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Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
Tuberculosis Care , ISTC training Module
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Tuberculosis Care , ISTC training Module

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Tuberculosis Care , ISTC training Module
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  • ISTC TB Training Modules 2009 Instructor’s Guide and Teaching Notes Module: Microbiologic Diagnosis of Tuberculosis ISTC Standards covered: [Standards reviewed only in terms of microscopy, culture, and drug susceptibility testing in Standards 2-6, 10, and 11.] Module Time: Approximately 60 minutes Alternate slides: Template slides to list local diagnostic testing resources and contact information, Introductory ISTC slides Interactive options: Ideas for interactive discussions are offered on many of the slides in this module. Participant discussion can enhance active learning, but will add more time to the lecture and must be planned for. Additional material: Slides containing related material may be found in the following module: Clinical presentation and diagnosis of TB. Test Questions: May be attached or inserted within presentation for discussion purposes, or alternatively, combined with questions from other modules to produce evaluation tool. The full text of the ISTC and all supporting references are available at www.istcweb.org Other useful Resources/References: Acid-fast direct smear microscopy training package, WHO/CDC/RIT/IUATLD/APHL, 2006. www.cdc.gov/dls/ila/acidfasttraining/default.aspx Management of Tuberculosis Training for Health Facility Staff, WHO Stop TB Partnership 2003. www.who.int/docstore/gtb/publications/training/management_of_tb/index.htm Pai M et al. New tools and emerging technologies for the diagnosis of tuberculosis: Part II. Active tuberculosis and drug resistance. Expert Rev. Mol. Diagn 2006; 6(3):423-432 Slide contribution: Ed Desmond, Ph.D., Microbial Diseases Lab, California Department of Public Health, Richmond, CA [Image Credits: Centers for Disease Control Public Health Image Library /Dr. George P. Kubica]
  • ISTC TB Training Modules 2009 [Review content of slide]
  • ISTC TB Training Modules 2009 Overview: [Review content of slide]
  • ISTC TB Training Modules 2009 Overview: [Review content of slide] [Image credit: World Lung Foundation/Jan van der Hombergh]
  • ISTC TB Training Modules 2009 Significance of microbiologic testing for public health goals and patient care: An important barrier to global TB control is the low rate of case detection. As part of the international WHO Stop TB Strategy, the overall target goal of 70% case detection of new smear-positive cases is dependent on appropriate access to and usage of microbiologic testing for M.tb . The direct significance to the patient is: [Review content of slide]
  • ISTC TB Training Modules 2009 Important points to be noted regarding sputum smear microscopy: For many areas of the world, sputum smear microscopy remains the most important and available test for the diagnosis of TB. Direct staining of unconcentrated specimens for smears is the most common practice, but sensitivity can be enhanced with chemical processing and concentration techniques or with the use of fluorescence microscopy. Quality assurance measures should be instituted and monitored routinely for all microscopy programs. [Transition: The following slides will review each of these important aspects.] [Image credit: World Lung Foundation/Jad Davenport]
  • ISTC TB Training Modules 2009 [Review content of slide] [Image credits: World Lung Foundation/Jad Davenport]
  • ISTC TB Training Modules 2009 [Review content of slide]
  • ISTC TB Training Modules 2009 Oil-immersion view of a Ziehl-Neelsen stained sputum smear with M.tb bacilli staining red with inflammatory cells stained blue. (Note: concentrated specimen). The photo shows the typical morphological features of M.tb . It is a slender, curved, beaded rod, often occurring in clumps. [Image Credit: CDC Public Health Image Library/Dr. George P. Kubica]
  • ISTC TB Training Modules 2009 Through a fluorescent microscope, the organisms will brightly fluoresce against a black background making them easy to identify (here with an Auromine-rhodamine stain). [Image credit: Microbial Diseases Laboratory, California Dept of Public Health/Ed Desmond, PhD]
  • ISTC TB Training Modules 2009 Fluorescence microscopy is widely used in many parts of the world. Advantages: A recent, comprehensive systematic review of 43 studies showed that fluorescence microscopy is more accurate. [Review content of slide] The combination of increased sensitivity with little or no loss of specificity makes fluorescence microscopy a more accurate test, although the increased cost and complexity might make it less applicable in many areas. For this reason, fluorescence staining is probably best used in centers with specifically trained and proficient microscopists, in which a large number of specimens are processed daily, and in which there is an appropriate quality control program. [Reference: Steingart KR et al. Fluorescence versus conventional sputum smear microscopy for tuberculosis: a systematic review. Lancet Infect. Dis. 2006;6(9):570-81]. [Image credit: World Lung Foundation/Pierre Virot]
  • ISTC TB Training Modules 2009 A variety of methods have been used to improve the performance of sputum smear microscopy. In general, the sensitivity of microscopy (as compared to culture) is higher with concentration by centrifugation and/or sedimentation (usually after pretreatment with chemicals such as bleach, NaOH, and NaLC)* or both, as compared to direct (unconcentrated) smear microscopy. A comprehensive, systematic review of 83 studies describing the effects of various physical and/or chemical methods for concentrating and processing sputum prior to microscopy found that concentration resulted in higher sensitivity (15-20%increase) and smear-positivity rate, when compared with direct smears. Centrifugation is more complex, requires electrical power, and may be associated with increased infection risk to laboratory personnel. Consequently, it is not clear that the advantages offset the disadvantages in low-resource settings. [ Interactive option: Ask participants if they know how their local facilities process their smears – direct vs. concentrated, chemical processing, and type of stain/microscopy available? Where are referral centers, what can be done there, when and how may specimens be sent?] *[Note: Bleach and sodium hydroxide (NaOH) are more toxic to oral flora than AFB and help to decontaminate the specimen. N-acetyl-L-cysteine (NALC) is a commonly used mucolytic agent that dissolves mucin and helps to liquefy respiratory specimens.] [Reference: Steingart KR, et al. Sputum processing methods to improve the sensitivity of smear microscopy for tuberculosis: a systematic review. Lancet Infect. Dis 2006; 6(10):664-74].
  • ISTC TB Training Modules 2009 [Review content of slide] [It is important to adjust according to your country’s National Tuberculosis Program procedures for sputum collection.]
  • ISTC TB Training Modules 2009 [Review content of slide] [It is important to adjust according to your country’s National Tuberculosis Program procedures for sputum collection.]
  • ISTC TB Training Modules 2009 The limitation of sputum smear microscopy is its sensitivity. As illustrated in the table: compared with culture, sputum smear microscopy is 68% sensitive in detecting M. tuberculosis. Of all specimens that are AFB positive nearly 86% are detected by examining one specimen and an additional 12% are found on the 2nd specimen; thus, the incremental yield of the 3 rd specimen is very low. A similar increment is found for the sensitivity of the 2 nd and 3 rd specimens. The yield is better with a single early morning specimen than with a spot specimen obtained at other times during the day. [Reference: Mase SR, et al. Yield of serial sputum specimen examinations in the diagnosis of pulmonary tuberculosis: a systematic review. Int J tuberc Lung Dis 2007;11(5): 485-95]
  • ISTC TB Training Modules 2009 The central importance of obtaining sputum for microscopic examination is the diagnosis of TB is reflected in the following ISTC Standards of Diagnosis (2, 3, 4, and 10). [Note: Guidelines have recently changed from three sputum smears to at least two sputum smears. The change is reflected above.] [Read Standard]
  • ISTC TB Training Modules 2009 [Read Standard]
  • ISTC TB Training Modules 2009 Repeat sputum microscopy plays an equally important role in the monitoring for response to treatment. [Read Standard – Note only partial Standard 10, limited to issue of sputum microscopy]
  • ISTC TB Training Modules 2009 Where quality-assured laboratory facilities are available, culture should be included in the algorithm for evaluating patients with negative sputum smears. Properly done, culture adds a significant layer of complexity and cost but also increases sensitivity, which should result in earlier case detection. Although the results of culture may not be available until after a decision to begin treatment has to be made, treatment can be stopped subsequently if cultures from a reliable laboratory are negative, the patient has not responded clinically, and the clinician has sought other evidence in pursuing the differential diagnosis as above. [Image credit: World Lung Foundation/Jad Davenport]
  • ISTC TB Training Modules 2009 Culture advantages: Culture of specimens has a higher sensitivity than microscopy and, at least in theory, can increase case detection (although this potential has not been demonstrated in low-income, high-incidence areas). While sputum microscopy is likely to be positive when there are at least 10,000 organisms per ml of sputum, at concentrations below 1000 organisms/ml, the chance of a positive smear is less than 10%. In contrast, a properly performed culture can detect far lower numbers of acid-fast bacilli with a detection limit of about 100 organisms per ml. Culture should be included in the algorithm for evaluating patients with negative sputum smears because culture results may confirm a TB diagnosis. Culture can also help to identify other mycobacterial species and allow for drug susceptibility testing (particularly important when drug-resistance is suspected).
  • ISTC TB Training Modules 2009 [Review content of slide] Because of the added complexity required for quality culture results, the test may only be available through referral. This should not discourage the use of culture when clinically indicated. It is very important that proper TB treatment be started empirically in suspicious cases, rather than delay treatment while awaiting culture confirmation. Early empiric treatment reduces both morbidity and transmission of M.tb .
  • ISTC TB Training Modules 2009 Culture methods using solid media is the reference standard. It is less technologically intensive and media can be made locally. [Review content of slide] [Note: Top image shows growth on Lowenstein Jensen slants and lower image is culture plate, also with Lowenstein Jensen media] [Image credits: San Francisco Public Health Laboratory]
  • ISTC TB Training Modules 2009 Liquid Media requires a much more sophisticated laboratory system, and where available, allows for a more rapid identification of culture positivity and increased sensitivity compared with solid media, and may also be used for drug-susceptibility testing. Liquid media, such as the BACTEC system utilize the release of radioactive CO2 from C-14 (carbon-14) labeled palmitic acid in the media to identify growth. The MGIT system, also using liquid medium, has the advantage of having growth detected by the appearance of fluorescence in a silicone plug at the bottom of the tube, thereby avoiding radioactivity. The use of either liquid technique can reduce time to culture results to 2-4 weeks, compared with 4-8 weeks for solid media. Because of the advantages over solid media, WHO has issued recommendation for phased implementation of liquid culture systems. Decisions to provide culture facilities for diagnosing TB and the methods to be used, clearly depend on financial resources, trained personnel, and the ready availability of reagents and equipment service. [Image credits: San Francisco Public Health Laboratory]
  • ISTC TB Training Modules 2009 Accurate differentiation of M.tb from other non-tuberculosis mycobacterium can be done in the following ways: [Review content of slide]
  • ISTC TB Training Modules 2009 Examples of identification by colony morphology: [Review content of slide] [Image Credits: CDC Public Health Image Library /Dr. George P. Kubica]
  • ISTC TB Training Modules 2009 [Review content of slide] Need to be aware that cross-contamination can occur and maintain suspicion for problems if the clinical scenario does not make sense and multiple positive results are found in specimens processed at the same time. Direct communication with laboratory can be very helpful and is the only way to investigate this possibility. Quality control is essential to the functioning of mycobacteriology laboratories. [Image – Example: use of same flask to add material to multiple specimens can lead to cross-contamination from one patient specimen to the next.] [Image credit: Microbial Diseases Laboratory, California Dept of Public Health/Ed Desmond, PhD]
  • ISTC TB Training Modules 2009 As noted in Standard 5, obtaining cultures, is particularly important in the diagnosis of suspected smear-negative TB. [Read Standard] * Abbreviated on slide (Note: Because the fluoroquinolones are active against M. tuberculosis complex, and thus may cause transient improvement in persons with tuberculosis, they should be avoided) [Note: Guidelines have recently changed from three sputum smears to at least two sputum smears. The change is reflected above.] It is important to remember that, on average, sputum smear microscopy is only approximately 50-60% sensitive when compared with culture.
  • ISTC TB Training Modules 2009 Cultures also have an important role in the diagnosis of TB in children. [Read Standard]
  • ISTC TB Training Modules 2009 Cultures play a particularly important role in children if smear-negative. [Read Standard]
  • ISTC TB Training Modules 2009 And as well as if extrapulmonary disease is present. [Read Standard] (continued)
  • ISTC TB Training Modules 2009 Once TB has been identified by culture growth, subcultures can be collected and tested for susceptibility to all of the first-line, and many of the second-line anti-TB drugs. Both solid and liquid culturing techniques may be used for drug-susceptibility testing (DST). The basic principle used in the test methods is that culture growth is attempted in the face of antibiotic challenge. If the organism is susceptible, it will not grow in the presence of the antibiotic. Example above shows agar plates divided into four segments. Each white disc has a different first-line drug impregnated onto it. Areas with no growth indicate drug susceptibility, areas with active growth indicate drug resistance. DST to the first-line drugs should be performed in specialized reference laboratories that participate in an ongoing, rigorous quality assurance program. DST for first-line drugs is currently recommended for all patients with a history of previous anti-TB treatment: patients who have failed treatment, especially those who have failed a standardized re-treatment regimen, and chronic cases are the highest priority. [Image credit: Microbial Diseases Laboratory, California Dept of Public Health/Ed Desmond, PhD]
  • ISTC TB Training Modules 2009 Important indicators for the use of drug-susceptibility testing are outlined in ISTC Standard 11. [Read Standard]
  • ISTC TB Training Modules 2009 [Read Standard] (continued)
  • ISTC TB Training Modules 2009 Tests to identify TB more rapidly then standard biochemical assays are available, but because of the high cost and technical requirements, are neither feasible or practical in many settings. The non-amplified DNA Probe tests are one example, and offer rapid identification of type of mycobacteria/species once growth is detected on culture. NAAT testing is used primarily on smear-positive sputum. These tests can be used directly on clinical specimens to verify the presence of M.tb with results in < 1 day. They have a high specificity and positive-predictive value for pulmonary disease, therefore allowing confidence in ruling-in disease, but sensitivity is lower and highly variable. In countries with high-endemic rates of TB, smear-positive results already confer a high likelihood of TB, and the added value of NAAT testing is questionable. The utility of NAAT testing may be more appropriate for low-burden, high-resource settings.
  • ISTC TB Training Modules 2009 It is clear that there is a great need for more rapid and accessible testing for TB. Other rapid or more advanced tests currently in use or still under investigation include: LAMP tests still being evaluated, but may be more promising for low-resource settings (do not require thermal cycler for PCR amplification). Immunologic tests are more readily available via commercial kits, but are not yet accurate enough to replace microscopy and culture.
  • ISTC TB Training Modules 2009 Another example of rapid diagnostic tests: HPLC is a highly technical and costly technique, and available in a limited setting even in high-resource areas.
  • ISTC TB Training Modules 2009 With the rising rates of MDR and XDR, the need for rapid and accurate identification of drug resistance is significant. Tests currently in use or under investigation include: Line probe assays: DNA from M.tb directly from sputum samples can be amplified and identified. Probes can both identify M.tb and test for drug resistance based on the presence of genetic mutations associated with specific drug resistance (isoniazid and rifampicin). Has a high sensitivity (>=97%) and specificity (>=99%) for rifampicin resistance. Overall accuracy for detection of MDR equally high at 99%, and retained when rifampicin resistance alone was used as a marker for MDR. The WHO has endorsed their use for both direct smear-positive sputum and culture specimens. - Image shows examples of GenoType MTDBRplus strips (Hain Lifescience, Nehren, Germany). [Resource: WHO Policy Statement, Molecular line probe assays for rapid screening of patients at risk of multi-drug resistant tuberculosis (MDR-TB), 2008. www.who.int ] Other rapid drug susceptibility tests – examples listed here are in use or under investigation in a more limited setting, most requiring more technical laboratory capabilities. [Image credit: Rapid Molecular Screening for Multidrug-Resistant Tuberculosis in a High-Volume, Barnard et al. Am. J. Respir. Crit. Care Med 2008; 177: 787-792]
  • ISTC TB Training Modules 2009 In summary, important points regarding the microbiologic diagnosis of TB discussed: [Review content of slide] [Continued] [Image credits: World Lung Foundation/Jad Davenport]
  • ISTC TB Training Modules 2009 (Continued) important points regarding the microbiologic diagnosis of TB discussed: [Review content of slide] [Image credits: Microbial Diseases Laboratory, California Dept of Public Health/Ed Desmond, PhD]
  • ISTC TB Training Modules 2009 And in summary, the International Standards reviewed: (abbreviated) [Review content of slide]
  • ISTC TB Training Modules 2009 [Continued - Review content of slide]
  • ISTC TB Training Modules 2009 [Continued - Review content of slide]
  • ISTC TB Training Modules 2009 [Continued - Review content of slide] [End]
  • ISTC TB Training Modules 2009 Alternate Slides: Offer additional options that may be added or substituted into module. See Facilitator’s Guide for further information.
  • ISTC TB Training Modules 2009 [ Optional slide: Use this slide to add specific information on where and what type of testing (those listed above or others) is available to the course participants.] [ Interactive option – ask participants what resources have they utilized, share views on how to optimize the proper use of the diagnostic tests available.]
  • ISTC TB Training Modules 2009 [ Optional slide: Use this slide to add in specific information who and how to contact local/regional/reference laboratory resources for questions].
  • ISTC TB Training Modules 2009 ISTC Training Modules 2008 The International Standards for Tuberculosis Care (ISTC): Are intended to unite public and private sectors in providing a uniformly accepted level of care for all patients with, or suspected of having, TB. Describes the essential elements of TB care that should be available everywhere. Provides a vehicle for mobilizing professional societies globally in support of TB programs. Serves as a powerful advocacy tool to ensure that the essential elements are available. [Image Credit: World Lung Foundation/Jad Davenport ]
  • ISTC TB Training Modules 2009 The ISTC consist of 21 evidence-based standards. The original 17 standards from 2006 were revised and renumbered in 2009. Standards differ from existing guidelines in that standards present what should be done, whereas, guidelines describe how the action is to be accomplished. To meet the requirements of the Standards, approaches and strategies, determined by local circumstances and practices and developed in collaboration with local and national public health authorities, will be necessary. There are many situations in which the level of care can, and should, go beyond what is specified in the Standards. The Standards should be viewed as a living document that will be revised as technology, resources, and circumstances change. Revisions to the original document released December 2005 are currently underway. Funded (Oct 1, 2004) by USAID via TBCTA, development was supervised by an international steering committee (28 members / 14 countries) chosen to represent perspectives relevant to tuberculosis care and control. The Standards are also intended to serve as a companion to and support for the Patients’ Charter for Tuberculosis Care developed in tandem with the Standards. A Handbook for using the International Standards for Tuberculosis Care is also available (2007). The Handbook presents suggestions and guidance, based mainly on country-level experiences, for using the ISTC as a tool to foster and guide the delivery of high-quality care by all practitioners providing TB care. [Resource: www.istcweb.org]
  • ISTC TB Training Modules 2009 The Standards are addressed to all healthcare providers, private and public, who care for persons with proven tuberculosis or with symptoms and signs suggestive of TB. Three categories of activities are addressed by the Standards: diagnosis, treatment, and public health responsibilities of all providers. The ISTC are intended to be complementary to local and national TB control policies that are consistent with the World Health Organization (WHO) recommendations. In many parts of the world there is great variability in the quality of tuberculosis care, and poor quality care continues to plague global tuberculosis control efforts. Effective engagement of all providers in providing high quality care in collaboration with TB control programs is key to the promotion of sound tuberculosis control.
  • ISTC TB Training Modules 2009 Questions: May be used for interactive discussion, course evaluation, or continuing medical education purposes. See Facilitator Guide for further information.
  • ISTC TB Training Modules 2009 Correct Answer: B
  • ISTC TB Training Modules 2009 Correct Answer: B
  • ISTC TB Training Modules 2009 Correct Answer: D
  • Transcript

    • 1. MicrobiologicDiagnosis ofTuberculosis Coordinator - Dr.T.V.Rao MDInstitution/organization Travancore Medical College, Kollam IndiaInternational Standards 2-6, 10, 11
    • 2.  I request all the Medical, Nursing and Health Care workers go through this informative programme on Tuberculosis created by  International standards for Tuberculosis  Please send to as many professionals as Possible to work with Tuberculosis on Scientific basis  Dr.T.V.Rao MD  Travancore Medical College, Kollam India  Copyright of ISTC TB trainingISTC TB Training Modules 2009
    • 3. 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 TBISTC TB Training Modules 2009
    • 4. 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 11ISTC TB Training Modules 2009
    • 5. Standards for DiagnosisISTC TB Training Modules 2009
    • 6. 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 treatmentISTC TB Training Modules 2009
    • 7. 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 essentialISTC TB Training Modules 2009
    • 8. 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 commonISTC TB Training Modules 2009
    • 9. 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 blueISTC TB Training Modules 2009
    • 10. Ziehl-Neelsen (ZN) StainISTC TB Training Modules 2009
    • 11. Auramine-rhodamine StainISTC TB Training Modules 2009
    • 12. 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):570-81ISTC TB Training Modules 2009
    • 13. Sputum Processing 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 Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74ISTC TB Training Modules 2009
    • 14. Specimen Collection and Transport  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 outdoorsISTC TB Training Modules 2009
    • 15. Specimen Collection and Transport  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 possibleISTC TB Training Modules 2009
    • 16. Performance of Sputum Microscopy Incremental Yield of Incremental Sensitivity Specimen smear specimens of smear specimens Number (of all smear-positive) (compared with culture) 1 85.8% 53.8% 2 11.9% 11.1% 3 2.4% 3.1% Total 100% 68.0% Average yield of single early morning specimen: 86.4% Average yield of single spot specimen: 73.9% Mase SR, Int J tuberc Lung Dis 2007;11(5): 485-95ISTC TB Training Modules 2009
    • 17. 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.ISTC TB Training Modules 2009
    • 18. 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 examinationISTC TB Training Modules 2009
    • 19. 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)ISTC TB Training Modules 2009
    • 20. Culture and Drug Susceptibility Testing 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.ISTC TB Training Modules 2009
    • 21. 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 testingISTC TB Training Modules 2009
    • 22. 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.ISTC TB Training Modules 2009
    • 23. 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 • OgawaISTC TB Training Modules 2009
    • 24. 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 • BACTEC MGIT Incubator • MGIT MGITISTC TB Training Modules 2009
    • 25. 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.tbISTC TB Training Modules 2009
    • 26. Culture: Identification of Mycobacteria Visual assessment of colony morphology: Smooth, buff-colored colonies suggestive of Mycobacterium avium complex Rough, buff-colored colonies suggestive of Mycobacterium tuberculosisISTC TB Training Modules 2009
    • 27. 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.ISTC TB Training Modules 2009
    • 28. 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.ISTC TB Training Modules 2009
    • 29. Standard 6: Culture in Children (1 of 3) 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 TB Training Modules 2009 Training Modules 2008
    • 30. Standard 6: Culture in children (2 of 3)  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 TBISTC TB Training Modules 2009 Training Modules 2008
    • 31. Standard 6: Culture in Children (3 of 3)  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.ISTC TB Training Modules 2009
    • 32. Culture: Drug Susceptibility Testing Methods for 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 drugISTC TB Training Modules 2009
    • 33. Standard 11: Drug Susceptibility (1 of 2) 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 resistanceISTC TB Training Modules 2009
    • 34. Standard 11: Drug Susceptibility (2 of 2)  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 appliedISTC TB Training Modules 2009
    • 35. 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 specimensISTC TB Training Modules 2009
    • 36. 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.ISTC TB Training Modules 2009
    • 37. 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 laboratoriesISTC TB Training Modules 2009
    • 38. 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 *GenoType MTDBRplus strips (Hain Lifescience)  Molecular beacons  Bacteriophage-based assays *Barnard et al. Am. J. Respir. Crit. Care Med 2008; 177: 787-792ISTC TB Training Modules 2009
    • 39. 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.ISTC TB Training Modules 2009
    • 40. 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 proceduresISTC TB Training Modules 2009
    • 41. 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. * Abbreviated versionsISTC TB Training Modules 2009
    • 42. Summary: ISTC Standards Covered* 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. * Abbreviated versionsISTC TB Training Modules 2009
    • 43. Summary: ISTC Standards Covered* 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. * Abbreviated versionsISTC TB Training Modules 2009
    • 44. 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 versionsISTC TB Training Modules 2009
    • 45. Alternate SlidesISTC TB Training Modules 2009
    • 46. 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  OtherISTC TB Training Modules 2009
    • 47. Resource Contact Information Laboratory testing: Microscopy/culture/DST [Name, addresses, e-mail, etc.] Specimen transport: [Name, addresses, e-mail, etc.]ISTC TB Training Modules 2009
    • 48. Purpose of ISTCISTC TB Training Modules 2009
    • 49. 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 CareISTC TB Training Modules 2009
    • 50. ISTC: Key Points  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 programsISTC TB Training Modules 2009
    • 51. QuestionsISTC TB Training Modules 2009
    • 52. 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 pretreatmentISTC TB Training Modules 2009
    • 53. 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 specimensISTC TB Training Modules 2009
    • 54. 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 microscopyISTC TB Training Modules 2009

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