Application of TaqMan Low-Density Arrays for Simultaneous Detection of Multiple Respiratory Pathogens

Abstract

The large and growing number of viral and bacterial pathogens responsible for respiratory infections poses a challenge for laboratories seeking to provide rapid and comprehensive pathogen identification. We evaluated a novel application of the TaqMan low-density array (TLDA) cards for real-time PCR detection of 21 respiratory-pathogen targets. The performance of the TLDA was compared to that of individual real-time PCR (IRTP) assays with the same primers and probes using (i) nucleic acids extracted from the 21 pathogen strains and 66 closely related viruses and bacteria and (ii) 292 clinical respiratory specimens. With spiked samples, TLDA cards were about 10-fold less sensitive than IRTP assays. By using 292 clinical specimens to generate 2,238 paired individual assays, the TLDA card exhibited 89% sensitivity (95% confidence interval [CI], 86 to 92%; range per target, 47 to 100%) and 98% specificity (95% CI, 97 to 99%; range per target, 85 to 100%) overall compared to IRTP assays as the gold standard with a threshold cycle (C T ) cutoff of 43. The TLDA card approach offers promise for rapid and simultaneous identification of multiple respiratory pathogens for outbreak investigations and disease surveillance.

Evaluation of two multiplex real-time PCR screening capabilities for the detection of Bacillus anthracis, Francisella tularensis and Yersinia pestis in blood samples generated from murine infection models

“…Therefore, there has been no driver for the manufacturer to optimize the cards for detection of bacterial DNA at low levels (equivalent to the femtogram scale). This and other factors, such as the stochastic distribution of target DNA at low concentrations (Taberlet et al, 1996), mean that the PCR assays on the card are generally 1 log less sensitive than the same PCRs in singleplex format, an observation found in other Array Card studies (Kodani et al, 2011). The architecture of the cards implies that increasing the number of assays to a given agent within an Array Card channel increases the chances of detecting that agent.…”

“…This would still require a clinician to order the tests at admission and an appropriate PCR platform and consumables being readily available in a hospital setting but the burden on the clinician in having to decide which specific tests to conduct would be reduced. For both the Array Card and the FilmArray platforms, cards and pouches for respiratory agents, which cause a large number of generic flu and fever-type diseases, have also been developed (Caliendo, 2011;Kodani et al, 2011). This could help rule in or out a large number of diseases that cause initial symptoms similar to those found with acute disease.…”

“…This compares to the results of our study for the detection rates (from samples shown to contain agent) of singleplex (60.4 %) against Array Card (33.3 %) PCRs, indicating that the Array Cards detected only 55.1 % of samples where a singleplex PCR-positive result was returned. Our study and that of Kodani et al (2011) indicate that the whole assay process (sample collection, DNA extraction and DNA purification) must be fully optimized to the Array Card system to obtain the best results. A recent study (Regan et al, 2012) indicated that a magnetic bead-based DNA extraction system reduced the effects of PCR inhibitors from blood culture fluids when compared with the silica column method used in this study.…”

“…The study of Kodani et al (2011) used Array Cards to test 292 clinical samples for 21 respiratory pathogen targets, with all samples processed using a magnetic bead-based DNA extraction method. In this study, the Array Cards detected 89 % of samples that were detected by equivalent singleplex PCR assays.…”

“…'do you contain agent X?' To act as a screening capability, where one sample is analysed by a panel of PCR assays for multiple agents, PCRs must be multiplexed, where multiple reactions are carried out in a single tube (Janse et al, 2010), or an engineering solution such as microfluidics (Kodani et al, 2011;Rachwal et al, 2012) or robotics (Hondow et al, 2011) must be found. When considering the advantages offered by PCR technology (sensitivity and specificity), approaches that mitigate the primary disadvantage (a biased assay) will help to unlock the full potential of PCR for diagnostic purposes.…”