Subtype Independent Sequencing of Low Level Viremia in HIV-1 Infected Patients

Tomas Mellberg, Jon Krabbe, Magnus Gisslén, Bo Svennerholm
Affiliates: Not submitted

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Background
In patients with low viral burden, sequencing is often problematic, yet clinically important. The aim of this study was to enable subtype independent sequencing of patients with low levels of plasma virus.

Methods
Plasma samples from 32 HIV-1 infected patients with known viral load were analyzed with an ultracentrifugation step prior to amplification and sequencing of the protease (PR) and reverse transcriptase (RT) regions. PCR and sequencing was performed using a combination of the commercially available ViroSeq® HIV-1 Genotyping System (Abbot) and an in-house nesting protocol. Eight different subtypes were represented. The median plasma HIV-1 RNA was 119 copies/ml (<20-966 copies/ml).

Results
Both PR and RT genes were successfully amplified in all samples with a plasma viral load above 100 copies/ml and in 9 out of 13 (69%) samples with a viral load below 100 copies/ml (figure 1A and B). Amplification of either gene in a sample was successful in 30 out of 32 samples (94%), figure 1C. Below 100 copies/ml, both genes were amplified in a single sample in 7 out of 13 patients (54%), figure 1D.
Sequencing was possible in seven out of eight (88%) for PR and in six out of eight (75%) for RT in subtype B patients. In subtype C, 11 out of 13 (85%) patients for PR and ten out of 12 (83%) for RT was successfully sequenced. Sequencing of PR was possible in all of CRF02_AG and CRF01_AE subtypes. In two of the CRF02_AG and in one of the CRF_AE subtypes, only PR sequencing was possible. One subtype F and one subtype G were included with successful sequencing for RT in both but negative for PR in the subtype F specimen. One patient with unsuccessful sequencing was previously known as subtype A.
Twelve out of 32 (38%) patients had drug resistance related mutations. The viral load in these patients ranged from <20-966 copies/ml and three patients had viral loads below 50 copies/ml.

Conclusion
We present a sensitive system for sequencing of low viral HIV-1 burden. These assays in combination are sub-type independent to a large extent and clinically important in settings with a variety of HIV-1 subtypes.

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