Rapid Detection of HIV-1 Proviral DNA for Early Infant Diagnosis Using Recombinase Polymerase Amplification

Overview

Journal mBio

Publisher American Society for Microbiology

Specialty Microbiology

Date 2013 Apr 4

PMID 23549916

Citations 105

Authors

David S Boyle

Dara A Lehman

Lorraine Lillis

Dylan Peterson

Mitra Singhal

Niall Armes

Mathew Parker

Olaf Piepenburg

Julie Overbaugh

Affiliations

Soon will be listed here.

Abstract

Early diagnosis and treatment of human immunodeficiency virus type 1 (HIV-1) infection in infants can greatly reduce mortality rates. However, current infant HIV-1 diagnostics cannot reliably be performed at the point of care, often delaying treatment and compromising its efficacy. Recombinase polymerase amplification (RPA) is a novel technology that is ideal for an HIV-1 diagnostic, as it amplifies target DNA in <20 min at a constant temperature, without the need for complex thermocycling equipment. Here we tested 63 HIV-1-specific primer and probe combinations and identified two RPA assays that target distinct regions of the HIV-1 genome (long terminal repeat [LTR] and pol) and can reliably detect 3 copies of proviral DNA by the use of fluorescence detection and lateral-flow strip detection. These pol and LTR primers amplified 98.6% and 93%, respectively, of the diverse HIV-1 variants tested. This is the first example of an isothermal assay that consistently detects all of the major HIV-1 global subtypes.

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References

Stevens W, Noble L, Berrie L, Sarang S, Scott L . Ultra-high-throughput, automated nucleic acid detection of human immunodeficiency virus (HIV) for infant infection diagnosis using the Gen-Probe Aptima HIV-1 screening assay. J Clin Microbiol. 2009; 47(8):2465-9. PMC: 2725647. DOI: 10.1128/JCM.00317-09. View

Grab D, Nikolskaia O, Inoue N, Thekisoe O, Morrison L, Gibson W . Using detergent to enhance detection sensitivity of African trypanosomes in human CSF and blood by loop-mediated isothermal amplification (LAMP). PLoS Negl Trop Dis. 2011; 5(8):e1249. PMC: 3149022. DOI: 10.1371/journal.pntd.0001249. View

Brown B, Darden J, Tovanabutra S, Oblander T, Frost J, Sanders-Buell E . Biologic and genetic characterization of a panel of 60 human immunodeficiency virus type 1 isolates, representing clades A, B, C, D, CRF01_AE, and CRF02_AG, for the development and assessment of candidate vaccines. J Virol. 2005; 79(10):6089-101. PMC: 1091694. DOI: 10.1128/JVI.79.10.6089-6101.2005. View

Violari A, Cotton M, Gibb D, Babiker A, Steyn J, Madhi S . Early antiretroviral therapy and mortality among HIV-infected infants. N Engl J Med. 2008; 359(21):2233-44. PMC: 2950021. DOI: 10.1056/NEJMoa0800971. View

Penazzato M, Prendergast A, Tierney J, Cotton M, Gibb D . Effectiveness of antiretroviral therapy in HIV-infected children under 2 years of age. Cochrane Database Syst Rev. 2012; (7):CD004772. DOI: 10.1002/14651858.CD004772.pub3. View

Gandelman O, Jackson R, Kiddle G, Tisi L . Loop-mediated amplification accelerated by stem primers. Int J Mol Sci. 2012; 12(12):9108-24. PMC: 3257119. DOI: 10.3390/ijms12129108. View

Germer J, Gerads T, Mandrekar J, Mitchell P, Yao J . Detection of HIV-1 proviral DNA with the AMPLICOR HIV-1 DNA Test, version 1.5, following sample processing by the MagNA Pure LC instrument. J Clin Virol. 2006; 37(3):195-8. DOI: 10.1016/j.jcv.2006.08.001. View

Kerr R, Player G, Fiscus S, Nelson J . Qualitative human immunodeficiency virus RNA analysis of dried blood spots for diagnosis of infections in infants. J Clin Microbiol. 2008; 47(1):220-2. PMC: 2620881. DOI: 10.1128/JCM.01521-08. View

Clouse K, Powell D, Washington I, Poli G, Strebel K, Farrar W . Monokine regulation of human immunodeficiency virus-1 expression in a chronically infected human T cell clone. J Immunol. 1989; 142(2):431-8. View

10.

Beck I, Drennan K, Melvin A, Mohan K, Herz A, Alarcon J . Simple, sensitive, and specific detection of human immunodeficiency virus type 1 subtype B DNA in dried blood samples for diagnosis in infants in the field. J Clin Microbiol. 2001; 39(1):29-33. PMC: 87674. DOI: 10.1128/JCM.39.1.29-33.2001. View

11.

Khamadi S, Okoth V, Lihana R, Nabwera J, Hungu J, Okoth F . Rapid identification of infants for antiretroviral therapy in a resource poor setting: the Kenya experience. J Trop Pediatr. 2008; 54(6):370-4. DOI: 10.1093/tropej/fmn036. View

12.

Goto M, Honda E, Ogura A, Nomoto A, Hanaki K . Colorimetric detection of loop-mediated isothermal amplification reaction by using hydroxy naphthol blue. Biotechniques. 2009; 46(3):167-72. DOI: 10.2144/000113072. View

13.

Panakitsuwan S, Yoshihara N, Hashimoto N, Miyamura K, Chotpitayasunondh T . Early diagnosis of vertical HIV infection in infants by rapid detection of immune complex-dissociated HIV p24 antigen. AIDS Patient Care STDS. 2001; 11(6):429-33. DOI: 10.1089/apc.1997.11.429. View

14.

Tetali S, ABRAMS E, Bakshi S, Paul M, Oyaizu N, Pahwa S . Virus load as a marker of disease progression in HIV-infected children. AIDS Res Hum Retroviruses. 1996; 12(8):669-75. DOI: 10.1089/aid.1996.12.669. View

15.

Curtis K, Rudolph D, Nejad I, Singleton J, Beddoe A, Weigl B . Isothermal amplification using a chemical heating device for point-of-care detection of HIV-1. PLoS One. 2012; 7(2):e31432. PMC: 3285652. DOI: 10.1371/journal.pone.0031432. View

16.

Rohrman B, Richards-Kortum R . A paper and plastic device for performing recombinase polymerase amplification of HIV DNA. Lab Chip. 2012; 12(17):3082-8. PMC: 3569001. DOI: 10.1039/c2lc40423k. View

17.

Braun M, Kabue M, McCollum E, Ahmed S, Kim M, Aertker L . Inadequate coordination of maternal and infant HIV services detrimentally affects early infant diagnosis outcomes in Lilongwe, Malawi. J Acquir Immune Defic Syndr. 2011; 56(5):e122-8. PMC: 3112277. DOI: 10.1097/QAI.0b013e31820a7f2f. View

18.

Niemz A, Ferguson T, Boyle D . Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 2011; 29(5):240-50. PMC: 3746968. DOI: 10.1016/j.tibtech.2011.01.007. View

19.

Benki S, McClelland R, Emery S, Baeten J, Richardson B, Lavreys L . Quantification of genital human immunodeficiency virus type 1 (HIV-1) DNA in specimens from women with low plasma HIV-1 RNA levels typical of HIV-1 nontransmitters. J Clin Microbiol. 2006; 44(12):4357-62. PMC: 1698424. DOI: 10.1128/JCM.01481-06. View

20.

Tang W, Chow W, Li Y, Kong H, Tang Y, Lemieux B . Nucleic acid assay system for tier II laboratories and moderately complex clinics to detect HIV in low-resource settings. J Infect Dis. 2010; 201 Suppl 1:S46-51. PMC: 2943734. DOI: 10.1086/650388. View