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Incidence of Meningococcal Meningitis Undetected by Culture in New York City

Arianne Ramautar, MPHNew York City Department of Health and Mental HygieneBureau 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:

Bacterial Viral Fungal Parasitic

Common Symptoms 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

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

Purpura Fulminans

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

Primary Objective To assess if NYC is missing cases of Nm meningitis

Testing culture negative reported cases of bacterial or aseptic meningitis

Secondary Objective 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

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 PCRDetect: Streptococcus pneumoniae, Streptococcus agalactiae, Haemophilus influenzae, and Methicillin-resistant 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 ProcessReal-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 Descriptive statistics

Comparison of available CSF samples versus unavailable Independent T-test Chi Square test

Detection of Nm in CSF samples Calculate sensitivity and specificity for real-time PCR

Where antibiotics were administered: Prior to lumbar puncture After lumbar puncture

Describe and explore organisms detected Calculate sensitivity for real-time PCR on confirmed cases of Nm

CSF Samples

Reports259

135 (52%)* available

124 unavailable

Results

* One sample was lost during transport

Comparison of sample characteristics

 

Available Specimen

(n=134)

Unavailable Specimen

(n=124)p-

value% Reported as MEX 74% (n=100) 82% (n=101) 0.23% Positive hospital culture 28% (n=37) 28% (n=34) 1.00

Mean Patient Age 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

WC Organism Identification

CSF Samples

Eligible Cases 259

135* available

61 due to bacterial

organisms

73 negative by culture, PCR

and 16S rRNA gene

124 unavailable

Results

No Neisseria meningitidis was identified!* One sample was lost during transport

*Where the hospital identified an organism when LP was >24 hours after start of antibiotics (PCR and 16S both negative):

Pseudomonas aeruginosa Corynebacterium Enterobacter

Suspected contaminants

LP after antibiotic therapy Examined cases where LP followed antibiotic therapy PCR was positive

LP hours after antibiotics PCR (+) Culture (+)

≤ 12 hours

> 12 hours

9/31 (29%)

5/16 (31%)

5/31 (16%)

7/16 (44%)*

LP before antibiotic therapy: PCR Sensitivity and SpecificityWe used cases where LP preceded antibiotic therapy

Culture is the “gold standard”

Culture & PCR found

same organism

Culture (+) PCR (-)

Culture (-) PCR (+)

Culture & PCR

negative

Real-time PCR yielded: Sensitivity: 80% Specificity: 89%

16 9

4 46

PCR (+)

Culture (+) Culture (-)

PCR (-)

Hospital

WadsworthCenter

LP before antibiotic therapy: PCR Sensitivity and Specificity

Includes PCR for Streptococcus pneumoniae, Streptococcus agalactiae,

Haemophilus influenzae, and Methicillin-resistant

Staphylococcus aureus

Fungus/Virus10

135 Samples Obtained

Concordant96

BacterialOrganisms

23

Negative73

Tube Broken1

Fungus/Virus10

135 Samples Obtained

Concordant96

BacterialOrganisms

23

Negative73

Tube Broken1

Hospital identified bacterial organisms detected by WC: Real-time PCR & 16S rRNA results

Streptococcus pneumoniae 11Staphylococcus aureus (includes 2 Methicillin-resistant) 3Streptococcus agalactiae 2Elizabethkingia meningoseptica 2Enterococcus sp. 2Staphylococcus epidermidis 1Escherichia coli or Shigella flexneri or Shigella dysenteriae 1Enterobacter kobei or Enterobacter cloacae or Enterobacter ludwigii, or Leclercia adecarboxylata 1

Fungus/Virus10

135 Samples Obtained

Discordant28

Hosp (+) WC (-)12

Hosp (+) WC (+)2

Hosp (-) WC (+)14

Tube Broken1

Fungus/Virus10

135 Samples Obtained

Discordant28

Hosp (+) WC (-)12

Hosp (+) WC (+)2

Hosp (-) WC (+)14

Tube Broken1

WC identified bacteria undetected by hospital culture

Streptococcus pneumoniae 5

Streptococcus agalactiae 2Viridans group streptococci (one intermedius and one salivarius) 2

Haemophilus influenzae 1

Stenotrophomonas maltophilia 1

Enterococcus faecium 1

Streptococcus cristatus 1

Klebsiella pneumoniae 1

Fungus/Virus10

135 Samples Obtained

Discordant28

Hosp (+) WC (-)12

Hosp (+) WC (+)2

Hosp (-) WC (+)14

Tube Broken1

Hospital identified bacteria undetected by WC

Streptococcus pneumoniae 2Enterobacter sp. 2Enterococcus sp. 2Viridans group streptococci 1Elizabethkingia meningoseptica 1Klebsiella pneumoniae 1Pseudomonas aeruginosa 1Corynebacterium sp. 1Klebsiella pneumoniae or Acinetobacter baumanii or Enterobacter cloacae 1

Fungus/Virus10

135 Samples Obtained

Discordant28

Hosp (+) WC (-)12

Hosp (+) WC (+)2

Hosp (-) WC (+)14

Tube Broken1

Hospital and WC conflicting identifications

Hospital Identified WC Identified

Staphylococcus epidermidis Klebsiella pneumoniae

Klebsiella pneumoniae MRSA

PCR Sensitivity

For confirmed cases of Nm Real-time PCR yielded a sensitivity of 100%

6 0Culture (+)

PCR (+) PCR (-)

For other bacterial organisms Real-time PCR yielded a sensitivity of 89%

Culture (+)

PCR (+) PCR (-)

Confirmed cases of Nm during the interval were also sent for real-time PCR testing

16 2

Summary

Nm was not found in any culture negative specimens

Bacterial organisms that were identified in this study PCR sensitivity was:

100% for confirmed Nm 89% for other organisms

Discussion

PCR sensitivity and specificity for detecting organisms after antibiotic therapy

Length of time on antibiotics? Not enough samples> 24 hours to evaluate (n=10).

Contaminant species were suspected where the hospital positively identified an organism >24 hours

16S rDNA sequencing Is useful in identifying unusual organisms

6 organisms identified by WC that hospital did not detect

Limitations

Sample size CSF availability & volume Tube sent for PCR and 16S rRNA gene may not have been same as

for culture

Specimen quality Degradation of sample during transport to WC

16S rRNA gene sequencing is usually performed on cultures Performed on primary specimens; novel application of method

Conclusion

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 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 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

Don Weiss Lola Arakaki Linda Steiner-Sichel Erlinda Amoroso Mike Antwi Paula Del Rosso Marie Dorsinville Prabhu Gounder Marci Layton Lan Li Laura Miller

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 Ramautarar2568@nyu.edu