Dr.T.V.Rao MD

ACID FAST STAINING IN

TUBERCULOSIS PRINCIPLES, PRACTICE, AND APPLICATIONS

DR.T.V.RAO MD 1

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 testing

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 treatment

• 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 4

• 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 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 background

DR.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.

8 DR.T.V.RAO MD

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 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

• Modifications

12 DR.T.V.RAO MD

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 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

DR.T.V.RAO MD 14

• 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 15

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% H2 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

ZIEHL-NEELSEN

STAIN

4 5 6

7

1 2 3

17 DR.T.V.RAO MD

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 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 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 countries

DR.T.V.RAO MD 20

HOW THE ACID FAST BACTERIA APPEAR

21 DR.T.V.RAO MD

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

• 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 23

• 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 24

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 25

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 26

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 27

HOW THE ACID FAST BACTERIA APPEAR

28 DR.T.V.RAO MD

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 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

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 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 background

DR.T.V.RAO MD 32

* 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 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 areas

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

AURAMINE STAIN

DR.T.V.RAO MD 35

AURAMINE-RHODAMINE STAIN

DR.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 areas

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

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 38

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

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 Standards

PURPOSE OF ISTC

DR.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 procedures

DR.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*

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 44

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 applied

DR.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 Care DR.T.V.RAO MD 47

• 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 48

• 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 49

• International

standards for

Tuberculosis

Care

REFERENCES

DR.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.com

DR.T.V.RAO MD 52