laboratory diagnosis of tuberculosis

Category: Entertainment

Presentation Description

No description available.


Presentation Transcript

Laboratory diagnosis of Tuberculosis :

Laboratory diagnosis of Tuberculosis Govind P. Sah Medical microbiology Lecture CMLT, 2 nd year

Tuberculosis or TB (short for tubercle bacillus):

Tuberculosis or TB (short for tubercle bacillus) Is a common and often deadly infectious disease caused by mycobacterium usually mycobacterium tuberculosis Genus mycobacteria is divided in to 1. Mycobacteria tuberculosis complex 2. NON Tuberculous Mycobacteria


CAUSITIVE AGENTS Mycobacterium tuberculosis complex M. tuberculosis M. bovis M. microti M. africanum


Pathogenesis Inhaled aerosols Engulfed by alveolar macrophages Bacilli replicate Macrophages die Infected macrophages migrate local lymph nodes Develop Ghon’s focus Primary complex Cell mediated immune response stops cycle of destruction and spread Viable but non replicating bacilli present in macrophages EVIDENCE OF INFECTION WITH M tuberculosis Chest x-ray / positive skin test

PowerPoint Presentation:

Types of specimens: 1.Pulmonary specimens -Sputum. -Gastric lavage -Transtracheal aspirations -Bronchoscopy -Laryngeal swabbing 2.Urine specimens 3.Tissue and body fluid specimens 4.Blood specimens 5.Wounds, skin lesions, and aspirates

Sputum collection :

Sputum collection Sputum collected in the morning before any meal Should not use oral antiseptics 2 sputum samples Sputum is not available then . Laryngeal swabs . Bronchial washings . In small children gastric Lavage

Observe to identify Sputum from Saliva:

Observe to identify Sputum from Saliva SPUTUM Specimens appear mucoid, even, blood stained. Contains many Polymorphonutrophils. SALIVA Appears clear, watery and frothy. Contains many squamous epithelial cells Absence of Polymorphoneutrophils.

Laboratory diagnosis:

Laboratory diagnosis Microscopy Culture Animal inoculation Nucleic acid technology Allergic tests

Laboratory Diagnosis:

Laboratory Diagnosis 1- Sputum smears stained by Z-N stain Two morning successive mucopurulent sputum samples are needed to diagnoise pulmonary TB . Advantage : - cheap – rapid - Easy to perform Disadvantages: - sputum ( need to contain 5000-10000 AFB/ ml.) - Young children, elderly & HIV infected persons may not produce cavities & sputum containing AFB.

Limitation of Microscopy for Tuberculosis.:

Limitation of Microscopy for Tuberculosis. Repeated sample examinations. load on technical staff. Training and dedication of Microscopist. The load of bacilli must be more than 10,000 / 1 ml of sputum. Low in sensitivity < 50 % Repeated requests for samples High drop out by patients, for repeated samples. Not dependable in pediatric age group.



What is Smear Positivity WHO:

What is Smear Positivity WHO All patients who have submitted two Specimens and found to be positive for identification of AFB

Grading :

Grading If the slide has Result Grading No. of fields to be examined More than 10 AFB /oil immersion field pos 3+ 20 1-10 AFB/ oil immersion field pos 2+ 50 10-99 AFB/100 oil immersion field pos 1+ 100 1-9 AFB/100 oil immersion field pos Scanty* 100 No AFB in 100 oil immersion field neg 100 *record actual number of bacilli seen in 100 fields

PowerPoint Presentation:

2 - Detecting AFB by fluorochrome stain using fluorescence microscopy: The smear may be stained by auramine-O dye. In this method the TB bacilli are stained yellow against dark background & easily visualized using florescent microscope . Advantages : - More sensitive - Rapid Disadvantages: - Hazards of dye toxicity - more expensive - must be confirmed by Z-N stain

Acid Fast Bacilli as seen under Fluorescent Microscope:

Acid Fast Bacilli as seen under Fluorescent Microscope

Why we need Florescent Microscopy:

Why we need Florescent Microscopy Useful when few bacilli are present. Increases the sensitivity in HIV patients with tuberculosis. Reduces the time needed for testing. About 15 times as many fields of view can be scanned by fluorescent microscopy than by Ziehl – Neelsen’method in the same period. Increases the sensitivity by 10 % Better conclusions with one or two specimens, unlike Ziehl Neelsen’s method needing 3 or > 3 specimens.

Processing Direct smear negative specimens:

Processing Direct smear negative specimens Sputum Microscopy can be improved with Sputum liquefaction, concentration and gravity sedimentation. Popular solvents Sodium hypochlorite. Sodium hydroxide. Ammonium sulphate N-acetyl-L-cysteine –sodium hydroxide.

Culturing Mycobacterium:

Culturing Mycobacterium Culturing for isolation of Mycobacterium spp continues to be a Gold standard, particularly in Developing countries. Need only 10 – 100 bacilli / 1 ml of sputum.

Recent facts on Culturing:

Recent facts on Culturing Useful in HIV infected patients with Tuberculosis. As even few bacilli can be grown in spite of smear negativity. But the specimens to be incubated for longer time as few bacilli are present.

Solid media:

Solid media Agar based 1. Middlebrook 7H10 and Middle brook 7H10 selective 2.Middlebrook 7H11 and Middlebrook 7H11 selective 3. Middlebrook biplate (7H10/7H11Sagar)

Solid media:

Solid media EGG BASED Lowenstein-Jensen (L-J) L-J with pyruvic acid L-J with iron

Solid media:

Solid media Cultures incubated at 35 °c in the dark in an atmosphere of 5% to 10% co2 and high humidity Cultures are examined weekly for growth Most isolates appear between 3 and 6 weeks a few isolates appear after 7 or 8 weeks After 8 wks of incubation negative cultures are reported

Mycobacterium tuberculosis:

Mycobacterium tuberculosis Dry , Rough , Raised , irregular colonies with wrinkled surface They are creamy white becoming yellowish or buff coloured on further incubation

Eight Week Growth of Mycobacterium tuberculosis on Lowenstein-Jensen Agar :

Eight Week Growth of Mycobacterium tuberculosis on Lowenstein-Jensen Agar

Commonly used liquid media systems to culture and detect the growth of mycobacterium:

Commonly used liquid media systems to culture and detect the growth of mycobacterium BACTEC 460 Mycobacteria growth indicator tube (MGIT) BACTEC MGIT 960 ( continuous growth monitoring systems)

Liquid media :

Liquid media Use of liquid media system reduces the turn-around time for isolation of acid-fast bacilli to approximately 10 days compared with 17 days or longer in conventional methods Once growth is detected in the liquid media , an acid fast stain is performed to confirm the presence of acid-fast bacilli

Recent Methods for Diagnosis:

Recent Methods for Diagnosis I – BACTEC 460 ( rapid radiometric culture system ) specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – labelled palmitic acid & PANTA antibiotic mixture. Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. The daily increase in GI output is directly proportional to the rate & amount of growth in the medium.

Advantages : :

Advantages : - Rapid (mycobacteria can be detected within 12 days.) - Determining drug susceptibility . - Specificity is very high Disadvantages : - Expensive - Hazards of using radioactive material.

II Mycobacteria Growth Indicator Tube (MGIT):

II Mycobacteria Growth Indicator Tube (MGIT) Tube contains modified Middlebrook 7H9 broth base with OADC enrichment & PANTA antibiotic mixture. All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood ) could be cultured on this type of media.

Principle of the procedure: :

Principle of the procedure: A fluorescent compound (which is sensitive to O 2 ) is embeded in silicone on the bottom of the tube. The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp.

The MGIT 960 System:

The MGIT 960 System The MGIT 960 system is a non-radiometric automated system that uses the MGIT media & sensors to detect the fluorescence. Advantages: -The system holds 960 plastic tubes which are continuously monitored. - Early detection as the machine monitoring & reading the tubes every hour.

Approach to identification :

Approach to identification Growth on solid or liquid media perform a acid-fast stain Several colonies on solid media are inoculated to middle brook 7H9 broth 5ml and incubated at 35 °c for 5 to 7 days with daily agitation to enhance the growth Either this broth or growth in primary liquid culture are inoculated all test media including biochemical tests and pigmentation and growth rate determinations

Biochemical tests:

Biochemical tests Niacin test P=Human tubercle bacilli: N=Bovine Aryl sulphatase test P= Atypical Mycobacteria Catalase – Peroxidase test Differentiate between typical and atypical Mycobacteria Nitrate reduction test P= M.tuberculosis N= M.bovis

Nucleic acid probes commercially available:

Nucleic acid probes commercially available M. tuberculosis complex M. avium complex M. avium M. intercellulare M. kansasii M. gordonae

III Polymerase Chain Reaction (PCR) & Gene probe:

III Polymerase Chain Reaction (PCR) & Gene probe Nucleic acid probes & nucleic acid amplification tests in which polymerase enzymes are used to amplify ( make many copies of specific DNA or RNA sequences extracted from Mycobacterial cells. Advantages: - Rapid procedure - High sensitivity (1-10 ( 3 – 4 hours) bacilli / ml sputum)

Disadvantages: :

Disadvantages : - Very expensive. - Require specialist training & equipments. - False positive results. - Can not differentiate between living & dead bacilli.

Susceptibility testing:

Susceptibility testing Resistance ratio Resistance is expressed as the ratio of the MIC of the test strain divided by the MIC for the standard strain for each drug


SUSCEPTIBILITY Proportion The extent of growth in the absence or presence of drug is compared and expressed as percentage If growth at the crictical concentration of drug is >1% , the isolate is considered clinically resistant

Susceptibility testing:

Susceptibility testing Radiometric The rate and amount of co2 produced in the absence or presence of drug is compared

Newer methods:

Newer methods Line probe assay for rapid detection of Rifampicin mutations High density DNA probes Luciferase gene technique

Non Specific Tests:

Non Specific Tests Tuberculin test ( Mantoux Test )

Tuberculin Test ( Mantoux Test ):

Tuberculin Test ( Mantoux Test ) Test to be interpreted in relation to clinical evaluation. Even the induration of 5 mm to be considered positive when tested on HIV patients. Lacks specificity.

Allergic test:

Allergic test 1. Mantoux test . 0.1 ml of PPD containing 5 TU is injected intradermally o flexor ascept of fore arm . Examine after 48 – 72 hrs . In duration of diameter 10 mm –p 5mm or less – N

Allergic test:

Allergic test 2. Multiple Heaf test 3. Disposable prongs carrying dried PPD (tine test ) Test become positive 4- 6 wks after infection or immunization Allergy wanes gradually and disappears after 4-5 yrs


uses In diagnosis of tuberculosis in infants and young children Measure of prevelance of infection Select the susceptibles

Dealing with Tuberculosis in HIV / AIDS patients.:

Dealing with Tuberculosis in HIV / AIDS patients. Diagnosing Tuberculosis in HIV infected is a priority and improve quality of Life

HIV/AIDS - Tuberculosis:

HIV/AIDS - Tuberculosis Consider the HIV status Identify the severity of Tuberculosis. Early use of chest radiography. Maximal number of sputum smear examinations. Sputum concentration methods to be encouraged even by smaller laboratories. Explore the use of Florescent Microscopy. All smear negative specimens should be cultured.

Extra pulmonary Tuberculosis:

Extra pulmonary Tuberculosis Poses several challenges, Yet no optimal, specific diagnostic methods

Extra pulmonary Tuberculosis:

Extra pulmonary Tuberculosis A real challenge to Clinicians and Laboratories. Optimal specimen collection a priority, Molecular Methods are growing need. Clinicians start drug regimes on empirical basis. Several serological tests for antibody determinations are evaluated.

Identification of Atypical Mycobacterium:

Identification of Atypical Mycobacterium A growing concern on infections with less known, uncommon Mycobacterium in immunosupreesed, an emerging infectious disease problem

Microscopy in Tuberculosis TODAY:

Microscopy in Tuberculosis TODAY In spite of several scientific, and molecular advances Microscopy in Tuberculosis continues to be back bone in Diagnosis.

authorStream Live Help