Clinical Evaluation of a Low Cost, In-House Developed Real-Time RT-PCR Human Immunodeficiency Virus Type 1 (HIV-1) Quantitation Assay for HIV-1 Infected Patients
Objectives HIV-1 viral quantitation is essential for treatment monitoring. An in-house assay would decrease financial barriers to access. Materials and Methods A real-time competitive RT-PCR in house assay (Sing-IH) was developed in Singapore. Using HXB2 as reference, the assay's primers and probes were designed to generate a 183-bp product that overlaps a portion of the LTR region and gag region. A competitive internal control (IC) was included in each assay to monitor false negative results due to inhibition or human error. Clinical evaluation was performed on 249 HIV-1 positive patient samples in comparison with the commercially available Generic HIV Viral Load assay. Correlation and agreement of results were assessed for plasma HIV-1 quantification with both assays. Results The assay has a lower limit of detection equivalent to 126 copies/mL of HIV-1 RNA and a linear range of detection from 100–1000000 copies/mL. Comparative analysis with reference to the Generic assay demonstrated good agreement between both assays with a mean difference of 0.22 log10 copies/mL and 98.8% of values within 1 log10 copies/mL range. Furthermore, the Sing-IH assay can quantify HIV-1 group M subtypes A–H and group N isolates adequately, making it highly suitable for our region, where subtype B and CRF01_AE predominate. Conclusions With a significantly lower running cost compared to commercially available assays, the broadly sensitive Sing-IH assay could help to overcome the cost barriers and serve as a useful addition to the currently limited HIV viral load assay options for resource-limited settings.
References
[1]
UNAIDS (2012) Global report: UNAIDS report on the global AIDS epidemic 2012. Available http://www.unaids.org/en/media/unaids/co？ntentassets/documents/epidemiology/2012/？gr2012/20121120_UNAIDS_Global_Report_201？2_en.pdf. Accessed: 12 Nov 2013.
[2]
World Health Organization (2013) Global update on HIV treatment 2013: Results, impact and opportunities. Available: http://www.who.int/hiv/pub/progressrepor？ts/update2013/en/index.html. Accessed: 19 Aug 2013.
[3]
Mellors JW, Mu？oz A, Giorgi JV, Margolick JB, Tassoni CJ, et al. (1997) Plasma viral load and CD4+ lymphocytes as prognostic markers of HIV-1 infection. Ann Intern Med 126: 946–954. doi: 10.7326/0003-4819-126-12-199706150-00003
[4]
Hammer SM, Eron JJ, Reiss P, Schooley RT, Thompson MA, et al. (2008) Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. Vol. 300. pp. 555–570. doi:10.1001/jama.300.5.555.
[5]
Sawe FK, McIntyre JA (2009) Monitoring HIV antiretroviral therapy in resource-limited settings: time to avoid costly outcomes. Clin Infect Dis 49: 463–465 doi:10.1086/600397.
[6]
Ford N, Roberts T, Calmy A (2012) Viral load monitoring in resource-limited settings: a medical and public health priority. AIDS 26: 1719–1720 doi:10.1097/QAD.0b013e3283543e2c.
[7]
Cohen GM (2007) Access to diagnostics in support of HIV/AIDS and tuberculosis treatment in developing countries. AIDS 21 Suppl 4: S81–S87 doi:10.1097/01.aids.0000279710.47298.5c.
[8]
Wilson D, Keiluhu AK, Kogrum S, Reid T, Seriratana N, et al. (2009) HIV-1 viral load monitoring: an opportunity to reinforce treatment adherence in a resource-limited setting in Thailand. Trans R Soc Trop Med Hyg 103: 601–606 doi:10.1016/j.trstmh.2008.11.007.
[9]
Oyomopito R, Lee MP, Phanuphak P, Lim PL, Ditangco R, et al. (2010) Measures of site resourcing predict virologic suppression, immunologic response and HIV disease progression following highly active antiretroviral therapy (HAART) in the TREAT Asia HIV Observational Database (TAHOD). HIV Med 11: 519–529 doi:10.1111/j.1468-1293.2010.00822.x.
[10]
Keiser O, Chi BH, Gsponer T, Boulle A, Orrell C, et al. (2011) Outcomes of antiretroviral treatment in programmes with and without routine viral load monitoring in Southern Africa. AIDS 25: 1761–1769 doi:10.1097/QAD.0b013e328349822f.
[11]
Munderi P, Grosskurth H, Droti B, Ross DA (2012) What are the essential components of HIV treatment and care services in low and middle-income countries: an overview by settings and levels of the health system? AIDS 26 Suppl 2: S97–S103 doi:10.1097/QAD.0b013e32835bdde6.
[12]
World Health Organization (2013) Consolidated guidelines on the use of antiretroviral drugs for treating and preventing HIV infection. Available: http://www.who.int/hiv/pub/guidelines/ar？v2013/en/. Accessed: 19 Aug 2013.
[13]
Rouet F, Chaix M-L, Nerrienet E, Ngo-Giang-Huong N, Plantier J-C, et al. (2007) Impact of HIV-1 genetic diversity on plasma HIV-1 RNA Quantification: usefulness of the Agence Nationale de Recherches sur le SIDA second-generation long terminal repeat-based real-time reverse transcriptase polymerase chain reaction test. J Acquir Immune Defic Syndr 45: 380–388 doi:10.1097/QAI.0b013e3180640cf5.
[14]
Steegen K, Luchters S, De Cabooter N, Reynaerts J, Mandaliya K, et al. (2007) Evaluation of two commercially available alternatives for HIV-1 viral load testing in resource-limited settings. J Virol Methods 146: 178–187 doi:10.1016/j.jviromet.2007.06.019.
[15]
HIV Sequence Compendium 2008. Carla Kuiken, Thomas Leitner, Brian Foley, Beatrice Hahn, Preston Marx, Francince McCutchan, Steven Wolinsky, and Bette Korber editors. 2008. Publisher: Los Alamos National Laboratory,Theoretical Biology and Biophysics, Los Alamos, New Mexico. LA-UR 08-03719. Accessed 31 August 2008.
[16]
Drosten C, Panning M, Drexler JF, H？nsel F, Pedroso C, et al. (2006) Ultrasensitive monitoring of HIV-1 viral load by a low-cost real-time reverse transcription-PCR assay with internal control for the 5′ long terminal repeat domain. Clinical Chemistry 52: 1258–1266 doi:10.1373/clinchem.2006.066498.
[17]
Bland JM, Altman DG (1986) Statistical methods for assessing agreement between two methods of clinical measurement. Lancet 1: 307–310. doi: 10.1016/s0140-6736(86)90837-8
Menu E, Truong TX, Lafon ME, Nguyen TH, Müller-Trutwin MC, et al. (1996) HIV type 1 Thai subtype E is predominant in South Vietnam. AIDS Res Hum Retroviruses 12: 629–633. doi: 10.1089/aid.1996.12.629
[21]
Motomura K, Kusagawa S, Kato K, Nohtomi K, Lwin HH, et al. (2000) Emergence of new forms of human immunodeficiency virus type 1 intersubtype recombinants in central Myanmar. AIDS Res Hum Retroviruses 16: 1831–1843 doi:10.1089/08892220050195793.
[22]
Buonaguro L, Tornesello ML, Buonaguro FM (2007) Human immunodeficiency virus type 1 subtype distribution in the worldwide epidemic: pathogenetic and therapeutic implications. J Virol 81: 10209–10219 doi:10.1128/JVI.00872-07.
[23]
Rouet F, Foulongne V, Viljoen J, Steegen K, Becquart P, et al. (2010) Comparison of the Generic HIV Viral Load？ assay with the Amplicor？ HIV-1 Monitor v1.5 and Nuclisens HIV-1 EasyQ？ v1.2 techniques for plasma HIV-1 RNA quantitation of non-B subtypes: The Kesho Bora preparatory study. J Virol Methods 163: 253–257 doi:10.1016/j.jviromet.2009.10.005.
[24]
Armas Cayarga A, Perea Hernández Y, González González YJ, Due？as Carrera S, González Pérez I, et al. (2011) Generation of HIV-1 and Internal Control Transcripts as Standards for an In-House Quantitative Competitive RT-PCR Assay to Determine HIV-1 Viral Load. Biotechnology Research International 2011: 1–10 doi:10.1001/jama.300.5.555.
[25]
Rekhviashvili N, Stevens W, Marinda E, Gonin R, Stevens G, et al. (2007) Clinical performance of an in-house real-time RT-PCR assay using a fluorogenic LUX primer for quantitation of human immunodeficiency virus type-1 (HIV-1). J Virol Methods 146: 14–21 doi:10.1016/j.jviromet.2007.05.024.
[26]
Rouet F, Liégeois F, Mouinga-Ondémé A, Kania D, Viljoen J, et al. (2011) Current challenges to viral load testing in the context of emerging genetic diversity of HIV-1. Expert Opin Med Diagn 5: 183–202 doi:10.1517/17530059.2011.566860.
[27]
Santoro MM, Perno CF (2013) HIV-1 Genetic Variability and Clinical Implications. ISRN Microbiol 2013: 481314 doi:10.1155/2013/481314.
[28]
Kalish ML, Korber BT, Pillai S, Robbins KE, Leo YS, et al. (2002) The sequential introduction of HIV-1 subtype B and CRF01AE in Singapore by sexual transmission: accelerated V3 region evolution in a subpopulation of Asian CRF01 viruses. Virology 304: 311–329. doi: 10.1006/viro.2002.1691
[29]
Lee CC, Sun YJ, Barkham T, Leo YS (2009) Primary drug resistance and transmission analysis of HIV-1 in acute and recent drug-na？ve seroconverters in Singapore. HIV Med 10: 370–377 doi:10.1111/j.1468-1293.2009.00698.x.
[30]
Ng OT, Munshaw S, Lamers SL, Chew KK, Lin L, et al. (2011) Molecular epidemiology of HIV type 1 in Singapore and identification of novel CRF01_AE/B recombinant forms. AIDS Res Hum Retroviruses 27: 1135–1137 doi:10.1089/AID.2010.0364.
[31]
Watanaveeradej V, Sirirattanaphoomee S, Chantratita W, Nitayaphan S, Viputtikul K, et al. (2005) Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe. J Med Assoc Thai 88 Suppl 3: S206–S213.
[32]
Promso S, Srichunrusami C, Utid K, Lulitanond V, Pairoj W, et al. (2006) Quantitative detection of human immunodeficiency virus type 1 (HIV-1) viral load by real-time RT-PCR assay using self-quenched fluorogenic primers. Southeast Asian J Trop Med Public Health 37: 477–487.
[33]
Pas S, Rossen JWA, Schoener D, Thamke D, Pettersson A, et al. (2010) Performance evaluation of the new Roche Cobas AmpliPrep/Cobas TaqMan HIV-1 test version 2.0 for quantification of human immunodeficiency virus type 1 RNA. J Clin Microbiol 48: 1195–1200 doi:10.1128/JCM.01832-09.
[34]
Church D, Gregson D, Lloyd T, Klein M, Beckthold B, et al. (2011) Comparison of the RealTime HIV-1, COBAS TaqMan 48 v1.0, Easy Q v1.2, and Versant v3.0 assays for determination of HIV-1 viral loads in a cohort of Canadian patients with diverse HIV subtype infections. J Clin Microbiol 49: 118–124 doi:10.1128/JCM.00685-10.
[35]
Panel on Antiretroviral Guidelines for Adults and Adolescents. Guidelines for the use of antiretroviral agents in HIV1infected adults and adolescents. Department of Health and Human Services. Available at http://aidsinfo.nih.gov/contentfiles/lvg？uidelines/AdultandAdolescentGL.pdf. Accessed: on 12 Nov 2013.
[36]
Doyle T, Smith C, Vitiello P, Cambiano V, Johnson M, et al. (2012) Plasma HIV-1 RNA Detection Below 50 Copies/mL and Risk of Virologic Rebound in Patients Receiving Highly Active Antiretroviral Therapy. Clinical Infectious Diseases 54: 724–732 doi:10.1093/cid/cir936.
[37]
Gandhi RT, Deeks SG (2012) Plasma HIV-1 RNA Levels During Antiretroviral Therapy: How Low Is Low Enough? Clinical Infectious Diseases 54: 733–735 doi:10.1093/cid/cir933.
[38]
Lee PK, Kieffer TL, Siliciano RF, Nettles RE (2006) HIV-1 viral load blips are of limited clinical significance. J Antimicrob Chemother 57: 803–805 doi:10.1093/jac/dkl092.
[39]
Garrett NJ, Apea V, Nori A, Ushiro-Lumb I, Oliver AR, et al. (2012) Comparison of the rate and size of HIV-1 viral load blips with Roche COBAS TaqMan HIV-1 versions 1.0 and 2.0 and implications for patient management. J Clin Virol 53: 354–355 doi:10.1016/j.jcv.2011.12.024.
