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.