Epidemiologic Laboratory Safety : Collection and Transport of Clinical Specimens

Epidemiologic Laboratory Safety : Collection and Transport of Clinical Specimens

The proper collection and transport of clinical specimens is critical for the isolation, identification, and characterization of agents that cause bacterial meningitis. Optimally, clinical specimens should be obtained before antimicrobial therapy commences in order to avoid loss of viability of the etiological agents.

Treatment of the patient, however, should not be delayed while awaiting collection of specimens or results from the laboratory and a specimen should be obtained in all suspect cases as bacterial pathogens can still be detected even after antimicrobial therapy has begun.N. meningitidis, S. pneumoniae, and H. influenzae are fastidious and fragile bacteria.

They are more reliably isolated if the clinical specimens are examined as soon as possible after collection. Cerebrospinal fluid (CSF) should be processed in a microbiology laboratory within 1 hour after collection or inoculated into Trans-Isolate (T-I) medium for transport to the laboratory if processing within 1 hour is not feasible. Blood specimens should be immediately inoculated into a blood culture bottle and transported to a microbiology laboratory as soon as possible for overnight incubation and growth of bacteria.

I. Biosafety

It is important to adhere to proper biosafety guidelines while handling potentially infectious clinical specimens in order to maintain a safe working environment for patients, health care workers, and laboratorians. Infection may be transmitted from patient to staff and from staff to patient during the procedures described below.

In addition to the agents that cause bacterial meningitis, the patient could have other bacterial or viral agents in either the CSF of blood and both are a great hazard and potentially lethal. Of particular importance are the viruses causing hepatitis and acquired immunodeficiency syndrome. To decrease the risk of transmission of these agents, the recommendations below should be followed:

1. Wear latex or nitrile gloves that are impermeable to liquids and change gloves between every patient.

2. Dispose of syringes and needles in a puncture-resistant, autoclavable discard container. Do not attempt to re-cap, shear, or manipulate any needle. A new sterile syringe and needle must be used for each patient.

3. For transport to a microbiology laboratory, place the specimen in a container that can be securely sealed. Wipe any bottles with CSF or blood on the outside thoroughly with a disinfectant, such as a 70% alcohol swab.

4. Do not use povidone-iodine on the rubber septum of a T-I or blood culture bottle.

5. Remove gloves and discard in an autoclavable container.

6. Wash hands with antibacterial soap and water immediately after removing gloves.

7. In the event of a needle-stick injury or other skin puncture or wound, wash the wound liberally with soap and water. Encourage bleeding.

8. Report a needle-stick injury, any other skin puncture, or any contamination of the hands or body with CSF to the supervisor and appropriate health officials immediately as prophylactic treatment of the personnel performing the procedure may be indicated.

II. Collection and transport of CSF

The collection of CSF is an invasive procedure and should only be performed by experienced personnel under aseptic conditions. If bacterial meningitis is suspected, CSF is the best clinical specimen to use for isolation, identification, and characterization of the etiological agents.

Suspected agents should include N. meningitidis, S. pneumoniae, and H. influenzae and other
pathogens in some cases.

A. Preparing for lumbar puncture

If possible, three tubes (1 ml each) of CSF should be collected for microbiology, chemistry, and cytology. If only one tube of CSF is available, it should be given to the microbiology laboratory.
Because the presence of blood can affect cultures of CSF, if more than one tube of CSF is collected from a patient, the first tube collected (which could contain contaminating blood from the lumbar puncture) should not be the tube sent to the microbiology laboratory.

The kit for collection of CSF should contain (Figure 1):

- Skin disinfectant: 70% alcohol swab and povidone-iodine
- Alcohol with concentrations greater than 70% should not be used because the increased concentrations result in decreased bactericidal activity. Do not use alcohol with glycerol added to it.
-Sterile gloves
-Be sure to check the expiration date.
-Sterile gauze
-Surgical mask
-Adhesive bandage
-Lumbar puncture needle
o 22 gauge/89 mm for adults
o 23 gauge/64 mm for children
-Sterile screw-cap tubes
-Syringe and needle
-Transport container
-T-I medium (if CSF cannot be analyzed in a microbiological laboratory immediately)
o T-I should be refrigerated at 4°C and added to the kit immediately before use in the field.
-Venting needle (only if T-I is being used)
-Instructions for lumbar puncture and use of T-I medium

B. Lumbar puncture procedure

Follow all appropriate biosafety precautions.

1. Gather all materials from the CSF collection kit and a puncture-resistant autoclavable container for used needles.

2. Wear surgical mask and sterile latex or nitrile gloves that are impermeable to liquids and change gloves between every patient.

3. Label the collection tubes with appropriate information: patient’s name, date and time of specimen collection, and Unique Identification Number. Be sure this number matches the number on both the request and report forms.

4. Ensure that the patient is kept motionless during the lumbar puncture procedure, either sitting up or lying on the side, with his or her back arched forward so that the head almost touches the knees in order to separate the lumbar vertebrae during the procedure (Figure 2).

5. Disinfect the skin along a line drawn between the crests of the two ilia with 70% alcohol and povidone-iodine to clean the surface and remove debris and oils. Allow to dry completely.

6. Position the spinal needle between the 2 vertebral spines at the L4-L5 level and introduce into the skin with the bevel of the needle facing up.

Accurate placement of the needle is rewarded by a flow of fluid, which normally is clear and colorless.

7. Remove CSF (1 ml minimum, 3-4 ml if possible) and collect into sterile screw-cap tubes. If 3-4 ml CSF is available, use 3 separate tubes and place approximately 1ml into each tube.

8. Withdraw the needle and cover the insertion site with an adhesive bandage. Discard the needle in a puncture-resistant, autoclavable discard container.

9. Remove mask and gloves and discard in an autoclavable container.

10. Wash hands with antibacterial soap and water immediately after removing gloves.

11. Transport the CSF to a microbiology laboratory within 1 hour for culture and analysis.

-If that is not possible, inoculate CSF into T-I medium (see Section I.C. below).

-If T-I is not available, incubate CSF at 35-37°C with ~5% CO2 (see Section I.D. below)

and store in an approved location if the laboratory is closed.

12. In the event of a needle-stick injury or other skin puncture or wound, wash the wound liberally with soap and water. Encourage bleeding.

13. Report a needle-stick injury, any other skin puncture, or any contamination of the hands or body with CSF to the supervisor and appropriate health officials immediately as prophylactic treatment of the personnel performing the procedure may be indicated.

C. Inoculating and transporting T-I medium

T-I is a biphasic medium that is useful for the primary culture of meningococci and other etiological agents of bacterial meningitis (S. pneumoniae and H. influenzae) from CSF,It can be used as a growth medium as well as a holding and transport medium. The preparation of T-I media is described in the Annex. T-I media should be stored at 4°C and warmed to room temperature (25°C) before use.

1. Label the T-I bottle with appropriate information: patient name, date and time of CSF inoculation, and Unique Identification Number. Be sure this number matches the number on both the request and report forms.

2. Use sterile forceps to pull the aluminum cover of a T-I bottle away from the rubber stopper and disinfect the stopper with 70% alcohol. Allow to dry.

-Do not use povidone-iodine as it may be carried into the medium by the passing needle and would inhibit growth of bacteria.

-Do not completely remove the aluminum cover.

3. Use a sterile syringe and needle to inoculate 0.5-1.0 ml of CSF into the T-I medium. The remaining CSF should be kept in the collection tube. It should not be refrigerated, but should be maintained at room temperature (20-25°C) before Gram staining and other tests. Discard the needle in a puncture-resistant, autoclavable discard container.

4. After inoculation, invert the T-I bottle several times to mix.

5. If transport to a reference laboratory is delayed (next day or longer), insert a venting needle (sterile cotton-plugged hypodermic needle) through the rubber stopper of the T-I bottle, which will encourage growth and survival of the bacteria.

-Be sure that the venting needle does not touch the broth.

6. Incubate inoculated T-I medium at 35-37°C with ~5% CO2 (or in a candle-jar) overnight or until transport is possible. If transportation is delayed more the 4 days, remove the vented TI bottle from the incubator or candle jar and place at room temperature until shipment.

7. Remove the venting needle and wipe the rubber stopper with 70% alcohol before shipping. It is essential to avoid contamination when sampling the bottles to obtain specimens aseptically.

8. If the T-I bottle can be transported to a reference laboratory the same day, do not vent the bottle until it arrives in the receiving laboratory. Upon arrival, vent the T-I bottle, incubate at 35-37°C with ~5% CO2 (or in a candle-jar), and observe daily for turbidity in the liquid phase for up to 7 days.

-If turbidity is observed, culture onto a blood agar plate (BAP) and a chocolate agar plate (CAP) immediately.

-If no turbidity is observed, culture onto a BAP and a CAP on day 4 and day 7.

-If T-I medium appears to be contaminated, selective media such as Modified Thayer- Martin and chocolate agar with bacitracin may be used.