DIAGNOSIS CLINICAL LAB LAB DIAGNOSIS 1.PUL TUBERCULOSIS •SAMPLE COLLECTION •MICROSCOPY •CONCENTRATION METHODS •CULTURE •SENSITIVITY TESTS •ANIMAL INOCULATION •NUCLEIC ACID TECHNOLOGY •IMMUNODIAGNOSIS Sputum •Samples collected---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 AFB 0 1-2 Seen in 300 fields 300 F 1-9 1-9 1-9 10 or more 100 F 10 F 1F 1F report AFB not seen Doubtful, repeat smear 1+ 2+ 3+ 4+ By fluorescent microscopy Advantages •Reliable method •Rapid screening for epidemiological purpose Disadvantages •Has only 30-70% sensitivity Concentration methods For microscopy For 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 0.00002 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 4ml of Middlebrook 7H12 broth with carbon 14 labeled palmitic acid •Clinical specimen inoculated with PANTA •Use radiometric method for growth detection BACTEC TB Instrument ADVANTAGES • rapid method •Increase no. of +ve cultures •Antibiotic sensitivity testing can be done DRAWBACK—very expensive 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 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.