DIAGNOSIS CLINICALLAB

LAB DIAGNOSIS

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

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

MICROSCOPY By Ziehl-Neelsen staining

ZN smear evaluation and AFB report No. of AFBSeen inreport 0300 fieldsAFB not seen FDoubtful, repeat smear F F F3+ 10 or more1 F4+

By fluorescent microscopy

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

Concentration methods For microscopyFor smear,culture & animal inoculation

MYCOPROSAFE

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

 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

Antibiotic sets for sensitivity testing.

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

 DISADVANTAGES: Cumbersome Costly Less sensitive than culture

 SAMPLES COLLECTED CSF Bone marrow &liver biopsy Blood Pus Pleural effusion--- exudate urine

CMI….manifests as 1. Delayed hypersensitivity 2. Resistance to infection

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

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

ALLERGIC TESTS OT PPD - S 1 TU= 0.01 ml of OT or mg of PPD-S MANTOUX TEST  METHOD  INTERPRETATION OF RESULT

HEAF TEST Multiple puncture testing For screening & surveys

Tine test Disposable prongs carrying dried PPD is used

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

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

Recent advances in detection of M.tb 1.Bactec 460 TB system Broth based growth system Medium contains 4 ml of Middlebrook 7 H 12 broth with carbon 14 labeled palmitic acid Clinical specimen inoculated with PANTA Use radiometric method for growth detection

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

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. 2.Rapid evaluation of drug susceptibility by bioluminescence assays Advantage---testing drug susceptibility Drawback---expensive & requires expertise

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

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.

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

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

RFLP patterns. Cluster I comprises strains 9, 12, 16, 28, 38, 14, a nd 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. tuberculosi s MT14323 reference strain.