Acid fast staining in Tuberculosis

ACID FAST STAINING IN TUBERCULOSIS PRINCIPLES, PRACTICE, AND APPLICATIONS Dr.T.V.Rao MDDR.T.V.RAO MD 1

MICROBIOLOGIC DIAGNOSIS OF TBOverview:• 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 testingDR.T.V.RAO MD 2

WHY MICROBIOLOGIC DIAGNOSIS OF TB IS IMPORTANTSignificance of microbiologic testing forpublic 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 treatmentDR.T.V.RAO MD 3

MICROBIOLOGIC 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.DR.T.V.RAO MD 4

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 essentialDR.T.V.RAO MD 5

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 6

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 backgroundDR.T.V.RAO MD 7

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. DR.T.V.RAO MD 8

Acid Fast Cell EnvelopeMycolic acid lipids Peptidoglycan-mycolic acid-arabinogalactan r r r r r r r r r r Cytoplasmic membrane Cytoplasm DR.T.V.RAO MD 9

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 10

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• Zeihl Neelsen’s- hot stain• Kinyoun’s-cold stain• ModificationsDR.T.V.RAO MD 12

EXAMPLE OF ACID-FAST BACTERIA Blue=Non acid-fast bacteria Red= acid fast bacteriaEach person will make a smear and Acid-Fast stain of a mixedbroth containing:Mycobacterium smegmatis (Gram +) &Staphlococcus epidermis (Gram +) DR.T.V.RAO MD 13

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 commonDR.T.V.RAO MD 14

ACID - FAST STAIN BASIC REQUIREMENTS• 1. Carbolfuchsin (Red)• 2. Acid Alcohol• 3. Counterstain with Methylene Blue• Acid - Fast Cells Red• Non Acid - Fast BlueDR.T.V.RAO MD 15

PROCEEDING WITH ZIEHL- NEELSEN PROCEDURE1. 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 wall3. Cover flooded smear with filter paper4. Steam for 10 minutes. Add more Carbol Fuchsin stain as needed5. Cool slide6. Rinse with DI water7. Flood slide with acid alcohol (leave 15 seconds). The acid alcohol contains 3% HCl and 95% ethanol or 20% H 2 SO4 • 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 16

1 2 3 4 5 6 7 ZIEHL-NEELSENDR.T.V.RAO MD STAIN 17

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 smear9. Rinse with DI water10. 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 minute11. 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 NocardiaDR.T.V.RAO MD 19

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 countriesDR.T.V.RAO MD 20

HOW THE ACID FAST BACTERIA APPEAR DR.T.V.RAO MD 21

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 22

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 outdoorsDR.T.V.RAO MD 23

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 possibleDR.T.V.RAO MD 24

STANDARD 2: SPUTUM MICROSCOPYStandard 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.DR.T.V.RAO MD 25

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 rateDR.T.V.RAO MD Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (10):664-74 26

STANDARDS 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 examinationDR.T.V.RAO MD 27

HOW THE ACID FAST BACTERIA APPEAR DR.T.V.RAO MD 28

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 toughDR.T.V.RAO MD 29

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 30

ZEIHL NEELSEN AND FLUOROCHROME MICROSCOPY AFB QUANTIFICATION SCALES System & Quantification Scale Fluor. & No. of AFB Brightf. & Brightf. & ATS Fluor. & ATS IUATLD/WHO per field IUATLD/WHO Scale Scale Scale Scale (1000x) (1000x) (200-250x) (200-250x)None Negative Negative Negative Negative1-2/300 fields Actual Actual Actual Actual1-9/100 fields Actual 1+ Actual Actual1-9/10 fields 1+ 2+ Actual 1+1-9/1 field 2+ 3+ 1+ 2+10-99/1field 3+ 4+ 2+ 3+>=100/1field 3+ 4+ 3+ 4+ DR.T.V.RAO MD 31

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 backgroundDR.T.V.RAO MD 32

FLUOROCHROME 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 performedDR.T.V.RAO MD 33

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 areasDR.T.V.RAO MD Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81 34

AURAMINE STAINDR.T.V.RAO MD 35

AURAMINE-RHODAMINE STAINDR.T.V.RAO MD 36

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 areasDR.T.V.RAO MD Steingart KR, et al. Lancet Infect. Dis. 2006; 6 (9):570-81 37

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.DR.T.V.RAO MD 38

INTERNATIONAL STANDARDS FOR TUBERCULOSIS CARE.The International Standards for Tuberculosis Care (ISTC) describe a widelyendorsed level of care that all practitioners should seek to achieve in managingindividuals who have, or are suspected of having, tuberculosis.The basic principles of care for people with, or suspected of having, tuberculosisare the same worldwide: a diagnosis should be established promptly;standardized treatment regimens should be used with appropriate treatmentsupport and supervision; response to treatment should be monitored; andessential public-health responsibilities must be carried out. Prompt and accuratediagnosis, and effective treatment are essential for good patient care andtuberculosis control. All providers who undertake evaluation and treatment ofpatients with tuberculosis must recognize that not only are they delivering careto an individual, but they are also assuming an important public-health function.DR.T.V.RAO MD 39

APPLICATION OF INTERNATIONAL STANDARDS FOR TUBERCULOSIS CARE (ISTC) STANDARDS BETTER CARE OF TUBERCULOSIS PATIENTS•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 StandardsDR.T.V.RAO MD 40

PURPOSE OF ISTCDR.T.V.RAO MD 41

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 proceduresDR.T.V.RAO MD 42

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 43

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.DR.T.V.RAO MD 44 s

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 45

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 appliedDR.T.V.RAO MD 46

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 CareDR.T.V.RAO MD 47

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 programsDR.T.V.RAO MD 48

PLAN 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.DR.T.V.RAO MD 49

REFERENCES• International standards for Tuberculosis CareDR.T.V.RAO MD 50

FOR CURRENT INTEREST ON INFECTIOUS DISEASES FOLLOW ME ON..DR.T.V.RAO MD 51

CREATED FOR MICROBIOLOGISTS AND HEALTH CARE WORKERS IN DEVELOPING WORLD Email doctortvrao@gmail.comDR.T.V.RAO MD 52

Acid fast staining in Tuberculosis

by tumalapalli venkateswara rao

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