ACID FAST STAINING IN TUBERCULOSIS

PRINCIPLES, PRACTICE, AND APPLICATIONS

Dr.T.V.Rao MD

DR.T.V.RAO MD

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

DR.T.V.RAO MD

WHY MICROBIOLOGIC DIAGNOSIS OF TB IS IMPORTANT

Significance of microbiologic testing for public health goals and patient care :

WHO global target of 70% case detection of new smearpositive 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

DR.T.V.RAO MD

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

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

DR.T.V.RAO MD

PRINCIPLES OF ZIEHLNEELSEN STAIN

The ZiehlNeelsen 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 ZiehlNeelsen carbolfuchsin, acid alcohol and methylene blue. Acid-fast bacilli will be bright red after staining.
DR.T.V.RAO MD

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

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

Acid Fast Cell Envelope

Mycolic acid lipids
r r

Peptidoglycan-mycolic acid-arabinogalactan
r r r r r r

r r

Cytoplasmic membrane

Cytoplasm
DR.T.V.RAO MD

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.

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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.
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AFB STAINING METHODS

Zeihl Neelsenshot stain Kinyouns-cold stain

Modifications
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EXAMPLE OF ACID-FAST BACTERIA

Blue=Non acid-fast bacteria
Red= acid fast bacteria

Each person will make a smear and Acid-Fast stain of a mixed broth 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 common
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ACID - FAST STAIN BASIC REQUIREMENTS

1. Carbolfuchsin (Red) 2. Acid Alcohol 3. Counterstain with Methylene Blue Acid - Fast Cells Red

Non Acid - Fast

Blue

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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 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.
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DR.T.V.RAO MD

ZIEHL-NEELSEN STAIN

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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 Loefflers Methylene Blue stain (counter stain). This stain adds blue color to non-acid fast cells!! Leave Loefflers Blue stain on smear for 1 minute
11. Rinse slide. Blot dry. 12. Use oil immersion objective to view.
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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
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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
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HOW THE ACID FAST BACTERIA APPEAR

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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?
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SPECIMEN COLLECTION AND TRANSPORT

Collect specimens in a laboratoryapproved, 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

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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 possible
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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 DR.T.V.RAO MD obtained.

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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

DR.T.V.RAO MD

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

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. chest radiographic findings suggestive of tuberculosis should have sputum specimens submitted for microbiological examination
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Standard 4: All persons with

DR.T.V.RAO MD

HOW THE ACID FAST BACTERIA APPEAR

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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
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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

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ZEIHL NEELSEN AND FLUOROCHROME MICROSCOPY

AFB QUANTIFICATION SCALES

System & Quantification Scale

No. of AFB per field Fluor. & Brightf. & ATS Brightf. & IUATLD/WHO Scale IUATLD/WHO Scale Scale (1000x) (1000x) (200-250x) Negative Actual Actual 1+ 2+ 3+ 3+ Negative Actual 1+ 2+ 3+ 4+ 4+ Negative Actual Actual Actual 1+ 2+ 3+ Fluor. & ATS Scale (200-250x) Negative Actual Actual 1+ 2+ 3+ 4+

None 1-2/300 fields 1-9/100 fields 1-9/10 fields 1-9/1 field 10-99/1field >=100/1field

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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
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FLUOROCHROME AFB MICROSCOPY

* More rapid and sensitive * Specificity : same with sufficient exprience * Equipment cost , bulbs, technical demands * for busy labs * External quality assessment should be done if this method is performed

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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
DR.T.V.RAO MD

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

AURAMINE STAIN

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AURAMINE-RHODAMINE STAIN

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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

DR.T.V.RAO MD

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

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.
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INTERNATIONAL STANDARDS FOR TUBERCULOSIS CARE.

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.

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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 Standards
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PURPOSE OF ISTC

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BETTER GOALS IN MICROBIOLOGIC DIAGNOSIS OF TB

Culture and drugsensitivity testing should be obtained, when feasible, for smearnegative TB and treatment failure. Quality assurance is essential for all TB diagnostic procedures
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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.
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Standard 5: The diagnosis of smear-negative pulmonary TB

SUMMARY: ISTC STANDARDS COVERED*

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.
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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).

SUMMARY: ISTC STANDARDS COVERED*

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.
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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.

SUMMARY: ISTC STANDARDS COVERED*

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
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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

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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 programs
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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 highquality 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.
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DR.T.V.RAO MD

REFERENCES

International standards for Tuberculosis Care

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Email

doctortvrao@gmail.com

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