Journal of Clinical Virology - Volume 82 - Supplement 1

S22

Abstracts / Journal of Clinical Virology 82S (2016) S1–S142

Abstract no: 103

Presentation at ESCV 2016: Poster 1

Direct fluorescent antibody, focus diagnostics

Simplexa

Flu A/B & RSV and multi-parameter

customized respiratory Taqman

Array Card

testing in immunocompromised patients

D. Steensels

1 ,

∗

, M. Reynders

P. Descheemaeker

, M.D. Curran

, F. Jacobs

O. Denis

, M.-L. Delforge

, I. Montesinos

Department of Clinical Microbiology, Erasme

Hospital, Université Libre de Bruxelles, Brussels,

Belgium

Department of Clinical Microbiology, AZ St-Jan

Brugge-Oostende Hospital, Brugge, Belgium

Public Health England, Clinical Microbiology and

Public Health Laboratory, Addenbrooke’s Hospital,

Cambridge, United Kingdom

Department of Infectious Diseases, Erasme Hospital,

Université Libre de Bruxelles, Brussels, Belgium

Background:

Molecular assays for diagnosis of influenza A,

influenza B, and respiratory syncytial virus (RSV) with short

turnaround time are of considerable clinical importance. These

viruses are clinically most important since they are the most fre-

quently encountered, since they cause substantial disease burden

and since a targeted treatment and prevention exists. In addition,

rapid and accurate diagnosis of a large panel of viral and atyp-

ical pathogens can be crucial for an appropriate and sometimes

life-saving clinical management in immunocompromised patients.

Objectives:

The goals of the present study are firstly to compare

the sensitivity and specificity of direct fluorescent antibody (DFA)

(Argene and Light Diagnostics), Simplexa

Direct assay system

and a customized Taqman

Array Card (TAC) testing for RSV, Flu A,

and Flu B. Secondly to compare the cost, turnaround time (TAT) and

diagnostic yield of different algorithms for the detection of respi-

ratory pathogens in this group of immunocompromised patients.

And finally to test the Simplexa

assay on BAL samples, which has

only been validated on nasopharyngeal swabs (NTS) so far.

Study design:

We collected 125 NTS and 25 BAL samples

from symptomatic immunocompromised patients. Samples for

which Simplexa

and TAC (premarket version Cambridge-Brugge)

results were discordant underwent further verification testing

using the multiplex real-time PCR assay FTD Flu/HRSV (Fast-track

Diagnostics), on the nucleic acid extract that was used for TAC test-

ing. The TAC assay is based on singleplex, reverse transcription

real-time PCR, targeting 24 viruses, 8 bacteria and 2 fungi simulta-

neously.

Results:

As expected, the overall sensitivity was significantly

lower for DFA testing than for the twomolecularmethods (

< 0.05).

However, when considering results for each pathogen separately,

the difference in performance between DFA and molecular meth-

ods was only statistically significant for Flu A. The Simplexa

direct test missed one RSV, one Flu A and two Flu B positive samples

in comparison to the TAC assay and verification PCR. One sample

was found strongly positive for Flu A by Simplexa

(Ct 15), but

was negative by viral culture, TAC and verification testing. Nev-

ertheless, the differences in individual and overall sensitivity and

specificity of Simplexa

testing were not significant compared to

TAC testing (

> 0.1). For BAL samples only (

= 25), the sensitivity

and specificity of the Simplexa

assay was 100%.

In total, DFA identified14 samples (9.3%) and Simplexa

testing

found 24 (16%) samples positive with one pathogen each. The TAC

assay identified 93 sampleswith one ormore respiratory pathogens

(62%). More than half (54%) of Simplexa

negative samples were

positive by TAC for other pathogens than RSV, Flu A and B. A co-

infection rate of 15.3% was found by TAC.

The estimated costs and TAT were 8.2

and 2 hours for DFA,

31.8

and1.5 hours for Simplexa

and56

and6 h for TAC testing.

Conclusions:

Based on these results, performing a first line

molecular method such as the Simplexa

test instead of DFA

would be necessary to obtain an acceptable overall sensitivity,

albeit at a higher cost generated in the laboratory. Performing the

TAC as a second line test for patients with a negative Simplexa

result would increase the diagnostic yield significantly, albeit at an

even higher cost.

http://dx.doi.org/10.1016/j.jcv.2016.08.041

Abstract no: 106

Presentation at ESCV 2016: Poster 2

Correlation between a new BioPlex

2200 ToRC

IgM assay and established commercial assays

for the detection of IgM antibodies to

T. gondii

rubella, and cytomegalovirus

J. Ford, C. Chung, M. Leos, H. Scholz, J. Vogel

Bio-Rad Laboratories, Hercules, CA, United States

Background:

Toxoplasmosis, cytomegalovirus (CMV) infection,

and rubella are diseases that can cause serious complications dur-

ing pregnancy. Traditionally, laboratories test for antibodies to

these diseases using manual methods such as enzyme immunoas-

say (EIA). EIA can be associated with challenges such as low

specificity, irreproducibility, low throughput, and high demand for

labor. As laboratories are moving towards a centralized and auto-

mated core lab, there has been a transition away from manual

testing towards fully automated test systems.

The improved Bio-Rad BioPlex 2200 ToRC IgM assay is a multi-

plex flow immunoassay, currently in development, employing an

array of

T. gondii

, Rubella, and CMV antigen coated beads as individ-

ual substrates in order to simultaneously detect antibodies reactive

to these pathogens in a single reaction vessel using a single sam-

ple. Since the BioPlex 2200 substrates are segregated by unique

fluorescent signatures, the presence of specific antibodies can be

individually determined in a single test.

Methods:

Samples from a presumptive positive population

(Toxo IgM

= 152, Rub IgM

= 133, and CMV IgM

= 153), a test

ordered sample population (Toxo IgM

= 259, Rub IgM

= 322,

and CMV IgM

= 325), a pregnancy sample population (Toxo IgM

= 479, Rub IgM

= 477, and CMV IgM= 476), and samples from a

normal population (Toxo IgM

= 941, Rub IgM

= 940, andCMV IgM

= 941) were analyzed with the new BioPlex 2200 ToRC IgM assay.

The

T. gondii

IgM results were compared to the BioMerieux Vidas

Toxo IgM assay. The rubella and CMV IgM results were compared

to the original BioPlex 2200 ToRC IgM assay. The improved BioPlex

2200 ToRC IgM was further evaluated for imprecision around the

cutoff.

Results:

Among the presumptive positive sample population,

the BioPlex 2200 ToRC IgM assay showed a positive agreement of

96.6%, 100.0%, and 100.0% for Toxo IgM, Rub IgM, and CMV IgM

respectively. The negative agreement for the combined pregnancy

and test ordered sample population was 98.2%, 99.9%, and 99.6%

for Toxo IgM, Rub IgM and CMV IgM respectively. The negative

agreement for normal population samples was 99.6% for all three

analytes. The prevalence of IgM antibodies in the normal popula-

tion samples was 0.6%, 1.6%, and 2.0% for Toxo IgM, Rub IgM, and

CMV IgM respectively. The imprecision for positive samples was

shown to be between 5.0% and 9.4% for Toxo IgM, 4.8% and 11.6%

for Rub IgM, and 5.0% and 11.8% for CMV IgM. The standard devi-