evaluation of rapid identification assays for viral

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evaluation of rapid identification assays for viral
Poster # T 71
EVALUATION OF RAPID IDENTIFICATION ASSAYS FOR VIRAL MARKERS AND DETERMINING OPTIMAL SAMPLE SUBMISSION
D. Hathaway, T. Hayes, D. Jaskot, J. Krahn, S. Milburn, A. Tirolese, R. Wheeler, and R. Lannigan
Virology Department, London Laboratory Services Group, London, Ontario
Abstract
Objective: Rapid detection of viral pathogens is a staple of most clinical Microbiology
laboratories. Methods to achieve this goal vary according to the depth of expertise
and equipment available in each of these laboratories. Products to suit most (if not
all) laboratory operational levels are available to provide timely and accurate clinical
information for the front line care provider.
Purpose: Laboratory protocols are continuously under pressure to provide rapid turnaround-time results coupled with a diverse scope of investigation. An evaluation of
current and prominent rapid antigen detection methodologies was evaluated. This
laboratories current protocol of direct fluorescent antibody investigation was compared
to lateral-flow immunoassays. A secondary investigation regarding optimal volume of
transport media utilized to submit a sample was also investigated.
Method: Published sample handling and processing testing protocols were followed
for both the rapid immunoassays techniques (Quidel: QuickVue / Meridian TRU FLU /
TRU RSV) and direct immunofluorescence as currently performed by the investigating
laboratory. Spiked respiratory samples were produced in-house and used to evaluate
the two differing technologies. A second aspect to the investigation was the volume of
transport media (Starplex Multitrans S-160) utilized to submit a patient sample.
Results: The comparison of differing technologies for a similar purpose illuminates
inherent advantages and disadvantages for each process. Most rapid immunoassays
kits on the market provide comparable results when it comes to sensitivity and specificity. Direct fluorescent techniques allow for expanded testing algorithms, but fall victim
to subjective evaluation and may also produce a false negative result for the patient
sample that only contains a minimal number of infected cells or a large percentage
of cell-free viruses. Analysis indicated that rapid immunoassays had a higher level
of sensitivity than DFA techniques, but both comparisons to individual manufacturers
products and volume of transport media utilized were not conclusive.
Conclusions: Rapid and accurate analysis of respiratory infections in the hospital
and community setting are of major concern. Depending on the scope and needs of
the laboratory service, differing technologies may be utilized. In the case of cell-free
viral samples the immunoassay technique has a distinct advantage over DFA, but the
scope of testing offered by DFA is a certain advantage if required. Sample volumes
related to transport media can, at the extremes, play a role but this needs to be evaluated by each testing site based on the extent of laboratory examination performed.
Objective
Diagnostic laboratories need to provide the critical patient information required to treat
or control the presence of infectious agents. Many new technologies have been introduced in the past few years to challenge some of the identified standard techniques
and some of the improving methodologies that have been established for years. Many
laboratories have chosen to refer certain work out or stand pat on techniques utilized
for many years without question. In light of some recent findings our laboratory decided
to evaluate some competing assay technologies, different manufacturer’s products and
differing sample submission conditions.
Purpose
The laboratory initiated an evaluation of current and common direct antigen detection
methods. The current testing protocol involves respiratory samples tested by direct
fluorescent techniques for eight common viral pathogens (Flu A/B, PF 1/2/3, AV, RSV
and MPV). Along with culture backup and introduction of molecular based assays the
laboratory identified failures in the last two consecutive External Quality Assurance challenges, which target DFA processes. In both cases the molecular method was able to
properly identify all the challenge material. On the last examination we were also aided
by the availability of some evaluation kits of lateral-flow immunoassays. It was also
discovered that some manufacturers of the rapid kit methods are evaluating a sample
submission volume of Viral Transport (Starplex Multitrans S-160, used by this lab) significantly reduced from the standard produced.
Results
Meridian Tru A+B, RSV
Method for swabs in transport media.
1) Remove conjugate tube from foil pouch – label tube, remove and discard cap.
2) Using a kit supplied transfer pipette, add 100 uL (second mark from the tip) of sample diluent. Swirl or vortex 10 seconds.
3) Add 100 uL of well mixed patient sample. Vortex or mix with pipette 10 seconds.
4) Add test strip and secure in place. Incubate at 20 – 25 degrees Celsius for 15 minutes. Read results within 1 minute.
QuickVue /RSV test
Method / transport media
1) Add extraction reagent to fill line on supplied test tube (250 uL)
2) Fill supplied pipette with patient sample. Note the pipette is designed to collect
and dispense the correct amount of liquid sample.
3) Add sample to tube. Swirl or shake. Wait one minute before placing test strip into tube.
4) Incubate 15 minutes at 20 – 25 degrees Celsius.
QuickVue Influenzae A + B
Method / transport media
1) Dispense all of the extraction reagent solution from the reagent tube. Gently swirl the extraction tube to dissolve its contents.
2) Fill the supplied pipette to uppermost notch. Add entire contents to tube.
3) Place test strip into tube. Incubate 10 minutes at 20 – 25 degrees Celsius.
Chart 2
Influenza A/B
Rapid Immunoassay Evaluation
RSV
Evaluation
Specimen
Panel – Lot
500557.026
Expected
Results
1 Neg
2 Pos
3 Pos
4 Pos
5 Neg
6 Neg
7 Pos
8 Neg
9 Neg
10 Pos
11 Neg
12 Neg
13 Neg
14 Pos
15 Pos
16 Pos
17 Neg
18 Pos
19 Neg
20 Pos
Methods
[email protected]
Typical Visual Observations:
The following charts summarize the findings from evaluating an EQA panel and spiked
samples created in the lab utilizing two different volumes of VTM.
Chart 1
A Joint Venture between London Health Sciences Centre and
St. Joseph’s Health Care Centre, London, Ontario, Canada
Quidel
QuickVue
RSV – Lot
705336
exp.
2010.07.31
Meridian
Tru RSV
- Lot
751330.022
exp.
2010.04.20
Neg
Pos
Pos
Pos
Neg
Neg
Pos
Neg
Neg
Pos
Neg
Neg
Neg
Pos
Pos
Pos
Neg
Pos
Neg
Pos
Neg
Pos
Pos
Pos
Neg
Neg
Pos
Neg
Neg
Pos
Neg
Neg
Neg
Pos
Pos
Pos
Neg
Pos
Neg
Pos
Flu A/B
Evaluation
Specimen
Panel – Lot
500556.024
Expected
Results
1 Neg
2 A Pos
3 A Pos
4 B Pos
5 Neg
6 Neg
7 B Pos
8 Neg
9 Neg
10 A Pos
11 Neg
12 Neg
13 Neg
14 B Pos
15 A Pos
16 A Pos
17 Neg
18 B Pos
19 Neg
20 B Pos
Quidel
QuickVue
Influenzae
A/B – Lot
705425 exp
2010.09.10
Neg
A Pos
A Pos
B Pos
Neg
Neg
B Pos
Neg
Neg
A Pos
Neg
Neg
Neg
B Pos
A Pos
A Weak Pos
Neg
B Pos
Neg
B Pos
Meridian
Tru Flu
Influenzae
A/B – Lot
751230.037
exp
2010.02.26
Neg
A Weak Pos
A Weak Pos
B Pos
Neg
Neg
B Pos
Neg
Neg
A Pos
Neg
Neg
Neg
B Pos
A Weak Pos
A Weak Pos
Neg
B Pos
Neg
B Pos
Conclusions
Strongly positive samples as indicated by immunoassay and DFA analysis
Respiratory Syncytial Virus
Evaluation of Transport Media Volume
Serial
Dilution
Quidel QuickVue
Starplex VTM 2.7 mL
Quidel QuickVue
Starplex VTM 1.0 mL
Meridian Tru Flu
Starplex VTM 2.7 mL
Meridian Tru RSV
Starplex VTM 1.0 mL
Neat
A
Pos
B
Pos
RSV
Pos
A
Pos
B
Pos
RSV
Pos
A
Pos
B
Pos
RSV
Pos
A
Pos
B
Pos
RSV
Pos
10^2
Weak
Pos
Weak
Pos
Pos
Pos
Pos
Pos
Pos
Weak
Pos
Neg
Pos
Pos
Pos
10^4
Weak
Pos
Weak
Pos
Pos
Pos
Pos
Pos
Neg
Weak
Pos
Neg
Weak
Pos
Weak
Pos
Weak
Pos
10^6
*Very
weak
POS
*Very
weak
POS
Neg
Weak
Pos
Weak
Pos
Pos
Neg
Neg
Neg
Neg
Neg
Neg
10^8
Neg
Neg
Neg
Trace
POS
Neg
Trace
POS
Neg
Neg
Neg
Neg
Neg
Neg
10^10
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
Neg
*Very weak positive… very difficult to read.
Strong positives and weak reactions which can be easily interpreted as negative by DFA
Many factors need to be considered when a testing facility adopts an algorithm for
the testing of patient samples. Commercial companies have recently expanded the
considerations regarding the direct detection of primarily viral pathogens, but will also
impact secondary analysis of expanded microorganisms by predominantly molecular based assays. Ease of performance and interpretation coupled with rapid turnaround-time favor the lateral flow immunoassays. Our study compared only two of
the kits on the market and there were distinguishable sensitivity differences when
in-house prepared materials were utilized. When External Quality Control (supplied
by Somagen Diagnostics Inc.) was utilized as test material, results were comparable.
Direct Fluorescent Antibody analysis allows the lab to easily expand a testing menu to
include other pathogens of choice, but is dependent on the availability of Fluorescent
Microscopy and expertise in interpretation. Also noted was the fact that some samples
submitted lack an adequate number of intact and appropriate cells for analysis which
lead to false negative or indeterminate conclusions, when compared to immunoassays or molecular techniques. This was primarily evident for Influenza B in this study,
but historically also included Influenza A and RSV. The amount of Viral Transport
used in sample collection has some noticeable effect on the positive/negative interpretation to a degree, but varied slightly between viruses analyzed and manufacturer
of kit used which did not qualify for statistical analysis. The determination of VTM
volume to utilize should be based on the extent of testing that will be performed post
direct analysis as our findings showed only some advantages to more concentrated
samples. Those labs still performing viral culture techniques or molecular testing for
both viral and bacterial targets may be limited to the larger volume, but should not witness significant reduction in performance.

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