Post on 09-May-2018
Laboratory Diagnosis of Meningitis
and Encephalitis
February 3rd, 2018
Kevin Alby PhD, D(ABMM)
Director, Clinical Microbiology
Hospital of the University of Pennsylvania
2
Outline
Overview and epidemiology of meningitis/encephalitis
Review of select current diagnostic assays
Future directions and other considerations
3
Meningitis
Estimates of
approximately 4000
cases and 500 deaths
annually in the US due
to bacteria
• Leading causes are S.
pneumoniae, GBS, N.
meningitidis, H. influenzae
and L. monocytogenes
Frequency of viral
meningitis in difficult to
calculate due to
insufficient diagnostic
testing
Figure : National Meningitis Association
4
Encephalitis
Approximately 60% of
cases do not have an
etiologic agent
Bacterial, viral, fungal,
parasitic causes have all
been documented
Many non-infectious
causes have also been
described
Figure: Encephalitis Society
5
Recommended algorithm for evaluation of encephalitis in adults
Venkatesan, Clin Infect Dis, 2013. 57(8):1114-28
6
Incidence of bacterial meningitis by
organism in the US 1997-2010
Castelblanco, Lancet Infect Dis, 2014. 14(9):813-9
7
Community onset meningitis
Analysis of 611 patients from
a Houston hospital over a 6
year span (2005-2010)
Acute vs sub-acute defined
on duration of symptoms
(<=5 or >5 days respectively)
Sulaiman, Medicine, 2017. 96(36):e7984
8
Healthcare associated neuroinvasive
infections in adults
Data from an 11 year period
at a single tertiary care US
hospital
Considered patients with
diagnosis of healthcare
associated meningitis or
ventriculitis
Srihawan, J Am Geriatr
Soc, 2017. 65(12):2646-
2650
9
Aseptic meningitis in children and adults
Shukla, J Clin Virol, 2017. 94:110-114
Inclusion = >5 cells/mm3 in CSF, negative Gram stain and culture
Exclusion = Positive CrAg, blood cultures, or signs of abscess
10
Viral ME in Chinese Children 2009-2012
Meningitis Encephalitis
Ai, BMC Infect Dis, 2017. 17(1):494
3 year multicenter prospective study
PCR for Enteroviruses and herpes multiplex (HSV, VZV, EBV, CMV, HHV-6)
CSF IgM for JEV. Serum IgM for TBEV, dengue, mumps, rubella
11
Enterovirus and parechovirus in children
285 children with suspected
EV infection
Blood, urine, stool, NP, and
CSF samples were tested via
RT-qPCR and viral culture
De Crom, J Clin Virol, 2013. 58:449-454
Performance of other samples for patients with meningitis (positive CSF)
12
The arboviruses – 2016 US incidence of
neuroinvasive disease
West Nile – 2,150
La Crosse – 35
Powassan – 22
Jamestown Canyon – 15
Saint Louis Encephalitis – 8
Eastern Equine Encephalitis – 7
3 unspecified California
serogroup virus cases
Burakoff, MMWR, 2018. 67(1):13-17
13
Other things to consider…
Cryptococcus
Histoplasma
Other bacteria, especially
with indwelling devices or
zoonotic exposures
Other fungi either native
or iatrogenic
Karakas, Science, 2014. 344(6187):992-97
14
Outline
Overview and epidemiology of meningitis/encephalitis
Review of select current diagnostic assays
Future directions and other considerations
15
Detection of bacterial ME
16
Improved detection of bacterial ME?
1326 Samples inoculated to agar plates and blood culture
bottles (Bactec)
Of 70 samples only positive via bottle, 30 were considered
definite or probable infections.
Bottle only included Candida, various Enterobacteriaeceae,
and Staphylococcus aureus.
Yoo (JCM 2016) study showed similar findings with BacT/Alert
Calderaro, Diagn Microbiol Infect Dis, 2016. 84(4):281-6
17
Diagnosis of fungal ME
18Litvintseva 2014 Clin Infect Dis 58(5):622-30
1-3-Beta-D-glucan as a marker in cases of
Exserohilum infection
19
Diagnosis of HSV Encephalitis
Diasorin Simplexa assay
• Binnicker et al 100 sample retrospective study showed 96.2%
sensitivity and 97.9% specificity compared to LDT
• Kuypers et al 318 sample retrospective study showed 87.5%/91.7%
positive agreement for HSV-1/HSV-2 respectively. Also demonstrated
a 99.0%/98.1% Negative agreement
– 12/14 discordant samples had a Ct >35
20
Multicenter evaluation of Xpert EV
Nolte, J Clin Microbiol, 2011. 49(2):528-33
21
FilmArray ME Panel
22
HUP evaluation of ME
17 previously positive frozen samples
• 5 HSV-2
• 8 VZV
• 4 Enterovirus
44 semi-prospective frozen samples
23
HUP ME results – Previous Positives
5 HSV -2
• All confirmed, no additional targets detected
4 Enterovirus
• All confirmed, no additional targets detection
8 VZV
• All confirmed
• 3 detected additional targets (HSV-2, CMV, H. influenzae)
• 1 (HSV-2) QNS for repeat
• 2 (CMV and H. influenzae) negative upon repeat ME testing
24
HUP Semi-prospective – Initial test results
0
5
10
15
20
25
30
35
40
45
50
Cx HSV VZV JCV Entero CMV EBV HHV-6
Co
un
t
Test
PosNeg
3 samples had growth upon culture
(2 Cryptococcus neoformans, 1 single colony S. hominis)
1 sample had a positive JCV PCR
25
HUP Semi-prospective – ME results
44 samples tested
• 36 negative for all targets
• 2 positive for Cryptococcus (both Cx confirmed)
• 1 positive for Enterovirus (not originally requested – PCR confirmed)
• 1 positive for HHV-6 (not originially requested – PCR confirmed)
• 1 positive for H. influenzae (not Cx confirmed)
• 1 positive for S. pneumoniae (not Cx confirmed)
26
Evaluation of ME Positive/Cx Negative
H. influenzae
• Repeat ME was negative
• WBC 34->12; Protein 34, Glucose 82,
• Pt presented with AMS and left-sided weakness
• Discharge Dx = Stroke
S. pneumoniae
• Repeat ME was negative
• WBC1->2; Protein 45, Glucose 73
• Pt hx of CTCL, admitted with status
• Discharge to hospice, no definitive dx, likely related to CTCL
27
Performance of ME RUO in patients with HIV
Rhein, Diagn Microbiol Infect Dis. 2016 84(3):268-273
*RUO panel included EBV
28
Co-infections and unexpected results
48 patients had residual CSF tested
15 samples positive by ME (RUO)
• 8 EBV
• 4 S. pneumoniae
• 3 HSV-2
• 3 VZV
• 1 HSV-1
• 1 Enterovirus
• 1 Cryptococcus
6 co-infections
• EBV + another pathogen
13 pathogens not originally found/tested in CSF or Blood
• 8 EBV
• 3 S. pneumoniae
• 2 HSV-2
• 2 VZV
Wooten, Ann Clin Microbiol Antimicrob. 2016, 15(1):26
29
Evaluation of ME on pediatric samples
Graf, Diagn Microbiol Infect Dis, 2017. 87(1):92-94
30
Evaluation of ME on pediatric samples
Graf, Diagn Microbiol Infect Dis, 2017. 87(1):92-94
31
Multi-center evaluation of ME panel
Leber, J Clin Microbiol, 2016. 54(9):2251-61
32FilmArray ME PI
Performance data from Package Insert
33FilmArray ME PI
Performance data from Package Insert
Study on S. pneumoniae LDT by Algayoudhi demonstrated 98%
specificity with 11% false positive detection rate (Pediatr Infect Dis J
36(9):833-836)
34
Multi-center evaluation of ME panel
Leber, J Clin Microbiol, 2016. 54(9):2251-61
35
Outline
Overview and epidemiology of meningitis/encephalitis
Review of select current diagnostic assays
Future directions and other considerations
36
Performance of Xpert MTB/RIF Ultra for
diagnosis of MTB
Bahr, Lancet Inf Dis, 2018. 18(1):68-75
37
Fast Track Diagnostics
CE Marked Kits
• Viral meningitis
– HSV 1/2, VZV, Mumps virus, Enterovirus, Parechovirus
• Bacterial meningitis
– N. meningitidis, S. pneumoniae, H. influenzae,
• Neonatal meningitis
– Streptococcus agalactiae, L. monocytogenes, E. coli
• “NEURO 9”
– CMV, EBV, Adenovirus, HSV 1/2, VZV, Enterovirus, Parechovirus,
HHV-6/7, Parvovirus
Evaluation of 470 CSF samples with BM and NEURO9 panels
• 21% positivity
• 25% Enterovirus, 22% Adenovirus, 15% S. pneumoniae
• Only 7/14 S. pneumoniae and 1/8 N. meningitidis recovered in culture
Akkaya, Clin Lab, 2017. 63(4):827-832
38
TaqMan Array for ME
Onyango, J Clin Microbiol, 2017. 55(7):2035-2044
39
Onyango, J Clin
Microbiol, 2017.
55(7):2035-2044
TaqMan Array
Performance
Tested 120 specimens
35 were known positive via
individual target real-time
PCR
40
Lightmix RT-PCR
Target Culture Lightmix
H. influenzae 0 0
L. monocytogenes 0 0
N. meningitidis 0 1
S. agalactiae 0 1
S. pneumoniae 6 8
E. coli 2 0
K. pneumoniae 1 0
S. marcescens 1 0
S. epidermidis 4 0
S. hominis 2 0
Testing of 220 CSF specimens via Lightmix multiplex PCR
Wagner, J Clin Microbiol, 2017 Epub
41
Using MALDI for Gram stain positive CSF?
Bishop, Clin Microbiol Infect, 2017. Epub
44 Samples
Extracted GS positive CSF using an formic acid/acetonitrile based extraction
Spotted on Vitek MS
GNR - 17/21 correct, 1 incorrect, 3 no ID
GPC - 1/20 correct, 2 incorrect, 17 no ID
Yeast - 0/2 correct, 1 incorrect, 1 no ID
GNDC – 0/1 correct, 1 no ID
42
Next-Generation Sequencing
43
Use of ME in neonates
Arora, Pediatr Infect Dis J, 2017. 36(7):685-687
44
Impact of Enteroviral Testing in Infants
Aronson, J Pediatr, 2017. 189:169-174
45
Use of Xpert EV outside of core virology lab
Ninove, Clin Microbiol Infect, 2011. 17:1890-94
46
Value of Rapid Testing
Retrospective study from CHLA looking at 363 cases of
negative HSV testing
• 182 had batched LDT HSV testing performed (Median TAT 23.6h)
• 181 had rapid sample to answer HSV testing performed (Median TAT
9.1h, p<0.001)
• Reduced median time from sample collection to discontinuation of
acyclovir by 17 hours
Van, J Clin Microbiol, 2017. 55(5):1557-1565
47
Value of selective testing
Wilen, J Clin Microbiol, 2015. 53(3):887-895
48
Value of selective testing
Criteria
• >10 WBC for immunocompetent patients
• Seasonal (April-October) Enterovirus testing
Wilen, J Clin Microbiol, 2015. 53(3):887-895
49
How do we interpret CNS testing results?
Labska, J Med Virol, 2015. 87(7):1235-40
50
Summary
Laboratory diagnosis of meningitis/encephalitis is critical for
patient care
Laboratory diagnosis of meningitis/encephalitis is
multifaceted with many complexities, not suitable to one stop
shopping
New assays are constantly being developed to aid in the
diagnosis of meningitis/encephalitis
51
Questions?
Comments?
Kevin.alby@uphs.upenn.edu