JOURNAL OF CLINICAL MICROBIOLOGY, Jan. 2009, p. 220–222
Copyright © 2009, American Society for Microbiology. All Rights Reserved.
Vol. 47, No. 1
Qualitative Human Immunodeficiency Virus RNA Analysis of Dried
Blood Spots for Diagnosis of Infections in Infants?
Ryan J. S. Kerr, Gray Player, Susan A. Fiscus, and Julie A. E. Nelson*
UNC Center for AIDS Research and Department of Microbiology and Immunology, University of North Carolina at Chapel Hill,
Chapel Hill, North Carolina 27599
Received 7 August 2008/Returned for modification 10 October 2008/Accepted 3 November 2008
The Gen-Probe Aptima human immunodeficiency virus type 1 (HIV-1) RNA assay was adapted for the
diagnosis of HIV infection in infants by using dried blood spots. The assay was 99% sensitive (128/129) and
100% specific (162/162). This may prove useful in resource-limited settings, since it precludes the need for a
phlebotomist and maintenance of a cold chain.
Diagnosis of human immunodeficiency virus type 1 (HIV-1)-
infected infants is essential for the evaluation of interventions
for the prevention of this transmission and for identifying in-
fants for initiation of therapy. Nucleic acid detection methods
are necessary for infant diagnosis due to the presence of ma-
ternal antibodies in the babies for up to 18 months after birth.
Dried blood spots (DBS) are an easy way to collect and ship
specimens for diagnostic testing of HIV and preclude the ne-
cessity of a phlebotomist and maintenance of the cold chain (2,
7). However, the limited volume of specimen (50 ?l) dried on
filter paper decreases the sensitivity of any assay. Current di-
agnostic methods that have been used on DBS include DNA
PCR (3, 14), the ultrasensitive p24 antigen assay (12), and viral
load (VL) testing (1, 4, 8). The lower limits of detection in
these RNA and DNA PCR detection methods were approxi-
mately 4,000 copies (cp)/ml (1, 4, 8, 14), likely due to the small
sample size of DBS.
The Gen-Probe Aptima HIV-1 RNA qualitative assay is
extremely sensitive for the detection of HIV in plasma (5). This
assay has been evaluated recently as being a more sensitive
screening tool for HIV-positive samples from a sexually trans-
mitted disease clinic than typical antibody testing followed by
pooled RNA testing (13). In addition, preliminary results from
our laboratory suggest that plasma specimens with VLs of ?20
cp/ml are detectable. Therefore, we evaluated this assay for use
with DBS from infants and children as a rapid and less-expen-
sive alternative to other assays for the diagnosis of HIV-1
infection in infants.
We formulated an elution buffer for the removal of blood
containing HIV from DBS (1 mM EDTA, 1 mM EGTA, 3%
lithium lauryl sulfate in phosphate-buffered saline). Optimiza-
tion of elution resulted in the following protocol: two 6-mm
circles were punched from each card and placed in a 2-ml
screw-top tube. The hole punch was cleaned between cards by
punching a clean Whatman 903 card twice. Elution buffer (525
?l) was added to each tube, and the specimens were rocked for
2 hours at room temperature. The specimens were spun down
for 30 s at 10,000 rpm in a microcentrifuge, and the filter paper
was removed from the tubes using disposable wooden appli-
cator sticks. The Aptima HIV-1 RNA qualitative assay was
performed according to the manufacturer’s instructions, using
500 ?l of the eluate.
Initial experiments with DBS made from whole blood spiked
with known quantities of HIV-1 indicated that our limit of
detection was near 400 cp/ml, which seemed appropriate given
the small sample volume represented by two punches from a
single spot (approximately 10 ?l plasma). However, once we
began testing DBS collected from adults with low VLs, the
detection limit for the Aptima assay fell to 20 to 200 cp/ml (Fig.
1). The lower detection limit with patient DBS, compared to
the spiked level of DBS, may be explained by the contribution
of cellular HIV RNA and possibly proviral DNA from infected
cells in the blood, since all nucleic acid is included in the
Aptima assay (a lower limit of 4 cp/reaction was achieved by
Pasternak et al.  by using peripheral blood mononuclear
cells). The real-time reverse transcription-PCR assay devel-
oped by Ou et al. (8) also involved isolation of total nucleic
acid, which is likely why that assay was also very sensitive (a
lower limit of approximately 8 cp/reaction or 4,000 cp/ml).
Cellular RNA is not measured in plasma testing, so the level of
RNA may be higher in whole blood due to the infected cells.
In all, we tested 291 DBS from infants and children born to
* Corresponding author. Mailing address: University of North Caro-
lina at Chapel Hill, 11-141 Lineberger Cancer Center, CB 7295,
Chapel Hill, NC 27599. Phone: (919) 843-9252. Fax: (919) 966-3015.
?Published ahead of print on 12 November 2008.
FIG. 1. Aptima results (reactive or nonreactive) from 39 DBS col-
lected from adults. VLs were determined using the Roche Monitor
v1.5 RNA assay (using either the standard or ultrasensitive method) on
plasma from the same blood collection that was used for the DBS.
Open circles, determined VLs; filled triangles, VLs under the lower
detection limit (50 cp/ml).
HIV-1-infected mothers (Table 1). The samples had been col-
lected as part of the prevention of mother-to-child transmis-
sion studies in South Africa (subtype C) (10–12); Malawi (sub-
type C); Tanzania (HIVNET 024; subtypes A, C, and D) (6),
Vietnam (CRF 01_AE); Dominican Republic, Haiti, and Trin-
idad (subtype B); and North Carolina (subtype B). The spots
had been made from 50 ?l whole blood per spot on Whatman
903 cards with blood drawn by heel stick. The samples were
dried at room temperature, sealed individually in plastic bags
with desiccant pouches, and stored at 4°C (for Vietnam and
North Carolina infants) or room temperature prior to testing.
The spots were stored in our laboratory for between ?1 year
(North Carolina) and 3 to 5 years (HIVNET 024) prior to
testing for this study. HIV status for the samples had originally
been determined by NucliSens HIV-1 QT manual VL assay
(bioMe ´rieux; DBS from Dominican Republic, Haiti, Trinidad,
Malawi, and Tanzania), DNA PCR assay (Roche Amplicor
v1.5; DBS from South Africa), or an in-house HIV assay (DBS
from Vietnam). All samples had been collected with appropri-
ate Institutional Review Board approval for each country, and
the UNC IRB also approved the study.
The Aptima assay was very sensitive for infant diagnosis
using DBS. One hundred twenty-nine of the 291 samples had
previously tested positive, and 128 of the 129 were reactive on
the Aptima assay (99.2% sensitivity) (Table 1). The one false-
negative infant specimen had been stored for 4 years at room
temperature and had the lowest VL of the HIVNET 024 sam-
ples tested (10,954 cp/ml in May 2003). We have shown in a
separate study that these DBS lost an average of 1 log10in VL
over this length of time (J. A. E. Nelson, A. M. Loftis, D.
Kamwendo, W. W. Fawzi, T. E. Taha, R. L. Goldenberg, and
S. A. Fiscus, submitted for publication). The 162 samples that
had previously tested negative were all nonreactive on the
Aptima assay (100% specificity) (Table 1).
The results of the present study indicate that DBS can be
used in the Gen-Probe Aptima HIV-1 RNA qualitative assay
for infant diagnosis without the loss of sensitivity or specificity
achieved by currently used methods. In our laboratory, we have
found that the Aptima assay is less expensive than the Nucli-
Sens HIV-1 QT manual assay (bioMe ´rieux, Inc.) for DBS, with
increased sensitivity and less labor involved, making it a good
choice for infant diagnosis from DBS. In addition, this very
sensitive assay would be ideal for the diagnosis of acute HIV
infection via DBS.
Overall support for the International Maternal Pediatric Adolescent
AIDS Clinical Trials Group (IMPAACT) was provided by the Na-
tional Institute of Allergy and Infectious Diseases (U01 AI068632), the
Eunice Kennedy Shriver National Institute of Child Health and Hu-
man Development, the National Institute of Mental Health, and the
NICHD International and Domestic Pediatric and Maternal HIV
Clinical Trials Network (N01-DK-9-001/HHSN2672008000001C). This
work was also supported by the UNC Center for AIDS Research (P30
The content is solely the responsibility of the authors and does not
necessarily represent the official views of the National Institutes of
DBS for this study were provided by Melissa Miller, Gayle Sherman,
Charles van der Horst, Annette Sohn, and Consuela Beck-Sague.
Technical assistance was provided by Takesha McMillion and Mark
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TABLE 1. Sources, subtypes, and Aptima results for infant DBS
Site of collectionPresumed subtype(s) Initial testing method
No. of Aptima-R samples/
no. of HIV-positive
No. of Aptima-NR samples/
no. of HIV-negative
Tanzania (HIVNET 024)c
A, C, D
Roche Monitor v1.5 (standard)
Total128/129 (99.2)162/162 (100)
aAptima-R, reactive in the Aptima assay; Aptima-NR, nonreactive in the Aptima assay.
bSee references 10, 11, and 12.
cSee reference 6.
dThe false-negative Aptima result was obtained with the sample in this group with the lowest VL.
VOL. 47, 2009NOTES221
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222 NOTESJ. CLIN. MICROBIOL.