CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB) – SOP No: CRM-SOP 1
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 2 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
TABLE OF CONTENTS
Acknowledgements 3 Introduction 5 Amendment Procedure 6 Process Flow Chart 7 1. Title 8 2. Purpose 8 3. Introduction 8 4. Scope 10 5. Staff Competency Requirements 10
6. Safety Instructions 10 7. Pre-Examination Procedures 12
7.1 Sample Type 12 7.2 Sample Collection 12 7.3 Sample Transport & Storage 12 7.4 Rejection Critiria 13
7.5 Relevant Clinical Information 13
8. Table of Media, Reagents, Materials & Equipment 14 9. Examination Procedures 15 9.1 Quality Control 15 9.2 Sample Processing 17 9.3 Culture 21
9.4 Identification 21 9.5 Susceptibility Testing 21 9.6 Sample Referral 22 10. Post-Examination Procedures 22
10.1 Interpretation of Results 22
10.2 Reporting 23 10.3 Sample Retention, Storage & Disposal 24 11. Limitations and Pitfalls of the Procedure 25 12. References 25 13. Appendices 27
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 3 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 4 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 5 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
INTRODUCTION
The Caribbean Regional Standard Methods include a variety This initiative should enable the region to implement a of standard, validated methods, produced as a single standardized and constructive method for ensuring that standard operating procedure (SOP) for use in a variety of validated methods are available for the region, and that they levels of microbiology laboratory service. It is intended that are updated as required. these methods provide detailed instructions for microbiology services for microbiological investigations, in order to Advantages of using regionally validated methods are to provide accurate, reliable and reproducible results which improve quality, make better use of resources, reduce costs, will have clinical utility. These methods may be adopted by enable central procurement & media preparation, facilitate laboratories within the region, or adapted, provided that such staff training and transfers due to horizontal integration, a adaptations use an evidence-based validation process. reduction in variability of service provision, an improved quality of surveillance data, and the purchase of appropriate These methods have been developed by the Caribbean equipment. A major advantage is that the availability of Regional Microbiology Standard Methods Drafting Group regional standard methods would assist microbiology (CSMDG) in response to a request by the Caribbean laboratories with documentation for accreditation Regional Microbiology Council (CRMC), which was set up by the CARIFORUM Project entitled ‘Strengthening of Medical Although the CSMDG has taken every care with the Laboratories in the Caribbean’ to strengthen specifically the preparation and issue of these standard procedures, and they microbiology services in the Caribbean Region. The Project have been validated regionally, nationally and internationally, was initiated in response to findings which indicated that the CSMDG, or any other organization, cannot be responsible there was an unacceptable level of error in laboratories within for the accuracy of any statement or representation made the region. External quality assessment results revealed that or the consequences arising from the use of or alteration to microbiology laboratories were not performing well and any information contained in them. These procedures are feedback from the region via laboratory staff, lab managers intended solely as a resource for practicing microbiology and directors was that they felt that guidance in microbiology professionals in the field, operating in the Caribbean region, requirements was required. and specialist advice should be obtained where necessary. If changes are made to the original publication, it must be The background for this initiative is a worldwide move to made clear where changes have been made to the original implement standards in all areas, which has now extended document. When referring to these SOPs in successive to include medical laboratories. As tourism is so vital to documentation, the CSMDG should be acknowledged. the region’s economy, the need for accurate diagnosis and treatment is paramount. It was accepted that there is a requirement for validated methods for accreditation purposes and providing validated standard methods will assist in the move towards accreditation.
The methods will be chosen for standardization by the Caribbean Regional Microbiology Council, and this selection will be based on a review of EQA results, most common and/ or critical tests. Part of the method standardization process will be an ongoing review and amendment procedure. The CSMDG consists of microbiology laboratory representatives from most of the CARIFORUM countries, all of whom were nominated to the task by the CRMC.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 6 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
AMENDMENT PROCEDURE
Controlled Document Reference CRM-SOP 14
Controlled Document Title Standard Operating Procedure for Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
Each Regional Standard Method should be reviewed annually by the Caribbean Standard Methods Drafting Group. Any amendments should be validated and authorized by an agreed process, and referenced.
Each Regional Standard Method has an individual record of amendments. The current amendments are listed on this page.
On issue of revised or new pages, each controlled document should be updated by the copyholder in the laboratory.
Amendment Issue Number Insert Page Section(s) Amendment Number / Date Discarded Issue Number involved
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 7 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
PROCESS FLOW CHART
SPUTUM MICROSCOPY TO DETECT ACID ALCOHOL FAST BACILLI (AAFB)
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 8 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
1. Title
Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB) .
2. Purpose
To ensure the correct validated procedure is followed for processing of sputum to detect AFB by microscopy, providing accurate, reliable and reproducible results having clinical utility.
3. Introduction
Tuberculosis (TB) in humans is caused predominantly by Mycobacterium tuberculosis and less often by other members of the M. tuberculosis complex (MTBC) including M. bovis and M. africanum. Mycobacteria other than tubercle (MOTT) bacilli are increasingly encountered as a cause of disease in humans. Not all those infected with tubercle bacilli develop disease, and not all those that are infected become infectious to others. Overt disease may develop months or years after the initial exposure. Tuberculosis may occur in virtually any organ of the body, but is most prominent in the lungs, where infection is characterised by granuloma formation.
Pulmonary tuberculosis
Initial infection occurs by inhalation of droplet nuclei. From this primary focus the organism may spread via the lymphatics to the lymph nodes and may reach the blood stream, infecting the lungs and other organs. The granulomatous lesions may heal but may contain viable organisms.
Diagnosis of pulmonary tuberculosis is usually based on a combination of chest X-ray findings, a positive AFB smear and/or culture, and a positive tuberculin skin test. Culture of the organism and subsequent susceptibility testing is important as many isolates are multi-dug resistant.
A positive tuberculin skin test usually occurs three to eight weeks after infection and indicates the development of cellular immunity and tissue hypersensitivity.
Non-pulmonary tuberculosis
Lymphadenitis is the most common form. Cervical lymph nodes are the most commonly infected although any lymph node may be involved.
Miliary tuberculosis describes all forms of progressive disseminated haematogenous tuberculosis.
Neuro-tuberculosis – Tuberculous meningitis is usually caused by the rupture of a sub-ependymal tubercle into the sub- arachnoid space rather than by direct haematogenous seeding of the meninges. The clinical findings usually begin with malaise, intermittent headache and low-grade fever, followed within 2 - 3 weeks by protracted headache, vomiting, confusion, meningism and focal neurological signs. Diagnosis of tuberculous meningitis relies heavily on clinical suspicion. Although the typical CSF picture in these cases is usually a raised white blood cell (WBC) count with a predominance of lymphocytes, cells may be absent or polymorphonuclear cells may predominate in some cases. CSF protein is usually elevated.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 9 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
3. Introduction continued
Gastrointestinal tuberculosis – diagnosis of gastrointestinal TB is often made endoscopically. Biopsy tissue from the organ involved yields the highest numbers of organisms for AAFB smear and culture.
Peritoneal TB may occur in two forms: the ascitic form is characterised by the presence of free fluid, the adhesive form resulting in fibrous adhesions and abdominal swelling.
Genitourinary tuberculosis is uncommon before puberty. The interval between infection and development of active renal disease is usually very long (years or even decades). Urinalysis will often show proteinuria, haematuria and sterile pyuria.
Bone, joint and spinal tuberculosis is usually a result of haematogenous spread to the bone from a primary pulmonary infection. Predisposing factors include compromised immunity and intravenous drug use.
Bacteraemia caused by Mycobacterium species is becoming increasingly common as the incidence of Human Immunodeficiency Virus (HIV) infection and Acquired Immune Deficiency Syndrome (AIDS) increases. Up to 63% of HIV positive patients with Mycobacterium tuberculosis infection have positive blood cultures. .
Multi-drug resistant tuberculosis (MDR-TB) In recent years, cases of single drug and multi-drug resistance in TB have been increasingly reported throughout the world. M. tuberculosis becomes drug resistant by spontaneous random mutation. Treatment regimens for MDR-TB tend to be less effective and more prolonged than therapy for drug sensitive TB.
Mycobacteria other than tubercle bacilli (MOTT) Mycobacteria other than tubercle bacilli have been recognised as causing human disease since the 1950s. MOTT are ubiquitous in nature, have a varied spectrum of pathogenicity for humans, are not transmitted person to person and are often resistant to classical anti-tuberculous chemotherapy. More than half of the ninety species are recognised as obligate or opportunistic pathogens of man or animals.
Detection of Acid Alcohol Fast Bacilli (AAFB) on Microscopy
Microscopy can provide a rapid, cost effective means of identifying AAFB in sputa and other samples so that early treatment may be considered. Fluorescent techniques are considered more sensitive then the conventional ZN.
AFB are difficult to stain because of the lipid content of their cell wall. For this reason the Ziehl - Neelsen (or Auramine-phenol) staining method is recommended. It is believed that phenol dissolves the lipid sufficiently to allow penetration of the primary stain - carbol fuchsin (or auramine) and that the heat helps the penetration process. The cell wall retains the primary stain even after exposure to the decolorizing agent, acid alcohol and the organisms are said to be acid and alcohol fast (AAFB = acid and alcohol fast bacilli).
A counter stain is used to stain the background material in a contrasting colour.
The detection of AAFB increases significantly when the sputum sample is first liquefied and decontaminated by either sodium hydroxide or sodium hypochlorite (bleach) followed by centrifugation.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 10 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
3. Introduction continued
Treatment with a 5% solution of sodium hypochlorite or bleach, followed by centrifugation or overnight sedimentation usually kills any AFB present and is therefore, safer for the processing Technologist. However as the organism is destroyed, the sample will not be suitable for culture.
Tuberculosis is re-emerging as an important public health issue, therefore rapid diagnosis is essential for appropriate intervention. Positive AAFB microscopy and relevant clinical symptoms are usually sufficient for initiating antituberculous treatment, pending culture results. Culture still remains the ‘gold standard’ as this permits formal identification and susceptibility testing of the organism.
4. Scope
This procedure provides detailed instructions for sputum microscopy to detect AAFB as offered by regional laboratories. It may be adopted or adapted by any laboratory as needed, provided that such adaptations use an evidence-based validation process.
5. Staff Competency Requirements
Laboratory personnel, trained and assessed to be competent to perform this procedure.
6. Safety Instructions
Please refer to CRM-SOP 20: Safety In The Microbiology Laboratory.
Keep all books, forms and papers away from technical work surfaces.
Level 2 containment; require all personal protective equipment (PPE).
Tuberculin skin testing
Laboratory personnel should be vaccinated against tuberculosis prior to working with suspected tuberculous samples to prevent laboratory acquired infection.
All new personnel should receive a two-step tuberculin skin test by the Mantoux procedure. If the tuberculin skin-test results are positive, a reference chest X-ray should be performed to exclude current infection.
Full Level 3 containment whenever possible. As a minimum requirement all work involving treatment and manipulation of the sputum sample and subsequent cultures should be performed in a biosafety cabinet because of the risk of aerosols.
Biosafety Cabinets should be maintained and decontaminated according to manufacturer’s instructions. Airflow should be monitored regularly to ensure correct functioning.
All samples should be treated as potentially hazardous. Efforts should be made to avoid, minimize or control practices which create potentially infectious aerosols. Common procedures which produce aerosols include opening the sample container,
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 10 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 11 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
6. Safety Instructions continued shaking or vortexing the sample with liquefying/decontaminating reagent, flaming of loops used to culture or make smears from samples. Wherever possible disposable loops should be used.
Bunsen burners should not be sited inside the biosafety cabinet as this causes disturbance of the airflow making it less effective.
Smears should be air dried whilst still inside the biosafety cabinet. Once dry they may be removed and fixed in a bunsen flame outside of the cabinet or placed on a slide heater at 65 – 75°C
Require all personal protective equipment (PPE) ie laboratory coat, gloves.
All microbiological samples for processing should be considered a potential source of transmissible infections so universal safety precautions should be observed.
Hands should be thoroughly washed with soap and water before and after handling all samples.
Disinfect all work surfaces with a suitable disinfectant freshly prepared 10% bleach solution prior to testing and after processing.
All samples and reagents should be properly discarded according to the current standards for disposal of hazardous waste.
Samples and culture plates should be autoclaved before finally discarding.
All health care workers (HCW) and laboratory personnel are responsible for their own safety and that of their co-workers. It is the duty of each supervisor, to provide the correct information and appropriate environment to the laboratory personnel carrying out the work.
Handle all glass slides with care. Mycobacteria remain viable even after heat fixing.
Ensure that all stains, reagents and chemicals are stored in a cool dry place in the Laboratory away from open flames, heat or direct sunlight. Handle with care and avoid direct skin contact. Adhere to advice on hazard labels on commercially obtained stains and reagents.
Sodium hydroxide is caustic. If the solution comes into contact with skin flush well with water.
Acid alcohol is flammable. Do not use near open flames.
Tuberculosis is transmitted in three ways:
Inhalation of infectious droplet nuclei (aerosols), containing tubercle bacilli, which produces a focus of infection in the alveoli of the lungs.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 11 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 12 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
6. Safety Instructions continued
Ingestion of contaminated material via the mouth, usually milk.
Direct inoculation, usually occurring among Health care workers caused by laboratory accidents e.g. through broken culture bottles, tubes and needlestick injury.
7. Pre-Examination Procedures
7.1 Sample Type
Expectorated sputum: should preferably contain purulent or mucopurulent material. However, samples should not be rejected if mucosalivary as these may also give positive microscopy results.
7.2 Sample Collection
For the initial diagnosis of tuberculosis, all samples should be fresh and should be taken, whenever possible, before the start of antituberculous therapy.
The patient should be given an appropriate sterile, wide-mouth, leak-proof container with screw cap lid and told to expectorate the sample into the container. This should be undertaken outside the building wherever possible (to prevent the spread of infection. to the patient should be asked to avoid contaminating the outside of the sample container with sputum when the sample is being collected. The outside of the container should be disinfected using a phenolic disinfectant, after the sample has been collected.
For labs without a certified level II BSC, the patient should be given a plastic wrap to cover the sample and a rubber band to secure plastic wrap after collection.
Patient must close container using the screw-capped lid, after covering and securing the plastic wrap, when used, with a rubber band.
Number of samples required.
Diagnostic: 3 early morning samples.
Follow up / Monitoring: A sample should be submitted 1 month and 5 months after antituberculous therapy started and at completion of therapy.
Ensure that all samples are clearly labeled with the patient’s name, number, date and time of collection, and the type of sample before promptly transporting to the laboratory for testing.
7.3 Sample Transport & Storage
Sample should be transported and processed as soon as possible. The sample should be processed and the result should be available within 24 hours of receipt of sample within the laboratory to correspond with CDC guidelines.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 12 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 13 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
7. Pre-Examination Procedures continued
If processing is delayed, refrigeration is preferable to storage at ambient/ room temperature for up to 48 hours.
Delays of > 48 hours are undesirable.
If sample is to be transported by air, compliance with IATA regulations is essential.
7.4 Rejection Criteria
• unlabelled or incorrectly labeled sample. • sample sent in unsterile or inappropriate container. • leaking/ contaminated samples. • sample not accompanied by properly completed requisition form. • sample > 48 hours old. • pooled samples.
7.5 Relevant Clinical Information
• chronic, productive cough. • night sweats, loss of weight. • travel to known (multi-drug resistant TB) MDR areas. • previous antituberculous therapy. • contact with known positive TB case. • antituberculous therapy, if any.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 13 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 14 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
8. Table of Media, Reagents, Materials and Equipment
Method Equipment Materials/ Reagents
Sample processing Lidded centrifuge with 50ml tube 50 mol plastic, screw capped conical tubes – all methods rotor and sealable buckets; minimum or universal containers + suitable racks Relative Centrifugal Force (RCF) of Disposable plastic pipettes 3000 x g Discard containers Disinfectant Biosafety cabinet Appropriate PPE Vortex mixer Sample processing As for ‘all methods’ above plus: 4.0 % – sodium hydroxide sodium hydroxide solution (NaOH) (modified Petroff’s) Sterile 0.067 M phosphate buffer (pH 6.8) method
Sample processing As for ‘all methods’ above plus: 2% N- – NALC - NaOH Acetyl-L Cysteine (NALC) + equal volume method 0.5N NaOH
Sterile 0.067 M phosphate buffer (pH 6.8)
Sample processing As for all methods above plus: – Bleach method 5% sodium hypochlorite ie household bleach (freshly diluted)
Smear preparation Vortex New, frosted end, glass slides Bunsen burner/spirit lamp Pencil Disposable Pasteur pipettes/loops 70% alcohol (ethanol) 5% phenol alcohol
Staining – Ziehl Staining rack Strong Carbol fuchsin Neelsen method Spirit lamp 1% Methylene blue or1% Malachite Green or 1% Brilliant Green Distilled water 3% (v/v) acid alcohol
Staining Fluorescent microscope Auramine-phenol stain –Fluorescence Staining racks 1% (v/v) acid alcohol Microscopy BSC 0.1% potassium permanganate or (Blair method) thiazine red Distilled Water
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 14 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 15 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
8. Table of Media, Reagents, Materials and Equipment continued
Preparation of Staining Reagents for Fluorescent Microscopy
Blair Method10 Dissolve 0.1g of Carefully add 0.5 ml of Dissolve 0.5g of Auramine O in 10ml of conc. HCL to 100ml of potassium permanganate 95% ethanol. Dissolve 70% ethanol in 100ml of distilled water 3.0g of phenol crystals in 87ml of distilled water. Mix Auramine and phenol solutions
9. Examination Procedures
9.1 Quality Control
Please refer to CRM-SOP 18: Media Preparation and Quality Control.
Please refer to CRM-SOP 21: Quality Control of Reagents and Tests.
Please refer to CRM-SOP 19: Propagation and Maintenance of Quality Control Organsims.
9.1.1 Purpose
• Positive and negative control smears should be included with each batch of staining or when a new batch of stain is being utilized. Ensure that stains are properly stored in a cool area away from heat and direct sunlight. • Check all stains just before use to ensure that they are not expired or do not show any signs of deterioration, contamination or precipitation. • Results of all QC tests should be recorded on the appropriate forms available in the department for that purpose. • Test results are acceptable for reporting only if the QC results are as expected. • Ensure that reagents are not used beyond the expiration dates. • Ensure that any water used to make up stains and reagents or to rinse slides during the staining process has been validated as free from environmental mycobacteria. If necessary filtered water should be used. • Refer to SOPs for stains and biochemical tests for further details on quality control.
9.1.2 Preparation of positive and negative control smears
Control smears for acid fast stains are commercially available. However in-house smears can be prepared as follows:
a. A previously confirmed positive patient sample demonstrating a light to moderate number of AAFB can be used to prepare positive control smears.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 15 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 16 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
b. Negative control smears can be prepared from a suspension of Escherichia coli equal to No.1 McFarland turbidity standard. c. Prepare smears, air dry and fix as described in 9.2.4 d. Label control smears as “AFB POS (or NEG) CONTROL” and date of preparation, source and preparer’s name. e. Test ‘new’ control smears against ‘old’ controls and document results before initiating use. f. Store in labeled slide boxes, preferably in a biosafety cabinet. Note: mycobacteria will still be viable on the positive slides. g. Run a positive and negative control smear with each batch of staining.
Control smears should be examined before patient smears to ensure validity of staining procedure.
9.1.3 Interpretation of QC results
ZN Stain
Positive Control - red or magenta bacilli against a blue or green background
Negative Control - blue or green bacilli
Auramine stain
Positive Control - yellow to orange fluorescence
Negative Control - no fluorescence
Record the results of the control smears: • If acceptable results, proceed with reading patient smears. • If unacceptable results, review procedures and reagent preparations before reading patient smears.
Unacceptable control slides include the following:
ZN Stain • Positive control demonstrates weakly staining or non-staining bacilli. Negative control remains red after decolorization • Stain deposit on either positive or negative control indicates poor filtration prior to use
Auramine stain • Background is not properly decolorized or demonstrates generalized weak fluorescence. Positive control does not demonstrate well defined, fluorescent bacilli. • Negative control demonstrates fluorescent bacilli. This may indicate a contaminated water supply with environmental mycobacterium.
After identifying and resolving the cause of any QC failure, repeat control and patient smears should be made and stained. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 16 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 17 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
9.2 Sample processing
Refer to table above for the appropriate materials and equipment for each section.
It is important to ensure that the rinsing water and the water that is used to process the samples or to make up the stain is not contaminated with environmental acid alcohol fast bacteria. This is frequently found in tap water and with the use of rubber tubing and may lead to false positive results. Filtered water should be used if necessary.
Process the sputum samples by one of the following methods, designed to homogenize, decontaminate and concentrate the sample:
9.2.1 Bleach Method (Appendix 1)
a. Carefully open the screw capped lid of sample container in a biosafety cabinet, while keeping the plastic wrap in place if used. b. Using a plastic pipette, add an equal volume of 5% household bleach through the plastic sheet, into the sputum sample c. Recap sample container tightly and shake briefly. d. Allow to sit at room temperature for 10-15 minutes (This time is sufficient for the bleach to destroy all mycobacterium in the sample, while still retaining the characteristic morphology). e. Do not leave the sample in contact with the bleach for longer than 2 hours as lysis of the cells may occur and result in false negative results. f. Transfer the sample/bleach mixture into a sterile 50mL, screw-capped centrifuge tube and fill up to the 50 mL mark with sterile normal saline or sterile distilled water. g. Centrifuge at 3000 x g for 15 minutes h. Decant supernatant leaving approximately 0.5ml of the fluid in the tube. i. Prepare at least 2 smears as in 9.2.4.
NOTE: This method renders the sample unsuitable for culture as the mycobacteria have been rendered non-viable.
9.2.2 NALC – Sodium Hydroxide Method (Appendix 2)
All work should be carried out in a biosafety cabinet, observing universal safety precautions.
a. Only one sample should be opened at a time to prevent cross contamination. b. Using a new plastic pipette for each patient sample, add a volume of the working NALC-NaOH solution (no more than 24 h old) equal to the volume of the sample, in a sterile 50 mL screw-cap centrifuge tube. If there is more than 5mL of sample this should be divided before the addition of the NALC-NaOH. c. Agitate the tubes on a vortex mixer for not more than 20 s. Invert the tubes to ensure that NALC-NaOH comes into contact with the entire inner surface of the tube. Note: Avoid excessive agitation, as NALC is unstable in the presence of oxygen. d. Let the tubes stand for 25 minutes at room temperature to homogenise and decontaminate the sample. e. Aliquot approximately 35ml of 0.067 M phosphate buffer (pH 6.8) from the stock container into sterile plastic containers or tubes. STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 17 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 18 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
f. Dilute each homogenised sample up to approximately 45 mL with one of the aliquots of the sterile buffer. This will reduce the continued action of the NaOH and lower the viscosity of the mixture. In order to avoid cross-contamination, use a single buffer aliquot per sample and ensure there is only one tube open at a time. g. Recap each 50mL tube tightly and invert several times to mix the contents. h. Centrifuge the tubes at 3000 x g for 15 minutes in a refrigerated centrifuge at 4°C in sealed centrifuge buckets. Centrifuge buckets should be loaded and unloaded in the biosafety cabinet and transported on a cart. i. Inside the BSC, open one tube at a time and decant the supernatant carefully into a splash-proof discard container containing concentrated disinfectant (concentrated bleach). A funnel lined with bleach soaked filter paper is placed in the top of the discard container to avoid splashing. The lip of the container may be blotted with a piece of blotting or filter paper to prevent any liquid running back and contaminating the outside of the container. Discard any blotting paper into the discard container. j. Supernatant is added until the final concentration of disinfectant in the discard container is 1% (pre-mark the container with levels). k. Vortex the sediment that is left in the tubes. Open one tube at a time. Use a sterile plastic pipette to remove a portion of the sediment remaining in the tube and prepare two smears on new, cleaned, unscratched glass slides. l. Please note persons using pre-prepared decontamination reagents should follow manufacturer’s instructions, and then make 2 smears.
9.2.3 Modified Petroff’s Method (Appendix 3)
a. Add an equal volume of 4% NaOH to the sputum sample and leave at room temperature for 25 minutes. Alternatively incubate at 35 – 37°C for 15 minutes. b. Vortex at regular intervals to homogenize. c. Centrifuge at 3000 x g for 15 min in a lidded centrifuge (e.g. Megafuge/Labofuge), preferably with sealed buckets. d. Open the sealed buckets in the biosafety cabinet in case of breakage of the container and subsequent aerosol formation. e. Decant the supernatant into a discard container, making sure that the outside of the container does not become contaminated. f. Neutralize the deposit with about 20 ml of sterile distilled water. g. Centrifuge again at 3000 x g for 15 min, decant supernatant as above and resuspend the deposit in any remaining liquid. h. Make 2 smears from the deposit as in 9.2.4
9.2.4 Smear Preparation (Appendix 5)
a. Clean slides with 70% alcohol. b. Label frosted end slides with patient I.D. using a pencil. c. Vigorously mix or vortex sample in a biosafety cabinet.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 18 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 19 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
d. Using a new disposable loop or sterile plastic pipette for each sample transfer a portion of the deposit onto the slide. e. Smear the material evenly over an area of 1 X 2 cm on the slide. Smears that are too thick or too thin are not acceptable. f. Discard the loop or Pasteur pipette in a disinfectant or biohazard container. g. Allow smears to air dry completely within the cabinet. Do not heat fix moist slides in a bunsen flame. h. Fix dried smears by placing on a slide warmer (2 hours) or by passing right side up through a bunsen flame (3-4 times). Do not overheat.
Smears may also be prepared from positive mycobacterial cultures and examined using the ZN stain as follows:
• Using a disposable loop, some of the growth from a Lowenstein – Jensen (LJ) slope may be taken and emulsified in a drop of phenol alcohol or distilled water. Continue e – h as above.
NOTE: 5% phenol ethanol may also be used for fixing smears. Flood slide with 5% phenol ethanol and leave for 15 minutes or overnight.
It may be prepared as follows:
56 ml. 90% phenol ( liquid phenol) 737 ml 95% ethanol 07 ml dist. water
a. Mix solution well and dispense to a small, labeled glass bottle. b. Label the reagent appropriately, including hazard labels and indicate the date of preparation.
Store inside the biosafety cabinet.
9.2.4 Ziehl Neelsen Stain (Appendix 4)
a. Arrange fixed slides on a staining rack. Ensure that the slides do not touch each other to prevent cross contamination. Do not use coplin jars for staining. b. Apply enough strong carbol fuchsin to cover the slides. c. Heat slides to steaming with an alcohol soaked cotton swab. Ensure there is a continual stream of water running into the sink to prevent accidental ignition of excess alcohol. Keep slides covered with hot, steaming carbol fuchsin for 5 minutes. Reflame as needed. Do not allow stain to dry on the slide; add more stain if needed. d. Wash slides gently with clean water to remove excess carbol fuchsin. e. Flood slides with 3% acid-alcohol to decolorize for approximately 3 minutes or until no further carbol fuchsin leaks from the smear. Wash slides with water. f. Flood slides with 1% methylene blue or malachite green to counter stain. Rinse with water, drain and dry. DO NOT BLOT.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 19 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 20 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
g. Examine the dried, stained smear with an oil immersion objective and transmitted light microscope. (total magnification: X100), After examining a positive smear, the objective should be cleaned before reading the next smear.
9.2.5 Fluorescent Stain: Blair Method (Appendix 6)
a. Arrange fixed slides on a staining rack. Ensure that the slides do not touch each other to prevent cross contamination. Do not use coplin jars for staining. b. Flood smear with Auramine O (1:10 v/v) solution and allow to stain for 15 minutes. Be sure that the staining solution remains on the smear. Do not apply heat to smear. Rinse smear with chlorine free water and drain. Chlorine may interfere with fluorescence, therefore, rinse with distilled or de-ionized water. c. Flood smear with 1% acid alcohol and decolourize for 2 minutes. d. Rinse again with water. e. Flood with 0.1% potassium permanganate or thiazine red and counter stain for 2 minutes.
NB. Time is critical with potassium permanganate because counter staining for a longer time may quench fluorescence of acid-fast bacilli.
9.2.6 Smear Examination
Before starting the examination, the Technologist must ensure that all elements of the microscope are correctly set. He/she should, in particular, check that the source of light is well regulated and focused; that the condenser is in the upper position with the diaphragm open and that the immersion objective lens and the eyepieces are clean.
9.2.9 Smear Reading: Ziehl Neelsen (Appendix7)
AFB positive smears from a new patient may be confirmed and co-signed by a second technologist ifin doubt.
A ZN stained smear should be examined with a medium power oil immersion objective for 10-15 minutes. A high power oil immersion objective can be used to confirm typical morphology.
• Examine the smear with the X10 objective to see the distribution of material. • Put a drop of immersion oil on the left edge of the stained smear. Care should be taken not to touch the smear with the end of the oil dispenser or the oil immersion objective lens because this could transfer AFB from one preparation to another. • Examine the whole smear using immersion oil and following a systematic reading pattern. eg. start reading the slide in the top left hand corner then scan laterally to the right top hand corner. • Move the stage up slightly and examine the next level down from right to left. • Repeat scanning as above until the whole smear has been examined from top to bottom. • Examine at least 100 fields.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 20 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 21 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
• Appearance: The arrangement of cells varies from “serpentine cords” to a uniform distribution of cells. Cellular morphology is variable with cells ranging in length from 1-10um and in width from 0.5-1.5um. Some Mycobacterium species appear almost coccoid and others fusiform. Staining may be regular or show granules or beading. • Tubercle bacilli stain as slightly curved, slender, red, bacilli, isolated in pairs or in groups, standing out clearly against the blue/green background. • Note any large numbers of clumps on the report, thus indicating that the actual number of AFB is larger than actually reported. • It is not possible to speciate mycobacteria using microscopy; report only the number of AAFB seen. • Several systems are used for reporting the results of acid fast microscopy. The suggested method is shown in the table in 10.2.1 • Counts of less than 3 AFB per 100 fields are not considered as positive. A second smear from the same sample may be stained and examined or another sample should be requested. • Examine the slides in sequence. No single technologist should examine more than 20 slides in a day. • Before examining the next slide, wipe the immersion oil objective lens with a piece of lens paper. • Store all slides. Slides should be stored sequentially for ease of retrieval and / or QC purposes.
9.2.10 Smear Reading: Fluorescence Microscopy
• Fluorochrome (FL) stained smears should be examined with a 20x or 40x dry objective. • Fluorochrome staining is more sensitive than ZN, but is less specific as it is more prone to false positivity due to fluorescing artefacts such as blood cells or yeast. If necessary fluorescence microscopy positive smears may be restained by ZN stain to confirm the presence of AAFB.
Appearance: AFB stained by a fluorescent stain will appear as bright yellow or orange, fluorescing bacilli against a dark background.
9.3 Culture
N/A.
9.4 Identification
See sections 9.2.9 and 9.2.10: Smear Reading.
9.5 Susceptibility Testing
N/A.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 21 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 22 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
9. Examination Procedures continued
9.6 Sample Referral
Applies to bleach treated samples only.
Samples with positive slides: request new samples for referral to the appropriate Reference Laboratory for confirmation of microscopy and mycobacterial culture.
If sample is to be transported by air, compliance with IATA regulations is essential.
All referrals must be accompanied by appropriately completed laboratory requisition forms.
Reference labs must be notified prior to sending samples, especially if an urgent sample is included in the shipment.
10. Post-Examination Procedures
10.1 Interpretation of Results
See 10.2
10.1.1 Smear Validation
Smear validation is a process of re-reading slides from a laboratory to assess performance.
The smears should constitute a statistically representative and random sample based on test volume in the laboratory and the expected performance that must be defined for each laboratory.
Sample size depends on the positivity rate, total number of negative slides processed each year and the expected performance (sensitivity).
Laboratories may calculate the number of slides to be referred as follows:
a. List the number of slides done per year (e.g. 363). b. List the number of positive slides per year (e.g. 23). c. List the number of negative slides per year (e.g. 363-23=340). d. Calculate the Slide positivity Rate (SPR) as follows: SPR= # of positive slides per year (23) X 100 = 6% (rounded up). Annual slide volume (363). e. Decide on acceptable limits of performance = 80%. f. Select appropriate sample size table (Table 1 below is for 80% sensitivity). g. Choose number of negative slides per year – first column. (340; however closest option is 200). h. Identify SPR from top of table (6%; closest option is 5%).
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 22 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 23 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
10. Post-Examination Procedures continued
i. Locate corresponding sample size. (eg. if the number of negatives is 200 and SPR is 5%, sample size is 108 slides per year. j. Decide on a convenient interval; such as 4 times/year. Therefore 108/4 = 27 slides to be referred every three months. Table 110
No of Negative Positivity Rate slides per year
5% 10% 15% 20% 25% 30% 200 108 72 54 42 35 30 500 155 88 61 46 37 31 1000 184 96 65 47 37 31 5000 214 104 68 48 37 31 50000 222 105 68 48 37 31
10.2 Slide Retention.
Slides should be stored consecutively.
If 91 slides have been processed during the last quarter and 27 slides need to be sent for validation, then a sampling interval has to be determined. The sampling interval is determined by dividing 91 by 27 = 3.4
Therefore collect every 3rd slide. If a slide is missing, select the next slide in the register irrespective of the result and continue systematically, using the sampling interval.
10.2 Reporting
Methods for reporting the number of AAFB observed when scanning at 100X and 1000X magnification.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 23 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 24 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
10. Post-Examination Procedures continued
10.2.1 Ziehl-Neelsen.
Number of AFB seen Recording/reporting
No of AFB per 100 immersion fields No acid-fast bacilli observed (no AFB per 100 fields) 1-9 AFB per 100 immersion fields Record exact figure (1-9 AFB per 100 fields) 10 – 99 per 100 immersion fields + or 1+ (10 -99 AFB per 100 fields) 1-10 AFB per field ++ or 2+ (1-10 AFB per field in 50 fields) More than 10 AFB per field +++ or 3+ (more than 10 AFB per field in 20 fields)
10.2.2 Fluorescence Method
Number of AFB seen Report 250X magnification 450X magnification
0 0 No AFB seen
1-2 AFB per 30 1-2 AFB per 70 F Doubtful: repeat test and request microscopic fields (F) further sample
1-9 AFB per 10 F 2-18 AFB per 50 F 1+
1-9 AFB per F 4-36 AFB per 10 F 2+
10 – 90 AFB per F 4-36 AFB per F 3+
> 90 AFB per F > 36 AFB per F 4+
10.3 Sample Retention, Storage & Disposal
Storage. Store the treated concentrate in fridge until smear stained and read. The whole of the original sample should have been used for processing.
Samples and all materials used should be placed in a red biohazard bag and autoclaved before final discarding according to the current standards for disposal of hazardous waste. Waste may also be sent for incineration.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 24 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 25 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
11. Limitations and Pitfalls of the Procedure
• Failure to follow staining procedure. • Presence of stain deposit making smear interpretation difficult, particularly with ZN. Stains should be filtered before use. • Environmental mycobacteria present in water used to make up reagents or washing slides during staining procedure – gives rise to false positives (including the negative control slide. • Cross contamination of negative samples with a positive sample from the same batch eg touching side of container when adding reagents during homogenization /decontamination or immersion oil dropper touching positive slide. • Blot drying- using bibulous paper. • Incomplete/improper examination of smear. • Incorrect interpretation. • Red/green colour blindness (ZN stain only). • A positive staining reaction provides only presumptive evidence of the presence of M. tuberculosis in the sample. • A negative staining reaction does not necessarily indicate that the culture will be negative. ZN staining is less sensitive than the fluorescent method. • For positive identification and susceptibility testing of M. tuberculosis, cultural methods must be employed.
12. References
1. CAREC SOP: AFB Smear Microscopy by bleach method in the Caribbean. 2. ‘Sample procedure for Processing and reporting AFB Smears’ Appendix A. 3. Kubica, G. P., W. E. Dye, M. L. Cohn, and G. Middlebrook. Sputum digestion and decontamination with N-acetyl-L- cysteine-sodium hydroxide for culture of mycobacteria. Am. Rev. Respir. Dis 1963;87:775-779. 4. Ratnam, S., and S. B. March. Effect of relative centrifugal force and centrifugation time on sedimentation of mycobacteria in clinical samples. J. Clin Microbiol 1986;23:582-585. 5. Roberts, G. D., E. W. Koneman, and Y. K. Kim. Mycobcterium, p. In: A. Balows. W.J. Hausler, Jr., K. L. Herrmann, H. D. Isenberg, and H. J. Shadomy (ed.), Manual of Clinical Microbiology, 5th ed. American Society for Microbiology, Washington, D. C. 1991. p301 - 339. 6. Kent P. T., and G. P. Kubica,. Public Health Myocbacteriology. A guide for the Level III Laboratory. U.S. Public Health Service Publication 1985. No. 86 - 21654. Center for Disease Control, Atlanta. 7. Section 3.4.4 In: Isenberg HD, editor. Clinical Microbiology Procedures Handbook. Vol 1. Washington D.C.: American Society for Microbiology. 8. N. Selvakumar, Fathima Rahman, Renu Garg, S. Rajasekaran, Nalini Sunder Mohan, K. Thyagarajan, V. Sundaram, T. Santha, Thomas R. Frieden, and P. R. Narayanan .2002. Evaluation of the Phenol Ammonium Sulfate Sedimentation Smear Microscopy Method for Diagnosis of Pulmonary Tuberculosis. Journal of Clinical Microbiology 40(8): 3017–3020. 9. Fluorescent – Auramine Rhodamine Staining retrieved on 8/10/06 from http://www2.provlab.ab.ca/bugs/mycob/afb_stain/ stainmeth.htm
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 25 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 26 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
12. References continued
10. B R Bird and B M Madison. Use of Fluorochrome Staining for Detecting Acid fast bacteria. In: Current laboratory Practice Series (CLPS).CDC. 2000. http://www.phppo.cdc.gov/dls/lta/TB_Fluorochrome/fluorochrome.pdf 11. Ängeby K.A.K, Hoffner S.E. and Diwan V.K. Should the ‘bleach microscopy method’ be recommended for improved case detection of tuberculosis? Literature review and key person analysis. The International Journal of Tuberculosis and Lung Disease 2004; 8: No. 7, p806-815 12. Health Protection Agency (2006). Investigation of samples for Mycobacterium species. National Standard Method CRM- SOP 40 Issue 5.
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 26 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 27 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 1 Procedure Flow Chart for Sputum Concentration: Bleach Method
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 27 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 28 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 2 Procedure Flow Chart for Sputum Concentration: NALC - Sodium Hydroxide Method
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 28 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 29 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 3 Procedure Flow Chart for Sputum Concentration: Petroff Method
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 29 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 30 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 4 Ziehl Neelsen Staining Technique
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 30 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 31 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 5 Direct Smear Preparation
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 31 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 32 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 6 Flourescent Stain: Blair Method
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 32 Sputum Microscopy to Detect Acid Alcohol Fast Bacilli (AAFB)
CARIBBEAN REGIONAL MICROBIOLOGY STANDARD OPERATING PROCEDURE
Title: Sputum Microscopy to Detect SOP No: CRM-SOP: 1 Acid Alcohol Fast Bacilli (AAFB) Version: 1 Page No: 33 of 33
Prepared By: Caribbean Regional Standard Effective Date: 1st September 2007 Methods Drafting Group Review Date: 1st September 2008
APPENDIX 7 Smear Reading: Ziehl Neelsen
STRENGTHENING OF MEDICAL LABORATORY SERVICES IN THE CARIBBEAN A CARIFORUM Project Funded by the European Union and Implemented by CAREC 33