Tuberculosis UG MBBS class

1. Tuberculosis –
integrated class
Sayantan Banerjee
AIIMS Kalyani

2. History of Tuberculosis
Tuberculosis- an ancient disease
“Captain among these men of death”
Signs of Skeletal TB have been found in
remains from Europe from Neolithic times
(8000 BC), ancient Egypt (1000 BC)
TB was recognized as a contagious disease
by the time of Hippocrates( 400 BC) –
termed as “phthisis”
German physician Robert Koch discovered
and isolated Mycobacterium tuberculosis in
1882

3. Key Facts of the Disease
• A total of 1.5 million people died from TB in 2020
• In 2020, there were an estimated 10 million new incident cases of TB
worldwide (WHO)
• Men 56 Lakh
• Women 33 Lakh
• Children 11 Lakh
• The five countries with the highest number of cases were India,
China, South Africa, Indonesia, and Pakistan
• An estimated 6.6 crore lives were saved through TB diagnosis and
treatment between 2000 and 2020

4. India has the highest burden of TB in the world
•Incidence of 27 lakh new cases in 2019
•Incidence rate of 199 cases per lakh
population

5. General characteristics
• Tubercle bacilli can remain viable for many years in the tissues of
healthy persons
• When they produce disease, it runs a chronic and protracted course
that gives ample time for transmission to susceptible hosts
• The infection can produce disease in a human being after decades of
dormancy
• It can produce an epidemic, when introduced into a population of
which only a small portion is immunologically protected by already
having been infected

6. M. tuberculosis complex (MTBC)
• The most common causative agent of TB in humans, Mycobacterium
tuberculosis, is a member of the M. tuberculosis complex (MTBC) which includes
six other closely related species:
• M. bovis - causes disease in cattle and spreads to humans through animal
contact and consumption of unpasteurized milk
• M. bovis BCG variant
• M. africanum - causes human TB in West Africa, up to 50% cases
• M. microti
• M. pinnipedii
• M. caprae
• M. canettii
All MTBC members are obligate pathogens and cause TB

7. M. tuberculosis
• Aerobic
• non–spore-forming
• nonmotile bacillus
• with a high cell wall content of high-molecular-weight lipids.
• Growth is slow - generation time being 15 to 20 hours,
compared with much less than 1 hour for most common
bacterial pathogens
• Visible growth takes from 3 to 8 weeks on solid media

8. Live culture colonies
in L J Medium
• The colonies of Mycobacterium on solid
culture media can be of various shades from
cream to yellow and deep orange color
• M. tuberculosis produces cream-colored buff,
rough colonies after 2–3 weeks inoculation on
Löwenstein–Jensen medium

9. Live culture colonies in LJ Medium
• Smooth colonies of
Mycobacterium
grown on
Löwenstein–Jensen
medium
• Rough colonies of
Mycobacterium grown
on Löwenstein–Jensen
medium

10. Mycobacterial Cell Wall

11. Acid-fast staining
• The term acid-fast bacilli (AFB) is practically synonymous with
mycobacteria, although Nocardia and some other organisms
are variably acid-fast
• The cell walls of mycobacteria, because of their high lipid
content, have the unique capability of binding fuchsin dye so
that it is not removed (destained) by acid–alcohol

12. Pulmonary Tuberculosis
• The lung is the most commonly affected organ in TB infection in the
immunocompetent host
• Lung involvement in subjects with active tuberculosis of 80%
• The lung is the portal of entry in the majority of cases of tuberculosis
• The first contact with the organism results in few or no clinical
symptoms or signs
• Ordinarily, the tubercle bacillus sets up a localized infection in the
periphery of the lung, where it has been deposited by inhalation
• Body defenses appear to have little effect on the organism until the
time of development of tuberculin hypersensitivity (4 to 6 weeks)

13. Pulmonary Tuberculosis
• Pulmonary tuberculosis frequently develops slowly, without a definite date
of onset
• Symptoms may be divided into two categories –
• Constitutional – fever, weight loss, fatigue
• Pulmonary – cough, expectoration (sputum production), hemoptysis, chest pain
• Characteristically, the fever develops in the late afternoon and may not be
accompanied by pronounced symptoms.
• With defervescence, usually during sleep, sweating occurs—the classic
“night sweats.”

15. Pulmonary Tuberculosis
• Apical-posterior localization with a tendency to cavitation and
progression is characteristic of pulmonary TB in adolescents and adults
• Apical localization of pulmonary TB (adults) –
• the hyperoxic environment of the apices and the aerobic nature of the
organism.
• Deficient lymphatic flow at the lung apices, especially the posterior apices,
where the pumping effect of respiratory motion is minimal.
• Deficient lymph traffic would favor retention of bacillary antigen and, when
hypersensitivity ensues, tissue necrosis.
• Elderly and progressive primary infection of childhood - often causes
nondescript lower lobe pneumonia

16. Extra Pulmonary Tuberculosis
Lymph node tuberculosis
Pleural effusion and empyema thoracis
Bone and joint tuberculosis
Genitourinary tuberculosis
Disseminated tuberculosis
CNS tuberculosis
Abdominal tuberculosis
Others

17. Tuberculous
Lymphadenitis
• Most common presentation of EPTB in both HIV
infected and non infected patients
• Accounts for nearly 35% of EPTB cases
• 50% cases involve peripheral lymph nodes
• Posterior cervical and supraclavicular sites are
commonly involved
• Cervical lymphadenopathy is common in HIV-negative
• Multifocal involvement common in HIV-positive

18. Tubercular Pleural Effusion
• Accounts for nearly 20% of EPTB cases
• Usually presents as an acute illness and symptom
duration ranges from a few days to few weeks
• A small sub pleural focus may rupture into the
pleural space
• Present with chest pain, breathlessness, cough
with expectoration, fever, and toxaemia
• Occasionally, tuberculosis empyema may present
as a chest wall mass or draining sinus tract

19. Skeletal TB
• Accounts for nearly 10% of EPTB
• Reactivation of hematogenous foci or
spread from adjacent paravertebral
lymph nodes
• Spine (40%), hips (13%), knee (10%)
commonly affected
• Lower thoracic and lumbar vertebrae
are commonly involved followed by
middle thoracic and cervical vertebrae

20. CNS TB & Tuberculosis meningitis
• Accounts for approx 1 - 5% of cases of EPTB
• Results from hematogenous spread of primary or pulmonary TB or
from rupture of a subependymal tubercle into subarachnoid space
• Pathological features of TBM:
• Inflammatory meningeal exudate
• ependymitis
• vasculitis
• encephalitis
• disturbance of CSF circulation and absorption
• With onset of meningitis - headache, neck rigidity and vomiting, fever
develops

21. CNS TB & Tuberculosis meningitis
• Focal neurological deficits and features of raised intracranial tension may
precede signs of meningeal irritation
• Focal or generalised seizures- 20 to 30 % of patients
• Cranial nerve palsies- 20 to 30 % of patients, 6th nerve involvement is
most common
• Hydrocephalus occurs in approximately two-third of cases
• Complete or partial loss of vision- a major complication of TBM
• Terminal illness is characterized by deep coma and decerebrate/
decorticate posturing

23. Brain Parenchymal Granuloma

24. Cobweb coagulum

25. Gastrointestinal Tuberculosis
• 70% of patients with advanced pulmonary disease acquired gastrointestinal TB
from swallowing infectious secretions
• Abdominal TB can present in any of the sites:
Most commonly -
• Peritoneum
• intestinal tract
• lymph nodes
Also may involve -
• Stomach
• hepatobiliary tree
• Pancreas
• perianal area

26. Latent TB infection - Definition
• The Latent Tuberculosis Infection (LTBI) has been defined as infection
with M. tuberculosis within granuloma, that remains in non
replicating state but retains its ability to come out of latency and
cause active TB if and when a disruption of the immune response
occurs.

27. Difference Between Latent TB Infection and
TB Disease
Latent Tuberculosis Active Tuberculosis
• Have no symptoms
• Do not feel sick
• Cannot spread TB to others
• Usually have a positive skin and IGRA
test
• Chest x-ray and sputum test usually
normal
•Symptoms include
a) bad cough that lasts longer than 2 weeks
b) pain in the chest
c) coughing up blood or sputum
d) weakness or fatigue
e) weight loss
f) no appetite
g) chills, fever, sweating at night
• May spread TB to others
• Usually have a positive skin and IGRA
test
• May have abnormal chest x-ray, and/or
positive sputum smear or culture

28. Tests available for LTBI
There are currently two major classes of tests used to identify
patients with latent tuberculosis:
1) The tuberculin skin tests include:
• Mantoux test
2) The IFN-γ (interferon-gamma release assay - IGRA) tests include:
• QuantiFERON-TB Gold
• T-SPOT TB

29. Increased susceptibility to TB
A. Nonspecific decrease in resistance
• < 5 yrs of age and close contact of TB
• Senescence
• Malnutrition
• Postgastrectomy state
• Diabetes mellitus
• Renal failure
• Smoking, alcohol & drug abuse
B. Decrease in resistance due to hormonal
effects
• Pregnancy
• Therapy with adrenocortical steroids
C. Decrease in local resistance
• Silicosis
D. Decrease in specific immunity
• Lymphomas, Head & Neck cancers
• Immunosuppressive therapy (anti TNF⍺,
corticosteroids)
• Sarcoidosis
• HIV infection
• Transplantation

30. HIV-TB
• TB- leading cause of HIV/AIDS related mortality globally
• Risk of TB 16-27 times higher in PLHA, almost 60% of TB was not
diagnosed/treated
• Frequently disseminated disease, patient asymptomatic and TB goes
undetected- need for screening
• Low immunity- less cavitation/ sputum production
• Difficulty in procuring sputum / respiratory samples
• Technical expertise required in tests such as Xpert, LPA

31. Diagnostic Array for Tuberculosis - pipeline

32. Specimen collection for Diagnosis
• Mycobacteria can be recovered from a variety of clinical specimen -
Respiratory specimens (sputum, bronchial washes,
bronchoalveolar lavage fluid, and bronchial biopsies)
Urine, feces, blood, cerebrospinal fluid;
Tissue biopsies; and
Deep-needle aspirations of virtually any tissue or organ
• Specimens that may contain normal bacterial flora should be
processed as soon after collection as possible to minimize the degree
of overgrowth with specimen contaminants

33. Specimen - Sputum
• Sputum samples collected by expectoration or by ultrasonic
nebulization are best obtained shortly after the patient awakens in
the morning, when mycobacteria are at their highest concentration
• Irregular and intermittent release of mycobacteria into the bronchial
lumen from mucosal ulcers or loculated cavities often results in a
variable pattern of recovery from respiratory secretions

34. Lab methods
• Digestion & Decontamination procedures for sputum :
• The high concentration of lipids in the cell wall of most
mycobacteria makes them more resistant to killing by strong
acid and alkaline solutions than other bacteria that may be
present in the specimen.
• Consequently, specimens likely to contain a mixed bacterial
microbiota are treated with a decontaminating agent to reduce
undesirable bacterial overgrowth and to liquefy mucus
a) Petroff’s method (NaOH)
b) NALC – NaOH method (N acetyl L Cystine)

35. Direct Smear Microscopy
• Ziehl-Neelsen (ZN) staining / Acid Fast
• Kinyoun stain – cold
• Fluorescent staining (Rhodamine/ Auramine O)
–5 X 103 to 5 X 104 bacilli / ml of sputum is required
for detection by smear
–Culture detects 10 to 100 viable organism

36. ZN Stained
smear showing
acid-fast bacilli

37. • Solid media
a) Egg based - L J, Petragnani, Dorset
b) Agar based - media are transparent; colonies observed in 10 to 12 days
in contrast to 18 to 24 days with opaque egg based media.
eg. Middle brook 7H11, Middle brook 7H10
Culture media

38. • LIQUID MEDIA –
Middle brook 7H9
• Rapid Culture Methods
BACTEC 460 radiometric (obsolete now)
BACTEC MGIT 960 fluorometric
BACTAlert 3D colourimetric

39. BACTEC MGIT 960
Mycobacterial Growth Indicator Tube

40. MGIT - Principles
• MGIT tube contains Middlebrook 7H9 liquid media and an
oxygen-quenched fluorochrome embedded in silicone at
bottom of the tube
• Bacterial growth = free oxygen is utilized and replaced with
carbon dioxide
• With depletion of free oxygen, the fluorochrome is no longer
inhibited, resulting in fluorescence within the MGIT tube when
visualized under UV light
• The intensity of fluorescence is directly proportional to the
extent of oxygen depletion.

41. MGIT …contd
• An antimicrobial mixture called PANTA™ (Polymyxin B,
Amphotericin B, Nalidixic Acid, Trimethoprim, Azlocillin) is used
for reducing the contamination of other bacteria
• MGIT system has also been used to detect drug susceptibility
against Antitubercular drugs – particularly for SIRE
(Streptomycin, Isoniazid, Rifampicin and Ethambutol)

42. MPT 64 Antigen test
• MPT64 is one of the major MTB complex culture filtrate protein
• This lateral flow test has been reported to identify the M.
tuberculosis complex from the MOTT in the Liquid Culture using the
mouse monoclonal anti-MPT64 antibody

43. Lab Diagnosis of Pulmonary TB
• Sample collection
• Sample decontamination and liquefaction
• Microscopy – ZN Staining / Fluorescence staining
• NAAT – GeneXpert / TrueNAAT
• Culture – Solid medium – LJ Medium
Liquid Medium – Bactec MGIT 960
• Drug Susceptibility – Solid media / liquid media / NAAT
• NAAT DST – Line Probe Assay, GeneXpert

44. Gene Xpert

45. Gene Xpert
• Single use cartridge based nucleic acid amplification test
(CBNAAT)
• Semi-quantitative, hemi-nested, real-time PCR, targeting the
81 bp Rifampin Resistance Determining Region (RRDR) of MTB
rpoB gene (96% of RR cases)
• Contains internal controls and five partially overlapping
fluorescent probes A, B, C, D and E

46. Time-to-result: 1 h 45 min
GeneXpert
DNA molecules are mixed
with dry PCR reagents
Sample is
automatically
filtered & washed
Ultrasonic lysis of filter-
captured organisms to
release DNA
Semi-nested real-time
amplification & detection
in integrated reaction tube
4
5
7
Concentrates bacilli &
removes inhibitors
1
2
Sputum liquefaction &
inactivation with 2:1 SR
Transfer of 2 ml
after 15 min
3
End of hands on work
6
Printable test result
Time-to-result: 1 h 45 min
GeneXpert
DNA molecules are mixed
with dry PCR reagents
Sample is
automatically
filtered & washed
Ultrasonic lysis of filter-
captured organisms to
release DNA
Semi-nested real-time
amplification & detection
in integrated reaction tube
4
5
7
7
Concentrates bacilli &
removes inhibitors
1
2
2
Sputum liquefaction &
inactivation with 2:1 SR
Sputum liquefaction &
inactivation with 2:1 SR
Transfer of 2 ml
after 15 min
3
End of hands on work
6
Printable test result
Gene Xpert

47. Line Probe Assay

48. Line Probe Assay
• Multiplexed DNA hybridization assay - can interrogate a
variety of amplified genetic targets in a single, relatively
simple test format
• Available test kits: MTBDRplus V1, MTBDRplus V2, Nipro
NTM+MTBDR, MTBDRs V1/V2
• WHO’s stand on LPA:
– 2008: Use of FL-LPA (Genotype MTBDRplus)
– 2016: Recommended the use of SL-LPA (Genotype
MTBDRs)