Diphtheria. Tuberculosis. Leprosy. Anaerobic infections. Spirochetes.

Lesson 1.

Theme: «Diphtheria. Tuberculosis. Leprosy.»

1. Corynebacterium diphtheriae.

 

Morphology.

The C.diphtheriae is a slender rod. Organization of cells – in hieroglyphs, latin letters: X, V, Y, L. The bacteria are pleomorphic. They are Gram positive, nonsporing, noncapsulated and nonmotile. Granules composed of polymetaphosphate are seen in the cells. Stained with methylene blue (Nasser stain), the granules take up a bluish purple colour and hence they are called metachromatic granules. They are also called volutin granules.

Cultural characteristics.

Growth is scanty on ordinary media. Enrichment with blood, serum or egg is necessary for good growth. The optimum temperature for growth is 37⁰C. Based on colonial morphology on the blood-tellurite medium and other properties, McLeod classified C.diphtheriae into three types: gravis (R-form of colonies), intermedius (S – R form) and mitis (S –form).

Biochemical reactions.

C.diphtheriae ferment with the production of acid, (but no gas) glucose,

sucrose. They have enzymes: urease and cystinase.

Antigenic structure.

1. O-antigen.

2. K-antigen (58 serovars).

Pathogenicity.

1. Adhesion (pili).

2. Enzymes: hyaluronidase, neurominidase, protease, DNA-ase.

3. Toxins:

·        Histotoxin (exotoxin).

·        Hemolysin.

·        Necrotoxin.

The toxigenicity of the C.diphtheriae depends on the presence in it of corynephages (tox+), which act as the genetic determinant controlling toxin production. Nontoxigenic strains may be rendered toxigenic by infecting them with

corynephage tox+ . This is known as lysogenic or phage conversion. The toxigenicity remains only as long as the bacteria is lysogenic (have corynephage tox+).

The diphtheria toxin consists of two fragments, A and B. Both fragments are necessary for the toxic effect. All the enzymatic activity of the toxin is present in fragment A. Fragment B is responsible for binding the toxin to the cells.

The site of infection may be:

1. faucial                      5. conjunctival

2. laryngeal                  6. genital-vulval, vaginal or prepucial

3. nasal                         7. cutaneous

4. otitic

The source of infection. patient or bacteriocarrier.

The mode of infection: inhalation, rarely by contact and ingestion.

Diphtheria is a toxemia. The bacteria remain confined to the site of entry, where they multiply and form the toxin. The toxin causes local necrotic changes and the resulting fibrinous exudate, together with the disintegrating epithelial cells, leucocytes, erythrocytes and bacteria, constitute the pseudomembrane, which is characteristic of diphtheritic infection. The mechanical complications of diphtheria are due to the membrane while the systemic effects are due to the toxin.

Laboratory diagnosis.

1. Bacteriological method (main). Material for diagnostics: nose swab and nasopharyngeal swab. Identification bacteria by morphologycal, cultural, biochemical properties and production histotoxin.

 For determination histotoxin (exotoxin) use: precipitin reaction in gels, EIA, reaction passive hemagglutination and genetic method – PCR.

Specific prophylaxis.

1. Adsorbate diphtheria inactivated toxin (toxoid).

2. Adsorbate diphtheria, tetanus inactivated toxin (toxoid).

3. Adsorbate pertussis, diphtheria, tetanus vaccine which consist of: killed B.pertussis vaccine, diphtheria and tetanus toxoid (triple vaccine)

4. Tetracoc vaccine – for prophylaxis diphtheria, tetanus, pertussis and poliomyelitis.

Specific treatment.

Antidiphtheritic antitoxic serum (from blood of house).

2. Mycobacterium.

Mycobacterium are cause different diseases:

·        Tuberculosis

·        Disease resembling tuberculosis

·        Leprae

The main agent of tuberculosis (in human) – Mycobacterium tuberculosis.

Morphology.

M.tuberculosis is a straight or slightly curved rods, occuring singly, in pairs or as small clumps. Sputum smears are stained by the Ziehl – Neelsen technique. They are acid fast bacteria, nonmotile, noncapsulated and nonsporing.

Cultural characteristics.

They are facultative anaerobes. The bacteria grow slowly. Colonies appear in about 3-4 weeks. Growth on solid media contain egg (Lowenstein). On Lowenstein media, M.tuberculosis forms dry, rough, colonies with a wrinkled surface. They are creamy white coloured.

Pathogenicity.

Cord – factor (antiphagocytic and cytotoxic action).

M.tuberculosis cause in human organism delayed hypersensitivity.

Source of infection: patient.

The mode of infection.

1. by direct inhalation of aerosolised bacteria contained in droplet nuclei of expectorated sputum.

2. prolonged contacts with patient.

3. by ingestion, for example, through infected milk.

The main clinical form of disease: pulmonary tuberculosis.

Pathogenesis.

Bacteria reaching the lungs are ingested by the alveolar macrophages. The essential pathology in tuberculosis is the production in infected tissues of a characteristic lesion, the tubercle. This is an avascular granuloma composed of a central zone containing giant cells, with or without caseation, and a peripheral zone of lymphocytes and fibroblasts.

Clinical forms of tuberculosis.

Clinical forms	Material for diagnostics
I. Primary.
1. Pulmonary tuberculosis	Sputum
2. Intestinal tuberculosis	Serum
3. Skin tuberculosis	Discharge from ulcer
II. Secondary.
1. Osteoarthritic tuberculosis	Arthritic fluid, serum
2. Renal tuberculosis	Urine, serum
3. Tuberculosis of genital system	Sperm, prostatic secretion, serum, biopsic of endometrium
4. Tuberculous meningitis	Liqour (cerebrospinal fluid)

Laboratory diagnosis.

1. Microscopic method:

·        Smears are stained by Ziehl-Neelsen technique. M.tuberculosisis acid-fast bacteria.

·        When several smears are to be examined daily, it is more convenient to use fluorescent microscopy. Smears are stained with auramine.

2. Bacteriological method (main). M.tuberculosis growth on Lowenstein media.

3. Allergological method (Mantoux test with tuberculin).

4. Biological method is used for cultivation of M.tuberculosis in laboratory animals (guinea pig) - rarely.

5. Serological method – reaction passive hemagglutination, EIA, complement fixation test (for diagnostics extrapulmonary tuberculosis).

6. Genetic method – PCR.

Specific prophylaxis.

Immunoprophylaxis is by intradermal injection of the live attenuated vaccine developed by Calmette and Guerin, the Bacilli Calmette Guerin, or BCG.

3. Mycobacterium leprae.

M. leprae – agent of leprosy.

Morphology.

M.leprae is a straight or slightly curved rod, nonmotile, noncapsulated and nonsporing. It is acid – fast (smears are stained by Ziehl-Neelsen technique). They are intracellular parasites with organization of microbe s cells – in sphere or parallel.

Cultural characteristics.

M.leprae don t grow on nutritional media.

They may be cultivated only in laboratory animals (armadillos).

Source of infection: patient.

The mode of entry may be either through the respiratory tract or through the skin.

Leprosy is a chronic granulamatous disease of humans primarily involving the skin, peripheral nerves and nasal mucosa but capable of affecting any tissue or organ.

The incubation period is very long and average 2 - 5 years. It has been estimated to vary from a few months to as long as 30 years.

The main clinical form of leprosy:

·        Lepromatous.

·        Tuberculoid.

·        Indeterminate.

Laboratory diagnosis.

1. Microscopic method – smears are stained by Ziehl – Neelsen technique. Material for diagnostics: scrape of mucous membrane of nose, puncture of lepromatous nodes.

2. Serological method – EIA.

3. Allergological method (Mitsuda test with lepromin).

Lesson 2.

Theme: «Nonsporing anaerobes. Clostridium».

1. Nonsporing anaerobes.

Anaerobic bacteria are normally present on the skin, mouth, nasopharynx and upper respiratory tract, intestines and vagina. It is can cause different disease after trauma, IDD, operative procedure.

The main nonsporing anaerobes.

Name	Morphology	The main types
I. Gram negative.
1. Bacteroides and Prevotella	Pleomorphic rods	B.fragilis P.melaninogenicus
2. Fusobacterium	Long, thin, or spindle shaped bacteria with pointed ends.	F.necroforum F.nucleatum
3. Veillonella	Diplococci (coffe-bean shaped)	V.parvula
II. Gram positive.
4. Propionibacterium	Rods	P.acnes
5. Eubacterium	Pleomorphic rods	E. foadans
6. Actinomyces	Thin filaments	A.israelii
7. Bifidobacterium	Pleomorphic rod that shows true and false branching	B.bifidum
8. Lactobacillus	Pleomorphic rods	L.casei
9. Peptococcus	Cocci, organization of cells – grape-like clusters	P.niger
10. Peptostreptococcus	Cocci, organization of cells – in chains	P.anaerobius

Pathogenicity.

1. Adhesion.

2. Capsule.

3. Enzymes (hyaluronidase, neurominidase, fibrinolysin).

4. Endotoxin.

Clinical forms.

1. Peritonitis, appendicitis, abscess of liver, abscess of lung.

2. Endometritis, septic abort.

3. Sepsis, endocarditis, osteomyelitis.

4. Phlegmon, abscess.

Laboratory diagnosis.

1. Microscopic method.

2. Bacteriological method (main).

Plates are incubated at 370C in an anaerobic jar with gas mixture (80%N₂, 10%H₂ and 10%CO₂).

Material for diagnostics: blood, pus, puncture from abscess, discharge from ulcer, etc.

2. Clostridium perfringens.

The main agent of gas gangrene: Clostridium perfringens. Rarely disease may be cause: Cl.novyi, Cl.septicum, Cl.histolyticum.

Morphology.

Gram positive rods. They are nonmotile, but capsulated and sporing. Spores are central or subterminal. Diameter of spores is greater than diameter of cell.

Cultural properties.

Kitta-Tarocci medium, Vilsone-Bler medium, medium with milk, Ceyssler agar are used for cultivation C.perfringens.

Antigenic structure.

Cl.perfringens strains are classified intosix types, A to F, based on the toxins they produce. Cl.perfringens types A is the predominant agent causing gas gangrene.

Pathogenicity.

1. a-toxin (lecithinase C)

·        Dermonecrotic action.

·        Lysis of erythrocytes.

·        Damage lipid membrane of nervous cells.

2. Collagenase, hyaluronidase, DNA-ase.

3. Enterotoxin (exotoxin). This toxin is responsible for the manifestations of food poisoning.

Natural reservoir of infection: soil.

The mode of infection: contact.

Clostridia usually enter the wounds along with implanted foreign particles such as soil. Gas gangrene is characteristically a disease of war. Infection may at times be endogenous.

Clinical forms of gas gangrene.

1. Necrosis of muscle tissues.

2. Accumulation of gas in damage tissues (Crepitus symptom).

3. Oedema of tissues.

4. Sepsis and toxic shock syndrome.

Laboratory diagnosis.

1. Bacteriological method (main).

2. Microscopic method. Gram stained films show large numbers of Gram positive rods in the absence of leucocytes.

Material for diagnostics:

·        Films from the muscles.

·        Exudates.

·        Necrotic tissue and muscle fragments.

·        Blood.

3. Clostridium tetani.

Morphology.

Gram positive rods. It is noncapsulated and motile by peritrichate flagella. The spores are spherical, terminal and bulging, giving the bacillus the characteristic «drumstick» appearance.

Cultural properties.

C.tetani growth on Kitta-Tarocci medium.

Pathogenicity.

Cl.tetani produces at least two distinct toxins – a hemolysin (tetanolysin) and a powerful neurotoxin (tetanospasmin).

Natural reservoir of infection: soil.

The source of infection: contact. C.tetani penetrate through damage skin and mucous membrane (scratch, abrasion on extremity).

Pathogenesis.

1. Deep wound and inflammatory response.

2. Anaerobic conditions.

3. Very limited infection: disease is an intoxication.

4. Toxin production locally, spread through body intraaxonally.

5. Causes spaatic paralysis, opisthotonus.

Laboratory diagnosis.

1. Bacteriological method (main).

2. Microscopic method.

3. Neutralisation test, reaction passive hemagglutination – for determination of toxin C.tetani.

Specific prophylaxis.

1. Planned

·        Adsorbate diphtheria, tetanus inactivated toxin.

·        Adsorbate pertussis, diphtheria, tetanus vaccine (triple vaccine).

·        Tetracoc vaccine.

4. Clostridium botulinum.

Morphology.

It is a Gram positive bacillus, noncapsulated, motile by peritrichate flagella, producing subterminal, oval, bulging spores.

Cultural properties.

C.botulinum growth on Kitta-Tarocci medium.

Antigenic structure.

Seven types of C.botulinum have been identified (A – G) based on the immunological difference in the toxins produced by them.

Pathogenicity.

1. Exotoxin (neurotoxin)

·        Termostable

·        Stable to gastric juice and intestinal enzymes.

Natural reservoir of infection: soil.

The source of infection.

1. by ingestion of contaminated food (fish, meat, vegetables).

2. contact (C.botulinum enter the wounds with soil).

Pathogenesis.

Toxin→ blood→ central nervous system→ damage of nuclei of craniocerebral nerves (III, IV, VI, VII, IX)→ appear paresis and paralyses of muscle.

Clinical symptoms.

Vomiting, thirst, constipation, ocular paresis, difficulty in swallowing, speaking and breathing.

Laboratory diagnosis.

Material for diagnosis:

·        Blood – for determination of toxin.

·        Feces – for determination of C.botulinum.

·        Vomitus – for determination of toxin.

·         

1. For determination toxin – neutralisation test, reaction passive     hemagglutination.

2. For determination C.botulinum – bacteriological method.

Lesson 3.

Theme: «Spirochetes».

1. Treponema pallidum.

Morphology.

Treponema – short with tight uniform spirals. They are noncapsulated and nonsporing. It is actively motile (by microfibrilla, which found under cell wall). Its motility can be seen under the dark-field microscope. By Romanovsky-Giemsa stain T.pallidum have light rose colour.

Cultural properties.

They are anaerobes. Growth on nutritional media very bad.

Pathogenecity.

1. Adhision.

2. Invasion.

T.pallidum – agent of syphilis.

Source of infection: patient.

The mode of infection.

·       sexual contact

·       transplacental transmission

·       intranatal

·       blood transfusion

Syphilis.

Primary syphilis – is the chancre (ulcer) at the site entry of the spirochete. The regional lymph nodes are swollen, discrete, rubbery and nontender.

Secondary syphilis set in 1-3 months after the primary lesion heals. During this interval the patient is asymptomatic. Roseolar or papular skin rashes and generalized lymphadenopathy are the characteristic lesions.

After the secondary lesions disappear, there is a period of quiescence known as «latent syphilis». Diagnosis during this period is possible only by serological tests. In many cases, this is followed by natural cure but in others, after several years, manifestations of tertiary syphilis appear. The main clinical symptoms of tertiary syphilis: gumma in organs and bone. Gumma is granuloma which can destroy and cicatrize.

Damage of nervous system named neurosyphilis (tabes dorsalis or general paralysis)

Innate syphilis.

The mode of infection: transplacental transmission.

·        Early innate syphilis. The main clinical symptoms: papular skin rashes, damage of mucous membrane of nose, hepatosplenomegaly, hydrocephaly.

·        Late innate syphilis:

1. Pathology of tooth.

2.Parenchymal keratitis.

3. Labyrinthiform deafness.

Laboratory diagnosis.

1. Microscopic method

·        Dark-field microscopy – for determination motility.

·        Light microscopy – use Romanovsky-Giemsa stain).

2. Serological method.

3. Genetic method – PCR.

Laboratory diagnosis of primary syphilis.

1-3 week of disease.

1. Microscopic method (main). Material for diagnostics: discharge from chancre.

2. Serological method (additional): immunofluorescence test, EIA – for determination IgM.

4 week of disease.

1. Serological method (main) – for determination IgG.

  Use next serological reactions:

·        Complement fixation test (Wassermann test) – with treponemal and cardiolipin antigens.

·        Reaction immobilization with Treponema from testicle of rabbit and complement.

·        EIA.

·        Reaction passive hemagglutination.

·        Immunofluorescence test.

2. Microscopic method (additional).

Laboratory diagnosis of secondary and tertiary syphilis.

1. Serological method (main) – for determination IgG.

2. Microscopic method (additional).

Laboratory diagnosis of innate syphilis.

1 – 3 month.

1. Serological method (main) – for determination of IgM in serum of newborn.

·        Immunofluorescence test.

·        EIA.

2. Microscopic method.

4 month.

Serological method – for determination of IgG in serum of newborn.

2. Borrelia.

Latin names: Borrelia recurrentis, B.duttoni, B.hispanica, B.persica, B.caucasica, B.burgdorferi.

Morphology.

They have spirals form (spirochetes), with irregular, wide, open coils. They are noncapsulated and nonsporing, but motile (by microfibrilla). By Romanovsky-Giemsa stain Borrelia have blue-violet colour.

Cultural characteristics.

They are obligate anaerobes. Cultivation is difficult.

Antigenic properties.

Borrelia readily undergoes antigenic variations in vivo and this is believed to be the reason for the occurrence of relapses in the disease.

Pathogenicity.

1. Invasion.

2. Endotoxin.

3. Antigenic variations.

B.burgdorferi – the causative agent of Lyme disease.

The source of infection: rodents, deer, elk.

The mode of infection.

Borrelia burgdorferi transmitted by the bite of Ixodid ticks.

Pathogenesis.

Lyme disease occurs in three stages:

1. Localised infection – appears as an expanding annular skin lesion (erythema migrans).

2. Disseminated infection – fever, headache, myalgia, arthralgia and lymphadenopathy.

3. Persistent infection – chronic arthritis, polyneuropathy, encephalopathy and acrodermatitis.

Laboratory diagnosis.

1. Microscopic method.

   Material for diagnistics: liquor, synovial fluid.

   Smears are stained by Romanovsky-Giemsa technique.

2. Serological method: immunofluorescence test (indirect), EIA.

3. Genetic method – PCR.

  Borrelia recurrentis – agent of epidemic relapsing fever.

The source of infection: patient.

The mode of infection.

Infection is transmitted to humans through the bite of louses.

Pathogenesis.

 After an incubation period of 2-10 days relapsing fever sets in as fever of sudden onset. During this period, borreliae are abundant in the patient s blood. The fever subsides in 3-5 days. After an afebrile period of 4-10 days during which borreliae are not demonstrable in blood, another bout of fever sets in. The borreliae reappear in blood during the relapsesof fever. The disease ultimately subsides after 3-10 relapses.

Laboratory diagnosis.

Material for diagnostics: blood (when rased temperature).

The main method for diagnostics: microscopic.

B.duttoni, B.hispanica, B.persica, B.caucasica – agent of endemic relapsing fever.

Reservoir of infection: rodents, ticks.

The mode of infection: infection is transmitted to humans through the bite of ticks.

3. Leptospira.

Leptospira interrogans: agent of leptospiral infection.

Morphology.

They are noncapsulated, nonsporing, but motile (by microfibrilla). By Romanovsky-Giemsa stain Leptospira have pink-violet colour.

 

Cultural characteristics.

Leptospira are microaerophilic. Optimum temperature for cultivation 28-30⁰C. Leptospires can be grown in media enriched with rabbit serum.

Pathogenicity:

1. High invasion (L.interrogans penetrate through slight damage skin and mucous membrane).

2. Endotoxin.

The source of infection: wild and domestic animals.

The mode of infection:

1. Contact. Humans are infected when the leptospires in water contaminated by the urine of carrier animals enters the body through cuts or abrasions on the skin or through intact mucosa of mouth, nose or conjunctiva.

2. Alimentary (through contaminated food).

Laboratory diagnosis.

1. Microscopic method. Material for diagnostics: blood, liquor, urine.

2. Bacteriological method.

3. Serological method (reaction agglutination and lysis of leptospira, CFT, RPHA, EIA).