Cossarini, Francesca MD*‡; Boeri, Enzo PhD†; Canducci, Filippo MD‡; Salpietro, Stefania MSC*; Bigoloni, Alba RN*; Galli, Laura MSC*; Spagnuolo, Vincenzo MD*‡; Castagna, Antonella MD*; Clementi, Massimo MD†‡; Lazzarin, Adriano MD*‡; Gianotti, Nicola MD*
*Department of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy; †Diagnostica e Ricerca San Raffaele, Milan, Italy; and ‡Universita' Vita-Salute San Raffaele, Milan, Italy.
Authors contribution: F.C. and N.G. developed the idea of the study. E.B., F.C., and M.C. performed genotyping. S.S., A.B., and F.C. collected patients' samples and clinical records. F.C., L.G., and N.G. analyzed the data. V.S., A.C., and A.L. helped collecting the data and followed patients. All authors contributed finalizing and reviewing the article. This is a collaborative research between the Department of Infectious Diseases and the Microbiology and virology Departments of our Scientific Institute. All authors contributed importantly in the management of patients, collecting their blood samples, collecting clinical data, performing and analyzing genotypes and clinical data.
Financial disclosure: No funds were received from either National Institutes of Health, Wellcome Trust, Howard Hughes Medical Institute, or others.
To the Editors:
In HIV-1-infected population, the estimated risk of acquiring a virus with resistance to at least one class between reverse transcriptase (RT) and protease (PRO) inhibitors ranges from 6% to 16% in United States and Europe.1-4 Usefulness of RT and PRO resistance testing was assessed by several studies showing suboptimal virologic response to antiretroviral therapy in patients with resistance mutations at baseline.5-8 International guidelines thus recommend performing RT and PRO genotyping before start treatment. Resistance testing for integrase and fusion inhibitors is generally not recommended before starting first-line therapy; however, it may become important when considering these compounds in the initial regimen. Prevalence of transmitted drug resistance (TDR) vary greatly over time and according to the geographic area, it is thus important to have updated data on the epidemiology of such phenomenon. Data so far available on the prevalence on resistance to integrase and fusion inhibitors in patients who never received these antiretrovirals generally come from drug-experienced patients or from subjects who were infected before the availability of such compounds in clinical practice.9-12 We aimed at investigating the prevalence of integrase and fusion inhibitors TDR in naive patients with a recent diagnosis of HIV-1 infection, during a period of time in which integrase and fusion inhibitors are in use.
We studied antiretroviral-naive patients who presented at our Clinic between June 2008 and March 2010 when genotyping for integrase in naive patients was implemented. HIV-1 RNA was extracted from patient's plasma samples using QIAamp viral RNA kit (Quiagen, Valencia, CA) and sequences were obtained as described elsewhere.13,14 Genotypes were interpreted according to either the Stanford algorithm or to the International AIDS Society mutation list (gp41 sequences).
Results are described as median [interquartile range (Q1-Q3)] or frequency (%). Fisher exact test and Student t test were applied.
Overall 79 patients had RT-PRO and integrase genotype available, of these patients 72 had also gp41 genotype evaluable. Patients were mainly young homosexual men with a recent diagnosis of HIV-1 infection (Table 1). Seventy-seven patients (97%) were male, 61 patients (77%) were men who have sex with men (MSM) and median age was 35 (29-43) years. Seventy-three (92%) had a B subtype virus and median time between last negative and first positive HIV-1 test was 375 (181-790) days.
A high proportion of patients presented RT or PRO mutations: 47 of 79 (60%) patients showed any RT or PRO associated mutation and 14 of 79 (18%) patients had reduced susceptibility to at least one of these drug classes. Nine patients (11%) showed nonnucleoside reverse transcriptase inhibitors resistance mutations, the most frequent being K103N (n = 6) and Y181C (n = 2). Seven patients (9%) showed NRTIs resistance mutations, the most frequent being L210M/V (n = 4), T215D/Y (n = 4), and K219Q (n = 2). Three patients (4%) showed PRO inhibitors resistance mutations, all of them harbored a virus bearing the L90M.
We did not observe any major raltegravir-associated mutation. Thirty (38%) patients had at least 1 polymorphism in the integrase sequence: S230N was the most frequently detected (n = 18, 60%); 9 patients (30%) had polymorphisms at position M154 (I = 6, L = 1, I/M = 2); 2 patients (7%) showed the I203M mutation. Two patients presented the Q95K and A128T substitutions respectively.
There was no association between RT or PRO-associated mutations and polymorphisms in the integrase sequences: integrase polymorphisms were found in 15 of 47 (32%) patients with and 15 of 32 (47%) patients without RT or PRO-associated mutations (P = 0.239). Furthermore, integrase polymorphisms were detected in 29 of 73 (40%) patients with B subtype and 1 patient of 6 (17%) with non-B subtype (P = 0.399).
Median time between HIV-1 infection diagnosis and genotyping was 76 (50-225) days. Fourteen of 38 patients (37%) who had genotype performed within 76 days and 16 of 41 patients (39%) who had genotype performed after 76 days showed integrase polymorphisms (P = 0.999). Similarly, RT-PRO resistance mutations were detected in 21 of 38 patients (55%) and 26 of 41 patients (66%) who had genotype performed before and after 76 days respectively (P = 0.499).
We also considered active syphilis as an additional indirect marker of recent HIV-1 infection. Syphilis was tested in 54 patients: 10 (19%) had active syphilis at HIV diagnosis. Integrase polymorphisms were observed in 4 (40%) patients with and 19 (43%) without active syphilis (P = 0.999). Five (50%) patients with and 27 (61%) patients without active syphilis had RT-associated or PRO-associated mutations (P = 0.723).
No difference in terms of HIV-1 RNA level and CD4+ cell counts was observed between patients with and without integrase polymorphisms (Table 1).
As a comparison, we tested whether having or not RT or PRO TDR could affect levels of HIV-1 RNA and CD4+ counts. First HIV-1 RNA and CD4+ count were 4.0 (3.5-4.7) and 4.5(3.9-4.7) log10 copies per milliliter, 475 (333-782), and 528 (402-691) cells per microliter in patients with and without RT or PRO TDR (P = 0.233 and P = 0.794).
We observed 1 enfuvirtide-associated resistance mutation (N42T), and 2 patients (3%) presented the L44M polymorphisms in the gp41 sequence. The patient with enfuvirtide-associated TDR carried a B subtype virus, did not show integrase polymorphisms, showed the L10I PRO mutation, and did not have syphilis test available.
In this population of recently diagnosed, HIV-1-infected subjects, we observed a high prevalence of RT-PROtransmitted drug resistance, one case of enfuvirtide-associated transmitted drug resistance, but no transmission of major integrase inhibitors-associated mutations.
In our population of mainly homosexual men, the proportion of RT-PRO drug resistance was somewhat higher than what observed in other studies,1-4 and a even higher proportion of patients showed any RT-PRO mutations.
We used a population-based sequencing for HIV-1 genotypes, we cannot exclude additional resistance mutations in the RT-PRO, gp41 or integrase sequences were present as minority species, as recently shown.15 Our results are however in line with those from other studies in which bulk sequencing was used.9-12 In addition, no difference in the proportion of patients showing RT-PRO-associated mutations or integrase polymorphisms was observed according to time between HIV-1 infection diagnosis and genotyping. Despite the rapid reversion to wild-type integrase upon discontinuation of integrase inhibitors,16,17 these findings argue against the possibility of bulk sequencing missing mutations due to reversion to wild-type.
Transmission of viral strains with enfuvirtide-associated resistance mutations was previously described,18 and prevalence of enfuvirtide-transmitted resistance mutations in enfuvirtide-naive antiretroviral-experienced patients is reported as high as 10.5%.19
Raltegravir was approved for clinical use in Italy in late 2007, and the prevalence of patients experiencing virologic failure is still low.20-23 In addition, transmission of a drug-resistant virus is generally less effective than transmission of a wild-type virus.24,25 This was shown for RT and PRO TDR, whether it holds true for integrase inhibitors still needs to be verified. Impact of integrase inhibitors resistance mutations on viral fitness does not seem to be univocal: raltegravir-resistant HIV-1 variants are described as highly fit. Nonetheless, the combination of other integrase inhibitors resistance mutations, such as N155H and E92Q, results in a reduced replication capacity of the double mutant compared to wild-type.14,26 Furthermore, in our population, the presence of integrase polymorphisms and RT-PRO TDR did not influence levels of CD4+ cells or HIV-1 RNA. Similar results were found by Pillay et al5 in the CASCADE cohort. Conversely, Kuritzkes et al8 found higher levels of CD4+ cells in naive patients harboring nonnucleoside reverse transcriptase inhibitors-resistant viruses.
Epidemiologic surveys also show conflicting data as follows: 1 previous study reported 2 sequences of naive patients obtained from the Los Alamos Database with evidence of N155H and Q148Q mutations, respectively.9 Two other reports from Brazil and from the Swiss and Western Australian HIV Cohorts did not detect integrase inhibitors TDR.11,12
We found 38% prevalence of polymorphisms in the integrase sequence. Comparisons with earlier reports is difficult since those studies analyzed datasets with sequences which were often obtained form public libraries and/or from patients with HIV-1 infection diagnosed before the introduction of raltegravir and other integrase inhibitors in clinical practice or trials.9-12
Although the most commonly substitutions we detected (S230, M154I/L, I203M) are described as naturally occurring polymorphisms,9-12 Q95K and A128T were also selected in vitro by integrase inhibitors.
A128T was described as an elvitegravir in vitro selected mutation, with small or no impact on susceptibility to integrase inhibitors,27 and is also as a naturally occurring polymorphism.9,12
Q95K was as well described as an accessory mutation selected in vitro by both raltegravir and elvitegravir with only small or no impact on integrase inhibitors resistance.26,28 Q95H/S polymorphisms were previously described in the Swiss and Western Australian Cohorts Study.12 This is the first report of Q95K as a naturally occurring polymorphism.
In conclusion, our study showed no evidence of transmission of major resistance mutation for integrase inhibitors in a population with high prevalence of RT and PRO inhibitors-transmitted drug resistance and potentially exposed to transmission of HIV-1 variants resistant to integrase inhibitors. By contrast, we showed transmission of enfuvirtide-transmitted drug resistance in such population. The earlier availability of enfuvirtide in clinical settings compared with integrase inhibitors, might explain these results, highlighting the importance of surveillance on local prevalence of HIV-1-transmitted drug resistance.
Francesca Cossarini, MD*‡
Enzo Boeri, PhD†
Filippo Canducci, MD‡
Stefania Salpietro, MSC*
Alba Bigoloni, RN*
Laura Galli, MSC*
Vincenzo Spagnuolo, MD*‡
Antonella Castagna, MD*
Massimo Clementi, MD†‡
Adriano Lazzarin, MD*‡
and Nicola Gianotti, MD*
*Department of Infectious Diseases, San Raffaele Scientific Institute, Milan, Italy
†Diagnostica e Ricerca San Raffaele, Milan, Italy
‡Universita' Vita-Salute San Raffaele, Milan, Italy
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