Laboratory diagnosis of staphylococcal and streptococcal infection.

Laboratory diagnosis of staphylococcal and streptococcal infection.

Laboratory diagnosis of meningococcal and gonococcal infection

 

Gram-positive cocci

There are several types of symbiosis between cocci and the human body. Saprophytic and conditionally pathogenic types of staphylococci and streptococci live on the skin, mucous membranes, and in the respiratory tract. Meningococci may be harboured for long periods in the nasopharynx, and faecal streptococci (enterococci) in the intestine.

When body resistance is lowered or the skin and mucous membranes are injured, these bacteria penetrate the body tissues and cause infection. The various cocci possess different organotropic ability. This is distinctly manifest in meningococci, and gonococci but less so in staphylococci and streptococci. Cocci belong to the families Micrococcaceae, Streptococcaceae Peptococcaceae, Neisseriaceae.

Staphylococci. The staphylococcus, Staphylococcus aureus, was discovered by R. Koch (18,78), and later isolated from furuncle pus by L. Pasteur (1880). It has been described as the causative agent of numerous suppurative processes by A. Ogston (1881), and has been studied in detail by F. Rosenbach (1884).

Morphology. Staphylococci are spherical in shape, 0.8-1 mem in diameter, and form irregular clusters resembling bunches of grapes  (Fig. 1). In smears from cultures and pus the organisms occur in short chains, in pairs, or as single cocci. Large spherical (L-forms) or very small (G-forms) and even filterable forms may be seen in cultures which have been subjected to various physical, chemical, and biological (antibiotics) factors.

 

Figure 1. Staphylococci.

 

Staphylococci are Gram-positive organisms which possess no flagella and do not form spores. A macrocapsule can be seen on ultrathin sections of Staphylococci isolated from infected mice. The nucleoid occupies most of the cytoplasm and is filled with DNA fibrils. The G+C content in DNA ranges between 30.7 and 39.0 per cent.

 

Cultivation. Staphylococci are facultative-anaerobes. They grow well on ordinary nutrient media with a pH of 7.2-7.4 at a temperature of 37 °C but do not grow at temperatures below 10 °C and above 45 °C. At room temperature with adequate aeration and subdued light – the organisms produce golden, white, lemon-yellow, and other pigments known as lipochromes. These pigments do not dissolve in water but are soluble in ether, benzene, acetone, chloroform, and alcohol. They are most readily formed on milk agar and potatoes at a temperature of 20-25° C.

 

On meat peptone agar Staphylococci produce well defined colonies with smooth edges, measuring from 1-2 to 2.5 mm in diameter. Under the microscope the course granular nature of the colonies can be seen, the latter are opaque and have a dense centre. Their colour epends on the pigment produced by the organisms. Besides the typical colony types, Staphylococci produce R-, G-, and L-forms.  Growth of Staphylococci in meat-peptone broth produces diffuse opacity throughout the medium and, subsequently, a precipitate. In some cases when there is sufficient aeration, the organisms form a pellicle on the surface of the broth. Staphylococci grow well on potatoes and coagulated serum. After 24-48 hours of incubation there is usually abundant growth along the inoculation stab and liquefaction of gelatin media. On the fourth or fifth day the gelatin medium resembles a funnel filled with  fluid.

On blood agar pathogenic Staphylococci cause haemolysis of the erythrocytes. Rabbit and sheep erythrocytes are the most sensitive to the staphylococcal haemotoxin.

Fennentative properties. Staphylococci produce enzymes which cause the lysis of proteins and sugars (see Table 1). There is no indole production in young cultures. The organisms liquefy gelatin, coagulate milk and occasionally serum, reduce nitrates to nitrites, produce urease, catalase, phosphatase, ammonia, and hydrogen sulphide. They ferment glucose, levulose, maltose, lactose, saccharose, mannitol, and glycerin, with acid formation. A connection has been revealed between arginase activity and the level of g-toxin formation.

Toxin production. Staphylococci produce a-, b-, d– and g-haemolysins which are characterized by lethal, haemolytic, and necrotic activity. Filtrates of staphylococcal broth cultures contain an enterotoxin which causes food poisoning on entry into the gastro-intestinal tract. Staphylococci excrete exofoliatines which cause staphylococcal impetigo and pemphigus neonatomm in children.

 

Leucocidin, a substance which destroys leucocytes, haematoblasts of the bone marrow and nerve cells, is also produced by the Staphylococci. The organisms also coagulate blood plasma. Their ability to coagulate, plasma is a stable property and is used for differentiating various strains. Coagulase is thermoresistant. It can  be isolated from staphylococcal broth cultures.

Staphylococci produce fibrinolysin which when added to a blood clot dissolves the latter within 24-48 hours.

The Staphylococci produce hyaluronidase which breaks down hyaluronic acid, a component of connective tissue.

Coagulase, fibrinolysin, lecithinase, hyaluronidasa and phosphatasa all belong to the group of enzymes possessing destructive properties. Lecithinase destroys the lecithin protective membranes of the colloidal particles of a substance found in human, sheep, and rabbit erythrocytes. An anticoagulant which inhibits blood coagulation has also been derived from the staphylococcal cultures. This staphylococcal anticoagulant is produced in exudates of inflamed tissues, occurring during staphylococcal infections. Haemagglutmins which cause the agglutination of rabbit erythrocytes have also been found in staphylococcal culture filtrates. Virulent Staphylococci inhibit the phagocytic activity of leucocytes.

Many microbiologists believe that the Staphylococci isolated from patients produce alpha-haemolysins, while the organisms pathogenic for animals (e. g. itesponsible for mastitis in cows) more often produce beta-haemolysin.

Staphylococcal exotoxin, inactivated by treatment with 0.3-0.5 per cent formalin at 37 °C for 7-28 days, and injected parenterally to humans and animals, stimulates the production of a specific antitoxin capable of reacting with the toxin.

Antigenic structure. Polysaccharides A and B have been obtained from a staphylococcal suspension by treating the latter alternately with acid and alkali and removing the proteins with trichloracetic acid.

Polysaccharide A was extracted from pathogenic strains isolated from patients with septicaemia, furunculosis, osteomyelitis, and acute conjunctivitis, etc. Polysaccharide B is found in avirulent, non-pathogenic strains. Polysaccharides A and B differ not only in their serological reactions but also in their chemical structures.

Antigen C, containing a specific polysaccharide, has been recently isolated. Staphylococcal polysaccharides demonstrate a marked type specificity. Even in a 1 :1000 000 dilution they give a distinct precipitin reaction. The protein antigen is common to all species and types of  staphylococci.

Three types (I, II, III) of staphylococci have been revealed by the agglutination test and precipitin reaction. However, quite a number of cultures are unsuitable for serological typing. Recent studies have revealed fifteen type-specific staphylococcal antigens.

Classification. Staphylococci are included in the class Bacteria, family Micrococcaceae, genus Staphylococcus.

According to the contemporary classification, staphylococci are subdivided into three species: S. aureus, S. epidermidis, and S. saprophyticus.

Differentiation of Staphylococci

Note: S, sensitive; R, resistant, NT, not tested.

 

Certain strains of the family Micrococcaceae are strict anaerobes. Peptococcus niger, Peptococcus anaerobius, Peptococcus asaccharolyticus and others are usually conditionally pathogenic for human beings. They live in the mouth mucosa, in the intestine, urogenital tract, and in other parts of the human body. In weakened individuals and in people suffering from chronic diseases the anaerobic micrococci may give rise to various diseases and complications.

Resistance. Staphylococci are characterized by a relatively strong resistance to desiccation, freezing, sunlight, and chemical substances. After desiccation they can survive for more than 6 months. Repeated freezing .and thawing do not kill the organisms. They survive for many hours under direct sunlight. Staphylococci maintain their viability for more than 1 hour at 70 °C. At a temperature of 80 °C they are destroyed within 10-60 minutes and at boiling point, they instantly perish. A 5 per cent phenol solution kills the organisms in 15-30 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant green which is used for treating pyogenic skin diseases caused by these organisms. Staphylococci possess high resistance to antibacterial agents, in 70-80 per cent of cases they are resistant simultaneously to 4-5 agents. Cross resistance to antibiotics of the macrolide group (erythromycin, oleandomycin, etc.) is encountered.

Pathogenicity for animals. Horses, cattle, sheep, goats, pigs, and, among laboratory animals, rabbits, white mice, and kittens are susceptible to pathogenic staphylococci.

An intracutaneous injection of a culture of pathogenic staphylococci produces inflammation and subsequent necrosis in the skin of the rabbit. An intravenous injection of a staphylococcal culture filtrate causes a condition similar to acute poisoning in rabbits, which is characterized by motonc excitation, respiratory disorders, convulsions, paralysis of the hind extremities, and sometimes, by diarrhoea and urine discharge. After complete fatigue the animal shortly dies.

Staphylococci or their toxin will cause vomiting, diarrhoea, and weakness in kittens if introduced per os or intraperitoneally. Functional disorders of the digestive tract arise owing to the effect of  tne enterotoxin which is distinguished from the other fractions of the staphylococcal toxin by its thermoresistance. It withstands a temperature of 100 °C for 30 minutes. The most reliable test for the presence of enterotoxin is an intravenous injection to adult cats.

Pathogenesis and diseases in man. Staphylococci enter the body through the skin and mucous membranes. When they overcome the lymphatic barrier and penetrate the blood, staphylococcal septicaemia sets in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of diseases caused by these organisms. Consequently, staphylococcal diseases should be regarded as toxinfections.

The development of staphylococcal diseases is also influenced by the resulting allergy which in many cases is the cause of severe clinical forms of staphylococcal infections which do not succumb to treatment.

Staphylococci are responsible for a number of local lesions in humans: hidradenitis, abscess, paronychia, blepharitis, furuncle, carbuncle, periostitis, osteomyelitis, folliculitis, sycosis, dermatitis, eczema, chronic pyodermia, peritonitis, meningitis, appendicitis, and cholecystitis.

Diabetes mellitus, avitaminosis, alimentary dystrophy, excess perspiration, minor occupational skin abrasions, as well as skin irritation caused by chemical substances, are some examples of the conditions conducive to the formation of pyogenic lesions of the skin and furunculosis.

In some cases staphylococci may give rise to secondary infection in individuals suffering from smallpox, influenza, and wounds, as well as postoperative suppurations. Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe diseases. Ingestion of foodstuffs (cheese, curds, milk, rich cakes and pastry, ice cream, etc.) contaminated with pathogenic staphylococci may result in food poisoning.

Staphylococci play an essential part in mixed infections, and are found together with streptococci in cases of wound infections, diphtheria, tuberculosis, actinomycosis, and angina.

The wide use of antibacterial agents, antibiotics in particular, led to considerable changes in the severity and degree of the spread of staphylococcal lesions. Growth in the incidence of diseases and intrahospital infections m obstetrical, surgical and children’s in-patient institutions, intensive spread of the causative agent, and increase in the number of carriers among the medical staff and population have beeoted in all countries of the world. Intrauterme and extrauterine contamination of children with staphylococci has been registered, with the development of vesiculopustular staphyloderma, pemphigus, infiltrates, abscesses, conjunctivitis, nasopharyngitis, otitis, pneumonia, and other diseases.

It has been established that staphylococci become adapted rapidly to chemical agents and antibiotics due to the spread of R-plasmids among these bacteria. The high concentration of drugs  in the body of humans and in the biosphere has resulted in essential disturbance in the microflora and the extensive spread of resistant strains possessing more manifest virulence. The L-forms of staphylococci are especially marked by increased degree of resistance to antibiotics.

Immunity. The tendency to run a chronic flaccid course or relapse is regarded as a characteristic symptom of staphylococcal infections. This peculiarity gives a basis for concluding that postmfectional immunity following staphylococcal diseases is of low grade and short duration.

Immunity acquired after staphylococcal diseases is due to phagocytosis and the presence of antibodies (antitoxins, precipitins, opsonins, and agglutinins).

The inflammation restricts the staphylococci to the site of penetration and obstructs their spreading throughout the body. At the centre of inflammation the organisms undergo phagocytosis. Neutralization of the staphylococcal toxin by the antitoxin is an important stage of the immunity complex. As a result of capillary permeability, the antitoxin penetrates from the blood into the inflammation zone and renders harmless the toxin produced by staphylococci. Thus, the phagocytic and humoral factors act together and supplement each other.

The presence in the human organs and tissues of antigens which are also common in the staphylococci (mimicry antigens) is among the causes of low immunity. This causes a state of immunological tolerance to staphylococci and their toxins, which provides favourable conditions for uninhibited reproduction of the causative agent in the patient’s body. The wide use of antibacterial agents promotes intensive selection of staphylococcal strains resistant to the natural inhibitors of the microorganism.

Laboratory diagnosis. Test material may be obtained from pus, mucous membrane discharge, sputum, urine, blood, foodstuffs (cheese, curds, milk, pastry, cakes, cream, etc.), vomit, lavage fluids, and faeces.

 

The material is examined for the presence of pathogenic staphylococci. Special rules are observed when collecting the material since non-pathogenic strains are widespread iature.

Laboratory studies comprise the determination of the main properties of the isolated staphylococci (i. e their morphologic, cultural and biochemical characteristics), as well as their virulence. For this purpose the following procedures are carried out. Smears are made from pus and stained by the Gram method. Pus is inoculated onto blood agar and meat-peptone agar containing crystal violet. In cases of septicaemia blood is inoculated into glucose broth.The isolated pure culture is tested for its haemolytic (by inoculation onto blood agar plates), plasmacoagulative (by inoculation into citrated rabbit plasma), and hyaluronidase activities. Virulence is determined in rabbits by intracutaneous injection of 400 million microbial cells. Necrosis develops at the site of injection within 24-48 hours.

 

Pigment production of the isolated culture is also taken into account. For revealing sources of infection, particularly food poisoning and outbreaks of sepsis in maternity hospitals, serological typing and phage typing are carried out. To ensure effective therapy the isolated cultures are examined for sensitivity to antibiotics.

In cases of food poisoning presence of the enterotoxin in th isolated  staphylococcal culture is tested for by intravenous injection of the culture filtrate to adult cats. In cases when intoxication is due to ingestion of the milk of a cow suffering from mastitis, the culture grown on starch medium is tested directly for toxin production as a means of detecting staphylococci of animal origin. When the causative agent cannot be detected (osteomyelitis and other diseases), the patients’ serum is tested for agglutinins.

Treatment. Staphylococcal diseases are treated with antibiotics (penicillin, phenoxymethyl penicillin, tetracycline, gramicidin, etc.), sulphonamides (norsulphazol, sulphazol, etc.), and antistaphylococcal gamma-globulin.

When tr eating patients suffering from staphylococcal infections one should bear in mind that it is necessary to relieve intoxication and improve the immunological defence forces of the body (infusion of glucose, plasma, blood transfusion, injection of cardiac stimulants).

In cases of chronic staphylococcal lesions specific therapy is recommended: autovaccines, staphylococcal anatoxin, antitoxic serum, and diphage containing staphylococcal and streptococcal phages.

Staphylococd produce strains resistant to sulphonamides, antibiotics, and bacteriophage, which advances the wide distribution of staphylococcal infection. This variability is of particular importance in the therapy of staphylococcal pyogenic diseases. The medical services produce semisynthetic preparations of penicillin and tetracycline which are effectively used for the treatment of these diseases.

Prophylaxis. The general precautionary measures include: hygiene in working and everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and excess perspiration, observance  of rules of hygiene in maternity hospitals, surgical departments, children’s institutions, industrial plants and enterprises, particularly canneries, observance of personal hygiene and frequent washing of hands in warm water with soap.

Routine disinfection of hospital premises (surgical departments, maternity wards) and bacteriological examination of the personnel for carriers of pathogenic staphylococci resistant to antibiotics  are also necessary.

To prevent pyoderma protective ointments and pastes are used at industrial enterprises. For the treatment of microtraumas, besides iodine tincture and alcohol solutions of aniline dyes, solutions are widely applied which dry in one or two minutes and form an elastic film protecting the wound surface from dirt and infection. Patients with bums are kept in soft (plastic) isolating compartments which protect them against the entry of microflora from the external environment. In some cases specific prophylaxis by means of immunization with the staphylococcal anatoxin may be recommended for individuals subject to injury or infection with antibiotic-resistant staphylococci.

 

Streptococci. 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.

 

Figure. Streptococci

 

 

 

Some differential signs of staphylococci are present in table.

 

To perform the plasmoagglutination test, plasma is poured into a test tube and pure staphylococcal culture is ground with a loop at a distance of 0,5 cm from the plasma surface. Then, using a loop, the plasma is transferred to the culture. A positive result is recog­nized by the appearance of agglutinate flakes on the tube wall.

To demonstrate DNase activity, a 24-bour staphylococcal culture is inoculated in the form of small plaques into a Petri dish contain­ing Hottinger’s agar and DNA^[1][1].

Twenty-four hours later 5 ml of 1N HCl solution is introduced into the dish and zones of clearing indicating the presence of DNase are counted in 3-5 min.

Lysozyme activity of staphylococci is considered to be an addi­tional indicator of pathogenicity. To demonstrate it, solid nutrient medium with bacterial suspension of Micrococcus lysodeikticus is inoculated in a patch-like manner with 24-hour agar culture of staphylococcus. Zones of lysis form around the colonies when lysozyme is produced. Under anaerobic conditions pathogenic staphylococci break mannitol to acid.

Examination of cultural phagovar is of great epidemiological significance with regard to identifying the source of infection. The main international set of staphylococcal bacteriophages consists of 21 types divided into four groups, Staphylococcal bacteriophages in two working dilutions 1 TD (test-dilution), which should be no less than 10 ^­­­-3, and 100 TD, not less than 10^–1, are the  ones most com­monly used for typing. Phagotyping is started with 1 TD, then, if the result has been negative, 100 TD is employed.

To diagnose staphylococcal diseases, one can use the  IHA test with an erythrocytic diagnosticum (red blood cells are sensitized with alpha-toxin of the staphylococcus), which allows the detection of antibodies.

Determination of sensitivity of the isolated staphylococci to antibiotics is essential for the purposeful and effective treatment of a given patient.

To isolate a haemoculture of staphylococci, sugar broth is inoculated with blood and incubated at 37 °C for 18-24 hrs. The  staphylococcus causes uniform cloudiness of the medium. On the second day of the study, prepare smears from the blood culture and streak the latter onto a meat-peptone agar slant and a blood agar plate to evaluate the haemolytic activity of staphylococci. On the third day, examine both the staphylococcal culture, which has grown on the agar slant, and the culture that has been isolated from the pus or other mate­rials.

In inoculation of exudate, pus from unruptured abscesses and phlegmons, meat-peptone agar or 5 per cent blood agar is employed. The inoculated cultures are placed into an incubator at 37 °C for 18-24 hrs, pure culture is isolated and then identified by the afore­mentioned methods.

In food intoxications the material to be studied is streaked onto plates with milk-salt and yolk-salt agar and also onto broth con­taining 1 per cent of glucose (for enrichment). Material contaminat­ed with extraneous microorganisms is inoculated onto blood agar containing 6-7 per cent of sodium chloride. Staphylococci demon­strate good growth and haemolytic action (they may propagate in the presence of 12 per cent of salt and 50 per cent of sugar) in this. medium; the development of enterobacteria and bacilli is inhibited, while Proteus is incapable of uninterrupted growth in the  form of a film.

The cultures are placed into an incubator at 37 °C. On the  next day, the colonies are examined, pure cultures are isolated, and staphylococci are identified. Pathogenic staphylococci responsible for food intoxications produce a golden, less commonly, white pigment, liquify gelatin, induce haemolysis, and coagulate plasma.

To establish the production of enterotoxin, the staphylococcal culture is streaked onto a special nutrient medium^[2][2]. The  inoculated cultures are placed into an exsiccator with 20 per cent of CO₂ and in­cubated at 37 °C for 3-4 days, and then filtered through Nos 3 and 4 membrane filters. The obtained filtrate (10-15 ml in total) is mixed with the equal amount of warm milk and fed to 1-2-month-old kit­tens or alternatively the filtrate is introduced into their stomach via a catheter. If enterotoxin is present, the kittens develop vomiting (the key sign of poisoning) in 30-60 min and diarrhoea in 2-3 hours, which persists for 2-3 days and may -result in death in grave cases. The filtrate may also be injected intraperitoneally, following its heating at 100 °C for 30 min to inactivate thermolabile fractions of a toxin. Currently, toxigenicity of isolated staphylococcal cultures is determined in vitro, making use of the  diffuse precipitation test in gel.

Pathogenic staphylococci may harbour in the air of operating-theatres, dressing rooms, post-delivery wards, and other hospital premises. To demonstrate them, air samples are inoculated on yolk-salt agar^[3][3].

Determination of the  staphylococcal antitoxin in the blood serum becomes of great importance in diagnosing chronic staphylococcal infections (osteomyelitis, septicopyaemia, etc.) when bacteriological studies conducted together with aggressive antibiotic therapy yield no results. The reaction is based on suppression of the haemolytic activity of the staphylococcal toxin by the patient’s serum antitoxin. For this purpose, add the patient’s serum diluted 1:5; 1:10; 1:15; 1:20, etc. to the staphylococcal toxin taken in a definite dose, mix thoroughly, and add 0.05 ml of rabbit erythrocytes to the resultant mixture. Incubate the test tubes for 1 h at 37 °C and for 1 h at room temperature, and then read the results.

The amount of serum with which the tested dose of toxin either retards or inhibits haemolysis is taken as the serum titre.

In clinically healthy children and individuals with a history of staphylococcal skin infection or staphylococcal infection that compli­cated surgical disease, the titre of the  staphylococcal antitoxin does not exceed 0,5-4 AU/ml. Patients with chronic staphylococcal infec­tions usually demonstrate higher titres.

 

Summary. GRAM-POSITIVE COCCI. The Gram-positive cocci are grouped together based on their Gram-stain reaction, thick cell wall composition, and spherical shape. Most of the organisms in these groups are members of the Micrococcaceae family. All of the organisms in these groups are non-endospore forming chemosynthetic heterotrophs.

 

 

 

 

 

 

 

 

 

 

 

MICROCOCCUS. Micrococcus is a Gram-positive, aerobic bacterium which is a member of the Micrococcaceae family. Micrococcus cells can be observed under the microscope as spherical cells forming pairs or clusters. If cultured in broth or outrient agar, the colonies may be red or yellow when observed unstained. Although these bacteria are a common human skin contaminant, they are relatively harmless to humans because they maintain a saprophytic lifestyle. They can also be found in freshwater environments or in soil. Three common species of Micrococcus are M. luteus, M. roseus, and M. varians.

LABORATORY INDICATIONS:

Catalase +

Oxidative action on glucose

Growth on mannitol

 

STAPHYLOCOCCUS. Clinically, the most important genus of the Micrococcaceae family is Staphylococcus. The Staphylococcus genus is classified into two major groups: aureus and non-aureus. S. aureus is a the cause of soft tissue infections, as well as toxic shock syndrome (TSS). It can be distinguished from other species of Staphylococcus by a positive result in a coagulase test (all other species are negative).

The pathogenic effects of Staphylococcus are mainly asssociated with the toxins it produces. Most of these toxins are produced in the stationary phase of the bacterial growth curve. In fact, it is not uncommon for an infected site to contaio viable Staphylococcus cells. The S. aureus enterotoxin causes quick onset food poisoning which can lead to cramps and severe vomiting. Infection can be traced to contaminated meats which have not been fully cooked. These microbes also secrete leukocidin, a toxin which destroys white blood cells and leads to the formation of puss and acne. Particularly, S. aureus has been found to be the causative agent in such ailments as pneumonia, meningitis, boils, arthritis, and osteomyelitis (chronic bone infection). Most S. aureus are penicillin resistant, but vancomycin and nafcillin are known to be effective against most strains.

Of the non-aureus species, S. epidermidis is the most clinically significant. This bacterium is an opportunistic pathogen which is a normal resident of human skin. Those susceptible to infection by the bacterium are IV drug users, newborns, elderly, and those using catheters or other artificial appliances. Infection is easily treatable with vancomycin or rifampin.

LABORATORY INDICATIONS:

Anaerobic glucose fermentation with acid production

Catalase +

Nitrate +

Coagulase +

STREPTOCOCCAL INFECTION. In diseases caused by pathogenic streptococci (Streptococcus pyogenes}, the material subjected to the study is pus, blood, mucosal secretions, urine, sputum, and cerebrospinal fluid. The procedure of obtaining the material to be examined is the same as in diseases caused by staphylococci.

Bacterioscopic examination. In Gram-stained smears from pus and mucosal secretions streptococci appear as short chains, less commonly as pairs or individual cocci. In the latter case streptococci cannot be distinguished from staphylococci, and they should be studied for other attributes.

Bacteriological study. Samples of pus, mucosal secretions, urine, sputum, and cerebrospinal fluid are inoculated onto Petri dishes with 5 per cent blood agar (defibrinated blood should be preferably used) and into a test tube with sugar broth. After an overnight incu­bation at 37 °C, the colonies and growth in the sugar broth are stud­ied. Streptococcus pyogenes develops with the formation of small flat rather dry granular colonies.

Streptococci producing beta-haemotoxin (streptolysin) form a zone of haemolysis around the colonies, while alpha-haemotoxin-producing streptococci are characterized by the appearance of green zones around the colonies as a result of methaemoglobin formation. There is no haemolysis in the absence of haemotoxin. In sugar broth strepto­coccal growth appears as flakes or granules on the bottom or walls whereas the medium remains transparent.

Smears from streptococcal colonies display no typical pattern of cocci in the form of chains. Cells are arranged singly, in pairs or in small aggregates. In liquid medium, a cultural smear exhibits typical chains of streptococci.

At further stages of examination the group and serovar of the streptococcus are determined.

Streptococcal groups (by Lancefield) are classified by the presence of a polysaccharide antigen which is revealed using the precipita­tion test with group sera (A, B, C, etc.).

Serovars (serotypes) of streptococci (by Griffith) are characterized by the presence of a specific protein antigen which is determined with the help of the slide agglutination test performed with typical agglutinating sera. For this purpose, a 24-hour broth culture of the isolated streptococcus is centrifuged and the sediment is diluted in 0,5-1 ml of isotonic sodium chloride solution. A drop of serum is mixed with a drop of the tested culture on a glass slide. The majority of pathogenic streptococci belong to group A.

To recover b-haemolytic streptococcus of group A, the immuno-fluorescence test may be employed. In this case a smear is prepared from the isolated streptococcal culture, fixed for 15 min with 95 per cent alcohol, and then stained with fluorescent serum for 15 min. After that, it is washed in running water, dried, and examined under the luminescent microscope.

Blood examination. The procedure of blood inoculation into sugar broth is the same as that employed in staphylococcal diseases. The presence of streptococci is indicated by the formation of floccular sediment on the bottom and haemolysis. Smears are characterized by long chains of streptococci. Microscopic examination allows a pre­liminary conclusion as to the presence or absence of streptococci. To detect haemotoxin, the culture is transferred onto a blood agar plate. Typical small colonies surrounded by a zone of haemolysis or a greenish halo develop in 24 hours (the third day of investigation). These findings permit the conclusion that the culture harbours S. pyogenes.

To isolate anaerobic streptococci (Peptestreptococcus anaerobius), which harbour the genital mucosa and may cause postpartal sepsis, blood is introduced into the Kitt-Tarozzi medium. Some strains of anaerobic streptococci form gas when they get into the liquid media.

Pathogenic streptococci show good growth in Garrod’s medium. It presents meat-peptone agar (pH 7,4) to which 5 per cent of blood and 0,1 per cent aqueous solution of gentian violet are added (0,2 ml per every 100 ml of meat-peptone agar). Growth of saprophytic air microflora and enterococci on Garrod’s medium is suppressed.

In order to determine pathogenic properties of streptococci, for­mation of toxins, in particular of fibrinolysin (streptokinase), is investigated. Human blood plasma is used for this purpose, which is obtained by adding 1 ml of 2 per cent citrate sodium solution to 10 ml of blood. Following sedimentation, the unstained plasma is separated and diluted in a 1 to 3 ratio. At the next stage, 0,5 ml of an 18–20-hour culture of the tested streptococcus and 0,5 ml of 0,25 per cent solution of calcium chloride are added. The test tubes are carefully shaken and placed in a water bath at 42 °C for 20–30 min.

A fibrin clot is formed during this period. The test tubes are left in the water bath for another 20 min. If the streptococcal culture pro­duces fibrinolysin, the clot is dissolved within 20 min.

As some strains of streptococci dissolve fibrin slowly, the test tubes are transferred from the water bath to an incubator two hours later, and the results are read on the following day.

Determination of the amount of superficial M-protein, which is observed only in pathogenic strains, may be considered as a method evaluating the virulence of streptococcal cultures. Young cultures are utilized to obtain hydrochloric extracts, and the content of M-protein is determined in the latter by the precipitation test.

No laboratory studies are usually used in the diagnosis of scarlet fever. Occasionally, secretion of the faucial mucosa is cultivated, and streptococci of various serogroups are isolated.

Haemolytic streptococci releasing a- and P-toxins may harbour the air of various hospital rooms. To demonstrate them, air samples are inoculated onto Garrod’s medium for the subsequent isolation and identification of pure culture.

In laboratory diagnosis, streptococci should be differentiated from enterococci (Streptococcus faecalis} which are characterized by the following specific features: ability to grow at temperatures vary­ing from 10° to 45 °C, resistance to high concentrations of sodium chloride, penicillin, and alkaline medium (pH 9.6). Enterococci occur in diseases affecting the duodenum, gallbladder, and urinary tract. Their presence in the environment serves as a criterion of faecal contamination of drinking water, sewage waters, and food-stuffs.

Serological diagnosis of streptococcal infections is conducted in patients with chronic diseases treated with large doses of antibiotic and sulphanilamide drugs, i.e., when isolation of the causative agent by bacteriological methods proves to be very difficult. It envis­ages identification in the blood of the streptococcal antigen and specific streptococcal antibodies to toxins, in particular to steptolysin 0.

The streptococcal antigen in the patient’s blood serum is demon­strated by complement fixation in the cold. For this test antistreptococcal immune serum is utilized, which is obtained through hyperim-munizing rabbits with streptococcal culture of serological group A. The antigen titre is assumed to be the maximum dilution of the serum tested, which induces the haemolytic inhibition of at least ++ intensity.

Isolation of antistreptolysin O contained in patients’ sera is based on its capacity to neutralize the haemolytic activity of streptolysin O. For this purpose the patient’s serum is diluted and streptolysin O (commercial drug) is added to the obtained dilutions. The mixture is incubated at 37 °C for 15 min and then 0,2 ml portions of rabbit erythrocytic suspension are added to all tubes. The tubes are reincubated for 1 h, and then the results are read. Positive cases are witnessed by the absence of haemolysis.

Techniques of identifying antibodies to other toxins released by streptococci, for example, to antistreptohyaluronidase, are also used for serological diagnosis.

 

INFECTION CAUSED BY STREPTOCOCCI OF PNEUMONIA (PNEUMOCOCCI). Streptococci of pneumonia (pneumococci), Streptococcus pneumoniae, are the causative agents responsible for croupous pneumonia, focal pneumonia, other respiratory diseases, creeping cornea! ulcer, suppurative processes in the middle ear and maxillary sinus, and also for sepsis and meningitis.

Material to be studied includes sputum, pus, cerebrospinal fluid, blood, and post-mortem organs.

Bacterioscopic examination. Two smears are made of the tested material (with the exception of blood); one is stained by the Gram method, the other by the Burri or Kozlovsky technique. Microorgan­isms and Indian ink placed on a glass slide are mixed by circular movements to prepare the conventional smear. Indian ink is di­luted with isotonic saline in a ratio of’ 1 to 4. The smear is dried in the air, left unfixed and stained for 2–3 min with formol-gentian violet (10 ml of 40 per cent formalin and 1,5 g of gentian violet). Formol-gentian violet may be substituted with 0,33 per cent aqueous fuchsine solution and 3 per cent alkaline solution of methylene blue. Microscopic examination reveals unstained capsules which contain violet bacterial cells against a black-gray background. Pneumococci are arranged in pairs, their cells are elongated, resembling a candle flame, and are surrounded with a capsule. Hence, micro­scopic findings seem to suggest the presence of the causative agent. Yet, it is bacteriological investigation that provides most reliable data.

Bacteriological examination. To obtain pure culture, 5-10 ml of blood is inoculated into a serum broth (1 part of serum and 3 parts of meat-peptone broth, pH 7,2–7,4) or into sugar broth, or into a special medium which contains (in 100 ml): 1,8–2,0 ml of agar-agar, 70–75 ml of Hottinger’s hydrolysate or casein hydrolysate (1.8–2.0 g/l of amine nitrogen), 20-25 ml of bovine heart hydrolysate (1,40–1,60 g/I of amine nitrogen), 4-5 ml of defibrinated horse blood 0,5-0,7 ml of baking yeast extract. After 18–24-hour incubation in a heating block, the culture is transferred onto a plate with 10 per cent blood agar. Cerebrospinal fluid is centrifuged and the deposit is inoculated onto a blood agar. Colonies of the pneumococcus re­semble those of the streptococcus: they are small, almost flat, non transparent, with a halo of a green colour or, less commonly, of haemolysis. Another characteristic sign is an impression in the centre of the colony.

As a rule, direct inoculation of the material onto nutrient media (pus, sputum) does not give a positive result since saprophytes, es­pecially saprogenous microorganisms present in them, inhibit the growth of pneumococci. For this reason, pus and sputum are treated before cultivation: clumps are collected and comminuted in a por­celain mortar, then 1 ml of isotonic saline is added, and the resul­tant mixture is injected intraperitoneally to albino mice. Mice are very susceptible to the pneumococcus and die of pneumococcal sep­ticaemia in 18-72 hrs. The mouse’s carcass is dissected and the blood from the heart, pieces of the internal organs, and peritoneal fluid are inoculated into a blood agar plate and a test tube with serum broth.

Morphological and cultural characteristics do not allow any clear-cut differentiation between the pneumococcus and Streptococcus viridans. To achieve this, one can employ the reaction of pneumo­coccal lysis by bile: 1 ml of broth culture is introduced into a sterile test tube and then 0.5 ml of bovine bile is added. Ten-fifteen min­utes of incubation in a heating block is enough to bring about complete lysis of the pneumococci. A tube containing bile-free broth culture serves as a control. Streptococcus viridans whose colonies resemble those of the pneumococcus are not dissolved by bile. If lysis is present, the tested material is inoculated onto the Hiss me­dium. In contrast to the streptococcus, the pneumococcus splits inulin with the formation of oxygen and forms ammonia from arginine.

The conducted study allows the final identification of the isolated microorganism. The factors to be taken into consideration are: a lancet shape of diplococci, the presence of the capsule in the native material, high virulence for albino mice, dissolvement by bile, and inulin splitting.

 

Summary. STREPTOCOCCUS. The Streptococcus genus consists of Gram-positive bacteria which appear as chains under microscopic observation. Members of Streptococcus can be aerobic, anaerobic, or microaerophilic. The organisms in this genus are characterized by a coccus appearance, a thick cell wall, and aerobic action on glucose. Four different classification systems exist:

CLINICAL

Pyogenic Streptococci

Oral Streptococci

Enteric Streptococci

HEMOLYSYS

alpha-hemolysis

beta-hemolysis

gamma-hemolysis

SEROLOGICAL-Lancefield (A-H), (K-U)

BIOCHEMICAL(physiological)

GROUP A. The first group in the Lancefield classification system includes only one species of Streptococcus, S. pyogenes. This particular opportunistic pathogen is responsible for about 90% of all cases of pharyngitis. A common form of pharyngitis is “Strep throat” which is characterized by inflamation and swelling of the throat, as well as development of pus-filled regions on the tonsils. Penicillin is usually administered to patients as soon as possible to quell the possibility of the infection spreading from the upper respiratory system into the lungs. Once in the lungs, the infection could give rise to pneumonia. Some cases also develop into rheumatic fever if left untreated. Other diseases linked to S. pyogenes are skin infections such as impetigo, cellulitis, and erysipelas.

LABORATORY INDICATIONS:

·        Catalase –

·        Beta-hemolysis

·        Bacitracin sensitive

 

GROUP B. The B classification of Lancefield also includes only one bacterium, S. agalactiae. For years this bacterium has been the causative agent in mastitis in cows. Currently, it has been found to be a cause of sexually transmitted urogenital infections in females. Although infection is easily treated with penicillin, proper diagnosis is necessary for womeearing labor because the infection can easily spread to the child via the birth canal.

LABORATORY INDICATIONS:

·        CAMP +

·        Beta-hemolysis

 

GROUP D. Type D Streptococcus is the next clinically important bacterium because of the multitude of diseases it is known to cause. Although many are harmless, the pathogenic strains cause complications of the human digestive tract. This group has recently been reclassified into two divisions: Enterococcus and non-Enterococcus. The Enterococci include E. faecalis, a cause of urinary tract infections, and E. faecium, a bacterium resistant to many common antibiotics. Diseases such as septicemia, endocarditis, and appendicitis have also been attributed to group D Strep. Steptococcus is part of he normal human fecal flora . Once identified, Group D Strep can be treated with ampicillin alone or in combination with gentamicin.

LABORATORY INDICATIONS:

·        Hydrolysis of bile esculin (dark brown medium) -this indicates the ability of the bacteria to tolerate bile

·        Growth in high salt concentration.

 

Gram-negative cocci

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. The G+C content in DNA ranges from 50.5 to 51.3 per cent.

 

 

 

Meningococci in cerebrospinal fluid. The leukocytes have engulfed (phagocytized) large number of the diplococci

 

 

In culture smears, small or very large cocci are seen singly, in pairs, or in fours. Meningococci may vary not only in shape but also in their Gram reaction. Gram-positive diplococci appear among the Gram-negative cells in smears.

Cultivation. 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. 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.

Toxin production. Meningococci produce toxic substances which possess properties of exo- and endotoxins. Disintegration of bacterial cells leads to the release of a highly toxic endotoxin. Meningococci readily undergo autolysis which is accompanied by accumulation of toxins in the medium. The meningococcal toxin is obtained by treating the bacterial cells with distilled water, or 10 N solution of soda, by heat autolysis, by exposure to ultraviolet rays.

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 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.

Pathogenicity for animals. Animals are not susceptible to the meningococcus iatural conditions. The disease can be produced experimentally in monkeys and rabbits by subdural injections of meningococci. Intrapleural and intraperitoneal infection of guinea pigs and mice results in lethal intoxication. Septicaemia develops in experimental animals only when large doses are injected.

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. Ion-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.

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. The G+C content in DNA is 49.5 to 49.6 per cent.

 

Drawing  of doughnut-shaped diplococci of Neisseria gonorrhoeae as they sometimes appear under the microscope.

 

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 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.

Toxin production. 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.

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.

Pathogenicity for animals. Gonococcus is not pathogenic for animals. An intraperitoneal injection of the culture into white mice results in fatal intoxication but does not produce typical gonorrhoea.

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 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.

 

Additional material about diagnosis of Neisseria diseases

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 cerebro­spinal 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.

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 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.

 

Students’ practical activities

1. To carry out precipitation test with a liquor from a patient with an epidemic cerebrospinal meningitis.

Iarrow test-tube pour 0.9 ml of cerebrospinal fluid and carefully, holding under an angle 45⁰, with Pasteur pipette slowly layer on the wall (separately for everyone) 0.1 ml of diagnostic antimeningococcal serum, which precipitated antigens of meningococci. The appearance of precipitate as the white ring on limit of two fluids the response is considered positive.

2. To carry out Bordeux – Gengou complement fixation test for diagnosis  of chronic gonorrhea.

 

The scheme of Bordeux – Gengou complement fixation test

 

In the final reading of the results the intensity of the reaction is expressed in pluses: (++++), a markedly positive reaction charac­terized by complete inhibition of haemolysis (the fluid in the tube is colourless, all red blood cells have settled on the bottom); (+++ , ++), positive reaction manifested by the intensification of the liq­uid colour due to haemolysis and by a diminished number of red blood cells in the residue; (+), mildly positive reaction (the fluid is intensely colourful and there is only a small amount of erythro­cytes collected on the bottom of the tube). If the reaction is nega­tive (–) there is a complete haemolysis, and the fluid in the tube is intensely pink (varnish blood).

The titer of serum is its biggest dilution, which causes complete (“+++” or “++++”) fixation of the complement.

 

References:

1.                     Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 1982, P197-216, 255-263.

2.                     Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed by Yu.S. Krivoshein, 1989, P. 76–84.

3. Wesley A.Volk et al. Essentials of Medical Microbiology. Lippincott – Raven Publishers, Inc., Philadelphia–New York.–1995, p. 349-358.