Incidence of Meningococcal
Meningitis Undetected by
Culture in New York City
{
Arianne Ramautar, MPH
New York City Department of Health and Mental Hygiene
Bureau of Communicable Disease
2012 CSTE Annual Conference (Breakout Presentation) Omaha, Nebraska, June 5, 2012
Background- Meningitis

Meningitis is the inflammation of the lining surrounding
the brain and spinal cord


Most often caused by an infectious agent
Can be:
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
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

Bacterial
Viral
Fungal
Parasitic
Common Symptoms





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
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Fever
Headache
Neck rigidity
Altered Mental Status
Photophobia
Sepsis/Shock
Petechiae
Purpura fulminans
Background- Neisseria meningiditis

One of the more common causes:
 Neisseria meningitidis (Nm)
 Can progresses rapidly
 Over 13 serogroups
 A, B, C, W-135, X, Y, Z
Purpura Fulminans

Prompt administration of antibiotics is key to survival

Nm is communicable person to person
 Potential to cause outbreaks
 Prophylaxis provided to exposed contacts to prevent
secondary cases
Background- Meningococcal meningitis in NYC

NYC: 60-80 rule-out Nm reports a year, about half
are actually Nm and meningitis occurs in ≈ 33%

New York City (NYC) case fatality rate has been
higher than the national average (19% v. ~10%)

One theory for the higher CFR is that there exist
unreported, culture negative cases of Nm
NYC Health Code and Universal reporting form
Meningitis can be reported as bacterial,
aseptic/viral or invasive meningococcal
disease
Objectives
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Primary Objective
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To assess if NYC is missing cases of Nm meningitis
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Testing culture negative reported cases of bacterial or aseptic
meningitis
Secondary Objective
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Assess the utility of nucleic acid testing on CSF
Describe and compare the bacterial organisms identified
by culture vs. nucleic acid tests
Methods- Study Design
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Inclusion criteria:

All reports of bacterial meningitis (MEX) and a sample of aseptic
meningitis (MAS) submitted to DOHMH between March 2011 and
March 2012

Available cerebrospinal fluid (CSF) specimen
Confirm CSF
specimen availability
Transport of CSF to
Wadsworth Center
State Public Health
Laboratory (WC)
Nucleic Acid
Testing
performed

Reporting hospital final culture results were obtained

Culture confirmed cases of Nm meningitis were included for
PCR quality assurance
Wadsworth Center Algorithm
Real-time PCR
• Detect Neisseria meningitidis
If negative, additional real-time
PCR
Detect: Streptococcus pneumoniae, Streptococcus
agalactiae, Haemophilus influenzae, and Methicillinresistant Staphylococcus aureus
If negative
• Broad range PCR of 16S rRNA gene to detect
the presence of bacterial DNA
If present
• 16S rRNA gene sequence analysis
Nucleic Acid Amplification Process
Real-time Polymerase Chain Reaction (PCR)
Amplifies a specific DNA sequence
Rapid method for bacterial identification
Effective after prior antibiotic therapy
16S rRNA gene sequencing
16S is a universal gene in bacteria
Sequencing is performed then compared to a database
There are over 90,000 nucleotide sequences known for the 16S
gene
Statistical Methods
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Descriptive statistics
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Comparison of available CSF samples versus unavailable
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Independent T-test
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Chi Square test
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Detection of Nm in CSF samples
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Calculate sensitivity and specificity for real-time PCR
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Where antibiotics were administered:
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Prior to lumbar puncture
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After lumbar puncture
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Describe and explore organisms detected
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Calculate sensitivity for real-time PCR on confirmed cases of Nm
Results
Reports
CSF
Samples
259
135 (52%)*
available
* One sample was lost during transport
124
unavailable
Comparison of sample characteristics
Available
Specimen
(n=134)
Unavailable
Specimen
(n=124)
74% (n=100)
82% (n=101)
0.23
28% (n=37)
28% (n=34)
1.00
34 years
35 years
0.86
% Male
44% (n=59)
51% (n=63)
0.17
% from Queens
39% (n=52)
39% (n=48)
0.78
% Winter
40% (n=54)
32% (n=40)
0.49
% Reported as MEX
% Positive hospital
culture
Mean Patient Age
pvalue
Results
Eligible
Cases
259
CSF
Samples
WC
Organism
Identification
135* available
61 due to
bacterial
organisms
124
unavailable
73 negative by
culture, PCR
and 16S rRNA
gene
* One sample was lost during transport
No Neisseria meningitidis was identified!
LP after antibiotic therapy
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Examined cases where LP followed antibiotic therapy
PCR was positive
LP hours after antibiotics
PCR (+)
Culture (+)
≤ 12 hours
9/31 (29%)
5/31 (16%)
> 12 hours
5/16 (31%)
7/16 (44%)*
*Where the hospital identified an organism when LP was >24
hours after start of antibiotics (PCR and 16S both negative):
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Pseudomonas aeruginosa
Corynebacterium
Enterobacter
Suspected contaminants
LP before antibiotic therapy:
PCR Sensitivity and Specificity
We used cases where LP preceded antibiotic therapy

Culture is the “gold standard”
Culture &
PCR found
same
organism
Culture (-)
PCR (+)
Culture (+)
PCR (-)
Culture &
PCR
negative
LP before antibiotic therapy:
PCR Sensitivity and Specificity
Includes PCR for
Streptococcus pneumoniae,
Streptococcus agalactiae,
Haemophilus influenzae, and
Methicillin-resistant
Staphylococcus aureus
Real-time PCR yielded:


Sensitivity: 80%
Specificity: 89%
Hospital
Culture (+)
Culture (-)
PCR (+)
16
9
PCR (-)
4
46
Wadsworth
Center
135
Samples
Obtained
Tube Broken
1
Concordant
96
Bacterial
Organisms
23
Negative
73
Fungus/Virus
10
135
Samples
Obtained
Tube Broken
1
Concordant
96
Bacterial
Organisms
23
Negative
73
Fungus/Virus
10
Hospital identified bacterial organisms detected
by WC: Real-time PCR & 16S rRNA results
Streptococcus pneumoniae
11
Staphylococcus aureus (includes 2 Methicillin-resistant)
3
Streptococcus agalactiae
2
Elizabethkingia meningoseptica
2
Enterococcus sp.
2
Staphylococcus epidermidis
1
Escherichia coli or Shigella flexneri or Shigella dysenteriae
Enterobacter kobei or Enterobacter cloacae or Enterobacter ludwigii,
or Leclercia adecarboxylata
1
1
Tube Broken
1
135
Samples
Obtained
Fungus/Virus
10
Discordant
28
Hosp (-) WC Hosp (+) WC Hosp (+) WC
(+)
(-)
(+)
2
12
14
Tube Broken
1
135
Samples
Obtained
Fungus/Virus
10
Discordant
28
Hosp (-) WC
(+)
14
Hosp (+) WC Hosp (+) WC
(+)
(-)
2
12
WC identified bacteria undetected by
hospital culture
Streptococcus pneumoniae
5
Streptococcus agalactiae
2
Viridans group streptococci (one intermedius and one
salivarius)
2
Haemophilus influenzae
1
Stenotrophomonas maltophilia
1
Enterococcus faecium
1
Streptococcus cristatus
1
Klebsiella pneumoniae
1
Tube Broken
1
135
Samples
Obtained
Fungus/Virus
10
Discordant
28
Hosp (-) WC Hosp (+) WC
(+)
(-)
14
12
Hosp (+) WC
(+)
2
Hospital identified bacteria
undetected by WC
Streptococcus pneumoniae
2
Enterobacter sp.
Enterococcus sp.
2
2
Viridans group streptococci
1
Elizabethkingia meningoseptica
1
Klebsiella pneumoniae
1
Pseudomonas aeruginosa
1
Corynebacterium sp.
Klebsiella pneumoniae or Acinetobacter baumanii or
Enterobacter cloacae
1
1
Tube Broken
1
135
Samples
Obtained
Fungus/Virus
10
Discordant
28
Hosp (-) WC Hosp (+) WC
(+)
(-)
14
12
Hosp (+) WC
(+)
2
Hospital and WC conflicting identifications
Hospital Identified
WC Identified
Staphylococcus epidermidis
Klebsiella pneumoniae
Klebsiella pneumoniae
MRSA
PCR Sensitivity
Confirmed cases of Nm during the interval were also
sent for real-time PCR testing
For confirmed cases of Nm
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Real-time PCR yielded a sensitivity of 100%
PCR (+)
Culture (+)
6
PCR (-)
0
For other bacterial organisms
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Real-time PCR yielded a sensitivity of 89%
PCR (+)
Culture (+)
16
PCR (-)
2
Summary
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Nm was not found in any culture negative specimens
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Bacterial organisms that were identified in this study
 PCR sensitivity was:
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100% for confirmed Nm
89% for other organisms
Discussion
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PCR sensitivity and specificity for detecting organisms after
antibiotic therapy
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Length of time on antibiotics? Not enough samples> 24 hours to
evaluate (n=10).
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Contaminant species were suspected where the hospital positively
identified an organism >24 hours
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16S rDNA sequencing
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Is useful in identifying unusual organisms
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6 organisms identified by WC that hospital did not detect
Limitations
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Sample size
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Specimen quality
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CSF availability & volume
Tube sent for PCR and 16S rRNA gene may not have been same as
for culture
Degradation of sample during transport to WC
16S rRNA gene sequencing is usually performed on cultures
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Performed on primary specimens; novel application of method
Conclusion
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New York City’s surveillance system is effective at capturing
cases of Nm meningitis
Due to the severe nature of the disease Nm diagnoses should be
rapid and comprehensive
The “gold standard” for Nm confirmation is a positive culture

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However, can take 24 hours or longer for results
PCR testing is an effective method that could be utilized routinely
Conclusion

PCR is currently available in research and state
laboratories

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Useful in cases where culture and Gram stain are negative or
inconclusive
There is room for expansion of test in clinical settings
Further studies in a clinical setting need to be undertaken to
establish real-time PCR and 16S rRNA gene:


Cost-effectiveness
Clinical utility
Acknowledgements
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Don Weiss
Lola Arakaki
Linda Steiner-Sichel
Erlinda Amoroso
Mike Antwi
Paula Del Rosso
Marie Dorsinville
Prabhu Gounder
Marci Layton
Lan Li
Laura Miller
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Sally Slavinski
Anna Smorodina
James Yea
Alice Yeung
Tanya Halse
Kimberlee Musser
Lillian Lee
Jennifer Rakeman
Nellie Dumas
Elizabeth Nazarian
Danielle Wrobleowski
Michelle Dickinson
Thank you!
Contact Information:
Arianne Ramautar
ar2568@nyu.edu