Comparison of multiplex PCR hybridization-based and singleplex real-time PCR-based assays for detection of low prevalence pathogens in spiked samples

- Standardized methods need to be established for evaluating low-prevalence pathogen diagnostic devices.
- Biothreat and emerging agents such as Francisella tularensis and Babesia microti are examples of low-prevalence pathogens.
- The effectiveness of standardized methods was demonstrated on two testing platforms, real-time PCR and TEM-PCR.
- Compared results from two types of sample matrix, healthy donor blood and blood from patients with signs of infection.
- Reproducibility of comparisons demonstrated the standardized methods are effective for evaluating diagnostic devices.

Molecular diagnostic devices are increasingly finding utility in clinical laboratories. Demonstration of the effectiveness of these devices is dependent upon comparing results from clinical samples tested with the new device to an alternative testing method. The preparation of mock clinical specimens will be necessary for the validation of molecular diagnostic devices when a sufficient number of clinical specimens is unobtainable. Examples include rare pathogens, some of which are pathogens posing a biological weapon threat. Here we describe standardized steps for developers to follow for the culture and quantification of three organisms used to spike human whole blood to create mock specimens. The three organisms chosen for this study were the Live Vaccine Strain (LVS) of Francisella tularensis, surrogate for a potential biothreat pathogen, Escherichia coli, a representative Gram-negative bacterium and Babesia microti (Franca) Reichenow Peabody strain, representing a protozoan parasite. Mock specimens were prepared with blood from both healthy donors and donors with nonspecific symptoms including fever, malaise, and flu-like symptoms. There was no significant difference in detection results between the two groups for any pathogen. Testing of the mock samples was compared on two platforms, Target Enriched Multiplex-PCR (TEM-PCR™) and singleplex real-time PCR (RT-PCR). Results were reproducible on both platforms. The reproducibility demonstrated by obtaining the same results between two testing methods and between healthy and symptomatic mock specimens, indicates the standardized methods described for creating the mock specimens are valid and effective for evaluating diagnostic devices.