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DIAGNOSIS

DIAGNOSIS. DIAGNOSIS. CLINICAL. LAB. LAB DIAGNOSIS. 1.PUL TUBERCULOSIS. SAMPLE COLLECTION MICROSCOPY CONCENTRATION METHODS CULTURE SENSITIVITY TESTS ANIMAL INOCULATION NUCLEIC ACID TECHNOLOGY IMMUNODIAGNOSIS. Sputum Laryngeal swabs Bronchial washings Gastric lavage.

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DIAGNOSIS

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  1. DIAGNOSIS

  2. DIAGNOSIS CLINICAL LAB

  3. LAB DIAGNOSIS

  4. 1.PUL TUBERCULOSIS • SAMPLE COLLECTION • MICROSCOPY • CONCENTRATION METHODS • CULTURE • SENSITIVITY TESTS • ANIMAL INOCULATION • NUCLEIC ACID TECHNOLOGY • IMMUNODIAGNOSIS

  5. Sputum Laryngeal swabs Bronchial washings Gastric lavage • Samples collected---- • Best collected-in morning before meal • If scanty-24hr sample taken

  6. MICROSCOPY By Ziehl-Neelsen staining

  7. ZN smear evaluation and AFB report

  8. By fluorescent microscopy

  9. Advantages • Reliable method • Rapid screening for epidemiological purpose Disadvantages • Has only 30-70% sensitivity

  10. Concentration methods For microscopy For smear,culture & animal inoculation

  11. MYCOPROSAFE

  12. CULTURE Conc material into IUAT-LJ medium Incubation at 37○C Examine twice weekly growth No growth _ve report after 8-12 wks

  13. SENSITIVITY TESTING • Absolute conc method • In which no. of media containing serial conc of drugs are inoculated(for MIC) • Resistance ratio method • 2 sets of media containing graded conc of drugs are inoculated • Proportion method • Indicates avg sensitivity of strain

  14. Antibiotic sets for sensitivity testing.

  15. ANIMAL INOCULATION • Conc specimen • Inoculated IM into thigh of 2 healthy guinea pigs 12 weeks old • Progressive loss of wt • Infected animals show + tuberculin test • 1 animal is killed after 4 weeks & autopsied • On autopsy • Caseous lesion at site of inoculation • Draining &internal LN are enlarged & caseous • Spleen enlarged with irregular necrotic areas • Tubercles seen in peritoneum

  16. contd.... • DISADVANTAGES: • Cumbersome • Costly • Less sensitive than culture

  17. 2. EXTRA PULMONARY TB • SAMPLES COLLECTED • CSF • Bone marrow &liver biopsy • Blood • Pus • Pleural effusion--- exudate • urine

  18. ALLERGY & IMMUNITY • CMI….manifests as • Delayed hypersensitivity • Resistance to infection

  19. s/c injection given to a normal guinea pig No immediate response After 10 – 14 days Nodule at site Breaks up to form ulcer Persists till animal dies of progressive TB

  20. KOCH PHENOMENON Guinea pig inj which has prior contact 4-6 wk before After 1-2 days indurated lesions at site Next day necrosis which forms shallow ulcer Heals rapidly without involving draining LN 3 COMPONENTS----LOCAL REACTION, FOCAL RESPONSE, SYSTEMIC RESPONSE

  21. ALLERGICTESTS • OT • PPD - S • 1 TU= 0.01 ml of OT or 0.00002mg of PPD-S MANTOUX TEST • METHOD • INTERPRETATION OF RESULT

  22. HEAF TEST • Multiple puncture testing • For screening & surveys

  23. Tine test Disposable prongs carrying dried PPD is used

  24. FALSE NEGATIVES • Miliary tuberculosis • Convalscents from viral infections like Measles • Lympho reticular malignancy • Immuno suppressive therapy • Inactive PPD preparation • Improper injection technique FALSE POSITIVES • Infection or exposure to atypical mycobacteria • BCG vaccine

  25. USES • Aids in diagnosing active infections in infants & young children • To measure prevalence of infection in an area • To select susceptibles • As an indication of successful vaccination

  26. Recent advances in detection of M.tb 1.Bactec 460 TB system • Broth based growth system • Medium contains 4ml of Middlebrook 7H12 brothwith carbon 14 labeled palmitic acid • Clinical specimen inoculated with PANTA • Use radiometric method for growth detection

  27. BACTEC TB Instrument ADVANTAGES • rapid method • Increase no. of +ve cultures • Antibiotic sensitivity testing can be done DRAWBACK—very expensive

  28. 2.Rapid evaluation of drug susceptibility by bioluminescence assays medium alone (+) or with INH (H), rifampin (R), or ethambutol (E). Erdman susceptible reference strain (ERD), a CDC INH-resistant strain (CDC-K), and clinical monoresistant (Z) and multidrug resistant (DS) strains. • Advantage---testing drug susceptibility • Drawback---expensive & requires expertise

  29. 3.Serological tests • To measure IgG antibody---ELISA • Ag-capture ELISA test---for mycobacterial lipoarabinomannan Ag

  30. 4.PCR-based tests Detection of PCR product of M. tuberculosis gene on polyacrylamide gel electrophoresis. The lane numbers are as follows: lane M marker of 50-bp ladder; lane 1,2,3, positive ; lane 4, negative; and lane 5, negative control.

  31. Automated nucleic acid amplification testPCR-based. For DNA extraction and amplification and detection of TB DNA and rifampin-resistance encoding mutations.

  32. Restriction Fragment Length Polymorphism (RFLP) • Cutting by restriction enzymes • Gel electrophoresis Uses • To know how many cases are due to reactivation of latent infection &how many are recent transmission • Epidemiological typing of strains

  33. RFLP patterns. Cluster I comprises strains 9, 12, 16, 28, 38, 14, and 40; cluster II, strains 3, 7, 8, and 20; cluster III, strains 31 and 24; cluster IV, strains 1 and 13; and cluster V, strains 37 and 36. Mt, M. tuberculosis MT14323 reference strain.  

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