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Diagnosis for tuberculosis(1).ppt2003


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Diagnosis for tuberculosis(1).ppt2003

  1. 1. <ul><li>Prepared by :- </li></ul><ul><li>Eriny Milad </li></ul><ul><li>Fatma Elbadry </li></ul>
  2. 2. <ul><li>1-History and clinical examination </li></ul><ul><li>2-Radiographic features </li></ul><ul><li>3-Bacteriolgic evaluation :- </li></ul><ul><li>a)Conventional diagnostic methods {smear,culture} </li></ul><ul><li>b)Immunologic diagnosis{ serological test ,T-cell assay e.g tuberculin test ,INFgamma assay} </li></ul><ul><li>c)New diagnostic methods{ BACTEC,nuclic acid amplification method,…} </li></ul>
  3. 3. <ul><li>radiographic examination of the chest is usually the first diagnostic study undertaken, after the history and physical examination . </li></ul><ul><li>In primary tuberculosis, as a recent infection, is seen as a middle or lower lung zone infiltrate , often associated with ipsilateral hilar adenopathy </li></ul><ul><li>. Atelectasis may result from compression of airways by enlarged lymph nodes. If the primary process persists beyond the time when specific cell-mediated immunity develops, cavitation may occur (so-called progressive primary tuberculosis). </li></ul>
  4. 5. <ul><li>Tuberculosis that develops many years after the original infection (endogenous reactivation) usually involves the upper lobes of one or both lungs. </li></ul><ul><li>Cavitation is common in this form of tuberculosis. </li></ul><ul><li>The most frequent sites are the apical and posterior segments of the right upper lobe and the apical-posterior segment of the left upper lobe. </li></ul><ul><li>Healing of the tuberculous lesions usually results in development of a fibrotic scar with shrinkage of the lung parenchyma and, often, calcification. </li></ul><ul><li>Involvement of the anterior segments alone is unusual. In the immunocompetent adult with tuberculosis, intrathoracic adenopathy is uncommon. </li></ul><ul><li>When the disease progresses, infected material may be spread via the airways (i.e., &quot;bronchogenic&quot; spread) into the lower portions of the involved lung or to the other lung. Erosion of a parenchymal focus of tuberculosis into a blood or lymph vessel may result in dissemination of the organism and a miliary pattern on the chest film . </li></ul>
  5. 6. <ul><li>the chest radiograph, although extremely valuable, cannot provide a definitive diagnosis of tuberculosis. </li></ul><ul><li>Because of the:- </li></ul><ul><li>1- Radiographic similarities among the other disorders in the differential diagnosis, </li></ul><ul><li>2- And because of the uncertainties in assessing disease activity and in determining the reasons for progressive radiographic changes, </li></ul><ul><li>careful microbiologic evaluation is always indicated. A nondiagnostic microbiologic evaluation should prompt a careful assessment for other causes of the radiographic abnormality. </li></ul>
  6. 7. <ul><li>Laboratory investigations:- (nonspecific) </li></ul><ul><li>High ESR </li></ul><ul><li>Anemia due to either hemoptysis or chronic illness </li></ul><ul><li>Lymphocytosis , leucopenia. </li></ul>
  7. 8. <ul><li>Active tuberculosis (TB) is diagnosed by detecting Mycobacterium tuberculosis complex bacilli in specimens from the respiratory tract (pulmonary TB) or in specimens from other bodily sites (extrapulmonary TB). </li></ul><ul><li>acid fast bacilli (AFB) smear microscopy and culture on Löwenstein-Jensen medium are still the“ gold standards ” for the diagnosis of active TB. </li></ul><ul><li>AFB smear microscopy and culture can also be used to monitor the effectiveness of treatment and can help to determine when a patient is less likely to be infectious. </li></ul><ul><li>Jacobus H. de Waard and JaiRobledo </li></ul>
  8. 10. <ul><li>Children :- </li></ul><ul><li>“ Fasting” gastric aspirates is the specimen of choice in the case of young children who cannot cough up phlegm </li></ul><ul><li>Gastric lavage fluid must be neutralized with sodium carbonate immediately after collection </li></ul><ul><li>(100 mg per 5-10 mL specimen). </li></ul><ul><li>2) Adults :- sputum, induced sputum, bronchoalveolar lavage or a lung biopsy. </li></ul>
  9. 11. <ul><li>three first-morning sputum specimens (not saliva)obtained after a deep, productive cough on non-consecutive days are usually recommended. </li></ul><ul><li>In patients who cannot produce it spontaneously, the sputum can be induced by inhalation of hypertonic saline solution. Otherwise, the specimen can be collected from bronchoscopy. </li></ul>
  10. 12. <ul><li>depend on the site of the disease. </li></ul><ul><li>The most common specimens received in the laboratory are biopsies, aspirates, pus, urine, </li></ul><ul><li>Normally sterile body fluids, including cerebrospinal fluid, synovial, pleural,pericardial, and peritoneal liquid. </li></ul><ul><li>Whole blood and/or bone marrow specimens are collected only if disseminated TB is suspected,mainly in patients with an underlying severe immunosuppressive condition such as AIDS </li></ul><ul><li>Bone biopsies are the specimen of choice when skeletal TB is suspected </li></ul><ul><li>AFB smear microscopy from body fluids is rarely positive and the whole sediment from concentrated specimens should rather be cultured </li></ul>
  11. 13. <ul><li>1-Ziehl-Neelsen (Ziehl-Neelsen is a hot acid-fast stain because the slide has to be heated during incubation with fuchsin) </li></ul><ul><li>2- Kinyoun (is a cold acid-fast staining procedure and therefore does not require heating) </li></ul><ul><li>3-fluorochrome procedure (primary staining is done with auramine/rhodamin ) </li></ul><ul><li>. The AFB fluoresce yellow against a counterstain of potassium permanganate when observed with a fluorescence microscope </li></ul>
  12. 14. <ul><li>Acid-fast microscopy is easy and quick, but it does not confirm TB diagnosis because mycobacteria other than M. tuberculosis are also AFB in the smear. </li></ul><ul><li>A positive culture for M. tuberculosis confirms the diagnosis of active disease. </li></ul><ul><li>For culturing of mycobacteria, two types of clinical specimens are considered:- </li></ul><ul><li>1-contaminated from non-sterile bodily sites(should be decontaminated befor inoculation to eliminate the associated flora ) </li></ul><ul><li>2-sterile from sterile body sites(inoculated directly onto the culture medium) </li></ul>
  13. 15. <ul><li>A ) Solid media </li></ul><ul><li>1- Löwenstein-Jensen (egg and also contain high concentrations of malachite green to overcome contamination with other bacteria) </li></ul><ul><li>2- Middlebrook 7H10 and 7H11 are ( agar-based media.) Middlebrook </li></ul><ul><li>media have been shown to achieve slightly higher isolation yields than egg-based media, but are considerably more expensive. </li></ul><ul><li>B ) liquide media </li></ul><ul><li>1- The BACTEC TB-460 </li></ul><ul><li>2- MGIT960 systems </li></ul><ul><li>All cultures reported positive for mycobacteria should be identified to the level of species using either biochemical or molecular methods </li></ul><ul><li>Reading of results </li></ul><ul><li>1- Egg-based media they produce characteristic non-pigmented colonies, with a general rough and dry appearance simulating breadcrumbs </li></ul><ul><li>2- On agar based media, the colonies appear flat, dry and rough with irregular edge . </li></ul>
  14. 18. <ul><li>Adenosine deaminase, also known as ADA, is an enzyme involved in the metabolism of purines. Its presence is needed for the breakdown of adenosine from food and for the turnover of nucleic acids in tissues. </li></ul><ul><li>Many articles and reviews have reported the utility of ADA determination in body fluids(spinal, pleural, ascitic,pericardial) </li></ul><ul><li>ADA determination is simple and cheap and also has a high positive predictive value, especially in high endemic countries. </li></ul><ul><li>The specificity is very high in fluids with a lymphocyte-to-neutrophil ratio higher than 0.75 </li></ul><ul><li>The suggested laboratory cut-off of ADA activity is 40 U/L for pleural, peritoneal and pericardial fluids and 10 U/L for cerebrospinal fluid. However different laboratories have established different cut-offs varying between 33 and 50 U/L. </li></ul>
  15. 19. <ul><li>Serological diagnosis:- </li></ul><ul><li>Basically, it uses the specific humoral and cellular immune responses of the host to infer the presence of infection or disease </li></ul><ul><li>Humoral immune responses detection:- </li></ul><ul><li>Early studies utilizing crude antigen preparations of M. tuberculosis showed seroreactive antibodies in TB patients. However, cross-reactions occurred in healthy individuals, elicited by commensal bacteria, environmental mycobacteria and BCG </li></ul><ul><li>A)Enzyme Linked Immunosorbent Assay (ELISA) </li></ul><ul><li>B)Rapid tests </li></ul>
  16. 20. <ul><li>tests based on the detection of specific antigens, such as the 38 kDa protein, have been developed and have been in use, primarily in developing countries </li></ul><ul><li>The 38 kDa antigen has shown the highest sensitivity and specificity </li></ul><ul><li>anti-38 kDa antibodies seem to be restricted to advanced TB , which is the main cause of TB transmission. </li></ul>
  17. 21. <ul><li>Immunochromatographic assays, also called lateral-flow tests or simply strip tests are a logical extension of the technology used in latex agglutination tests </li></ul><ul><li>The benefits of rapid test:- </li></ul><ul><li>• User-friendly format </li></ul><ul><li>• Very short time to test result </li></ul><ul><li>• Long-term stability over a wide range of climates </li></ul><ul><li>• Relatively inexpensive to make </li></ul>
  18. 23. <ul><li>1-Tuberculin skin test </li></ul><ul><li>2-Interferon-gamma determination </li></ul><ul><li>Enzyme-linked immunospot for interferon-gamma </li></ul><ul><li>Quantiferon-TB test </li></ul>
  19. 25. Tuberculin skin test ▪ TST has been used to identify patients actively infected with TB, ▪ to measure the prevalence of infection in a community, and to select susceptible or high-risk patients for BCG vaccination . How to do TST : ▪ TST works by intradermally injecting 0.1 mL of 5 TU PPD on the forearm. (Mantoux's method) On examination, after 48-72 hours, a positive reaction is indicated by erythema and induration of > 10 mm in size. Erythema (redness) alone is not taken as a positive reaction . ▪ To detect persons at high risk for LTBI or at high risk to progress to disease
  20. 27. ☻ A positive TST result is a manifestation of type IV delayed hypersensitivity. □ False-positive test findings can occur in ; ● cross-reactions caused by non-tuberculous (atypical) mycobacterial infection ● bacille Calmette-Gu´ erin (BCG) vaccination.
  21. 28. Causes of False-Negative Tuberculin Skin Test 1: Factors Releated to the Person Being Tested ● Concurrent infections    ● Human immunodeficiency virus ● Other viral infections (measles, mumps, chickenpox) ● Bacterial diseases (typhoid fever, brucellosis, typhus, leprosy, pertussis, overwheiming tuberculosis) ● virus vaccination (measles, mumps, polio) ● Diseases affecting lymphoid organs (Hodgkin's disease, lymphoma, chronic lymphocytic leukemia, sarcoidosis) ● Age (newborns, elderly) ● Metabolic conditions (advanced renal failure, malnutrition) ● Recent surgery ● Certain medications (e.g.,TNF- α antagonists, corticosteroids and other immunosuppressive drugs), will suppress the IV response and T-lymphocyte function
  22. 29. 2 : Factors Related to the Test ● Loss of antigen potency (exposure to heat and/or light) ● Incorrect administration (too little antigen, deep injection) ● Incorrect reading (inexperienced reader, reader bias, recording error
  23. 30. <ul><li>An in vitro T-cell-based assays tests for diagnosis of latent TB infection : </li></ul><ul><li>- QuantiFERON TB gold Test </li></ul><ul><li>- T-Spot Test. </li></ul><ul><li>These whole-blood assays measure IFN- γ production by previously sensitised lymphocytes in response to M.tuberculosis-specific protein antigens ESAT6 and CFP-10 </li></ul>
  24. 31. <ul><li>Some studies have shown that: </li></ul><ul><li>-Compared with TST these tests have higher specificity. </li></ul><ul><li>-Correlate better with exposure to tuberculosis. </li></ul><ul><li>-Have less cross-reactivity with the BCG vaccine & environmental mycobacteria. </li></ul><ul><li>NB: Yet there is no evidence for the use of these tests in young children at present. </li></ul>
  25. 32. Enzyme-linked immunospot for interferon-gamma The ELISPOT assay for diagnosis of M. tuberculosis infection is based on the rapid detection of T cells specific for M. tuberculosis antigens. IFN-γ released in vivo from these cells can be detected by the extremely sensitive ELISPOT In a preliminary trial, this test was positive in 96 % of 47 TB patients and in 85 % of 26 persons presumed to have latent TB. The ELISPOT test was negative in 26 BCG-vaccinated control subjects, and this specificity implies a major advantage over TST ELISPOT offers a more accurate approach than TST for the identification of individuals who have latent TB infection . Each such T cell gives rise to a dark spot and the readout is the number of spots. The T cells enumerated by the ELISPOT assay are effector cells that have recently encountered antigen in vivo and can rapidly release IFN-γ when re- exposed to the antigen
  26. 33. Quantiferon-TB test The IFN-γ secreted by T-cells into the plasma is measured by ELISA to indicate the likelihood of TB infection . Different studies demonstrated that the QuantiFERON-TB test was comparable to TST in its ability to detect latent TB infection. also showed that the QuantiFERON-TB test was less affected by BCG vaccination QuantiFERON-TB was approved by the Food and Drug Administration (FDA) of the United States (US) in 2001. In 2003, the US Centers for Disease Control and Prevention released guidelines for using the QuantiFERON®-TB Test in the diag- nosis of latent M. tuberculosis infection ,
  27. 34. QuantiFERON-TB Gold , This in vitro test detects the release of interferon-gamma (IFN-γ) from lymphocytes of sensitized persons when their blood is incubated with peptide mixtures simulating two M. tuberculosis proteins called ESAT-6 and CFP-10 . the TB-specific antigens ESAT-6 and CFP- 10 that are only present in M. tuberculosis and are absent from all strains of M.bovis (BCG) and most environmental mycobacteria ,
  28. 35. LIMITATIONS IFN γ assays are expensive tests and their higher cost appears to limit their wider applicability, especially in resource-limited settings and developing countries,. The ELISPOT test is not yet suitable for widespread use, because it is costly and requires isolation of mononuclear cells, a procedure that is not performed in clinical laboratories .
  29. 36. RECENT METHODS FOR DIAGNOSIS <ul><li>I – BACTEC 460 ( rapid radiometric culture system ) </li></ul><ul><li>specimens are cultured in a liquid medium (Middle brook7H9 broth base )containing C 14 – labelled palmitic acid & PANTA antibiotic mixture. </li></ul><ul><li>Growing mycobacteria utilize the acid, releasing radioactive CO 2 which is measured as growth index (GI) in the BACTEC instrument. </li></ul><ul><li>The daily increase in GI output is directly proportional to the rate & amount of growth in the medium. </li></ul>
  30. 37. <ul><li>P ---- Polymyxin B </li></ul><ul><li>A ---- Amphotericin B </li></ul><ul><li>N ---- Nalidixic acid </li></ul><ul><li>T ---- Trimethoprim </li></ul><ul><li>A ---- Azlocillin </li></ul><ul><li>The antibiotic mixture inhibits the growth of contaminating bacteria. </li></ul>
  31. 38. <ul><li>- Rapid (mycobacteria can be detected within 12 days.) </li></ul><ul><li>- Determining drug susceptibility . </li></ul><ul><li>Differentiating between TB complex & NTM by NAP test. </li></ul><ul><li>- Specificity is very high </li></ul><ul><li>Disadvantages: </li></ul><ul><li>- Expensive </li></ul><ul><li>- Hazards of using radioactive material. </li></ul>
  32. 39. <ul><li>Members of M.tuberculosis complex do not grow in the presence of p-nitro- ∞ -acetylamine </li></ul><ul><li>-ß-hydroxypropiophenone. </li></ul><ul><li>If 5 ug of NAP is added to actively growing culture in 12B medium vial the growth of M. tuberculosis complex is inhibited while Mycobacteria other than tuberculosis (NTM) do not demonstrate significant inhibition. </li></ul>
  33. 40. <ul><li>Tube contains modified Middlebrook 7H9 broth base with oleic acid, albumin, dextrose, and catalase (OADC) enrichment & PANTA antibiotic mixture. </li></ul><ul><li>All types of clinical specimens, pulmonary as well as extra-pulmonary ( except blood ) could be cultured on this type of media. </li></ul>
  34. 41. <ul><li>A fluorescent compound (which is sensitive to O 2 ) is embeded in silicone on the bottom of the tube. </li></ul><ul><li>The actively respiring microorganisms consume the oxygen & allow the fluorescence to be observed using UV trans-illuminator lamp. </li></ul>
  35. 42. <ul><li>The MGIT 960 system is a non-radiometeric automated system that uses the MGIT media & sensors to detect the fluorescence. </li></ul><ul><li>Advantages: </li></ul><ul><li>-The system holds 960 plastic tubes which are continuously monitored. </li></ul><ul><li>- Early detection as the machine monitoring & reading the tubes every hour. </li></ul>
  36. 43. <ul><li>Nuclic 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. </li></ul><ul><li>Advantages: </li></ul><ul><li>- Rapid procedure - High sensitivity (1-10 </li></ul><ul><li>( 3 – 4 hours) bacilli / ml sputum) </li></ul>
  37. 45. <ul><li>- Very expensive. </li></ul><ul><li>- Require specialist training & equipments. </li></ul><ul><li>- False positive results. </li></ul><ul><li>- Can not differentiate between living & dead bacilli. </li></ul>
  38. 46. <ul><li>- Patient’ s sputum is mixed with myco-bactriophage. </li></ul><ul><li>- A virucide is added which destroy any phages outside the TB bacilli. </li></ul><ul><li>- Lysis of cells & release of phages after replication within the tubercle bacilli. </li></ul><ul><li>- Non-pathogenic mycobacteria are added & the sample incorporated in agar mixture( over night incubation) </li></ul><ul><li>- Zones of clearing indicate that patient’ s sputum contained viable M. tuberculosis. </li></ul>
  39. 49. <ul><li>As regards the time: </li></ul><ul><li>The MGIT had the shortest mean time to positivity at 13.3 days, compared with 14.8 days for the BACTEC 460 system & 25.6 days for L J medium. </li></ul>
  40. 50. <ul><li>As regards the no. of culture yield : </li></ul><ul><li>The best yield, was with BACTEC 460, followed by BACTEC MGIT 960 , & then with L J medium. </li></ul><ul><li>As regards contamination rate: </li></ul><ul><li>L J medium (17%) had the highest contamination rate (Tortoli E, Cichero P,Et al. 1999) then the MGIT 960 ( 10.0% ) </li></ul><ul><li>Compared with radiometeric system (3.7%) </li></ul>