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  • lusufredy Lusubilo Malakbungu Oct. 10, 2014

    GPC,Yeast cells, GND in a CSF sample..what to do to rule out the possible organism in a lab with limited biochemical test.

  • yogandree yogandree ramsamy Oct. 27, 2014

    PROCESSING AND INTERPRETATION OF CEREBROSPINAL FLUID SPECIMENS
    Ramsamy Y , Mlisana K

    INTRODUCTION
    Bacterial meningitis is the result of an infection of the meninges. Identification of the
    infective agents is one of the most important functions of the microbiology laboratory. CSF from a patient suspected of meningitis is a specimen that requires immediate processing to determine the causative agent.

    SAFETY
    Universal safety precautions should be followed.
    Specimens must be processed in the biological safety cabinet.
    Laboratory personnel must wear appropriate protective equipment.

    MATERIALS
    Reagents
    Gram stain reagents
    India ink
    Cryptococcal Antigen test kit

    Media
    Chocolate agar
    Horse blood agar 5%
    MaConkey agar -Only if Gram negative organisms are seen on the Gram stain
    Horse blood agar 10%
    Amikacin blood agar
    Mueller Hinton agar
    Brain Heart Infusion Broth
    Thioglycollate if ANO2 suspected
    5% Sheep blood Mueller Hinton agar
    Sabouraud Dextrose agar
    Dnase agar
    Mannitol salt agar
    Haemophilus test agar

    Antibiotic discs
    Optochin 5ug – ID of strep pneumo
    Pen E-Test strips – susceptibility testing –S.pneumoniae and N. meningitidis
    CTX E Test strips - susceptibility testing – S. pneumoniae and N. meningitidis
    Oxacillin 1ug - susceptibility testing for S. pneumoniae
    Gram neg. sensitivity panel
    ST/STR sensitivity panel
    HI/Pneumo/Neiss sensitivity panel
    ESBL set (Cefotaxime, Augmentin, Ceftazidime)
    Cefoxitin 30µg – Determination of Stahylococcus aureus susceptibility (MSSA vs MRSA)

    SPECIMEN COLLECTION & RECEPTION
    Samples should be collected prior to commencement of antimicrobial therapy.
    CSF samples are obtained by lumber puncture, ventricular taps and shunts are removed via the reservoir unit.
    One plain tube with no additive is sufficient for Microbiology. Ideally 1.5 – 2ml is required for routine testing.

    SPECIMEN PROCESSING: CSF AND VENTRICULAR TAPS
    Label media, tubes and slides with the lab number of the specimen.

    MACROSCOPIC APPEARANCE
    Note gross appearance of CSF i.e. Clear and colourless, turbid, blood stained or xanthochromic. Also note presence of fibrin clot and/or blood clot.
    If a fibrin clot is present a cell count will be performed and the result will be treated with reserve.
    A sample with a blood clot is unsuitable for a cell count, proceed with Gram stain and culture.
    If specimen is blood stained record the colour of supernatant after centrifugation.
    If after centrifugation a “turbid trace” specimen has a clear supernatant with a red button of RBC’s this must be reported as blood stained and not as turbid.

    CELL COUNT
    Label a clean glass tube with the laboratory number.
    Using a sterile pasteur pipette place 1 drop of CSF stain in tube.
    Using a sterile pasteur pipette add 9 drops of CSF in the same tube.
    Mix contents by shaking gently.
    Place a cover slip over a Neubauer counting chamber and using a capillary tube fill chamber with stained sample.
    Examine the preparation under an ordinary light microscope using the 10X and perfor a CSF cell count (Information on CSF processing and cell count will be put onto the Global Health Network site shortly. )

    CULTURE AND MICROSCOPY
    Concentrate the CSF sample by centrifugation at 3000 rpm for 10 minutes.
    Label sterile tube, sterile glass slide and media with laboratory number.
    Aseptically decant the supernatant into a sterile capped tube.
    Vortex the sediment for a few seconds to re-suspend the pellet.
    Do not use a pipette to mix the sediment, because the bacteria and cells may adhere to the sides of the tube and cause false negative findings.
    Using a sterile Pasteur pipette inoculate Choc and BA plates with a free falling drop of CSF, as well as BHI broth if required.
    Use a sterile Pasteur pipette to prepare a smear for Gram stain by placing a free falling drop of CSF sediment onto a slide.
    Streak out plates using a sterile loop.
    Incubate BA and Choc agar at 37 _C in 5% CO2 for 18 – 24 hours.
    Incubate BHI broth aerobically at 37 _C for 18 – 24 hours.
    Set up direct antimicrobial susceptibility tests and additional media according to standard procedure if organisms are seen on microscopy. Refer to list below.
    Discard all biological waste as described in standard procedures.

    ADDITIONAL MEDIA REQUIRED BASED ON MORPHOLOGY AND GRAM REACTION OF THE ORGANISM

    Gram positive cocci in clusters: - DNA agar
    (? Staph) - MSA ( mannitol salt agar )
    - ST/STR sens on MH (Mueller Hinton agar)
    - Cefoxitin disc on MH (Mueller Hinton agar)

    Gram positive cocci in pairs/chains: - Optochin disc on BA(Blood agar)Pneumococcus is sensitive to Optochin. - ST/STR sens on mueller Hinton + Sheep blood )agar
    If Pneumococcus suspected also set up:
    - Pneumo sensitivities on MHSB agar
    - Oxacillin 1ug disc on MHSB
    - Pen, CTX E-Test on MHSB
    Gram negative diplococci: - Neiss sens on MHSB (Mueller Hinton + Sheep blood
    agar)

    Gram negative bacilli? Enterobacteriacea: -Culture on Mackonkey agar
    - Gram neg. sensitivities and ESBL on MH
    (Mueller Hinton agar)

    Gram negative bacilli? Haemophilus: - Chocolate and Mackoney
    - HI sens on Haemophilus test media agar

    Appearance of anaerobes: - 10% BA incubated anaerobically 48hrs
    - Amikacin blood agar anaerobically 48hrs
    - Thioglycolate broth O2 48hrs

    GRAM STAIN
    Quantify presence/absence of pus cells/organisms:-
    Not detected (0)
    Few (1 – 5)
    Moderate (6 – 15)
    Many (>16)
    Examine the Gram stained smear for organisms on an ordinary light microscope using the 10X and 100X oil immersion objectives.
    Record results on patient’s worksheet. If a cell count was not performed comment on presence of and semi-quantitate white blood cells. If possible report if there is a predominance of polymorphs or lymphocytes.
    Notify the pathologist/registrar if organisms are seen on the gram stain.

    INDIA INK PREPARATION (ONLY PERFORMED IF REQUIRED)
    If yeast cells are seen on the counting chamber and/or gram stain determine and record
    whether they morphologically resemble Cryptococcus neoformans. If unsure an India ink
    preparation should be done to confirm the presence of a capsule. India ink does not stain
    cellular material therefore it provides a black background against which the capsules of
    C. neoformans may be visualized.
    Place one drop of India ink on a slide.
    Add one drop of CSF sediment using a sterile Pasteur pipette and mix gently.
    Place cover slip over and examine on an ordinary light microscope using the 10X and
    40X objectives.

    EXAMINATION OF CULTURES
    Examine media plates for growth and broth for turbidity.
    If no visible growth/turbidity is observed re-incubate plates and broth for a further 24 hours.
    If no growth/turbidity is observed after additional incubation, re-incubate Choc and Blood agar plates for an additional 5 days. Discard broth as per standard procedures.
    Report as “No growth after 48 hours”
    If turbidity is observed make a smear from broth by placing a drop on a glass slide. Dry and heat fix, gram stain and examine microscopically for organisms.
    If organisms are seen subculture broth and identify all isolates and perform antimicrobial susceptibility tests according to standard procedures.(See protocol on identification and susceptibility testing of micro-organisms)
    If growth occurs identify all isolates and perform antimicrobial susceptibility tests according to standard procedures .(See protocol on identification and susceptibility testing of micro-organisms)
    Record the types and semi-quantitate growth as scanty 1+, 2+, 3+.
    Quantitation of growth on plates:- scanty- growth on initial inoculum
    1+ - growth on 1st quadrant
    2+ - growth on 2nd quadrant
    3+ - growth on 3rd quadrant
    Read and record direct antimicrobial susceptibility tests. All antimicrobial susceptibility tests are repeated as per SOP.
    Plates with no growth after 7 days incubation are discarded as per standard procedures

    If growth occurs after 7 days incubation, identify isolates and perform antimicrobial susceptibility tests according to standard procedures.

    ANTIMICROBIAL SUSCEPTIBILITY TESTING
    Antibiotic susceptibility testing is done on the automated VITEK 2 system. Refer to SOP of VITEK 2 System. Alternatively it may be done manually using the Kirby Bauer disc diffusion method according to CLSI guidelines. Read and record direct antimicrobial susceptibility tests.
    Perform beta lactamase tests on all H. influenzae and N. meningitidis isolates.
    Perform standardised antimicrobial susceptibility testing on all CSF isolates if requested.

    CRYPTOCOCCAL ANTIGEN TEST
    Performed when requested/ordered, using CSF supernatant.

    Method: Cryptococcal antigen latex agglutination
    Systems (Meridian)

    CULTURE FOR TB
    Culture for TB is done when requested by ward or departmental medical staff.
    If a separate specimen is not received the deposit may be forwarded to the TB laboratory.
    Record on the worksheet that the specimen has been sent to the TB lab in the space provided.

    CULTURE FOR YEAST
    Growth of organisms resembling yeast on the culture plate. Gram stain and India Ink performed – exclude Cryptococcus
    If yeast resembling Candida – determine if yeast is a Candida albicans or a non albicans Candida spp. _ Perform a germ tube test
    C. albicans are germ tube positive and Non albicans candida spp are germ tube negative

    REFERENCES
    Lisa Anne Shimeld, Anne T. Rodgers, Essentials of Diagnostic Microbiology, Delmar.
    Isenberg, Clinical Microbiology Procedures Handbook, Volume 1.

    ADDITIONAL INFORMATION
    Common causes of meningitis
    1. < 1 month (neonate)
    Enteric GNB
    Streptococcus agalactiae
    Listeria monocytogenes
    Herpes simplex virus 2
    2. 3 mths - 5 yrs
    H. influenzae
    S. pneumoniae
    N. meningitidis
    TB
    Viral - enterovirus and mumps

    3. > 5 yrs - Adults
    S. pneumoniae
    N. meningitidis
    TB
    Cryptococcus, Viral

    4. Immunocompromised patients
    TB
    Cryptococcus
    Any organism

  • yogandree yogandree ramsamy Oct. 27, 2014

    I hope this helps. Please feel free to ask more questions. I will assist wherever I can.

    Regards
    Yogandree

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