title: Quantitative recovery of proviral HIV-1 DNA from leukocytes by the Dried Buffy Coat Spot method for real-time PCR determination.
abstract: The current recommended method for diagnosing HIV-1 in newborns infected vertically and in adults, during the "window period", is the detection of proviral HIV-1 DNA within leukocytes (buffy coat). This study describes a new portable Dried Buffy Coat Spot (DBCS) assay able to provide a quantitative proviral HIV-1 DNA recovery from the buffy coat. Fifty blood samples were collected from HIV-positive children and processed for DBCSs. Total DNA and proviral DNA were normalised to β-globin and HIV-1 pol genes. Assay sensitivity and specificity were evaluated against the whole blood dried blood spot (DBS) method. Both procedures, using automatic DNA extraction, were compared to a standard whole blood DNA manual extraction. DNA recovery from whole blood was nearly equivalent to that of the DBCS-based extraction, while DBS-based extraction was 10-fold less sensitive. The detection rate of proviral HIV-1 DNA with DBCS assay was equivalent to whole blood manual extraction (100% concordance), but DBS-extracted samples showed limited concordance (44%). The DBCS assay may prove to be more feasible in resource-limited settings. It may represent a simple and robust point-of-care assay for HIV screening of children, for whom a reference test is still lacking.
authors.affiliation: Microbiology Unit, Children's Hospital and Research Institute Bambino Gesù, Piazza Sant'Onofrio 4, Rome, Italy. marco.rossidegasperis@opbg.net
authors.forename: Marco
authors.initials: M
authors.lastname: Rossi de Gasperis
authors.forename: Maria Daniela
authors.initials: MD
authors.lastname: Caione
authors.forename: Carlo
authors.initials: C
authors.lastname: Concato
authors.forename: Ersilia
authors.initials: E
authors.lastname: Fiscarelli
authors.forename: Nicoló
authors.initials: N
authors.lastname: Di Pietro
authors.forename: Vittorio
authors.initials: V
authors.lastname: Salotti
authors.forename: Lorenza
authors.initials: L
authors.lastname: Putignani
authors.forename: Donato
authors.initials: D
authors.lastname: Menichella
authors.forename: Francesco
authors.initials: F
authors.lastname: Callea
mesh_terms: D000293:Adolescent; D052160:Benzothiazoles; D058621:Blood Buffy Coat / Q000821:virology*; D001774:Blood Chemical Analysis / Q000379:methods*; D002648:Child; D002675:Child, Preschool; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics / Q000302:isolation & purification; D003959:Diamines; D015751:Genes, pol; D015497:HIV-1 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D007223:Infant; D007962:Leukocytes / Q000821:virology*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification*; D011804:Quinolines; D012680:Sensitivity and Specificity; D055815:Young Adult; D055544:beta-Globins / Q000235:genetics
chemical_list: D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D055544:beta-Globins; C098022:SYBR Green I