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Premium member Presentation Transcript Acid fast staining in tuberculosis principles, practice, and applications: Dr.T.V.Rao MD Acid fast staining in tuberculosis principles, practice, and applications Dr.T.V.Rao MD 1Microbiologic Diagnosis of TB: Microbiologic Diagnosis of TB Dr.T.V.Rao MD 2 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 testingWhy Microbiologic Diagnosis of TB is important: Why Microbiologic Diagnosis of TB is important Dr.T.V.Rao MD 3 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 treatmentMicrobiologic Diagnosis of TB: 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. Microbiologic Diagnosis of TB Dr.T.V.Rao MD 4Sputum 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 Sputum Smear Microscopy Dr.T.V.Rao MD 5Principles of Ziehl–Neelsen stain : Principles of Ziehl–Neelsen stain The Ziehl–Neelsen stain , also known as the acid-fast stain , was first described by two German doctors; Franz Ziehl (1859 to 1926), a bacteriologist and Friedrich Neelsen (1854 to 1898), a pathologist. It is a special bacteriological stain used to identify acid-fast organisms, mainly Mycobacteria. Mycobacterium tuberculosis is the most important of this group, as it is responsible for the disease called tuberculosis (TB) along with some others of this genus. It is helpful in diagnosing Mycobacterium tuberculosis since its lipid rich cell wall makes it resistant to Gram stain. It can also be used to stain few other bacteria like Nocardia . The reagents used are Ziehl–Neelsen carbolfuchsin, acid alcohol and methylene blue. Acid-fast bacilli will be bright red after staining. Dr.T.V.Rao MD 6Principle of acid fast staining: Principle of acid fast staining Primary stain binds cell wall mycolic acids Intense decolonization does not release primary stain from the cell wall of AFB Color of AFB-based on primary stain Counterstain provides contrasting background Dr.T.V.Rao MD 7Mycobacterium are Acid Fast Bacilli: Mycobacterium are Acid Fast Bacilli Mycobacterium are Gram-resistant (waxy cell walls), non-motile, pleomorphic rods, related to the Actinomyces. Most Mycobacteria are found in habitats such as water or soil. However, a few are intracellular pathogens of animals and humans. Mycobacterium tuberculosis , along with M. bovis , M. africanum , and M. microti all cause the disease known as tuberculosis (TB) and are members of the tuberculosis species complex. Each member of the TB complex is pathogenic, but M. tuberculosis is pathogenic for humans while M. bovis is usually pathogenic for animals. 8 Dr.T.V.Rao MDSlide 9: r r r r r r Acid Fast Cell Envelope Cytoplasm r r r r Peptidoglycan-mycolic acid-arabinogalactan Cytoplasmic membrane Mycolic acid lipids Dr.T.V.Rao MD 9Mycobacteria structure: Mycobacteria structure Contain large amount of fatty waxes (mycolic acid) within their cell wall resist staining by ordinary methods Require a special stain for diagnostic Acid Fast stain. Dr.T.V.Rao MD 10Ziehl-Neelsen stain : Ziehl-Neelsen stain Ziehl-Neelsen staining is used to stain species of Mycobacterium tuberculosis that do not stain with the standard laboratory staining procedures like Gram staining. The stains used are the red colored Carbol fuchsin that stains the bacteria and a counter stain like Methylene blue or Malachite green. Dr.T.V.Rao MD 11 AFB Staining Methods: AFB Staining Methods Zeihl Neelsen’s-hot stain Kinyoun’s-cold stain Modifications 12 Dr.T.V.Rao MDExample of Acid-Fast bacteria: Example of Acid-Fast bacteria Each person will make a smear and Acid-Fast stain of a mixed broth containing: Mycobacterium smegmatis (Gram +) & Staphlococcus epidermis (Gram +) Blue=Non acid-fast bacteria Red= acid fast bacteria Dr.T.V.Rao MD 13Sputum Microscopy: Direct Smears: 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 Dr.T.V.Rao MD 14Acid - Fast Stain basic requirements : 1. Carbolfuchsin (Red) 2. Acid Alcohol 3. Counterstain with Methylene Blue Acid - Fast Cells Red Non Acid - Fast Blue Acid - Fast Stain basic requirements Dr.T.V.Rao MD 15Proceeding with Ziehl- Neelsen Procedure : Proceeding with Ziehl- Neelsen Procedure 1. Make a smear. Air Dry. Heat Fix. 2. Flood smear with Carbol Fuchsin stain Carbol Fuchsin is a lipid soluble, phenolic compound, which is able to penetrate the cell wall 3. Cover flooded smear with filter paper 4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed 5. Cool slide 6. Rinse with DI water 7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains 3% HCl and 95% ethanol or 20% H 2 SO 4 The waxy cell wall then prevents the stain from being removed by the acid alcohol (decolorizer) once it has penetrated the cell wall. The acid alcohol decolorizer will remove the stain from all other cells. Dr.T.V.Rao MD 16Ziehl-Neelsen stain: Ziehl-Neelsen stain 4 5 6 7 1 2 3 17 Dr.T.V.Rao MDZiehl- Neelsen Procedure (continued): Ziehl- Neelsen Procedure (continued) 8. Tilt slide 45 degrees over the sink and add acid alcohol drop wise (drop by drop) until the red color stops streaming from the smear 9. Rinse with DI water 10. Add Loeffler’s Methylene Blue stain (counter stain). This stain adds blue color to non-acid fast cells!! Leave Loeffler’s Blue stain on smear for 1 minute 11. Rinse slide. Blot dry. 12. Use oil immersion objective to view. Dr.T.V.Rao MD 18 Acid-Fast Stain how it works: Acid-fast cells contain a large amount of lipids and waxes in their cell walls primarily mycolic acid Acid fast bacteria are usually members of the genus Mycobacterium or Nocardia Therefore, this stain is important to identify Mycobacterium or Nocardia Acid-Fast Stain how it works Dr.T.V.Rao MD 19Bright-field Techniques: Bright-field Techniques Hot Ziehl-Neelsen in practice Most reliable more visible AFB stronger color Cold methods : Kinyoun, Tan Thiam Hok… less laborious but also less robust higher concentration fuchsin, longer staining time errors !! not recommended for low-income countries Dr.T.V.Rao MD 20How the Acid fast bacteria appear: How the Acid fast bacteria appear 21 Dr.T.V.Rao MDSelecting a ideal Sputum Sample: Selecting a ideal Sputum Sample W What is a good sample? What is saliva? Good sample = yellow? mucous fluid? Discharge from the bronchial tree May contain solid or purulent substances Minimal amounts of oral/ nasal material May contain macrophages and other cells indicative of infectious disease Follow-up examination samples? Dr.T.V.Rao MD 22Specimen 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 outdoors Specimen Collection and Transport Dr.T.V.Rao MD 23Specimen 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 possible Specimen Collection and Transport Dr.T.V.Rao MD 24Standard 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 . Standard 2: Sputum Microscopy Dr.T.V.Rao MD 25Sputum 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 Sputum Processing Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74 Dr.T.V.Rao MD 26Standards 3 & 4: Sputum Microscopy: Standard 3 : For all patients (adults, adolescents, and children) suspected of having extra pulmonary 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 Standards 3 & 4: Sputum Microscopy Dr.T.V.Rao MD 27How the Acid fast bacteria appear: How the Acid fast bacteria appear 28 Dr.T.V.Rao MDMicroscopic Reading:: Microscopic Reading: Red slender rods on blue background accept only typical shape, at least some depends condition of microscope! Light binocular, mechanical stage, good optics 100x oil immersion objective, 10x eyepieces Requires: patience, sincerity AFB microscopy is not difficult but tough Dr.T.V.Rao MD 29Advantages and Disadvantage of Acid Fast Bacteria : Advantages and Disadvantage of Acid Fast Bacteria Advantages: Acid-fast cells contain a large amount of lipids and waxes in their cell walls, making them relatively impermeable and resistant to many disinfectants Also enables resistance to desiccation, antibiotics, and other toxins Disadvantage: Waxes delay nutrient uptake, so cells grow slower Ziehl-Neelsen Method is used to stain acid-fast bacteria Dr.T.V.Rao MD 30Zeihl Neelsen and Fluorochrome Microscopy AFB Quantification Scales : Zeihl Neelsen and Fluorochrome Microscopy AFB Quantification Scales System & Quantification Scale No. of AFB per field Brightf. & IUATLD/WHO Scale (1000x) Brightf. & ATS Scale (1000x) Fluor. & IUATLD/WHO Scale (200-250x) Fluor. & ATS Scale (200-250x) None 1-2/300 fields 1-9/100 fields 1-9/10 fields 1-9/1 field 10-99/1field >=100/1field Negative Actual Actual 1+ 2+ 3+ 3+ Negative Actual 1+ 2+ 3+ 4+ 4+ Negative Actual Actual Actual 1+ 2+ 3+ Negative Actual Actual 1+ 2+ 3+ 4+ Dr.T.V.Rao MD 31Sputum Microscopy: Fluorochrome Stain: 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 Dr.T.V.Rao MD 32Fluorochrome AFB Microscopy: More rapid and sensitive Specificity : same with sufficient expérience Equipment cost , bulbs, technical demands for busy labs External quality assessment should be done if this method is performed Fluorochrome AFB Microscopy Dr.T.V.Rao MD 33Fluorescence Microscopy: 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 Dr.T.V.Rao MD 34Auramine Stain: Auramine Stain Dr.T.V.Rao MD 35Auramine-rhodamine Stain : Auramine-rhodamine Stain Dr.T.V.Rao MD 36Fluorescence Microscopy: 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 Dr.T.V.Rao MD 37Culture 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. Culture and Drug Susceptibility Testing Dr.T.V.Rao MD 38international standards for tuberculosis care. : international standards for tuberculosis care. Dr.T.V.Rao MD 39 The International Standards for Tuberculosis Care (ISTC) describe a widely endorsed level of care that all practitioners should seek to achieve in managing individuals who have, or are suspected of having, tuberculosis . The basic principles of care for people with, or suspected of having, tuberculosis are the same worldwide: a diagnosis should be established promptly; standardized treatment regimens should be used with appropriate treatment support and supervision; response to treatment should be monitored; and essential public-health responsibilities must be carried out. Prompt and accurate diagnosis, and effective treatment are essential for good patient care and tuberculosis control. All providers who undertake evaluation and treatment of patients with tuberculosis must recognize that not only are they delivering care to an individual, but they are also assuming an important public-health function.Application of international standards for tuberculosis care (Istc) standards better care of tuberculosis patients: Application of international standards for tuberculosis care (Istc) standards better care of tuberculosis patients Dr.T.V.Rao MD 40 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 StandardsPurpose of ISTC: Purpose of ISTC Dr.T.V.Rao MD 41Better goals in Microbiologic Diagnosis of TB: Better goals in Microbiologic Diagnosis of TB 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 Dr.T.V.Rao MD 42Summary: ISTC Standards Covered*: 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 extra pulmonary 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. Dr.T.V.Rao MD 43Summary: ISTC Standards Covered*: Summary: ISTC Standards Covered* s 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. Dr.T.V.Rao MD 44Summary: ISTC Standards Covered*: 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. Dr.T.V.Rao MD 45Summary: ISTC Standards Covered*: 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 Dr.T.V.Rao MD 46ISTC: Key Points: 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 Dr.T.V.Rao MD 47ISTC: 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 programs ISTC: Key Points Dr.T.V.Rao MD 48Plan of action by who : Successful implementation of the Global Plan depends on implementation of the new 6-point STOP TB strategy recommended by WHO. This strategy promotes use of the new International Standards for Tuberculosis Care to engage all care providers (including those in the private sector) in delivering high-quality care. It specifically addresses HIV-associated TB, MDR-TB and other challenges, and strengthens human rights and health systems. However, the plan also relies on new diagnostic tests, new drugs and TB vaccines being developed by or before 2015. Plan of action by who Dr.T.V.Rao MD 49References : International standards for Tuberculosis Care References Dr.T.V.Rao MD 50For current Interest on Infectious diseases follow me on..: For current Interest on Infectious diseases follow me on.. Dr.T.V.Rao MD 51Created for Microbiologists and health care workers in Developing world: Created for Microbiologists and health care workers in Developing world Email firstname.lastname@example.org Dr.T.V.Rao MD 52 You do not have the permission to view this presentation. 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