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