epidemic was caused by contamination of water source and water route of transmission. Strains identical to ones detected in humans were isolated from water of surface well in zone of water intake. Genome and VNTR-analysis of ctxAB- tcpA+ vibrios that caused outbreak in Rostov region in 2005 showed that they differed from ctxAB- tcpA- and ctxAB- tcpA+ vibrios isolated previously during and beyond of outbreaks from patients, carriers and environment and formed separate group with certain genotype. These results confirms conclusions of epidemiological analysis about imported cause of recent outbreak.

[Detection of lipase in Vibrio cholerae serogroup O1 in reaction of volumetric agglomeration].

Study showed that El-Tor strains of V. cholerae isolated from different sources produce lipase for hemolysis after cultivation during 24 h on meat-peptone broth independently from their toxigenic and hemolytic abilities. Study of 3- and 4-hours broth cultures of vibrios revealed possibility to differentiate between hemolytic nontoxigenic strains and toxigenic nonhemolytic ones. Using antilipaze diagnostic kit it was possible to differentiate El-Tor vibrios from vibrios of classic biovar basing on lipase production 24 h after cultivation on meat-peptone broth that was evident in El-Tor vibrios but not in classic biovar strains.

[Specificity of lectin receptors of cholera vibrios].

Spectrum of carbohydrate specificity of lectin receptors of epidemically significant cholera vibrios (ctx(+) tcp(+) Hly(-)) as well as non epidemic hemolytic variants with or without tcp A gene (ctx(-) tcp(-) Hly(+), ctx(-) tcp(+) Hly(+)) was studied under the carbohydrates-mediated inhibition of hemagglutination between human erythrocytes of four blood groups and sheep erythrocytes. It was demonstrated that in toxigenic cultures lectin receptors specific for glucose, mannose, sacharose, lactose dominate whereas receptors specific for aminosugars are virtually absent. The latter are detected in hemolytic vibrios that can explain their ecologic flexibility.

[Development of antilipase immunoglobulin polymer diagnosticum for the detection of Vibrio cholerae eltor, possessing hemolytic and lipase activity].

The possibility of using a heterogeneous, but structurally similar antigen--the commercial preparation of Pseudomonas sp. lipase (Sigma, USA)--for the development of polymer diagnosticum aimed at determination of lipase production in cholera vibrios was shown. The new diagnosticum (antilipase antibodies) on a polymer carrier was used in the serological volume agglomeration test for the detection of hemolytic atoxigenic V. eltor, obtained from environmental, objects, which produced lipase in 80% of cases. The differentiating capacity of the diagnosticum was confirmed on 120 V. eltor cultures isolated from environmental objects. The newly developed diagnosticum makes it possible to determine the lipase activity in cholera vibrios of different biovars and serovars.

[Dynamics of the reversible transition of Vibrio cholerae into the uncultivable state in the presence of organic and inorganic microcosmic components].

The dynamics of the transition of V. cholerae into the uncultivable state in distilled, river and tap water, containing organic and inorganic components added, was studied. As additives, potassium nitrate, potassium phosphate, magnesium sulfate, ammonium chloride, lysine, alpha-ketoglutarate, succinic acid, catalase were used. The study of the influence of biotic factors on transition into the uncultivable state was carried out in the presence of one-celled green algae Scenedesmus quadricauda or infusoria Paramecium caudatum. The linear dependence of speed of transition into the uncultivable form on the concentration of cells was noted. The composition of the microcosmic medium was also found to have some influence on the speed of transition into the uncultivable form and on the reversibility of this process. The presence of organic substances, such as peptone solution or destroyed cells of phyto- and zooplankton, in the microcosmic medium prolonged the time of transition into the uncultivable form and produced a positive effect on the capacity of the population to reversion. In respect of live biotic components, no such dependence was found. Inorganic additives prolonged the time of transition into the uncultivable state, but did not promote reversion.

[Cholera at the beginning of the XXI century. Prognosis].

The worldwide epidemiological situation in cholera El Tor at the beginning of this century is presented; among its characteristic features are continued extensive epidemics and outbreaks in African and Asian countries with cases of import of this infection to other continents. Outbreaks caused by a new variant of the infective agent of cholera, Vibrio cholerae O139, are still registered at limited territories in the countries of South-East Asia. In some CIS countries (Azerbaijan, Kazakhstan and Russia) unstable situation in cholera is still preserved due to cases of infection import mainly from Asian countries, as well as to the isolation of epidemically insignificant haemolysin-positive and haemolysin-negative V. cholerae O1 and O139, containing no ctx and tcpA genes, from surface water reservoirs and other environmental objects. In Russia prognosis for cholera is still unfavorable.

[Activity of 22 antibacterials against O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world].

Analysis of antibioticograms of 390 O1 and O139 serogroup Vibrio cholerae strains isolated from humans within 1927-2005 in various regions of the world showed that the strains of V. cholerae isolated within 1927-1966 were susceptible to 22 antibacterials, the strains isolated within 1938-1993 possessed 1-3 resistance markers and the strains isolated within 1994-2005 had 3-8 resistance markers including resistance to fluoroquinolones. All the strains of O139 serogroup V. cholerae isolated in 1993 and 1994 possessed 3 resistance markers. Studies on albino mice with generalized experimental cholera due to the V. cholerae eltor 1 strain (P-18826, 2005) isolated from a cholera patient, which was highly resistant to nalidixic acid, streptomycin, ampicillin and trimethoprim/sulfamethoxazole and showed cross resistance to fluoroquinolones (ciprofloxacin, ofloxacin, pefloxacin and norfloxacin) and moderate resistance to ceftriaxone and cefotaxime, revealed that the only efficient antibiotics were tetracyclines and aminoglycosides (except streptomycin). The investigation demonstrated an extension of the antibiotic resistance spectra of the epidemically significant strains of the cholera pathogen and the necessity of using antibacterial drugs in strict accordance with the antibioticograms in emergent prophylaxis and therapy of cholera and immediate replacement of the drug by a more active one.