title: Pilot study of a rapid and minimally instrumented sputum sample preparation method for molecular diagnosis of tuberculosis.
abstract: Nucleic acid amplification testing (NAAT) enables rapid and sensitive diagnosis of tuberculosis (TB), which facilitates treatment and mitigates transmission. Nucleic acid extraction from sputum constitutes the greatest technical challenge in TB NAAT for near-patient settings. This report presents preliminary data for a semi-automated sample processing method, wherein sputum is disinfected and liquefied, followed by PureLyse(®) mechanical lysis and solid-phase nucleic acid extraction in a miniaturized, battery-operated bead blender. Sputum liquefaction and disinfection enabled a >10(4) fold reduction in viable load of cultured Mycobacterium tuberculosis (M.tb) spiked into human sputum, which mitigates biohazard concerns. Sample preparation via the PureLyse(®) method and a clinically validated manual method enabled positive PCR-based detection for sputum spiked with 10(4) and 10(5) colony forming units (cfu)/mL M.tb. At 10(3) cfu/mL sputum, four of six and two of six samples amplified using the comparator and PureLyse(®) method, respectively. For clinical specimens from TB cases and controls, the two methods provided 100% concordant results for samples with 1 mL input volume (N = 41). The semi-automated PureLyse(®) method therefore performed similarly to a validated manual comparator method, but is faster, minimally instrumented, and can be integrated into TB molecular diagnostic platforms designed for near-patient low-resource settings.
authors.affiliation: Claremont BioSolutions, LLC, Upland, CA.
authors.forename: Tanya M
authors.initials: TM
authors.lastname: Ferguson
authors.affiliation: University of Washington, Department of Environmental and Occupational Health Sciences, Seattle, WA.|Seattle Biomedical Research Institute, Seattle, WA.
authors.forename: Kris M
authors.initials: KM
authors.lastname: Weigel
authors.affiliation: University of Washington, Department of Environmental and Occupational Health Sciences, Seattle, WA.|Seattle Biomedical Research Institute, Seattle, WA.
authors.forename: Annie
authors.initials: A
authors.lastname: Lakey Becker
authors.affiliation: Seattle Biomedical Research Institute, Seattle, WA.|University of Santo Tomas Graduate School, Manila, Philippines.
authors.forename: Delia
authors.initials: D
authors.lastname: Ontengco
authors.affiliation: Public Health - Seattle &King County, TB Control Program, Seattle, WA.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Narita
authors.affiliation: Keck Graduate Institute of Applied Life Sciences, Claremont, CA.
authors.forename: Ilya
authors.initials: I
authors.lastname: Tolstorukov
authors.affiliation: Claremont BioSolutions, LLC, Upland, CA.
authors.forename: Robert
authors.initials: R
authors.lastname: Doebler
authors.affiliation: University of Washington, Department of Environmental and Occupational Health Sciences, Seattle, WA.|Seattle Biomedical Research Institute, Seattle, WA.
authors.forename: Gerard A
authors.initials: GA
authors.lastname: Cangelosi
authors.affiliation: Keck Graduate Institute of Applied Life Sciences, Claremont, CA.
authors.forename: Angelika
authors.initials: A
authors.lastname: Niemz
mesh_terms: D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics / Q000302:isolation & purification; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation / Q000379:methods; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D013183:Sputum / Q000382:microbiology*; D014376:Tuberculosis / Q000175:diagnosis* / Q000382:microbiology*; D014397:Tuberculosis, Pulmonary / Q000175:diagnosis / Q000382:microbiology