LessonN2: LABORATORY DIAGNOSIS OF STREPTOCOCCAL INFECTIONS1.Scientifically methodical ground of themeStreptococci produce ...
(3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causesnecrosis in...
develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar theyform small,...
microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and itsidenti...
while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negativereact...
No streptococci, streptococcal antigen or antibodies to them are found in some patients and penicillin therapyproves ineff...
8. Laboratory diagnostics of streptococcal and pneumococcal diseases.        5. Students Practical activities:        1. T...
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Bohomolets Microbiology Lesson #2

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Bohomolets Microbiology Lesson #2

  1. 1. LessonN2: LABORATORY DIAGNOSIS OF STREPTOCOCCAL INFECTIONS1.Scientifically methodical ground of themeStreptococci produce a wide variaty of infections, ranging from pharyngitis and cellulitis to sepsis. They also cantrigger immunologic desodes such as reumatic fever and acute glomerulonephritis. 2.Educational purpose STUDENTS MUST KNOW: 1. Structure, tinctorial and cultural properties of streptococci. 2. Antigenic structure of streptococci, their classification. 3. Fermentative properties and toxin production of streptococci. 4. Differentiation of streptococci. 5. Main clinical forms of streptococcal infections. 6. Methods of laboratory diagnosis of streptococcal infections and main preparations for specific prophylaxisand treatment of streptococcal infections STUDENTS SHOULD BE ABLE TO: – investigate separated colonies on YSA ( yolk salt agar) – stain the smears by Gram’s technique; – make microscopical examination of the smears; – subcultivate culture from MPB(meat pepton broth) onto blood agar to obtaining isolated colonies – subcultivate separated colonies from YSA onto MPA 3.Chart of topic content. The streptococcus (Streptococcus pyogenes) was discovered by T. Billroth (1874) in tissues of patients witherysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston describedthe organisms in studies of suppurative lesions (1881). A pure culture of the organism was isolated by F. Fehleisen(1883) from a patient with erysipelas and by F. Rosenbach (1884) from pus. Streptococci belong to the familyStreptococcaceae. Morphology. Streptococci are spherical in shape, 0.6 to 1 mem in diameter, and form chains.They are non-motile (although motile forms are encountered), do not form spores and are Gram-positive. Some strains arecapsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in shortchains, while in smears from broth cultures they form long chains or clusters. The capsule is clearly demonstrated at the end of the phase of logarithmic growth. The microcapsule is seenon ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule thereis a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which formsinvaginations directed into the cytoplasm. The cytoplasm is microgranular and contains ribosomes, some inclusions, and vacuoles with an electron-dense material resembling volutin granules. The nucleoid occupies most of the cytoplasm. Division of the cellbegins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of thecell begins before the previous one is completed, as the result of which dumbbell-like cells form. The G+C contentin DNA ranges from 34.5 to 38.5 per cent. Cultivation. Streptococci are facultatively aerobic, and there are also anaerobic species. The optimaltemperature for growth is 37° C, and no growth occurs beyond the limits of 20-40° C for enterococci the limits are10-45 °C). The organisms show poor growth on ordinary meat-peptone agar, and grow well on sugar, blood, serum andascitic agar and broth, when the pH of the media is 7.2-7.6. On solid media they produce small (0.5-1.0 mm indiameter), translucent, grey or greyish-white, and granular colonies with poorly defined margins. Somestreptococcal strains cause haemolysis on blood agar, others produce a green coloration surrounding the colony 1-2mm in diameter as result a conversion of haemoglobin into methaemoglobin, while others do not cause any changein the erythrocytes. On sugar broth medium growth is in the form of fine-granular precipitates on the walls and atthe bottom of the tube and only rarely does the broth become turbid. Fermentative properties. Streptococci are non-proteolytic, do not liquefy gelatin, and do not reduce nitratesto nitrites. They coagulate milk, dissolve fibrin, ferment glucose, maltose, lactose, saccharose, mannitol (not alwaysconstantly), and break down salicin and trehalose, with acid formation. Toxin production. Streptococci produce exotoxins with various activities: (1) haemolysin (haemotoxin, 0- and S-streptolysm) which loses its activity after 30 minutes at a temperatureof 55 °C; disintegrates erythrocytes; produces haemoglobinaemia and haematuria in rabbits following intravenousinjection; (2) leucocidin which is destructive to leucocytes; occurs in highly virulent strains and is rendered harmlessby a temperature of 70 °C 1
  2. 2. (3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causesnecrosis in other tissues, particularly in the hepatic cells; (4) erythrogenic toxin produces inflammation in humans who have no antitoxins in their blood; (5) Streptococcus pneumoniae produces alpha-hae molysin secretedinto the culture fluid and beta-haemolysin which is released after lysis of the streptococci. Other substances produced by streptococci are harmful enzymes. They include hyaluronidase (whichfacilitates the spread of the organisms throughout the tissues and organs of the affected animal), fibrinolysin,desoxyribonuclease, ribqnuclease, proteinase, amylase, lipase, and diphosphopyridine nucleotidase. Streptococcalphages possess transduction properties and may be responsible for increased toxigenicity of C. diphtheriae andincreased virulence of other bacteria occurring in association with streptococci. Endotoxins, characterized by their thermoresistance and specific activity, are responsible for the pathogenicproperties of streptococci together with the exotoxins and aggressive enzymes. Antigenic structure. The study of the antigenic structure of streptococci is based on serologic examinations.F. Griffith used th e agglutination test, while R. Lancefield employed the precipitin reaction with an extract of abroth culture precipitate. Four antigenic fractions were recovered from streptococci: the type-specific protein (M- and T-substances);group-specific polysaccharide (C-substance), and nucleoprotein (P-substance). The M-substance is a protein whichconfers type specificity, virulence, and immunogenicity. The T-substance contains 0-, K-, and L-antigens. The C-substance is a polysaccharide common to the whole group of haemolytic streptococci. The P-substance belongs tothe nucleoprotein fraction, being non-specific for haemolytic streptococci; it contains nucleoproteins common toother groups of streptococci, as well as staphylococci. Group A and, partly, group C and G streptococci possess extracellular antigens: streptolysin 0, a proteinwhich causes erythrocyte haemolysis, and streptolysin S, a lipoprotein complex possessing erythrocytolyticactivity. Classification. By means of the precipitation reaction founded on the detection of group specificcarbohydrates, streptococci are subdivided into groups which are designated by capital letters from A to H andfrom K to T. Five out of the 21 known Streptococcal species cannot be related to any antigenic group. Nine species are ofinterest for medical microbiology; their brief characteristics are shown in Table 13. The haemolytic streptococci, rec overed from sick human beings, weresubdivided by F. Griffith into 51serovars. He attributed 47 serovars to group A, serovars 7, 20, and 21 to group C, and serovar 16 to group G. Streptococcus faecalis (enterococci) are pleomorphic oval cells which occur in pairs or in short chains.Some are oval or spear-shaped in form. The organisms are from 0.5 to 1 mem in diameter. Haemolytic types(Streptococcus faecalis) and organisms liquefying gelatin (S. faecalis, var. liquefaciens) are found. According totheir antigenic structure, enterococci are divided into six 0-groups among which there are strains with K-antigens(capsular antigens). Some enterococcal and lactic streptococci possess identical antigens. On solid mediaenterococcal growths form a thin pellicle with smooth edges. On sugar broth they produce turbidity and precipitate.Certain enterococci are highly motile. Some of the strains produce a yellow pigment, and the pathogenicenterococci produce fibrinolysin. The organisms grow at tempera hires ranging from 10 to 45 °C. They are resistantto high temperature (e. g. withstand exposure to 60 °C for half an hour). Enterococci can be grown in brothcontaining 6.5 per cent common salt at pH 9.6 and on blood agar containing 40 per cent bile or an equivalentamount of bile salts. They ferment glucose, maltose, lactose, mannitol, trehalose, salicin, and inulin, with acidformation. They reduce and coagulate litmus milk in the presence of 0.1 per cent methylene blue. Enterococcidiffer from other streptococci in their ability to grow over a wide range of temperatures (10-45 °C) and in amedium of pH 9.6, in their resistance to high concentrations of salt and to penicillin (a number of strainsshowgrowth in media containing 0.5-1 U of antibiotic per 1 ml of media). All enterococci decarboxylate tyrosine. Enterococci inhabit the small and large intestine of man and warm-blooded animals. The organisms possessproperties antagonistic to dysentery, enteric fever, and paratyphoid bacteria, and to the coli bacillus. In the childsintestine the enterococci are more numerous than the E. coli. In lesions of the duodenum, gall bladder, and urinarytract enterococci are found as a result of dysbacteriosis. Isolation of enterococci serves as a criterion ofcontamination of water, sewage, and foodstuffs with faeces. Streptococcus pneumonias (Diplococcus pneumoniae) belongs to the family Streptomycetaceae. For manyyears it was called pneumococcus. These are lanceolate or slightly elongated cocci measuring up to 0.5-1.25 memin diameter and occurring in pairs, sometimes as single organisms or arranged in chains. In the body of humans andanimals they have a capsule: they are Gram-positive, but young and old cultures are Gram-negative. The organismsare non-motile and do not form spores. S. pneumoniae is a facultative anaerobe, the optimum temperature forgrowth is 37° C and the organism grows at 28-42 °C. The organisms are poorly cultivated on ordinary media but 2
  3. 3. develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar theyform small, rounded succulent colonies on a green medium. On sugar broth they produce turbidity and aprecipitate. The organisms grow readily on broth to which 0.2 per cent of glucose is added. They usually do notform capsules on artificial media, but the addition of animal protein to fluid medium promotes the formation of acapsule. There are 80 variants which are agglutinated ony by the corresponding type sera. Among farm animals the young ones (calves, piglets, and lambs) and in vivariums guinea pigs may contractpneumonia. Among experimental animals albino mice and rabbits are most susceptible to the disease. Variants I, II,and III S. pneumoniae cause lobar pneumonia in man, which is characterized by an acute course occurring incycles. The organisms may also cause septicaemia, meningitis, affection of the joints, ulcerative endocarditis, otitis,peritonitis, rhinitis, highmoritis, creeping comeal ulcer, tonsillitis, and acute catarrhs of the upper respiratorypassages. Of special interest are the alpha-streptococci (the viridans group). They are responsible forhaemometamorphosis on blood agar (greenish discoloration of the media) and produce no soluble haemolysin. Theorganisms of this group usually ferment raffinose and do not ferment mannitol. They are always isolated from themouth and throat of healthy people and have low virulence for humans and animals. The viridans streptococci arefound in pyogenic and inflammatory lesions of the teeth and gums and are responsible for subacute endocarditis. Anaerobic streptococci (Peptostreptococcus putridus, Peptostreptococcus anaerobius, and others) are thecause of severe puerperal septic infections (puerperal sepsis). They are isolated from pyogenic and gangrenouslesions which have a putrefactive odour. Resistance. Streptococci live for a long time at low temperatures, are resistant to desiccation, and survive formany months in pus and sputum. When exposed to a temperature of 70 °C, they are destroyed within one hour. A3-5 per cent phenol solution kills the organisms within 15 minutes. Pathogenicity for animals. Cattle, horses, and, among laboratory animals, rabbits and white mice aresusceptible to the pathogenic streptococci. The virulence of streptococci is tested on rabbits. The animals are infected by rubbing a culture suspensioninto a scratch made on the skin of the ear or on the back. This results in a local inflammation with the appearanceof hyperaemia and swelling. An intravenous injection of pathogenic streptococci causes septicaemia or selectivelyaffects the lungs, liver, kidneys, or joints. Pathogenesis and diseases in man. The pathogenesis of streptococcal infections is brought about by the effectof the exotoxin and the-bacterial cells.The reactivity of the infected body and its previous resistance play animportant part in the origin and development of streptococcal diseases. Such diseases as endocarditis, polyarthritis,highmoritis, chronic tonsillitis, and erysipelas are associated with abnormal body reactivity, hyperergia. Thiscondition may persist for a long period of time and serve as the main factor for the development of chronicstreptococcal diseases. With an exogenous mode of infection streptococci invade the human body from without (from sick people,and animals, various contaminated objects and foodstuffs). They gain access through injured skin and mucousmembranes or enter the intestine with the food. Streptococci are mainly spread by the air droplet route. When thenatural body resistance is weakened, conditionally pathogenic streptococci normally present in the human bodybecome pathogenic. Penetrating deep into the tissues they produce local pyogenic inflammations, such asstreptoderma, abscesses, phlegmons, lymphadenitis, lymphangitis, cystitis, pyelitis, cholecystitis, and peritonitis.Erysipelas (inflammation of the superficial lymphatic vessels) and tonsillitis (inflammation of the pharyngeal andtonsillar mucosa) are among the diseases caused by streptococci. Invading the blood, streptococci produce a seriousseptic condition. They are more commonly the cause of puerperal sepsis than other bacteria. Streptococci may cause secondary infections in patients with diphtheria, smallpox, whooping cough,measles, and other diseases. Chronic tonsillitis is attributed to the viridans streptococci and adenoviruses.Contamination of wounds with streptococci during war results in wound suppurations, abscess formation,phlegmons, and traumatic sepsis. Immunity. Immunity acquired after streptococcal infections is of low grade and short duration. Relapses oferysipelas, fre quent tonsilitis, dermatitis, periostitis, and osteomyelitis occur as a result of sensitization of the body.This is attributed to low immunogenic activity and high allergen content of the streptococci, as well as to thepresence of numerous types of the organisms against which no cross immunity is produced. Immunity following streptococcal infections is of an anti-infectious nature. It is associated with antitoxic andantibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitatephagocytosis. Laboratory diagnosis. Test material is obtained from the pus of wounds, inflammatory exudate. tonsillarswabs, blood, urine, and foodstuffs. Procedures are the same as for staphylococcal infections. Tests include 3
  4. 4. microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and itsidentification. Blood is grown on sugar broth if sepsis is suspected. Virulence is tested on rabbits by anintracutaneous injection of 200-400 million microbial cells. Toxicity is determined by injecting themintracutaneously with broth culture filtrate. The group and type of the isolated streptococcus and its resistance to the medicaments used are alsodetermined. In endocarditis there are very few organisms present in the blood in which they appear periodically.For this reason blood in large volumes (20-50 ml) is inoculated into vials containing sugar broth. If possible, theblood should be collected while the patient has a high temperature. In patients with chronic sepsis an examinationof the centrifuged urine precipitate and isolation of the organism in pure culture are recommended. Besides, the group and type of the isolated streptococcus are identified by means of fluorescent antibodies.Serological methods are also applied to determine the increase in the titre of antibodies, namely streptolysins O andantihyaluronidase. Treatment. Usually penicillin is used. For penicillin-resistant strains,and when penicillin is contraindicated,streptomycin, and erythromycin are required. Vaccine therapy (autovaccines and polyvalent vaccines) and phagetherapy are recommended in chronic conditions. In some countries diseases caused by beta-haemolytic streptococci of groups A, C, G, and H and by alpha-streptococci (endocarditis) are treated with anti-infectious (antitoxic and antibacterial) streptococcal sera togetherwith antibiotics and sulphonamides. Prophylaxis. Streptococcal infections are prevented by the practice of general hygienic measures atfactories, childrens institutions, maternity hospitals, and surgical departments, in food production, agriculturalwork, and everyday life. Maintaining appropriate sanitary levels of living and working conditions, raising the cultural level of thepopulation, and checking personal hygiene are of great importance. Since streptococci and the macroorganism share antigenic structures in common and because streptococci aremarked by weak immunogenic ability and there are a great number of types among them which do not possess theproperty of producing cross immunity, specific prophylaxis of streptococcal diseases has not been elaborated.Vaccines prepared from M-protein fractions of streptococci are being studied. Role of Streptococcus in the Aetiology of Scarlet Fever Scarlet fever has long been known as a widespread disease but at the present time its aetiology has not yetbeen ascertained. Four different theories were proposed: streptococcal, allergic, viral, and combined (viral-streptococcal). Most scientists and medical practitioners favoured the streptococcal theory. G. Gabrichevsky in1902 was the first to point out the aetiological role of the haemolytic streptococcus in scarlet fever. Usually herecovered the organisms from the pharynx of scarlet fever patients and from blood contained in the heart of thosethat had died of the disease. In 1907 he prepared vaccine from killed scarlet fever haemolytic streptococcalcultures. This vaccine was widely used for human vaccination. In 1905 I. Savchenko, cultivating scarlet fever streptoco cci, obtained the toxin and used it forhyperimmunization of horses. The antitoxic antiscarlatinal serum was effectively used for treating people sufferingfrom scarlet fever. Data presented by Gabrichevsky and Savchenko concerning the streptococcal theory were confirmed bystudies carried out in 1923-24 by G. Dick and G. Dick and by many other scientists. The streptococcal aetiology of scarlet fever is supported by the following arguments: (1) all people sufferingfrom scarlet fever are found to harbour in their throats haemolytic streptococci which are agglutinated by the seraof convalescents; (2) a subcutaneous injection of the scarlet fever toxin into susceptible people (volunteers) in somecases is followed by the appearance of a characteristic skin rash, vomiting, fever, tonsillitis, and other scarlatinalsymptoms; (3) an intracutaneous injection of the toxin into susceptible children produces a local erythematous andoedematous reaction; the toxin produces no reaction in children who had previously suffered from scarlet fever andwere im-mune to the disease; (4) if 0.1 ml of antitoxic antistreptococcal serum or convalescent serum is introducedinto the skin of a scarlet fever patient in the area of the rash, the latter turns pale (is extinguished); (5) hyper-immunization of animals with the scarlet fever toxin leads to the production of antitoxins, and a neutralizationreaction takes place between the toxin and antitoxins; (6) therapy with antitoxic sera and prophylaxis withcombined vaccines consisting of the toxin and haemolytic streptococcal cells result in the appearance of less severecases and decrease in morbidity and mortality. At present many investigators accept the streptococcal theory in scarlet fever aetiology. In postwar years thistheory has been confirmed by a number of investigations. Arguments against the streptococcal theory are asfollows: (1) people inoculated with scarlet fever streptococci or their toxins do not always display the characteristicsymptoms of the disease, e. g. there is no peeling, only rarely are there instances of tonsillitis, and phlegmon,sepsis, and erysipelas occasionally develop; (2) in severe hypertoxic forms the antitoxic ssswm has little effect, 4
  5. 5. while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negativereaction with susceptible children and produces a positive reaction with those who are immune; (4) immunityacquired after scarlet fever is very stable and of long duration, while that acquired after other streptococcal diseasesis unstable, of short duration, and is frequently accompanied by an increased susceptibility to streptococci. It is assumed that scarlet fever is caused by group A beta-haemolytic streptococci which possess M-antigenand produce erythrogenic exotoxin. People become infected by the air droplet route. Sick people, convalescents,and carriers of the causative agent of scarlet fever are all sources of infection. The disease is most commonlyencountered in children from 1 to 8 years of age. The causative agent sometimes enters the body through wounds on the skin and mucous membranes of thegenitalia. This form of scarlet fever is known as extrabuccal or extrapharyngeal (traumatic, combustion, surgical,and puerperal). Certain objects (e. g. utensils, toys, books, etc.) as well as foodstuffs (e. g. milk), contaminated byadult carriers, may also be sources of infection. Of great importance in the epidemiology of scarlet fever are thepatients with atypical, unrecognizable forms of the disease. In its initial stage scarlet fever is chiefly characterizedby intoxication, while in the second stage it is accompanied by septic and allergic conditions. Scarlet fever produces a relatively stable immunity. Reinfections are very rare. They have increased innumber in the last years as a result of wide use of antibiotics which reduce the immunogenic activity of thepathogen and its toxin. Data concerning the correlation between a positive Dick test and susceptibility to scarlet fever provideevidence of the antitoxic nature of immunity acquired after scarlet fever. Children from 1 to 5 years are mostsusceptible. Scarlet fever is recognized mainly by its clinical course and on epidemiological grounds. Laboratorydiagnosis for the detection of haemolytic streptococci and their typing is employed only in certain cases. Thismethod is of no practical value since haemolytic streptococci are often isolated from people with various diseasesand frequently from healthy individuals. The phenomenon of rash extinguishmenf is employed as an auxiliary diagnostic method. In the case ofscarlet fever, the rash at the site of injection will disappear within 12-20 hours and the skin will turn pale. Certain physicians apply the Dick test with the thermolabile fraction of the toxin. The diagnosis of scarletfever is verified to a certain extent if on a second injection of the toxin a positive Dick test reverts to a negativereaction. Scarlet fever may also be diagnosed by detecting precipitins in the urine (urine precipitation test). A layer oftype-specific streptococcal sera or convalescent serum is transferred onto freshly filtered urine of patients in thefirst days of the disease. The appearance of a greyish-white ring at the interface of the two fluids designates apositivereaction. Scarlet fever patients are treated with penicillin, tetracycline, sulphonamides (norsulphazol, etc.), andgamma-globulin from human blood. The wide use of antibiotics has led to a significant decrease in the morbidityand mortality rate of scarlet fever and to a milder course of the disease. This fact also confirms the definite roleplayed by haemolytic streptococci in the aetiology and pathogenesis of scarlet fever, since it is known that theseorganisms are extremely sensitive to penicillin and other antibiotics. In the recent years, however, an increase in theincidence of scarlet fever and a more severe course of the disease are noted. Prophylaxis consists of early diagnosis, isolation of patient s and hospitalization in the presence ofepidemiological and clinical indications. Extremely hygienic cleaning and ventilation and observance of correcthospital regime are also necessary. If cases of scarlet fever occur in childrens institutions, the children concernedmust be isolated. Debilitated children who have been in contact with scarlet fever patients must be injected with1.5-3.0 ml of human serum gammaglobulin. Role of Streptococcus in the Aetiology of Rheumatic Fever The majority of authors maintain that rheumatic fever develops as a result of the body becoming infected bygroup A beta-haemolytic streptococci. Acute or chronic tonsillitis and pharyngitis produce a change in theimmunological reactivity of the body and this gives rise to characteristic clinical symptoms and a pathologicalreaction. It should be noted, however, that recent research shows the leading role in rheumatism of virus agentswith persisting properties. The active phase with an acute and subacute course is attended with virusaemia.Rheumatism is characterized by the virus remaining in the body for a long period of time: the viral antigenpenetrates the leucocytes and sensitizes them. Diminution of the specific and non-specific reactions to the virusleads to tolerance of the body. Autoimmune reactions are encountered in rheumatism. Streptococci and otherexogenous and endogenous factors contribute to exacerbation of rheumatism. Virusaemia is almost always revealedin the patients during exacerbation. Identical viruses are detected in the mothers blood and in the blood ofpremature infants, stillborns, and in infants who die soon after birth. 5
  6. 6. No streptococci, streptococcal antigen or antibodies to them are found in some patients and penicillin therapyproves ineffective. On the grounds of this, it is considered that the diagnosis of rheumatism should be made andpenicillin prescribed only if the presence of streptococcal infection is sufficiently verified. The prevalence of rheumatic fever depends on the time of the year. The highest number of cases occur inOctober-November and March- April. Acute and chronic tonsillitis, pharyngitis, and catarrh are also most prevalentin these months. The allergic reaction produced in the body as a result of re-invasion by antigens (streptococcal exo- andendotoxms, autoantigens, and complexes consisting of streptococcal toxins and components of tissue and bloodproteins of sick people) is an important factor in the pathogenesis of the disease. It is known that blood ofindividuals who has suffered from a streptococcal infection contains antibodies against beta-haemolyticstreptococci. In 1 -3 per cent of these cases the formation of antibodies does not produce immunity, and asecondary invasion of the body by specific and non-specific antigens leads to the development of hyperergia.Experiments have shown that streptococci bring about the formation of autoantigens which cause the production ofautoantibodies in the body. These autoantibodies are responsible for lesions in certain tissues and organs. Studies of high-molecular gamma-globulins and their complexes in rheumatic fever have shown that thenormal human gamma-globulin contains two fractions (7S and 19S) which differ in their pre cipitate constant. Themajority of the common antibodies are associated with the 7S gamma-globulin fraction, while isoagglutinins, Rh-agglutinins, and complement-fixing antibodies are contained in the 19S fraction. The gamma-globulin fraction, richin 19S, is found to contain the rheumatoid factor. An interaction has been demonstrated between the rheumatoidfactor and the antigen-antibody precipitate, the latter possessing antigenic properties. Thus, in its reaction with theantigen-antibody complex the rheumatoid factor behaves as the complement. Alternatively, the rheumatoid factormay act as an antibody to gamma-globulin or to the antigen-antibody complex, the latter acting as an antigen. Antigen-antibody reactions result in the injury of the interstitial connective tissue, release of histamine, andinflammation. Disturbances of coordination in the hypophysis-adrenal system are encountered in rheumatic fever.For this reason supporters of the Selye theory consider rheumatic fever to be an adaptational disease. However, theabove mentioned aspects on the mechanism of rheumatic fever can by no means cover all the complex processesinvolved in the pathogenesis of this disease. According to its clinical course, rheumatic fever is differentiated into active and inactive phases. The activephase is characterized by acute rheumocarditis without valvular defects and relapsing rheumocarditis accompaniedby valvular defects, polyarthritis, chorea, pleuritis, peritonitis, nephritis, he patitis, pneumonia, lesions in the skinand subcutaneous tissue, eyes, and other systems. The inactive phase develops in the form of rheumaticmyocardiosclerosis, heart defects, and conditions following extracardial affections. Three periods can be distinguished during the development of rheumatic fever: (1) period of acutestreptococcal infection and initial sensitization; (2) penod ofhyperergic reactions, resulting frominteractionbetween antigens and antibodies, which are accompanied by pnmary rheumatic polyarthritis or carditis; (3) periodof stable allergic reacts ity accompanied by pronounced manifestations of parallergy and autosensitization,profound and stable immunogenic disturbances, and relapses. Laboratory diagnosis is made on the basis of determination of an increase in antistreptolysin,antifibrinolysin, and antihyaluronidase titres and detection of C-reactive protein. Treatment of rheumatic patients is accomplished by several measures aimed at desensitization of the body,abatement of inflammatory conditions, recovery of normal body reactivity, condition of the nervous system, anddisturbed processes, and control of local infections. Prophylaxis includes prevention of streptococcal infections, strengthening of general resistance, and creationof favourable conditions for everyday life and work. In addition, all people suffering from rheumatic fever andthose susceptible to the disease should be given prophylactic treatment with penicillin and tetracycline preparationsin spring and autumn.4. Student’s independent study program 1. Morphology, tinctorial properties of Streptococcus. Cultivation. 2. Antigenic structure and classification of Streptococci by R. Lancefield. Antigenic structure ofPneumococci. Main species of streptococci, which are caused human diseases. 3. Types of streptococci according to growth onto blood agar. 4. Toxin production and pathogenicity of streptococci. Peculiarities of toxin production and virulence factorsof pneumococci. 5. Ecology and spreading of streptococci. 6. Role of streptococci in the aetiology of Rheumatic fever and Scarlet Fever. Crossed antigens ofstreptococci and human tissues. 7. Ecology and route of spreading of pneumococci. A role of pneumococci in human pathology. 6
  7. 7. 8. Laboratory diagnostics of streptococcal and pneumococcal diseases. 5. Students Practical activities: 1. To familiarize with cultivation methods of streptococci (blood MPA, suger MPB etc.). 2. Examinate separated colonies on yolk-salt agar which grown after cultivate specimen (pus), make smear,stain it by Gram’s technique. Subcultivate colonies in MPB. 3. To estimate bacterial growth in sugar MPB after inoculation of blood patient with sepsis. Subcultivateculture on blood MPB. 4. To familiarize with biological preparations for active and passive prophylaxis and treatment ofstreptococcal infections. 6.Control questions and test: 1. Streptococci have such properties and characteristic: a – they occur in bunches; b – they occur in chain; c – Gram-positive; d – they stain by Neisser’s technique; e – they are capable of sporulation. 2. Pneumococci have such properties: a – they occur in chain; b – they occur in pairs; c – Gram-negative; d – form a capsule; e – Gram-positive. 3. Cultivation of streptococci: a – in MPB form fine granule precipitate; b – they produce diffuse opacity in MPB; c – grow onto MPA; d – grow on sugar MPA; e – grow on blood MPA. 4. In dependence from the growth onto blood agar streptococci are divided into: a – alpha-hemolytic; b – non-haemolytic; c – beta-haemolytic; d – hemophilic; e – fibrinolytic. 5. The streptococci cause: a – sepsis; b – Rheumatic Fever; c – Scarlet Fever; d – wound infection; e – toxinfections; 6. In a pathogenesis of streptococcal diseases matter: a – crossed antigens; b – toxins; c – enzymes of aggression; d – spores; e – adhesins. 7. Pneumococci have such properties: a –capsular antigens; b – produce hemolysins; c – produce perforins; d – produce leukocidin; e – anticomplementary activity. 8. Laboratory methods of diagnosis of pneumococcal infection: a – bacteriological; b – serological; c – bioassay; d – examination of toxins; e – allergic method. Real-life situations to be solved: 1. A patient was admitted to the hospital with initial diagnosis “Panaritium of third finger, phlegmon of righthand. Sepsis”. A. What tested material is it necessary to send to the bacteriological laboratory for examination? B. Make up the scheme of diagnosis of sepsis. 2. From the stomatopharynx of the boy with chronic tonsillitis was isolated the culture of spherical bacteria.They formed short chains in the smears. A. What bacteria they are? B. What is it necessary to determine before administration of antibiotics? C. To what danger children with untreated tonsillitis are exposed? 3. From the patient with suspicion on a croupous pneumonia were revealed gram-positive lanceolatediplococci with capsule. A. What material was taken for this purpose? B. What microbes are there in the smear? C. What fermentative properties allow you to consider these microbes as Streptococcus pneumoniae.7. List of literature:1. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology, , Lugansk, 2004,chapter15, p.11-19. 7

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