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4.1 Medical Microbiology > Laboratory Diagnosis of Meningitis > Flashcards

Laboratory Diagnosis of Meningitis Flashcards

1
Q

What is the specimen of choice for meningitis investigation, what is the gold standard?

A

Cerebrospinal fluid (CSF)

The specimen should be obtained before patient is put on antimicrobial therapy

The specimen should be transported rapidly to the lab

CSF should NOT be refrigerated

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2
Q

What are some specimens other than CSF for investigation of meningitis?
(5)

A

Blood cultures -> these tend to also be positive -> think of where the meningitis originates

Skin scrapings -> only used in meningococcal meningitis -> rapid, helpful, often positive

Wound swabs ->only helpful in trauma/chronic cases

Urine or sputum for tuberculosis meningitis

Blood -> looking for antiodies, antigens, raised CRP etc etc

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3
Q

How should CSF be collected?
(3)

A

CSF is normally collected sequentially into three or more separate containers -> and should be labelled in accordance of draw

The first and last samples are sent for micro and the second one is sent to bio for glucose and protein quantification

At least 1 ml per tube is necessary

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4
Q

What is different about the collection of a CSF for Mycobacterium investigation?

A

We need to take as much CSF as posible -> as large a volume as possible

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5
Q

What counts are done on CSF?

A

A white cell count and a red cell count

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6
Q

Why do we carry out a serial red cell count on our CSFs?

A

To determine whether a haemorrhagic CSF is due to a traumatic tap or a subarachnoid haemorrhage

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7
Q

How can you tell between a haemorrhagic sample and a traumatic CSF?

A

The uniform bloodstaining of all samples suggests previous haemorrhage into the subarachnoid space

Reducing bloodstaining in sequentially obtained samples suggest bleeding induced by the tap procedure

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8
Q

What are some safety considerations to handling CSF?

A

Biological Safety hood required due to the possibiltiy of N. meningitidis

A cat 3 lab is required for any M. tuberculosis meningitis

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9
Q

How should a CSF specimen be collected?

A

Collect sample before antimicrobial therapy (if possible)
Send sample immediately to the lab and process urgently
Minimal volume of 1ml required but more required for tuberculosis
Insufficient volumes must be prioritised
Collect sequentially into 3 separate containers

Other samples such as a pharyngeal swab, blood for PCR and FBC should be considered

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10
Q

Why do we always take a blood sample for query meningitis?

A

As we often query sepsis as the cause of meningitis

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11
Q

What is the first thing that a scientist does when receiving a CSF?

A

Specimen should be signed in
Describe the appearance of the CSF

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12
Q

How should you describe the appearance of a CSF?

A

Comment on turbidity -> indicater of wbcs
Comment on degree of haemorrhage -> rbcs
Any sign of xanthochromia/straw-coloured
Any cobwebs indicative of TB?

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13
Q

Why do we send a CSF sample to biochemistry?

A

Quantification of glucose and protein
-> used to differentiate between viral and bacterial meningitis

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14
Q

What do we do after describing the appearance of a CSF?

A

We carry out a complete cell count:
- rbcs
- cell count for wbcs (neutrophils vs lymphocytes)

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15
Q

What do we do after carrying out a cell count on a CSF sample?

A

We centrifuge the sample

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16
Q

Why do we centrifuge our CSF samples?

A

We centrifuge the sample as a means of concentrating any bacterial cells which would be present -> increases likelihood of us being able to cuture an organism

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17
Q

What do we do after we centrifuge a CSF sample?

A

Gram (can do further confirmatory testing based on what we see microscopically)
Culture on blood and chocolate agar (nothing every grew on these in the Mater)
We do molecular detection using the film array
We can send a sample out to a reference lab for antigen testing etc to confirm ID

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18
Q

What molecular detection methods do we do on the CSF?

A

We use the film array

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19
Q

What are some downfalls of molecular methods of detecting meningitis

A

The film array is not quantiative

We need to culture regardless for DST and for epidemiology

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20
Q

Why do we carry out grams for CSF?

A

A lot of the time nothing will grow on the blood and choocalte agar due to patients already being on antimicrobial treatment

Because of this we can use the gram to confirm ID of organism -> lets us confirm what we get on molecular

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21
Q

In general what six things do we do on CSF

A

Macroscopic appearance: colour and clarity

Cell count

Centrifugation

Gram stain

Culture on blood and choc

Further tests based on microscopy results

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22
Q

Talk about the macroscopic examination of CSF

A

CSF should be clear and colourless = normal

Cloudy/turbid sample is abnormal -> relates to wbcs, rbcs and microorganisms

Colour is related to haemorrhage or pigment

NB: a CSF sample may contain abnormal numbers of cells while still appearing “clear”

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23
Q

How many white cells are required to make a CSF sample cloudy?

A

50 WBCs and the CSF will start to loose clarity

200 WBCs and the CSF will look turbid

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24
Q

What is considered a normal white cell count in adults

A

<5/ul

Any elevation in this is abnormal

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25
When carrying out a cell count what might you notice other than wbcs
RBCS: these are not diagnostic of meningitis but they should still be reported Microorganisms may also be seen and their presence recorded e.g. Listeria, pneumococci, candida or cryptococcus
26
What is a normal CSF wcc for neonates, 1-4yr olds, 5 yrs-puberty and adults?
0-30 cells in neonates 0-20 cells in 1-4 year olds 0-10 cells in 5 year olds to puberty 0-5 cells in adults
27
What is a normal erythrocyte count for newborns vs adults?
Newborn = 0-675 cells x 10^6/L Adults = 0-10 cells x 10^6/L
28
What is the normal protein in csf of neonates <6days old vs others?
0.7 g/L in neonates <6days old 0.2-0.4 g/L in others
29
When would you not be concerned with a csf wcc of 35?
a wcc of 35 is perfectly normal in any baby less than 1
30
In what cases will lymphocytes in CSF predominate over PNMs?
Viral meningitis Fungal meningitis Tuberculous meningtisi You might also see more lymphs in early stage listeria meningitis
31
How do we centrifuge our CSF samples?
Centrifuge 1,200g for 5-10 minutes if query TB centrifuge at 3,000g for 15-20 minutes Aseptically remove supernatant Resuspend deposit Retain supernatant and store at 40 degrees celsius
32
What is the one reasons why we would not centrifuge a csf sample?
only done if you have more than half a ml
33
What is xanthochromia?
The yellow colouration of the supernatant of centrifuged CSF
34
What causes xanthochromia?
Metabolism of products of RBC breakdown Increased CSF protein concentration Bilirubin staining
35
How should we quantify xanthochromia?
It should be determined accurately only by spectrophotometry
36
After centrifugation, why might the supernatant be pink and what does this mean?
This may be due to oxyhaemoglobin This confirms pre-tap intracranial haemorrhage After 24 hours this degrades to bilirubin and xanthochromia increases to peak at 36-48 hours
37
What is the gold standard for meningitis diagnostics
culturre
38
What factors affect meningitis culture
Antimicrobial therapy Very small nnumbers of bacteria bacteria may be fastidious low volume of csf => low numbers of bacteria special culture may be required for Mycobacterium, Candida and crypto
39
What kinds of culture can we put up for meningitis?
Always: - blood agar in 5-10% CO2 - chocolate agar in 5-10% CO2 Blood anaerobically Broth Possibly mycobacterial media is query TB Possibly fungal media (SDA) if query fungus
40
What kinds of culture can we put up for meningitis?
Always: - blood agar in 5-10% CO2 - chocolate agar in 5-10% CO2 Blood anaerobically Broth Possibly mycobacterial media is query TB Possibly fungal media (SDA) if query fungus
41
Talk about the gram stain for meningitis
It can be invaluable to diagnose meningitis in minutes It can be technically difficult as organisms are often intracellular organisms - will need to look in wbcs etc You can concentrate the gram by adding drop on drop of centrifuged concentrate to slide
42
What would gram-positive diplococci indicate?
S. pneumoniae
43
What would gram negative coccobacilli mean?
Haemophilus
44
What would gram negatve diplococci indicate?
N. meningitidis
45
What would gram positive cocci in chains indicate
S. agalactiae
46
What would gram positive bacilli indicate
Listeria but these are rarely visible - intracellular
47
What are the two most likely yeast in csf?
Candida Cryptococcus
48
What special stains might be used on CSF?
Wet prep and india ink for cryptococcus Ziehl Neelson for tb
49
What is the recommended preparation for the white cell differential?
Cytospin + Leishman differential wcc: - polymorph:lymphocyte ratio
50
What are some exceptions to our wcc limits for meningitis?
Neonates, will be higher Viral and TB meningitis -> polys may predominate in early infection Neutropenic patients - still work up even if low neuts
51
Why is csf protein levels not diagnostic?
Elevated protein is not diagnostic of meningitis It may indicate the duration of the infection or a poor prognosis
52
What is normal glucose levels?
60% of blood glucose level usually Its between 2.2-3.3 m.mol/L
53
What is the one bacterial meningitis where there is not a decrease in glucose?
Listeria meningitis -> its usually 60%
54
What antimicrobial susceptibility should be carried out for meningitis
Should use standard EUCAST methods Choose antimicrobials based on microorganism identified Should test for B-lactamase production by H. influenzae
55
How should we treat meningitis?
Intravenous antimicrobials
56
What are the five methods of diagnosing meningitis
Gram stain Culture PCR test Serology Antgen test
57
How dependent is sensitivity of blood and culture on antimicrobial use
If no prior antibiotic use: - CSF culture is 95% sensitive - Blood culture is 50% sensitive If prior antibiotic use: - Blood sens is only 5% - CSF -> nothing really grows
58
Give a specific reason for the need for clinical need for molecular detection of samples in CSF
In a study of 103 patients in the UK with clinically defined meningitis only 13% of cases were positive on CSF cultures Our reference lab in 2019 carry out a study of N.meningitidis cases whereby 40% were detected by PCR methods only i.e. culture negative
59
What is the high clinical impact of meningitis and encephalitis
High morbidity High mortality Significant health care costs Rapid treatment is associated with improved outcome Current gap in diagnostics: identification of the causing pathogen Time-consuming (culture based) Sue to pre-treatmenet sometimes unsuccessful
60
Talk about the use of multiplex meningitis encephalitis platforms for the detection of meningitis
Syndromic testing - multiplex nested PCR Reverse transcription - cDNA used Broad coverage without the need to select specific tests Enhanced ability to detect co-infections Increased sensitivity Higher throughput but not really cause the film array can only process one sample at a time Reduced sample volume requirements
61
What bacteria is the biofire film array capable of detecting?
E. Coli K1 H. influenzae L. monocytogenes N. meningitidis S. agalactiae S. pneumoniae
62
What viruses are detectable by the biofire meningitis/encephalitis panel? (7)
Cytomegalovirus Enterovirus Herpes simplex virus 1 Herpes simplex virus 2 Human herpes virus 6 Human paraechovirus Varicella zoster
63
What fungi are detectable by the filmarray
Yeasy Cryptococcus neoformans Cryptococcus gattii
64
What are the three main benefits of the biofire?
Everything happens in one single pouch, very easy to use Very small amount of specimen needed ideal for CSF Very well controlled - multiple on board controls
65
What are the three steps to the film array?
1st stage PCR 2nd stage PCR DNA melting analysis
66
What are the steps of the 1st stage PCR
Reverse transcription of viral RNA into cDNA Multiplex nested PCR Enrich for the target nucleic acid in the sample Diluted
67
Wha are the steps to the 2nd stage of pcr on the film array
Primers are nested or internal to the specific products of the 1st stage multiplex PCR
68
What is DNA melting analysis on the biofire
Fluoresence in each well of the array is monitored and analysed to generate a melt curve This curve tells is if the organism is detected or not
69
What are the controls on the biofire
RNA process control: - RNA transcript from Schizosaccharomyces pombe - Lysis purification RT PCR 1st stage 2nd stage melt analysis - A positive control indicates that all steps carried out in the film array ME pouch were successful - this controls the PCR amplification steps A PCR2 control - DNA target - a positive result indicates that the 2nd stage PCR was successful Both controls must work or we will get an invalid result
70
What organisms are we missing from the biofire?
Were missing organisms that occur in shunt ingections such as S. aures, CNS, P acnes etc
71
What are the advantages of the Biofire?
High sensitivity with a low limit of detection for pathogens Short hands-on-time in sample preparation Short assay turn around time, overall about 3 hours Assessment of the most common community acquired pathogens
72
What are the disadvantages of the Biofire
Diagnostic gaps for apthogens e.g. staphylococci for shunt infections No information on antibiotic resistance No quantification Costs
73
What organisms does the BIOFIRE strugle to detect?
Group B streps -> culutre should be used to confirm these as they are often missed on molecular platforms
74
What is the main issue with the BIOFIRE?
Too many false positives: for viral, fungal and bacterial agents -> S. pneumoniae false positives frequent, as well as E. Coli K1 and S agalactiae -> Viral false positives in herpes frequent Issues with detection of Group B streps
75
What bacteria does the BIOFIRE struggle to detect?
It can miss Group B streps S. pneumoniae False Positive E. Coli K1 false positives S. agalactiae false positive -> i.e streps and E. Coli K1
76
What viruses does the BIOFIRE struggle to detect and why?
Herpes viruses These can be tricky to diagnose as some patients might have an initial infection while others migh have a latent infection False positives are associated with latent infection
77
What might cause so many S. pneumoniae false positives?
It was suggested that lab staff should wear masks etc to prevent S. pneumoniae contamination Suggested that a degree of these positives were jut to lab staff however this is unlikely as it was seen across a lot of labs
78
What are the limitations of the FilmArray?
Lack of clinical details on all patients -> cannot tell if latent or active infection for Herpes etc High clinical suspicion is required Positive bacterial and cryptococcus results should be correlate with Gram stain results and cultures as well as clinial findings -> discrepant molecular results should be witheld pending further investigation Confirmatory testing should be carried out e.g. PCR for HSV
79
Why should we confirm any organisms IDd on the BIOFIRE, give an example of why?
There was a case where a patient was diagnosed with HSV-1 on the biofire but was later diagnosed with MTB 7 days later The HSV-1 diagnosis was not confirmed by PCR methods -> this should have been done as antimicobrial therapy was 7 days late for patient etc etc
80
For th HSV/TB case what should have been done?
The gram stain should have been checked for correlation Clinical symptoms should have been looked at - they wouldnt have matched Confirmatory testing should have been carried out
81
What is the sensitivity of CSF culture, why so low?
Sensitivty of 13% Low due to antimicrobials
82
Compare the BIOFIRE to Reference lab, what is the main advantage of both
BIOFIRE only takes abour 70mins for a result while the reference lab takes up to 5.6 days The reference lab has a lower detection rate

4.1 Medical Microbiology (30 decks)

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