1. USMF, Microbiology, Virusology &Immunology ( Natalia Florea )
TUBERCULOSIS,
DIPTHERIA AND
WHOOPPING COUGH

2. Classification of
Mycobacterium:
Mycobacterium tuberculosis
Mycobacterium bovis
Mycobacterium avium
Mycobacterium ulcerans
Mycobacterium kansasii …
Mycobacterium leprae

3. Morphology & Identification
The mycobacterium are rod-shaped non spore
forming, aerobic bacteria that do not stain readily
but, once stained, resist de colorization by acid
or alcohol and are therefore called “acid-fast”
bacilli. Mycobacterium are rich in lipids. It cause
chronic diseases producing lesions of the
infectious granuloma type. The Ziehl-Neelsen
technique of staining is employed for
identification of acid-fast bacteria. In sputum or
sections of tissue, mycobacterium can be
demonstrated by yellow-orange fluorescence
after staining with fluorochrome stains (eg,
auramine, rhodamine).

4. Pathology: Two principal lesions of
mycobacterium tuberculosis is Exudative
type- this consists of an acute inflammatory
reaction, with edema fluid, and develops,
rapidly spreads to the lymphatic and regional
lymph nodes. In tissue often heals rapidly.
The lymph node undergoes massive
caseation, which usually calcifies. The
tuberculin test becomes positive. And
Productive type- when fully developed, this
lesion, a chronic granuloma.

5. Mycobacterium tuberculosis infects the lung, and
is distributed systemically within macrophages
and survives intracellularly. Inhibition of
phagosome-lysosome fusion and resistance to
lysosomal enzymes have both been suggested
to play a role. Cell-mediated immunity develops
which causes infiltration of macrophages and
lymphocytes with development of granulomas
(tubercles). The disease can be diagnosed by
skin testing for delayed hypersensitivity with
tuberculin (also know as protein purified purified
from Mycobacterium tuberculosis, PPD). A
positive test does not indicate active disease;
merely exposure to the organism.

6. Laboratory diagnosis of tuberculosis
The presence of acid fast bacteria in
sputum is a rapid presumptive test for
tuberculosis (link to method). Subsequently,
when cultured, M. tuberculosis will grow
very slowly producing distinct non-
pigmented colonies after several weeks. M.
tuberculosis can be differentiated from most
other mycobacterium by the production of
niacin. A rapid alternative to culture is
polymerase chain amplification (PCR).

7. Growth Characteristics: Mycobacterium are
obligate aerobes. Increased CO2 tension
enhances growth. Ordinarily, mycobacterium
grow in clumps or masses because of the
hydrophobic character of the cell surface.
Biochemical activities are not characteristic,
and the growth rate is much slower than that
of most bacteria. Incubation of the inoculated
media is continued for up to 8 weeks.
Isolated bacteria should be tested for drug
susceptibility. Virulent strains of tubercle
bacilli form microscopic “cord factor” in which
acid-fast bacilli are arranged in parallel
chains.

8. Tuberculosis is usually treated for
extensive time periods (6-9 months or
longer) since the organism grows
slowly and may become dormant. By
using two or more antibiotics (including
rifampicin and isoniazid), the possibility
of resistance developing during this
extended time is minimized.
M. tuberculosis causes disease in
healthy individuals and is transmitted
man-man in airborne droplets.

9. Three types of media are employed.
1. Simple synthetic media,
2. Oleic acid-albumin media,
3. Complex organic media
(Lowenstein Jensen )

10. Immunity: In the course of primary
infection, the host also acquires
hypersensitivity to the tubercle
bacilli. This is made evident by
the development of a positive
tuberculin reaction. Antibodies
form against a variety of the
cellular constituents of the
tubercle bacilli.

11. The BCG vaccine (Bacillus de
Calmette et Guerin, an attenuated
strain of M. bovis) has not been
effective. In the Russia, where the
incidence of tuberculosis is low,
widespread vaccination is not
practiced. Indeed immunization
(resulting in a positive PPD test)
is felt to interfere with diagnosis.

12. Tuberculosis is usually treated for
extensive time periods (6-9 months or
longer) since the organism grows
slowly and may become dormant. By
using two or more antibiotics (including
rifampicin and isoniazid), the possibility
of resistance developing during this
extended time is minimized. M.
tuberculosis causes disease in healthy
individuals and is transmitted man-
man in airborne droplets.

13. Tuberculin skin test
purified protein derivative
injected, sensitized T cells
react giving delayed
hypersensitivity reaction
BCG vaccine attenuated strain
Efficacy questionable, interferes with skin test

14. 2004 the Tuberculosis of
respiratory bodies
Average statistic disease on 100.000
population = 90 on Republic Moldova,
In Chisinau = 97 on 100.000 population
Children's disease in city’s 0-16 years
= 0,15 cases on 1000 children
Total 3238 cases of disease have
been registered in 2004 years

15. Leprosies mycobacterium and their
morpho-biological characters
The organism does not grow in
culture media. However, it grows well
in the armadillo (which has a low body
temperature), allowing production of
M. leprae antigens and pathogenesis
studies. M. leprae has traditionally
been identified on the basis of acid-
fast stains of skin biopsies and clinical
picture.

16. M. leprae is the causative agent of leprosy
(Hansen's Disease), a chronic disease
often leading to disfigurement.. It is rarely
seen in the Russia but common in the
third world. The organism infects the skin,
because of its growth at low temperature.
It also has a strong affinity for nerves. In
"tuberculoid" leprosy, there are few
organisms due to control by active cell-
mediated immunity. In "lepromatous"
leprosy, due to immuno-suppression by
the organism, the opposite is found.
Treatment with antibiotics is effective and
the overall disease incidence worldwide is
down. Lepromin is used in skin testing.

17. Classification of Corynebacterium
diphtheria, biovariants:
C. diphtheria
C. pseudo diphthericum
C. ulcerans
C. xerosis
C. haemolyticum (pyogenes)
C. hofmannii

18. Corynebacterium are gram-positive rods,
non-motile and non spore-forming, that often
posses club shaped ends and irregularly
staining granules. They are often in
characteristic arranjaments in forms V or N.
Irregularly distributed within the rod (often
near the poles) are granules straining deeply
with aniline dyes (metacromatic granules)
that give the rod a beaded appearance.
Several species form part of the normal flora
of the human respiratory tract, other mucous
membranes, and skin. Corynebacterium
diphtheria produces a powerful exotoxin that
causes diphtheria in humans.

19. Morfology
.

20. The principal human pathogen of the
group is C. diphtheria. In nature it occurs
in the respiratory tract, in wounds, or on
the skin of infected persons or normal
carriers. It is spread by droplets or by
contact to susceptible individuals; virulent
bacilli then grow on mucous membranes
and start producing toxin. All toxigenic C.
diphtheria are capable of elaborating the
same disease-producing exotoxin. The
factors that control toxin production in
vivo are not well understood.

21. Diphtheria toxin is absorbed into the mucous
membranes and causes destruction of
epithelium and a superficial inflammatory
response. The necrotic epithelium becomes
embedded in exuding fibrin and red and white
cells, so that a grayish “pseudo membrane” is
formed – commonly over the tonsils, pharynx,
or larynx. Any attempt to remove the pseudo
membrane exposes and tears the capillaries
and thus results in bleeding. The regional
lymph nodes in the neck enlarge, and there
may be marked edema of the entire neck. The
diphtheria bacilli within the membrane
continue to produce toxin actively, resulting
often in paralysis of the soft palate, eye
muscles, or extremities.

23. Colonization of the upper respiratory tract
(pharynx and nose) and less commonly skin with
C. diphtheria can lead to diphtheria. The
organism does not produce a systemic infection.
However, in addition to a pseudo membrane
being formed locally (which can cause choking)
systemic and fatal injury results primarily from
circulation of the potent exotoxin (diphtheria
toxin). The latter begins over a period of a week.
Thus treatment involves rapid therapy with anti-
toxin. The gene for toxin synthesis is encoded on
a bacteriophages (the tox gene).
Corynebacterium not infected with phage, thus
do not generally cause diphtheria.

24. Colony on the culture medium

25. Culture: On coagulated serum medium, the
colonies are small, granular, and gray, with
irregular edges. On blood agar containing
potassium tellurite, the colonies are gray to black
because the tellurite is reduced intracellular.
The 3 types of C. diphtheria typically have the
following appearance on such media:
var gravis – nonhemolytic, large, gray, irregular,
striated colonies;
var mitis – hemolytic, small, black, glossy,
convex colonies;
var intermedius– nonhemolytic, small colonies
with characteristics between the 2 extremes.

26. If is suspect the diphtheria: Swabs from the
nose, throat, or other suspected lesions must be
obtained before antimicrobial drugs are
administered. Smears stained with alkaline
methylene blue or Gram’s stain show beaded
rods in typical arrangement. Inoculate a blood
agar plate, a Loeffler slant, and a tellurite plate,
and incubate all 3 at 37oC. Unless the swab can
be inoculated promptly, it should be kept
moistened with sterile horse serum so the bacilli
will remain viable. In 12-18 hours, the Loeffler
slant may yield organisms of typical “diphtheria
like” morphology. In 36-48 hours, the colonies on
tellurite medium are sufficiently definite for
recognition of the type of C. diphtheria.

27. C. diphtheria are identified by growth
on Loeffler is medium followed by
staining for metachromatic bodies
(polyphosphate granules, Babes-
Ernst bodies). Characteristic black
colonies are seen on tellurite agar
from precipitation of tellurium on
reduction by the bacteria. Production
of exotoxin can be determined by in
vivo or in vitro tests.

28. Such tests are really tests for toxigenicity
of an isolated diphtheria like organism.
In vivo test – a culture is emulsified and
4 ml is injected subcutaneously into each
of 2 guinea pigs, one of which has
received 250 units of diphtheria antitoxin
intraperitoneally 2 hours previously. The
unprotected animal should die in 2-3
days, whereas the protected animal
survives.

29. In vitro test – a strip of filter paper
saturated with antitoxin is placed on an
agar plate containing 20% horse serum.
The cultures to be tested for toxigenicity
are streaked across the plate at right
angles to the filter paper. After 48 hour is
incubation, the antitoxin diffusing from the
paper strip has precipitated the toxin
diffusing from the toxigenic cultures and
resulted in lines radiating from the
intersection of the strip and the bacterial
growth.

30. Tissue culture test – the
toxigenicity of C.diphtheriae
can be shown by
incorporation of bacteria into
an agar overlay of cell culture
monolayer. Toxin produced
diffuses into cells bellow and
kills them.

31. Immunity in diphtheria determining the
antitoxic immunity by the Schick reaction
(in vivo) and IHAR (in vitro). Resistance
to the disease depends largely on the
availability of specific neutralizing
antitoxin in the bloodstream and tissues.
Thus, the treatment of diphtheria rests
largely on rapid suppression of toxin-
producing bacteria by antimicrobials and
the early administration of specific
antitoxin against the toxin formed by the
organisms at their site of entry and
multiplication.

32. Antitoxic immunity to diphtheria may be
active or passive. The relative amount of
antitoxin that a person posses at a given
time can be estimated in one of 2 ways:
Titration of Serum for Antitoxin Content:
Serum is mixed with varying amounts of
toxin and the mixture injected into
susceptible animals. The greater the
amount of toxin neutralized, the higher
the concentration of antitoxin in the
serum.

33. Schick Test : This test is based on the fact that
diphtheria toxin is very irritating and results in a
marked local reaction when injected
intradermally unless it is neutralized by
circulating antitoxin. One Schick test dose
(amount of standard toxin that, when mixed with
0.001 unit of the US Standard diphtheria
antitoxin and injected intradermally into a guinea
pig, Will induce a 10-mm erythematous reaction)
is injected into the skin of one forearm and an
identical amount of heated toxin is injected into
the other forearm as a control. (Heating for 15
minutes at 60oC destroys the effect of the toxin.)

34. The test should be read at 24 and 48
hours and again in 6 days and
interpreted as follows:
Positive reaction ( susceptibility to
diphtheria toxin, absence of adequate
amounts of neutralizing antitoxin; less
than 0,01 Lf units/ml) – Toxin produces
redness and swelling that increase for
several days and then slowly fade,
leaving a brownish pigmented area.
The control site shows no reaction.

35. Negative reaction (adequate amount of
antitoxin present: usually in excess of
0,02 Lf units/ml) – Neither injection site
shows any reaction.
Pseudo reaction - Schick test reactions
may be complicated by hypersensitivity
to materials other than the toxin
contained in the injections. A pseudo
reaction shows redness and swelling on
both arms which disappear
simultaneously on the second or third
day. It constitutes a negative reaction.

36. Combined reaction – begins
like a pseudo reaction, with
redness and swelling at both
injection sites; the toxin later
continues to exert its effects,
however, whereas the reaction
at the control site subsides
rapidly. This denotes
hypersensitivity as well as
relative susceptibility to toxin.

37. Diphtheria is now a disease of almost historic
importance due to effective immunization of
infants (in conjunction with pertussis and
tetanus, DPT) with a toxoid (inactive toxin)
which causes production of neutralizing
antibodies.
However, colonization is not inhibited and
thus C. diphtheria is still found in the normal
flora. Immunity can be monitored with the
Schick skin test. Treatment in non-immune
individuals primarily involves injection of anti-
toxin. Antibiotics are also administered at this
time.