# Presentation: 140152

## Session 2B Lab Diagnosis

- Third National Planning Meeting for Surveillance, Prevention
- and Control of West Nile Virus in the United States
- March 22, 2002

## Rob LanciottiCDC DVBID

- Current techniques for diagnostics and new developments in molecular diagnostics

## Serological Assays for West Nile Virus

- Notes:
- Assay for a variety of agents, not just WN
- for IgM ELISA
- 80%: WN P/N>2X SLE P/N
- 20%: Atypical serological profile
-     or secondary flavivirus infection
-         best to do full profile of
-         serological tests (IgM, IgG,
-         SNPR when appropriate) , not
-         just 1 assay to determine
-         flavivirus etiology

## Recommended Tests for WN Virus

- Oral swabs ~ brain tissue assay

## Virus/Antigen Detection Assays for West Nile Virus

- Notes:
- A variety of assays available, not all the same sensitivity
- VecTest & antigen ELISA ~sensitivity
- VecTest detected~60% Taqman +
- Standard RT-PCR~80% Taqman+

- Notes: Panel consensus is that cell culture is a valuable tool for the detection of additional & “unknown” viruses & to detect additional target viruses and should not be abandoned in testing algorithms

## Diagnostic & Reference SectionTaqMan & NASBA Assays 

- Multiplex screening

## Laura Kramer Wadsworth Center, NY

- Automating assays to deal with
- large sample sizes-
- a case study;

## RT-PCR assays on avian tissue

**RT-PCR assays on avian tissue**

**Number**

**Week of year, 2001**

- Notes: a sample is positive only if confirmed in 2 separate assays, therefore the number of assays exceeds the number of samples.
- During height of season, ~1000 assays per week-must automate to stay current

## High Throughput Testing

- Automated Nucleic Acid Workstation
- Automates sample and reaction preparation for nucleic acid analysis
**increase in productivity **
**cost efficient**
**high quality of product**
**decreased cross-contamination**
**consistency and reproducibility**

- Notes: Must validate instrument by comparison testing; large capital outlay, savings in labor (tech time), reagents/supply costs similar in manual and automated

## Summary of High Throughput  Techniques

- Submission of sample data to laboratory on Excel spreadsheets
- High capacity mixer mill
- Robotic workstation for RNA extraction and real time RT-PCR setup

- Notes: computer data entry is very time consuming; data can be submitted in excel and imported into data base;
- mixer mill greatly speeds up sample trituration and prevents cross contamination; can triturate in either cell culture diluent or directly in lysis buffer for molecular assay

## Susan WongWadsworth Center, NY

- Is ELISA challenged by the
- arbovirus IFA test and
- new technologies?

## Advantages of ELISA

- More objective readout than by IFA
- Spectrophotometric reading O.D. of microplate wells
- Time efficient
- 96 well plates can be  read in a few minutes
- partially automated by automatic pipetting stations
- Signal amplification by reporter enzyme
-  Analytic sensitivity  of the MAC-ELISA provides greater window of detection (often + close to onset)
- MAC-ELISA suitable for testing cerebral spinal fluids as well as serum samples

## How does arbovirus screening with IFA slides stack up for WNV surveillance of humans?

- It depends....
- Comparative sensitivity of assays
- Time period over which IgM and IgG are present at detectable levels
- Purpose of the surveillance efforts
- What other complementary surveillance programs are available (dead bird, mosquito, other vertebrates)

- Notes: MRL IFA is screen for IgG or IgM antibody to alpha (EEE,WEE) , flavi (SLE) & bunya (CAL) viruses using infected VERO cells fixed on a slide.  Requires experienced tech

## Detection Windows of Opportunity

**30**
**25**
**20**
**15**
**10**
**  **
**  ****5**
**  **
**  ****0**

**0          50         100        150        200        250        300        350        400**

- IFA IgG
- IFA IgM
- MAC - ELISA “Window”
- IgG - ELISA “Window”

**Serum MACELISA P/N**

- Cut Off

**Serum IgG ELISA P/N**

- Notes: IFA less capable of detecting IgM response

## Suspension Array Technology Sequence of Events

-         Notes: Assay in early development:
- antigen is covalently bound to a polystyrene bead; incubate with patients serum and PE-labeled anti-antibody; pass through flow cell with laser activation to detect antibody binding

## There are a variety of laboratory assays for arbovirus detection/isolation and antibody detection.  
These assays differ in sensitivity, specificity and appropriateness of use
Many new instruments and technologies are available.  Cost and labor needs are significant factors in selection.
All instruments and assays must be validated before implementation into routine testing.

- There are a variety of laboratory assays for arbovirus detection/isolation and antibody detection.
- These assays differ in sensitivity, specificity and appropriateness of use
- Many new instruments and technologies are available.  Cost and labor needs are significant factors in selection.
- All instruments and assays must be validated before implementation into routine testing.