This powerpoint focuses on the overview of laboratory diagnosis of lower respiratory tract infections including pulmonary tuberculosis.

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ShyamKumarMishra
Shyam Kumar Mishra
Assistant Professor,
Institute of Medicine
Laboratory
diagnosis of LoWEr
rEsPiratory traCt
infECtions inCLuding
tubErCuLosis

2. RESPIRATORY TRACT:
ANATOMICAL STRUCTURE
 It is utmost important to be familiar with the
anatomic structure of the thoracic cavity, so that
specimens collected from various sites in the
lower respiratory tract are appropriately
processed by the laboratory.
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3. ANATOMY OF RESPIRATORT TRACT
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4. INITIATION………
 Aspiration of colonizing flora into the alveoli
 Inhalation of aerosols
 Hematologic seeding of the lung from a distant
focus
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5.  When paediatric patients with cystic fibrosis (CF)
have respiratory infections (exacerbations), the
trigger is most commonly related to an increased
growth of bacteria that are chronically present in
the airway.
 However, in adult patients with COPD, the cause
of respiratory exacerbations is usually related to
the acquisition of new strains of bacteria, rather
than an increase in the number of bacteria
present when clinically stable.
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6. 1- Bronchitis.
A- Acute:
 Usually caused by viruses.
 In infants & preschool children is Bordetella pertussis.
B- Chronic:
It is defined by clinical symptoms in which excessive mucus production
leads to coughing up sputum on most days during at least 3 consecutive
months for more than 2 successive years.
2- Bronchiolitis.
It is the inflammation of smaller diameter bronchiolar epithelial surfaces.
It is an acute viral LRTI that primarily occurs during the first 2 years of
life.
Diseases of the Lower Respiratory Tract
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7. 2- Pneumonia
 Community-Acquired Pneumonia:
patients are believed to have acquired infection outside the hospital
setting.
 The etiology of acute pneumonias is strongly dependent on age. More
than 80% of pneumonias in infants & children are caused by viruses,
whereas 10%-20% of pneumonias in adults are viral.
 Hospital-Acquired Pneumonia
 It is the leading cause of death among patients with nosocomial infections
(as high as 50% mortality among patients in intensive care units).
Diseases of the Lower Respiratory Tract
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8. RESPIRATORY TRACT
PATHOGENS
 Definitive Respiratory Tract Pathogens
 Haemophilus influenzae
 Streptococcus pneumoniae
 Mycobacterium tuberculosis
 Mycoplasma pneumoniae
 Chlamydia trachomatis
 Chlamydophila pneumoniae
 Bordetella pertussis
 Legionella spp.
 Pneumocystis jiroveci
 Nocardia spp.
 Histoplasma capsulatum
 Coccidioides immitis
 Cryptococcus neoformans
 Blastomyces dermatitidis
 Viruses ( Respiratory syncytal viruse, human metapneumovirus,
adenoviruses, enteroviruses, Hantavirus, herpes simplex virus, influenza
and parainfluenza viruses, Rhinoviruses, SARS etc.)
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9. RESPIRATORY TRACT
PATHOGENS
 Rare Respiratory Tract Pathogens
 Francisella tularensis
 Bacillus anthracis
 Yersinia pestis
 Burkholderia pseudomallei
 Coxiella burnetti
 Chlamydophila psittaci
 Brucella spp.
 Pasteurella multocida
 Klebsiella rhinoscleromatis
 Varicella-zooster viruse (VZV)
 parasites
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11. PATHOGENESIS
 Host Factors
 Nasal hairs
 Convoluted passage
 Mucus lining
 Secretary IgA
 Nonspecific antibacterial substances
 Cilia
 Reflexes such as coughing, sneezing & swallowing.
 Microorganism Factors
 Adherence
 Colonization
 Fimbriae
 Toxins
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12. LABORATORY DIAGNOSIS
 Specimen collection and Transport
A. Sputum
1. Expectorated
 Food should not have been ingested for 1-2 hours before
expectoration.
 Mouth should be rinsed with saline or water just before
expectoration.
 The patient should be standing, if possible or sitting upright in bed.
 He or she should take deep breath to full the lungs, and empty then in one
breath, coughing as hard and as deeply as possible.
 Sputum brought up should be spit into screw capped container.
 Visually inspect the specimen.
 Tighten the cap of the container and send immediately to lab
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13.  Sputum of less than
2ml should not be
processed unless
obviously purulent
 Only 1 sputum per
24hr submitted
SPUTUM COLLECTION
Dr.T.V.RaoMD
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14. TRANSPORTATION OF
SPUTUM
Transportation in <2 hr is
recommended with refrigeration if
delays anticipated.
Handle all samples using universal
precautions.
Perform Gram stain and plant
specimen as soon as possible
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15. 2. Induced
 Assisted by respiratory therapy technician.
 Using postural drainage & thoracic percussion to
stimulate production of acceptable sputum.
 Aerosol induced specimen:
 Breathing of aerosolized droplets of a solution
containing 15% NaCl and 10% glycerin for
approximately 10 min. or until a strong cough
reflex is initiated.
 No pre-screening required (Although these
specimen appear watery resembling saliva but
they contain material directly from alveolar
spaces ).
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16. SPECIMEN COLLECTION AND
TRANSPORT
3. Gastric aspirate:
 For isolation of Acid-Fast bacilli.
 Inability to produce sputum.
 Nasogastric tube is inserted into the stomach and contests are
withdrawn.
 The relative resistance of mycobacteria to acidity allows them to
remain viable for short period.
 Acidity of content is neutralized.
B. Endotracheal or Tracheostomy suctions specimens.
 Collected in Lukens trap.
 Tracheostomy aspirates should be treated as sputum.
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17. ENDOTRACHEAL OR
TRACHEOSTOMY SUCTIONS
SPECIMENS
Fig: Collection of sputum Fig: Lukens Trap
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18. SPECIMEN COLLECTION AND
TRANSPORT
C. Bronchoscopy
 Fibreoptic devices
 Bronchial washings
 Bronchial aspirates
 Broncoalveolar lavage BAL (100-300ml NS is infused).
 Protected bronchial brushing samples.
 Broncoalveolar lavage (BAL)
 100-300ml NS is infused.
 Estimated that: 1 million alveoli are sampled during this process.
 Significant correlation : “Between”
Acute bacterial pneumonia and >103
-104
bacterial colonies
per mililitres of BAL fluid.
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19. BRONCHOALVEOLAR LAVAGE
(BAL)
SPECIMEN ACCEPTABILITY
 Microscopic examination of Gram-
stained smear
 Acceptable
<1% of cells present are squamous
epithelial cells
 Unacceptable
>1% of cells present are squamous
epithelial cells
Thorpe JE et. al. 1987. Bronchoalveolar lavage for diagnosing acute bacterial
pneumonia. J. Infect. Dis. 155:855-861
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20. SPECIMEN COLLECTION AND
TRANSPORT
 Mini-BAL:
 Bedside, Non-bronchoscopic.
 Using metras catheter.
 20ml or less saline is infused.
 Protected bronchial brushing:
 Can collect 0.001 to 0.01 ml of material.
 Material can be suspended in 1 ml of broth dilution with vigorous
vortexing.
 Inoculate into culture media using 0.001 ml calibrated inoculating
loop.
 Correlation to infection:
 ≥ 1000 CFU/ml of broth diluent.
 ≥10^6
CFU/ml of original specimen.
 Have been considered to correlate with infection.
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21. SPECIMEN COLLECTION AND
TRANSPORT
D. Transtracheal Aspirates(TTA):
 Reduces the likelihood of contamination by upper respiratory
tract flora.
 Inserting small plastic catheter into the trachea via a needle
previously inserted through the skin and cricothyroid membrane.
E. Other invasive procedure:
 Thoracentesis (In pleural empyema).
 Open lung biopsy.
F. Blood culture:
 Only 20% of patients requiring hospitalization due to pnuemonia
are Blood culture positive.
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22. CRITERIA FOR
REJECTING SAMPLES
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Mismatch of information on the label and the
request
Inappropriate transport temperature
Excessive delay in transportation
Inappropriate transport medium
specimen received in a fixative
dry specimen
sample with questionable relevance
Insufficient quantity
Leakage

23. SPECIMEN PROCESSING
 The purulent material which contains most of the relevant
pathogens, is usually embeded in clear mucoid secretion.
 If homogenised:
 Every drop & loopful of it will contains some of pathogen.
 Suitable for quantitative examinations.
 Dithiothreitol or Buffered Pancreatin
 Mix and incubate equal volumes of the sputum and a solution of
dithiothreitol or buffered pancreatin.
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24. SPECIMEN PROCESSING
I. Dithiothreitol
 Mix rapidly on a vortex mixer for 15 seconds and stand for 15
minutes at ambient temperature.
 Or mix gently and continuously on a machine that tilts to & fro
placed for 30 minutes in an incubator at 37ºC.
I. Pancreatin
 Incubate for 30 min. at 37ºC with continuous or occasional
shaking.
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25. MICROSCOPY
▓ Gram’s stain ▓10 % KOH ▓Calcofluor white stain ▓PASstain.
Fig: Gram’s stain of sputum
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(LPF)

26. MICROSCOPY
Screening by Gram’s stain
 Acceptable criteria:
 <10 epithelial cells /LPF.
 >25 WBCs/LPF ( Except in leucopenic patients).
 Reasonable rejection criteria:
 >10 epithelial cells /LPF.
 Rejection criteria for ETA:
 >10 epithelial cells /LPF.
 Or no organism seen under oil immersion.
 In Legionella pneumophila
 Should not be subjected to screening by gram’s stain since sputum may
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27. MICROSCOPY
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Fig:Neutrophils in gram’s stain Fig: Artifacts in gram’s stain
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28. CALCULATION OF COMPOSITE Q-
SCORE
Use a specimen with a Composite Q score of +1, +2 or +3 for the additional
tests. Tests are performed on a specimen with a Composite Q score of <+1
only if specifically ordered by the physician.
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32. INOCULATION:
 Inoculated onto MA and BA at 37ºC.
 CA at 37ºC in increased carbondioxide condition.
 5ug optochin (Ethyl hydrocupriene dihydrocloride to screen
for S. pneumoniae.
 5-20 Unit (10U) Bacitracin for H. influenzae.
 Only specimens obtained by percutaneous aspiration
(including transtracheal aspiration) and by protected
bronchial brush are suitable for anaerobic culture.
 Buffered charcoal-yeast extract (BCYE) agar for Legionella
spp.
 PC 0r OFPBL agars for B. cepacia.
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33. PROCESSING SPECIMENS FOR
CULTURE
 Process specimens in biological safety cabinet, as
aerosol can result in laboratory-squired respiratory
infections.
 Process all specimens as rapidly as possible, especially
specimen from emergency department, and inpatients.
Select the most purulent or most blood-tinged portion
of the specimen. Significant growth above the cutoff
should be reported; however if more than one pathogen
is isolated than it is suggestive of oropharyngeal
contamination and clinical correlation should be done
before reporting the samples.
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35. CONTAMINATION WITH
ORAL FLORA INTERFERES
RESULTS
 Because of contaminating oral flora ,sputum
specimens, specimens obtained by bronchial
washing, and lavage tracheostomy, or
endotracheal tube aspirates are not inoculated
to enriched broth or incubated anaerobically.
Only specimens obtained by percutaneous
aspiration (including trans tracheal
aspiration )and by protected bronchial brush
are suitable for anaerobic culture: he latter
must be done quantitatively for proper
interpretation.
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36. IDENTIFICATION OF
MICRORGANISM
 Identification of microorganism is based on
the gram’s staining, colonial characters,
biochemical tests and specific anti-sera if
required.
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37. OTHER SPECIMENS
 Urine for antigen detection
 Blood for serology
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39. OPTOCHIN SENSITIVITY TEST

40. “DRAUGHTSMAN” APPEARANCE

41. BILE SOLUBILITY TEST

42. LABORATORY DIAGONOSIS
COLONY PROCEDURE /Rapid presumptive test
-This method works well on large or mucoid colonies
-Select a well-isolated single colony from a blood or chocolate
agar plate.
-Circle the colony on the bottom of the Petri dish to locate
- Place one drop of 2% sodium deoxycholate directly on the
colony.
-Incubate at 37°Cfor up to 30 minutes. Do not invert the plate.
The lid may be left slightly ajar to aid evaporation
- When the reagent has dried examine the area for lysis or
disintegration of the original colony
 Positive result: Colony lysed or disintegrated
 Negative result: No change

43. QUELLUNG REACTION (NEUFELD
QUELLUNG RXN)
 Word origin: German quellung for "swelling"
 quellung phenomenon
Increase in opacity and visibility of the capsule of
capsulated organisms exposed to specific agglutinating
anticapsular antibodies
 Apparent swelling of the capsule upon binding of
homologous antibody due to change in refractive index
 reagent - antipneumoccal rabbit sera (omniserum)is used
against 90 sero- types of S.pnuemoniae from State Serum
Institute, DK2300,Copenhagen, Denmark)

44. SEROTYPING
 different serotypes causes different diseases
• Serotyping can be done by:
- Quellung reaction(described by Neufeld in 1902), classical method
 Agglutination of the pneumococci with type specific antiserum
 Capillary Precipitation of SSS with type specific antiserum
 Coagglutination
 dot blot assay
 counterimmuno-electrophoresis (CIEP)

45. Properties Pneumococcus Viridans streptococci
Morphology Capsulated,lanceolate-
shaped diplococci
Non-capsulated,oval or
round cells in short chains
Colony on BA Raised/flat initially later
“draughtsman”
Dome-shaped
Broth culture Uniform turbidity granular with powdery
deposit
Bile solubility test + -
Optochin sensitivity Sensitive Resistant
Inulin fermentation + -
Quellung reaction + -
Mice/rabbbit pathogenicity Pathogenic Non -pathogenic

46. NUCLEIC ACID TECHNIQUE
 Polymerase chain reaction
 Samples : blood, serum, CSF and respiratory samples,
body fluids
 Gene used as primers
 The pneumolysin gene,
 pneumococcal surface adhesin protein (PsaA) gene,
 cell wall autolysin (LytA) protein gene
 DNA/DNA hybridization

47. CASE STUDY…….
 A 16-month-old boy was admitted with fever, lethargy and
trouble breathing. A diagnosis of pneumonia was made by
physical examination. The child had recently been to
Panama and at that time was treated with ceftriaxone for
cough and fever. His fever continued, despite treatment.
On admission, he was given erythromycin therapy.
Thacheal aspirate and blood cultures were obtained, but
the respiratory specimen contained numerous epithelial
cells and yielded normal respiratory flora on culture. A
pleural aspirate and blood cultures were positive with
Streptococcus pneumoniae, which was reisistant to
erythromycin and penicillin and intermediate in
susceptibility to ceftriaxone. The patient was given high
doses of ceftriaxone and vancomycin and responded to
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48. QUESTIONS..???
 What criteria are used in laboratories to reject sputum and
tracheal aspirates for culture?
 If greater than 10 squamous epithelial cells per low-power field
are seen in a gram stain but the smear also has numerous white
blood cells (greater than 25/LPF), should the specimen be rejected
for culture?
 In cases of pneumococcal pneumonia, what percentage of blood
and sputum cultures is positive for S. pneumoniae?
 The organism was reported as resistant to penicillin and
intermediate in susceptibility to third-generation cephalosporins .
How does the laboratory perform testing for this organism?
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49. SPECIAL CONSIDERATION TO
PULMONARY TUBERCULOSIS
 Recovery of mycobacteria from clinical samples is more
time consuming procedure than for normal pathogenic
bacteria, requiring as it does the following steps:
1. Homogenization
2. Centrifugation
3. Neutralization
4. Inoculation
5. Incubation for at least 6wks
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50. CULTURAL
CHARACTERISTICS
(M. TUBERCULOSIS)
 M. tuberculosis is an obligate aerobe while M.
bovis is microaerophilic on primary isolation
(aerobic on subculture)
 Grows slowly (G.T. 15-20 hours)
 Optimum temperature 370
C
 pH 6.4-7.0
 Does not grow <250
C and >400
C
 Increased carbondioxide (5-10%) enhances
growth
 Grows more luxuriantly in culture (eugonic)
than M. bovis (dysgonic)

51.  Addition of 0.5% glycerol improves the growth of
M. tuberculosis but has no effect on or even
impair the growth of M. bovis.
 Sodium pyruvate helps the growth of both types
 Human tubercle bacilli do not grow in presence of p-
nitrobenzoic acid (500 mg/l), unlike other slow growing
nonchromogens.
 Highly susceptible even to traces of toxic substance
like fatty acid in culture media which can be
neutralized by serum albumin or charcoal.

52. CULTURE MEDIA
 Solid media:
 Egg containing – Lowenstein-Jensen media
Petragnani media
Dorset egg media
Ogawa media
 Blood containing – Tarshis media
 Serum containing – Loeffler’s serum slope
 Potato containing – Pawlowsky’s media
 Liquid media:
 Dubo’s media
 Middlebrook’s media 7H9
 Proskauer and Beck’s media
 Sula’s and Sauton’s media
 Kirchner media

53.  To prevent overgrowth by contaminants,
Cocktail of antibiotics
Polymyxin B
Amphotericin B
Nalidixic acid
Trimethoprim
Azlocillin
PANTA
Polymyxin B
Amphotericin B
Carbenicillin
Trimethoprim
PACT

54. COLONY CHARACTERS
 Solid media:
M. tuberculosis:
 Colonies appear after 2-3 weeks.
 Colonies first appear as small, dry,
friable, rough and granular, creamy
white (Rough, tough, buff)
 Typical colonies have a flat irregular
margin and look like a cauliflower in the
center.
M. bovis:
 Colonies appear after 3-6 weeks
 Colonies are smooth, translucent and
pyramidal

55. QUANTIFICATION SCALE FOR
MYCOBACTERIAL GROWTH ON AGAR
PLATES (ATS, 2000)
No. of colonies seen Quantity reported
No colonies seen
<50 colonies
50-100 colonies
100-200 colonies
200-500 colonies (almost confluence)
>500 colonies (confluence)
Negative
Report actual number seen
1 +
2 +
3 +
4+

56.  Liquid media:
Bacilli grow either on surface as pellicle or as floccules
throughout the medium due to hydrophobic nature of their
cell wall lipid.
Generally, virulent strains often grow as twisted rope like
colonies called serpentine cords??????
Diffuse bacterial growth is obtained by adding a detergent
Tween 80.

57.  Lowenstein-Jensen media
 Fresh whole eggs
 Defined salts
 Glycerol
 Potato flour
 Asparagine
 Malachite green

58.  Middlebrook 7H10
Defined salts
Vitamins
Cofactors
Glycerol
Oleic acid
Albumin
Dextrose
Catalase
Malachite green

59.  Ogawa media
 Potassium dihydrogen phosphate
 Sodium glutamate
 Glycerine
 2% malachite green
 Distilled water
 Whole egg homogenate

60.  Niacin test- Niacin test is positive with human
type and negative with bovine type of bacilli.
 Aryl sulphatase test- Aryl sulphatase test is
positive with atypical mycobacteria.
 Neutral red test- Neutral red test is positive with
virulent strains of tubercle bacilli while avirulent
strains are negative.
 Catalase-peroxidase test-
 Atypical mycobacterial strains - strongly catalase
positive
 Tubercle bacilli - only weakly positive for catalase
 Tubercle bacilli - peroxidase positive
 Atypical mycobacteria – peroxidase negative
 Catalase and peroxidase activity are lost when tubercle
bacilli become INH resistant.
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61.  Amidase test- Amidase test differentiates
mycobacteria by its ability to split amides. A
useful pattern is provided by testing five
amides, acetamide, benzamide, carbamide,
nicotinamide and pyrazinamide.
 Nitrate reduction test-
 M. tuberculosis +
 M. bovis -

62. LABORATORY DIAGNOSIS
Sample is collected according to the site of infection
 Primary tuberculosis- 3 early morning sputum
samples, laryngeal swab, bronchial washings,
stomach washings in children
Transport medium for preserving M. tuberculosis in sputum specimens –
Cetylpyridinium chloride – Sodium chloride (CPC-NaCl) transport medium.

63. Conventional techniques
 Sputum microscopy
Acid fast stains:
 Ziehl Neelsen stain
 Kinyoun’s modification of ZN stain
 Auramine-Rhodamine stain
Sputum culture
Mantoux skin test

64.  Direct methods
Smear
Culture
Phage based assays
Phenotypic methods- lipid analysis
Molecular methods- Probes, PCR
 Indirect methods
Serology- ELISA, Latex agglutination
ELISPOT Assays
Demonstration of biological products
 ADA estimation
 Tuberculostearic acid test
 Bromide partition test

65. SPUTUM SMEAR EXAMINATION
 Most widely applicable, most reliable ( WHO )
 Minimum 3 samples (spot- early morning-spot)
 Sensitivity- 50-70%

66. DISADVANTAGES OF SMEAR
EXAMINATION
 Lower limit of detection - 104
bacilli/ml
Sample
 Cannot determine viability
 Cannot identify species
 False positive/ False negative result

67. INTERNATIONAL UNION AGAINST
TUBERCULOSIS (IUAT)
If definite pink bacilli are not seen – AFB not found
If 1 or 2 bacilli in the entire field – Doubtful, repeat for another
sample
If 1-9 AFB/100 fields – AFB found (exact number)
10-100 AFB/ 100 fields – AFB found (+)
1-10 AFB/ field – AFB found (++)
> 10 AFB/ field – AFB found (+++)

68. DIGESTION, CONCENTRATION
AND CULTURAL METHODS
 Homogenization – To release the mycobacteria
from the body fluid or tissue in which they are
contained
 Decontamination – To kill the contaminants or
else they will overgrow the medium

69. 1. Petroff’s method
Equal vol. of Sputum and 4% NaOH
370
C – 30 mins with occasional shaking
Centrifugation 3000g – 20-30mins
Discard the supernatant
Neutralize the deposit with 8% HCl in presence of
phenol red indicator
Centrifugation
Deposit
 Inoculated on to the culture media
 Smear preparation
 Animal inoculation
 Molecular methods

70. 2. Modified Petroff’s method
 Sputum + Double vol. of 4% NaOH
 R.T. – 15mins with occasional shaking
 Centrifugation 3000g – 15mins
 Discard the supernatant
 15 ml saline or water + sediment
 Centrifugation 3000g – 15mins
 Deposit – inoculated on to the culture media

71. 3. N-acetyl-L-Cysteine-NaOH method
4. Zephiran (Benzalkonium chloride)-TSP method

72. ANIMAL INOCULATION
 Intramuscular inoculation of 0.5 ml bacterial suspension into the
thigh of 12 week old guinea pig
 The tuberculin test becomes positive after 3-4 weeks and there
will be progressive loss of weight of the animal
 The animal is autopsied after 6wks or 12 wks.
 Results:
 Caseous lesion at the site of inoculation
 Enlarged inguinal LN
 Tubercules in the lungs
 Splenomegaly
The identity of the bacteria is confirmed by demonstration of
AFB from the lesions to exclude the Y. pseudotuberculosis,
Brucella as their lesions macroscopically simulate tubercles.
ROOT INDEX OF VIRULENCE

73. CDC CLASSIFICATION OF
TUBERCULIN REACTION
An induration (palpable raised hardened area of skin)
of more than 5–15 mm (depending upon the person's
risk factors) to 10 Mantoux units is considered a
positive result, indicating TB infection.
5 mm or more is positive in
HIV-positive person,
Patients with organ transplants and other immunosuppressed
patients
10 mm or more is positive in
Recent arrivals (less than 5 years) from high-prevalent countries
Residents and employees of high-risk congregate settings (e.g.,
prisons, nursing homes, hospitals, homeless shelters, etc.)
Persons with clinical conditions that place them at high risk (e.g.,
diabetes, prolonged corticosteroid therapy, leukemia, end-stage renal
disease, chronic malabsorption syndromes, low body weight, etc)
15 mm or more is positive in
Persons with no known risk factors for TB

74.  Recent advances
 Serological diagnosis
 IgM and IgG antibody to mycobacterial antigens (ELISA, LA)
 IgG and IgA antibody against the mycobacterial antigen A60 in
patient with EPT
 Radiometric BACTEC 460 TB method
 MGIT 960 Mycobacterial detection system
 Chemical detection of biological compounds
 Adenosine deaminase
 Tuberculostearic acid test
 Bromide partition test
 HPLC
 Mycobacteriophage typing
 Genotyping methods

75. BACTEC
 Organisms multiply in the broth and metabolize
14
C – containing palmitic acid substrate producing
radioactively labelled 14
CO2 in the atmosphere
that collects above the broth in the bottle.
 The BACTEC instrument withdraws the 14
CO2 –
containing atmosphere and measures the amount
of radioactivity present which is converted
proportionally to a quantitative growth index
(GI).

76. BACTEC MGIT-960

77. -It is based on a glass tube containing a modified
Middlebrook 7H9 broth enriched with OADC and
PANTA antibiotic mixture.
-A fluorescent compound is embedded at the bottom
of tube.
- The fluorescent compound does not fluoresce in the
presence of oxygen, but it fluoresces following
depletion of oxygen by actively respiring organisms
as a result of mycobacterial growth.
Mycobacterial growth indicator tube
(MGIT) system

78. CULTURE METHODS
Modern method Conventional
Detection 14 days 28 days
Susceptibility 3 wks 8 wks
Sensitivity 95% 84%
Contamination 4% 5%

79. LYSIS-CENTRIFUGATION BLOOD
CULTURE SYSTEM
- The recovery of mycobacterium from peripheral blood and bone
marrow samples may be improved by releasing the intracellular
mycobacterial cells into the blood culture broth, increasing the rate and
reducing the time of recovery.
- In the lysis- centrifugation blood culture method, blood is put into a
tube containing an anticoagulant and a lysing agent to effect rupture of
both erythrocytes and neutrophils.
- Following centrifugation of the tube, the sediment is inoculated into
the appropriate culture media.
- This method has increased both the yield and shortened the time of
recovery of mycobacteria from blood cultures.

80. LIPID ANALYSIS
 Mycolic acid profiles
 HPLC- Confirmation of isolate , directly
from clinical specimen

81. OTHER TESTS
 Immunomagnetic separation of M. tuberculosis
(Murtagh et al 1995)
 Magnetic polystrene microspheres coated with antibody to
mycobacteria are added to the specimen and mixed together
 The beads and bound organisms are collected by using a
magnet and detected by microscopic examination with the use
of conventional method.
 It improves mycobacterial detection level by 2-3 times more
than conventional method.
 Used for CSF, Sputum.

82.  Mycodot antibody test
 Purified lipoarabinomannan is used as an antigen
and it detects antimycobacterial antibody levels
likely to be found in those with active diseases
 Specificity 97-98%
 Sensitivity 70-75%

83. URINARY ANTIGEN DETECTION
 LAM antigen
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84. MOLECULAR DIAGNOSIS OF M.
TUBERCULOSIS
 Nucleic acid probes
 Amplification techniques
PCR assay
Transcription mediated Assay
Strand displacement amplification
Q β replicase gene amplification
Spoligotyping

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86. DNA MICROARRAY

87. NUCLEIC ACID PROBES
 DNA probes
 M. tuberculosis complex, M. avium
 Not sensitive enough (104
organisms)
 Ribosomal RNA based probes
 Target r- RNA
 M. tuberculosis , M. avium, M. gordonae
 Chemiluminiscent system

88. PCR ASSAYS
 Target
 IS 6110 (10-20 times repeated in genome)
 25% of Indian population lack IS 6110
 PCR ( ROCHE AMPLICOR)
 Target gene 16S-rRNA (584 bp)
Reproducible, sensitive, specific
Can detect 1-10 organisms

89. DRAWBACKS OF PCR ASSAY
 Dead bacilli are detected.
 False Positive due to contamination.
 No prognostic value.

90. DEMONSTRATION OF BIOLOGICAL
PRODUCTS
Adenosine Deaminase Assay
 Lymphocytic enzyme - Increase in TB
 Surrogate marker for extrapulmonary
tuberculosis in Pleural, pericardial, CSF
 Sensitivity & specificity > 90%
 Limitations - False +

91. ADA TEST

92. FAST PLAQUE ASSAY

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