Establishment and evaluation of a loop-mediated isothermal assay (LAMP) for the semi-quantitative detection of HIV-1 group M virus in blood and plasma

Odari, Eddy Okoth (2015): Establishment and evaluation of a loop-mediated isothermal assay (LAMP) for the semi-quantitative detection of HIV-1 group M virus in blood and plasma. Dissertation, LMU München: Medizinische Fakultät

Abstract

The past decade has witnessed a dramatic increase of anti-retroviral treatment of Human Immunodeficiency Virus (HIV) infected patients in many African countries. Due to costs and sophistication of currently available commercial viral load assays, little attention has been paid to therapy monitoring through measurement of plasma viral load, a challenge that could reverse achievements already made against HIV/AIDS infection. Loop-mediated isothermal amplification (LAMP) has been shown to be simple, rapid and cost-effective, characteristics which make this assay ideal for viral load monitoring in resource limited settings. The aim of this study was to establish and evaluate LAMP for quantitative detection of HIV-1 group M virus in blood and plasma. Cell culture supernatants of HIV-1 subtype B (IIIB and MVP899-87) viruses were used to optimize reaction conditions and to test primer suitability. Together with HIV-1 M non-B subtypes, HIV-1 group O and HIV-2, the cell culture supernatants were used to evaluate the performance of LAMP, to generate a model for viral load estimation and to establish the limits of the assay. A panel of 467 clinical samples was analyzed (282 plasmas and 121 dry blood spots from Kenya and 112 plasmas from Germany) and the results obtained by LAMP were compared to those generated by the Abbott Real Time HIV-1 assay, an established commercial viral load quantification test. A linear regression equation was generated from time to detection values and used to estimate the viral loads of the samples by the LAMP assay. Kenyan samples were tested in Nairobi and Munich. LAMP primers targeting the integrase of the pol gene were found to be the most suitable compared to further 3 primer sets tested. Lower limit of detection (LLOD) of 1,200 copies/mL and lower limit of quantification (LLOQ) of 9,800 copies/mL were determined as suitable thresholds for quantitative estimations of the LAMP viral loads. Sensitivities of 82 and 86% (Kenyan samples) and 93% (German samples) and specificities of 99 and 100% were realized with plasma samples. The study also realized a sensitivity of 76% and specificity of 77% with dry blood spot samples from Kenya. In conclusion, LAMP assay shows obvious potential for diagnostic application in semi-quantification of HIV-1 group M viral load in resource limited countries. However there is a need for further improvement of primers in respect to detection of HIV-1 non-B viruses and evaluation of dry blood spot samples to ensure that more reliable results are obtained.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: LAMP assay, HIV integrase primer,semi-quantitative, viral load, Kenya
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 18. März 2015
• 1. Bericht­erstatter:in: Koszinowski, Ulrich