development of quantitative hiv-2 viral load rt-pcr assay alfred a. ngwa, phd
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Development of quantitative HIV-2 viral load RT-PCR assay Alfred A. Ngwa, PhD Molecular Diagnostic Services MRC Laboratories, The Gambia 16-01-13. Background. Increased access to ART in resources in recent years Challenges High Cost of Rx Monitoring - PowerPoint PPT PresentationTRANSCRIPT
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Development of quantitative HIV-2 viral load RT-PCR assay
Alfred A. Ngwa, PhDMolecular Diagnostic ServicesMRC Laboratories, The Gambia
16-01-13
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Background• Increased access to ART in resources in
recent years Challenges• High Cost of Rx Monitoring• Inadequacy of trained health Personnel • Lack of Infrastructure Results • Inadequate Immunological & Virological
Monitoring of patients on ART
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Commercially available Nucleic acid based HIV-1 Viral load tests
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Branched DNA (bDNA) Assay for Quantifying HIV-1 (Versant HIV-1 RNA 3.0)
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Nucleic Acid Sequence-Based Amplification (NASBA) Reaction
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
TaqMan Real-Time PCR
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Background- HIV2 VL issues
• Monitoring the treatment response of patients infected with HIV-2 is more difficult than monitoring people infected with HIV-1.
• No licensed HIV-2 viral load assay is available yet.
• Viral load assays used for HIV-1 are not reliable for monitoring HIV-2.
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Background- HIV2 VL issues
• Diagnosis in children• Sampling in children and adults• Low viral loads in HIV2• Poor sensitivity of current assays in detecting and
quantifying• Cost• Need for point of care
• Limited expertise • Limited equipment
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Background- Remaining issues
• Inadequate infrastructure & expertise• Subtype variations• Both HIV-1 & HIV-2 prevalent in West Africa and there is
no commercial VL assay for HIV-2• Direct virological monitoring is achieved by measuring
HIV viral load• Therefore, robust & cheap VL assays are needed to
monitor viral control in clinical trials/intervention programmes
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
MRC; ELONA STRUCTURE
J Clin Microbiol. 2008 June; 46(6): 2088–2091.Quality Control Assessment of Human Immunodeficiency Virus Type 2 (HIV-2) Viral Load Quantification Assays: Results from an International Collaboration on HIV-2 Infection in 2006Florence Damond,1* Antoine Benard,2 Jean Ruelle,3 Abraham Alabi,4 Bernd Kupfer,5 Perpetua Gomes,6 Berta Rodes,7 Jan Albert,8 Jürg Böni,9 Jeremy Garson,10 Bridget Ferns,10 Sophie Matheron,11 Geneviève Chene,2 Françoise Brun-Vezinet,1 and for the ACHIEV2E (a Collaboration on HIV-2 Infection) Study Group
J Clin Microbiol. 2011 Oct;49(10):3491-7. doi: 10.1128/JCM.02389-10. Epub 2011 Aug 3.An international collaboration to standardize HIV-2 viral load assays: results from the 2009 ACHI(E)V(2E) quality control study.Damond F, Benard A, Balotta C, Böni J, Cotten M, Duque V, Ferns B, Garson J, Gomes P, Gonçalves F, Gottlieb G, Kupfer B, Ruelle J, Rodes B, Soriano V, Wainberg M, Taieb A, Matheron S, Chene G, Brun-Vezinet F; ACHI(E)V(2E) Study Group.
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
ELONA; In-house chemiluminecense assay
• Funding from EDCTP in 2004 and Project commenced in February 2005
• Developed a colorimetric format of an RT-PCR Assay for quantifying HIV RNA in human plasma
• Principle
• The Assay is a quantitative reverse-transcribed PCR of the long terminal repeat (LTR) sequence of HIV in which test samples are quantified by comparison with a standard curve
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
ELONA; In-house chemiluminecense assay
• Basic Procedure of Assay:• Extraction of RNA from Patient’s plasma • Reverse transcription of RNA to cDNA• PCR with specific HIV LTR primers• Detection of DNA product by ELONA Unique features of the Assay:• Inclusion of internal calibrator to compensate for RNA
loss, inhibition, RT-PCR & makes assay competitive• Simple Technique• Use of common Lab Equipment• Affordable
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
ELONA; In-house chemiluminecense assay
• The standard curve is generated from tissue culture supernatant of either (1) HIV-1 virus (Strain U455) or (2) HIV-2 virus (strain CBL23) grown in 8166 cells.
• To control for extraction efficiency and non-specific inhibition of reverse transcription and PCR, an internal control (IC) is included. This is a 1kb synthetic RNA sequence, derived from the LTR of HIV-1/HIV-2 containing the PCR primer binding sequences but replacing the probe binding sequence with a random sequence.
• Detection and quantification of test and control PCR products is made in an Enzyme-Linked Oligonucleotide Assay (ELONA) and results are expressed as HIV-1 or HIV-2/IC ratios for each sample, standard or control.
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
ELONA
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Primers and probes; LTR
• P1; ATAAAAWTTAGTTTTTAGTAGTTCCACAAWTTGTT
• P2; ACAAAAACYYTTCAGCACYTWGAGAAWCAA
• P3; ATAKGGAAGTGGCWACAAAAACYYTTC
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
HIV-2 standard trial-2
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Standard curve; Dried Plasma Spot
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
MRC Capacity- Molecular diagnostic labs
• 6 real-time PCR machines• 384well ViiA7• QuantStudio
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Next steps
• Cross validation on the West African platform; WAPHIR• Development of QT_NASBA• Isothermal amplification based
• RPA; TwistDX• HDA
• Advantages• No need for cycling• No Nucleic acid amplification• Quantitative• POC
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Battery powered real time Fluorometer
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Validation
• Sampling in Dakar, Senegal and Ciao, Guinea Bissau• Plasma• Dried plasma spots and Dried blood spots from
children on Whatman 3MM and FDA (NPHL, Gambia)• Exchange of samples and controls between Dantec and
MRC Gambia• Training workshop in both institutions• MSc degree
MRC Unit, The Gambia Leading scientific research to save lives and improve health across the developing world
Acknowledgment
• Dr Assan Jaye (WAPHIR)• Ms Alberta Davis• Dr Abraham Alabi• Dantec HIV team• MRC Mol Diagnostic team• WAPHIR/WANETAM