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Acknowledgements
STRATEGIES TO OVERCOME INVALID RESULTS WITH THE
RESPIFINDER® SMART 22 ASSAY USED FOR DIAGNOSIS OF
RESPIRATORY TRACT INFECTIONS
Peeters B., Micalessi MI., Van Meensel B., Smismans A., Frans J.
Laboratory of Clinical Microbiology, Imelda Hospital, Bonheiden, Belgium.
Correspondence: [email protected]
OBJECTIVES
The RespiFinder® SMART 22 assay (PathoFinder) is a multiplex PCR, which enables the detection and differentiation of 18 respiratory viruses as well as
4 bacteria in 6 h. An internal control (IC) is included in the assay to assess amplification inhibition. A result is reported as invalid when the internal control
is not amplified and a negative result is obtained for the 22 respiratory pathogens.
This assay, which is performed in our daily routine, exhibits an inhibition rate of 5.9% (55/949). To circumvent false negative results due to inhibition, the
manufacturer recommends sample pretreatment with dithiothreitol and/or dilution of the sample extract. This study investigates how inhibition can be
resolved without repeating the test, using a second RespiFinder SMART 22 reagent panel and delaying patient diagnosis.
METHODS AND RESULTS
Nasopharyngeal eSwab
(Copan) 200 µl
Extraction in 100 µl on
Nuclisens EasyMAG
(bioMérieux)
Reverse
Transcription + Preamplification
Timeframe
40 min
130 min
Hybridization with
specific probes for
every pathogen
Fluorescence
detection during
melting on
RotorGene Q
(Qiagen)
Ligation/
Amplification/
Melting curve
70 min
145 min
Figure 1: RespiFinder SMART 22 workflow
The complete workflow from extraction untill analysis of the results is demonstrated in Figure 1. An IC is added to every sample before extraction to check
for inhibition and extraction efficiency and a negative sample is added to every run to check for contamination and to allow a correct threshold setting.
After analysis of the results, positive samples show a peak at a certain temperature in the ROX or Cy5 channel. Each peak is specific for a certain
respiratory pathogen.
The experimental set-up and results are given in Table 1.
Freeze-thaw cycle
Experiment 1
Experiment 2
Experiment 3
Invalid Samples:
Invalid Samples:
Positive Samples:
IC negative (N=10)
IC negative (N=4)
Pathogen positive, IC positive (N=3)
-70°C 15h
-70°C 1h
-70°C 30 min.
Aliquot 1 (200 µl)
Aliquot 2 (200 µl)
-70°C 15h
IC signal after freezethaw cycle
+
+
+
+
Pathogen signal after freezethaw cycle
2/10 positive
4/4 negative
4/4 negative
No significant effect on Ct-value
8/10 negative
Table 1: Experimental set-up and results
We assume that the inhibitor present in the sample is either a protease which exerts its activity on the PCR enzymes or is a non-protease allosteric PCRinhibitor. Allosteric inhibitors can result in competitive binding of reagents when the relative concentration of the inhibitor is extremely high compared to the
target.
CONCLUSION
Invalid results for the RespiFinder SMART 22 assay may be avoided by subjecting all samples to a one-time freeze thaw cycle with a freezing
step (-70°C) of at least 30 min. Freezing samples prior to analysis does not affect sensitivity of the respifinder test. This sample pretreatment
provides a fast, low-cost and easy to apply solution to prevent amplification inhibition and to obtain higher turnaround times. The inhibition
mechanism occurs in both positive and negative samples.
Acknowledgements
The author would like to thank PathoFinder BV for providing all reagents and the RespiFinder SMART 22 kits to conduct this study.