Previously Untreated Patients with Virus Samples Containing Transmitted K103N
HIV-1 RT and protease sequences were obtained from 1,334 ARV-naïve patients between August 1998 and December 2007. 40 of the 42 (3.1% of the total) patients with the RT mutation K103N had no other detectable drug-resistance mutations. 24 of these 40 patients had plasma HIV-1 RNA levels ≥4.5 log copies/ml. We identified cryopreserved aliquots of plasma for 17 of the samples with sufficiently high plasma HIV-1 RNA levels. From 13 of these cryopreserved aliquots, we successfully extracted ≥100 cDNA virus templates for UDPS.
shows the plasma HIV-1 RNA levels, CD4 counts, RT mutations detected by SGRT, and RT mutations detected by solely by UDPS. The median CD4 count was 326 (range: 3 to 832). The median plasma HIV-1 RNA level was 5.2 log copies/ml. Five patients had plasma HIV-1 RNA levels between 4.5 to 4.9 log copies/ml, four had between 5.0 to 5.6, and another four had ≥5.7 log copies/ml respectively. All virus samples belonged to subtype B. Five of the 13 patients had been infected within the year prior to presentation. The duration of infection for the remaining eight patients was not known. SGRT detected a median of six amino acid differences (range: 4 to 14) from the consensus B sequence. In addition to K103N, the RT sequence from one patient had the accessory NNRTI-resistance mutation P225H.
Clinical Characteristics and Ultradeep Pyrosequencing (UDPS) Results in 13 ARV-Naive Patients with HIV-1 Samples with the RT Mutation K103N
UDPS confirmed each of the mutations detected by direct PCR sequencing in an unmixed form and 15 of the 19 mutations detected by SGRT as part of an electrophoretic mixture. UDPS detected a median of 6 mutations that were not detected by direct PCR sequencing (range: 0 to 10). The median number of additional silent mutations present in ≥2.0% of sequence reads was 11 (range: 2 to 39) and the median number of residual consensus wildtype amino acids was 1 (range: 0 to 10). The one sample without any additional mutations detectable by UDPS was obtained in a patient undergoing primary HIV-1 infection (PID 22127). That sample, however, did have seven silent minor variants detected only by UDPS.
The polymorphic etravirine-associated mutations V90I and V106I were each detected in one sample. No major etravirine-associated mutations were detected at a level above 0.5%. Five samples had one or more NRTI or NNRTI-resistance mutation including 30062 which had four NRTI-resistance mutations: M184V+L210W+T215Y+219Q and 8048 had one NRTI-resistance mutation K65R and one NNRTI-resistance mutation P225H. No NNRTI-resistance mutations as high as 0.5% with the exception of K103S (a likely K103N revertant) which was detected in 0.6% and 0.9% of reads from samples 8048 and 28010, respectively. The sample from PID 30062 – which contained four NRTI-resistance mutations – also had the following minority protease inhibitor (PI) resistance mutations: M46I (0.8%), I84V (0.9%), and L90M (0.9%) (data not shown). No other minority PI-resistant variants were detected.
Six of the patients with transmitted K103N (16387, 22138, 25590, 27791, 27834, 30074) were treated with a regimen containing ritonavir-boosted atazanavir (n=4) or lopinavir (n=2) in combination with TDF + 3TC, FTC or ddI. These patients experienced complete virological suppression for a median of 3 years (range: 2 to 4 years) through January 2009. Five patients were untreated for a median of 3.5 years (range: 2 to 5 years) through January 2009. One patient (8048) was treated with LPV in combination with d4T + abacavir for seven years but was poorly adherent and eventually developed the LPV resistance mutations (I54V and V82A). One patient (26412) was untreated for 3 months and then lost to follow-up.
NNRTI-Experiencing Patients with Virus Samples Containing Acquired K103N Mutations
Between August 1998 and December 2007, HIV-1 RT and protease sequences were obtained from 1,057 NNRTI-experienced patients. By SGRT, 437 (41%) of these patients had a plasma sample containing K103N obtained while receiving a failing NNRTI-containing regimen including 160 patients (15%) whose virus samples exhibited no other major NNRTI-resistance mutations. Of these 160 plasma samples, 32 (20%) had plasma HIV-1 RNA levels ≥4.5 log copies/ml. Cryopreserved aliquots were available for 26 samples. For 20 of these aliquots, we successfully extracted ≥100 cDNA virus templates.
shows the plasma HIV-1 RNA levels, CD4 counts, RT mutations identified by SGRT, and RT mutations detected by solely by UDPS. The median CD4 count was 188 (range: 27 to 464). The median plasma HIV-1 RNA level was 4.9 log copies/ml (range 4.5 to 5.7). Eleven patients had plasma HIV-1 RNA levels between 4.5 and 4.9, five had 5.0 to 5.6, and another four had ≥5.7 log copies/ml, respectively. All virus samples belonged to subtype B. Direct PCR sequencing identified a median of 8.5 positions (range: 2 to 15) with differences from consensus B sequence. The accessory NNRTI-resistance mutations P225H (n=3), V108I (n=1), A98G (n=1), and H221Y (n=1) were detected in six of the samples. Twelve samples had established NRTI-resistance mutations including M184V/I in 9 samples and T215F/Y in 6 samples.
Clinical Characteristics and Ultradeep Pyrosequencing (UDPS) Results in 20 NNRTI-Treated Patients with Plasma Samples Containing the RT Mutation K103N
UDPS confirmed each of the mutations detected by SGRT in an unmixed form and 29 of the 37 mutations identified by SGRT as part of an electrophoretic mixture. UDPS also uncovered a median of 5.5 mutations that were not detected by direct PCR sequencing (range 0 to 11). A median of eight additional silent mutations (range 1 to 19) were present in ≥2.0% of sequence reads and there was a median of one residual consensus wildtype amino acids (range: 0 to 6). The one sample in which UDPS did not detect any additional mutations had four silent minor variants identified only by UDPS in ≥8.5% of sequence reads.
UDPS detected one or more NNRTI (n=12) or NRTI (n=12) resistance mutation in 14 of 20 samples: 10 samples had both NRTI and NNRTI-resistance mutations, two had NNRTI-resistance mutations alone, and two had NRTI-resistance mutations alone. Among the 12 samples with NNRTI-resistance mutations, seven had a total of 11 major etravirine-resistance mutations including L100I (n=2), K101E (n=2), Y181C (n=2), G190A/S (n=5). Four samples had a total of five accessory etravirine-resistance mutations (V90I, V106I, V179D) including two of the seven with major mutations. Ten of the 12 samples had a total of 14 non-etravirine resistance mutations including V108I (n=6), P225H (n=3), K101N (n=1), Y188C (n=1). Y188F (n=1), H221Y (n=1), P236L (n=1).
In conclusion, significantly more patients with acquired K103N than with transmitted K103N were infected with viruses containing nonpolymorphic etravirine-resistance mutations (7/20 vs. 0/13; p=0.03; Fisher's Exact Test). Furthermore, the overall number of nonpolymorphic etravirine-resistance mutations were significantly higher among those with acquired vs transmitted K103N (13 vs 0 mutations; p=0.02; Wilcoxon Rank Sum Test). However, there was no significant difference in the number of polymorphic etravirine-resistance mutations (V90I, V106I, and V179D) between those with acquired and transmitted K103N (4/20 vs 2/13; p=NS).