A COMPARISON OF WEST NILE VIRUS DETECTION METHODS (TaqMan RT-PCR, VecTest Antigen Assay and RAMP method) IN COOK COUNTY, ILLINOIS, 2002-2009

Nina M. Krasavin, Richard L. Lampman, Daniel R. Bernard and Barry AltoUniversity of Illinois, Illinois Natural History Survey, Medical Entomology Program, Champaign, IL

y = -8.1648x + 239.53R2 = 0.93

y = -11.045x + 283.09R2 = 0.91

y = -8.8321x + 245.49R2 = 0.82

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TaqMan RT-PCR Cycle Threshold

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COMPOSITE

RESULTS

Influence of TaqMan cycle threshold on VecTest detection of West Nile virus.Over 90% of the variation in the percent VecTest detection of TaqMan positive pools could be explained by cycle threshold based on linear regressions for both MADs. Between cycles 18 and 28 there was a linear decrease in VecTest detection. Detection above CT of 28 was rare and below 18 it was rarely missed by VecTest.

AcknowledgementsData from Cook Co. was provided by Mike Szyska, Tom Anderson &, Paul Geery.

I’d like to thank all the personnel in the Medical Entomology Program that helped process the samples,

as well as helped with data entry. Specifically, Jen Johnson & Millon Blackshear helped process and run the mosquito samples

MATERIALS AND METHODS

Relative Sensitivity of VecTest based on TaqMan Cycle Threshold

AmplificationRAMP comparison to TaqMan (TQ) cycle threshold (CT).RAMP values were negatively correlated to TQ CT.Previous field studies indicated the relationship was often poor when field specimens were used. This may be related to the processing of the samples (MAD test results showed a wide cloud of points around the regression line and they were not evenly distributed.

WNV surveys by VecTest and RAMP (RT-PCR TaqMan as Gold Standard)1. The average false negative for VecTest was 51.9%, which was larger than the false negative rate

of 32.8% for RAMP

2. A comparison of sensitivity and selectivity demonstrated RAMP had 4 times greater sensitivity

than VecTest. The specificity for the two tests were comparable.

3. False positives were initially high until MADs removed any blooded mosquitoes and started to

double centrifuge mosquito samples. Debris appeared to affect RAMP values.

4. RT-PCR continued to provide early warning for positives, although RAMP had a much improved

sensitivity.

Cook Co. MAD Year

Total Pools

Total Positive

Percent PCR +

VecTest+ PCR -

VecTest– PCR +

VecTest+ PCR +

% VecTest "False -"

DVMAD 2002 449 214 47.7% 3 93 121 43.5%2003 710 135 19.0% 2 81 54 60.0%2004 665 160 24.1% 1 102 58 63.7%2005 640 209 32.7% 3 115 94 55.0%2006 1031 290 28.1% 9 156 134 53.8%2007 1673 321 19.2% 3 216 48 74.5%2008 1480 137 9.3%2009 1624 51 3.1%

NWMAD 2002 342 92 26.9% 3 29 63 31.5%2003 966 94 9.7% 0 51 43 54.2%2004 821 257 31.3% 5 115 142 44.7%2005 1102 480 43.6% 19 139 341 28.9%2006 1563 650 41.6% 18 366 292 56.3%2007 857 177 20.5% 9 101 76 57.1%2008 751 110 14.6%2009 800 61 7.3%

Table 1. VecTest vs RT-PCR for Cook County MAD's 2002-2009

* Switched to RAMP Testing* See Table 2

* Switched to RAMP Testing* See Table 2

Composite Comparison RT-PCR to RAMP

Combined NWMAD + DVMADy = -0.0124x + 33.328

R2 = 0.3263

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DVMAD 2007 1673 19.2% 57 14 11 46 19.3%2008 1480 9.3% 133 2 67 66 50.4%2009 1624 3.1% 49 13 11 38 22.4%

NWMAD 2007 857 20.5%2008 751 14.6% 96 2 35 61 36.5%2009 800 7.3% 51 4 18 33 35.3%

RAMP- PCR +

*no ramp data available

RAMP+ PCR +

% RAMP "False -"

Table 2. RAMP vs RT-PCR for Cook County MAD's 2007-2009Cook Co.

MAD Year Total Pools

PCR + & RAMP Tested

Percent PCR +

RAMP+ PCR -

Sensitivity and Specificity Test

DVMAD ran both VecTest and RAMP assays during the transition from one testing method to the other. This allowed us to do a direct comparison of RT-PCR to theVecTest and RAMP in 2007, while eliminating operator and annual variation. The data was used to calculate the sensitivity and specificity of the two tests from the DVMAD results and our confirmations.

PCR - PCR + Total PCR - PCR + Total VEC + 3 48 51 RAMP + 14 46 60VEC - 1052 216 1268 RAMP - 283 11 294 Totals  1055 264 1319  Totals  297 57 354

VecTest RAMP Estimated

ValueLower Limit

Upper Limit

Estimated Value

Lower Limit

Upper Limit

Prevalence 0.200 0.179 0.223 Prevalence 0.161 0.125 0.204Sensitivity 0.182 0.138 0.235 Sensitivity 0.807 0.677 0.895Specificity 0.997 0.991 0.999 Specificity 0.953 0.920 0.973

Table 3. DVMAD 2007 Side by Side Comparison of VEC Test and RAMP Techniques

95% Confidence Interval

95% Confidence Interval

POOLING

COLLECTION

SORTING

Protocol for mosquito handlingin WNV/SLE surveillance

light/CO2 trap gravid trap

New Jersey trap

date, collection site,species, number, sex

mosquitoes, 50 max, in 2.0 ml plastic tubes, label tubes,

Temp. Critical! (keep on ice)

Biorobot 9600 (Qiagen)

TaqMan RT-PCR system (7000 pictured)

VecTest Strips

Controllines

WNV Positive

line

RAMP Machine

Testing by MAD’s

RNA Extraction

RT-PCR Testing

ABSTRACT Pools of Culex pipiens and Culex restuans from two Cook County mosquito abatement districts, Desplaines Valley and Northwest (DVMAD and NWMAD), were assayed for West Nile virus by either VecTest strips or RAMP slide assays and later tested for (WNV) RNA by TaqMan RT-PCR. The annual pattern of percent WNV positive mosquitoes from the 2 areas (RT-PCR) demonstrated a sustained period of high incidence from 2002 to 2006, which subsequently declined to a relatively low percentage by 2009. We were able to use the MAD test results by VecTest and RAMP to make a comparison between those tests and RT-PCR Taqman (assigned the role of Gold Standard for WNV presence). The RAMP test was more sensitive than the VecTest (fewer false negatives); however, both tests had a high specificity(few false positives). There were initial problems with the RAMP test related to the sample source (blooded mosquitoes were discarded) and processing (consistency of RAMP values improved with double centrifugation of the samples). The VecTest, based on its lower sensitivity, probably gives a better approximation of infectious vectors, whereas as RAMP and RT-PCR, based on enhanced sensitivity, tend to provide a better early warning of the transmission onset.