bohomolets microbiology lesson #2

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LessonN2: LABORATORY DIAGNOSIS OF STREPTOCOCCAL INFECTIONS 1.Scientifically methodical ground of theme Streptococci produce a wide variaty of infections, ranging from pharyngitis and cellulitis to sepsis. They also can trigger 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 prophylaxis and 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 with erysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston described the 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 family Streptococcaceae. 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 are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, 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 seen on ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule there is a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which forms invaginations 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 cell begins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of the cell begins before the previous one is completed, 1

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Page 1: Bohomolets Microbiology Lesson #2

LessonN2: LABORATORY DIAGNOSIS OF STREPTOCOCCAL INFECTIONS

1.Scientifically methodical ground of themeStreptococci produce a wide variaty of infections, ranging from pharyngitis and cellulitis to sepsis. They also can trigger 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 prophylaxis

and treatment of streptococcal infectionsSTUDENTS 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 with

erysipelas and wound infections and by L. Pasteur and others (1880) in patients with sepsis. A. Ogston described the 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 family Streptococcaceae.

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 are capsulated. In smears from cultures grown on solid media the streptococci are usually present in pairs or in short chains, 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 seen on ultrathin sections, which forms in the phases of logarithmic and stationary growth. Under the microcapsule there is a three-layer cell wall 100-300 nm thick and then a three-layer cytoplasmic membrane which forms invaginations 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 cell begins with protrusion of the cytoplasmic membrane after which a septum forms in the middle. New division of the cell begins before the previous one is completed, as the result of which dumbbell-like cells form. The G+C content in DNA ranges from 34.5 to 38.5 per cent.

Cultivation. Streptococci are facultatively aerobic, and there are also anaerobic species. The optimal temperature for growth is 37° C, and no growth occurs beyond the limits of 20-40° C for enterococci the limits are 10-45 C).

The organisms show poor growth on ordinary meat-peptone agar, and grow well on sugar, blood, serum and ascitic 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 in diameter), translucent, grey or greyish-white, and granular colonies with poorly defined margins. Some streptococcal strains cause haemolysis on blood agar, others produce a green coloration surrounding the colony 1-2 mm in diameter as result a conversion of haemoglobin into methaemoglobin, while others do not cause any change in the erythrocytes. On sugar broth medium growth is in the form of fine-granular precipitates on the walls and at the 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 nitrates to nitrites. They coagulate milk, dissolve fibrin, ferment glucose, maltose, lactose, saccharose, mannitol (not always constantly), 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 temperature

of 55 C; disintegrates erythrocytes; produces haemoglobinaemia and haematuria in rabbits following intravenous injection;

(2) leucocidin which is destructive to leucocytes; occurs in highly virulent strains and is rendered harmless by a temperature of 70 C

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(3) lethal (dialysable) toxin which produces necrosis in rabbits when injected intracutaneously; it also causes necrosis 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 (which

facilitates the spread of the organisms throughout the tissues and organs of the affected animal), fibrinolysin, desoxyribonuclease, ribqnuclease, proteinase, amylase, lipase, and diphosphopyridine nucleotidase. Streptococcal phages possess transduction properties and may be responsible for increased toxigenicity of C. diphtheriae and increased virulence of other bacteria occurring in association with streptococci.

Endotoxins, characterized by their thermoresistance and specific activity, are responsible for the pathogenic properties 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 a broth 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 which confers 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 to the nucleoprotein fraction, being non-specific for haemolytic streptococci; it contains nucleoproteins common to other groups of streptococci, as well as staphylococci.

Group A and, partly, group C and G streptococci possess extracellular antigens: streptolysin 0, a protein which causes erythrocyte haemolysis, and streptolysin S, a lipoprotein complex possessing erythrocytolytic activity.

Classification. By means of the precipitation reaction founded on the detection of group specific carbohydrates, streptococci are subdivided into groups which are designated by capital letters from A to H and from K to T.

Five out of the 21 known Streptococcal species cannot be related to any antigenic group. Nine species are of interest 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 51 serovars. 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 to their 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 media enterococcal 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 pathogenic enterococci produce fibrinolysin. The organisms grow at tempera hires ranging from 10 to 45 C. They are resistant to high temperature (e. g. withstand exposure to 60 C for half an hour). Enterococci can be grown in broth containing 6.5 per cent common salt at pH 9.6 and on blood agar containing 40 per cent bile or an equivalent amount of bile salts. They ferment glucose, maltose, lactose, mannitol, trehalose, salicin, and inulin, with acid formation. They reduce and coagulate litmus milk in the presence of 0.1 per cent methylene blue. Enterococci differ from other streptococci in their ability to grow over a wide range of temperatures (10-45 C) and in a medium of pH 9.6, in their resistance to high concentrations of salt and to penicillin (a number of strainsshow growth 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 possess properties antagonistic to dysentery, enteric fever, and paratyphoid bacteria, and to the coli bacillus. In the child's intestine the enterococci are more numerous than the E. coli. In lesions of the duodenum, gall bladder, and urinary tract enterococci are found as a result of dysbacteriosis. Isolation of enterococci serves as a criterion of contamination of water, sewage, and foodstuffs with faeces.

Streptococcus pneumonias (Diplococcus pneumoniae) belongs to the family Streptomycetaceae. For many years it was called pneumococcus. These are lanceolate or slightly elongated cocci measuring up to 0.5-1.25 mem in diameter and occurring in pairs, sometimes as single organisms or arranged in chains. In the body of humans and animals they have a capsule: they are Gram-positive, but young and old cultures are Gram-negative. The organisms are non-motile and do not form spores. S. pneumoniae is a facultative anaerobe, the optimum temperature for growth is 37° C and the organism grows at 28-42 C. The organisms are poorly cultivated on ordinary media but

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develop readily on serum or^ascitic agar at pH 7.2-7.6 as small colonies 1.0 mm in diameter. On blood agar they form small, rounded succulent colonies on a green medium. On sugar broth they produce turbidity and a precipitate. The organisms grow readily on broth to which 0.2 per cent of glucose is added. They usually do not form capsules on artificial media, but the addition of animal protein to fluid medium promotes the formation of a capsule. 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 contract pneumonia. 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 in cycles.

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 respiratory passages.

Of special interest are the alpha-streptococci (the viridans group). They are responsible for haemometamorphosis on blood agar (greenish discoloration of the media) and produce no soluble haemolysin. The organisms of this group usually ferment raffinose and do not ferment mannitol. They are always isolated from the mouth and throat of healthy people and have low virulence for humans and animals. The viridans streptococci are found 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 the cause of severe puerperal septic infections (puerperal sepsis). They are isolated from pyogenic and gangrenous lesions which have a putrefactive odour.

Resistance. Streptococci live for a long time at low temperatures, are resistant to desiccation, and survive for many months in pus and sputum. When exposed to a temperature of 70 C, they are destroyed within one hour. A 3-5 per cent phenol solution kills the organisms within 15 minutes.

Pathogenicity for animals. Cattle, horses, and, among laboratory animals, rabbits and white mice are susceptible to the pathogenic streptococci.

The virulence of streptococci is tested on rabbits. The animals are infected by rubbing a culture suspension into a scratch made on the skin of the ear or on the back. This results in a local inflammation with the appearance of hyperaemia and swelling. An intravenous injection of pathogenic streptococci causes septicaemia or selectively affects the lungs, liver, kidneys, or joints.

Pathogenesis and diseases in man. The pathogenesis of streptococcal infections is brought about by the effect of the exotoxin and the-bacterial cells.The reactivity of the infected body and its previous resistance play an important 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. This condition may persist for a long period of time and serve as the main factor for the development of chronic streptococcal 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 mucous membranes or enter the intestine with the food. Streptococci are mainly spread by the air droplet route. When the natural body resistance is weakened, conditionally pathogenic streptococci normally present in the human body become pathogenic. Penetrating deep into the tissues they produce local pyogenic inflammations, such as streptoderma, abscesses, phlegmons, lymphadenitis, lymphangitis, cystitis, pyelitis, cholecystitis, and peritonitis. Erysipelas (inflammation of the superficial lymphatic vessels) and tonsillitis (inflammation of the pharyngeal and tonsillar mucosa) are among the diseases caused by streptococci. Invading the blood, streptococci produce a serious septic 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 of erysipelas, 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 the presence 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 and antibacterial factors. The antitoxins neutralize the streptococcal toxin and together with the opsonins facilitate phagocytosis.

Laboratory diagnosis. Test material is obtained from the pus of wounds, inflammatory exudate. tonsillar swabs, blood, urine, and foodstuffs. Procedures are the same as for staphylococcal infections. Tests include

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microscopy of pus smears, inoculation of test material onto blood agar plates, isolation of the pure culture and its identification. Blood is grown on sugar broth if sepsis is suspected. Virulence is tested on rabbits by an intracutaneous injection of 200-400 million microbial cells. Toxicity is determined by injecting them intracutaneously with broth culture filtrate.

The group and type of the isolated streptococcus and its resistance to the medicaments used are also determined. 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, the blood should be collected while the patient has a high temperature. In patients with chronic sepsis an examination of 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 and antihyaluronidase.

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 phage therapy 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 together with antibiotics and sulphonamides.

Prophylaxis. Streptococcal infections are prevented by the practice of general hygienic measures at factories, children's institutions, maternity hospitals, and surgical departments, in food production, agricultural work, and everyday life.

Maintaining appropriate sanitary levels of living and working conditions, raising the cultural level of the population, and checking personal hygiene are of great importance.

Since streptococci and the macroorganism share antigenic structures in common and because streptococci are marked by weak immunogenic ability and there are a great number of types among them which do not possess the property 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 FeverScarlet fever has long been known as a widespread disease but at the present time its aetiology has not yet

been ascertained. Four different theories were proposed: streptococcal, allergic, viral, and combined (viral-streptococcal). Most scientists and medical practitioners favoured the streptococcal theory. G. Gabrichevsky in 1902 was the first to point out the aetiological role of the haemolytic streptococcus in scarlet fever. Usually he recovered the organisms from the pharynx of scarlet fever patients and from blood contained in the heart of those that had died of the disease. In 1907 he prepared vaccine from killed scarlet fever haemolytic streptococcal cultures. This vaccine was widely used for human vaccination.

In 1905 I. Savchenko, cultivating scarlet fever streptoco cci, obtained the toxin and used it for hyperimmunization of horses. The antitoxic antiscarlatinal serum was effectively used for treating people suffering from scarlet fever.

Data presented by Gabrichevsky and Savchenko concerning the streptococcal theory were confirmed by studies 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 suffering from scarlet fever are found to harbour in their throats haemolytic streptococci which are agglutinated by the sera of convalescents; (2) a subcutaneous injection of the scarlet fever toxin into susceptible people (volunteers) in some cases is followed by the appearance of a characteristic skin rash, vomiting, fever, tonsillitis, and other scarlatinal symptoms; (3) an intracutaneous injection of the toxin into susceptible children produces a local erythematous and oedematous reaction; the toxin produces no reaction in children who had previously suffered from scarlet fever and were im-mune to the disease; (4) if 0.1 ml of antitoxic antistreptococcal serum or convalescent serum is introduced into 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 neutralization reaction takes place between the toxin and antitoxins; (6) therapy with antitoxic sera and prophylaxis with combined vaccines consisting of the toxin and haemolytic streptococcal cells result in the appearance of less severe cases and decrease in morbidity and mortality.

At present many investigators accept the streptococcal theory in scarlet fever aetiology. In postwar years this theory has been confirmed by a number of investigations. Arguments against the streptococcal theory are as follows: (1) people inoculated with scarlet fever streptococci or their toxins do not always display the characteristic symptoms 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,

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while the serum of convalescents gives better results; (3) the skin toxin test (Dick test) sometimes gives a negative reaction with susceptible children and produces a positive reaction with those who are immune; (4) immunity acquired after scarlet fever is very stable and of long duration, while that acquired after other streptococcal diseases is 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-antigen and 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 commonly encountered 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 the genitalia. 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 by adult carriers, may also be sources of infection. Of great importance in the epidemiology of scarlet fever are the patients with atypical, unrecognizable forms of the disease. In its initial stage scarlet fever is chiefly characterized by 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 in number in the last years as a result of wide use of antibiotics which reduce the immunogenic activity of the pathogen and its toxin.

Data concerning the correlation between a positive Dick test and susceptibility to scarlet fever provide evidence of the antitoxic nature of immunity acquired after scarlet fever. Children from 1 to 5 years are most susceptible.

Scarlet fever is recognized mainly by its clinical course and on epidemiological grounds. Laboratory diagnosis for the detection of haemolytic streptococci and their typing is employed only in certain cases. This method is of no practical value since haemolytic streptococci are often isolated from people with various diseases and frequently from healthy individuals.

The phenomenon of rash 'extinguishmenf is employed as an auxiliary diagnostic method. In the case of scarlet 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 scarlet fever is verified to a certain extent if on a second injection of the toxin a positive Dick test reverts to a negative reaction.

Scarlet fever may also be diagnosed by detecting precipitins in the urine (urine precipitation test). A layer of type-specific streptococcal sera or convalescent serum is transferred onto freshly filtered urine of patients in the first days of the disease. The appearance of a greyish-white ring at the interface of the two fluids designates a positivereaction.

Scarlet fever patients are treated with penicillin, tetracycline, sulphonamides (norsulphazol, etc.), and gamma-globulin from human blood. The wide use of antibiotics has led to a significant decrease in the morbidity and mortality rate of scarlet fever and to a milder course of the disease. This fact also confirms the definite role played by haemolytic streptococci in the aetiology and pathogenesis of scarlet fever, since it is known that these organisms are extremely sensitive to penicillin and other antibiotics. In the recent years, however, an increase in the incidence 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 of epidemiological and clinical indications. Extremely hygienic cleaning and ventilation and observance of correct hospital regime are also necessary. If cases of scarlet fever occur in children's institutions, the children concerned must be isolated. Debilitated children who have been in contact with scarlet fever patients must be injected with 1.5-3.0 ml of human serum gammaglobulin.

Role of Streptococcus in the Aetiology of Rheumatic FeverThe majority of authors maintain that rheumatic fever develops as a result of the body becoming infected by

group A beta-haemolytic streptococci. Acute or chronic tonsillitis and pharyngitis produce a change in the immunological reactivity of the body and this gives rise to characteristic clinical symptoms and a pathological reaction. It should be noted, however, that recent research shows the leading role in rheumatism of virus agents with 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 antigen penetrates the leucocytes and sensitizes them. Diminution of the specific and non-specific reactions to the virus leads to tolerance of the body. Autoimmune reactions are encountered in rheumatism. Streptococci and other exogenous and endogenous factors contribute to exacerbation of rheumatism. Virusaemia is almost always revealed in the patients during exacerbation. Identical viruses are detected in the mother's blood and in the blood of premature infants, stillborns, and in infants who die soon after birth.

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No streptococci, streptococcal antigen or antibodies to them are found in some patients and penicillin therapy proves ineffective. On the grounds of this, it is considered that the diagnosis of rheumatism should be made and penicillin 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 in October-November and March- April. Acute and chronic tonsillitis, pharyngitis, and catarrh are also most prevalent in these months.

The allergic reaction produced in the body as a result of re-invasion by antigens (streptococcal exo- and endotoxms, autoantigens, and complexes consisting of streptococcal toxins and components of tissue and blood proteins of sick people) is an important factor in the pathogenesis of the disease. It is known that blood of individuals who has suffered from a streptococcal infection contains antibodies against beta-haemolytic streptococci. In 1 -3 per cent of these cases the formation of antibodies does not produce immunity, and a secondary 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 of autoantibodies 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 the normal human gamma-globulin contains two fractions (7S and 19S) which differ in their pre cipitate constant. The majority 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, rich in 19S, is found to contain the rheumatoid factor. An interaction has been demonstrated between the rheumatoid factor and the antigen-antibody precipitate, the latter possessing antigenic properties. Thus, in its reaction with the antigen-antibody complex the rheumatoid factor behaves as the complement. Alternatively, the rheumatoid factor may 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, and inflammation. 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, the above mentioned aspects on the mechanism of rheumatic fever can by no means cover all the complex processes involved in the pathogenesis of this disease.

According to its clinical course, rheumatic fever is differentiated into active and inactive phases. The active phase is characterized by acute rheumocarditis without valvular defects and relapsing rheumocarditis accompanied by valvular defects, polyarthritis, chorea, pleuritis, peritonitis, nephritis, he patitis, pneumonia, lesions in the skin and subcutaneous tissue, eyes, and other systems. The inactive phase develops in the form of rheumatic myocardiosclerosis, heart defects, and conditions following extracardial affections.

Three periods can be distinguished during the development of rheumatic fever: (1) period of acute streptococcal infection and initial sensitization; (2) penod ofhyperergic reactions, resulting frominteraction between antigens and antibodies, which are accompanied by pnmary rheumatic polyarthritis or carditis; (3) period of 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, and disturbed processes, and control of local infections.

Prophylaxis includes prevention of streptococcal infections, strengthening of general resistance, and creation of favourable conditions for everyday life and work. In addition, all people suffering from rheumatic fever and those susceptible to the disease should be given prophylactic treatment with penicillin and tetracycline preparations in 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 of

Pneumococci. 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 factors

of pneumococci.5. Ecology and spreading of streptococci.6. Role of streptococci in the aetiology of Rheumatic fever and Scarlet Fever. Crossed antigens of

streptococci and human tissues.7. Ecology and route of spreading of pneumococci. A role of pneumococci in human pathology.

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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. Subcultivate

culture on blood MPB. 4. To familiarize with biological preparations for active and passive prophylaxis and treatment of

streptococcal 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 right

hand. 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 lanceolate

diplococci 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.

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