RU2348039C1 - Method for prediction of suppuration of unicameral echinococcus cyst in noninvasive children - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention is related to medicine and method for prediction of suppuration of unicameral echinococcus cyst in noninvasive children. Substance of invention includes extraction of DNA from lymphocytes of peripheral venous blood, performance of HLA-typing, if allele DQB1*02 is detected in genotype, risk of development of echinococcosis complications related to cyst suppuration is predicted in examined patient.

EFFECT: provides for prediction of suppuration of unicameral echinococcosis cyst with high accuracy.

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Description

The present invention relates to medicine, namely to pediatric surgery, and can be used to identify risk groups for the development of complications of single-chamber echinococcosis associated with suppuration of cysts in invasive children.

With echinococcosis, one of the most severe and widespread parasitic diseases, the clinical implementation of invasion, the occurrence of complications and relapses are still not fully understood. The frequency of deaths with echinococcosis is 0.9-8%. The most common cause of death are complications associated with suppuration and subsequent breakthrough of the cyst.

The mismatch between the level of echinococcus invasion in different species of potential hosts suggested genetic determinism in the development of invasion (Ozeretskovskaya NN Cestodoses-zoonoses - an urgent global problem. // Medical parasitol. And par. Diseases. 2001. P. 52-59) . A study in Europe showed an association of HLA B8, DR3, DQ2 haplotypes, and in China HLA DR4 with severe alveolar echinococcosis (Godot V, Harraga S, Beurton I., et al. Resistance / susceptibility to Echinococcus multilocularis infection and cytokine profile in people .II. Influence of the HLA B8, DR3, DQ2 haplotype. Clin Exp Immunol 2000. V.121. P.491-98). Determination of the frequency of distribution of HLA class 1 antigens showed a high incidence of B5 and B18 with hydatidosis (cystic, single chamber) and A1, B35 with alveolar echinococcosis (Scherbakov AM, Monche Barredo P.A. Distribution of HLA system antigens among patients with echinococcosis. // Honey parasitol. 1989. No. 6. P.75).

Thus, based on the detection of associations of the alleles of the genes of the main histocompatibility complex (HLA) with the development of invasion, a genetic predisposition to the development of a specific clinical form of echinococcosis was determined.

The authors in the medical and patent literature did not find information about the popularity of the method for predicting the complications of hydatid echinococcosis associated with suppuration of cysts in invasive children.

The clinical diagnosis of echinococcosis complicated by suppuration in children presents significant difficulties, since the picture of the disease most often differs by polymorphism and does not have clear differential diagnostic signs. Conventional laboratory tests in most cases are not very specific, the diagnosis of echinococcosis complicated by suppuration is established by instrumental methods of investigation (ultrasound, CT, X-ray) often in advanced cases. Timely diagnosis of complications is also important in determining the nature and methods of surgical intervention.

When studying the nature of the distribution of HLA alleles in patients with cystic echinococcosis of children living in the regions of the South Urals (Republic of Bashkortostan), it was found that with echinococcosis complicated by suppuration of the cyst, the DQB1 * 02 allele is more common than with the uncomplicated course of the disease (46.1% against 21.6% with an uncomplicated form of the disease; χ2 = 4.59; p = 0.03; OR = 3.10). The relative risk of developing complications of echinococcosis for individuals carrying this gene allele was RR = 2.21. The cause of suppuration of the cyst is considered the death of the larvocysts of Echinococcus granulosus due to incompatibility with HLA antigens (the phenomenon of genetic restriction).

Thus, genetic testing of HLA polymorphism in invaded echinococci, it is very likely (OR = 3.10) to predict the possibility of suppuration of the cyst before the onset of severe clinical symptoms of the disease.

The objective of the invention is to develop an objective, highly informative method for predicting the complications of cystic echinococcosis associated with suppuration of cysts in invaded children, based on the identification of molecular genetic markers. The method is available for reagents and equipment.

EFFECT: obtaining criteria for predicting the development of suppurative cysts in patients with cystic echinococcosis.

The method is as follows.

The material for the study is DNA samples isolated from peripheral venous blood lymphocytes in patients with echinococcosis of children by the phenol-chloroform extraction method described by Mathew S.S. (The isolation of high molecular weight eucariotic DNA // Methods in Molecular Biology / Ed. J. M. Walker. - New York., London. - 1984. - Vol.2. - P.31-34).

1. Blood in a test tube with a preservative is thoroughly mixed and poured into a 100 ml centrifuge beaker, 50 ml of chilled lysis buffer containing 320 mM sucrose, 1% Triton X-100 solution, 5 mM MgCl ₂ , 10 mM Tris HCl (pH) are added there. 7.6).

2. The mixture is centrifuged for 20 minutes at 4000 rpm.

3. The supernatant is drained, 8 ml of 25 mM EDTA, pH 8.0 are poured onto the resulting precipitate, and suspended.

4. To the suspension add 0.8 ml of 10% SDS and proteinase K (concentration - 10 mg / ml). The lysis mixture is left overnight in an incubator at a temperature of 38 ° C.

DNA extraction is carried out in the following order.

1. For deproteinization, 0.5 ml of 5M sodium perchlorate and 8 ml of phenol saturated with 1 M Tris HCl are added to the lysate to a pH of 7.8.

2. The mixture is centrifuged at 3000 rpm for 10 minutes.

3. Select the aqueous phase containing DNA, RNA and non-denatured proteins.

4. The selected phase is treated with a mixture of phenol-chloroform (1: 1), and then with chloroform.

5. The preparations are precipitated with two volumes of 96% ethanol.

6. The resulting DNA precipitate is dissolved in 1.5 ml of deionized H ₂ O; the solution is stored at t-20 ° C.

Subsequently, the obtained DNA is used as a polymerase chain reaction (PCR) template for amplification of the desired fragment.

A standard set of reagents for typing the HLA DQB 1 gene (PROTRANS, Germany) is used. The specificity of the DQB1 gene was amplified in a reaction mixture containing 35 μl of genomic DNA (concentration of 50-300 ng / μl), 1.1 μl of Taq polymerase (5 U / μl), 46 μl of R buffer, 93 μl of Y buffer. Reaction mixture 10 μl are distributed into 16 eppendorfs, on the bottom of which oligonucleotide primers are applied. The amplification mode is as follows: initial denaturation at 94 ° C for 2 min; 10 cycles, including denaturation at 94 ° С - 10 sec, annealing of primers and DNA synthesis at 65 ° С - 1 min; 20 cycles, including denaturation at 94 ° С - 10 sec, annealing of primers at 61 ° С - 50 sec; DNA synthesis at 72 ° C for 30 sec. After amplification, DNA fragments are separated electrophoretically in a 1% agarose gel, stained with ethidium bromide solution and analyzed in transmitted ultraviolet light on a transilluminator. As an electrolyte for electrophoresis, 0.5X borate buffer (0.089 M TrisHCl, pH 7.9; 0.089 M boric acid, 0.002 EDTA with pH 8.0) is used. The amplification product is visible as a luminous strip of red-orange color.

The identified alleles of the HLA DQB1 gene are classified according to the generally accepted nomenclature (Nomenclature for factors of the HLA system, 1995 / JGBodmer, SGEMarsh, TDAlbert et al. // Hum. Immunol. 1995. V.43. P.149-164 )

To quantify the predisposition to the development of echinococcosis, the odds ratio (OR) is calculated for the above genetic marker. To do this, use the formula proposed by Bland (Bland, J.M. The odds ratio / J.M. Bland, D.G. Altman // Br. Med. J. - 2000. - Vol. 320. - P.1468):

OR = (a × d) / (b × c),

where a is the number of individuals with, b is the absence of a marker (HLA allele) among patients with complicated echinococcosis; c and d are the number of individuals, respectively, with the presence and absence of a marker among individuals with uncomplicated course of the disease. An increased risk of development was observed at OR> 1.

We examined children (N = 100), patients with cystic echinococcosis, who received surgical treatment at the Republican Children's Clinical Hospital (Ufa). The age of patients is from 3 to 16 years. The main share of the examined individuals was children of school age (71%). The distribution by gender was as follows: 57% boys, 43% girls. Liver damage was observed in 47% of patients, lung - 30%, combined lung and liver damage - 23%. The diagnosis was established on the basis of clinical, instrumental (ultrasound, CT, radiography) methods and was confirmed after surgery by histological examination of the cyst membrane. The control group consisted of 95 healthy individuals, residents of the southern Urals. The age of individuals in the control group ranged from 5 to 16 years. The groups of examined patients and controls were comparable by age, gender and place of residence.

A study of the distribution pattern of HLA DQB 1 alleles in patients depending on the clinical form and severity of the disease showed statistically significant differences. It was found that the most common (46.1%) in the group of patients with complicated (suppuration of the cyst) echinococcosis is the allele DQB1 * 02; in the group of patients with uncomplicated course, this allele was detected in 21.6% of cases, that is, more than 2 times less likely (χ2 = 4.59; p = 0.03; OR = 3.10). The allele DQB1 * 07 (3.8% versus 18.9% in the uncomplicated form of the disease; p = 0.12) is most rare in suppurative echinococcosis.

The results show that the presence of the HLA allele DQB1 * 02 in the genotype is a predisposing factor to the development of cystic echinococcosis complicated by suppuration of the cyst in children, therefore it can be used as a genetic risk marker. The determination of prognostic markers is of great practical importance, since suppurative echinococcosis is characterized by the spread of the inflammatory response from the cyst to the adjacent tissue of the affected organ, and there is a need for intensive pre- and postoperative conservative therapy to prevent cirrhotic changes.

Example 1. Girl Z., 9 years old, was admitted to the surgical department with complaints of general weakness, weight loss, temperature up to 38 ° C, pain in the right hypochondrium.

Objectively: satisfactory condition; skin and visible mucous membranes are pale. In the right hypochondrium pain is determined, the abdomen is not swollen, soft. The liver protrudes from the edge of the costal arch by 2 cm. In the lungs, vesicular breathing. Heart sounds are rhythmic, muffled, pulse 98 per minute. Blood test: er - 3.4 × 10 ¹² / l, l - 10.5 × 10 ⁹ / l, e - 3%, s - 48%, lph - 32%, m - 9%, ESR - 29 mm / hour. Ultrasound of the abdominal organs revealed a fluid volumetric formation of a round shape located in the III segment of the liver, size 39 × 34 mm with clear even contours, hyperechoic inclusions inside and suspension.

A study on the proposed methodology. Found: is a carrier of the HLA allele DQB1 * 02. In relation to suppuration of an echinococcal cyst, the prognosis is poor. Emergency surgery and intensive pre- and postoperative conservative therapy is recommended to prevent cirrhotic changes.

Laparoscopic echinococcectomy was performed. A turbid parasitic fluid was revealed in the cavity of the cyst. Histological examination of the chitinous membrane revealed inflammatory changes.

Example 2. Boy R., 11 years old, was admitted to the surgical department with complaints of abdominal pain. After hospitalization and examination, the diagnosis was made: echinococcal cyst of the right lobe of the liver. The ultrasound data revealed in the right lobe (6-7 segments) an oval-shaped fluid formation with clear even contours, 78 × 53 mm in size. Before the operation, a study was carried out according to the proposed technique. Found: is a carrier of the HLA allele DQB1 * 07. In relation to the development of suppuration of the cyst, the prognosis is favorable. Postoperative revision of the cyst showed no signs of suppuration.

Claims (1)

A method for predicting suppuration of a single-chamber echinococcal cyst in invasive children, characterized in that DNA is isolated from peripheral venous blood lymphocytes, HLA typing is performed, and if a DQB1 * 02 allele is detected, the risk of developing suppuration of the cyst in the subject is predicted.