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ShyamKumarMishra
Shyam Kumar Mishra
Assistant Professor,
Institute of Medicine
Laboratory
diagnosis of LoWEr
rEsPiratory...
RESPIRATORY TRACT:
ANATOMICAL STRUCTURE
 It is utmost important to be familiar with the
anatomic structure of the thoraci...
ANATOMY OF RESPIRATORT TRACT
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ShyamKumarMishra
INITIATION………
 Aspiration of colonizing flora into the alveoli
 Inhalation of aerosols
 Hematologic seeding of the lung...
 When paediatric patients with cystic fibrosis (CF)
have respiratory infections (exacerbations), the
trigger is most comm...
1- Bronchitis.
A- Acute:
 Usually caused by viruses.
 In infants & preschool children is Bordetella pertussis.
B- Chroni...
2- Pneumonia
 Community-Acquired Pneumonia:
patients are believed to have acquired infection outside the hospital
setting...
RESPIRATORY TRACT
PATHOGENS
 Definitive Respiratory Tract Pathogens
 Haemophilus influenzae
 Streptococcus pneumoniae
...
RESPIRATORY TRACT
PATHOGENS
 Rare Respiratory Tract Pathogens
 Francisella tularensis
 Bacillus anthracis
 Yersinia pe...
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PATHOGENESIS
 Host Factors
 Nasal hairs
 Convoluted passage
 Mucus lining
 Secretary IgA
 Nonspecific antibacterial ...
LABORATORY DIAGNOSIS
 Specimen collection and Transport
A. Sputum
1. Expectorated
 Food should not have been ingested fo...
 Sputum of less than
2ml should not be
processed unless
obviously purulent
 Only 1 sputum per
24hr submitted
SPUTUM COLL...
TRANSPORTATION OF
SPUTUM
Transportation in <2 hr is
recommended with refrigeration if
delays anticipated.
Handle all sam...
2. Induced
 Assisted by respiratory therapy technician.
 Using postural drainage & thoracic percussion to
stimulate prod...
SPECIMEN COLLECTION AND
TRANSPORT
3. Gastric aspirate:
 For isolation of Acid-Fast bacilli.
 Inability to produce sputum...
ENDOTRACHEAL OR
TRACHEOSTOMY SUCTIONS
SPECIMENS
Fig: Collection of sputum Fig: Lukens Trap
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SPECIMEN COLLECTION AND
TRANSPORT
C. Bronchoscopy
 Fibreoptic devices
 Bronchial washings
 Bronchial aspirates
 Bronco...
BRONCHOALVEOLAR LAVAGE
(BAL)
SPECIMEN ACCEPTABILITY
 Microscopic examination of Gram-
stained smear
 Acceptable
<1% of ...
SPECIMEN COLLECTION AND
TRANSPORT
 Mini-BAL:
 Bedside, Non-bronchoscopic.
 Using metras catheter.
 20ml or less saline...
SPECIMEN COLLECTION AND
TRANSPORT
D. Transtracheal Aspirates(TTA):
 Reduces the likelihood of contamination by upper resp...
CRITERIA FOR
REJECTING SAMPLES
22
Mismatch of information on the label and the
request
Inappropriate transport temperature...
SPECIMEN PROCESSING
 The purulent material which contains most of the relevant
pathogens, is usually embeded in clear muc...
SPECIMEN PROCESSING
I. Dithiothreitol
 Mix rapidly on a vortex mixer for 15 seconds and stand for 15
minutes at ambient t...
MICROSCOPY
▓ Gram’s stain ▓10 % KOH ▓Calcofluor white stain ▓PASstain.
Fig: Gram’s stain of sputum
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(LP...
MICROSCOPY
Screening by Gram’s stain
 Acceptable criteria:
 <10 epithelial cells /LPF.
 >25 WBCs/LPF ( Except in leucop...
MICROSCOPY
27
Fig:Neutrophils in gram’s stain Fig: Artifacts in gram’s stain
ShyamKumarMishra
CALCULATION OF COMPOSITE Q-
SCORE
Use a specimen with a Composite Q score of +1, +2 or +3 for the additional
tests. Tests ...
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INOCULATION:
 Inoculated onto MA and BA at 37ºC.
 CA at 37ºC in increased carbondioxide condition.
 5ug optochin (Ethyl...
PROCESSING SPECIMENS FOR
CULTURE
 Process specimens in biological safety cabinet, as
aerosol can result in laboratory-squ...
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CONTAMINATION WITH
ORAL FLORA INTERFERES
RESULTS
 Because of contaminating oral flora ,sputum
specimens, specimens obtain...
IDENTIFICATION OF
MICRORGANISM
 Identification of microorganism is based on
the gram’s staining, colonial characters,
bio...
OTHER SPECIMENS
 Urine for antigen detection
 Blood for serology
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OPTOCHIN SENSITIVITY TEST
“DRAUGHTSMAN” APPEARANCE
BILE SOLUBILITY TEST
LABORATORY DIAGONOSIS
COLONY PROCEDURE /Rapid presumptive test
-This method works well on large or mucoid colonies
-Select...
QUELLUNG REACTION (NEUFELD
QUELLUNG RXN)
 Word origin: German quellung for "swelling"
 quellung phenomenon
Increase in o...
SEROTYPING
 different serotypes causes different diseases
• Serotyping can be done by:
- Quellung reaction(described by N...
Properties Pneumococcus Viridans streptococci
Morphology Capsulated,lanceolate-
shaped diplococci
Non-capsulated,oval or
r...
NUCLEIC ACID TECHNIQUE
 Polymerase chain reaction
 Samples : blood, serum, CSF and respiratory samples,
body fluids
 Ge...
CASE STUDY…….
 A 16-month-old boy was admitted with fever, lethargy and
trouble breathing. A diagnosis of pneumonia was m...
QUESTIONS..???
 What criteria are used in laboratories to reject sputum and
tracheal aspirates for culture?
 If greater ...
SPECIAL CONSIDERATION TO
PULMONARY TUBERCULOSIS
 Recovery of mycobacteria from clinical samples is more
time consuming pr...
CULTURAL
CHARACTERISTICS
(M. TUBERCULOSIS)
 M. tuberculosis is an obligate aerobe while M.
bovis is microaerophilic on pr...
 Addition of 0.5% glycerol improves the growth of
M. tuberculosis but has no effect on or even
impair the growth of M. bo...
CULTURE MEDIA
 Solid media:
 Egg containing – Lowenstein-Jensen media
Petragnani media
Dorset egg media
Ogawa media
 Bl...
 To prevent overgrowth by contaminants,
Cocktail of antibiotics
Polymyxin B
Amphotericin B
Nalidixic acid
Trimethoprim
Az...
COLONY CHARACTERS
 Solid media:
M. tuberculosis:
 Colonies appear after 2-3 weeks.
 Colonies first appear as small, dry...
QUANTIFICATION SCALE FOR
MYCOBACTERIAL GROWTH ON AGAR
PLATES (ATS, 2000)
No. of colonies seen Quantity reported
No colonie...
 Liquid media:
Bacilli grow either on surface as pellicle or as floccules
throughout the medium due to hydrophobic natur...
 Lowenstein-Jensen media
 Fresh whole eggs
 Defined salts
 Glycerol
 Potato flour
 Asparagine
 Malachite green
 Middlebrook 7H10
Defined salts
Vitamins
Cofactors
Glycerol
Oleic acid
Albumin
Dextrose
Catalase
Malachite green
 Ogawa media
 Potassium dihydrogen phosphate
 Sodium glutamate
 Glycerine
 2% malachite green
 Distilled water
 Who...
 Niacin test- Niacin test is positive with human
type and negative with bovine type of bacilli.
 Aryl sulphatase test- A...
 Amidase test- Amidase test differentiates
mycobacteria by its ability to split amides. A
useful pattern is provided by t...
LABORATORY DIAGNOSIS
Sample is collected according to the site of infection
 Primary tuberculosis- 3 early morning sputum...
Conventional techniques
 Sputum microscopy
Acid fast stains:
 Ziehl Neelsen stain
 Kinyoun’s modification of ZN stain
...
 Direct methods
Smear
Culture
Phage based assays
Phenotypic methods- lipid analysis
Molecular methods- Probes, PCR
...
SPUTUM SMEAR EXAMINATION
 Most widely applicable, most reliable ( WHO )
 Minimum 3 samples (spot- early morning-spot)
 ...
DISADVANTAGES OF SMEAR
EXAMINATION
 Lower limit of detection - 104
bacilli/ml
Sample
 Cannot determine viability
 Canno...
INTERNATIONAL UNION AGAINST
TUBERCULOSIS (IUAT)
If definite pink bacilli are not seen – AFB not found
If 1 or 2 bacilli in...
DIGESTION, CONCENTRATION
AND CULTURAL METHODS
 Homogenization – To release the mycobacteria
from the body fluid or tissue...
1. Petroff’s method
Equal vol. of Sputum and 4% NaOH
370
C – 30 mins with occasional shaking
Centrifugation 3000g – 20-...
2. Modified Petroff’s method
 Sputum + Double vol. of 4% NaOH
 R.T. – 15mins with occasional shaking
 Centrifugation 30...
3. N-acetyl-L-Cysteine-NaOH method
4. Zephiran (Benzalkonium chloride)-TSP method
ANIMAL INOCULATION
 Intramuscular inoculation of 0.5 ml bacterial suspension into the
thigh of 12 week old guinea pig
 T...
CDC CLASSIFICATION OF
TUBERCULIN REACTION
An induration (palpable raised hardened area of skin)
of more than 5–15 mm (depe...
 Recent advances
 Serological diagnosis
 IgM and IgG antibody to mycobacterial antigens (ELISA, LA)
 IgG and IgA antib...
BACTEC
 Organisms multiply in the broth and metabolize
14
C – containing palmitic acid substrate producing
radioactively ...
BACTEC MGIT-960
-It is based on a glass tube containing a modified
Middlebrook 7H9 broth enriched with OADC and
PANTA antibiotic mixture.
...
CULTURE METHODS
Modern method Conventional
Detection 14 days 28 days
Susceptibility 3 wks 8 wks
Sensitivity 95% 84%
Contam...
LYSIS-CENTRIFUGATION BLOOD
CULTURE SYSTEM
- The recovery of mycobacterium from peripheral blood and bone
marrow samples ma...
LIPID ANALYSIS
 Mycolic acid profiles
 HPLC- Confirmation of isolate , directly
from clinical specimen
OTHER TESTS
 Immunomagnetic separation of M. tuberculosis
(Murtagh et al 1995)
 Magnetic polystrene microspheres coated ...
 Mycodot antibody test
 Purified lipoarabinomannan is used as an antigen
and it detects antimycobacterial antibody level...
URINARY ANTIGEN DETECTION
 LAM antigen
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MOLECULAR DIAGNOSIS OF M.
TUBERCULOSIS
 Nucleic acid probes
 Amplification techniques
PCR assay
Transcription mediated...
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DNA MICROARRAY
NUCLEIC ACID PROBES
 DNA probes
 M. tuberculosis complex, M. avium
 Not sensitive enough (104
organisms)
 Ribosomal RN...
PCR ASSAYS
 Target
 IS 6110 (10-20 times repeated in genome)
 25% of Indian population lack IS 6110
 PCR ( ROCHE AMPLI...
DRAWBACKS OF PCR ASSAY
 Dead bacilli are detected.
 False Positive due to contamination.
 No prognostic value.
DEMONSTRATION OF BIOLOGICAL
PRODUCTS
Adenosine Deaminase Assay
 Lymphocytic enzyme - Increase in TB
 Surrogate marker fo...
ADA TEST
FAST PLAQUE ASSAY
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Laboratory diagnosis of Lower respiratory tract infection including tuberculosis

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Laboratory diagnosis of Lower respiratory tract infection including tuberculosis

  1. 1. 1 ShyamKumarMishra Shyam Kumar Mishra Assistant Professor, Institute of Medicine Laboratory diagnosis of LoWEr rEsPiratory traCt infECtions inCLuding tubErCuLosis
  2. 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. 2 ShyamKumarMishra
  3. 3. ANATOMY OF RESPIRATORT TRACT 3 ShyamKumarMishra
  4. 4. INITIATION………  Aspiration of colonizing flora into the alveoli  Inhalation of aerosols  Hematologic seeding of the lung from a distant focus 4 ShyamKumarMishra
  5. 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. 5 ShyamKumarMishra
  6. 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 6 ShyamKumarMishra
  7. 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 7 ShyamKumarMishra
  8. 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.) 8 ShyamKumarMishra
  9. 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 9 ShyamKumarMishra
  10. 10. 10 ShyamKumarMishra
  11. 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 11 ShyamKumarMishra
  12. 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 12 ShyamKumarMishra
  13. 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 13
  14. 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 14
  15. 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 ). 15 ShyamKumarMishra
  16. 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. 16 ShyamKumarMishra
  17. 17. ENDOTRACHEAL OR TRACHEOSTOMY SUCTIONS SPECIMENS Fig: Collection of sputum Fig: Lukens Trap 17 ShyamKumarMishra
  18. 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. 18 ShyamKumarMishra
  19. 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 19
  20. 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. 20 ShyamKumarMishra
  21. 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. 21 ShyamKumarMishra
  22. 22. CRITERIA FOR REJECTING SAMPLES 22 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. 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. 23 ShyamKumarMishra
  24. 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. 24 ShyamKumarMishra
  25. 25. MICROSCOPY ▓ Gram’s stain ▓10 % KOH ▓Calcofluor white stain ▓PASstain. Fig: Gram’s stain of sputum 25 ShyamKumarMishra (LPF)
  26. 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 be scant and watery, with few or no host cells. 26 ShyamKumarMishra
  27. 27. MICROSCOPY 27 Fig:Neutrophils in gram’s stain Fig: Artifacts in gram’s stain ShyamKumarMishra
  28. 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. 28
  29. 29. 29 ShyamKumarMishra
  30. 30. 30
  31. 31. 31 ShyamKumarMishra
  32. 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. 32 ShyamKumarMishra
  33. 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. 33
  34. 34. 34 ShyamKumarMishra
  35. 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. 35
  36. 36. IDENTIFICATION OF MICRORGANISM  Identification of microorganism is based on the gram’s staining, colonial characters, biochemical tests and specific anti-sera if required. 36 ShyamKumarMishra
  37. 37. OTHER SPECIMENS  Urine for antigen detection  Blood for serology 37 ShyamKumarMishra
  38. 38. 38 ShyamKumarMishra
  39. 39. OPTOCHIN SENSITIVITY TEST
  40. 40. “DRAUGHTSMAN” APPEARANCE
  41. 41. BILE SOLUBILITY TEST
  42. 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. 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. 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. 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. 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. 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 therapy. 47 ShyamKumarMishra
  48. 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? 48 ShyamKumarMishra
  49. 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 49 ShyamKumarMishra
  50. 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. 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. 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. 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. 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. 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. 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. 57.  Lowenstein-Jensen media  Fresh whole eggs  Defined salts  Glycerol  Potato flour  Asparagine  Malachite green
  58. 58.  Middlebrook 7H10 Defined salts Vitamins Cofactors Glycerol Oleic acid Albumin Dextrose Catalase Malachite green
  59. 59.  Ogawa media  Potassium dihydrogen phosphate  Sodium glutamate  Glycerine  2% malachite green  Distilled water  Whole egg homogenate
  60. 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. 60 ShyamKumarMishra
  61. 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. 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. 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. 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. 65. SPUTUM SMEAR EXAMINATION  Most widely applicable, most reliable ( WHO )  Minimum 3 samples (spot- early morning-spot)  Sensitivity- 50-70%
  66. 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. 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. 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. 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. 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. 71. 3. N-acetyl-L-Cysteine-NaOH method 4. Zephiran (Benzalkonium chloride)-TSP method
  72. 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. 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. 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. 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. 76. BACTEC MGIT-960
  77. 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. 78. CULTURE METHODS Modern method Conventional Detection 14 days 28 days Susceptibility 3 wks 8 wks Sensitivity 95% 84% Contamination 4% 5%
  79. 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. 80. LIPID ANALYSIS  Mycolic acid profiles  HPLC- Confirmation of isolate , directly from clinical specimen
  81. 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. 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. 83. URINARY ANTIGEN DETECTION  LAM antigen 83 ShyamKumarMishra
  84. 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
  85. 85. 85 ShyamKumarMishra
  86. 86. DNA MICROARRAY
  87. 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. 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. 89. DRAWBACKS OF PCR ASSAY  Dead bacilli are detected.  False Positive due to contamination.  No prognostic value.
  90. 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. 91. ADA TEST
  92. 92. FAST PLAQUE ASSAY
  93. 93. 93 ShyamKumarMishra

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