Comparison of HIV viral load between

Dry Blood Spot (DBS) and plasma samples

using Abbott Real Time HIV-1 PCR

Dr. Tin Tin Htay

Senior Consultant Microbiologist
National Health Laboratory
Myanmar
 Background

Global target for HIV: 90-90-90 (UNAIDS 2014)

• Identify 90% of all people living with HIV,
• Treat 90% of those identified as infected
• Achieve virologic suppression in 90% of treated
individuals by 2020
• Viral load testing should be performed
– within 6 months after initiating ART
– at 12 months and
– at least every 12 months
 Background (cont:)

• In recent years, no. of HIV patients on ART has

dramatically increased
• Early detection of therapeutic failure is major
challenge for Management of HIV patients on ART
– optimize ART efficacy
– prevent accumulation of resistance mutations
• WHO encouraged routine VL monitoring in patients
on ART to identify virological failure
 Background (cont:)

• For routine VL monitoring in remote area, there is

difficulties in specimen storage, transportation, human
resources limitation and insufficient laboratory
infrastructure
• Currently, dried blood spot (DBS) sample may be
considered as an alternative way to determine HIV viral
load, especially in resource limited and remote areas
• Need to validate the VL results using DBS samples
compare to plasma from same patient
 DBS samples
Advantages:
Ø easy to perform
Ø can be kept ambient temperature for 2 weeks
before freezing
Ø can transfer through postal service
Ø not require laboratory equipment (eg. Centrifuge)
Disadvantages:
Ø more time for DBS sample processing
Ø higher detection level (839 copies/ml)
 Background (cont:)
In 2017,
• Prevalence of HIV in adults – 0.57% (227,000)
• HIV patients on ART – 146,826
• Laboratories for HIV VL monitoring using Abbott
machine in 3 large cities (Yangon, Mandalay, Magwe)
• Before implementing HIV VL monitoring using DBS
samples, need to evaluate VL >1000 copies/ml from
HIV positive patients with DBS compared to plasma
in routine testing condition using the existing Abbott
machine
 Aim

• To study the HIV viral load of DBS samples and

plasma samples from both HIV positive and
negative patients using Abbott real-time
polymerase chain reaction (RT-PCR) machine
Objectives
• To determine the HIV viral load from DBS
samples using Abbott machines
• To determine the HIV viral load from plasma
samples using Abbott machines
• To compare the HIV viral load results of DBS and
plasma samples
 MATERIALS AND METHODS
Study Design
Clinical and Laboratory based cross-sectional
descriptive study

Study Period
January to June 2018

Place of Study
§ Latha AIDS/STD clinic in Yangon, Myanmar
§ Virology Section, NHL, Yangon, Myanmar
Study Population
HIV positive and negative people tested with Myanmar
HIV testing algorithm
Ø ELISA, Unigold and Stat Pak
Sample Size
§ 60 people who are HIV positive
§ 30 people who are HIV negative
 Sample collection and transportation
• 4 ml of venous blood in EDTA tube for VL
• 2 ml of venous blood in Plain tube for HIV
serology
Transported within 2 hours with cold chain to
Virology Section, NHL, Yangon
 Specimen processing for HIV serology

• Whole blood in plain tube was centrifuged at

1600rpm x 10 mins
• Serum samples were tested using:
– Bio-Rad GenScreen HIV 1/2 Ag/Ab ELISA
– Trinity Biotech Unigold
– Chembio Stat-Pak
 Specimen processing for DBS Samples
• 70 μL of whole blood were dropped on each of
(12 millimetre) circles of perforated Whatman
903 paper cards using a calibrated pipette
• Leave on drying rack at ambient temperature
overnight
• Packed individually in zip-lock bags with
desiccant sachets
• DBS cards were stored at -40°C until viral load
was measured
 Specimen processing for the plasma samples

• Remaining blood was centrifuged at 2000rpm x

20 mins
• Plasma samples were collected in 2 cryotubes
and stored at -40°C until viral load was
measured
 Procedure for nucleic acid extraction
Nucleic acid extraction from dried blood spot
• One circle of dried blood spot was placed into a
master mix tube with 1300 μL of DBS buffer
• Samples were swirled and incubated for 30 mins in
a heating block at 55˚C
• Samples were directly loaded on the Abbott
m2000sp instrument
• The extraction and master mix preparation was
done in the Abbott m2000sp instrument according
to the standard protocol
 Nucleic acid extraction from plasma
• The stored plasma samples were thawed and placed
at 2-8˚C before testing
• Centrifuged at 2000g x 5 mins in refrigerated
centrifuge at 4˚C
• 800 ml of plasma from each patient was placed
directly into the Abbott m2000sp instrument
• The extraction and master mix preparation in the
Abbott m2000sp instrument according to the
standard protocol
– 0.6 mL HIV-1 RNA principle of nucleic acid extraction and
amplification
• In each test run for assay validation, an RNA
sequence unrelated to the HIV-1 target sequence,
as an internal control, a negative control (HIV-1-
negative human plasma) and a low and a high
positive control were included

Abbott m200sp
machine
 Procedure for real time PCR (RT PCR)
Amplification
• Two sets of oligonucleotide primers and probes
– One specific for amplifying and detecting HIV-1 RNA and
the other for internal control RNA
– Target sequence for HIV-1 is pol integrase region

Abbott m2000rt
machine
 Procedure for real time PCR (RT PCR)

Detection
• The amplification cycle, at which fluorescent signal is
detected, is proportional to the log of the HIV-1 RNA
concentration present in the original sample by the
Abbott m2000rt instrument

Quantitation
• The quantitation of the HIV-1 RNA in specimens and
controls were calculated from the stored calibration
curve, and the results were automatically reported
on the m2000rt workstation
Calibration curve of Calibration curve of
HIV viral load in DBS HIV viral load in Plasma
 Data management

• Data entry was done by Microsoft Office Excel

2010 and data analysis was done by Statistical
Package for Social Sciences (SPSS) version 18.0
Limit of Detection for HIV Viral Load with
Abbott RT PCR (instruction manual insert)

Lower Limit of Upper Limit of

Detection Detection
(copies/ml) (copies/ml)
Plasma 40 > 107
DBS 839 > 107
 Negative Control Low Positive Control High Positive Control

HIV viral load in Dried Blood Spot HIV viral load in plasma
(less than 2.92 log copies per ml) (less than 1.6 log copies per ml)
 Agreement between dried blood spot and plasma
samples for detectable and undetectable HIV viral load

Plasma samples
Dried blood spot Total
samples No. of samples No. of samples
with detectable with undetectable
viral load viral load
No. of samples with
detectable viral load 60 0 60

No. of samples with

undetectable viral 0 30 30
load
Total 60 30 90
n = 90
n = 90
Correlation between viral load from plasma and dried blood spot
- Each data point represents an individual study sample
- The line indicates the best fit of the data to a linear regression
Agreement between detectable viral load in dried
blood spot and plasma
 Policy and practice implications

• HIV viral loads in DBS and in plasma were analysed

by Pearson’s correlation, it was found that good
correlation between them (R2 = 0.8952) although
the lower limit of HIV viral load in DBS was a little
bit high
• Showed good performance to detect the treatment
failure with set point of above 1,000 HIV copies / ml
 Policy and practice implications (cont:)

• The obtained log transformed values of detectable

viral load were analysed by Bland Altman analysis
• It was found that 95% (57/60) was within the 95%
limits of agreement (mean ± 1.96 SD)
– Ranged from 0.501 to 0.557 log copies of HIV RNA
 Policy and practice implications (cont:)

• According to the above key findings, DBS samples

can be used in resource limited and remote areas as
an alternative to plasma samples for HIV VL
• It was hoped that the results from this study can be
useful in some extent to improve access to viral load
determination in HIV infected patients leading to
monitor effectiveness of antiretroviral treatment and
to support early detection of treatment failure and
drug resistance
 Acknowledgements

v Prof. Dr. Htay Htay Tin, DyDG (Lab), NHL, Myanmar

v Dr. Latt Latt Kyaw, Head of Virology Section, NHL
v Dr. Win Win Yee, SCS Microbiologist, NHL
v Dr. Wai Yin Nwe, Microbiologist
v NHL HIV section staffs
v Abbott technical group
v ASCP/CDC
v NRL
Thank You