Method for predicting lambliasis and its flow

FIELD: medicine, parasitology.

SUBSTANCE: one should carry out immunoenzymatic assay to detect diagnostic optic density and that of labeled immune complex in a plot's hole with tested serum measured in conventional units at wave length being 492 nm. One should calculate coefficient of antibodies concentration measured in conventional units by the following formula: CAC = (Odtsh - Odd) x 100, where CAC - coefficient of antibodies concentration, Odtsh - optic density of the hole with tested serum, Odd - diagnostic value of optic density, 100 - coefficient of serumal dilution. By CAC value one should detect the titer of antibodies to Lamblia intestinalis antigens to interpret results of the trial. The method enables to study the dynamics of disease flow.

EFFECT: higher efficiency and accuracy of diagnostics.

1 ex, 1 tbl

 

The invention relates to medicine, namely to infectious diseases, in particular to Parasitology.

Giardiasis is a pervasive invasion. In the Russian Federation annually about 110,000 cases of giardiasis, 80% of which are children under 14 years. In the etiological structure of diarrhea share of giardiasis varies from 3% to 8% of cases. Proven role Lamblia intestinalis in allergization of an organism, development duodenojejunal, in addition lambliasis invasion is associated with a wide spectrum of diseases of the gastrointestinal tract: chronic gastroduodenitis in 76,5%, pancreatitis is 15.7%, enterocolitis - 4,0%, reflux-esophagitis - 3.8% of cases.

As a counterpart, the authors propose a microscopic method for the diagnosis of giardiasis (Clinical laboratory analyst. Volume II. Private analytical technologies in the clinical laboratory / edited Riv. - M.: Labelform-RAMLA, 1999. - 352 C.). Of the investigated material (faeces) are prepared by two native drug under the cover glass. One of the preparations stained with Lugol solution. Microscopy of preparations carried out with a dry system high magnification. In the unpainted drug identify motile vegetative stage of Giardia, painted in the drug - cysts of protozoa.

The disadvantages of microscopic method for the diagnosis of giardiasis can about the bear underestimation of the frequency of selection of Giardia faeces, the dependence of the availability of various forms of the pathogen from stool consistency. In this regard, the effectiveness of the microscopic method is not sufficiently high, and the time of diagnosis is prolonged, so as to confirm negative results, it is necessary microscopic examination of faeces with an interval of 1 week for 4-5 weeks.

A prototype of the proposed method, according to the authors, is a method for detection of antibodies to antigens of Giardia ELISA using the test system Giardia-at-strip” (JSC “Vector-best, Novosibirsk region, palzoo). The method is to detect serum antibodies to antigens Lamblia intestinalis. In the course of the study in the interaction of the studied samples of blood sera (dilution 1:100) in the holes tablets with immobilized antigens of Giardia is the binding of specific antibodies and the formation of complex antigen-antibody on the surface of the hole. After the removal is not bound peroxidase serum components and added to wells conjugate antibodies against human immunoglobulins with horseradish peroxidase is the inclusion of the enzyme label in the immune complex. As a result of enzymatic reactions carried out in the wells, washed of excess conjugate, substrate solution for peroxidase, hydrogen peroxide and ortof kilindini, formed colored product, the optical density of which is proportional to the concentration of antibodies in the test serum sample to antigens of Giardia. The optical density (OD) of labeled immune complex in the holes tablets register with the analyzer colorimetric immunoassay at a wavelength of 492 nm. In the tablet, in addition to working holes, which make the test serum, put controls: 2 holes contribute positive control sample, 2 wells negative control sample, 2 wells (control conjugate) solution for dilution of sera. The subsequent conduct of the reaction in the control wells not differ from that in the working hole. Assessment results hold only if the average OD of the control conjugate does not exceed the value of 0.2; OP in the wells with the control negative sample (OP-) not more than 0,3; and OP in the wells with positive control sample has a value of not lower than 0.8. Diagnostic OP (OPd) is calculated by the formula: OPd=1,48×OP-+0,04. The result of the ELISA test is considered positive if the OD labeled immune complex in the hole that used the investigated serum (OPstudies.) in dilution 1:100, equal to or more diagnostic OP (OPd). Because the error ELISA method extending t is 10-15% when the OD value studies.15%less than OPdthe result is considered equivocal and re-study.

The disadvantages of prototype method for the diagnosis of giardiasis include determining a presence of antibodies to antigens of Giardia (one dilution 1:100) - that is only a qualitative method of accounting for the results of ELISA, but not investigated the amount of specific antibodies (titre). Obvious in this situation the approach - the study investigated serum not one, but several dilutions, for example from 1:100 to 1:3200 with twofold dilutions, increases the cost of analysis is a multiple of the number of dilutions. In the absence of quantitative data in the prototype method, there is no possibility of assessing the severity of the immune response to the pathogen, the dynamics of the disease. In addition, it is impossible to evaluate the effectiveness of selected chemotherapy drugs and therapy of diseases in General.

The authors suggest the following method for the diagnosis and course of giardiasis. Setting ELISA for the presence of antibodies in the serum to antigens of Giardia using the test system Giardia-at-strip” is similar to the prototype. The OD is measured at the same wavelength. Calculation ODdheld on the above formula. However, to account for the result of the study calculates the difference between OPstudies.and OPdto area is multiplied by 100, with the aim of obtaining values of the concentration ratio of antibody to antigens of Giardia, which the authors propose to call the ratio of the concentration of the antibodies (ADC). The value of the ADC is proportional to the concentration of specific antibodies in the serum. ADC is calculated by the formula ADC=(OPstudies.OPd)×100, where 100 is the factor of dilution of serum.

Along with the definition of the coefficient of concentration of the antibodies (qualitative reaction in a single dilution of 1:100) in the study of blood sera for antibodies to antigens of Giardia using the test system Giardia-at-strip” authors conducted and the determination of the titer of antibodies (quantitative research). These parallel studies were conducted on 150 samples of blood sera of patients with complaints of the gastrointestinal tract, allergic diseases and indirect signs of giardiasis. The control group consisted of blood samples from 30 healthy individuals. The blood samples of the examined individuals in an amount not less than 0.5 ml were taken from a finger on an empty stomach. The serum was carefully separated from the clot of red blood cells and impurities. Serum was stored at a temperature of 6±2°C for no more than 48 hours prior to the study. The survey data processing was performed using the computer program statistical analysis 3.03 patch BIOSTAT (S.A.lantz, 1997), was used criterion linear regression and correlation.

In 94 samples of the experimental serum samples the ADC value from 101 to 205 units, and the antibody titers of 1:800-1:3200, the result is regarded as positive; in 24 samples experienced serum - ADC value from 11 to 100 conventional units and antibody titer of 1:200-1:400, the result is regarded as positive, as the patients had symptoms of gastro-intestinal tract, eosinophilia; in 14 patients with ADC from 11 to 100 conventional units, and the antibody titer of 1:200-1:400 the results of the study regarded as a carrier, as there were no symptoms of gastro-intestinal tract and eosinophilia; 18 samples experienced blood sera obtained the ADC value from 6 to 10 conventional units and a titer of 1:100, which is interpreted as equivocal result. In the control group in all samples of blood sera value ADC ranged from 0 to 5 conventional units, the antibody titer less than 1:100.

Obtained a statistically significant correlation between ADC and antibody levels has implications for the interpretation of research results: a negative result is questionable - carriage - positive result, which is necessary for the treating physician.

The proposed method for the diagnosis and course of giardiasis cost-effective, on the Kolka at the same cost for conducting EIA in a single dilution of serum gives a quantitative result (titer of specific antibodies), and not just the presence of these antibodies, which allows you to monitor the effectiveness of treatment, be used to diagnose the phenomenon of rise in antibody titer in paired sera.

Clinical example.

To s Paul, born in 1995 (history No. 2512) was admitted to the medical Department of the regional children's clinical hospital with complaints of pain in the abdomen, in the paraumbilical area, bloating, bad breath. History of frequent colds (up to two times per month) long lasting, prolonged fever (up to 37°C in the evening for three months). The examination found - underweight, pale skin, jaundice-cyanotic tint, pronounced periorbital shadows, cyanosis of nasolabial triangle. Posterior cervical lymph nodes increased to 1 cm in diameter, painless, Espanya surrounding tissues. Tongue thickly coated with white bloom. The abdomen is painful in the paraumbilical area, in the right hypochondrium and the projection point of the gall bladder. The liver acts from under the edge of the costal arch on 1 see Spleen not palpated. In the study of blood on presence of antibodies to antigens of Giardia using ELISA using the proposed method obtained the ratio of the concentration of the antibodies 202 conventional units, which corresponds to a titer of 1:3200 and is regarded as a positive result is at. Diagnosed with giardiasis assigned treatment tiberal, choleretic, enzyme preparations, herbal medicine. During the control study through month determined the concentration ratio of antibody 48 conventional units, which corresponds to a titer of 1:200 and was interpreted as a positive result, because the symptoms were still present, although expressed were weaker - cyanosis of nasolabial triangle was absent, periorbital shadows were poorly marked, the posterior cervical lymph nodes are enlarged to 0.3 cm in diameter, temperature is normal, according to authorities without features, appetite, weight gain of 1 kg, body weight deficit persists. Continued therapy choleretic, enzyme preparations, herbal medicine. Two months later, during the control study determined the concentration ratio of antibody 5 conventional units, which corresponds to the antibody titer less than 1:100 and was regarded as a negative result. The child had no complaints, skin normal color, lymph nodes did not even royalty, bodies without features, weight 3 kg. Thus, when the proposed method for the diagnosis and course of giardiasis in spite of a positive laboratory result for the presence of antibodies to antigens of Giardia after treatment tiberal would the and continued pathogenetic therapy without the use of antiparasitic drugs, because the antibody titer decreased by 16 times, which speaks to the effectiveness of antimicrobial therapy. - Control study, conducted over two months, confirmed the correctness of the chosen medical tactics - continuation of pathogenetic therapy without any additional prescription drugs with known side effects.

A method for the diagnosis of giardiasis and its trends, including enzyme-linked immunosorbent assay and determination of diagnostic optical density and the optical density of labeled immune complex in the hole of the tablet with the test serum, measured in arbitrary units at a wavelength of 492 nm, characterized in that the counting factor antibody concentrations measured in arbitrary units according to the formula: ADC = (Openssl - PDX)×100, where ADC is the coefficient of concentration of the antibodies, Openssl optical density of the wells with the test serum, PDX - diagnostic value of optical density, 100 - coefficient of dilution of the serum; and the value of the ADC to determine the titer of antibodies to antigens Lamblia intestinalis and interpret the results of the study: the ADC value from 0 to 5 conventional units corresponds antibody titer less than 1:100 and the result is regarded as negative, the ADC value from 6 to 10 conventional units corresponds to a titer of 1:100 and the ISS is adowanie considered questionable, the ADC values from 11 to 50 conventional units and from 51 to 100 conventional units comply with antibody titers of 1:200 and 1:400 respectively, and the result is regarded as positive with a diagnosis of giardiasis in the presence of symptoms of gastro-intestinal tract and eosinophilia or as carriers of Giardia in their absence, the ADC values from 101 to 135 conventional units, from 136 to 170 units, and from 171 to 205 conventional units comply with antibody titers of 1:800, 1:1600 and 1:3200, respectively, and the test result is considered positive.



 

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