Materials and Methods

1
Materials and Methods
No. 39
REAL-TIME RT-PCR BASED ASSAY ON BLOOD CLOT
SPECIMENS FOR DIAGNOSIS OF HIV-1 INFECTION IN
CHILDREN, MALAWI
CDC, Mail Stop D-12 1600 Clifton Road Atlanta, GA
30333 cyang1_at_cdc.gov 404-639-4975
Hua Yang1, Rita Helfand2, Desiree Witte3, Ashley
C. LaMonte2, Yashieka Blount1, Robin Broadhead3,
Felicity Cutts4, Richard Fudzulani3, Chin-Yih
Ou1, Renu B. Lal1, Chunfu Yang1
1Divisions 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
Summary

• 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-PCR-based
  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 gt 18 months of age
  from children born to HIV-infected 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.

Results

• 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
  -20C freezer for HIV-1 viral sequence detection.

• 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.
• 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.
• 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.
• Based on the excellent sensitivity and
  specificity of the assay, we then tested 144
  children who didnt 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.

Sensitivity of the Assay
RT-PCR positive (month)
No. of specimens (34)
6
9
12
18
20
24
30
HIV rapid tests positive 18 months
10


9



4


Digestion of blood clots with streptokinase and
extraction total nucleic acid
Real Time RT-PCR


3

2
For a 25 µL of real-Time RT-PCR Reaction
2

Blood clot specimens
1

1. Add 12.5 µL of QuantiTect Probe RT-PCR Master Mix
2. 0.25 µL of /Quantitect RT Mix
3. 1 µL of HIV-1-specific primer and probe mix
4. 1 µL of Human RNAse P primer and probe mix
5. 0.25 µL of RNase-free water

Add 300 µL of PBS
1



1


Add 20 units/vial of Streptokinase

1
Incubate at 37C for O/N
15 µL
Distribution of HIV-1 RT-PCR positivity based on
the first positive RT-PCR
Add 10 µL of total nucleic acid extract
Extract total nucleic acid with QIAamp Blood DNA
mini kit using 200 µL of the O/N digests
Real Time RT-PCR 50C, 30 minutes 95C, 15
minutes 60 cycles at 95C for 15 second, 52C
for 1 minutes
Conclusions

• The duplex, one tube, 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.
• 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.

Introduction
Primers and Probes

• 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 real-time 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.

Gene
Sequences
Functions
HIV-1 LTR
5tgcttaagcctcaataaagcttgccttga
Forward Primer
Specificity of the assay
HIV-1 LTR
5tctgagggatctctagttaccag
Reverse Primer
No. Specimen Negative Positive HIV-1 sequence amplified
Rapid tests months 24 24 (100) 0
RT-PCR 24 23 (95.8) 1 (4.2) Yes
HIV-1 LTR
5Fam-aagtagtgtgtgcccgtctgt-Qsy-7
Probe
RNaseP
5 agatttggacctgcgagcg
Internal Control Farward Primer
RNaseP
5gagcggctgtctccacaagt
Internal Control Reverse Primer
This child was HIV-1 real-time RT-PCR positive
at 6, 12, 18 and 24 months.
RNaseP
5Hex-ttctgacctgaaggctctgcgcg-Qsy-7
Probe