Aims
To demonstrate standardized methods for spiking pathogens into human
matrices for evaluation and comparison among diagnostic platforms.
Methods and Results
This study presents detailed methods for spiking bacteria or
protozoan parasites into whole blood and virus into plasma. Proper methods
must start with a documented, reproducible pathogen source followed by steps
that include standardized culture, preparation of cryopreserved aliquots,
quantification of the aliquots by molecular methods, production of
sufficient numbers of individual specimens and testing of the platform with
multiple mock specimens. Results are presented following the described
procedures that showed acceptable reproducibility comparing in-house
real-time PCR assays to a commercially available multiplex molecular
assay.
Conclusions
A step by step procedure has been described that can be followed by
assay developers who are targeting low prevalence pathogens.
Significance and Impact of Study
The development of diagnostic platforms for detection of low
prevalence pathogens such as biothreat or emerging agents is challenged by
the lack of clinical specimens for performance evaluation. This deficit can
be overcome using mock clinical specimens made by spiking cultured pathogens
into human matrices. To facilitate evaluation and comparison among
platforms, standardized methods must be followed in the preparation and
application of spiked specimens.
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.
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