Use of competitive polymerase chain reaction to determine HIV-1 levels in response to antiviral treatments.
OBJECTIVE
To develop a competitive polymerase chain reaction technique with which to evaluate the usefulness of HIV-1 level as a marker of response to antiviral treatment.


METHODS
HIV-1 sequences were assessed by competitive polymerase chain reaction in four subjects participating in a double-blind study of monotherapy versus combination therapy with nucleoside analogues.


METHODS
We inserted a mutant construct of the HIV-1 pol sequence into a commercial vector, enabling us to generate known amounts of mutant DNA and RNA for competitive polymerase chain reaction. To measure HIV-1 DNA copies in cells, the mutant DNA fragments were allowed to compete in a 10-fold dilution series with a constant amount of nucleic acid from the subject. To measure HIV-1 RNA copies in plasma, in vitro synthesized mutant RNA was added in a 10-fold dilution series to a constant amount of subject RNA and copy DNA was synthesized. DNA and copy DNA were used as the input for nested pol polymerase chain reaction. Mutant and wild-type amplimers were discriminated by size.


RESULTS
The competitive polymerase chain reaction technique has been validated in model experiments and can be used over a broad range (at least 6 logs) of levels. Three of the four subjects showed a decline of 1 log in proviral DNA levels in cells after beginning antiviral treatment. All four showed a decline of at least 1 log in viral RNA levels in plasma, but this decline was transient in one subject.


CONCLUSIONS
The HIV-1 sequence level is a useful marker in antiviral treatment studies.
Bruisten
S M
SM
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Koppelman
M H
MH
Roos
M T
MT
Loeliger
A E
AE
Reiss
P
P
Boucher
C A
CA
Huisman
H G
HG
D000998:Antiviral Agents / Q000627:therapeutic use*; D001667:Binding, Competitive; D015415:Biomarkers / Q000097:blood; D004279:DNA, Viral / Q000097:blood / Q000235:genetics; D004311:Double-Blind Method; D015751:Genes, pol; D015658:HIV Infections / Q000188:drug therapy* / Q000276:immunology / Q000382:microbiology*; D015497:HIV-1 / Q000187:drug effects / Q000235:genetics* / Q000302:isolation & purification*; D006801:Humans; D016296:Mutagenesis; D016133:Polymerase Chain Reaction / Q000379:methods*; D011533:Proviruses / Q000235:genetics / Q000302:isolation & purification; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D015203:Reproducibility of Results
D000998:Antiviral Agents; D015415:Biomarkers; D004279:DNA, Viral; D012367:RNA, Viral