Detection of drug-resistant human immunodeficiency virus
The invention relates to methods of detecting a drug resistant HIV in a subject. The methods include detecting mutations associated with drug resistance in an HIV 2-LTR circle DNA molecule obtained from a cell of an HIV-positive subject, e.g., an HIV-1-positive human.
1. A method of evaluating the efficacy of a treatment for modulating or stopping replication of HIV in a subject, the method comprising:
a) administering a treatment to a subject;
b) obtaining a biological sample from the subject,
c) selectively purifying extrachromosomal DNA comprising an HIV 2-LTR circle from the sample,
d) specifically amplifying a region of 2-LTR circular genome comprising a sequence, wherein a mutation of said sequence confers drug resistance, and
e) detecting a drug-resistance mutation in the amplified region of the HIV 2-LTR circle,
wherein the presence or absence of a drug-resistance mutation in the HIV 2-LTR circle is indicative of the efficacy of the treatment for modulating or stopping replication of HIV in the subject.
2. The method of claim 1 , wherein the region of the 2-LTR circular genome is amplified using a polymerase chain reaction.
3. The method of claim 1 , wherein the treatment comprises administering to the subject at least one HIV reverse transcriptase inhibitor.
4. The method of claim 1 , wherein the treatment comprises administering to the subject at least one HIV protease inhibitor.
5. The method of claim 1 , wherein the treatment comprises administering to the subject at least one HIV reverse transcriptase inhibitor and at least one HIV protease inhibitor.
6. The method of claim 1 , wherein the subject is an HIV-1-positive mammal.
7. The method of claim 1 , wherein the subject is a non-human primate.
8. The method of claim 1 , wherein the subject is a human.
9. The method of claim 1 , wherein the subject is a human subject in a clinical trial.
10. The method of claim 1 , wherein the biological sample comprises a peripheral blood mononuclear cell.
11. The method of claim 1 , wherein HIV viral RNA is not detected in the blood of the subject.
12. A method for evaluating the efficacy of a treatment for treating a subject infected with HIV, the method comprising:
a) administering a treatment to a subject;
b) obtaining a biological sample from the subject,
c) selectively purifying extrachromosomal DNA comprising an HIV 2-LTR circle from the sample,
d) specifically amplifying a region of 2-LTR circular genome comprising a sequence, wherein a mutation of said sequence confers drug resistance, and
e) detecting a drug-resistance mutation in the amplified region of the HIV 2-LTR circle,
wherein the presence or absence of a drug-resistance mutation in the HIV 2-LTR circle is indicative of the efficacy of the treatment.
13. The method of claim 12 , wherein the region of the 2-LTR circular genome is amplified using polymerase chain reaction.
14. The method of claim 12 , wherein the treatment comprises administering to the subject at least one HIV reverse transcriptase inhibitor.
15. The method of claim 12 , wherein the treatment comprises administering to the subject at least one HIV protease inhibitor.
16. The method of claim 12 , wherein the treatment comprises administering to the subject at least one HIV reverse transcriptase inhibitor and at least one HIV protease inhibitor.
17. The method of claim 12 , wherein the presence of a drug resistance mutation indicates that the treatment is sub optimally effective.
18. The method of claim 12 , wherein the subject is an HIV-1-positive mammal.
19. The method of claim 12 , wherein the subject is a non-human primate.
20. The method of claim 12 , wherein the subject is a human.
21. The method of claim 12 , wherein the subject is a human subject in a clinical trial.
22. The method of claim 12 , wherein the biological sample comprises a peripheral blood mononuclear cell.
23. The method of claim 12 , wherein HIV viral RNA is not detected in the blood of the subject.
24. The method of claim 1 , wherein specifically amplifying a region of 2-LTR circular genome comprises performing two rounds of amplification using a polymerase chain reaction-based method.
25. The method of claim 12 , wherein specifically amplifying a region of 2-LTR circular genome comprises performing two rounds of amplification using a polymerase chain reaction-based method.
26. The method of claim 1 , wherein the region of 2-LTR circular genome that is specifically amplified comprises a portion of a reverse transcriptase gene or a protease gene that is sufficient to detect the presence of a drug-resistance mutation in the gene.
27. The method of claim 12 , wherein the region of 2-LTR circular genome that is specifically amplified comprises a portion of a reverse transcriptase gene or a protease gene that is sufficient to detect the presence of a drug-resistance mutation in the gene.