Lesson N 3: LABORATORY DIAGNOSIS OF MENINGOCOCCAL AND GONOCOCCAL INFECTION

1.Scientifically methodical ground of theme Neisseriacae are Gram-negative diplococci. Although difficult to differentiate on morphological and cultural character, these two pathogens are associated with entirely different diseases. Neisseria meningitidis is the cause of a range of diseases, of which acute purulent meningitis (variously called epidemic cerebrospinal meningitis, cerebrospinal fever or, because of a purpuric rush is sometimes present, spotted fever) and an acute septicemic illness with a petechial rash but without meningitis are commonest. About one-third of cases of meningococcal disease present as septicemia, most other being of meningiti. The term meningococcal infection embraces these and other syndromes associated with the organism. N. gonorrhoeae is the cause of sexually transmitted disease gonorrhea. In the newborn may give rise to a purulent conjunctivitis, ophthalmia neonatorum and in young girls a vulvovaginitis.

2.Educational purposeSTUDENTS MUST KNOW:1. Structure, cultivation and biological properties of meningococci. 2. Antigenic structure, serogroups and serovars of meningococci.3. Epidemiology of diseases. The clinical forms of meningococcal infections, pathogenesis.

Immunity.4. Laboratory diagnosis of meningococcal infections.5. Specific prophylaxis and therapy of meningococcal diseases.6. Structure, cultivation and biological properties of gonococci.7. Pathogenesis and diseases in man. Immunity.8. Laboratory diagnosis of gonococcal infections.9. Prophylaxis and treatments of gonorrhea.STUDENTS SHOULD BE ABLE TO:– read the result of complement fixation test for serological diagnosis of gonorrhea; – make smear and stain it by Gram’s and Neisser’s method;– value incomplete phagocytosis of gonococci and memingococci;– value of microbial growth on different media;– carry out determining of microbial sensitivity to antibiotics;– create the schemas of diagnosis of meningococcal and gonococcal infections.

3.Chart of topic content.Meningococci. The meningococcus (Neisseria meningitidis) was isolated from the cerebrospinal

fluid of patients with meningitis and studied in detail in 1887 by A. Weichselbaum. At present the organism is classified in the genus Neisseria, family Neisseriaceae.Morphology. The meningococcus is a coccus 0.6-1 mcm in diameter, resembling a coffee bean, and is found in pairs The organism is Gram-negative. As distinct from pneumococci, meningococci are joined longitudinally by their concave edges while their external sides are convex.

Spores, capsules and flagella are not formed. In pure cultures meningococci occur as tetrads (in fours) and in pus they are usually found within and less frequently outside the leukocytes In culture smears, small or very large cocci are seen singly, in pairs, or in foursCultivation. The meningococcus is an aerobe or facultative anaerobe and does not grow on common media. It grows readily at pH 7.2-7.4 on media to which serum or ascitic fluid has been added. Optimum temperature for growth is 36-37 C and there is no growth at 22° C. Growth is facilitated by 5-10 per cent CO2 and high humidity. On solid media the organisms form fine transparent colonies measuring 2-3 mm in diameter. In serum broth they produce turbidity and a precipitate at the bottom of the test tube, and after 3-4 day's, a pellicle is formed on the surface of the medium. Meningococci can be adapted to simple media by repeated subculture on media with a gradual change from the optimum protein concentration to media containing a minimal concentration of proteins.

Fermentative properties. Meningococci do not liquefy gelatin, cause no change in milk, and ferment glucose and maltose, with acid formation.

Virulenсe factors. Meningococci have 3 important virulence factors: polysaccharide capsule that enables organism to resist phagocytosis, endotoxin, which causes fever, shock, and other, and immunoglobulin A(Ig A) protease, which , by cleaving sescretory Ig A, helps the bacteria to attach to the membranes of respiratory tract.

Antigenic structure and classification. Meningococci were found to contain three fractions: carbohydrate (C) which is common to all meningococci, protein (P) which is found in gonococci and type

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III S. pneumoniae, and a third fraction with which the specificity of meningococci is associated. According to the International Classification, four groups of meningococci are distinguished, groups A, B, C, and D. Recently the number of types has increased to seven, but only the first two are dominant.

The organisms are characterized by intraspecies variability. A change of types takes place at certain times.

Resistance. The meningococcus is a microbe of low stability, and is destroyed by drying in a few hours. By heating to a temperature of 60° C it is killed in 10 minutes, and to 80 C, in 2 minutes. When treated with 1 per cent phenol, the culture dies in 1 minute. The organism is very sensitive to low temperatures. Bearing this in mind, test material should be transported under conditions which protect the meningococcus against cooling.

Pathogenesis and diseases in man. People suffering from meningococcal infection and carriers are sources of diseases. The infection is transmitted by the air-droplet route. The causative agent is localized primarily in the nasopharynx. From here it invades the lymph vessels and blood and causes the development of bacteriemia. Then as a result of metastasis the meningococci pass into the meninges and produce acute pyogenic inflammation in the membranes of the brain and spinal cord (nasopharyngitis, meningococcaemia, meningitis).

The disease usually arises suddenly with high temperature, vomiting, rigidity of the occipital muscles, severe headache, and increased skin sensitivity. Later paresis of the cranial nerves develops due to an increase in the intracranial pressure. Dilatation of the pupils, disturbances of accommodation, as well as other symptoms appear. A large number of leukocytes are present in the cerebrospinal fluid, and the latter after puncture escapes with a spurt because of the high pressure.

In some cases meningococcal sepsis develops. In such conditions the organisms are found in the blood, joints, and lungs. The disease mainly attacks children from 1 to 5 years of age. Before the use of antibiotics and sulphonamides the death rate was very high (30-60 per cent).

The population density plays an important part in the spread of meningitis. During epidemic outbreaks there is a large number of carriers for every individual affected by the disease. In non-epidemic periods the carrier rate increases in the spring and autumn. Body resistance and the amount and virulence of the causative agent are significant. Depending on these factors, the spread of infection is either sporadic or epidemic.

Meningitis can also be caused by other pathogenic microbes (streptococci, E. coli, staphylococci, bacteria of influenza, mycobacteria of tuberculosis, and certain viruses). These organisms, however, cause sporadic outbreaks of the disease, while meningococci may cause epidemic meningitis.

Immunity. There is a well-developed natural immunity in humans. Acquired immunity is obtained not only as a result of the disease but also as the result of natural immunity developed during the meningococcal carrier state. In the course of the disease agglutinins, precipitins, opsonins, and complement-fixing antibodies are produced. Recurring infections are rare.

Laboratory diagnosis. Specimens of cerebrospinal fluid, nasopharyngeal discharge, blood, and organs obtained at autopsy are used for examination.

The following methods of investigation are employed: (1) microscopic examination of cerebrospinal fluid precipitate; (2) inoculation of this precipitate, blood or nasopharyngeal discharge into ascitic broth, blood agar, or ascitic agar; identification of the isolated cultures by their fermentative and serologic properties; differentiation of meningococci from the catarrhal micrococcus (Branhamella catarrhalis) and saprophytes normally present in the throat. The meningococcus ferments glucose and maltose, whereas Branhamella catarrhalis does not ferment carbohydrates, and Neisseria sicca ferments glucose, levulose, and maltose; (3) performance of the precipitin reaction with the cerebrospinal fluid.

Treatment. Antibiotics (penicillin, oxytetracycline, etc.) and sulphonamides (streptocid, methylsulphazine) are prescribed.

Prophylaxis is ensured by general sanitary procedures and epidemic control measures (early diagnosis, transference of patients to hospital), appropriate sanitary measures in relation to carriers, quarantine in children's institutions. Observance of hygiene in factories, institutions public premises, and lodgings, and prevention of crowded condition are also obligatory. An antimeningococcal vaccine derived from the C/B serogroup is now under test. It contains specific polysaccharides.

The incidence of meningitis has grown recently. The disease follows a severe course and sometimes terminates in death.

Gonococci. The causative agent of gonorrhoea and blennorrhoea (Neisseria gonorrhoeae) was discovered in 1879 by A. Neisser in suppurative discharges. In 1885 E. Bumm isolated a pure culture of the organism and studied it in detail. Gonococci belong to the genus Neisseria, familyNeisseriaceae.

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Morphology. Gonococci are morphologically similar to meningococci. The organism is a paired, bean-shaped coccus, measuring 0.6-1 mcm in diameter. It is Gram-negative and occurs inside and outside of the cells. Neither spores nor flagella are formed. Under the electron microscope a cell wall, 0.3-0.4 mcm in thickness, surrounding the gonococci is visible. Pleomorphism of the gonococci is a characteristic property. They readily change their form under the effect of medicines, losing their typical shape, and growing larger, sometimes turning Gram-positive, and are found outside the cells.

In chronic forms of the disease autolysis of the gonococci takes place with formation of variant types (Asch types). Usually gonococcal cells varying in size and shape are formed. The tendency toward morphological variability among the gonococci should be taken into account in laboratory diagnosis. L-forms occur under the effect of penicillin.

Cultivation. The gonococcus is an aerobe or facultative anaerobe which does not grow on ordinary media, but can be cultivated readily on media containing human proteins (blood, serum, ascitic fluid) when the pH of the media is in the range of 7.2-7.6. The optimum temperature for growth is 37° C, and the organism does not grow at 25 and 42° C. It is essential to provide 5-10 per cent CO2.It also requires an adequate degree of humidity. Ascitic agar, ascitic broth, and egg-yolk medium are the most suitable media. On solid media gonococci produce transparent, circular colonies, 1-3 mm in diameter . Cultures of gonococci form a pellicle in ascitic broth, which in a few days settles at the bottom of the test tube.

Fermentative properties. The gonococcus possesses low biochemical activity and no proteolytic activity. It ferments only glucose, with acid formation.

Virulenсe factors The gonococci do not produce soluble toxin (exotoxin). An endotoxin is released as a result of disintegration of the bacterial cells. This endotoxin is also toxic for experimental animals. Pili are most of the important virulence factors, because they mediate attachment to mucosal cel surface and are antiphagocytic. Piliated gonococci are usually virulent, whereas nonpiliated strains are avirulent. Ig A protease can hydrolyze secretory Ig A, which could otherwise block attachment to the mucosa. Gonococci have no capsules.

Antigenic structure and classification. The antigenic structure of gonococci is associated with the protein (O-antigen) and polysaccharide (K-antigen) fractions. No group specific or international types of gonococci have been revealed. Gonococci and meningococci share some antigens in common.

Resistance. Gonococci are very sensitive to cooling. They do not survive drying, although they may live as long as 24 hours in a thick layer of pus or on moist objects. They are killed in 5 minutes at a temperature of 56 °C, and in several minutes after treatment with a 1 : 1000 silver nitrate solution or 1 per cent phenol.

Pathogenesis and diseases in man. Patients with gonorrhoea are sources of the infection. The disease is transmitted via the genital organs and by articles of domestic use (diapers, sponges, towels, etc). The causative agent enters the body via the urethral mucous membranes and, in women, via the urethra and cervix uteri. Gonorrhoea is accompanied by acute pyogenic inflammation of the urethra, cervix uteri, and glands in the lower genital tract. Often, however, the upper genito-urinary organs are also involved. Inflammations of the uterus, uterine tubes, and ovaries occur in women, vulvovaginitis occurs in girls, and inflammation of the seminal vesicles and prostata in men. The disease may assume a chronic course. From the cervix uteri the gonococci can penetrate into the rectum. Inefficient treatment leads to affections of the joints and endocardium, and to septicaemia. Gonococci and Trichomonas vaginalis are often found at the same time in sick females. The trichomonads contain (in the phagosomes) gonococci protected by membranes against the effect of therapeutic agents. Gonococcus is responsible for gonorrhoeal conjunctivitis and blennorrhea in adults and newborn infants.

Immunity. The disease does not produce insusceptibility and there is no congenital immunity. Antibodies (agglutinins, precipitins, opsonins, and complement-fixing bodies) are present in patients' sera, but they do not protect the body from reinfection and recurrence of symptoms. Phagocytosis in gonorrhoea is incomplete. The phagocytic and humoral immunity produced in gonorrhoea is incapable of providing complete protection, so, in view of this fact, treatment includes measures which increase body reactivity. This is achieved by raising the patient's temperature artificially.

Laboratory diagnosis. Specimens for microscopic examination are obtained from the discharge of the urethra, vagina, vulva, cervix uteri, prostate, rectal mucous membrane, and conjunctiva. The sperm and urine precipitates and filaments are also studied microscopically, Smears are stained by Gram's method and with methylene blue by Loeffler's method). Microscopy is quite frequently an unreliable diagnostic method since other Gram-negative bacteria, identical to the gonococci, may be present in the material under test. Most specific are the immuno-fluorescence methods (both direct and indirect). In the

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direct method the organisms under test are exposed to the action of fluorescent antibodies specific to gonococci. In the indirect method, the known organisms (gonococci) are treated with patient's serum. The combination of the antibody with the antigen becomes visible when fluorescent antiserum is added.

If diagnosis cannot be made by microscopic examination, isolation of the culture is carried out. For this purpose the test material (pus, conjunctival discharge, urine precipitate, etc.) is inoculated onto media. The Bordeux-Gengou complement-fixation reaction and the allergic test are employed in chronic and complicated cases of gonorrhea.

Treatment. Patients with gonorrhoea are prescribed antibiotics (bicillin-6, ampicillin, monomycin, kanamycin) and sulphonamides of a prolonged action. Injections of polyvalent vaccine and autovaccine as well as pyrotherapy (introduction of heterologous proteins) are applied in complicated cases.

Improper treatment renders the gonococci drug-resistant, and this may lead to the development of complications and to a chronic course of the disease.

Prophylaxis includes systematic precautions for establishing normal conditions of everyday and family life, health education and improvement of the general cultural and hygienic standards of the population.

In the control of gonorrhoea great importance is assigned to early exposure of sources of infection and contacts and to successful treatment of patients.

The prevention of blennorrhea is effected by introducing one or two drops of a 2 per cent silver nitrate solution into the conjunctival sac of all newborn infants. In certain cases (in prematurely born infants) silver nitrate gives no positive result. Good results are obtained by introducing two drops of a 3 per cent penicillin solution in oil into the conjunctival sac. The gonococci are killed in 15-30 minutes.

In spite of the use of effective antibiotics the incidence of gonorrhoea tends to be on the increase in all countries (Africa, America, South-Eastern Asia, Europe, etc.). The number of complications has also increased: gonococcal ophthalmia of newborn infants (blennorrhea), vulvovaginitis in children, and inflammation of the pelvic organs (salpingitis) and sterility in women. The rise in the incidence of gonorrhoea is caused by social habits (prostitution, homosexualism, etc.), inefficient registration of individuals harbouring the disease, deficient treatment, and the appearance of gonococci resistant to the drugs used.

The WHO expert committee has recommended listing the gonococcal infection among infectious diseases with compulsory registration and making a profound study of the cause of the epidemic character of gonococcal diseases in certain African countries. Stricter blennorrhea control measures, and elaboration of uniform criteria of clinical and laboratory diagnosis, and treatment of gonococcal infection and more efficient methods for determining the sensitivity of circulating gonococci to various drugs are also recommended by the committee.4. Student’s independent study program

1. Structure and biochemical properties of meningococci. Cultivation. Virulenсe factors and pathogenicity of causative agents. Resistance in an external environment.

2. Characterize antigenic structure, serogroups and serovars of meningococci.3. Epidemiology of diseases. The clinical forms of meningococcal infections, pathogenesis.

Immunity.4. Laboratory diagnosis of epidemic cerebrospinal meningitis: a – describe the features of receipt of material for examination;b – microscopical method of diagnosis; c – characteristic of main stages of bacteriological method of diagnosis; d – role of serological tests (precipitation test, counter immunoelectrophoresis, radioimmune

methods) as express methods of diagnosis of epidemic cerebrospinal meningitis.6. Specific prophylaxis and therapy of meningococcal diseases.7. Structure, staining, biochemical properties of gonococci. Cultivation.8. Virulence factors of gonococci and mechanism of their penetration into organism.9. Resistance of gonococci.10. Pathogenesis and diseases in man. Immunity.11. Bacterioscopic method of diagnosis of gonorrhea. The method of receipt of tested material.12. Value of bacteriological method of diagnosis of gonorrhea.13. Diagnostic value of a Bordeux–Gengou test.14. Main methods of provocations in diagnosis and treatment of gonorrhea.15. Prophylaxis and treatments of gonorrhea

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5. Students Practical activities:

1. To study morphology of meningococci and gonococci in museum smears (incomplete phagocytosis). 2. To study peculiarities of gonococci and meningococci growth on nutrient media.

3. To familiarize with biological preparations, which are used for diagnosis, specific prophylaxis and treatment of gonococcal and meningococcal diseases.

4. To describe the features of growth of bacteria on blood agar, make smear, stain it by Gram’s technique, create scheme of identification of isolated streptococci culture.

5. To continue diagnostic of staphilococcal infection. Examine bacterial growth in MPA. Subcultivate culture in Hiss’s media, blood agar and plasma. Start determining of isolated culture sensitivity to antibiotics and phagotype.

MENINGOCOCCAL INFECTION. Meningococcal infection is caused by meningococci (Neisseria meningitidis). The material to be tested is secretions from the nasal portion of the throat, cerebrospinal fluid, blood, and scrapings from elements of the haemorrhagic rash on the skin.

Cerebrospinal fluid is collected into a sterile tube to be inoculated onto nutrient media or to be promptly sent (without allowing it to cool down) to the laboratory. This requirement is necessitated by the fact that meningococci are very sensitive to temperature fluctuations.

Mucosal secretions in the nasal portion of the throat are collected with a special swab bent at a definite angle. The best results are obtained when the nasopharyngeal mucus is immediately streaked onto solid nutrient media. To achieve the maximal separation of bacterial cells, 2-3 plates with medium are utilized. If the material is to be studied 3-5 hrs after the collection, it is inoculated onto a liquid nutrient medium (casein hydrolysate of fermentative splitting, which contains 1.5 g/1 of amine nitrogen and 250 U/ml of ristomycin) and then placed in a 37 C water bath. Thereafter, it is streaked onto serum agar and placed into an incubator.

Bacterioscopic examination of cerebrospinal fluid and blood permits detection of the causative agent. If the cerebrospinal fluid looks like pus, smears are prepared without its preliminary treatment whereas in the presence of only mild turbidity the cerebrospinal fluid is centrifuged and the deposit is used to make smears. The latter are stained with aniline dyes (aqueous solution of basic fuchsine, methylene blue) since the Gram staining method is associated with alteration in the formed elements of the cerebrospinal fluid and a large number of artefacts. Meningococci appear as bean-shaped diplococci situated within the leukocyte cytoplasm and touching each other with concave edges. A tender capsule is quite a frequent finding. In meningococcaemia meningococci may be demonstrated in blood smears. A thick-drop (film) preparation is made, stained for 2-3 min with aqueous solution of methylene blue without fixation, washed in tap water, and dried in the air. On a light blue background of the preparation one can see dark blue leukocytes with numerous small dark-blue cocci arranged in clusters, pairs, and singly in and around leukocytes.

Rapid diagnosis is performed by means of gel precipitation, counter-immunoelectrophoresis with group precipitating antisera or radioimmunoassay and based on the detection in the patient's cerebro-spinal fluid or blood of the specific meningococcal antigen.

Bacteriological examination. The cerebrospinal fluid or its sediment is cultured simultaneously with conducting bacterioscopic study. The meningococcus grows on special nutrient media containing native protein (serum broth and agar). One can also use Hottinger's agar containing 0.15 per cent of insoluble starch, which does not change the cultural, fermentative, and agglutinating properties of the causative agent. It is preferable that the cerebrospinal fluid be cultured after centrifugation at 3500 X g for five minutes. Some 0.3-0.5 ml of the material is taken from the bottom and 2-3 drops are placed on the surface of heated nutrient medium. The inoculated culture is incubated at 37 °C and in conditions of elevated CO2 contents. To do it, place onto the lid of a sterile Petri dish a sheet of filter paper soaked with 1.5-2.0 ml of 10 per cent pyrogallic acid and then cover it with a second sheet moistened with 1.5-2.0 ml of 20 per cent solution of sodium hydrocarbonate. The inoculated dish is covered with the lid containing the paper sheets and inverted (lid downward). The remainder of the cerebrospinal fluid is utilized for counter-immunoelectrophoresis.

On the second day of incubation at 37 °C, the growth is studied for its cultural properties. Meningococci form small, round, convex, and transparent colonies. Smears made of these colonies display polymorphic diplococci and tetracocci. The microscopic picture is so diverse that it creates the impression of unpure culture. The colonies are subcultured onto a serum agar slant.

On the third day of investigation, the isolated culture is agglutinated with meningococcal sera.

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Prior to the use of sulpha nil amide drugs and antibiotics, it is necessary to determine the serovar of the meningococcus responsible for the disease since treatment is based on specific meningococcal sera. The agglutination test in Noble's modification is currently employed for determining the meningococcal serovar with an epidemiological purpose. Three-drop portions of thick suspension of microorganisms are poured into three test tubes, then three-drop aliquots of undiluted or diluted 1:10 meningococcal serum of A, B, and C serovars are added to them. The mixture is shaken for 2-4 min, then 10-20 drops of isotonic sodium chloride solution are added to each test tube, and the results are read.

To assay the fermentative activity of pure culture, it is transferred to media with lactose, glucose, maltose, sucrose, and fructose. Meningococci ferment glucose and maltose with the production of acid. The culture is also streaked onto a 5 per cent yolk agar and serum agar containing 5 per cent sugar. After a 48-hour incubation, 1 drop of Lugol's solution is put on the surface of the grown colonies. The appearance of brownish staining indicates polysaccharide splitting. Neisseria are identified by the oxidase test which consists in the following. On the colony formed on the serum agar place a drop of the freshly-prepared 1 per cent solution of hydrochloric paradiethylphenylendiamine. As a result, colonies possessing oxidase activity turn pink and then black. Such colonies are transferred to a serum agar for further investigation.

To differentiate between the meningococcus and non-pathogenic Neisseria (Neisseria catarrhalis), the ability of the latter to grow on simple nutrient media and to form colonies at room temperature (22 °C) is utilized.

To demonstrate the meningococcus in the blood, introduce 5-10 ml of blood obtained from a vein under sterile conditions into vials with 50 ml of broth containing 0.1 per cent of agar-agar. Subculture onto a serum agar 24 hours later. The procedures of isolation and identification of the cultures are the same as in the examination of cerebrospinal fluid.

Indirect haemagglutination with erythrocytes sensitized with group-specific polysaccharides is employed for serological diagnosis.

GONOCOCCAL INFECTION. The causative agent of gonorrhoea is the gonococcus (Neisseria gonorrhoeae), which is morphologically similar to the meningococcus. Bacterioscopic, bacteriological, and serological techniques are employed for the diagnosis of this disease.

Bacterioscopic examination is the main method for diagnosing acute gonorrhoea and blennorrhea. The material for examination is taken from the urethra in the following manner: wipe the urethral opening with cotton wool moistened with sterile physiological salt solution, press with your finger onto the posterior wall of the urethra in the outward direction (in females the forefinger is inserted into the vagina for this purpose), and express a drop of pus. The secretion from the prostate is obtained by prostatic massage. The secretion of the cervical mucosa is collected with a swab, following intravaginal introduction of Cusco's speculum. In patients with blennorrhea conjunctival secretion is removed with a loop and spread over a glass slide. The preparation is stained with alkaline solution of methylene blue and with the Gram stain (two smears). Upon microscopic examination gonococci appear as bean-shaped Gram-negative diplococci positioned outside or inside the cells (neutrophilic granulocytes) similar to meningococci.

Gram's staining allows differentiation of the gonococci from other bacteria. To ensure a more distinct outline of the gonococci, smears should be fixed by dimethylsulphoxide (dimexide). Pour dimexide on the smear until it is completely dry and then stain it.

Since the examined material may also contain other Gram-negative bacteria resembling the gonococci, both direct and indirect immunofluorescence methods are employed. In the direct immunofluorescence test the smears are treated with fluorescent antibodies against gonococci, in the indirect one, gonococci and the patient's serum are used. Conjugation between the antibody and the antigen becomes evident when a fluorescent serum against human globulins is added.

Bacteriological examination is carried out when the study of smears reveals either no gonococci or only their atypical, altered forms. In view of extreme sensitivity-of the gonococcus to temperature the material tested should not be transported. Moreover, the gonococcus is very sensitive to disinfectants, so it is advisable that 1 to 2 days before culturing the patients should temporarily discontinue the use of disinfectants and antibacterial drugs.

The material is inoculated immediately after its collection onto plates with a protein-containing meat-peptone agar. Ascitic-free media with casein digest, yeast autolysate, and native cattle serum are widely utilized for this purpose. Inclusion into the nutrient medium of ristomycin and poIymixin M (10 U/ml) significantly enhances gonococcal growth. Prior to inoculation, the nutrient medium should be

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heated in an incubator. To facilitate better growth of the gonococci, the inoculated plates are placed into an exsiccator with a CO2 concentration amounting to 10 per cent.

A 24-hour incubation at 37 °C brings about the formation of transparent, with smooth edges, convex, mucoid colonies of the gonococcus, which resemble drops of dew. Pure culture is isolated and identified. Biochemically, the gonococcus shows weak activity and breaks down only glucose with the formation of acid. To determine oxidase activity, the culture is introduced into yolk medium (to 100 ml of protein-containing meat-peptone agar add 1.5 g of glucose, 6 ml of phenol red solution, and 15 ml of egg yolk). Agglutination with specific serum does not always yield positive results because the gonococcus has many serovars and the serum may contain only low titres of the appropriate agglutinins.

Serological diagnosis is resorted to in chronic gonorrhoea when the patient has no discharge, and bacterioscopic and bacteriological examinations are impossible. In such cases the complement-fixation test with the patient's blood serum or indirect immunofluorescence is used. A gonococcal vaccine or a special antigen prepared of killed (by variable methods, with antiformin being the most common one) gonococci is employed as the antigen.

6.Control questions and test:Choose the correct answers: 1. Meningococci have such properties: a – gram-negative; b – lancet-shaped form; c – tetracocci; d

– diplococci; e – stain with methylene blue. 2. Cultivation of meningococci: a – grow on МPA and in МPB; b – require the presence of

carbohydrates; c – grow on media with serum; d – give good growth on Endo’s and Levin’s media; e – on agar with serum form fine transparent colonies.

3. For differentiation between meningococci and gonococci such tests are used: a – fermentation of lactose b – fermentation of glucose and maltose; c – morphological properties; d – motility; e – hemolysis test. 4. Antigenic structure of meningococci and their classification: a – they are classified into serogroups A, B, C, D, Х; b – determination of serovars is necessary for treatment of the patients; c – most strains from outbreaks are A, B, C serovars; d – determination of serovars is necessary for epidemiological analysis; e – serovars are determined by surface antigens.

5. Choose diseases, which are caused by meningococci: a – epidemic cerebrospinal meningitis; b– nasopharyngitis; c – furunculosis; d – meningococcaemia, e-pneumonia.

6. Methods of laboratory diagnosis of meningococcal infections: a – bacteriological; b – serological; c – allergic; d – hemolysis test; e – inoculation of yolk salt agar.

7. In bacteriological diagnosis of an epidemic cerebrospinal meningitis such material is used: a – lavage waters of the stomach; b – liquor; c – samples of washings from skin surface; d – vomitive masses; e – urine.

8. Main methods of diagnosis of epidemic cerebrospinal meningitis: a – biological; b – allergic; c – bacteriological; d – precipitation test; д – Coombs’ test.

9. Gonococci have such properties: a – paired bean-shaped cocci; b – lancet-shaped cocci; c – gram-positive; d – gram-negative; e – have fimbriae.

10. Cultivation and biochemical properties of gonococci: а – require special media; b – are cultivated on serumal and ascitic media; c – are cultivated on sugar MPA; d – fermentate glucose; e – fermentate glucose and maltose. 7. List of literature: 1. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, part 2, Lugansk,

2004,Chapter16, p.20-28.

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