Improved HIV-1 viral load determination based on reverse transcriptase activity recovered from human plasma.
A more sensitive version of ExaVir Load, a test that utilizes reverse transcriptase (RT) activity from virions in plasma to determine HIV-1 viral load, is described. The virions were immobilized on a gel that was washed, followed by lysis of the virions, elution of purified RT, and finally RT activity determination. The changes made to the original test were: (1) improved washing of the immobilized virions by addition of a non-lytic detergent to the wash buffer, (2) improved virion lysis procedure, including changes in salt, detergent and pH, (3) the use of larger sample volumes in the RT assay, and (4) prolonged RT reaction time. The alterations gave a tenfold increased sensitivity compared to the original version. The correlation between RT load by the current test and RNA PCR was the same as previously (r=0.90). Using colorimetric product detection, the average detection limit in a panel of 262 patient plasma from Stockholm was 0.5 fg RT/ml, corresponding to approximately 170 RNA copies/ml. None of 54 HIV-1 RNA negative samples exhibited RT. The amount of RT load positive samples were 19% for samples containing 50-400 RNA, 71% for samples with 400-1,500, and 100% among samples with >8,000 copies/ml (according to Roche Amplicor). The sensitivity could be increased further using fluorimetric detection. In conclusion, the modifications of the test described result in an important increase in sensitivity. It can now be regarded as a competitive alternative method for HIV viral load determinations.
Malmsten
Anders
A
Department of Genetics and Pathology, Uppsala University, Uppsala, Sweden.
Shao
Xing-Wu
XW
Sjödahl
Staffan
S
Fredriksson
Eva-Lena
EL
Pettersson
Ingvar
I
Leitner
Thomas
T
Källander
Clas F R
CF
Sandström
Eric
E
Gronowitz
J Simon
JS
D005470:Fluorometry; D015658:HIV Infections / Q000821:virology*; D054303:HIV Reverse Transcriptase / Q000097:blood* / Q000302:isolation & purification; D015497:HIV-1 / Q000502:physiology*; D006801:Humans; D012367:RNA, Viral / Q000097:blood; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013019:South Africa; D013053:Spectrophotometry; D013223:Statistics as Topic; D013548:Sweden; D019562:Viral Load / Q000379:methods*
D012367:RNA, Viral; D054303:HIV Reverse Transcriptase