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Development of quantitative HIV-2 viral load RT-PCR assay Alfred A. Ngwa, PhD

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

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Development of quantitative HIV-2 viral load RT-PCR assay Alfred A. Ngwa, PhD

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  1. Development of quantitative HIV-2 viral load RT-PCR assay Alfred A. Ngwa, PhD Molecular Diagnostic Services MRC Laboratories, The Gambia 16-01-13

  2. 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

  3. Commercially available Nucleic acid based HIV-1 Viral load tests

  4. Branched DNA (bDNA) Assay for Quantifying HIV-1 (Versant HIV-1 RNA 3.0)

  5. Nucleic Acid Sequence-Based Amplification (NASBA) Reaction

  6. TaqMan Real-Time PCR

  7. 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.

  8. 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

  9. 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

  10. 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 2006 Florence 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 ClinMicrobiol. 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.

  11. 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

  12. 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

  13. 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.

  14. ELONA

  15. Primers and probes; LTR • P1; ATAAAAWTTAGTTTTTAGTAGTTCCACAAWTTGTT • P2; ACAAAAACYYTTCAGCACYTWGAGAAWCAA • P3; ATAKGGAAGTGGCWACAAAAACYYTTC

  16. HIV-2 standard trial-2

  17. Standard curve; Dried Plasma Spot

  18. MRC Capacity- Molecular diagnostic labs • 6 real-time PCR machines • 384well ViiA7 • QuantStudio

  19. 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

  20. Battery powered real time Fluorometer

  21. 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

  22. Acknowledgment • DrAssanJaye (WAPHIR) • Ms Alberta Davis • Dr Abraham Alabi • Dantec HIV team • MRC Mol Diagnostic team • WAPHIR/WANETAM

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