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Surveillance of HIV variants in Cameroon and evaluation of their impact on HIV diagnostics

Surveillance of HIV variants in Cameroon and evaluation of their impact on HIV diagnostics. Indira K. Hewlett, Ph.D Chief, Laboratory of Molecular Virology, CBER, FDA. Genetic diversity of HIV. Two major types of HIV: HIV-1 and HIV-2 3 distinct HIV-1 groups identified to date:

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Surveillance of HIV variants in Cameroon and evaluation of their impact on HIV diagnostics

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  1. Surveillance of HIV variants in Cameroon and evaluation of their impact on HIV diagnostics Indira K. Hewlett, Ph.D Chief, Laboratory of Molecular Virology, CBER, FDA

  2. Genetic diversity of HIV • Two major types of HIV: HIV-1 and HIV-2 • 3 distinct HIV-1 groups identified to date: • M (major), O (outlier) and N (non-M, non-O) • Group M consists of many subtypes of viruses (A-H) and group O (3 prototype classes) • 5 major HIV-2 subtypes • Increasing numbers of circulating recombinant forms (CRFs) of HIV-1

  3. Mosaic structure of HIV circulating recombinant forms

  4. CRF01_AE Worldwide distribution of predominant HIV-1 group M subtypes and CRFs CRF14_BG Cameroon B Adapted from Thomson et al. Lancet Infect Dis 2002.

  5. HIV genetic diversity: diagnostic implications • 1994: Schable et al. from CDC tested 9 sera of HIV group O from Cameroon, Africa with 10 different FDA licensed tests • 2/9 sera were not detected by 3 assays used for blood screening; 1/9 was not detected by 5/10 tests • Tests that detected all 9 sera were based on whole virus lysate (WVL) antigens • Some tests based on synthetic peptides or recombinant antigens did not detect all sera

  6. Diagnostic implications- con’t • CDC study caused heightened awareness of impact of HIV genetic diversity on sensitivity of diagnostic tests • FDA requested manufacturers to modify tests for detection of HIV-1 group O by including group O antigens • FDA considered need for in-house research in HIV diversity

  7. Methods of detection Target • EIA antibodies • Western Blot antibodies • IFA antibodies • P24 ELISA HIV antigen • NAT HIV RNA

  8. Study goals • Evaluate ability of existing and new blood screening, rapid and other diagnostic tests to detect and quantitate HIV variants • Determine appropriate strategies to modify tests for sensitive detection of HIV variants • Investigate co-infection of HIV with HHV-8 • Study HIV and HHV-8 epidemiology in Cameroon blood bank setting • Characterize and genotype emerging HIV variants • Develop reagents (panels) for standardizing new tests for variant HIV

  9. CBER HIV surveillance efforts • CBER initiated dialogue with Cameroonian investigators in 1998/99 and CDC to study HIV diversity in-country • Cameroon has all known HIV genetic variants • Interagency agreement (3-5 yr.) with CDC for cooperative surveillance developed last year • Additional collaboration with Johns Hopkins and DoD

  10. Study Plan and Methods • Blood samples were collected from sites around Yaounde, tested by a rapid HIV assay used to screen blood donors in Cameroon. Plasma were shipped to FDA • At LMV samples were tested by FDA licensed HIV antibody, antigen and nucleic acid tests (NAT) • Confirming HIV infection of Discordant samples using in-house test • Genotyping HIV positive samples • Samples were also tested for HHV-8 antibodies and its co-infection with HIV

  11. Results A total 239 blood samples were evaluated. There are 149 HIV positive samples and 90 HIV negative samples. Six different FDA approved tests were used: EIA (2) IFA (1) Western Blot (1) NAT (2)

  12. Results

  13. Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 Discordant result CE01 R R ++ Pos R <LDL CE17 NR NR ++ neg NR <LDL CE29 NR R + Ind NR <LDL CE33 NR NR ++ neg NR <LDL LT11 NR NR - Pos NR <LDL LT13 R R + neg nt <LDL LT21 NR NR + Pos R <LDL CE62 NR R ++ Ind NR <LDL CE64 NR NR - neg NR <LDL CE84 NR NR - Ind NR <LDL LT61 R R +/- Pos R 5.1x103 CE164 R Ind - neg NR <LDL CE182 NR NR - Pos NR <LDL LT130 R R - Ind Ind <LDL

  14. Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 Discordant result CE11 NR R - Ind NR <LDL CE18 NR NR - neg NR <LDL CE39 NR R nd neg NR <LDL CE76 NR R nd neg NR <LDL CE87 NR R nd neg NR <LDL LT01 NR R - neg NR <LDL LT36 R NR - neg NR <LDL LT16 NR NR - neg R<LDL LT05 NR NR - neg R <LDL CE107 R R nd Pos R 7.5X104 CE223 R R nd Pos R 2.4X103

  15. Viral load in HIV positive samples

  16. No HIV group O was detected by using an in-house ELISA. • None of the samples were detected by the p24 antigen test • In-house PCR is performed to aid the determination of HIV infection

  17. Investigate discordant samples to identify new HIV variant. • Searched for new HIV variant through genetic characterization (Subtype determination) 239 plasma samples from urban areas around Yaounde % RT-PCR + Set #1: n= 41 80.5 Set #2: pos. n=59 78 neg. n=40 0 Set #3: pos. n=48 48 neg. n=51 1.9

  18. Methods PCR amplification: env (gp41) and gag (p17) Sequencing: PCR product forward and reverse PCR primers ABI prism 310 Genetic Analyzer Editing: Sequence Navigator software Alignment: CLUSTAL W software Fhylogenetic Analysis: Matrix distance (2 Kimura parameters) Neiborg-joining (tree) Bootstrap (fidelity of the tree) Maximum likelihood

  19. SET #1 DNA Matrix Distances O4 O1 O3 O2 GP41 (n=33) 1000 O N CPZ-2 CPZ-3 M N2 N1 A3 976 1000 A4 A5 LT34 LT02 652 LT051 CPZ-1 LT28 A6 LT04 A1 LT32 LT22 A2 CRF02 AG4 CRF02 AG3 LT17 CE38 LT26 LT07 CE01 LT03 CE41 CE42 LT25 CRF02 AG2 LT33 CE32 LT09 LT19 G3 J2 J1 G2 G4 LT10 CRF02 AG1 B3 B2 LT27 LT24 CE46 LT31 B1 G1 LT29 CRF11 AGJ3 B4 CE09 CRF11 AGJ4 CE35 CRF11-AGJ1 CE34 CPZ-5 D4 D1 D3 D2 CRF11 AGJ2 C1 CE03 C2 LT06-gp4 C4 F1-3 K2 C3 F1-1 CRF01 AE3 CRF01 AE1 H1 CRF01 AE-U H2 K1 CRF01 AE2 LT141 F1-2 H3 F2-1 0.1 CE36 F2-2

  20. DNA Matrix Distances SET #1 A3 A (5, 15.2%) A4 GP41 (n=33) LT05 A5 LT02 CRF02_AG (18, 54.5%) LT34 LT28 A6 CRF02 AG4 A1 CRF02 AG3 LT32 A2 LT25 LT22 LT17 LT04 LT07 LT26 CE41 CE38 J CE42 CRF02 AG2 LT03 G (3, 9.1%) CE01 CE32 LT19 LT09 J2 G3 LT10 B J1 LT33 G2 LT24 G4 CRF02 AG1 B2 B3 CE46 LT27 974 LT31 B1 G1 LT29 922 B4 208 CRF11 AGJ3 CRF11 AGJ4 817 498 737 CRF11_AGJ (4, 12.1%) CE35 CE09 CRF11-AGJ1 CE34 D (1, 3%) D4 D1 D3 D2 CRF11 AGJ2 995 C1 LT06 C2 CE03 C4 F1-3 K2 C3 F1-1 C CRF01 AE3 CRF01 AE1 H1 CRF01 AE4 K1 H2 LT14 CRF01 AE2 F1-2 CRF01_AE K H3 F2-1 CE36 H F (2, 6.1%) F2-2 0.1

  21. Maximum Likelihood SET #1 C G (3, 9.1%) H C1 GP41 (n=33) C2 F (2, 6.1%) H3 AGJ2 LT06 K C4 C3 CE09 H2 CRF11_AGJ (4, 12.1%) H1 AGJ3 CE34 F2-2 CE36 CE03 K1 G3 F1-2 K2 G1 AGJ4 LT27 G2 J F2-1 AGJ1 LT14 F1-1 J2 J1 F1-3 LT31 G4 D (1, 3%) LT29 D4 CE35 D2 D1 D3 CRF01_AE AE2 B2 LT22 AE3 AE1 AE-4 A6 B3 B1 B A5 A1 LT33 A2 LT19 B4 LT25 LT34 AG1 A4 AG2 LT10 CE32 CE01 CE42 A (4, 12.1%) A3 LT07 LT24 AG3 LT02 LT05 LT17 CE41 LT26 LT03 CRF02_AG (19, 57.6%) AG4 LT04 CE38 CE46 LT09 LT32 LT28 0.1

  22. DNA Matrix Distances SET #1 P17 (n=23) O2 CPZ2 CPZ1 N2 N N1 O O4 961 O3 1000 719 503 O1 1000 CPZ3 CE03 LT05 LT26 LT28 LT04 LT31 LT24 CRF02 AG4 CRF01 AE2 CFR02 AG1 CRF02 AG2 CRF01 AE4 LT02 CRF01 AE3 CRF01 AE1 CRF02 AG3 CE09 CRF11 AGJ2 LT33 CE34 CE32 LT25 CRF11 AGJ1 CE41 LT29 CRF11 AGJ4 LT03 CRF11 AGJ3 LT06 CE38 LT27 LT32 A1-2-AF069 G3 A1-4 A1-1-AF004 A1-3 G4 M A2-1-AF286 G1 G2 H1 B2 H3 H2 B3 B1 J2 B4 D3 D2 D4 J1 LT22 D1 CE35 C2 F1-1 C3 C4 K2 LT14 F1-3 F1-2 F2-1 F2-2 K1 C1 0.1

  23. O N DNA Matrix Distances SET #1 p17 (n=23) CRF01_AE (5, 21.7%) CRF02 AG2 CE03 LT28 CRF02 AG4 LT05 LT26 CFR02 AG1 CRF01 AE4 LT04 CRF02_AG (10, 43.5%) LT24 LT31 CRF01 AE3 CRF01 AE2 CRF02 AG3 LT02 CRF01 AE1 CRF11_AGJ (2, 8.7%) CE09 CE34 CRF11 AGJ2 LT33 CE32 LT25 CRF11 AGJ1 CE41 LT29 CRF11 AGJ4 LT03 CE38 CRF11 AGJ3 G (1, 4.3%) 980 540 LT06 LT32 175 LT27 A1-2 A A1-4 860 493 G3 A1-1 535 956 A1-3 G4 650 889 720 G1 A2-1 778 G2 533 585 H1 512 955 B2 H B3 H3 B1 H2 1000 B J2 B4 D3 D2 D4 J J1 LT22 D (1, 4.3%) D1 CE35 C2 F1-1 C3 C4 LT14 F1-3 K2 F1-2 C (1, 4.3%) F2-1 F (1, 4.3%) F2-2 0.1 K K1 C1

  24. 0.1 Maximum Likelihood SET #1 B p17 (n=23) J D (1, 4.3%) B4 C (1, 4.3%) C3 H2 J1 B2 H3 B3 C2 D4 D1 J2 H1 B1 C4 CE35 D2 F1-3 D3 K1 F1-1 C1 LT22 G (1, 4.3%) F1-2 K G2 A2-1 G1 K2 G3 LT27 G4 F2-1 LT14 A1-1 A1-4 F (1, 4.3%) A1-2 AG3 LT26 LT06 AG2 LT28 F2-2 AG4 LT29 CE38 AGJ4 LT32 LT03 H CE32 CE34 CRF02_AG (11, 47.8%) LT33 AGJ2 AG1 AGJ1 A1-3 AE2 AGJ3 CE41 CE09 O N LT25 LT02 AE1 AE3 CRF11_AGJ (2, 8.7%) LT24 A LT05 AE4 LT04 LT31 CE03 CRF01_AE (5, 21.7%)

  25. 8 CRF_02-AG 1 A 1 F 10 samples amplified gp41 but not p17: SUBTYPE DETERMINATION: SET DATA #1 70% with VL>1000 RNA cop/ml Gp41 A AG AGJ D F G P17 AE C AG AE AGJ AE D F G AG AE n 2 1 10 1 2 1 1 1 1 1 1 23 samples amplified both gp41 and p17: gp41 (AGJ) p17 (?)

  26. SUBTYPE DETERMINATION: SET DATA #2 6 CRF02_AG 1 A 7 samples amplified gp41 but not p17: 85% with VL>1000 RNA cop/ml Gp41 A AG AGJ G F2 P17 A AE AG AE G F2 AGJ AG G F2 F2 n 3 1 25 1 1 1 1 1 3 1 1 39 samples amplified both gp41 and p17:

  27. SET #3 DNA Matrix Distances O1 O3 GP41 (n=24) O2 LT110-gp41 CE208-gp41 LT117-gp41 CE133-gp41 CE173-gp41 AG4 LT165-gp41 LT89-gp41 CE202-gp41 LT160-gp41 CE180-gp41 CE217-gp41 CE145-gp41 CE198-gp41 LT115-gp41 AG3 A2-2 CE129-gp41 CE175-gp41 CE177-gp41 LT107-gp41 AG1 CE207-gp41 CE187-gp41 A2-1 CE174-gp41 CE115-gp41 B1 LT88-gp41 AG2 A1-3 B2 A1-1 B3 A1-2 CE107-gp41 D3 D1 N2 D2 AE3 AE1 C1 AE2 G1 G2 LT129-gp41 C2 AGJ1 CE189-gp41 AGJ2 H1 J1 N1 H2 AGJ3 F1-2 K2 J2 F1-1 K1 F2-1 0.1 F2-2

  28. SET #3 DNA Matrix Distances LT110 LT165 CE217 LT89 CE208 CE133 LT117 CRF02_AG (20, 83.3%) GP41 (n=24) CE145 CE173 CE202 AG4 LT160 CE180 LT115 CE198 CE177 A (1, 4.2%) CE207 AG3 CE175 A2-2 LT107 AG1 CE187 A2-1 B1 CE174 B LT88 B2 AG2 A1-3 B3 A1-1 A1-2 CE107 D3 D (1, 4.2%) D1 D2 AE3 CRF01_AE C1 AE1 AE2 G1 C G2 C2 LT129 AGJ1 CE189 H1 G (1, 4.2%) AGJ2 J1 H2 AGJ3 J2 F1-2 K2 H F1-1 K1 K F2-1 F 0.1 F2-2

  29. Maximum Likelihood G (2, 8.3%) AE3 AE2 G1 AE1 LT129 AGJ3 AGJ2 CE189 H2 H1 C2 G2 AGJ1 C1 J1 J2 K2 K1 F1-1 A2-1 F1-2 A2-2 F2-2 A (1, 4.2%) F2-1 LT107 D3 D1 D2 B2 A1-1 A1-2 CE107 B1 LT165 CE173 D (1, 4.2%) A1-3 B3 LT89 AG1 CE202 LT160 CE208 AG4 LT117 CE145 AG2 LT110 LT115 CE217 CE133 AG3 CRF02_AG (20, 83.3%) CE207 LT88 CE174 CE175 CE187 CE177 CE198 CE180 SET #3 GP41 (n=24) 0.1

  30. CRF02_AG SUMMARY Subtype % 5.8 0 0 1.9 2.9 5.8 5.8 70 4.8 3 A B C D F G CRF01_AE CRF02_AG CRF011_AGJ AC AF GF

  31. Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE01 R R ++ Pos R <LDL CE17 NR NR ++ neg NR <LDL CE29 NR R + Ind NR <LDL CE33 NR NR ++ neg NR <LDL LT11 NR NR - Pos NR <LDL LT13 R R + neg nt <LDL LT21 NR NR + Pos R <LDL CE62 NR R ++ Ind NR <LDL CE64 NR NR - neg NR <LDL CE84 NR NR - Ind NR <LDL LT61 R R +/- Pos R 5.1x103 CE164 R Ind - neg NR <LDL CE182 NR NR - Pos NR <LDL LT130 R R - Ind Ind <LDL Discordant result AG

  32. Sample ID HIV ½ EIA Abbot Bio-Rad HIV-1 IFA HIV-1 Western HIV-1 NAT Gen-Probe Roche 1.5 CE11 NR R - Ind NR <LDL CE18 NR NR - neg NR <LDL CE39 NR R nd neg NR <LDL CE76 NR R nd neg NR <LDL CE87 NR R nd neg NR <LDL LT01 NR R - neg NR <LDL LT36 R NR - neg NR <LDL LT16 NR NR - neg R<LDL LT05 NR NR - neg R <LDL CE107 R R nd Pos R 7.5X104 CE223 R R nd Pos R 2.4X103 Discordant result D

  33. SUMMARY AND CONCLUSIONS • Six different FDA licensed tests detected additional HIV positive specimens. However, discordant results are not rare among all six tests when testing African samples. • EIA results from different manufactures do not always agree with each other. WB may not resolve some discordant results. • Current licensed HIV NAT can detect most of the subtypes and recombinant HIV variants. The sensitivity may be different between manufacturer’s tests. • HIV viral load varies in Cameroon blood donors, the majority viral load around 103-105 RNA/ml.

  34. SUMMARY AND CONCLUSIONS – con’t • CRF02_AG was the most prevalent viral strain found in Cameroon samples (70%) • New CRF AC, AF and GF were found • All the subtypes and CRF identified and sequenced to date in Cameroonian plasma samples are detected by FDA licensed HIV NAT assays.

  35. FUTURE DIRECTIONS • Establish primary isolates from these plasma samples • Genotyping of the virus will be continue performed • Analyze discordant and negative samples with degenerate primers and PCR Enhance RT assay (PERT) to identify emerging retroviruses • Optimize new technologies including Microarray for identification of HIV groups and subtypes • Samples collected from blood banks outside Yaounde and in rural areas will be analyzed in future studies

  36. Acknowledgements Cameroon Ministry of Health Leopold Zekeng Bih Awazi LMV/DETTD/OBRR Indira Hewlett Owen Wood Sherwin Lee Stephen Kerby Sylvester Daniel Ana Machuca Jinjie Hu Dr. Hsia CDC Thomas Folks Marcia Kalish

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