diagnosis clinicallab lab diagnosis 1.pul tuberculosis sample collection microscopy concentration...
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DIAGNOSIS
DIAGNOSIS
CLINICAL LAB
LAB DIAGNOSIS
1.PUL TUBERCULOSIS
•SAMPLE COLLECTION•MICROSCOPY•CONCENTRATION METHODS•CULTURE•SENSITIVITY TESTS•ANIMAL INOCULATION•NUCLEIC ACID TECHNOLOGY•IMMUNODIAGNOSIS
•Samples collected----SputumLaryngeal swabsBronchial washingsGastric lavage•Best collected-in morning before
meal•If scanty-24hr sample taken
MICROSCOPY
By Ziehl-Neelsen staining
ZN smear evaluation and AFB reportNo. of AFB Seen in report0 300 fields AFB not
seen
1-2 300 F Doubtful, repeat smear
1-9 100 F 1+
1-9 10 F 2+
1-9 1 F 3+
10 or more 1 F 4+
By fluorescent microscopy
Disadvantages
•Has only 30-70% sensitivity
Advantages
•Reliable method•Rapid screening for epidemiological purpose
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
SENSITIVITY TESTING
Absolute conc methodIn 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 methodIndicates avg sensitivity of strain
Antibiotic sets for sensitivity testing.
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
contd....
DISADVANTAGES:• Cumbersome• Costly• Less sensitive than culture
2. EXTRA PULMONARY TB
SAMPLES COLLECTED• CSF• Bone marrow &liver biopsy• Blood• Pus• Pleural effusion--- exudate• urine
ALLERGY & IMMUNITY
• CMI….manifests as 1. Delayed hypersensitivity2. 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
METHODINTERPRETATION OF RESULT
HEAF TEST
•Multiple puncture testing •For screening & surveys
Tine test
Disposable prongs carrying dried PPD is used
FALSE NEGATIVES
Miliary tuberculosisConvalscents from viral infections like MeaslesLympho reticular malignancyImmuno suppressive therapyInactive PPD preparationImproper injection technique
FALSE POSITIVESInfection or exposure to atypical mycobacteriaBCG vaccine
USES
Aids in diagnosing active infections in infants & young children
To measure prevalence of infection in an areaTo 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
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, 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.