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Sputum DigestionDecontamination for Mycobacteriology Culture

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Sputum DigestionDecontamination for Mycobacteriology Culture

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Sputum Digestion/Decontamination for Mycobacteriology Culture- Guidelines

Author: Peggy Coulter/ Patricia Document Number: Pro67-C-15-G

    Charache, MD; Effective (or Post) Date: 10 Feb 09

    Review History Date of last review: 11 May 2010

    Reviewed by: Heidi Hanes

    SMILE Comments: This document is provided as an example only. It must be revised to accurately reflect your lab’s specific processes and/or specific protocol requirements. Users are directed to countercheck facts when considering their use in other applications. If you have any questions contact SMILE.

Background Information:

    Specimens submitted for Mycobacteriology culture are most frequently obtained from body sites where other pathogens and/or normal flora reside. Because mycobacteria are slow growing and require long incubation times these contaminating organisms can overgrow in cultures, blocking the ability to detect the presence of the mycobacteria. Digestion and decontamination procedures are used in processing sputum for examination and culture of sputum specimens. This step performs two major functions: 1) sputum is liquefied (digestion), permitting the mycobacteria to be released from the thick sputum so that they can be subsequently concentrated by centrifugation, and 2) contaminating normal flora is preferentially killed (decontamination). Unfortunately, it must also be recognized that the strong alkaline reagent usually used for the decontamination step is also toxic to mycobacteria. The degree to which the mycobacteria in the specimen are killed is a function of the concentration of the alkali used, the length of time over which the organisms are exposed, and the temperature at which the exposure occurs. Appropriate digestion and decontamination procedures, culture media, and conditions of incubation must be selected to facilitate optimal recovery of the mycobacteria. These considerations are especially important for paucibacillary disease, i.e. sputum from patients who present with non-cavitary disease, who excrete only small numbers of organisms in their sputum. These populations include patient with early infections, and those who are HIV-infected. The success of digestion/decontamination depends upon the following considerations:

    ; The concentration of organisms present in a given specimen;

    ; the resistance of tubercle bacilli to the concentration of the strongly alkaline or acidic

    digesting solutions used;

    ; the length of time the mycobacteria are exposed to the digestant;

    ; the temperature of the room, in which the exposure is carried out

    ; the amount of heat generated by the specimen centrifugation step; and

    ; the efficiency of the centrifuge used in the sedimentation step.

    Several methods are available for digestion and decontamination of sputum specimens for culturing of Mycobacteria, but not all are acceptable for all specimens or for all systems used to culture for mycobacteria subsequently. The choice must be based upon technical capability, and upon the quality and type of equipment, supplies and reagents available. Optimally for quality control reasons, only a single method should be used in a given laboratory. Whichever method is used, meticulous care must be taken to prevent laboratory cross-contamination of specimens SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    during processing. Risk of contamination occurs especially when digestant and/or buffer are added subsequently to a batch of tubes. A single positive culture for M. tuberculosis can lead to diagnosis of tuberculosis, and a false-positive culture affects not only clinical management of the patient, but also epidemiological investigations and public health controls. Major Liquefaction/Decontamination Processing Methods:

    N-Acetyl-L-Cysteine-Sodium Hydroxide (NALC-NaOH) Method

    NALC (a mucolytic agent) combined with sodium hydroxide is the preferred method for the digestion step because it is the least toxic to the mycobacteria, and therefore provides the highest yield of positives. NALC perform the liquefaction step, and permits the use of a lower, (1%) concentration of NaOH than that required when NALC is omitted. Sodium citrate is also included to bind heavy metal ions, which, if present in the specimen, can inactivate the acetyl-cysteine. The digestant must be made fresh daily due to the rapid loss of acetyl-cysteine activity when the compound is in solution.

A variety of specimen types may be processed using this method, making it particularly useful

    for processing specimens from patients with few organisms, as seen in non cavitary disease which includes patients with concurrent HIV infections.

NaOH Method

    Higher concentrations of sodium hydroxide can both liquefy sputum, and concurrently kill the contaminating normal flora however, it is more toxic to mycobacteria. Sodium hydroxide should be used at the lowest concentration that will effectively digest and decontaminate the specimen. If excessive contamination occurs with the use of 2% NaOH, increase the concentration to 3 or 4% rather than increasing the time of exposure. Harsh decontamination can kill 20 to 90% of the mycobacteria in a clinical specimen.

Resources

    1. Cernoch, P.L. et al. (1994). Cumitech 16A: Laboratory diagnosis of the Mycobacterioses.

    ASM Press. Washington, DC.

    2. Forbes, B.A., et al (2007). Laboratory detection and identification of mycobacteria; Proposed

    guideline. CLSI document M48-P. Clinical and Laboratory Standards Institute, Wayne, PA. 3. Kent , P.T. and Kubica, G. P. (1985). Public Health Mycobacteriology. A Guide for the

    Level III Laboratory. U.S. Dept. Health and Human Services. Center for Disease Control. 4. SA Healthinfo, Tuberculosis Part III: Culture homogenization and decontamination.

    Obtained from the World Wide Web on 14 March 2007 at http://www.sahealthinfo.org/tb

    5. Weitzman, I. (2007) p 7.1.2.1-7.1.2.9. In H.D. Isenberg (ed.) Clinical Microbiology

    Procedures Handbook American Society for Microbiology, Washington, D.C.

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

     Sputum Digestion/Decontamination for Mycobacteriology Culture - SOP

    Peggy Coulter MDE, MT (HEW) Document Effective

    International QA/QC Coordinator Number Date Author(s), Name &

    Title Patricia Charache, MD; Pro6.7--15 30 Sep 08

    Consultant

    Approved By Name, Title Signature Date

    SOP Annual Name, Title Signature Date Review

     Version # [0.0] Revision Date Description (notes)

    Revision [dd/mm/yy]

    History

    Distributed Name (or location) # of copies Name (or location) # of copies Copies to

Associated Forms:

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Purpose

    There are several approved methods for the digestion-decontamination procedure. The two most useful for processing sputum are outlined below. Both can be used for liquid culture media as well as solid media, while most other methods kill the mycobacteria when used for liquid systems.

Pre-analytic Procedure

    Refer to the following procedures:

    1. Mycobacteriology Laboratory Specimen Collection/Receiving

    2. Safety in the BSL-3 Laboratory

    3. Use of the Bio-Safety Hood in the Mycobacteriology Laboratory

    4. Mycobacteriology Culture

Abbreviations Used:

    ; AFB = Acid Fast Bacilli

    ; BSC = Bio-Safety Cabinet

    ; SOP = Standard Operating Procedure

Analytic Procedure

    Specimen Information:

    Specimen collection- Refer to Mycobacteriology Laboratory Specimen Collection/Receiving

    SOP.

    Specimen types

    ; Specimens that require decontamination include sputum, bronchial secretions, washings, or

    biopsies, skin, soft tissue, gastric lavage, stool specimens, urines and all other specimens

    from sites contaminated with normal microbial flora.

    ; Sterile sites not requiring decontamination include CSF, bone marrow, blood, pleural fluid,

    and sterile biopsy sites. These should be processed following specimen handling in the

    Mycobacteriology Culture SOP.

    ; Specimens should be collected prior to the initiation of antimicrobial therapy. ; When a specimen is determined to be unacceptable, a repeat specimen must be requested.

    o Unacceptable specimens include unlabeled or inadequately identified specimens, dry

    swabs, and those received in previously used containers, containers that are non-

    sterile or cannot be tightly sealed.

    o Specimens held unrefrigerated prior to processing for greater than one hour may lead

    to bacterial overgrowth at levels that prevent detection of mycobacteria

    o Sputum specimens collected at different times must never be pooled as this greatly

    increases the contamination rates.

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Specimen storage

    ; Specimens should be delivered to the laboratory as soon as possible to avoid overgrowth by

    contaminants and normal respiratory flora.

    ; Specimens not processed within one hour of collection must be refrigerated at 2 8? C.

    ; If gastric lavage specimens are to take longer than 4 hours prior to processing, 100 mg. of

    sodium carbonate must be added to the container to neutralize the high acidity of the

    specimen.

Reagents/Media:

    Reagents used for digestion/decontamination are dependent on precise adherence to the required procedures. Directions for preparing reagents are included in Appendix A.

Required Supplies:

    1. 50 ml conical centrifuge tubes

    2. Vortex mixer

    3. Mechanical mixer (optional)

    4. Centrifuge- capable of speed 3,0003,500 RCF (relative centrifugal force), fixed angle rotor

    with aerosol-free safety centrifuge cups

    5. Funnel and waste container filled 1/3 full with an approved disinfectant solution 6. Disposable sterile pipettes

    7. Slide warmer set between 65 and 75?C

    8. Glass microscope slides (a frosted end for labeling with a pencil is useful)

Quality Control:

    Monitor the percentage of contaminated specimens for quality assurance. If too low, mycobacterial recoveries are compromised due to excessive toxicity; if too high, procurement and processing practices require review.

    Process four to six sputum samples following the digestion/decontamination steps and use the pellet to inoculate a blood agar plate, as well the mycobacteriology media normally used. ; Check the blood agar plate after 24-48 hour incubation to observe bacterial contamination.

    Only few or no contaminants should be present.

    ; The acceptable range of specimen contamination is 3 5%.

    ; A contamination rate of less than 3% suggests overly harsh decontamination. ; A contamination rate of greater than 5% growth suggests inadequate decontamination,

    incomplete digestion, reagent and/or media contamination or environmental contamination.

Note: Fresh specimens will have a lower contamination rate whereas contamination rates of 5

    10% will be reached when specimens take several days and/or not kept refrigerated during transportation.

    Record Quality Control growth results on a monthly basis.

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Test Method Instructions:

    1. Refer to Safety in the BSL-3 Laboratory before performing these procedure steps.

    2. Prepare fresh working decongestant/decontamination solution described in Appendix A. 3. Prepare the BSC for use following the Use of the Bio-Safety Hood in the Mycobacteriology

    Laboratory SOP. All specimen processing steps except centrifugation must be performed

    inside the BSC.

    4. If the specimen was not collected in a sterile, labeled 50 ml disposable centrifuge tube,

    transfer aliquots of the specimen to a labeled 50 ml conical tube. No more than 10 ml of

    specimen may be processed per conical tube. The remaining sample should be transferred to

    second tube and processed. Repeat for all patient specimens.

    5. Label each tube with patient name, date and identification number.

    NALC-NaOH Method:

    6. To the first specimen add an equal amount of fresh working NACL-NaOH solution to the

    tube and vortex the mixture for 10-15 seconds. Start the timer for 15 minutes after adding the

    solution to the first tube in the batch of 6-8 tubes. Repeat this step for each specimen in the

    batch.

    7. Allow the specimens to stand the entire15 minutes or shake vigorously on a mechanical

    shaker.

    8. During the incubation time check each specimen by slightly tilting the tube and observing for

    liquefaction.

    9. After the digestant has remained in the first tube for 15 minutes, begin with the first

    specimen and fill the first tube to the 50 ml mark with phosphate buffer by slowly pouring

    the buffer down the side of the tube avoiding splashing or contamination. Tighten the cap and

    wipe the outside of each tube with the disinfectant soaked towel, then invert the tube several

    times to mix thoroughly. Repeat this step on each of the remaining specimens in the batch. 10. Centrifuge tubes for 15 minutes at 3,000-3,500 RCF.

    11. Pour off the supernatant through a funnel into a waste container filled 1/3 full with approved

    disinfectant solution. (Pour slowly so as not to disturb the pellet and be sure to not touch the

    funnel while pouring. Touching the funnel can allow for cross contamination among

    specimens.) Wipe the lip of the conical tube with a disinfectant soaked towel and then recap. 12. Using a sterile disposable transfer pipette, resuspend the pellet with 1-2 mL of phosphate

    buffer.

    13. Inoculate the media with the suspension following the Mycobacteriology Culture SOP.

    14. With the same pipette, place a drop of the suspension onto a clean, labeled glass microscope

    slide. Prepare a smear over an area of 1 by 2 cm of the slide.

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Alternate Sodium Hydroxide Method:

    6. To the first specimen add an equal amount 2% NaOH solution to the tube and vortex the

    mixture for 10-15 seconds. Start the timer for 15 minutes after adding the solution to the first

    tube in the batch of 6-8 tubes. Repeat this step for each specimen in the batch. 7. Allow the specimens to stand the entire15 minutes or shake vigorously on a mechanical

    shaker.

    8. Fill the first tube to the 50 ml mark with sterile distilled water or sterile 0.85% NaCL by

    slowly pouring the buffer down the side of the tube avoiding splashing or contamination.

    Tighten the cap and wipe the outside of each tube with the disinfectant soaked towel, then

    invert the tube several times to mix thoroughly. Repeat this step on each of the remaining

    specimens in the batch.

    9. Centrifuge tubes for 15 minutes at 3,000-3,500 RCF.

    10. Pour off the supernatant through a funnel into a waste container filled 1/3 full with approved

    disinfectant solution. (Pour slowly so as not to disturb the pellet and be sure to not touch the

    funnel while pouring. Touching the funnel can allow for cross contamination among

    specimens.) Wipe the lip of the conical tube with a disinfectant soaked towel and then recap. 11. The sediment may be tested with a drop of phenol red indicator solution. Add 2 N HCl

    dropwise until the color of the sediment turns from red to persistent yellow. The use of the

    lower 2% NaOH may eliminate this step.

    12. Using a sterile disposable transfer pipette, resuspend the pellet with sterile 0.85% NaCL or

    sterile distilled water.

    13. Inoculate the media with the suspension following the Mycobacteriology Culture SOP.

    14. With the same pipette, place a drop of the suspension onto a clean, labeled glass microscope

    slide. Prepare a smear over an area of 1 by 2 cm of the slide.

Method Limitations:

    ; The procedure is dependant on strict adherence to recommended techniques, timing,

    temperature, and biochemical requirements. Any deviation from the SOP will not provide

    appropriate clinical care.

    ; The NaOH procedure is very robust and may kill up to 60% of tubercle bacilli in clinical

    specimens, and may give a false negative result, especially in cases of paucibacillary disease

    as seen in early disease, or in many HIV positive patients.

    ; Additional contributory factors such as heat build-up in the centrifuge step may also kill

    tubercle bacilli.

Post-analytic Procedure

    Specimen Retention:

    Store processed specimens in AFB refrigerator for as long as space allows (approximately two

    weeks).

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Calculations:

    Contamination rates are calculated monthly to monitor the digestion process of the lab.

Expected Values:

    Contamination rates of 3 5 % are considered to be in the normal range.

Interpretation of Results:

    Contamination rates higher than 5 % should be investigated to determine whether equipment, reagent or personnel are causing the high rates. Data is included in the monthly quality assurance report.

References:

    1. Cernoch, P.L. et al. (1994). Cumitech 16A: Laboratory diagnosis of the Mycobacterioses.

    ASM Press. Washington, DC.

    2. Clinical Laboratory Standards Institute (CLSI). Clinical Laboratory Technical Procedure

    Manuals; Fourth Edition. CLSI Document GP2-A4 (ISBN 1-56238-458-9). Clinical and

    Laboratory Standards Institute, Wayne, PA

    3. Forbes, B.A., et al (2007). Laboratory detection and identification of mycobacteria; Proposed

    guideline. CLSI document M48-P. Clinical and Laboratory Standards Institute, Wayne, PA. 4. Kent , P.T. and Kubica, G. P. (1985). Public Health Mycobacteriology. A Guide for the

    Level III Laboratory. U.S. Dept. Health and Human Services. Center for Disease Control. 5. SA Healthinfo, Tuberculosis Part III: Culture homogenization and decontamination.

    Obtained from the World Wide Web on 14 March 2007 at http://www.sahealthinfo.org/tb

    6. Weitzman, I. (2007) p 7.1.2.1-7.1.2.9. In H.D. Isenberg (ed.) Clinical Microbiology

    Procedures Handbook American Society for Microbiology, Washington, D.C.

SMILE Document 8 August 2008

    SMILE

    Johns Hopkins University

    Baltimore, MD USA

    Appendix A:

    NALC-NaOH Method Reagents

    Stock NaOH-Sodium Citrate Solution:

    Solution One- Sodium Citrate:

     Sodium citrate dehydrate……….. .29 g

     Distilled water …………………...to 1,000 ml

Solution Two- Sodium Hydroxide:

     NaOH pellets……………………..40 g

     Distilled water…………………….to 1,000 ml

    1. Add an equal volume of solution one to an equal volume of solution two. 2. Label the bottles of reagent with the name of the reagent, the date of preparation, expiration

    date and the initials of preparer.

    3. May be stored at room temperature.

Working NALC-NaOH Solution:

    1. Just before use combine .25 g of NALC to 50 ml volume of NaOH-Sodium Citrate stock

    solution. (Adjust proportions as needed for higher volumes). 2. Keep the reagent refrigerated, and use within 24 hours of preparation.

Commercial digestant (Snap n Digest or Mycoprep)

    1. Simply break the snap vials inside the bottle.

    2. Use immediately

    Sodium Hydroxide (Modified Petroff) Method Reagents

    2% sodium hydroxide (NaOH) solution:

    Sodium hydroxide pellets (20g)

    Distilled or deionized water 1000mL

    1. Dissolve NaOH in distilled or deionized water and sterilize by autoclaving at 121 C for 15

    minutes.

    2. Label the bottles of reagent with the name of the reagent, the date of preparation, expiration

    date and the initials of preparer.

    3. Store at room temperature.

Sterile saline:

    Sodium chloride pellets (0.85g)

    Distilled water 100mL

    1. Dissolve NaCl in distilled water and sterilize by autoclaving 121 for 15 minutes. 2. Label the bottles of reagent with the name of the reagent, the date of preparation, expiration

    date and the initials of preparer.

    3. Store at room temperature.

    SMILE Document 8 August 2008

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