The presentation about Molecular diagnostic of anthrax.the diagnostic based on convention method and immunological method

1. MOLECULAR
DIAGNOSTICS OF Bacillus
anthracis
JEGANATHAN C
IV –YEAR (2014-2019)
Dept Of Biomedical Science
Bharathidasan university
jeganathanbms@gmail.com
9626307988

2. INTRODUCTION
Anthrax (splenic fever), is an acute
infectious disease caused by the
bacteria Bacillus anthracis.
Highly lethal in some forms.
Primarily a disease of domesticated
and wild animals, particularly
herbivores.
Humans become infected when
brought into contact with diseased
animals.

3. Bacillus anthracis
 Gram-positive rods with square end
shape, tending to form long chains
(Bamboo cane like)
 Non motile
 1 - 1.2µm in width x 3 - 5µm in length.
 Originates from the Greek word
anthrakis ( (ἄνθραξ), meaning coal.
 Normally rest in spore form in the soil,
and can survive for decades in this state.
 The spores are ellipsoidal, located in the
middle of the vegetative form, without
enlarging the original form.
 Weapon of bioterrorism.

4. TYPES OF ANTHRAX
 Cutaneous (skin) anthrax
 Inhalational anthrax(also known
pulmonary anthrax)
 Gastrointestinal anthrax

7. DIAGNOSIS
 Conventional Method
 Molecular Method (immunologic
method)

8. CONVENTIONAL METHOD
 Direct Gram’s-stained smear of a skin
lesion (vesicular fluid or eschar), csf, or
blood showing encapsulated, broad,
gram-positive bacilli.
◦ - Indicators of growth apparent on
sheep’s-blood–agar cultures —
nonhemolytic colonies and large,
nonmotile, nonencapsulated, gram-
positive, spore-forming
◦ rods. Growth does not occur on
MacConkey agar.

9. ◦ Confirmatory diagnostic tests are
performed at a level B laboratory of the
Laboratory Response Network for
Bioterrorism (LRN), where the growth
of
◦ virulent strains on nutrient agar in the
presence of 5 percent carbon dioxide
(or other basal mediums supplemented
with 0.8 percent sodium bicarbonate)
◦ produces heavily encapsulated bacilli
that may be visualized
◦ with India-ink staining.

10. - Susceptibility to lysis by gamma phage
or
◦ - direct fluorescence-antibody staining
of cell-wall polysaccharide
◦ antigen

12.  B. anthracis is a non-fastidious
organism and can grow on simple
laboratory media. They are facultative
anaerobes. The optimum temperature
for growth is 37°C and the pH, 7.0–7.4.

13. CULTURE
CHARACTERISTICS
 Nutrient agar- After overnight
incubation at 37oC colonies are large
2–3 mm in diameter, irregular, raised,
dull, opaque and grayish white with
‘frosted glass’ (ground glass)
appearance
 Sheep blood agar- On sheep blood
agar the colonies are nonhaemolytic
2-3mm in diameter

15. SELECTIVE MEDIA
 PLET medium- PLET (polymyxin-
lysozyme-EDTA-thallous acetate) is
a selective media used for the
isolation of Bacillus anthracis from
contaminated specimens. PLET Agar
inhibits most contaminating organisms
and spore-formers closely related to
B.anthracis , such as B. cereus

16.  It consists of heart infusion agar with
polymyxin, lysozyme, ethylene diamine
tetra acetic acid (EDTA) and thallous
acetate. After incubation at 37°C for 36–
48 hours, the colonies of B. anthracis
are 1–3 mm, roughly circular, creamy
white with ground-glass texture

18. SPECIAL FEATURES
 Susceptibility to penicillin G- B.
anthracis is almost always susceptible to
penicillin, as shown by susceptibility to
penicillin G 10 units discs on Mueller-
Hinton agar. In contrast the non-
pathogenic Bacillus species are more
generally resistant to penicillin.
 Susceptibility to gamma bacteriophage-
Gamma Phage has the ability to lyse B.
anthracis grown aerobically on blood or
other nutrient agar and rarely lyses any
other Bacillus species.

19. MOLECULAR METHOD
 - In cases of cutaneous anthrax, antibodies to
protective antigen or to the capsule develop
in 68–92% of Patients.
 - In one study of 12 patients with confirmed
cutaneous anthrax, 11 had a positive titre (>
1/128) to protective antigen by
electrophoretic immunotransblotting and

 - 11 were positive (cut off point, 1/32) for
anticapsule antibodies as measured by
enzyme linked immunosorbent assay.
Samples were taken six weeks after
development of the disease

21. PATHOGENESIS
 Major virulence factors are encoded
on two virulence plasmids, pXO1 and
pXO2.
pX01:
 184.5 kilobase pairs (kbp) in size
 codes for the genes that make up the
secreted exotoxins.
82.7 kDa protective antigen (PA)
90.2 kDa lethal factor (LF)
88.9 kDa oedema factor (OF)

22. pXO2:
 95.3 kbp in size
 Codes for three genes capB, capC,
and capA
 Capsule inhibits phagocytosis of
vegetative anthrax bacilli

23.  A skin test using an extract from an
attenuated strain of B.anthracis is
available and is diagnostic in 82% of
patients 1 to 3 days and in 99% of
patients 4 weeks after the onset of
symptoms.

24. PCR METHOD
 Sample DNAs were used as template in a
PCR containing primers that
 amplify portions of the B. anthracis capA,
capB, capC, cya, lef, and pag genes.
 These genes are located on the two large B.
anthracis plasmids, pX01 and pX02, required
for pathogenicity
 Samples were also analyzed by using
primers that amplify chromosomal B.
anthracis-specific DNA sequences.

26. ANOTHER TECHNIQUES
 Immunochromatographic lateral flow
assay
 Time resolved fluorescence
 Immunomagnetic separation and
electrochemiluminescence assay