PROOF OF CAUSATION OF THE DISEASE BY KOCH’S POSTULATES

Students’ seminarFACULTY OF MEDICINE

UNIVERSITY OF PERADENIYA SRI LANKA

• Discriminating between organisms and associating organisms with particular diseases

• formulated a set of conditions that needed to be met in order to ‘prove’ a causal relationship

1843-1910

Koch's postulates 1. The microorganism must be found in abundance in all organisms

suffering from the disease, but not in healthy organisms.

2. The microorganism must be isolated from a diseased organism and grown in pure culture

3. The cultured microorganism should cause disease when introduced into a healthy organism.

4. The microorganism must be re-isolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent.

Which germ?Which disease?

Signature of organism

1. chronic or minor conditions 2. multiple causes 3. pathogen can't be grown 4. no suitable animal model5. Long incubation period

Why may it be difficult to prove causation using Koch’s postulates ?

"Molecular Koch's Postulates":Stanley Falklow (1988)

1. Identify gene (or gene product) responsible for virulence determinant

2. Show gene present in strains of bacteria that cause the disease

3. Not present in avirulent strains

4. Disrupting the gene reduces virulence

5. Introduction of cloned gene into avirulent strain confers virulence.

6. The gene is expressed in vivo

7. Specific immune response to gene protects

• Guillain Barre Syndrome• Hepatitis A• Boil• Leprosy (Hanson’s disease)• Tetanus• Typhoid fever• Yellow fever• Rheumatic Fever

Guillain Barre Syndrome & Campylobacter jejuni

Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter jejuni at 42 C

Scanning electron microscope image of Campylobacter jejuni, illustrating its corkscrew appearance and bipolar flagella.

Evidence of link between campylobacter jejuni and GBS

•For more than 100 years, a variety of preceding infectious diseases have been described in association with GBS.•But the relation with Campylobacter jejuni was discovered recently.•However gastrointestinal illnesses occurring in up to 20% of GBS patients were recognized many decades ago.•Campylobacter infection was first reported as a potential cause of GBS in 1982 in a 45-year-old man who developed severe GBS with irreversible neurologic damage 2 weeks after a gastrointestinal illness caused by Campylobacter infection .

Guillain Barre Syndrome & Koch’s postulates

Koch’s postulates can not be applied to prove the etiological relationship between GBS and Campylobacter jejuni

Koch's postulates 01 In GBS only 40 % of the cases Campylobacter jejuni can be isolated as an antecedent event But in the other cases no such association is found.Campylobacter can be found in patients with no evidence of GBS and in patients with gastroenteritis.

Koch's postulates 02 Only 40% of cases this can be done. It requires special culture media for culture and special conditions. (E.g. Blood-free, charcoal-based selective medium agar (CSM) at 420C)

Organism will also be absent in stool by the time the patient gets the disease.

Koch's postulates 03 It does not cause GBS in all cases only few cases progress in to Guillan Barre syndrome.Only 1:1000 cases will get GBS from C.jejuni infection.

Koch's postulates 04

Development of GBS can be related to the inoculation of C. jejuni in to a healthy individual or it can be a purely an incidental cause.So organism isolation is not always definite.

Guillan Barre Syndrome & Koch’s postulates

Blood-free, charcoal-based selective medium agar (CSM) for isolation of Campylobacter jejuni at 42 C

Scanning electron microscope image of Campylobacter jejuni, illustrating its corkscrew appearance and bipolar flagella.

A patient with Guillain Barre syndrome showing acute flaccid paralysis of lower limbs with areflexia

Robert Koch

Guillan Barre Syndrome & Koch’s postulates

Koch’s postulates can not be applied to prove the etiological relationship between GBS and Campylobacter jejuni

1. Koch's postulates 01 The microorganism must be found in abundance in all organisms suffering from the disease, but should not be found in healthy animals.

In GBS only 40 % of the cases Campylobacter jejuni can be isolated as an antecedent event but in the other cases no such association is found. Campylobacter can be found in patients with no evidence of GBS and in patients with gastroenteritis

2. Koch's postulates 02 The microorganism must be isolated from a diseased organism and grown in pure culture.

Only 40% of cases this can be done. It requires special culture media for culture and special conditions. E.g. Blood-free, charcoal-based selective medium agar (CSM) at 42 C

3. Koch's postulates 03 The cultured microorganism should cause disease when introduced into a healthy organism.

It does not cause GBS in all cases only few cases progress in to Guillan Barre syndrome.

4. Koch's postulates 04 The microorganism must be reisolated from the inoculated, diseased experimental host and identified as being identical to the original specific causative agent

Development of GBS can be related to the inoculation of C. jejuni in to a healthy individual or it can be a purely an incidental cause. So organism isolation is not always definite.

The Discovery of Hepatitis A HAV is a Heat stable, Picornavirus (ssRNA) Mode of transmission by Fecal-oral route Water & food borne disease HAV causes inflammation of the liver Signs & symptoms

Jaundice, Fever, Abdominal pain, Nausea ,Diarrhoea,Sharp pain in right upper quadrant of the abdomen.

During Hippocrates's era - Infectious icterus 1956, Dr. Saul Krugmann - Labelled the of two forms of hepatitis

By their aetiological agents (In 1956 Dr. Krugman conduct a study among mentally retarded, disabled children in Willow brook State School (New York), and he fed & injected infected serum to the children. And ethics of his experiment aroused controversy & it was censured by NY senate.) 1973, Feinstone & Purcell - identified Hepatitis A virus –

1st visualized by Immune electron microscopy

HAV & Koch’s postulates • First postulate –

90% of infected children <= 5 yrs are AsymptomaticNot all the subjects who have infected get the symptoms, infants

• Second postulate –Pure cultures for the viruses were not available during that time. Nowadays HAV grows in primate cell cultures.

(chimpanzees/marmosets)• Third postulate –

No evidence of disease by Pure cultured virus.BUT ;

1944, McCollam, Transmission of the infected serum to volunteers1956, Dr.Saul Krugman, feed & inject infected serum to the

mentally retarded, disabled children in Willow brook State School (New York) and they developed the hepatitis

• Fourth postulate –HAV grows in primate cell cultures. After serial passage, strains

may lose virulence for the liver.

BOIL

• Sub-epidermal collection of pus

• Usually arises around a hair follicle

• This is caused by a bacteria called staphylococcus aureus

Staphylococcus aureus

Koch’s postulates - Proved1. The organism must be present in all cases of

disease.2. The bacteria must be isolated from the

diseased host and grown in pure culture.3. The specific disease must be reproduced

when a pure culture of bacteria is inoculated into a healthy susceptible host.

4. The bacteria must be recoverable from the experimentally infected host.

1878 – Koch saw - staphylococci in pus from boils .

1884 – Rosenbach studied through pure culture and identified Staphylococcus aureus

1885 – Garre rubbed Staphylococcus aureus on the skin of his arm and was able to produce boils.

Leprosy (Hanson’s disease)

1. The micro-organism must regularly be isolated from cause of the illness.2. It must be grown in pure culture in vitro.3. When such a pure culture is inoculated in to susceptible animal species,

the typical disease must result.4. From such experimentally induced disease, the micro-organism must

again be isolated.

1. 1873- Dr.Gerhard Hansen discovered Mycobacterium leprae.2. 1968- Research started on in vivo culturing of the bacterium, in

Armadillos.3. When such an in vivo-cultured organism is inoculated into susceptible

species the particular disease results.4. The particular microbe can be isolated from such a diseased animal.

Koch’s Postulates Evidence/Exceptions

Disease :- Tetanus

Name of Microbe :- Clostridium tetani

Genus of the microbe :- Genus Clotridium

• Widely distributed

• Rod shaped

• Spore forming

• Gram-positive

• Anaerobic

Spores (cultivated fields)

Vegetative microbe

Toxin – tetanospasmin (Tetanospasmin toxemia)

Necrotic & Poorly perfused wounds

Contamination

Colonization

Infection

Common Features (Genus - Clostridum )

Postulate 01 :- agree

- Not included in ‘Normal Microbiota’ (Primary pathogen)

- Production of Highly toxic Tetanospasmin

- When colonized always cause Tetanus

Postulate 02 :- agree

- Can be grown in ‘Pure Culture’ – Needs special conditions

- Very little number of organisms

- Difficulty to locate the place of colonization

Postulate 03 :- agree

Should be inoculated under special conditions

Well perfused and living tissue

No Colonization tissue O2

Postulate 05 :- Does not agree

Very small amount of toxin Disease or death

Little or Inadequate production of the antigen

No immune response when inoculated again

Postulate 04 :- agree

- Can be grown again by specimens taken from experimentally . inoculated patients

- (same principles apply as postulate 02)

Typhoid Fever (Salmonella typhi)

• Characterised by– Sustained fever– Headache– Malaise– Anorexia– Constipation– Diarrhea– Non productive cough– Rose spots

• Number of organisms needed - 103

• Transmitted by– Faecally contaminated

water and food– Via the fecal-oral route

• Affects the – Reticuloendothelial

systemTyphoid Mary !

Koch’s Postulates & TyphoidKoch’s Postulate Agree/

DisagreeReason

1. Presence of microbe at the lesion site and absent in all healthy animals

(Agree) Typhoid MaryTony Labella

2. Ability to isolate from the site and culture

Agree HEK agarMacConkey’s agarRed slant and yellow butt in TSI

3. Ability to reproduce the disease in a susceptible experimental animal

Agree Chimpanzee’s throat By Elie Metchnikoff (1911)

4. Ability to recover from the experimental animal

- -

YELLOW FEVER

• Introduction• History• Virus & vector• How it affects humans• Clinical features

KOCH

Relationship with Koch’s Postulates

• Found in both affected & unaffected people• Is grown in a variety of cultures.• May or may not produce disease.• Isolated from diseased persons.

Virus:

Why cannot it be proved by Koch's postulates?

Can it be proved by Koch's postulates?Can't.........

Is the organism (Streptococcus pyogenes) present in all the affected sites (joints, heart, CNS)?

Can the organism be isolated from the affected sites (joints, heart, CNS)?

If the isolated and cultured organism inoculated in to a susceptible person or animal, does it always cause rheumatic fever?

Rheumatic Fever

How was causation proved?

1960 – Stollerman notedOutbreak of type A streptococcal upper respiratory tract infection followed by outbreak of Rheumatic fever ¹

Streptococcus is not resistant to penicillin and it has not developed into resistant forms!

Pathogenesis of Rheumatic fever?Is Streptococcus pyogenes the only causative agent?

Epidemiological studies

Another observation:

But... Penicillin has clearly failed to eradicate Rheumatic fever ²

Reference: 1 & 2 http://www.heart.bmjjournals.com/cgi/content/full/91/1/3