Laboratory diagnosis of tuberculosis pract.

Lab diagnosis approach

1. LABORATORY DIAGNOSIS OF TUBERCULOSIS By – DR.D.W.DESHKAR – DEPARTMENT OF MICROBIOLOGY D.Y.PATIL EDUCATION SOCIETY INSTTUTION DEEMED TO BE UNIVERSITY

2. Learning Objectives: Is a major health problem world wide. Largest cause of “DEATHS” from a single infectious disease. HENCE “ACCURATE” & “EARLY” diagnosis is required for its effective “MANAGEMENT”. Diagnose people with infectious TB Monitor the progress of treatment

3. This involves – Demonstrating the bacillus in the lesion by microscopy Isolating it in culture by transmitting the infection to the experimental animals. Demonstrating hypersensitivity to tuberculoprotein Using molecular diagnostic methods.

4. 1. Collection of Specimen – a) In Pulmonary tuberculosis – Sputum collected in a clean wide mouthed container. - Two sputum specimens – spot ( on the same day of visit) and early morning sample (collected on next day). - Laryngeal swab, Bronchial washing may also be collected where sputum is not available. - In children – who tend to swallow the sputum gastric lavage may be examined.

5. 1. Collection of Specimen b) In Extra – Pulmonary tuberculosis (EPTB) – Vary depending on site of involvement – - Lymph node aspirate - Pleural fluid - Urine - Synovial fluid - Cerebrospinal fluid (CSF) - Pus from cold abscess - Tissue biopsies

6. 2. Digestion, decontamination and concentration of specimen - Specimens from non sterile sites need prior treatment for digestion ( to liquefy thick pus cells & homogenization), decontamination (to inhibit normal flora) & concentration( to increase the yield) Processing to be done in class II biosafety cabinet. Petroff’s method- ( Sputum + 4% NaOH equal volume + Incubation (37 C) with frequent shaking, till becomes clear ( 20 min.) – centrifuged ( at 3000 rpm) for 20 min. – sediment neutralized with N/10HCl) indicator is phenol red. – used for smear, culture & animal inoculation.

7. 2. Digestion, decontamination and concentration of specimen - NALC ( N – acetyl L – cysteine ) + 2% NaOH – Better than Petroff’s NALC liquefies sputum & NaOH kills normal flora. The sample is neutralized with buffer and concentrated by centrifugation. It is also compatible with culture in automated systems. For smear microscopy, formalin or hypochlorite may be used as mucolytic agent. Not useful for culture

8. 3. Direct microscopy by acid – fast staining - Ziehl Neelsen (ZN) technique : Direct or concentrated smears of sputum are examined for AFB. Sputum microscopy – the most reliable single method in the diagnosis & control of TB. Smears are prepared from thick purulent part of the sputum, dried, heat fixed & stained by Ziehl Neelsen (ZN) technique . Reveals -Long slender, beaded, less uniformly stained red coloured acid fast bacilli. ZN smear evaluation and AFB report as per RNTCP guidelines- RNTCP No. of bacilli seen / Oil - Field Result Grading No. of fields > 10 / OF +ve +++ 20 1 – 10 / OF +ve ++ 50 10 – 99 / 100 OF +ve + 100 1 – 9 / 100 OF +ve Scanty 100 No AFB in 100 OF -ve 1000

9. 3. Direct microscopy by Auramine rhodomine stain – When several smears are to be examined daily , conveniently use Fluorescence Microscopy. The smears are stained with auramine phenol or auramine rhodomine fluorescent dyes , acid – alcohol as decolourizer and potassium permanganate as counter stain and examined under ultraviolet illumination, the bacilli appear as bright rods against the dark background. 3. Direct microscopy by Kinyoun modification of acid fast staining – A cold method where heating of the stain is not employed. Increasing conc. of phenol acid and increasing the duration of staining.

10. 4. Culture : take 6 – 8 weeks Solid Media – Egg based Media – Lowenstein Jensen (L J), Petragnani, Dorset egg media. L.J. Medium – Most widely used & recommended by International Union Against Tuberculosis. It consists of hen’s egg, mineral salt, glycerol, asparagine & malachite green ( selective) Colony Appearance – Rough, dry, raised, wrinkled, buff coloured colonies – M.tuberculosis ( Eugonic growth) White smooth moist colonies (Dysgonic growth) - M.bovis

11. 4. Conventional Culture Techniques – Other Solid Media – Blood based – Tarshis medium Serum based – Loffler medium Potato based – Pawlowsky medium Agar based – Middlebrook 7H11 & 7H10. Conventional Culture Techniques – Liquid Media – Not generally employed for routine culture, but are used for drug sensitivity testing, preparation of antigens & vaccine. Examples – Middlebrook 7H9, Dubos, Proskauer, Sula & Sauton media. = virulent strains produce long serpentine cords in liquid media. Avirulent strains grow in dispersed manner.

12. 5. Automated Culture Methods – 1. BACTEC MGIT ( M ycobacteria G rowth I ndicator T ube) – Uses an oxygen sensitive fluorescent compound dissolved in the broth . The Mycobacteria quenches the oxygen and the organism fluoresce. 2. B AC T/ Alert – Uses the principle of colorimetric detection of pH change in the medium which occurs due to CO 2 liberated by the growth of M.tuberculosis . 3. ESP system – Detects a change of pressure in the medium, due to the production of CO 2 by M.tuberculosis . 4. BACTEC 460 - was first semi automated method. Was based on the use of radioisotopes to detect growth. – Not in use currently.

13. SPECIES NIACIN TEST ARYL-SULPH---ATASE TEST NITRATE REDUC---TION TEST CATAL---ASE TEST PEROX---IDASE TEST TWEEN 80 HYDRO--LYSIS TEST TELLURI--TE REDUCTION TEST GROWTH ON TCH PYRAZI-NAMIDASE TSET UREASE TEST M tuberculosis + - + Weakly + + - +/- + + + M bovis - - - - - - +/- - - + M Africanum - - - - - - - +/- - + 10%cynogen bromide + 4% Aniline in 96% ethanol added to suspension of M.tuberculosis culture on L J = A canary yellow colour develops - + Reaction. 6 Biochemical Reactions

14. Growth on L J Medium Rapid growth (within 7 days) Growth on MacConkey Aryl sulphatase test M.Fortuitum complex Tellurite reduction M.pheli M. smegmatis Slow growth Type of growth M.tuberculosis Niacin Pigment Scanty smooth flat colonies In light In dark No Pigment Group – I Photochromogens Group – III Non - chromogens Group – II Scotochromogens Rabbit pathogenicity -ve BCG +ve M.bovis Identification of tubercle bacilli and related Mycobacteria

15. 7. Anti – tubercular sensitivity tests – Phenotypic Methods – Commonly used methods are Proportion method – Gold standard method. The ratio of colonies growing on drug containing medium to the number of colonies growing on the drug free medium indicates the proportion of drug resistant bacilli present in the bacterial proportion. Less popular phenotypic methods – Absolute concentration method Resistance ratio method

16. 7. Anti – tubercular sensitivity tests – Phenotypic Methods – Automated systems such as BACTEC MGIT are used now a days which mainly detect resistance to Rifampicin

17. 7. Anti – tubercular sensitivity tests – Molecular methods – PCR based assays Line probe assay DNA microarray Gene expert

18. 8. Animal Inoculation – Using Guinea pig and rabbit. 9. Diagnosis of Latent tuberculosis – - By tuberculin test ( eg Monteux test), & Interferon  assay.

19. 10. Molecular methods – Nested PCR using IS6110 primer. Other genes that are targeted – 65kDa, 38kDa. Line Probe assay Transcription Mediated Amplification (TMA) Strand displacement amplification (SDA) Nucleic acid sequence based amplification (NASBA) Ligase chain reaction.

20. THANKS