No. 39
REAL-TIME RT-PCR BASED ASSAY ON BLOOD CLOT SPECIMENS FOR DIAGNOSIS OF
HIV-1 INFECTION IN CHILDREN, MALAWI
1
Yang ,
2
Helfand ,
3
Witte ,
2
LaMonte ,
1
Blount ,
Hua
Rita
Desiree
Ashley C.
Yashieka
4
3
1
1
1
Felicity Cutts , Richard Fudzulani , Chin-Yih Ou , Renu B. Lal , Chunfu Yang
1Divisions
Robin
3
Broadhead ,
CDC, Mail Stop D-12
1600 Clifton Road
Atlanta, GA 30333
cyang1@cdc.gov
404-639-4975
of HIV/AIDS Prevention, NCHSTP and 2Division of Viral and Rickettsial Diseases, NCID, CDC, Atlanta; 3University of Malawi;
4London School of Hygiene and Tropical Medicine
Abstract
Materials and Methods
• Participants are part of a study of Measles vaccination of HIV-1 infected and –
uninfected children born to HIV-1 infected mothers from outside of Blantyre, Malawi.
• One milliliter of blood was collected by fingerstick at 6, 9, 12, 18-24 and 30-36 months
of age. HIV rapid tests, Unigold and Determine were performed on blood collected ≥
18 months.
• Sera were collected for anti-Measles antibody testing, the remaining blood clots were
stored at -20°C freezer for HIV-1 viral sequence detection.
Digestion of blood clots with streptokinase
and extraction total nucleic acid
Real Time RT-PCR
For a 25 μL of real-Time RT-PCR Reaction
Blood clot specimens
1.
2.
3.
4.
5.
Add 300 μL of PBS
Add 20 units/vial of
Streptokinase
Incubate at 37°C for O/N
Add 12.5 μL of QuantiTect Probe RT-PCR Master Mix
0.25 μL of /Quantitect RT Mix
1 μL of HIV-1-specific primer and probe mix
1 μL of Human RNAse P primer and probe mix
0.25 μL of RNase-free water
Sensitivity of the Assay
HIV rapid
tests
positive ≥ 18
months
No. of specimens
(34)
10
9
4
3
2
2
1
1
1
1
RT-PCR positive (month)
9
12
18
20
24
6
+
+
+
30
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
Extract total nucleic acid with
QIAamp Blood DNA mini kit
using 200 μL of the O/N digests
Real Time RT-PCR:
50°C, 30 minutes;
95°C, 15 minutes;
60 cycles at 95°C for 15 second, 52°C for 1 minutes
Primers and Probes
Distribution of HIV-1 RT-PCR positivity
based on the first positive RT-PCR
Sequences
Functions
HIV-1 LTR
5’tgcttaagcctcaataaagcttgccttga
Forward Primer
5’:tctgagggatctctagttaccag
Reverse Primer
HIV-1 LTR
5’Fam-aagtagtgtgtgcccgtctgt-Qsy-7
Probe
RNaseP
5’ agatttggacctgcgagcg
Internal Control Farward Primer
RNaseP
5’gagcggctgtctccacaagt
Internal Control Reverse Primer
RNaseP
5’Hex-ttctgacctgaaggctctgcgcg-Qsy-7
Probe
 Based on the excellent sensitivity and
specificity of the assay, we then tested 144
children who didn’t have HIV antibody tests
due to unavailable specimens ≥ 18 months.
We found 32 had two positive RT-PCR, 28
had one positive RT-PCR; 61 had two
negative RT-PCR and 23 had one negative
RT-PCR.
Conclusions
60
40
20
 The duplex, one tube, one step real-time RTPCR used here was both sensitive and
specific in detecting true HIV-1 infection from
blood clot specimens in children born to HIV1-infected mothers under the age of 18
months.
17.6
2.9
2.9
12
18
5.9
2.9
20
24
30
Month of specimen collection
Specificity of the assay
HIV-1 LTR
 We also tested 24 children with negative HIV
antibody tests. 23 of them had negative HIV-1
RT-PCR results while one child gave positive
RT-PCR results at 6, 12, 18 and 24 months.
HIV status of the child was further confirmed
by the amplification and sequencing of the
gag gene.
67.6
0
Gene
 We tested 34 children who were HIV antibody
positive the age of ≥18 months using two
rapid tests, Unigold and Determine, and all of
them had positive RT-PCR results, giving a
100% testing sensitivity.
80
6
• Mother to child HIV-1 transmission (MTCT) is the most
common route of HIV-1 infection in children despite the
advantage of ARV.
• Because of maternal antibodies, HIV-1 infection in
children born to HIV-1 infected mothers under the age of
18 months is diagnosed by detecting HIV-1 viral
sequences.
• Although HIV-1 DNA PCR assay for HIV-1 diagnosis is
commercially available, it is expensive and labor intensive
for resource-limited countries.
• Here we report an adaptation of a duplex, one-step realtime RT-PCR assay for detection of HIV-1 total nucleic
acid using blood clot specimens for the diagnosis of HIV-1
infection in children born to HIV-1 infected mothers.
 In this study, we used a duplex, one-tube,
one step real-time RT-PCR assay to detect
HIV-1 infection from blood clot specimens
collected from children born to HIV-1 infected
mothers from Blantyre, Malawi.
15 μL
Add 10 μL of total nucleic acid extract
Introduction
Summary
Results
Percentage of infection
• Background: Mother to child HIV-1 transmission is the most
common route of HIV-1 infection in children. HIV antibody
testing is unreliable for children born to HIV-infected mothers
under 18 months of age due to maternal antibodies. Therefore,
in children younger than 18 months of age, HIV-1 infection is
diagnosed by detecting HIV-1 viral sequences. We have
developed a duplex, one-tube, one-step real-time RT-PCRbased assay for HIV-1 diagnosis. In this study, we evaluated
this assay for its ability to detect HIV infection in blood clot
specimens in children born to HIV-1-infected mothers under
the age of 18 months in Malawi.
• Methods: We collected 1ml of blood by fingerstick at 6, 9, 12,
18-24, and 30-36 months of age during a study of measles
vaccination of HIV-infected and -uninfected children outside of
Blantyre, Malawi. We performed HIV rapid antibody testing on
blood collected > 18 months of age from children born to HIVinfected mothers. Antibody positivity ≥ 18 months was taken
as the indicator of HIV-1 infection in children. Total nucleic
acids were extracted from blood clot specimens following
digestion with streptokinase, and real-time RT-PCR was
performed using the primers in the HIV-1 LTR region. We
tested blood clots from 34 children with positive HIV antibody
results and 24 of 146 children with negative HIV antibody
tests.
• Results: To evaluate the sensitivity and specificity of the
assay, we first tested 34 children who were consistently HIV
antibody positive ≥ 18 months of age and all of them had
positive RT-PCR results from samples taken on at least two
time points. We then tested 24 HIV-1 antibody negative
children. Of the 24 children, 23 were HIV-1 negative by RT-PCR
while one had positive HIV-1 RT-PCR at 6, 12, 18 and 24
months and undetectable antibody at 18 and 24 months by
two rapid HIV tests. HIV infection was confirmed in this child
by amplification of the HIV-1 gag region and sequencing.
• Conclusion: The one tube and one step Real-time RT-PCR
used here was both sensitive and specific in detecting true
HIV-1 infection from blood clot specimens in children born to
HIV-1-infected mothers under the age of 18 months.
No. Specimen
Negative
Positive
Rapid tests ≥
months
24
24
(100%)
0
RT-PCR
24
23
(95.8%)
1*
(4.2%)
HIV-1 sequence
amplified
Yes
* This child was HIV-1 real-time RT-PCR positive at 6, 12, 18 and 24 months.
 This study confirms that blood clot
specimens can be used for the detection of
HIV viral sequences, opening a new avenue
for HIV detection in children in terms of
specimen specification.