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

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.