diagnosis clinicallab lab diagnosis 1.pul tuberculosis sample collection microscopy concentration...

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.