title: Standardized methods to generate mock (spiked) clinical specimens by spiking blood or plasma with cultured pathogens.
abstract: OBJECTIVE
  To demonstrate standardized methods for spiking pathogens into human matrices for evaluation and comparison among diagnostic platforms.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: M
authors.initials: M
authors.lastname: Dong
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: C
authors.initials: C
authors.lastname: Fisher
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: G
authors.initials: G
authors.lastname: Añez
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: M
authors.initials: M
authors.lastname: Rios
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: H L
authors.initials: HL
authors.lastname: Nakhasi
authors.affiliation: Office of In Vitro Diagnostics and Radiological Health (OIR), Division of Microbiology Devices, Center for Devices and Radiological Health (CDRH), FDA, Silver Spring, MD, USA.
authors.forename: J P
authors.initials: JP
authors.lastname: Hobson
authors.affiliation: Office of Biodefense, Research Resources, and Translational Research, Division of Microbiology and Infectious Diseases (DMID), National Institute of Allergy and Infectious Diseases (NIAID), Bethesda, MD, USA.
authors.forename: M
authors.initials: M
authors.lastname: Beanan
authors.affiliation: Diatherix Laboratory, Huntsville, AL, USA.
authors.forename: D
authors.initials: D
authors.lastname: Hockman
authors.affiliation: Diatherix Laboratory, Huntsville, AL, USA.
authors.forename: E
authors.initials: E
authors.lastname: Grigorenko
authors.affiliation: Laboratory of Emerging Pathogens, Division of Emerging and Transfusion Transmitted Diseases, Center for Biologics Evaluation and Research (CBER), Food and Drug Administration (FDA), Silver Spring, MD, USA.
authors.forename: R
authors.identifier: http://orcid.org/0000-0001-8409-2501
authors.initials: R
authors.lastname: Duncan
mesh_terms: D016470:Bacteremia / Q000175:diagnosis*; D001769:Blood / Q000382:microbiology* / Q000469:parasitology / Q000821:virology; D006801:Humans; D018512:Parasitemia / Q000175:diagnosis*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D014766:Viremia / Q000175:diagnosis*
keywords: blood-borne; mock specimens; molecular quantification; pathogen spiking; standardized methods
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