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Detection of low frequency HIV-1C drug resistant variants in treatment
exposed patients
Harriet Okatch, Vlad Novitsky, Awo Osafo-Addo,Max Essex
Harvard Scool of Public Health651 Huntington av, Boston, MA, 02115
The monitoring of development and transmission of drug resistant HIV-1 is of importance
especially for optimization of treatment strategies. However, conventional methods for
genotypic analyses only allows detection of viral strains if they exist at greater than 30 %
of the viral population. However, low frequency variants may be predictors of failure of
antiretroviral therapy and therefore, their prevalence should be determined in patients
receiving treatment. The aim of this study is to detect low frequency viral variants in
HIV-1C infected patients by single genome sequencing 1 . This study utilizes samples from
a longitudinal study in which patients are followed every two months for a period of
thirty-six months. At each visit, CD4 and plasma viral load is monitored. Bulk sequencing
of plasma RNA is carried out when patients experience virological failure. The method
employed, determines proviral load by real time PCR, after which, the sample is diluted to
contain one DNA copy per 5 µl. Nested PCR's are carried out and 20 sequences
generated per time points prior to time point when virological failure is observed.Drug
resistant variants were successfully detected in proviral DNA four months earlier than
virological failure is observed. The method employed also detected viral mutants that had
been missed by the conventional bulk sequencing and detected viral variants that were
present at as low as 5%. Although the full clinical significance is undetermined, the ability
to detect drug resistant mutations at earlier time points than conventional bulk sequencing
can lead to intervention of treatment and allow for optimization of treatment strategies.
The presence of low frequency viral variants can be used as predictors of virological
failure.
1.S. Palmer et al. Multiple, Linked Human Immunodeficiency Virus Type ! Drug Resistance
Mutations in Treatment-Experienced Patients Are Missed by Standard Genotype Analysis.
J. Clin. Microbiol., 2005; 43(1) 406-413.
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