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Your name Institution/organization Meeting Date International Standards 2-6, 10, 11 Microbiologic Diagnosis of Tuberculosis.

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Presentation on theme: "Your name Institution/organization Meeting Date International Standards 2-6, 10, 11 Microbiologic Diagnosis of Tuberculosis."— Presentation transcript:

1 Your name Institution/organization Meeting Date International Standards 2-6, 10, 11 Microbiologic Diagnosis of Tuberculosis

2 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB Objectives: At the end of this presentation, participants will be able to:  Understand the important role of sputum smear microscopy in the diagnosis of TB  Describe the various methods of sputum staining and processing, and identify methods that may enhance results  Recognize the role of culture and drug sensitivity testing in the diagnosis and management of TB

3 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB Overview:  Significance of microbiologic testing in TB care  Sputum staining and processing Direct smears, unconcentrated Fluorochrome staining and fluorescence microscopy Concentration and chemical processing Specimen collection and transport  Culture and drug-susceptibility testing  Rapid diagnostic testing International Standards 2, 3, 4, 5, 6, 10, and 11

4 ISTC TB Training Modules 2009 Standards for Diagnosis

5 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB Significance of microbiologic testing for public health goals and patient care:  WHO global target of 70% case detection of new smear-positive cases  Rapid and accurate case detection coupled with effective treatment is essential to reduce the incidence of TB  Failure to perform a proper diagnostic evaluation before initiating treatment potentially: Exposes the patient to the risks of unnecessary or wrong treatment May delay accurate diagnosis and proper treatment

6 ISTC TB Training Modules 2009 Sputum Smear Microscopy  Sputum smear microscopy is the most important test for the diagnosis of pulmonary TB in many areas of the world  Direct smears (unconcentrated specimen) are most common  Fluorescence microscopy and chemical processing can increase sensitivity  Assessment of laboratory quality is essential

7 ISTC TB Training Modules 2009 Sputum Microscopy: Direct Smears Direct smears of unconcentrated sputum:  Fast, simple, inexpensive, widely applicable  Extremely specific for M. tuberculosis in high-incidence areas  Ziehl-Neelsen staining (carbol fuchsin type) most common

8 ISTC TB Training Modules 2009 Sputum Smear Microscopy Carbolfuchsin-based stains  Utilize a regular light microscope  Must be read at a higher magnification  Two types: Ziehl-Neelsen and Kinyoun. Both use carbolfuchsin/phenol as the primary dye  Smear is then decolorized with acid (HCI) alcohol and counter-stained with methylene blue

9 ISTC TB Training Modules 2009 Ziehl-Neelsen (ZN) Stain

10 ISTC TB Training Modules 2009 Sputum Microscopy: Fluorochrome Stain Fluorochrome stain  Fluorochrome stained smears require a fluorescent microscope  Generally read at 250X-450X magnification which allows rapid scanning of the smear  Auramine-rhodamine is an example of such a stain where the AFB appear yellow against a black background

11 ISTC TB Training Modules 2009 Auramine-rhodamine Stain

12 ISTC TB Training Modules 2009 Fluorescence Microscopy Advantages:  More accurate: 10% more sensitive than light microscopy, with specificity comparable to ZN staining  Faster to examine = less technician time Disadvantages:  Higher cost and technical complexity, less feasible in many areas Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81

13 ISTC TB Training Modules 2009 Sputum processing for optimizing smear results (vs. direct smear of unconcentrated sputum):  Concentration by centrifugation and/or sedimentation  Chemical pretreatment (e.g., bleach, NaOH, NaLC) for decontamination and digestion  Usually both  Higher sensitivity (15-20% increase) and higher smear positive rate Sputum Processing Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74

14 ISTC TB Training Modules 2009  Collect specimens in a laboratory- approved, leak-proof container  Label all containers (name and date collected)  Collect specimens prior to initiation of therapy  Infection Control: Consider the safety of other patients and healthcare workers Collect sputum in well-ventilated area, preferably outdoors Specimen Collection and Transport

15 ISTC TB Training Modules 2009  Minimize contamination of specimens by: Instructing the patient to rinse mouth with water before collection Transport the specimen to the lab as soon as feasible after collection  Keep specimens refrigerated if possible Specimen Collection and Transport

16 ISTC TB Training Modules 2009 Mase SR, Int J tuberc Lung Dis 2007;11(5): 485-95 Average yield of single early morning specimen: 86.4% Average yield of single spot specimen: 73.9% Specimen Number Incremental Yield of smear specimens (of all smear-positive) Incremental Sensitivity of smear specimens (compared with culture) 185.8%53.8% 211.9%11.1% 32.4%3.1% Total100%68.0% Performance of Sputum Microscopy

17 ISTC TB Training Modules 2009 Standard 2: Sputum Microscopy Standard 2: All patients (adults, adolescents, and children who are capable of producing sputum) suspected of having pulmonary TB should have at least two sputum specimens obtained for microscopic examination in a quality- assured laboratory. When possible, at least one early morning specimen should be obtained.

18 ISTC TB Training Modules 2009 Standards 3 & 4: Sputum Microscopy Standard 3: For all patients (adults, adolescents, and children) suspected of having extrapulmonary TB, appropriate specimens from the suspected sites of involvement should be obtained for microscopy, culture, and histopathological examination. Standard 4: All persons with chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination

19 ISTC TB Training Modules 2009 Standard 10: Sputum Microscopy Standard 10*: Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months). If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed. (* Partial Standard 10)

20 ISTC TB Training Modules 2009 Although sputum microscopy is the first bacteriologic diagnostic test of choice, both culture and drug susceptibility testing (DST) can offer significant advantages in the diagnosis and management of TB. Culture and Drug Susceptibility Testing

21 ISTC TB Training Modules 2009 Culture: Advantages  Higher sensitivity than smear microscopy (culture can make diagnosis despite fewer bacilli in specimen)  If TB suspected and sputum smears are negative, culture may provide diagnosis  Allows for identification of mycobacterial species  Allows for drug susceptibility testing

22 ISTC TB Training Modules 2009 Culture: Disadvantages  Cost  Technical complexity  May take weeks to get results  Requires ongoing quality assurance  Therefore, more likely to be found in major referral centers. Avoid delaying appropriate TB treatment in suspicious cases while awaiting results.

23 ISTC TB Training Modules 2009 Culture: Solid Media  Solid media have the advantage that organisms (colonies) can be seen on the surface of the medium  Types most commonly used are: Lowenstein-Jensen: egg-based Middlebrook 7H 10 or 7H11: agar-based Ogawa

24 ISTC TB Training Modules 2009 MGIT Incubator Culture: Liquid Media  More sophisticated equipment  Faster detection of growth  Higher sensitivity than solid media  Can also be used for drug- susceptibility testing  Two examples: BACTEC MGIT BACTEC

25 ISTC TB Training Modules 2009 Culture: Identification of Mycobacteria Growth characteristics (preliminary ID)  Preliminary indication of M.tb can be determined from colony characteristics Rate of growth Colonial morphology Pigmentation Biochemical tests  There is a battery of 8 – 12 biochemical tests used to differentiate M.tb within the genus  Nitrate reduction and niacin production are definitive for M.tb

26 ISTC TB Training Modules 2009 Smooth, buff-colored colonies suggestive of Mycobacterium avium complex Rough, buff-colored colonies suggestive of Mycobacterium tuberculosis Culture: Identification of Mycobacteria Visual assessment of colony morphology:

27 ISTC TB Training Modules 2009 Culture: Cross-Contamination  Be aware that faulty technique can lead to laboratory cross- contamination of specimens (difficult to verify without access to more technical testing).  Adequate quality control is an essential component of any mycobacteriology laboratory.

28 ISTC TB Training Modules 2009 Standard 5: Culture in Smear- Standard 5: The diagnosis of sputum smear- negative pulmonary TB should be based on the following criteria: At least two negative sputum smears (including at least one early morning specimen) Chest radiography findings consistent with TB Lack of response to a trial of broad-spectrum antimicrobial agents (*avoid fluoroquinolones) For such patients, sputum cultures should be obtained. In persons are seriously ill or have known or suspected HIV infection, the diagnostic evaluation should be expedited and if clinical evidence strongly suggests TB, a course of antituberculosis treatment should be initiated.

29 ISTC TB Training Modules 2009 Standard 6: Culture in Children Standard 6: In all children suspected of having intrathoracic (i.e., pulmonary, pleural, and mediastinal or hilar lymph node) TB, bacteriological confirmation should be sought through examination of sputum (by expectoration, gastric washings, or induced sputum) for smear microscopy and culture. ISTC Training Modules 2008 (1 of 3)

30 ISTC TB Training Modules 2009 Standard 6: Culture in children  In the event of negative bacteriological results, a diagnosis of TB should be based on: The presence of abnormalities consistent with TB on chest radiography A history of exposure to an infectious case, evidence of TB infection (positive tuberculin skin test or interferon gamma-release assay), and Clinical findings suggestive of TB ISTC Training Modules 2008 (2 of 3)

31 ISTC TB Training Modules 2009 Standard 6: Culture in Children  For children suspected of having extrapulmonary tuberculosis, appropriate specimens from the suspected sites of involvement should be obtained for microscopy and for culture and histopathological examination. (3 of 3)

32 ISTC TB Training Modules 2009 Culture: Drug Susceptibility Testing  Agar proportion method: Compares growth on solid agar media with and without one of the four primary drugs (on discs)  Broth based (BACTEC, MGIT): Liquid broth is inoculated with each test drug; growth in vial indicates resistance to that drug Methods for susceptibility testing

33 ISTC TB Training Modules 2009 Standard 11: Drug Susceptibility Standard 11: An assessment of the likelihood of drug resistance, based on history of prior treatment, exposure to a possible source case having drug- resistant organisms, and the community prevalence of drug resistance, should be obtained for all patients.  Drug susceptibility testing should be performed at the start of therapy for all previously treated patients  Patients who remain sputum smear-positive at completion of 3 months of treatment and patients who have failed, defaulted from, or relapsed following one or more courses of treatment should always be assessed for drug resistance (1 of 2)

34 ISTC TB Training Modules 2009 Standard 11: Drug Susceptibility  For patients in whom drug resistance is considered to be likely, culture and testing for susceptibility/resistance to at least isoniazid and rifampicin should be performed prompltly  Patient counseling and education should begin immediately to minimize the potential for transmission  Infection control measures appropriate to the setting should be applied (2 of 2)

35 ISTC TB Training Modules 2009 Rapid Diagnostic Testing Nucleic acid probe tests (non-amplified) to identify organisms grown in culture:  DNA probe tests are species or complex specific Commercial probes are available for M.tb complex, MAC, M. kansasii and M. gordonae Nucleic acid amplification tests (NAAT):  These tests are designed to amplify and detect DNA specific to M.tb  Enables direct detection of M.tb in clinical specimens

36 ISTC TB Training Modules 2009 Other Rapid Diagnostic Tests  Loop-mediated isothermal amplification (LAMP) Rapid, simplified NAAT still under investigation May be more feasible in lower resource settings  Immunological tests Serologic tests for antibody, antigens, and immune complexes; not currently accurate enough to replace microscopy and culture.

37 ISTC TB Training Modules 2009 Other Rapid Diagnostic Tests  High performance liquid chromatography (HPLC) HPLC uses a liquid chromatography method to identify mycobacteria based on their mycolic acid profiles (cell wall composition) The equipment is expensive and is usually reserved for larger, specialized, reference laboratories

38 ISTC TB Training Modules 2009 Rapid Drug Susceptibility Tests  Line-probe assays Identifies M.tb and genetic mutations associated with INH and RIF resistance Can be used directly on sputum specimens, results within 1-2 days  Molecular beacons  Bacteriophage-based assays *GenoType MTDBRplus strips (Hain Lifescience) *Barnard et al. Am. J. Respir. Crit. Care Med 2008; 177: 787-792

39 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB Summary:  Smear microscopy plays a central role in the diagnosis and management of tuberculosis.  It is important to understand the aspects of specimen handling and processing that can ensure or enhance accurate results.

40 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB Summary (cont.):  Culture and drug- sensitivity testing should be obtained, when feasible, for smear-negative TB and treatment failure.  Quality assurance is essential for all TB diagnostic procedures

41 ISTC TB Training Modules 2009 * Abbreviated versions Summary: ISTC Standards Covered* Standard 2: All TB suspects should have at least 2 sputum specimens obtained for microscopic examination (at least one early morning specimen if possible). Standard 3: Specimens from suspected extrapulmonary TB sites should be obtained for microscopy, culture and histopathological exam. Standard 4: All persons with chest radiographic findings suggestive of TB should have sputum specimens submitted for microbiological examination.

42 ISTC TB Training Modules 2009 Summary: ISTC Standards Covered* * Abbreviated versions Standard 5: The diagnosis of smear-negative pulmonary TB should be based on the following: at least two negative sputum smears (including at least one early morning specimen); CXR finding consistent with TB; lack of response to broad-spectrum antibiotics (avoid fluoroquinolones), and culture data. Empiric treatment if severe illness. Standard 6*: In all children suspected of having intrathoracic and extrapulmonary TB, specimens (sputum, extrapulmonary tissue) should be obtained for microscopy, culture, and histopathological (tissue) examination. TB diagnosis should be based on chest radiography, history of TB exposure, positive TB test, and suggestive clinical findings if bacteriologic studies are negative.

43 ISTC TB Training Modules 2009 Summary: ISTC Standards Covered* * Abbreviated versions Standard 10 (partial): Response to therapy in patients with pulmonary tuberculosis should be monitored by follow-up sputum smear microscopy (2 specimens) at the time of completion of the initial phase of treatment (2 months). If the sputum smear is positive at completion of the initial phase, sputum smears should be examined again at 3 months and, if possible, culture and drug susceptibility testing should be performed.

44 ISTC TB Training Modules 2009 Summary: ISTC Standards Covered* Standard 11: An assessment of the likelihood of drug resistance, based on history of prior treatment, exposure to a possible source case having drug-resistant organisms, and the community prevalence of drug resistance, should be obtained. DST should be performed at the start of therapy for all previously treated patients For patients in whom drug resistance is considered to be likely, culture and testing for susceptibility/resistance to at least isoniazid and rifampicin should be performed promptly Patient counseling and education should begin immediately to minimize the potential for transmission Infection control measures appropriate to the setting should be applied * Abbreviated versions

45 ISTC TB Training Modules 2009 Alternate Slides

46 ISTC TB Training Modules 2009 Resource Availability The following tests are available at _________:  Smear microscopy Direct smear / light microscopy Fluorescence microscopy Concentration/chemical processing  Culture Solid media Liquid media  Drug susceptibility testing  Other

47 ISTC TB Training Modules 2009 Resource Contact Information Laboratory testing: Microscopy/culture/DST [Name, addresses, e-mail, etc.] Specimen transport: [Name, addresses, e-mail, etc.]

48 ISTC TB Training Modules 2009 Purpose of ISTC

49 ISTC TB Training Modules 2009 ISTC: Key Points  21 Standards (revised/renumbered in 2009)  Differ from existing guidelines: standards present what should be done, whereas, guidelines describe how the action is to be accomplished  Evidence-based, living document  Developed in tandem with Patients’ Charter for Tuberculosis Care  Handbook for using the International Standards for Tuberculosis Care

50 ISTC TB Training Modules 2009  Audience: all health care practitioners, public and private  Scope: diagnosis, treatment, and public health responsibilities; intended to complement local and national guidelines  Rationale: sound tuberculosis control requires the effective engagement of all providers in providing high quality care and in collaborating with TB control programs ISTC: Key Points

51 ISTC TB Training Modules 2009 Questions

52 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB 1. All of the following can increase sensitivity of sputum smear microscopy except: A.Fluorescence microscopy B.Sputum collection after the start of anti-tuberculosis treatment C.Concentration by centrifugation and/or sedimentation D.Chemical pretreatment

53 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB 2. A 37 year-old man with diabetes presents with clinical symptoms highly suspicious for TB. Two sputum smears are negative. The patient collected the specimens ten days before he brought them back and kept them in a cool area of the house (no refrigeration). Which of the following statements is most correct? A.Two negative smears predict that a culture would be negative, and therefore a culture offers no further diagnostic advantage and need not be obtained B.A lack of response to broad-spectrum antimicrobial agents and a chest film suggestive of TB, would together suggest a diagnosis of smear-negative TB C.The delay in transport and lack of refrigeration for the sputum specimens are unlikely to have a negative effect on results D.Six sputum specimens for smear microscopy would have tripled the sensitivity for diagnosing TB compared to two specimens

54 ISTC TB Training Modules 2009 Microbiologic Diagnosis of TB 3. Advantages of culture for TB compared to sputum microscopy alone include all of the following except: A.Obtaining a positive culture can allow for drug-susceptibility testing B.Culture can allow for identification of non-tuberculous mycobacterium species C.Culture has a higher sensitivity than smear microscopy for diagnosing TB. D.Culture, particularly by liquid media, can be faster than smear microscopy


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