No increase in protease resistance and a decrease in reverse transcriptase resistance mutations in primary HIV-1 infection

Rates of antiretroviral resistance in recently transmitted virus in Sydney, Australia fluctuated over the past decade, influenced by treatment trends. Current rates of drug resistance are not high in historical terms or compared with those reported. Rates of resistance to reverse transcriptase inhibitors peaked in the mid-1990s, fell dramatically with the introduction of combination therapy and appear to have plateaued at 10–15% over the past 3 years. Primary resistance mutations in the protease gene are still rare. The development of drug resistance mutations is a major obstacle to effective long-term therapy for HIV [1,2]. Several studies have suggested an increasing prevalence of drug-resistant virus among newly HIV-1-infected patients [3–6]. The complete suppression of HIV-1 could be compromised if therapy-naive patients already harbour virus with resistance mutations [7]. Furthermore, the incomplete suppression of viral replication promotes the development of broader drug resistance [2,7]. In order to determine changes in the prevalence of drug-resistant isolates in recently transmitted virus over the past decade, a retrospective study was conducted on the basis of samples collected by the NSW HIV Reference laboratory at St Vincent's Hospital, Sydney. This laboratory diagnoses the majority of cases of newly acquired HIV in the Sydney Metropolitan area. All patients presenting with acute primary HIV-1 infection between January 1992 and November 2001, who had a sterile plasma sample stored at −70°C and who had received no antiretroviral therapy before the first plasma sample was collected, were included in the study. Acute primary infection was defined as the presence of p24 antigenaemia or less than four bands on Western blot. In all 185 subjects were identified. The viral genotype from plasma viral RNA was successfully determined on all subjects using standard methodologies (Visible Genetics, Inc., Toronto, Ontario, Canada). Samples were collected early in primary infection. The median time from the first positive test for acute primary infection and the collection of samples for resistance testing was one day (range 0–13 days). Both viral loads (mean 3 485 781 copies/ml) and CD4 cell counts (mean 537 cells/ml) were high, consistent with acute primary infection. Some 97.3% samples came from men; 86.5% reported homosexual contact as their major risk factor. Their mean age was 37 years. A total of 22% of samples were collected before the introduction of protease inhibitors into this community in 1996. Mutations were designated as primary or secondary according to published criteria [2]. At least one mutation associated with resistance was detected in 21.6% (40/185) of reverse transcriptase (RT) sequences and 51.4% (95/185) of protease sequences. Mutations associated with resistance to nucleoside analogue reverse transcriptase inhibitors (NRTI) were found in 18.4%, whereas those associated with resistance to non-NRTI were found in 2.7%. No patient had combined NRTI and non-NRTI mutations. Six primary RT mutations were found: M41L (8.1%), T69N (2.2%), K70R (3.2%), K103N/R (2.2%), M184V (0.5%), and T215Y (3.2%). The levels of RT resistance dropped precipitously upon the introduction of highly active antiretroviral therapy into the transmitting community (Fig. 1a). Dividing the population into those presenting pre- and post-January 1996, the date taken to represent the timing of introduction of protease inhibitors [8], revealed a significant decrease in the frequency of primary NRTI resistance mutations from 29.3 to 9.0% (P = 0.008). Similarly, there were significant decreases in the frequency of the individual RT mutations: M41L (17.1 to 5.6%; P < 0.0008), D67N (4.9 to 0%; P < 0.03), K70R (12.2 to 0.7%; P < 0.0008), and T215Y (12.2 to 0.7%; P < 0.0008). Combinations of the three most commonly found mutations (M41L, K70R, and T215Y) confer high level zidovudine resistance [9–12]. Combined M41L and T215Y declined from 12.2 to 0% (P < 0.05). Mutations consistent with possible reversion from resistance mutation T215Y, 215C (n = 2) and 215D (n = 3), [9,10] were all seen post-1995.Fig. 1.: The year by year frequency of primary and secondary mutations in the reverse transcriptase gene (a) and protease gene (b). ―◆― Primary mutation; –░– secondary mutation.Only three primary resistant mutations (D30N, V82I, and L90M) were detected in protease. None were found in combination, all occurred in the past 18 months of the study period. All other resistance mutations in protease were secondary mutations with the most common being L63P (29.2%), V77I (15.7%), and L10I/V (12.3%). Fig. 1b shows the number of primary and secondary mutations seen in protease each year. The frequency of secondary protease resistance mutations remained unchanged despite the introduction of protease inhibitors. There was no change in the proportion of protease resistance mutations represented by any single secondary mutation. These observations add weight to the existing data suggesting that these so-called secondary resistance mutations are more likely to be naturally occurring polymorphisms [7,13–15]. Phylogenetic analysis was performed using software from the program manual for the Wisconsin Package, version 8, (Genetics Computer Group, Madison, WI, USA) on the 131 samples collected up until December 2000. All sequences were subtype B. Fifty-three of the sequences fell into 20 clusters supported by highly significant bootstrap values (> 70%). Fourteen resistant strains occurred as a part of clusters, indicating possible transmission of the same or closely related resistant strain, whereas 19 were sporadic. Seven of the clusters showed evidence of the transmission of RT resistance (data not shown, available from authors on request). The large number of different clusters and the number of unclustered sequences indicate that the population surveyed is probably representative of transmission patterns in Sydney, and is not biased by either multiple transmissions from a single individual or the preferential transmission of particular virus with some advantage in transmission or fitness. Several recent reports have suggested that the transmission of drug-resistant mutants is becoming more frequent [3,6,16–19], raising concern regarding the future effect of treatment of not only individuals but also of populations. Many of the studies were relatively small, based on samples collected up to 36 months after infection, making it impossible to be sure that the viral sequence at the time of sampling reflected transmitted virus. Many were based on data from the mid to late 1990s, so no comparison can be made with pre-existing rates of resistance mutations [7,13,20,21]. Furthermore, no attempt was made to take changes in treatment regimes into account or to show that the sample was representative. This study attempts to address each of these deficiencies. Our samples were taken early in infection, often coincident with the diagnosis of acute primary HIV infection. Approximately a quarter of the samples are from the period before the introduction of protease inhibitors into the Australian population. The phylogenetic analysis suggests that these samples are representative. The rates of antiretroviral therapy use derived from the same communities suggest that the decrease seen in transmitted RT resistance is coincident with the uptake of combination therapy by the transmitting community [22]. Contrary to data published in several other studies, the current rate of resistance in transmitted virus is not high in this population, either in historical terms or in comparison with other populations with similar demographics. Early sampling makes it unlikely that this lower rate is derived from reversion to wild type upon the removal of drug pressure in the newly infected host. The relatively low rates of resistance seen in this study may be explained by attributing most transmission to untreated individuals. This may be in addition to the decreased fitness for transmission associated with certain resistance mutations. The introduction of highly active antiretroviral therapy appears to have reduced the incidence of resistance mutations in the population as a whole, as well as in individuals.

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