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Journal of Virological Methods 170 (2010) 121–127

Contents lists available at ScienceDirect

Journal of Virological Methods

journa l homepage: www.e lsev ier .com/ locate / jv i romet

rotocols

uantitative recovery of proviral HIV-1 DNA from leukocytes by the Dried Buffyoat Spot method for real-time PCR determination

arco Rossi de Gasperisa, Maria Daniela Caionea, Carlo Concatoa, Ersilia Fiscarelli b, Nicoló Di Pietroc,ittorio Salotti c, Lorenza Putignania,∗, Donato Menichellaa, Francesco Calleab

Microbiology Unit, Children’s Hospital and Research Institute “Bambino Gesù”, Piazza Sant’Onofrio 4, 00165 Rome, ItalyDepartment of Pathology, Children’s Hospital and Research Institute “Bambino Gesù”, Piazza Sant’Onofrio 4, 00165 Rome, ItalyGeneDia, Via Lombarda, 169/A – 55013 Lammari, Lucca, Italy

rticle history:eceived 30 March 2010eceived in revised form 7 September 2010ccepted 13 September 2010vailable online 21 September 2010

eywords:IV-1 (human immunodeficiency virus

a b s t r a c t

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). Thisstudy describes a new portable Dried Buffy Coat Spot (DBCS) assay able to provide a quantitative proviralHIV-1 DNA recovery from the buffy coat.

Fifty blood samples were collected from HIV-positive children and processed for DBCSs. Total DNA andproviral DNA were normalised to ˇ-globin and HIV-1 pol genes. Assay sensitivity and specificity wereevaluated against the whole blood dried blood spot (DBS) method. Both procedures, using automatic

ype 1)roviral HIV-1 DNABS (Dried Blood Spot)BCS (Dried Buffy Coat Spot)uman ˇ-globinIV-1 pol

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 DBCSassay was equivalent to whole blood manual extraction (100% concordance), but DBS-extracted samplesshowed limited concordance (44%).

ove tassay

The DBCS assay may prand robust point-of-care

. Introduction

Several studies have reported on HIV infection contracted byhildren in the third trimester of pregnancy or during deliveryDunn et al., 1995; Kuhn et al., 1997; De Cock et al., 2000; Lehmannd Farquhar, 2007). Throughout the first month of life, PCR tests,mployed to detect the presence of integrated proviral HIV DNA,ay provide false negative results given that the small number of

eukocytes infected by the virus is potentially lower than thresh-ld sensitivity. Nevertheless, international guidelines suggest annitial test within the first 24 h of life (Udeh et al., 2008) because

ndividuals positive for HIV-1 at this early stage have probably con-racted the infection in utero and the infection may progress rapidlyOwens et al., 1996). Therefore, detection of HIV-1 at an early stages extremely important in order to begin treatment, to prevent

∗ Corresponding author. Tel.: +39 06 68592598; fax: +39 06 68592218.E-mail addresses: marco.rossidegasperis@opbg.net (M. Rossi de Gasperis),

ariadaniela.caione@opbg.net (M.D. Caione), carlo.concato@opbg.net (C. Concato),rsilia.fiscarelli@opbg.net (E. Fiscarelli), ndipietro@genedia.it (N. Di Pietro),ittorio@genedia.it (V. Salotti), lorenza.putignani@opbg.net (L. Putignani),enichella@opbg.net (D. Menichella), francesco.callea@opbg.net (F. Callea).

166-0934/$ – see front matter © 2010 Elsevier B.V. All rights reserved.oi:10.1016/j.jviromet.2010.09.012

o be more feasible in resource-limited settings. It may represent a simplefor HIV screening of children, for whom a reference test is still lacking.

© 2010 Elsevier B.V. All rights reserved.

opportunistic infections and to discourage behaviour that wouldlead to transmission of the infection (Klein et al., 2003).

The Dried Spot (DS) method is a good manageable technique thatuses a special filter paper for sampling, safe transport and preserva-tion of specimens. Whole blood, bone marrow or buffy coat, afterspotting on a paper matrix, are air-dried for at least 3 h to allowDNA capture. Afterwards, the DNA remains stable for an indeter-minate period during storage at room temperature and can be usedreadily for diagnostic tests in reference laboratories even in regionsor countries distant from those of origin as reported formerly formetabolic disorders in neonatal screening (Guthrie, 1980; Irie et al.,1975).

By 2007, 30 low- and medium-income countries had adopteda new blood collection method for HIV diagnostic tests calledDried Blood Spot (DBS) (Children and AIDS, 2008), which has beenemployed increasingly in different screening programs and diag-nostic methods in developing countries (Ngo-Giang-Huong et al.,2008; Zhan et al., 2008; Jacob et al., 2008; Somi et al., 2008; Creek

et al., 2008; Mehta et al., 2009; Jangam et al., 2009).

Over the last few years, DBS method has also been used for thediagnosis of other viral infections, e.g., CMV and EBV (McDade etal., 2000; Barbi et al., 2006; Yamagishi et al., 2006; Soetens et al.,2008; Walter et al., 2007).

122 M. Rossi de Gasperis et al. / Journal of Virological Methods 170 (2010) 121–127

Table 1Calculation of DNA yields obtained by manual and automatic extraction methods from whole blood and buffy coat samples.a

Extraction method Procedure DNA yield (ng/�l) Reference

Whole bloodManual extraction (standard) Elution in 100 �l from 200 �l 40–80 EZ1® DNA Handbook, Qiagen, III editionAutomatic extraction (DBS) Elution in 100 �l from 4 punched-out discs

(3.5 mm/each)b5.4–10.8 EZ1® DNA Handbook, Qiagen, III edition

Automatic extraction (optimised DBS) Elution in 50 �l from 2 punched-out discs(5.0 mm/each)b

11.0–22.2 This work

Buffy coatManual extraction (standard) Elution in 100 �l from 75 �l 65–145 EZ1® DNA Handbook, Qiagen, III editionAutomatic extraction (DBCS) Elution in 100 �l from a 11 mm filter paper

disc234.6–77.3 This work

Automatic extraction (optimised DBCS) Elution in 50 �l from 2 punched-out discs 48.0–107.4 This work

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a DBS, Dried Blood Spot; DBCS, Dried Buffy Coat Spot.b An 11-mm filter paper disc corresponds to 40 �l of spotted whole blood or buff

After sample collection by DBS, PCR-based procedures, coupledo manual, semi-automatic or completely automated DNA extrac-ion, can be easily performed to detect viral DNA (Stevens et al.,008; Alvarez-Munoz et al., 2005; Jennings et al., 2005; Li et al.,004; Karlsson et al., 2003; Lilian et al., 2010).

A procedure based on Dried Buffy Coat Spot (DBCS) is describedor the detection of proviral HIV-1 DNA is described. The methods especially suitable for low proviral lymphocyte integration suchs that observed in newborn babies, infants or during the “windoweriod” in adult patients.

DBCS sampling may represent a high performance method foroint-of-care (POC) recovery of HIV-1 provirus compatible withdvanced DNA detection assays often accessible only in referenceentres. It could provide a new diagnostic tool for monitoring provi-al content especially in infants living in areas with high povertyates and poor access to specialised health care structures, whichre factors that contribute to the increasing global burden of HIVnfection in children in developing countries.

. Materials and methods

.1. Patients

From January 2008 to December 2008, blood samples were col-ected from two cohorts of patients infected with HIV. The firstohort was composed of 30 in-patients (1–25 years old, mean age4 years) admitted to the Division of Immunology and Infectiousiseases of Children’s Hospital and Research Institute “Bambinoesù” of Rome. The second cohort was made up of 20 children

1–10 years of age, mean age 4 years) among the 150 childrenositive for HIV and residing at the Mission “Villaggio della Sper-nza”, Dodoma, Tanzania. The observational study was approved byhe Ethics Committee of Children’s Hospital and Research InstituteBambino Gesù” (study No. 331, protocol No. 272 CM/vp) whichroduced dedicated forms for informed consent signed by chil-ren’s parents or guardians.

.2. Sample processing

Pre-test and test phases of the processing of blood samples werearried out under the same conditions for both Italian and Africanohorts of patients. Fifty whole blood specimens (5 ml/each) wereaken by venipuncture using a combined syringe-butterfly devicequipment, without changing any procedure used regularly for

cquiring blood for HIV RNA monitoring in children, and processedmmediately in situ. After withdrawing blood in air conditioned

ithdrawal rooms, all blood samples were collected immediately in2EDTA-containing vacutainer tubes (Beckton Dickinson, Franklinake, NJ), and processed as follows: after mixing by repeated inver-

fraction volume

sion, 40 �l of whole blood were spotted onto an FTA ELUTE MicroCard (Whatman, Clifton, NJ), an aliquot of 200 �l was used for theleukocyte cell count with an automated SYSMEX SF 3000 Analyser(Dasit, Cornaredo, Milan) and another 200 �l aliquot was collectedfor whole blood DNA manual extraction. In order to prepare buffycoat fractions, samples were allowed to settle spontaneously for120 min at 37 ◦C in an incubator (Memmert, Büchenbach, Ger-many). After separation from red cells, plasma was transferredinto 1.5-ml safe-lock tubes (Eppendorf, Hamburg, Germany) andcentrifuged at 1000 × g for 10 min at RT in a 5417 R Eppendorf cen-trifuge (Eppendorf). After removing supernatant, 40 �l of buffy coatpellet was recovered, re-suspended by vortexing for 10 s, spottedon an FTA ELUTE Micro Card (Whatman) and air-dried at RT for atleast 24 h, before in situ processing or before transport for remotemolecular determination. Particularly, spotting from whole bloodand buffy coats was performed by filling 11-mm diameter discswith 40 �l of each sample for both DBS and DBCS assays (Table 1).

Two 5-mm discs were produced by punching them from the11-mm disc as a modification of the standard protocol (EZ1®

DNA Handbook, Qiagen, III edition) (Table 1) previously describedfor the Biorobot EZ1 DSP automated extraction system (Qiagen,Hilden, Germany). The steps from blood withdrawing to spottingand punching took approximately 2 h, 20 min and 3 min, for eachsample, respectively. Once spotted, the discs were stored at roomtemperature. Afterwards, punches were processed for DNA auto-matic extraction as described below.

2.3. DNA extraction and amplification

DNA from whole blood samples (200 �l) was eluted in 100 �lof elution buffer following a manual extraction protocol (QIAampDNA Blood Mini Kit, Qiagen). Automatic extraction from DBS andDBCS, after punching two 5-mm discs (Table 1), was performed byusing a DNA dried blood card (V1.066054157) on a Biorobot EZ1DSP (Qiagen) and a DNA tissue kit (Qiagen). The extraction tookapproximately 45 min for a set of six samples up to the elutionstep, performed in 50 �l of buffer. Samples were stored at −20 ◦Cuntil usage.

All real-time PCR reactions were performed in duplicate withthe use of SET UP HIV Integrated GD 200 (GeneDia, Lammari, Lucca,Italy) and SYBR Green I MyIQ Single-Color detection systems (BIO-RAD Laboratories, Hercules, CA, USA).

2.4. Detection of ˇ-globin gene

Genomic DNA recovery for each sample type (whole blood, DBSand DBCS) was assessed by monitoring the internal amplificationcontrol (IAC) ˇ-globin by real-time PCR. The critical threshold (CT),defined as the number of PCR cycles required for the fluorescent

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M. Rossi de Gasperis et al. / Journal o

ignal of the amplification to cross the threshold, was calculatedn order to compare DNA extraction recovery from the three sam-les. ˇ-Globin detection was performed by employing the followingrimers: ˇ-globin sense (5′-ACA CAA CTG TGT TCA CTA GC-3′) and-globin antisense (5′-CAA CTT CAT CCA CGT TCA CC-3′) (Cook etl., 2000). The PCR mix (up to 50 �l) contained 15 �l of Real-Timeuffer including SYBR Green I (GeneDia), 3 �l of 25 mM MgCl2GeneDia), 1 �l of both ˇ-globin primers (Eurogentec, Seraing, Bel-ium), 0.5 �l of DNA Taq Plus (GeneDia), 5 �l of extracted DNA and4.5 �l of DNAse-RNAse free H2O (GeneDia). After a denaturationtep at 94 ◦C for 7 min, two amplification cycles (30 s at 94 ◦C, 1 mint 55 ◦C and 1 min at 72 ◦C) and forty additional cycles (30 s at 94 ◦C,0 s at 55 ◦C, 30 s at 72 ◦C), including a reading step at 490 nm for5 s at 79 ◦C, were run. The latter step, performed by a temperatureradient from 65 to 95 ◦C ramped at a rate of 0.3 ◦C per 10 s, pro-ided the specific ˇ-globin melting curve. The PCR products werelectrophoresed on a 3% agarose gel in 1X Tris–acetate–EDTA (TAE)uffer [50 mM Tris–HCl (pH 8.0), 20 mM sodium acetate, 2 mMa2EDTA] and visualized by ethidium bromide (Sigma–Aldrich,ilan, Italy) staining. The sizes of the amplified PCR products were

stimated by comparison with the MVIII DNA ladder (Fermentasnternational, Burlington, Canada). The resulting DNA fragments

ere visualized on a Gel Doc XR System and analysed using theoftware Quantity One 4.6.3 (Bio-Rad Laboratories).

.5. Detection of proviral HIV-1 DNA

DNA extracted from whole blood by the manual method androm spots was amplified through nested-PCR by using outer andnner degenerate primers targeting integrase of HIV-1 pol geneBruzzone et al., 2010). For the first PCR round, the reaction mixup to 50 �l) contained 15 �l of Real-Time Buffer including SYBRreen I (GeneDia), 6 �l of 25 mM MgCl2 (GeneDia), 1 �l of both poluter sense and antisense primers (Eurogentec), 0.5 �l of DNA Taqlus DNA Polymerase (GeneDia), 5 �l of extracted DNA and 21.5 �lf DNAse-RNAse free H2O (GeneDia). After a denaturation step at4 ◦C for 7 min, two amplification cycles (30 s at 94 ◦C, 1 min at 52 ◦Cnd 1 min at 72 ◦C) and twenty further cycles (30 s at 94 ◦C, 30 s at2 ◦C and 30 s at 72 ◦C) were run. For the second nested-PCR round,he reaction mix (up to 50 �l) contained 15 �l of Real-Time Bufferncluding SYBR Green I (GeneDia), 6 �l of 25 mM MgCl2 (GeneDia),�l of both pol inner sense and antisense primers, 0.5 �l DNA Taqlus DNA Polymerase (GeneDia), 2 �l of amplified DNA from therst round and 24.5 �l of DNAse-RNAse free H2O (GeneDia). After aenaturation step at 94 ◦C for 7 min, two amplification cycles (30 st 94 ◦C, 1 min at 52 ◦C for and 1 min at 72 ◦C) and thirty furtherycles (30 s at 94 ◦C, 30 s at 52 ◦C, 30 s at 72 ◦C), including a readingtep at 490 nm for 15 s at 77 ◦C, were run. The latter step, performedy a temperature gradient from 65 to 95 ◦C ramped at a rate of.3 ◦C per 10 s, provided the specific pol melting curve. PCR prod-cts were electrophoresed on a 3% agarose gel in 1X TAE buffernd visualized by ethidium bromide (Sigma–Aldrich) staining. Theizes of the amplified PCR products were estimated by comparisonith the MVIII DNA ladder (Fermentas International). The result-

ng DNA fragments were visualized on a Gel Doc XR System andnalysed using the software Quantity One 4.6.3 (Bio-Rad Laborato-ies).

All samples were processed for viral-load content by using theranched-DNA assay bDNA HIV-1 RNA 3.0 (Siemens, NY, USA) andersantTM 340 molecular system (Bayer, NY, USA).

.6. Specificity assay

To assess specificity, eight samples, which were collected ran-omly within a sample group tested as HIV antibody-negative,ere subjected to whole blood manual and DBS, DBCS automatic

ogical Methods 170 (2010) 121–127 123

DNA extraction procedures using the same experimental settingsof positive samples.

2.7. Statistical analysis

Inter-assay correlation and regression for the IAC ˇ-globin andthe pol-based assays, performed on DNA obtained from whole blood(manual extraction) and from DBS and DBCS (automated extrac-tion), were assessed by a macro-extended version of Excel 2003and by the MedCalc ver. 7.3.0.1 software according to previouslydescribed methods (Twomey and Kroll, 2008).

3. Results

3.1. Detection of ˇ-globin gene

The IAC ˇ-globin signal was employed to assess the presenceand the quality of extracted DNA and to quantitatively compareDBS and DBCS DNA recovery obtained by automatic methods versuswhole blood manual extraction. No signal failures were observedfor the extracted DNA, indicating (i) high quality DNA, as inferredby spectrophotometric determinations, (�260/280 was ∼1.8 for allsamples; data not shown) and (ii) absence of PCR inhibitors. The ˇ-globin CT revealed nearly equivalent DNA extraction recovery fromboth whole blood (manual) and DBCS (automated), with a CT for thewhole blood plot ranging from 16 to 21 and a CT for the DBCS plotranging from 17 to 23 (Fig. 1, panel A). The major variation betweenwhole blood and DBCS ˇ-globin CT (18 versus 23), a potential 10-folddifference in DNA quantification, was observed only for 2 out of 50samples (33 and 41). However, all the ˇ-globin CT values obtainedfor DNA extracted from DBS (Fig. 1, panel A) were considerablyhigher (range 23–29), with the least difference up to 100-fold andthe greatest up to 1000-fold between determinations. The lowestquantitation observed for the DBS extracted DNA resulted indepen-dent of any single clinical sample processing and DNA extractionsetting.

Statistical analysis showed high inter-assay reproducibility forthe DNA extraction protocols from whole blood and DBCS sam-ples. Mean CT values were 18.80 ± 0.92 SD and 20.35 ± 1.67 SD forthe manual and the DBCS extractions, respectively. DBS extrac-tion correlated with a reduction of DNA yield up to 100-foldcompared to the manual extraction method, as inferred by aCT of 25.86 (±1.12 SD). Coefficient of variation (CV) for DBCS-based automated extraction appeared proximate (8.2) to thoseobtained for DBS (4.33) and whole blood (4.89) manual extractionmethods.

The ˇ-globin melting curve produced a single amplification peakat 82.5 ◦C ± 0.5 ◦C, which corresponded to a specific amplicon bandof 110 bp of expected size (data not shown).

3.2. Detection of proviral HIV-1 DNA

The CT values obtained after the second round of the HIV-1pol-based nested-PCR, which was performed on DNA automati-cally extracted from the entire sample panel (n = 50), were plottedin order to compare both DBCS- and DBS-based assay sensitiv-ity to the whole blood-based assay. Panel B of Fig. 1 illustratesthe comparison amongst the pol CT plots for the three assays. Theentire set of 50 samples tested positive by both DBCS and wholeblood extraction methods, thus providing a 100% of concordance

(50/50). Therefore, both procedures provided a comparable 100%sensitivity in the detection of the 50 positive samples. However,the pol gene-based assay showed only 22 out of 50 positives forthe DBS extracted samples, revealing a limited concordance (44%)with the whole blood-based assay (Fig. 1, panel B). The correla-

124 M. Rossi de Gasperis et al. / Journal of Virological Methods 170 (2010) 121–127

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ig. 1. Quantitative detection of DNA obtained by whole blood-, DBS- and DBCS-baased nested-PCR (internal amplification control, IAC) performed on DNA extractedIV-1 pol-based nested-PCR performed on DNA extracted from the HIV-positive pand pol CT for DBS DNA automatic extraction; (�) ˇ-globin and pol CT for DBCS DNA

ion plot between CT values obtained for the pol-based assays, fromBCS and whole blood extracted manually DNA, showed a good lin-arity range (y = 0.5691x + 9.1456) with a slope (formula = 0.5691,= 7.3209, p < 0.0001), an intercept (formula = +9.1456, t = 6.5068,< 0.0001) significantly different from zero and a regression coef-cient R2 = 0.5275 (Fig. 2, panel A). An algorithm with no specialssumptions regarding the distribution of the samples and theeasurement errors was employed to assess the linear regres-

ion between DBCS and whole blood sample sets irrespective ofhe sample set distribution (Passing and Bablok, 1983). The scatterlot (Fig. 2, panel B) showed a regression line (y = 0.7665x + 6.0515)ith a slope and intercept ranging from 0.6193 to 1.0046 and from

.4734 to 8.2916, respectively (95% CI), and the plot’s linearity wasssessed by Cusum test (p < 0.05). A contained divergence from theheoretical identity line (y = x) was observed for the regression linebtained for the comparison of the two assays (Fig. 2, panel B)hich indicates a considerable agreement between the sensitivities

f DBCS and whole blood methods.For DBS-based assay, the correlation plot against the whole

lood-based assay showed a linear regression (y = 1.7960x + 5.5717)ith a slope (formula = 1.7960, t = 3.2667, p = 0.0020), an intercept

formula = +5.5717, t = 0.5605, p = 0.5778) and a regression coeffi-ient R2 = 0.1819 (Fig. 2, panel C). Therefore, the linear regressionor DBS-based assay versus the manual extraction data showed

breakdown in the linearity range compared to the regressionbtained for DBCS assay versus the whole blood extraction data.ith the aim to improve the correlation between the DCS and thehole blood-based assay, the 28 samples assessed as negative by

ol CT values (Fig. 2, panel D) were removed from the DCS data set

xtraction techniques. (Panel A) Plot of CT obtained after the 2nd round of ˇ-globin-the HIV-positive patient panel. (Panel B) Plot of CT obtained after the 2nd round ofpanel. ♦, ˇ-globin and pol CT for whole blood DNA manual extraction; (�) ˇ-globin

atic extraction. A CT value ≥50 indicates the sample is negative for HIV.

and the correlation re-run. Even in this case, the linear regressionequation (y = 0.3252x + 16.2357) with a slope (formula = 0.3252,t = 2.8359, p = 0.0102) and intercept (formula = +16.2357, t = 8.4677,p < 0.0001) reached a regression coefficient R2 = 0.2868 (Fig. 2, panelD), which is still lower than the coefficient R2 = 0.5275 obtained forthe DBCS versus the whole blood correlation plot.

3.3. Specificity assay

The pol gene melting curve produced a single amplification peakat 79 ◦C ± 0.5 ◦C (Fig. 3, panel A), which corresponded to a singlespecific amplicon band of 349 bp (Fig. 3, panel B). The eight samplestested as HIV antibody-negative, from which DNA was manuallyextracted from whole blood and automatically from both DBS andDBCS, were characterised by CT ≥ 50 for the pol assay. The IAC ˇ-globin curve provided for DBCS samples CT values (16.54–21.28)which almost overlapped the CT values (16.25–20.52) obtained forthe whole blood samples. On the contrary, the CT values obtainedfor DBS samples ranged from 24.80 to 28.92, confirming a reduc-tion of DNA yield of more than 100-fold compared to the manualextraction method (Table 2).

4. Discussion

In the experimental setting, the HIV-1 proviral determinationworkflow was based on the punching of two 5-mm discs for bothDBS and DBCS procedures instead of using four 3.5-mm discs aspreviously described (EZ1® DNA Handbook, Qiagen, III edition).

M. Rossi de Gasperis et al. / Journal of Virological Methods 170 (2010) 121–127 125

Fig. 2. Regression plots of CT values obtained after the 2nd round of HIV-1 pol-basednested-PCR performed on DNA extracted from the HIV-positive patient panel forDBCS (panels A and B) and DBS (panels C and D) automatic extraction (axis Y) versusthe manual method (axis X). (Panel A) Linear regression for DBCS versus standardblood extraction data. (Panel B) Scatter plot illustrating the correlation betweenDBCS and manual extraction data obtained by using Passing and Bablok algorithm.Solid line shows the linear regression interpolated between the upper and lowercurves (95% CI). Dotted line indicates the theoretical identity line (y = x) for the twomethods. (Panel C) Linear regression for DBS versus standard blood extraction data.(Panel D) Linear regression for DBS versus standard extraction data after removal ofthe 28 samples assessed as negative by pol CT values.

Fig. 3. Amplification plot of the HIV-1 pol-based nested-PCR for the DBCS extracted

samples. (Panel A) Melting curves of the 2nd round of HIV-1 pol-based nested-PCRare reported for three different clinical samples (1–3) selected randomly within thepositive sample set. (Panel B) The sizes of the amplified PCR products (1–3) areestimated by comparison with the DNA MVIII ladder on a 3% agarose gel.

The strategy was adopted for convenience; however, a theoreti-cal evaluation of the automatic DNA extraction from DBS and DBCSprovided an estimation of DNA yield ranging from 11.0 to 22.2 and48.0 to 107.4 ng/�l, respectively, after recovering in 50 �l of elutionbuffer (Table 1). Consistently, the IAC ˇ globin assay, performed onDNA automatically extracted from the buffy coat discs, provided aset of CT values that were closely related to the CT values obtainedfor the reference manual extraction from whole blood (Fig. 1). Thissuggests a high degree of purity (quality) of the extracted DNA fromDBCS and the absence of inhibitory effects in the real-time PCRchemistry.

The ˇ-globin real-time results noticeably showed that DNArecovery from DBCS was very similar to the whole blood DNAyield obtained by manual extraction (Fig. 1, panel A). On the otherhand, lower DNA quantities appeared to be recovered by DBS proce-dures. However, irreversible DNA absorption by the filter should beregarded theoretically as a potential factor affecting DNA yield fromDBS, especially considering the absence of proviral DNA degrada-tion in the eluates during the drying process.

Furthermore, the proviral DNA was detected and quantified by apol-based real-time PCR method and provided overlapping curves

for both whole blood DNA manual and DBCS automatic extractions,while the DBS-based extraction appeared less sensitive (Fig. 1,panel B). This actually reflects the integration of the proviral HIV-1DNA within the buffy coat fraction, which contains a total quantityof DNA at least 10 times higher than the amount of DNA present in

Table 2Test specificity assigned by the IAC ˇ-globin assay on negative samples.

Sample CT

Whole bloodmanual extraction

DBS automaticextraction

DBCS automaticextraction

1 18.58 25.22 18.022 18.62 28.92 19.703 16.25 24.60 16.544 16.44 25.24 17.465 18.08 25.95 19.496 20.52 27.78 20.727 18.63 25.80 21.288 17.12 24.80 21.12

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26 M. Rossi de Gasperis et al. / Journal o

he same quantity of whole blood (EZ1® DNA Handbook, Qiagen,II edition). A similar specificity, indicated by the absence of pol-pecific amplification in negative samples, was inferred for bothBS and DBCS procedures (Table 2).

The correlation between DBCS and whole blood data setsas characterised by a large regression coefficient (Fig. 2, panel). Passing and Bablok algorithm (1983) showed only moderateivergence from the theoretical identity line suggesting a robustgreement between DBCS and the whole blood assays (Fig. 2, panel). Furthermore, a plot of DBS assay against the whole blood-ased assay revealed a small regression coefficient (R2 = 0.1819),hich was still negligible after removing the 28 negative samples

R2 = 0.2868) (Fig. 2, panel C and panel D). This result suggests thathe latter DBS method is rudimentary for proviral HIV-1 DNA deter-

ination. In fact, it may lose 56% of the entire data set and lead ton underestimation of potential positive samples that would oth-rwise be assessed and diagnosed correctly by a DBCS-based assay.mportantly, no correlation was observed between patient age andBCS assay sensitivity, which would suggest the likely applicationf this assay for HIV-1 diagnosis in both infants and children.

These data show that DBCS DNA extraction performance is veryimilar to the manual one from whole blood, whereas extractionrom DBS decreases significantly the DNA yield and the pol-basedssay sensitivity for proviral DNA determination. This evidenceay suggest that DNA quantities recovered from DBS are often

nsufficient for HIV-1 provirus detection despite using a nested-CR-based determination method. In contrast, DBCS, coupled to aighly specific detection system such as the Sybr Green I (Fig. 3)ay provide a rapid, sensitive and suitable assay for proviral HIV-DNA determination from buffy coat fractions. Indeed, the entirerocedure from blood withdrawing to DBCS spotting takes approx-

mately 2 h, 20 min for each sample, and, after air-drying at RT,emote determinations can be easily performed. Combined discunching and automatic extraction may take up to approximately1 min, while the additional pol-based real-time PCR and the IAC ˇ-lobin assay do not exceed a turn-around-time (TAT) of 1 h, 30 minnd 1 h, 10 min for each sample, respectively.

Taken together, the present results seem to suggest that remoteiagnoses of HIV-1 provirus by DBS may be enhanced consider-bly by procedures designed to spot leukocyte buffy coat, especiallyhen combined to a real-time PCR assay based on a SYBR Green Ietection system. The assay described above may represent a tool toperate under basic field conditions in resource-limited settings. Itay represent an affordable, simple and robust point-of-care assay

or HIV screening but, especially, an accurate option for diagnosisf HIV in children.

cknowledgments

We wish to thank Dr. Laura Arboit and Dr. Marco Costantinior supporting the EZ1 automatic extractor set-up and Dr. Raffaeleaffa for his useful suggestions regarding statistical analyses. Welso like to thank Dr. Giuseppe Pontrelli and Dr. Paolo Palma ofhe Division of Immunology and Infectious diseases, Dept of Paedi-trics, Bambino Gesù Children Hospital.

We acknowledge Dr. Andreina Santoro for her thorough Englishevision of the manuscript.

We also would like to thank Sister Rosaria Gargiulo of the Reli-ious Congregation of “Suore Adoratrici del Sangue di Cristo” andather Vincenzo Boselli of the Religious Congregation of “Padri

issionari del Preziosissimo Sangue” who are responsible for the

are offered to the children at the Mission “Villaggio della Sper-nza”, Dodoma, Tanzania. We especially thank Sister Caterinaonci, Mother Superior of the Congregation of “Suore Adoratriciel Sangue di Cristo” for her remarkable support to the study.

ogical Methods 170 (2010) 121–127

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