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Persistence of a sexually transmitted highly resistant HIV-1: pol quasispecies evolution over 33 months in the absence of treatment
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[Research Letters]
AIDS: Volume 20(17) 14 November 2006 p 2231-2233
Neifer, Stefana; Somogyi, Sybilleb; Schlote, Frankc; Berg, Thomasa; Poggensee, Gabrieleb; Kuecherer, Claudiab
aMedical Laboratory Prenzlauer Berg, Berlin, Germany
bRobert Koch Institut, Berlin, Germany
cDr Schlote and colleagues medical practice, Berlin, Germany
Abstract
The evolution of a sexually transmitted multiresistant HIV-1 in a linked transmission chain was followed for 33 months to assess its potential to persist in the absence of treatment. The multiresistant HIV reverted slowly to wild type in reverse transcriptase (positions 44, 67, 74, 118) rendering the virus only susceptible to lamivudine/emtricitabine. Persistence of the replication-competent resistant HIV increases its potential to spread further and strengthens the importance of resistance testing in newly infected patients.
The transmission of drug-resistant HIV strains may undermine the success of HAART [1-3] in reducing the morbidity and mortality of the infection [4,5]. Different transmission rates (5-27%) of drug-resistant HIV strains in countries where antiretroviral therapy is established have been described [6-8]. Here we report the evolution of a multidrug-resistant HIV-1 strain in a newly infected patient by clonal analysis of the viral pol region over 33 months in the absence of treatment.
A 43-year-old man (index patient) presented in November 2001 with a symptomatic primary HIV infection, together with his HIV-1-positive partner (source patient) who had been seropositive since 1992 and treated with different combinations of antiretroviral drugs. In February 2002, when seroconversion of the index patient was complete, his viral load was 16 000 copies/ml and HIV isolated from plasma was multidrug resistant, with a predicted intermediate to high resistance level to all known antiretroviral drugs. At that time the actual regimen of the source patient was stavudine, didanosine, indinavir, ritonavir and efavirenz, but had failed with a viral load of 49 000 geq/ml.
The viral load, CD4 cell count, and genotypic resistance analysis [9] were continuously monitored. The HIV quasispecies of the index and the source patient were analysed by sequencing seven to 13 clones (pol fragment cloned in pBSSK+; Stratagene, USA) at 2, 19 and 28 months after the seroconversion of the index patient. Taking into account the date of primary infection, follow-up of the evolution of the index strain was for 33 months. Genotypic resistance was predicted using the Stanford algorithm ( http://hivdb.stanford.edu , version 4.0). CLUSTAL W-alignment, DNADIST (Kimura-2-Parameter) and the neighbour-joining method (Phylogeny Inference Package, version 3.573c) were used for phylogenetic analysis of the sequences. HIV subtype reference sequences ( http://www.hiv.lanl.gov , release 2004) and sequences from HIV strains derived from patients with a documented seroconversion [7,8] were included.
The HIV-1 subtype B strains of the source and index patient were closely related (bootstrap value 100%). The transmission link was also strongly supported by a population sequence identity of 99.1% compared with other known linked (99.4 ± 0.5%) and unlinked (94.2 ± 0.5%) HIV subtype B strains of the same transmission group and transmitted in the same year (data not shown) [10]. Despite their close relationship, all clonal sequences of the source were separated from the index sequences in a distinct subcluster in the tree topology (bootstrap value 94%).
The CD4 cell number and viral load of the index patient were stable at approximately 400 cells/ml and 35 000 copies/ml during follow-up.
Intermediate to high level resistance to all three classes of antiretroviral drugs of the index strain was predicted as a result of the presence of the following resistance mutations: L10I, I54V, L63A, A71V, V77I, V82T, L90M, I93L in the protease gene causing resistance to all protease inhibitors and M41L, E44D, D67N, L74V, V118I, L210W, T215D and K219R in the reverse transcriptase (RT) gene mediating for resistance to all nucleoside reverse transcriptase inhibitors and K101E, Y181V and G190S resulting in resistance to all non-nucleoside reverse transcriptase inhibitors.
The genotypic resistance profiles of both HIV strains were nearly identical. Only five resistance-associated amino acid positions of the protease (33, 48, 63, 82) and RT (215) genes differed between the source and index strain. In the protease gene the index strain carried the wild-type substitutions F33L and V48G and two other resistance-associated mutations, P63A and S82T, than identified in the source strain. At position 215 of the RT gene tryptophane was replaced by the revertant substitution aspartic acid (Y215D). The different viral quasispecies in the index patient could be caused by mutations occurring in the absence of selective drug pressure [6], or may reflect differences between the transmitted semen- and plasma-derived viral quasispecies of the source [11]. Interestingly, all viral clones derived from plasma of the two patients were distinct from each other, supporting the compartment theory.
The sequence divergence of the index quasispecies was very low (mean identity 98.3 ± 0.7%) and did not increase over time. At the three sequential blood sampling dates of quasispecies analysis in April 2002 (71%), September 2003 (54%) and June 2004 (75%) of the clonal sequences, respectively, matched the population sequence. After the evolution of the transmitted HIV for 28 months after seroconversion, a slow increase of clonal sequences encoding wild-type substitutions at RT positions 44 and 67 to 75% and at positions 74 and 118 to 38% was observed (Table 1). In the protease gene the resistance-associated V82T mutation was replaced by the resistance-associated V82A substitution (Table 1). The evolution observed in the clonal analysis of the quasispecies reflected the changes observed in the population sequences. Sensitive wild-type clones were never detected at any time in either patient.
During the therapy-naive course of infection the index virus mutated only at five of 19 resistance-associated positions (protease 82; RT 44, 67, 74, 118). The substitution of the protease resistance mutation from 82T (ACC) to 82A (GCC) could be a step on the way to the wild-type 82V (GTC). Reversion of the four RT positions to wild type rendered the virus susceptible only to lamivudine and emtricitabine (loss of E44D), and had no significant impact on the resistance level predicted for other nucleoside reverse transcriptase inhibitors and non-nucleoside reverse transcriptase inhibitors. During almost 3 years of follow-up only 21% (n = 4) of 19 resistance mutations reverted to wild type. None of the reversions reached 100% at the clonal level. All clonal sequences encoded at least 16 of the 19 resistance-associated mutations.
In conclusion, the transmission and persistence of a replication-competent and highly resistant HIV-1 strain leaves the patient with little promising treatment options once his CD4 lymphocytes continue to decline. The persistence and high stability of resistant HIV upon transmission in the absence of competing wild type and treatment was shown previously [12-16], and is in contrast to the rapid reappearance of sensitive wild-type variants, which has been described as a consequence of treatment interruptions [17]. The potential of resistant virus to persist could contribute to further spread and strengthens the importance of resistance surveillance in newly infected patients. In agreement with actual treatment guidelines, patients newly infected with HIV-1 should generally be tested for genotypic antiretroviral drug resistance [18].
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