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SOP Co-ordinator (document control, formatting, reference manager) ... Raised CRP. History revisited. Any thoughts? 42. Added History (Rob's ) ... – PowerPoint PPT presentation

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Title: HPA Presentation 2 white background


1
INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
National Standard Methods CSF SOP 27 How well
did we do in practice? 2 case histories
examined Evaluations and Standards Laboratory
Centre for Infections Sheffield Teaching
Hospitals 20 May 2005
Valerie Bevan Trevor Winstanley Rob Townsend
2
INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
3
Evaluations and Standards LaboratoryOrganisation
Chart
April 2005
4
Infrastructure to producing Standard
Methods(Valeries slide)
  • Staff in Standards Unit
  • Director 10
  • Head of Standards Unit (80)
  • Two staff write, edit documents, co-ordinate
    process
  • SOP Co-ordinator (document control, formatting,
    reference manager)
  • Information Officer (20 - issues passwords,
    places docs on website)
  • Office Manager (10 - organizes meetings, sends
    out all documents for meetings)
  • Working Groups
  • Medical, clinical and biomedical scientists
  • National AMM, CVN, ACM, IBMS, SGM, UK wide,
    Pathlinks, Leeds, other networks
  • All day meetings every two months
  • Virology Working group
  • FWE group

5
Standards Unit (Valeries slide)
  • SOPs current position
  • 109 clinical microbiology
  • 37 food water and environmental
  • 50 media
  • 42 guidance notes

6
Global access Over 1000 passwords issued for the
Extranet site Password requests from 48
countries outside of the UK
Access to National SOPs (Valeries slide) New
website! http//hpa-standardmethods.org.uk
Versions in Adobe PDF www.HPA.org.uk www.evalua
tions-standards.org.uk Versions in Microsoft
Word Password protected Drafts SOPs for review
6 Over to Trevor
7
Investigation of Cerebrospinal FluidNational
Standard Method
1st case history
  • Dr Trevor Winstanley
  • Royal Hallamshire Hospital
  • Sheffield UK

8
Specimen (Trevors slide)
  • Cerebrospinal fluid
  • Tuberculosis and HIV infection endemic in Somalia
  • Unknown HIV status
  • Specimen and request form labelled Category 3
  • increased risk of containing organisms that may
    cause severe human disease and present a serious
    hazard to laboratory workers

9
Patient (Trevors slide)
  • 1994
  • Male, 9 y
  • Somalian
  • In England for 3 months
  • Headaches, vomiting, fever, diplopia
  • ? tuberculous meningitis

10
Specimen (Trevors slide)
  • Cerebrospinal fluid
  • Tuberculosis and HIV infection endemic in Somalia
  • Unknown HIV status
  • Specimen and request form labelled Category 3
  • increased risk of containing organisms that may
    cause severe human disease and present a serious
    hazard to laboratory workers

11
Safety (Trevors slide)
  • Risk assessment laboratory safety protocols
    followed
  • Class 1 biological safety cabinet at CL3
  • capture and retain airborne particles and protect
    laboratory worker
  • Centrifuge with sealed buckets
  • opened in Class 1 cabinet
  • All media appropriately labelled

Over to Val
12
Tuberculous meningitis (Valeries slide)
  • CSF may be infiltrated with lymphocytic cells
  • Insidious clinical manifestations
  • Rare in the UK
  • Considered in areas of high TB prevalence and
    in patients from high risk groups
  • Examination for AAFB or mycobacterial genome if
    specific indication
  • Rapid tests are useful if positive, but culture
    remains the gold standard for diagnosis
  • HG3, CL3
  • Think of other unusual organisms

13
Examination of CSF for meningitis (Valeries
slide)
  • Complete cell count
  • Differential leucocyte count
  • Examination of Gram-stained smear
  • Culture for pathogens
  • Determination of glucose and protein concentrat
    ions
  • PCR where appropriate

14
Normal CSF values (Valeries slide)
12 Over to Trevor
15
Cell Count Results (Trevors slide)
  • Total cell count 440 x 106 / L
  • White cell count 400 x 106 / L (0-10)
  • Differential 40 polymorphs
  • 55 lymphocytes
  • 5 undifferentiated
  • Protein 0.1 g/L (0.2-0.4)
  • Glucose 1.6 mmol/L ( 60 plasma)

16
Results (Trevors slide)
  • Ziehl Neelsen film negative
  • Mycobacterium culture negative
  • India ink stain negative
  • Cryptococcal Ag test negative
  • Sabouraud culture negative
  • Gram film No organisms seen

17
Culture (centrifuged deposit) (Trevors slide)
  • Bacteria
  • Columbia BA (5) ANO2 _at_ 37oC
  • Heated CBA AER CO2 (7) _at_ 37oC
  • Hartleys digest broth 0.1 glucose _at_ 37oC
  • Cryptococcus neoformans
  • Sabouraud agar AER _at_ 30oC
  • Mycobacterium spp.
  • Löwenstein-Jensen Kirschners

18
Further tests (Trevors slide)
  • Aliquot mixed with equal volume of buffered
    formalin (4) and left for 10 min
  • CSF cell counts
  • Protein and glucose (Clinical Chemistry)
  • Cryptococcal Ag test

19
Culture media, conditions and organisms
(Valeries slide) For all specimens
Broth cultures are not recommended as a
significant positive yield is rarely achieved and
contamination is frequent
20
Broth Cultures? (Valeries slide)
  • Broth cultures are not recommended
  • Significant positive yield is rarely achieved and
    contamination is frequent (Dunbar et al, 1998)

Over to Trevor
21
Bacterial culture results (Trevors slide)
  • CL3 accommodation
  • Columbia BA ANO2 negative after 48 hours
  • Chocolate CBA CO2 negative after 6 days
  • Hartleys Digest Broth clear at 6 days
  • Sub-cultured in Class 1 safety cabinet
  • Columbia BA ANO2
  • Chocolate CBA CO2

Over to Val
22
Broth sub-cultures (Trevors slide)
  • No growth _at_ 24 h
  • 1 mm diameter colonies _at_ 48 h aerobic

23
Gram film (Trevors slide)
  • Small (0.5 µ) Gram negative cocci
  • Oxidase positive ? Neisseria sp.
  • N.meningitidis is in Hazard group 2
  • At this time, local policy did not dictate the
    use of full
    containment level 3 conditions

24
Susceptibility tests (Trevors slide)
  • No growth on Lysed Blood Agar after 24 h
  • Identity as Neisseria sp. questioned
  • Cocco-bacillary forms seen on Gram film

25
Biochemistry (Trevors slide)
  • Rapid urease test positive
  • Provisionally identified as Brucella sp.

26
Biochemistry (Trevors slide)
  • We did not attempt further identification
  • Others have misidentied Brucella sp. as Moraxella
    phenylpyruvica
  • Confirmed as B.melitensis by Portsmouth PHL

27
Since broth sub-culture (Trevors slide)
  • Cultures processed on open bench
  • Provisional identification of Brucella sp. caused
    alarm
  • an understatement
  • infectious aerosol 10-100 organisms
  • 7 laboratory workers handled cultures
  • A further 4 handled specimens

28
What did we do? (Trevors slide)
  • Took base-line sera
  • Post-exposure prophylaxis of doxycycline
    rifampicin (3 weeks)

29
Consequences (Trevors slide)
  • Pharmacy ran out of rifampicin
  • The mens urinal turned red!
  • Luckily, nobody was infected
  • We learned some lessons

30
Brucella sp. (Trevors slide)
  • Zoonosis
  • B.melitensis (goats and cattle) B.suis (swine)
    B.abortus (cattle) B.canis (dogs)
  • Mediterranean Europe and Africa, Middle East,
    India, Central Asia, Mexico, Central and South
    America

31
Transmission (Trevors slide)
  • Ingestion, inhalation
  • Direct contact
  • cuts and abrasions, mucous membranes
  • Laboratory exposure
  • aerosolisation
  • Bioterrorism
  • high morbidity, protracted illness

Over to Val and Rob
32
INVESTIGATION OF CEREBROSPINAL FLUID BSOP 27
Over to Rob
33
A Clear Case ofMeningitis?
2nd case history
  • Dr Rob Townsend MBCHB MSc DTMH
  • Microbiology SpR/Lecturer
  • Sheffield Teaching Hospitals

34
The Patient (Robs slide)
  • Mrs JM 30 Yr Old Admitted Royal Hallamshire
    Hospital, Sheffield, 2001
  • Approx 24 hours History of
  • Headache
  • Neck Stiffness
  • Photophobia

35
History (Robs slide)
  • No Rash noted
  • Denies any prior symptoms
  • No dysuria
  • No sore throat
  • Came on over hours the previous day
  • Other family members well
  • No pets
  • No recent holiday

36
Examination (Robs slide)
  • Patient was noted to be Pyrexial
  • Looked unwell but GCS 15/15
  • Examination
  • Neck stiffness clinically
  • No rash
  • No Papilloedema
  • Remainder of examination unremarkable

37
Investigations (Robs slide)
  • Raised WCC on full blood count
  • Blood cultures sent
  • Lumbar Puncture performed
  • Which Looked Like.

38
Gin Clear ! (Robs slide)
39
Lumbar Puncture (Robs slide)
  • Cell count
  • Total cell count lt1/hpf
  • White cell count lt1/hpf
  • Protein 0.5g/l
  • Any Thoughts?

40
Treatment (Robs slide)
  • Given Cefotaxime in AE prior to ward admission
  • Cefotaxime continued BUT Given CSF result
  • ? Stop as it may be viral?
  • ? Stop as it may not be meningitis?
  • Continued Cefotaxime over night to review next day

41
Next Day (Robs slide)
  • Blood cultures CSF no growth
  • But Post Antibiotic
  • Reviewed by Consultant
  • Clinically meningitis
  • Raised CRP
  • History revisited
  • Any thoughts?

42
Added History (Robs slide)
  • No recent holiday BUT only been in UK 6/12
  • Previously from Zimbabwe
  • History of recurrent infections inc
  • Respiratory infections
  • Candidiasis
  • Telephoned micro. Please could you do a Gram
    Film?
  • Not in our protocol but as you asked.

43
(Robs slide)
44
Conclusions (Robs slide)
  • Pneumococcal meningitis on gram film
  • Cultures No growth (prior antibiotics)
  • Requires 14 days treatment
  • Later tests
  • HIV ve
  • CD4 count lt100
  • Importance of Gram films in immunesuppressed
    patients with no cells they may have no cells!
  • Completed 14 days and made good recovery
  • 1 month later started Anti-retrovirals

45
How did standard methods fair?(Valeries slide)
  • Brucella covered in SOP
  • Broth Cultures?
  • Broth cultures are not recommended
  • Significant positive yield is rarely achieved and
    contamination is frequent (Dunbar et al, 1998)
  • Gram stain? (refer to BSOP SP 8)
  • CSF SOP states do Gram-staining method on ALL
    specimens except
  • clotted specimens (see below)
  • routine neurological specimens unless leucocyte
    counts are raised

19
46
About Psychrobacter phenylpyruvicus(Valeries
slide)
  • Rods, often coccobacilli. Usually occur in
    planes with one plane of division. Microscopy
    can differentiate Brucella species (very small
    coccobacilli) from P. phenylpyruvicus
  • Brucella species can be misidentified as P.
    phenylpyruvicus in some commercial identification
    kits

47
Where do we go from here with National SOPs?
(Valeries slide)
  • Future work
  • Front end clinical algorithms for bacteriology
    AND virology
  • Standards for reporting?
  • Reconsider format?
  • What should we emphasise?
  • How do we evaluate methods?
  • How do we encourage people to comment?

48
(Valeries slide)
  • Your help is still needed!

We have a new website to make things easier for
you
49
(No Transcript)
50
  • www.hpa.org.uk
  • www.evaluations-standards.org.uk
  • http//hpa-standardmethods.org.uk
  • valerie.bevan_at_hpa.org.uk

Acknowledgements to all in Evaluations and
Standards Laboratory Laboratory staff at
Hallamshire Patients
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