June 3, 2003 1

Hema Kapoor MD. SM (NRM)

Virology Section manager

Bureau of laboratories

Michigan Department of community Health

Lansing Michigan

Laboratory Issues and West Nile Virus

June 3, 2003 2

Laboratory Issues and West Nile Virus

ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV

outbreak in MichiganPlans for 2003-Lab perspective

June 3, 2003 3

The arboviruses

Arthropod-borne viruses(mosquitoes, sand-flies, fleas, ticks, lice, etc)

Enveloped RNA viruses -4 familiesFlaviviridae-WNV isolated in 1937 in

west Nile district of Uganda in Eastern Africa.

June 3, 2003 4

Arboviral encephalitides

Mosquitoeastern equine encephalitis (EEE)western equine encephalitis (WEE) St. Louis encephalitis (SLE) La Crosse (LAC) Ticks Powassan

June 3, 2003 5

WNV Transmission

Dead end host

Dead end host

New modes of transmission

June 3, 2003 6

Laboratory Issues and West Nile Virus

ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV

outbreak in MichiganPlans for 2003-Lab perspective

June 3, 2003 7

DAYS POST ONSETDAYS POST ONSET

1 2 3 4 5 6 7 8 9 10-5 -4 -3 -2 -1 0

IgM

IgG

ELISAELISAP/NP/N#pfu/ml#pfu/ml

WNviremia

150

illnessillness

Immune Response in WNV Infection

Ref: 4th National WNV Conf. 2003 www.cdc.gov

June 3, 2003 8

Laboratory Diagnosis of Human cases

• SpecimensCerebrospinal fluid (CSF)CSF and Serum combinationSera- Acute and Convalescent (obtained

at least 8 d and 22 d post onset respectively)

Least preferred single serum sample

Ref: Antibody Capture ELISA (IgM & IgG) Protocol. CDC Fort Collins, colorado

CDC Neutralization Test Protocol. CDC Fort Collins, colorado

June 3, 2003 9

Laboratory Diagnosis of Human cases contd

Ref: Antibody Capture ELISA (IgM & IgG) Protocol. CDC Fort Collins, colorado

CDC Neutralization Test Protocol. CDC Fort Collins, colorado

•Laboratory Tests

Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgM).

Capture enzyme-linked immunosorbent assay (MAC-ELISA-IgG) and

Plaque Reduction Neutralization Test (PRNT)

June 3, 2003 10

Serological Testing Criteria at Michigan

CSF Paired sera(Ac. at least 8 days and conv at least 22 days post onset)

Single serum

•P/N ratio >/= 10 positive

•P/N 2-10 equivocal and requested for a serum sample.

•P/N ratio >/=5 presumptive. •P/N 2-5 equivocal.

•IgG Tests -A four-fold rise in titre to distinguish a recently acquired infection from a past infection.

•PRNT was performed on all specimens showing a four fold increase in IgG titer.

•P/N ratio- same as in paired sera.

•A convalescent serum requested on equivocals .

•PRNT To rule out the cross- reactions between WNV and other arbovirus infections (SLE, EEE and CGV).

•MAC-ELISA IgM performed in Singlet. Positive MAC-ELISA repeated in duplicate

June 3, 2003 11

West Nile Virus Nucleic Acid Amplification Tests (NAAT) Diagnostic Testing

Higher persistent levels of WNV RNA found in immuno-compromised patients

Improved sensitivity with better quality of diagnostic samples or virus concentration methods?

WNV RNA(-) results are meaningless

June 3, 2003 12

Identifying viremic / antibody negative “window phase” patients

Low levels WNV RNA found in clinically ill, immuno- competent patients (i. e., WN fever)

West Nile Virus Nucleic Acid Amplification Tests (NAAT) Diagnostic Testing

June 3, 2003 13

Laboratory Issues and West Nile Virus

ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV

outbreak in MichiganPlans for 2003-Lab perspective

June 3, 2003 14June 3, 2003June 3, 2003

55

94

240

266

319

270 270289

333

193

144

231

7055

422311 16 20 20

91

61 65

38 38

12 16 14 620

0

112

0

50

100

150

200

250

300

350

11-Aug

18-Aug

25-Aug

01-Sep

08-Sep

15-Sep

22-Sep

29-Sep

06-Oct

13-Oct

20-Oct

27-Oct

03-Nov

10-Nov

17-Nov

24-Nov

Weeks

Nu

mb

ers

total positive

Total Specimens (Jan, 02-Dec, 02)-2910

Weekly WNV Testing (Aug-Nov 02)

June 3, 2003 15

Virology section and intersection staff worked collectively 6 days a week

How we handled the 2002 outbreak in the laboratory ?

Triaged the samples

June 3, 2003 16

CSF at MDCH 0 day

IgM ELISA 1st Run Report

via EPIC

NegNotificationVia EPIC toSubmitter,

LDH & EPI 4 day

Pos

Investigation

Confirmed positive reportsVia

EPIC 5 6day

IgM ELISA 2nd Run

pos

Quantity Sufficient

pos

Equivocal

Request a convalescent serum sample

QNS

To report a Probable case

request a serum sample

June 3, 2003 17

Convalescent serumNo

PRNT on acute serum (Convalescent sample not possible )

Pos

Acute Serum at MDCH (0day)

NegIgM ELISA 1st runReport Via EPIC

No 4x increase

4x rise in titer

Yes

PRNT on pair

Confirmed positive reportsVia

EPIC

Arboviral

Panel

IgM ELISA 2nd run

Reported a presumptive positive (LAB) & probable case (EPI)Via EPIC with a request for a convalescent serum

Pos

Pos

Pos

Investigation

Notification via EPIC to

Sub, LHD & EPI

June 3, 2003 18

Single serum with documented CNS symptoms or paired sera without 4X increase in titer

IgM ELISA (+) PRNT (-)

Test for SLE, EEE and CGV

Probable WNV Case

Neg

Yes

Single serum collected too early (0-8 d) post onset

No

Paired sera

Yes

No WNV Case

June 3, 2003 19

Laboratory Issues and West Nile Virus

ArbovirusesLaboratory DiagnosisInvestigation of 2002 WNV

outbreak in MichiganPlans for 2003-Lab perspective

June 3, 2003 20

Plans for 2003

Human Surveillance No triaging of samples Test whole panel Two LT positions in virology for WNV testing Cross training IgM capture ELISA testing on CSF and Paired serum Attempt culture on for WNV

June 3, 2003 21

Issues with Persistence of IgM antibodies

Getting Ready for summer of 2003 contd

June 3, 2003 22

Longevity of Human WN Virus-Reactive IgM in Serum

Days P.I.

N Positive MAC-ELISA Total (%)

Ave. P/N (Range)Positive

(%)Equivocal

200 22 13 (60) 4 17 (77) 6.0 (3.0-10.8)

300-400

21 9 (43) 2 11 (52) 4.0 (31.-6.5)

500 12 5 (42) 2 6 (60) 5.0 (3.1-6.9)

Ref: 4th National WNV Conf. 2003 www.cdc.gov

June 3, 2003 23

Hold and request for the Convalescent serum

PRNT on acute serum

Pos

No

CNS symptoms +

Approval of Virology Lab Manger

Neg IgM ELISA 1st Run

IgM ELISA 2nd RunPos

Pos

Report

Acute Serum at MDCH

2003 Testing

Yes

4x rise in titer

PRNT (IgG) on pair

Confirmed positive reportsVia

EPIC

No 4x increaseArboviral

Panel

June 3, 2003 24

Persistence of IgM antibodies

In CSF-No studies

Published-47d

MDCH observations-Three cases with igM positive

110d

141d

199d

June 3, 2003 25

To Sum Up……. Human Cases ?

Over wintering

adult Culex mosquitoes