Mycobacterium Mycobacterium tuberculousis Mycobacterium leprae

1. Mycobacterium

2. Mycobacterium
● Mycobacterium belongs to the family Mycobacteriaceae
● They are non- motile, non-sporing, noncapsulated, weakly gram-positive,
straight or slightly curved rod-shaped bacteria, which are obligate aerobes.
● All species under Mycobacterium are acid fast that is they resist
decolourization by weak acid. This is due to the presence of mycolic acid in
cell wall
● They sometimes show branching filamentous forms resembling fungal
mycelium and form pellicles in liquid media hence named Mycobacterium
meaning fungi like bacteria.
● Mycobacteria can be classified into:
○ M. Tuberculosis complex: lt is responsible for tuberculosis in man.
○ M. leprae (Hansen's bacilli): lt causes leprosy.
○ Nonluberculous mycobacteria (NTM): this diverse group of mycobacteria
occasionally cause opportunistic human infection, e.g. M. knnsasii.

3. MYCOBACTERIUM TUBERCULOSIS COMPLEX
● M. tuberculosis complex causes tuberculosis.
● M.tuberculosis complex includes:
○ M. tuberculosis (human tubercle bacillus)
○ M. bovis (bovine tubercle bacillus)
○ M. Caprae (closely related to M. Bovis)
○ M. africanum (isolated from few West African cases)
○ M. microti ('vole' bacillus, rare and less virulent)
○ M. pinnipedii (infects seals in the Southern hemisphere and recently isolated from
humans)
○ M. canetti
● Among all, M. tuberculosis is the most common cause of tuberculosis in man.

4. MYCOBACTERIUM TUBERCULOSIS
● They are non- motile, non-sporing, noncapsulated, gram-positive, acid fast
rod-shaped bacteria, which are obligate aerobes.
● They are the most common cause of tuberculosis in man.
● The source of infection of M. tuberculosis may be- (1) human (e.g. cases of
pulmonary tuberculosis), (2) bovine source ( e.g. consumption of
unpasteurised infected milk).
● M. tuberculosis is mainly transmitted by inhalation of droplet. Other rare
transmission includes swallowing of sputum, consumption of unpasteurised
infected milk, direct inoculation.

5. Pathogenesis
The sequence of pathogenic events that take place are as follows:
1. Droplet nuclei containing tubercle bacilli from infectious patients are inhaled.
A small fraction of it reaches the alveoli.
2. Phagocytosis by macrophages. The bacilli get engulfed by the macrophages.
3. Survival inside the macrophages: the bacterial cell wall impairs
phagosomelysosome fusion and starts to replicate inside the macrophage.
4. The macrophage eventually ruptures and releases its bacillary contents which
infect other phagocytes and the cycle continues.

6. Clinical Manifestations
Tuberculosis (TB) is classified as pulmonary and extrapulmonary forms.
Pulmonary Tuberculosis {PTB)
It accouts for 80% of all cases of Tuberculosis (TB). It can be further categorized
into primary or post-primary (secondary) types.
The initial infection (exogenous) with M. tuberculosis is referred to as a primary
infection. Subsequent disease in a previously sensitized person, either from an
exogenous source or by reactivation (endogenous) of a primary infection, is
known as postprimary (secondary or reinfection) tuberculosis

7. Primary Tuberculosis
Primary tuberculosis is the initial infection by tubercle bacilli in a host. It usually affect children
The site of the initial infection is usually the lung,located in the lower lobe or the lower part of the
upper lobe to form the initial lesion or Ghon focus.
Some bacilli are carried in phagocytic cells to the hilar lymph nodes where additional foci of
infection develop. The Ghon focus, together with the enlarged hilar lymph nodes, form the primary
complex.
It may be asymptomatic or may present with fever, productive cough, and occasionally
hemoptysis, night sweating, Weight loss.
In the majority of cases lesions heal spontaneouslyand Primary complex becomes calcified (Ranke
complex).
In immunocompromised, bacilli are seeded by further lymphatic and hematogenous dissemination
in many organs and tissues, including other parts of the lung.

8. Postprimary (Secondary) Tuberculosis
The postprimary (secondary or adult) type of tuberculosis is due to reactivation
of latent infection or exogenous reinfection.
Reactivation TB occurs when there is an alteration or a suppression of the
cellular immune system in the infected host that favors replication of the bacilli
and progression to disease.
This commonly affects adults.
Unlike primary pulmonary TB , here apical segment of upperlobe is commonly
affected. Calcified nodules are formed (Assmann focus).
Symptoms are similar to primary TB but more pronounced.
Two special features of secondary tuberculosis are the presence of caseous
necrosis and of cavities, which may rupture into blood vessels, spreading
mycobacteria throughout the body. These rarely heals spontaneously.

9. Extrapulmonary Tuberculosis (EPTB)
EPTB results from hematogenous dissemination of tubercle bacilli to various
organs.
the sites commonly involved are :
Tuberculous lymphadenitis: It is the most common form, accounting for 35% of
all EPTB cases.The most common sites are posterior ceivical and supraclavicular
lymph nodes.
Renal tuberculosis
Skeletal tuberculosis : Weight-bearing joints, such as spine (Pott’s disease is
most common), hips and knees are commonly affected.

10. Gastroinlestinal tuberculosis: Terminal ileum and cecum are the most common sites
involved. The route of spread may be due to swallowing of sputum wilh direct seeding
or hematogenous spread.
Tuberculous pericarditis: usually seen in elderly.
Miliary or disseminated tuberculosis: Hematogenous spread of tubercle bacilli results
in the formation of yellowish 1-2 mm size granulomatous lesions resembling millet
seeds (thus termed as miliary) in various organs. It is more common in HIV-infected
people.
Tuberculous skin lesions
Tuberculous meningitis and tuberculoma
Pleural tuberculosis:

11. LABORATORY DIAGNOSIS
Specimen
In pulmonary TB
● sputum (2 specimens-spot and early morning),
● gastric aspirate (in children)
In EPTB - specimen varies depending on site
● CSF - shows spider web clot
● Bone marrow and liver biopsy specimens from miliary tuberculosis and blood from
those with HIV coinfec tion are useful for culture
● Pus from tuberculous abscesses
● Urine sample
● Tissue Biopsy

12. Microscopy
Gram Staining: Gram positive rods are seen
Ziehl-Neelsen staining: Acidfast bacilli are seen as pink brightened rods, while
background is blue. It has been estimated that at least 50,000 to 1,00,000 acidfast
bacilli should be present per ml of sputum to get positive report. A negative report
should not be given till at least 100 fields are examined.
Fluorescent microscopy: Smears are stained with auramine phenol and
examined under ultraviolet light and bacilli appear bright rods against dark
background.

14. Culture
Tubercle bacilli are slow growing due to long generation time (10-15 hours). Hence,
inoculated culture media are incubated at 37°C, for 6-8 weeks. Media are incubated
aerobically.
Tubercle bacilli are highly fastidious and can grow only in enriched culture media, such as:
Lowenstein-Jensen media (egg based media) : It is the most widely used and
recommended media. M.tuberculosis produces typical rough, tough and buff colored
colonies
Dorset egg media
Loeffler serum slope ( serum based)

15. Liquid media :
They are not generally employed for routine culture, bur are used for drug
sensitivity resting and preparation of antigens and vaccines.
Examples include Middlebrook 7H9, Dubos etc
Virulent strains tend to form long serpentine cords in liquid media.
Disadvantage of culture is that it is time consuming, Minimum 8 weeks of
incubation is needed to give a negative report.

16. Automated Culture Methods
They use liquid broth such as Middlebrook 7H9, supplemented with:(1) OADC
enrichment growth media (Oleicacid, Albumin, Dextrose and Catalase); and (2)
PANTA antibiotic mixture (Polymyxin B,amphotericin B, nalidixic acid,
trimethoprim and azlocillin). They can detect growth within 2-3 weeks. The
disadvantage is that they are very expensive.

17. Blochemical Identification
Mycobacterium tuberculousis is positive for :
● Niacin test
● Nitrate reduction test
● Pyrazinamidase test
They are weakly catalase positive.

18. PCR test : it is mainly used in diagnosis of extra pulmonary TB where bacteria is less
in number. It also has the added advantage of taking less time than culture.
Technique like geneXpert are used to identify drug resistance.
Others:
ESR is markedly elevated.
In TB meningitis , CSF will show cobweb coagulum, elevated CSF pressure, raised
protein and decreased glucose.

19. Tuberculin Skin Test ( Mantoux test)
The principle of this test is delayed (Type IV) hypersensitivity reaction. The test
is based on the fact that persons infected with tubercle bacilli develop
hypersensitivity to the proteins of the organism.
Purified protein derivative (PPD) is the skin test reagent used.
In the Mantoux test, 0.1 ml of PPD containing 5 Tuberculin Unit is injected
intradermally on the flexor aspect of the forearm with a tuberculin syringe and
when properly performed, will produce a discrete pale elevation of the skin.

20. Tuberculin tests should be read 48 to 72 hours after injec tion. The reading is
based on the presence or absence of induration, which may be determined
visually and by palpation.
Indura tion of diameter 10 mm or more is considered positive, 5 mm or less
negative and 6 to 9 mm is of doubtful significance because it may be due to other
mycobacterial infection.
A positive tuberculin test indicates infection with tubercle bacillus or BCG
immunization, recent or past, with or without clinical disease.
It is used to diagnose active infection in infants and young children, indication of
successful BCG vaccination.

21. TREATMENT
Treatment of tuberculosis aims to:
● Interrupt transmission by rendering patients non-infectious.
● Prevent morbidity and death by curing patients.
● Prevent the emergence of drug resistance.
● Prevent relapse.
The bactericidal drugs, along with the bacteriostatic drug constitute the first line
drugs in antituberculous therapy. As per the National TB Elimination Program
(NTEP) treatment of drug sensitive TB is by usage of combination of 4 drugs
namely Isoniazide (H), Rifampicin (R) , Pyrazinamide (Z), Ethambutol (E) for a
period of 8 weeks ( intensive phase) followed by a continuation phase of 16 weeks
consisting of Isoniazide (H), Rifampicin (R) ,Ethambutol (E).

22. DOTS strategy (DirectlyObserved Treatment, Short course) is recommend by
RNTCP and WHO. Here, the strategies used are:
The entire treatment course is supervised to improve the patient's compliance.
Treatment response is also monitored by periodic sputum smear microscopy.
To ensure compliance a single daily dose of all first line antitubercular drugs is
preferred.

24. Multidrug-Resistant Mycobacterium tuberculosis
multidrug resistance (MDR) strain are the one that is at least resistant to rifampicin
(R) and isoniazid (H).
Another serious condition extensively drug resistanttuberculosis (XDRTB) has
emerged recently. XDRTB is due to M. tuberculosis strains which are resistant to
any fluoroqui nolone and at least one of three injectable second line drugs
(capreomycin, kanamycin and amikacin) in addi tion to isoniazid and rifampicin.

25. Prophylaxis
BCG (Bacille Calmette-Guérin) vaccine
The vaccine contains live avirulent bacilli.
This is a bovine strain of tubercle bacillus rendered completely avirulent by culturing repeatedly.
BCG vaccine should be administered soon after birth failing which it may be given at any time during the
first year of life.
The vaccine is given intradermally over deltoid region.
BCG may not protect from the risk of tuberculosis infection but gives pro tection to infants and young
children against the more serious types of the disease, such as meningitis and dis seminated tuberculosis
Immunity lasts for 10 to 15 years. After vaccination a Tuberculin test may last positive for 3-7 years.
BCG also confers immunity against leprosy.

26. NONTUBERCULOUS MYCOBACTERIA

27. NONTUBERCULOUS MYCOBACTERIA (NTM)
NTM are diverse group of mycobacteria that are isolated from birds, animals, and
from environmental sources, such as soil arid water.
They are opportunistic pathogens, occasionally associated with human infection.
Man to man transmission is not known.
Non-tuberculous mycobacteria (NTM) have been classified into four groups (by
Runyon) based on pigment production and rate of growth.
1. Photochromogens
2. Scotochromogens
3. Non -Photochromogens
4. Rapid growers

28. Photochromogens
● They produce pigments only when the colonies are exposed to light.
● This group contains the following pathogens:
a. M.marinum : lt is acquired from water sources. lt typically causes papules or ulcers known as
swimming pool granuloma or fish tank granuloma.
b. M. kansasii : It causes chronic pulmonary disease resembling tuberculosis in elderly and
immunocompromised with pre-existing lung disease.
Scotochromogens
● They produce pigments (yellow, orange or red) even when cultures are
incubated in dark, but intensity of color may increase on exposure to light.
● M. scrofulecum: It causes scrofula (cervical lymphadenitis) in children.
● M.gordonae : it is a common contaminant in clinical specimens

29. Nonphotochromogens
● They do not produce any pigments.
● M. avium and M. intracellulare: They are opportunistic pathogens, especially
in HIV infected people with low CD4 T-cell count. They cause Lymphadenitis,
respiratory infection and disseminated disease.
● M. xenopi: it has been isolated from hospital water supplies, and associated
with nosocomial pulmonary disease outbreaks.
● M. ulcerans: It is the agent of Burulli ulcer. Lesions are typically painless
ulcers and nodules that become necrotic later.
Rapid Growers
● This group of NTM grow in culture within 1 week of incubation. Examples
include:
● M. Fortitum and ,M. chelonae: they cause post-trauma injection abscess and
catheter- related infections.
● Arylsulfatase test is Positive for all rapid growers.

31. Laboratory Diagnosis
Specimens: Sputum, lymph node aspirate, pus or exudate, biopsy from skin
lesions are the usual specimens, depending on the type of infection.
Microscopy by ZN staining: Shows red acid fast bacilli.
Culture on LJ media: LJ media are incubated in dark and light separately for
distinguishing between phtochromogens and scotochromogens.

32. Treatment
Most environmental mycobacteria are resistant to the usual antituberculosis
drugs although infections often respond to various combinations of these drugs.
Pulmonary disease caused by M. avium complex or M. kansasii may respond
to prolonged treatment with macrolide (clarithromycin or azithromycin) rifampicin,
isoniazid and ethambutol.

33. MYCOBACTERIUM LEPRAE

34. MYCOBACTERIUM LEPRAE
Mycobacterium leprae is the causative agent of leprosy.
M. leprae is not cultivable either in artificial culture media or in tissue culture; hence it does not follow
the Koch's postulates.
Compared to tubercle bacilli, they are less acid fast and can resist up to 5% sulfuric acid. ( Other
mycobacteria are resistant to 20%sulfuric acid)
In smears made from skin lesions, they appear in groups, called cigar-like bundles of bacilli present
inside lipid laden macrophages called virchow's lepra cells.
Thet have a tendency to grow in cooler area of the body; hence, the clinical manifestations are largely
confined to the skill, peripheral nerves, upper respiratory tract, anterior eyes, and testes.
Lepra bacillus has a long generation lime of 12-13 days.

35. Clinical Manifestations
Leprosy is a chronic granulomatous disease of humans, primarily inolving
cooler parts of the body but are capable of affecting any tissue or organs causing
bony deformities and disfigurements in untreated cases.
M. leprae has mulliple routes of transmission. Portal of entry is either nose or skin.
Leprosy is not highly communicable. Intimate and prolonged contact is necessary
for transmission.
Leprosy has a long incubation period, an average of 3-5 years.
Leprosy is a bipolar disease. Under any classification scheme, lepromatous and
tuberculoid cases are the two extreme poles of the disease.
Following start of treatment or alteration of host immunity, the leprosy category of
patient changes from one type to another type.

36. Tuberculoid leprosy is seen in patients with high degree of resistance. The lesions
are few, well demarcated consisting of macular anesthetic patches. Bacilli are scanty
in the lesions (Paucibaclllary disease). Neural involvement occurs early.Most
common nerves involved are ulnar nerve. Cell mediated immunity is adequate and
lepromin test is positive. Prognosis is good.
Lepromatous leprosy, where host resistance is very low, the lepromin test is
negative and prognosis is poor. Here we find infiltrating skin lesion. Large number
(Multibacillary disease) of acid fast bacilli are found in large clumps (globi) inside the
macrophages (lepra cells). Peripheral nerve trunk becomes involved with the progress
of diseases. There may be erythematous patch and diffuse infiltrate of nodular lesions.
Lesions on face produce leonine facies appearance, Loss of eye brow/lashes may
occur late.

37. Borderline type refers to lesions possessing characteristics of both tuberculoid
and lepromatous lesion. It may shift to lepromatous or tuberculoid depending upon
chemotherapy or change in host resistance.
The indeterminate type is early unstable tissue reaction which is not
characteristic of lepromatous or tuberculoid type. In many persons the lesion heals
spontaneously. In others, lesion may progress to tuberculoid or lepromatous type.
Ridly and Jopling have introduced leprosy into 5 groups: Tuberculoid (TT),
Borderline tuberculoid (BT), Borderline (BB), Borderline lepromatous (BL), and
lepromatous (LL)

39. Deformities
About 25% of untreated cases develop deformities in due course of time which may arise
due to-
(l) nerve injury leading to muscle weakness or paralysis, or
(2) disease process (facial deformities or loss of eyebrow), or
(3) infection or injury (ulcers).
Common deformities include:
Face: Leonine facies, sagging face, loss of eyebrow/eye lashes, saddle nose and corneal
ulcers.
Hands: Claw hand and wrist drop
Feet: Foor drop, clawing of toes, inversion of foot, and plantar ulcers.

40. Lepra Reactions
Acute exacerbations occur through out
the course of leprosy called lepra
reaction which are of two types :
Lepra Reaction type-1
Lepra Reaction type-2

41. Lepra Reaction type-1
● This occurs in borderline cases, occurring spontaneously or more often
during chemotherapy.
● It is a cell mediated immune (Type 4 reaction) reaction, with an influx of
lymphocytes into lesions, and a shift to tuberculoid morphology (termed as
reversal reactions)
● The lesions develop erythema and swelling, along with pain and tenderness.
● It may rapidly cause severe and permanent nerve damage.
● Patient usually respond well to glucocorticoid.

42. Lepra Reaction Type-2
This is an immune-complex reaction ( Type 3 reaction) seen only in lepromatous and
borderline lepromatous cases, usually a few months after institution of chemotherapy.
The most common feature is crops of painful erythematous papules, which become
nodular termed as erythema nodosum leprosum (ENL). Constitutional disturbances
like fever, arthritis, iridocyclitis, orchitis and painful neuritis are common.
The immunological basis of type 2 is vasculitis associ ated with the deposition of
antigen-antibody complexes.
Treatment is started wich glucocorticoids. Thalidomide should be initiated for
nonresponsive or recurrent cases.

43. Laboratory Diagnosis
Specimen Collection
Total six samples are collected; four from skin (forehead, cheek, chin and
bunock), one from ear lobe and nasal mucosa.
Biopsy from the thickened nerves and nodular lesions may be necessary in some
cases.
Microscopy
The smears are stained by Ziehl-Neelsen technique by using 5% sulfuric acid for
decolorizarion. Under oil immersion objective, red acid fast bacilli are seen,
arranged singly or in groups ( cigar like bundles) within macrophages (Lepracell).
The viable bacilli stain uniformly and the dead bacilli are fragmented, irregular or
granular.

45. Animal Inoculation
M.lepra cannot be cultivated on artificial media.
Injection of ground tissue from lepromatous nodules or nasal scrapings from
leprosy patient into the foot pad of mouse produces typical granuloma at the site
of inoculation within 6 months. Nine-banded armadillo is another animal used for
inoculation of material.
The lesions which develop in these animals can be identified by histological
examination and Ziehl-Neelsen staining.

46. Lepromin Test
it demonstrates the delayed hypersensitivity reaction and an intact CMl against the
lepra antigen.
In this 0.1mL of lepromin antigen is injected intradermally to inner forearm and
reading is taken at two occasions; at 48 hours and 21 days.
Fernandez reaction: reading is taken at 48hrs. Red area of >10 mm diameter is
considered as positive, which indicates past exposure to lepra bacilli. However, it
does not indicate active infection.
Mitsuda reaction: Reading is taken after 2ldays. Positive test is indicated by a
nodule formation of>5 mm size at the site of inoculation which ulcerates later on.it
indicates that the patient's CMI is intact.

47. Uses of lepromin test:
The late reaction is a useful tool to measure the immune status (CMl) of the
individual. Lepromin test is said to be positive only when the CMI is intact.
it can be used for:
Classifying lesions of leprosy: In TT patients with intact CMI the test is strongly
positive. Gradually the positivity becomes weaker as the patient progresses
towards lepromatous end. In LL patients; the test is negative indicating a low
CMI.Assessing prognosis: Intact CMJ (as in TT patients) indicates good
prognosis. Assessing resistance to leprosy in individuals: lepromin negative
persons are at higher risk of developing multibacillay leprosy than lepromin
positive persons.

48. TREATMENT
Because of risk of development of drug resistance to single drug WHO
recommends mukidrug therapy (MDT) for treatment of leprosy.
Recommended drugs: Dapsone, rifampicin and clofazimine.
In Paucibacillary, treatment is for 6 months while in multibacillary treatment is upto
1 year or till smear negative.
Prophylaxis:
No specific vaccine is available. ( Some study claim BCG to provide some
protection)
Dapsone can be given to high risk contacts of lepromatous patients.