In highly developed countries, more than 60% of acute gastroenteritis is caused by viruses from the Reoviridae family . Among them, rotaviruses dominate . The genus Rotavirus includes human rotaviruses and viruses that cause diarrhea in animals. The latter are non-pathogenic for humans. Human rotaviruses cause mainly gastroenteritis in children under three years of age and the elderly. The causative agent is transmitted by the fecal-oral route through household contacts. The high resistance of viruses in the environment contributes to the development of outbreaks of gastroenteritis, especially in winter (up to 90%). For protein antigens, rotaviruses are divided into 4 serovariants .
Material for laboratory diagnostics
The material for laboratory diagnosis is stool and blood. Fecal samples are taken in the first hours and days of illness in sterile penicillin vials and sent to the virology laboratory in ice containers. A 10-20% suspension in Hanks solution is prepared , centrifuged for 30 minutes at 3000 rpm. The centrifugate is transferred into a sterile vial, add Freon 113 or 1000 IU / ml of penicillin and 500 IU / ml of streptomycin, placed in the refrigerator for 10-12 hours. Blood for serological reactions is taken in the first 2-3 days of illness and after 12-14 days. Laboratory diagnosis of viral diarrhea is based on the rapid detection of viruses in the feces using electron and immune electron microscopy, an indirect hemagglutination reaction with antibody erythrocyte viral diagnostics . The enzyme-linked immunosorbent assay in the solid-phase variant is also used for the coaglutination reaction , the method of cloned RNA probes and the polymerase chain reaction. In the early days of the disease, the content of rotaviruses in copromaterials reaches 106-108 per 1 g, therefore, their detection by direct electron microscopy is possible without additional concentration. A drop of the test material is applied to special films of nitrocellulose and microscopic at a magnification of 50,000. The method is sensitive and reliable, allows you to quickly detect the virus and examine its morphology. If 5-6 virions are found in almost every field of view, the diagnosis of rotavirus gastroenteritis can be reliably confirmed . The method of immunoelectronic microscopy is of even greater diagnostic value . Its principle is that the immune antiviral serum is added to the fecal suspension illuminated by low-speed centrifugation, kept for 60 minutes at room temperature and another 12 hours at 4 ° C, then centrifuged at 15,000 rpm to precipitate viral particles and immune complexes. The precipitate is resuspended in 2-3 drops of distilled water, contrasted in phosphoric tungsten acid and examined under an electron microscope. Characteristic accumulations of rotaviruses are found in the preparations . The high efficiency of the method is practically independent of the initial concentration of viruses in the studied samples.
Virological studies to isolate rotaviruses in cell cultures or laboratory animals in routine virological laboratories are not carried out . The enzyme immunoassay is most commonly used in the diagnosis of viral gastroenteritis. Its principle is; that the wells of the polystyrene plate are first sensitized with antibodies against rotaviruses , and then an illuminated feces suspension is added to them, incubated for 1 hour in an incubator. An antigen-antibody complex is formed in the wells after washing three times with phosphate buffer and manifested by the addition of antiglobulin serum conjugated to the enzyme. After incubation, chromogenic substrates are added in a thermostat until a colored reaction product appears. Analysis is carried out using an enzyme-linked immunosorbent analyzer. In small laboratories, a reverse indirect hemagglutination reaction is often performed using an antibody rotavirus erythrocyte diagnosticum . It is easy by the method of formulation, although it is less sensitive than the reaction enzimmichenih antitel.V practice laboratory diagnosis of viral infections are also administered test koaglyutinatsii for indicating rotavirus antigen in the stool of patients. A drop of a suspension of staphylococci ( Cowan strain 1) sensitized with rotavirus serum is applied to a glass slide and a drop of centrifuged copromaterial is added . In the presence of rotaviruses , agglutination of staphylococci loaded with antibodies occurs after 30-60 s. A very sensitive way to quickly diagnose viral gastroenteritis is a method for detecting virus-specific RNAs using molecular hybridization. It is based on the hybridization of labeled RNA probes fixed on nitrocellulose filters. Labeling is carried out with biotin or an enzyme, which greatly simplifies and cheapens the method compared to radioisotope labels.
Rotavirus RNA Assay
The analysis of rotavirus RNA, despite its high cost, is increasingly being introduced into practice, especially after the development of the polymerase chain reaction method. The latter is a sensitive and specific diagnostic method, which, of course, is a new generation diagnostic test, the so-called “gold standard”. However, the formulation of this reaction still requires highly qualified researchers, expensive reagents and sophisticated equipment.
Serological diagnosis is aimed at identifying specific antibodies in paired sera taken at a two-week interval. For this purpose, various neutralization or precipitation tests and an indirect immunofluorescence reaction are used . But enzyme-linked immunosorbent assay has been widely used recently. Previously, for the serodiagnostics, CSCs were widely used using pre-selected feces of patients with gastroenteritis or calf rotaviruses as antigen . But in its sensitivity and specificity, it is significantly inferior to the method of enzyme immunoassay. To detect specific antibodies to rotaviruses, they also put inhibition of indirect hemagglutination with paired sera. Gastroenteritis in children can also cause caliciviruses , astroviruses and Norwalk viruses . All of them are excreted with feces in the first 2-3 days of the disease, do not reproduce in cell cultures or are poorly reproduced without cytopathic effect. For laboratory diagnosis of gastroenteritis caused by these viruses, mainly using the method of immune electron microscopy. There are no other diagnostic methods available for virology laboratories.