Quantitative recovery of proviral HIV-1 DNA from leukocytes by the Dried Buffy Coat Spot method for real-time PCR determination.
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.
Rossi de Gasperis
Marco
M
Microbiology Unit, Children's Hospital and Research Institute Bambino Gesù, Piazza Sant'Onofrio 4, Rome, Italy. marco.rossidegasperis@opbg.net
Caione
Maria Daniela
MD
Concato
Carlo
C
Fiscarelli
Ersilia
E
Di Pietro
Nicoló
N
Salotti
Vittorio
V
Putignani
Lorenza
L
Menichella
Donato
D
Callea
Francesco
F
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
D052160:Benzothiazoles; D004279:DNA, Viral; D003959:Diamines; D009930:Organic Chemicals; D011804:Quinolines; D055544:beta-Globins; C098022:SYBR Green I