Quick, Cheap, and Easy Method for HIV-1 Human Blood Screening.
Authors: Rozmyslowicz Tomasz , Wroblewski Krzysztof , Moodley Jaynathan , Gaulton Glen N. 
Published: October 2019

Abstract
When performed under strict conditions of temperature and pH, Perchloric acid (PCA) extraction is claimed to be a reliable method of providing cytosolic composition without quantitative or qualitative changes. We applied this technique to human lymphocytes, and obtained 31P-NMR spectra of both infected and uninfected cell extracts. Our studies show that the phosphomonoester (PME) to phosphodiester (PDE) ratio in uninfected cell extracts is up to three times higher than in extracts of HIV-1 infected cells. While PME signals include mainly phosphocholine and phosphoethanolamine, the PDE was identified mainly as glycerophosphorylcholine. Changes are clearly visible within 8–12 hours after infection. We conducted our initial studies on cells infected in vitro with HIV-1. We used the cell lines U87, MAGI, SupT1 and CEM, and primary lymphocytes isolated from blood of healthy donors (obtained from Bergen Community Regional Blood Center, Paramus, NJ) infected in vitro with HIV-1 type IIIB (X4-type) or 89.6 (X4/R5-type) at an M.O.I. of 0.01. The infection of these cells was confirmed by PCR analysis. The mean PDE/PME ratio of 30 uninfected lymphocyte samples was 0.482 ± 0.094 while the mean PDE/PME ratio of 30 infected samples was 0.206±0.056. Prior to extraction all cells were isolated from dead ones using Ficoll procedure, then cell pellets containing at least 5×106 cells were placed on ice and extracted with PCA. Subsequently, the KHCO3 neutralized extract was analyzed using phosphorus NMR spectroscopy. The mechanisms that determine the content of phosphodiesters and their modulation by the presence of HIV-1 comprise another target for future investigation, but its analytical value is clearly visible. Lastly, to determine whether the observation of reduced PDE/PME ratio was applicable in the analysis of HIV-1 sero-positive patients, we analyzed, in a blinded fashion, the 31P-NMR spectra of lymphocyte extracts obtained from six HIV-1 infected individuals. HIV-1 infected cells and plasma were isolated from subjects enrolled in the AIDS clinic at the University of Durban, South Africa. These individuals constitute both HIV-1 infected asymptomatic and AIDS patients, and were previously untreated by any regimen for HIV-1 infection. This population was confined to individuals aged 18–50 and consisted of ∼50% male participants, of which 100% were Black. The mean PDE/PME ratio for these samples was 0.203 ± 0.076, and again all six samples fell below the mean PDE/PME ratio of uninfected cells minus Standard Deviation. Statistical analysis was then conducted among all of these groups: uninfected (group 1), in-vitro infected (group 2) and in-vivo infected (group 3), using the Student t-test for unpaired samples. For group 1 vs. group 2 the t = 5.34, probability = 0.000040, for group 2 vs. group 3 the t = 0.142, probability = 0.89, and for group 1 vs. group 3 the t = 4.88, probability = 0.00011. The method developed here is self-calibrating (thus it measures the ratio of two blood extract components), easy to implement, and allows for measurements to be done automatically, using methods which are much more sensitive than NMR spectroscopy- for example HPLC. This technology is also much faster and cheaper than any currently used screening technique.