Diphtheria Bacilli (Study Notes) | Microbiology

Let us make an in-depth study of the diphtheria bacilli. The below given article will help you to learn about the following things:- 1. Morphology of Diphtheria Bacilli 2. Cultivation of Diphtheria Bacilli 3. Pathogenesis and Diseases in Man 4. Therapeutic use of Diphtheria Antitoxin 5. Necessity for Early Diagnosis and Therapy 6. Immunity to Diphtheria and 7. Application to Nursing.

Diphtheria (Corynebacterium Diphtheriae):

Corynebacterium diphtheriae, the causal agent of diphtheria (G.diphtheria-skin, membrane) is usu­ally confined to the respiratory tract. It is rarely found in any part of the body. In general, it is a good exam­ple of respiratory tract pathogen. Diphtheria bacilli was first discovered in 1883-84 by German physicians (Kleb and Loeffler) in bacterial specimens from the throats of patients. These bacilli are sometimes spoken of as Klebs – Loeffler or K-L bacilli.

Morphology of Diphtheria Bacilli:

C. diphtheriae(L. Coryna-club) are pleomorphic, Gram-positive, club shaped bacilli look­ing like Chinese letters. When they are stained by polychrome methylene blue or a selective stain (Neisser or Albert-Laybourn stain), the volutin (metachromatic granule) stains very darkly purple in contrast with the brown or green colouration. So the rods show ‘beaded’ or ‘barred’ appearance. C.diphtheriae are non-motile, non-sporing and non- capsulate. These morphological and staining peculi­arities are very useful in the immediate bacteriologi­cal diagnosis of diphtheria.

Cultivation of Diphtheria Bacilli:

Corynebacteria are aerobes, grow best at 37°C on Loeffler serum slopes, on blood po­tassium tellurite (KTe) agar media on which three types of colony have been recognised as characteris­tic of different biological types designated C. diphtheriae gravis, and mitis have been applied in virtue of the association of these types with severe and mild forms, respectively, of the disease. The gravis type produces relatively large greyish black, flat, lusterless colonies exhibiting often “daisy head” formation. The mitis type yields a convex, small, translucent colony. The intermedius type is represented by rela­tively small, black, lusterless colonies with domed centre and flat, irregular margins like a “poached egg”.

Toxin Production:

Diphtheria bacilli produce a powerful exotoxin with specialised toxic properties, while they remain localised at the site of infection, the diffusible toxin is absorbed into the blood stream and leads to various systemic disturbances of diph­theria (in particular myocardial damage) and to such sequelae as post-diphtheria paralysis.

All strains of diphtheria bacilli, produce the same toxin but it has been claimed that two distinct substances designated by A and B enter into its constitution. Substance A is toxin which is lethal to guinea pig; whereas the sub­stance B is not definitely toxic per se but acts by pro­moting the spread of substance A in the tissues. This toxin is unstable and its potency diminishes on expo­sure to sunlight.

The toxin has two components: toxic and antigenic components. When toxic component is removed, it has only antigenic property which can be used for immunization. The conversion of toxin into toxoid can be carried out most effectively by adding 0.3 per cent of formalin and incubating the toxin with formalin for two to three weeks at 37°C. The detoxified or converted toxin is known as “toxoid”. The toxoid is a valuable immunizing agent for active immunization.

Test for toxigenicity. It is important to deter­mine whether diphtheria bacilli isolated are virulent, i.e., toxigenic to guinea pig. The virulence test is usu­ally done by intradermal injection of pure culture into guinea pig.

Gel diffusion test. The production of toxin by a virulent strain of C.diphtheriae can be determined by the line of precipitation formed, when toxin meets antitoxin in the agar gel diffusion technique (Elek test). Diphtheroids non-pathogenic corynebacteria (C. pseudodiphtherieum and C. xerosis) are frequently found in the normal throat and nose and often mis­taken for C. diphtheriae. The pathogenic activity of C. diphtheriae can be determined by toxigenicity and gel diffusion tests.

Resistance:

C. diphtheriae are relatively resist­ant to harmful environmental factors.They survive for one year on coagulated serum, for two months at room temperature and for several days on children’s toys. They remain viable in the membrane of diph­theria patients for long periods, particularly when the membranes are not exposed to light.They are killed at a temperature of 63°C for 30 minutes (Pasteurization) and by a one per cent phenol solution in ten minutes.

Transmission of Diphtheria:

Patients suffering from the disease and carriers are the sources of in­fection in diphtheria. The disease is transmitted by an air droplet route and sometimes with dust particles. The transmission by various objects (toys, dishes, hooks, towels, handkerchiefs etc.) and foodstuffs (milk, cold dishes etc.) contaminated with C. diphtheriae is also possible. The carriers play an es­sential part in the epidemiology of diphtheria.

Infec­tion is spread by close contact, i.e., schools, fomites and institutions but the actual mode of transmission has not yet been clearly defined. Spread of the con­taminated dust and fomites play an important part than direct droplet infection. Nasal cases and carriers have been recognised as dangerous sources of in­fection. The carriers may be segregated and the susceptible may be protected by combined active- passive immunization. Carriers and cases of diphthe­ria have become rare in communities subjected to mass immunization.

Pathogenesis and Diseases in Man:

In human beings pseudo membranes contain­ing a large number of C. diphtheriae and other bacte­ria are formed at the site of entry of the causative agent (e.g., nose, throat, pharynx, trachea, conjunc­tiva, skin, vulva, vagina and wounds).The exotoxin is of greatest importance in pathogenesis of diphthe­ria. C. diphtheriae is capable of producing hyaluronidase and invading the blood and tissues of patients.

For this reaction, diphtheria is considered to be toxinfection. The toxin produces diphtherial inflam­mation and necrosis in the mucous membrane or skin. On being absorbed, the toxin affects the nerve cells, cardiac muscle and parenchymatous organs and cause severe toxaemia. Deep changes take place in the cardiac muscle, blood vessels, adrenals and in the central and peripheral nervous system.

According to the site of the lesions, faucial diph­theria and diphtheric croup occur most frequently and nasal diphtheria somewhat less frequently. The incidence of diphtheria of the eye, ears, genital or­gans and skin are relatively rare. Faucial diphtheria constitutes more than 90 per cent of all the diphthe­ria cases and the nasal diphtheria takes the second place. The infection becomes more severe in the pres­ence of the pathogenic streptococci and staphyloco­cci. Sometimes, non-toxic diphtheria corynebacterium (diphtheroid) and anaerobic corynebacterium may be responsible for some cases of diphtheria.

Therapeutic use of Diphtheria Antitoxin:

The Unit of Antitoxin:

The potency of diphtheria antitoxin is stated in units. The unit of antitoxin is the amount that counteracts strictly over 100 minimal lethal doses (MLD) of diphtheria toxin. An MLD of toxin is the least amount of toxin necessary to kill a guinea pig in four to five days. A unit of antitoxin, therefore, neutralizes a little over 100 MLD of toxin. The unit is used to express the dosage of antitoxin just as milligrams are used for ordinary medicines.

A strong artificially concentrated diphtheria antitoxin contains 2,000 to 2,500 units per ml. Dosage may range from 20,000 to 100,000 units, depending on age, weight, severity of the attack and time after on-set. The same basic principles are applicable for the use of other antitoxins (tetanus, gas gangrene etc.), though units and dosages are somewhat different for each kind of antitoxin.

Necessity for Early Diagnosis and Therapy:

Any uncomplicated infectious disease caused by toxin producing bacteria can be cured by adminis­tration of sufficient quantities of specific antitoxin in early (first day after onset) stage. The serum therapy is said to be effective if it can neutralize the toxin before it could damage the body cells. Even few hours’ delay in administrating the antitoxin may cre­ate a problem between life and death.

Immunity to Diphtheria:

(a) Passive immunity. A small dose of anti­toxin (500 to 2,000 units) will usually pre­vent the development of diphtheria in those exposed to the disease. This is called a prophylactic or preventive dose. The immunity lasts for two to three weeks only. This is a good representative of the tem­porary nature of passive artificial immu­nity. This type of preventive measure is usually undertaken to those immediately exposed to a case of diphtheria, e.g., pub­lic health nurse and doctor.

Serum Sickness may sometimes develop after the administration of antitoxin, because of the mani­festation of allergy to the foreign protein in the horse serum containing the antitoxin. The use of horse se­rum sensitizes the patient, usually for life time, to other antisera derived from horses (e.g., tetanus antitoxin), hence it can be used judiciously for prophylaxis until unless it is indicated.

Active immunity:

Individuals having little or no antitoxin in the blood stream, e.g., as indicated by a positive Schick test, may be actively immunized by the injection of one of the following preparations:

1. Formol-Toxoid (FT) diphtheria toxin is ren­dered non-toxic by formalin. The recommended dose is three intramuscular or deep subcutaneous injec­tions, each of 1 ml, at four weekly intervals. Recent studies proved it is a poor antigen.

2. Toxoid-Antitoxin floccules (TAF). A suspen­sion of the precipitate floccules formed when toxoid and antitoxin are mixed in appropriate “neutralizing” amounts. Its tendency to cause reactions is slight and a good immunity follows the injection of three doses, each of 1 ml given at intervals of four weeks. TAF has been recommended for immunization of adults or adolescents.

3. Alum Precipitated Toxoid (APT). A suspen­sion of washed precipitate produced by the addition of small amount of aluminium hydroxide to toxoid is gradually liberated from the site of injection. Reac­tions are negligible in children under eight years, but in older children and adults they may be somewhat more severe than with TAF :Two doses, each of 0.5 ml, are given at an interval of four weeks, but the interval may be lengthened, e.g., to 3-8 months with­out any loss of antibody response to the second dose.

4. Another alum containing prophylactic is a suspension of purified toxoid absorbed on hydrated aluminium phosphate (PTAP) and this preparation is more potent and more reproducible antigen than APT. However, with alum-absorbed antigens, there is slight risk of provocation poliomyelitis in susceptible indi­viduals.

When an outbreak of diphtheria has occurred in a school or institution, the susceptible contacts should, after preliminary Schick testing, be given passive im­munity by the injection of a small dose of antitoxin (500-1,000 units) followed by active immunization which may be given immediately or after two weeks.

Triple vaccine or triple toxoid (DPT). It is more common practice to combine diphtheria and tetanus toxoids with pertussis (whooping cough). So the vac­cine is a triple vaccine (DPT) or triple toxoids. The three mixed anti­gens, tetanus, diphtheria and pertussis, act together than anyone of the three above antigens. One prod­uct, which includes also Salk, (killed) poliovirus vac­cine, has been introduced. All are very effective.

Primary and Secondary stimulus:

The two or three initial doses in infancy constitute a primary stimulus. Antibodies formed due to primary stimulus may circulate in the blood and disappear with time. The person who had primary stimulus may respond quickly to secondary stimulus which may be due ac­tual infection: tetanus, diphtheria, pertussis or polio. The previously immunized person is usually able to respond quickly with the production of specific anti­bodies. Immunized persons may rarely get diphthe­ria, pertussis, tetanus, poliomyelitis, even though they did not have a booster dose at all.

Therefore, every child receives at least primary stimulus with multiple antigens early in life and a second or “booster” stimu­lus a year later and again at the time of entering the school. Antibodies derived from immune mother’s blood may protect the infant up to 2-4 months of age against many diseases (including diphtheria).There- fore, children are naturally protected against diph­theria by mother antibodies against diphtheria; but it is essential to immunise artificially after this period.

Schick Test:

For the first time Viennese physi­cian put into practice the Schick test. This test can be used to detect the presence or absence of significant amount of diphtheria antitoxin the blood and can be carried out by injecting a minute quantity of diph­theria toxin (1/50 MLD — minimal lethal dose) intradermally.

If the person has no antitoxin in the blood (i.e., is not immune), a red area or wheal ap­pears at the site of injection within 24 hours and spreads for a centimeter or more. This reaction is due to irritating effect of the toxin on the cells of skin. In five days, the area becomes slightly pigmented and later scale is formed and it falls off. This test should not be read earlier than fifth day of the injection, be­cause a temporary irritation (pseudo reaction), caused by foreign substance present in the toxin, persists. An area that persists after five days and undergoes the changes is a positive reaction (i.e., the person has insufficient antitoxin in the blood and may be sus­ceptible to diphtheria if under fifteen years of age).

If an individual has antitoxin in his blood, this antitoxin will neutralize the toxin injected in the Schick test and the skin remains normal. This is a negative Schick reaction and it indicates that the individual has sufficient antitoxin (about 0.01 ml. unit per ml. of blood) to protect him from diphtheria. This testis harm­less, painless and leaves no scar.

Immunization of Nurse:

At the beginning of the training, every student nurse should be protected compulsorily against diphtheria by active immunization, though her first year training does not mainly involve with the nursing care of the contagious dis­ease patients.

Application to Nursing:

The nurse should know very well that she may get infected while caring for a diphtheria patient. Therefore, she must wear the mask for her own pro­tection from this highly infectious disease. The respi­ratory secretions and saliva are highly infectious, so all these materials and contaminated objects should be destroyed or completely burnt.

The professional nurse should have knowledge that no antiseptics (e.g., in the form of gargles etc.) must be applied within twelve hours before the col­lection of samples. The lesion in the throat can be clearly viewed by depressing the tongue with tongue depressor or table spoon, then to obtain a specimen of the throat secretion or lesion, she must rub with a sterile throat swab over the affected area in the fau­cial region of the throat or where there is no defi­nitely localised lesion, over the pseudo membrane of the pharynx and tonsils.

She should label the throat swab test tube with the patient’s name, the date and the time. If the patient has been on antibiotic or sulphonamides within five days, this information should be sent to the laboratory with the throat swab since these drugs may inhibit the growth in the cul­ture.

If the diphtheria patient is admitted in a special or isolation ward of the hospital, and whenever the same patient is discharged, the terminal disinfection of the room should be carried out under the observa­tion of the well trained intelligent knowledgeable nurse. This technique is always followed in the hospi­tal.

School nurse should assist the school doctor in the segregation, isolation and immunization proce­dure during an epidemic of the diphtheria in the school or other institution. Besides she should be very careful to see that the nasal secretion and saliva are properly disposed off.