HIVAIDS Surveillance and Targeted Epidemiological Studies Plan

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HIV-1 Drug Resistance Genotyping Dried Blood
Spots Compared with Plasma
Paul Sandstrom National HIV and Retrovirology
Laboratories Centre for Infectious Disease
Prevention and Control, Public Health Agency of
Canada
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The Problem

• While plasma and serum specimens are considered
  the gold standard for genotyping, they are not
  practical for many resource-limited settings and
  unsuitable for serving remote populations.
• logistic difficulties,
• lack of a cold chain
• lack of secure sample transport/transport of
  dangerous goods ,
• requirement for trained phlebotomist
• restricted field collection may introduce
  sampling bias
• Expense
• Laboratory required for pre-storage specimen
  processing
• Compliance from research subjects

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DBS in HIV Surveillance and Monitoring

• DBS have been reported in peer reviewed
  scientific
• literature to be suitable for
• HIV serology
• HIV molecular diagnostics
• HIV viral RNA quantification (viral load).
• CD4 lymphocyte enumeration
• However because DBS remain non traditional
  specimens for commercial kit
• based testing, proficiency testing programs will
  be required.

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Objectives

• To assess the feasibility of genotyping HIV-1
  from DBS collected in the field and stored under
  extreme environmental conditions
• Concordance between sequences as well as DR
  genotypes obtained from plasma vs DBS
• No attempt to delineate the nature of the genetic
  material (RNA vs DNA) being preferentially
  amplified from DBS.

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Study Design

• Part of a large WHO survey to evaluate prevalence
  of HIVDR in 10 testing and counselling (TC)
  sites in Central Mexico
• Prospectively collected paired plasma and DBS
  from consecutive newly diagnosed, ARV-naïve HIV
  Mexican subjects (n558).
• Whole blood (EDTA anticoagulated) was spotted
  onto SS 903 cards, dried at room temperature and
  stored with desiccant at 37C and 87 humidity
  for 3 months in sealed plastic bags.

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Laboratory MethodsAmplification and Sequencing
of DBS

• Amplification of the protease and RT regions of
  the pol gene based on an in house nested RT-PCR
  method.
• Target template was amplified in two overlapping
  pieces using Qiagen OneStep RT-PCR
• Nested PCR was performed using AmpliTaq Gold
  (ABI) in 50ul reactions containing 5ul RT-PCR
  product
• Viral load determination in the matched plasma
  specimen for those DBS that could not generate
  PCR products (Amplicor HIV-1 Monitor)

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Primers used to amplify the protease and RT
regions
DBS specimens not producing PCR products using a
first primer set were attempted with a second set
under the same condition indicated above.
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MethodsAmplification and Sequencing of DBS

• All specimens for which PCR products for both the
  protease and the RT regions could be generated
  were sequenced (BigDye v3.1 (ABI) and run on an
  ABI Prism 3100 analyzer).
• Sequences were aligned to HXB2 pol reference
  sequence using SeqScape software (ABI) and a
  contiguous sequence generated.
• Sequences were queried against the Stanford
  University HIV Drug Resistance Database

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Phylogentic analysis confirmed that all matched
plasma and DBS specimens originated from the
same individual and that no PCR contamination
occurred
Neighbor-Joining with bootstrap resampling
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Results

• 90.1 of all DBS specimens could be amplified in
  at least one region (compared to 100 for
  plasma).
• 78.2 amplified in both PR and RT regions
• For DBS which could not be amplified (n14),
  plasma viral load ranged from 4,580 to gt100,000
  copies/ml
• All DBS that produced a PCR product were
  successfully sequenced.
• Excellent agreement between paired DBS/plasma
  sequences (99.90 complete concordance across
  entire sequence).

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Major mutations associated with resistance
detected in 101 matched DBS and plasma specimens

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ConclusionGenotyping

• For specimens in which drug resistance mutations
  were identified, detection of mutant amino acids
  were completely concordant in 94.2 of pairs,
  with the remaining differences being represented
  as partial discordances
• May represent an intrinsic randomness of results
  generated during the sequencing of non-clonal PCR
  products
• In the case of DBS nucleic acid extracts, the
  relative contribution of archival variants
  contained within the proviral DNA reservoir may
  be responsible for the generation of mixed base
  positions not observed in plasma.

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ConclusionsSuccess Rate-PCR

• The observed differences between DBS and plasma
  amplification rates may be due to the independent
  or combined effects of several factors including
• Differences in primers and amplification
  conditions employed at each site
• Lower sample volume used for the extraction of
  DBS (100 ul) as compared to plasma (200-500 ul)
• The partial degradation of RNA could have
  occurred under extreme storage conditions.

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Conclusions General

• DBS appear to be a promising public health tool
  for HIVDR surveillance of treatment-naïve
  subjects, especially in regions where specimens
  might be exposed to severe environmental
  conditions.
• More study is needed to validate DBS for patient
  monitoring.
• Suitability of DBS for commercial genotyping
  assays remains under investigation

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Acknowledgments

• World Health Organization
• Silvia Bertagnolio
• Donald Sutherland
• Instituto Nacional Ciencias Medicas, México
• Luis Soto-Ramirez
• Roberto Rodriguez
• Monica Viveros
• Luis Fuentes
• BC Centre for Excellence
• Richard Harrigan
• Theresa Mo
• Public Health Agency of Canada
• Richard Pilon
• Linda Ares