Uso clínico de Maraviroc (MRV) en pacientes con infección por VIH-1: diseño de una nueva estrategia para la determinación de la sensibilidad clínica a MRV mediante una exposición a corto plazo al fármaco. Evolución inmunovirológica y seguridad a largo plazo de un régimen antirretroviral conteniendo MRV

UNIVERSIDAD DE SEVILLA Uso clínico de Maraviroc (MRV) en pacientes con infección por VIH-1: diseño de una nueva estrategia para la determinación de la sensibilidad clínica a MRV mediante una exposición a corto plazo al fármaco. Evolución inmunovirológica y seguridad a largo plazo de un régimen antirretroviral conteniendo MRV. Departamento de Medicina, Universidad de Sevilla Doctorando: Miguel Genebat González Directores de Tesis Doctoral: Manuel Leal Noval y Ezequiel Ruiz-Mateos Carmona El Dr. Manuel Leal Noval, Profesor Asociado del Departamento de Medicina de la Universidad de Sevilla, y el Dr. Ezequiel Ruiz-Mateos Carmona, Doctor por la Universidad de Sevilla, CERTIFICAN QUE: Don Miguel Genebat González, con DNI 28.618.912-N y Licenciado en Medicina por la Universidad de Sevilla, ha realizado bajo su dirección el trabajo “Uso clínico de Maraviroc (MRV) en pacientes con infección por VIH-1: diseño de una nueva estrategia para la determinación de la sensibilidad clínica a MRV mediante una exposición a corto plazo al fármaco. Evolución inmunovirológica y seguridad a largo plazo de un régimen antirretroviral conteniendo MRV”, para optar al grado de doctor en formato compendio de publicaciones. En Sevilla a 15 de septiembre de 2010, Directores de Tesis Doctorando Dr. Manuel Leal Noval Miguel Genebat González Dr. Ezequiel Ruiz-Mateos Carmona “Si no conozco una cosa, la investigaré” Louis Pasteur “Cuando Dios borra, es que va a escribir algo ” Jacques Benigne Bousset (clérigo francés, siglo XVII) …a mis padres e hijos AGRADECIMIENTOS Antes de nada quiero expresar mi agradecimiento a mis directores de tesis, los doctores Manuel Leal y Ezequiel Ruiz-Mateos, personas por las que siento verdadera admiración profesional y cariño personal. En primer lugar Manolo: mi maestro, mi amigo…y para colmo el abuelo de mis hijos, una persona apasionada por su profesión y que transmite esa pasión, a la que le gusta la Medicina y hace que a los demás nos guste. Gracias por enseñarme a aplicar en la cabecera del enfermo lo que aprendíamos en el Harrison, ya desde ese lejano tercer curso de carrera, teniéndole respeto pero nunca miedo a un problema clínico; y gracias también por exigirme siempre más, por apretarme las tuercas y por obligarme todos los días a explicarnos las cosas que veíamos en la consulta, dándole siempre las vueltas hasta que salía un estudio para delante. En segundo lugar Ezequiel: mi maestro de laboratorio, esa persona que siempre con una sonrisa y nunca con un mal gesto (aunque el domingo anterior hubiese perdido el Cádiz, o sea, casi todos los domingos) estaba dispuesta a abandonar su tarea para explicar aspectos moleculares y virológicos a un “clínico torpe”. Gracias a los dos por vuestras enseñanzas, estando al lado vuestro se aprende exponencialmente y es un honor teneros como directores de esta tesis que lleva vuestro sello. Agradezco también el apoyo del resto de miembros del Laboratorio de Inmunovirología: Sarita, predecesora doctora por compendio de publicaciones, Mª Mar “la grande”, Mª Antonia (la abuela de mis hijos), el dueto Marita-Gema, Yoli, Antonio, Kawthar, Alejandro, Conchita y la exiliada Natalia. Gracias a todos porque sin duda alguna sois parte activa de esta tesis y gracias por hacer comprensible para “clínicos torpes” los seminarios de los miércoles. Gracias también a Ilde, el otro clínico del grupo y amigo, una mente privilegiada para la clínica y la investigación, que tiene hueco también para darlo todo en el Cohiba. Mi más sincero agradecimiento también al Servicio de Enfermedades Infecciosas, en la figura de su Jefe de Servicio Dr Jerónimo Pachón, por facilitar mi presencia en la asistencia a los pacientes de la consulta y desarrollar la labor investigadora que ha tenido como fruto final esta tesis y las diferentes publicaciones. Por último, gracias a Marien, Inma y a las enfermeras del Hospital de Día de Enfermedades Infecciosas por su paciencia y comprensión, por su ayuda en la planificación y extracción de muestras a los pacientes que han participado en estos estudios. Fuera del ámbito científico quiero que mis amigos tengan el merecido lugar en esta tesis, porque permiten desconectar, son un apoyo fundamental en momentos complicados y te regalan una sonrisa cuando más lo necesitas. No quiero dejarme a nadie fuera, así que por grupos quiero daros las gracias a los churrasqueros, los amigos del colegio y del equipo de fútbol, los tolanos, los compañeros de carrera y de MIR, los compañeros de profesión en Medicina Interna primero y en Urgencias después…Gracias también al colegio Portaceli y a la comunidad jesuita, por transmitirme los valores cristianos y enseñarme mucho más que meros conceptos teóricos. Por último, las personas a las que más debo: mi FAMILIA. Lo que pasa es que no hay espacio para nombrar a todos ni palabras para agradecer todo lo que tengo que agradecer, porque en mi formación y educación han influido padres, hermanos, tíos, primos y sobrinos de una forma trascendental. Desde mis abuelos hasta mi sobrina Reyitas (que nacerá si Dios quiere en unos meses), gracias a todos por ayudarme a ser doctor en la Universidad más difícil de todas: la vida. Pero evidentemente merecen un lugar de honor mis hermanos, por regalarme un día a día lleno de felicidad, y mis padres, por habernos dado la vida y por haber construido un hogar lleno de felicidad, cariño, comprensión, respeto…gracias de verdad porque si hoy estoy aquí es fundamentalmente por vosotros. Finalmente, gracias a Dios por haber puesto en mi vida a Elena, una mujer llena de vitalidad, inteligente, apoyo incondicional en los buenos y malos momentos y que es lo mejor de cada día; gracias Señor también por haberme regalado a dos niños maravillosos, Perico y Teresita, y gracias a vosotros porque me habéis perdonado tardes de parque y piscina para que esta tesis se llevara a cabo. ÍNDICE BIBLIOGRAFÍA GLOBAL 1. Pallela FJ Jr, et al. Declining morbidity and mortality among patients with advanced human immunodeficiency virus infection. HIV Outpatient Study Investigators. N Engl J Med 1998;338:853-60. 2. Lalezari JP, et al. Enfuvirtide, an HIV-1 fusion inhibitor, for drug-resistant HIV infection in North and South America. N Engl J Med 2003;348:217585. 3. Steigbigel ST, et al. Raltegravir with optimized background therapy for resistant HIV-1 infection. N Engl J Med 2008;359:339-54. 4. Hardy WD, et al. Two-year safety and virologic efficacy of maraviroc in treatment-experienced patients with CCR5-tropic HIV-1 infection: 96-week combined analysis of MOTIVATE 1 and 2. J Acquir Immune Defic Syndr 2010 [Epub ahead of print]. 5. Katlama C, et al. Efficacy and safety of etravirine in treatment-experienced, HIV-1 patients: pooled 48 week analysis of two randomized, controlled trials. AIDS 2009;23:2289-300. 6. Berger EA, et al. Chemokine receptors as HIV-1 coreceptors: roles in viral entry, tropism and disease. Annu Rev Immunol 1999;17:657-700. 7. Hammer SM, et al. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA 2008;300:555-70. 8. Whitcomb JM, et al. Development and characterization of a novel singlecycle recombinant-virus assay to determine human immunodeficiency virus type 1 co-receptor tropism. Antimicrob Agents Chemother 2007;51:556-575. 9. Reeves JD, et al. An enhanced-sensitivity Trofile HIV coreceptor tropism assay for selecting patients for therapy with entry inhibitors targeting CCR5: A review of analytical and clinical studies. J Viral Entry 2009;3:94-102. 10. Schürmann D, et al. Antiviral activity, pharmacokinetics and safety of vicriviroc, an oral CCR5 antagonist, during 14-day monotherapy in HIVinfected adults. AIDS 2007;21:1293-1299. 11. Landovitz RJ, et al. Phase II study of vicriviroc versus efavirenz (both with zidovudine/lamivudine) in treatment-naive subjects with HIV-1 infection. J Infect Dis 2008;198:1113-22. 12. Soulié C, et al. Primary genotypic resistance of HIV-1 to CCR5 antagonists in CCR5 antagonist treatment-naïve patients. AIDS 2008;22:2212-4. 13. Raymond S, et al. Development and performance of a new recombinant virus phenotypic entry assay to determine HIV-1 coreceptor usage. J Clin Virol 2010;47:126-30. 14. Rozera G, et al. Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte subpopulations. Retrovirology 2009;6:15. 15. Poveda E, et al. Design and validation of new genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 antagonists. J Antimicrob Chemother 2009;63:1006-10. 16. Recordon-Pinson P, et al. Evaluation of the genotypic prediction of HIV-1 coreceptor use versus a phenotypic assay and correlation with the virological response to maraviroc: the ANRS Geno Tropism Study. Antimicrob Agents Chemother 2010;54:3335-40. OBJETIVOS Los objetivos de esta Tesis Doctoral, abordados en las diferentes publicaciones, fueron los siguientes: 1. Desarrollar una herramienta clínica (MCT) rápida y sencilla que permita determinar la sensibilidad clínica a MRV de un paciente. Correlation between Trofile® test and virological response to a short-term Maraviroc exposure in HIV-infected patients (J Antimicrob Chemother 2009). 2. Establecer las tasas de discordancia entre MCT y Trofile® y la posterior evolución inmunovirológica de los pacientes con discordancia entre ambos métodos. Discordance rates between Trofile® test and shortterm virological response to maraviroc (Antivir Res 2010, en revisión). 3. Estudiar la eficacia inmunovirológica y la seguridad a largo plazo (48 semanas) de un régimen TARGA de rescate que contenga MRV asociado a otros antirretrovirales en una población con elevada tasa de coinfección por VHC. Long-term immunovirological effect and tolerability of a maraviroc-containing regimen in routine clinical practice (Curr HIV Res 2010). MATERIAL Y MÉTODO, RESULTADOS, DISCUSIÓN Y BIBLIOGRAFÍA Correlation between the Trofile w test and virological response to a short-term maraviroc exposure in HIV-infected patients Miguel Genebat1, Ezequiel Ruiz-Mateos1, Juan A. Leo ´n2, Alejandro Gonza ´lez-Serna1, Ildefonso Pulido1, Inmaculada Rivas1, Sara Ferrando-Martı ´nez1, Berta Sa ´nchez3, M a A ´ngeles Mun ˜oz-Ferna ´ndez4and Manuel Leal1* 1 Laboratory of Immunovirology, Biomedicine Institute of Seville (IBIS), Service of Infectious Diseases, Virgen del Rocı ´o University Hospital, Seville, Spain; 2 Paediatrics Service, Virgen del Rocı ´o University Hospital, Seville, Spain; 3 Service of Immunology, Virgen del Rocı ´o University Hospital, Seville, Spain; 4 Molecular Immunobiology Laboratory, Gregorio Maran ˜o ´n University Hospital, Madrid, Spain Received 26 June 2009; returned 8 July 2009; revised 9 July 2009; accepted 19 July 2009 Objectives: The current validated assay to determine tropism of HIV variants is Trofile w , which has some limitations. The aim of this work was to correlate the virological response to a short-term maraviroc exposure with Trofile w . Methods: From 1 July 2008 to 1 March 2009, 34 consecutive HIV-infected patients with detectable viral load during the last 6 months began an 8 day exposure to maraviroc (MCT group); six HIV-infected patients without antiretroviral therapy received no treatment (control group). Plasma viral load was evaluated on days 0, 2, 5 and 8. Baseline Trofile w was performed in MCT group patients. The maraviroc clinical test (MCT) was considered positive if viral load was undetectable (<40 HIV-RNA copies/mL) or a reduction  1 log 10 HIV-RNA copies/mL was achieved after 8 days of maraviroc exposure. Results: Global concordance between MCT and Trofile w was 93.5%. In patients with R5 virus according to Trofile w , MCT was positive in 19/20 (concordance 95%); in patients with dual/mixed virus, MCT was negative in 10/11 (concordance 90.9%). An additional phenotypic tropism assay was performed in patients with discordance between MCT and Trofile w , being concordant with MCT in both cases. Three patients showed a non-reportable Trofile w result, and all of them achieved undetectability after MCT. Conclusions: A clinical approach like short-term maraviroc exposure could be an additional resource to genetic and phenotypic HIV tropism assays. This clinical approach shows high concordance with Trofile w , and could allow patients with non-reportable results by Trofile w to benefit from maraviroc therapy. Keywords: CCR5 antagonists, tropism assays, antivirals Introduction Maraviroc is currently the only commercialized drug of a new antiretroviral family: the CCR5 receptor antagonists. According to actual guidelines, maraviroc is indicated in pretreated and viraemic patients who have been shown to be infected with R5 virus. 1 In order to determine the tropism of HIV variants in HIV-infected patients, the current validated assay is Trofile w (Monogram BioSciences, San Francisco, CA, USA). 2 However, this assay has some limitations, such as high cost, prolonged time to obtain a confirmed result, availability (samples need to be sent to San Francisco), a variable proportion of ‘non-reportable’ results and limited access in developing countries. Besides, discordance in two consecutive Trofile w results before being exposed to CCR5 inhibitors has been observed in 10% of patients. 3,4 Moreover, it has been recently described that 7% of patients with R5 virus according to the tropism assay showed primary mutations in the V3 loop that could be involved with maraviroc resistance; 5 hence, patients with potential resistance to maraviroc could receive treatment with this CCR5 antagonist if the tropism assay alone is considered. These limitations may exclude some patients with virological failure from using maraviroc as part of a rescue regimen. ..................................................................................................................................................................................................................................................................................................................................................................................................................................... *Corresponding author. Tel: þ34-955013291; Fax: þ34-955013292; E-mail: [email protected] Journal of Antimicrobial Chemotherapy (2009) 64, 845–849 doi:10.1093/jac/dkp293 Advance Access publication 11 August 2009 ..................................................................................................................................................................................................................................................................................................................................................................................................................................... 845 #The Author 2009. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For Permissions, please e-mail: [email protected] Therefore, additional resources different from Trofile w to assay HIV tropism, such as other phenotypic and genotypic assays, are being explored, but no clinical approaches have been developed. Various genotypic analyses have been examined, but caveats, such as high specificity but low sensitivity to classify X4 virus, are being observed. 6,7 More recently, correlation between genotypic and phenotypic assays has been described, but the combination of these methods is still expensive, time consuming and requires sophisticated laboratories. 8,9 We propose a clinical approach [maraviroc clinical test (MCT)] in which the virological response to short-term exposure to maraviroc may predict the indication for maraviroc use. Hence, the objective of our study was to correlate the result of MCT with the result of Trofile w . Methods Patients From 1 July 2008, a prospective study was started in the Infectious Diseases Department at Virgen del Rocio University Hospital (Seville, Spain). To participate in this study, inclusion criteria were: (i) persistently detectable viral load (.50 HIV-RNA copies/mL) during the last 6 months; (ii) no highly active antiretroviral therapy (HAART) modification in the last 6 months; (iii) no HAART reintroduction in the last 6 months in patients under previous supervised treatment interruption (STI); (iv) no previous treatment with co-receptor antagonists; and (v) available future therapeutic options apart from maraviroc. Up to 1 May 2009, 34 consecutive HIV-infected patients were included (MCT group). Nineteen of these patients were under STI and treatment was required due to low CD4þcell count; the other 15 patients were under HAART, despite which viral load was persistently detectable at least during the last 6 months. In order to determine the intra-patient variability of the viral load, six HIV-infected patients without any antiretroviral therapy (three of them were naive and the other three were under STI) were used as controls (the control group). Patients, or legal guardians for those patients under 18 years old, had given written informed consent and the Ethical Committee of the Hospital approved the study. Intervention Patients in the MCT group began an 8 day exposure to maraviroc at a dose of 300 mg twice daily, adjusted if necessary for associated antiretrovirals. In the MCT group, patients under STI (n¼19) were exposed only to maraviroc during MCT, while patients not under STI (n¼15) received maraviroc with the previous failing regimen. Control group patients (n¼6) received no treatment. Patients in both groups were evaluated prospectively on days 0, 2, 5 and 8, analysing plasma viral load at each timepoint. Trofile w was performed for MCT group patients from blood samples obtained not more than 12 weeks before starting MCT, and in 24/34 (70.6%) the date of starting MCT was the same as Trofile w . MCT was considered positive if a significant viral load reduction, defined as a reduction of 1 log 10 HIV-RNA copies/mL or an undetectable viral load (,40 HIV-RNA copies/mL), was achieved on day 8 after the addition of maraviroc. Once the result of MCT was obtained, a new HAART regimen was started according to the following criteria: (i) previous genotype resistance testing results; (ii) previous antiretroviral exposure; and (iii) response to MCT, in order to include maraviroc or not in the new HAART. Laboratory tests Plasma HIV-1 RNA was measured in fresh samples by quantitative PCR (COBAS Ampliprep/COBAS Taqman HIV-1 test; Roche Molecular Systems, Basel, Switzerland) according to the manufacturer’s instructions. Determination of HIV-1 co-receptor usage (i) Trofile w : circulating virus was tested from the plasma of patients using the PhenoSense HIV Entry assay for co-receptor tropism (Monogram Biosciences Inc., South San Francisco, CA, USA). Enhanced sensitivity Trofile w was employed for all samples. (ii) Tropism of virus isolates from patients: frozen peripheral blood mononuclear cells (PBMCs) of the patients from HIV BioBank integrated in the Spanish AIDS Research Network (RIS) were co-cultured with phytohaemagglutinin (PHA; Boehringer Ingelheim)-stimulated HIV–hepatitis C virus (HCV)-uninfected donor PBMCs, as previously described. 10 Briefly, cells were cultured in medium supplemented with interleukin-2 (IL-2; R&D Systems) and cell-free supernatants were harvested and used for further infection assays. Co-receptor usage was determined modifying the technique used previously, 10 using U87 cell lines (U87-CD4þCCR5þand U87-CD4þCXCR4þ) that were infected with cell-free virus supernatant overnight and maintained for 6 days and tested for HIV-1 RNA copies/mL as described above. We used the X4-tropic NL4.3 strain and the R5-tropic BaL strain as positive controls. In this way, the measurement of HIV-1 RNA copies/mL in the supernatant of the cultures of X4 and R5 cell lines showed the tropism of the virus isolates of the patients. Statistical analysis Statistical analyses were performed using the Statistical Package for the Social Sciences software (SPSS 16.0; SPSS, Inc., Chicago, IL, USA). Differences between groups were analysed using the Mann– Whitney U-test. Means and minimum–maximum ranges were used to describe continuous variables or means and 95% confidence intervals (CI) when stated. All differences between groups with P,0.05 were considered statistically significant. Results Correlation between MCT and Trofile w The baseline characteristics of MCT group patients are shown in Table 1. Mean viral load evolution during MCT in control group patients, patients with an R5 Trofile w result (R5 patients) and patients with a dual/mixed Trofile w result (D/M patients) is shown in Figure 1. No significant viral load modification was observed in control group patients during MCT, similar to viral load evolution in D/M patients; however, a mean viral load reduction of 0.18, 0.76 and 1.41 log 10 HIV-RNA copies/mL was observed in R5 patients on days 2, 5 and 8 of MCT, respectively. Response to MCT and the result of the Trofile w test in every patient is shown in Table 2. A global concordance of 93.5% Genebat et al. 846 between MCT and Trofile w was observed. In R5 patients the MCT was positive in 19/20 (4 patients achieved undetectability and 15 achieved a viral load reduction of .1 log 10 HIV-RNA copies/ mL); so, concordance between Trofile w and MCT in R5 patients was 95%. Another phenotypic method to detect HIV tropism 10 was performed in the ‘non-concordant’ patient and the result was D/M (97.63% of the virus was R5 and 2.37% was X4), concordant with MCT but not with Trofile w . In D/M patients, no significant viral load reduction was observed in 10/11 (concordance 90.9% between MCT and Trofile w ). In this ‘non-concordant’ case, when HIV tropism was assayed by the other phenotypic method the result was R5 (100% of the viruses were R5), again concordant with MCT. Finally, three patients showed a non-reportable Trofile w result, and all of them achieved undetectability after MCT therapy. Every patient completed maraviroc exposure with no significant increase in liver enzymes during MCT (data not shown) and the drug was well tolerated, except for one patient who showed dizziness during the first 2 days after starting MCT. Immunovirological evolution with rescue therapy after MCT Those patients with a positive MCT started rescue therapy containing maraviroc plus an optimized HAART, while those patients with a negative MCT started a rescue therapy without maraviroc, independent of the Trofile w result. Up to now, no virological rebound has been observed in any patient once optimized therapy was started. Mean CD4þgain and HIV-RNA viral load reduction after a mean follow-up of 15 weeks once the rescue therapy was started (with or without maraviroc) is shown in Figure 2; a progressive CD4þincrease and a mean viral load reduction of .2log 10 HIV-RNA copies/mL was observed. No significant differences were observed between patients on maraviroc and patients not including maraviroc as part of the new rescue HAART (data not shown). Seven patients started the rescue therapy having achieved undetectability after MCT (7/34, 20.59%); the percentage of patients on rescue therapy with undetectable viral load during follow-up is shown in Figure 3; 15/16 patients (93.75%) achieved undetectability (limit of detection: 40 HIV-RNA copies/mL) and 100% showed ,200 copies/mL at week 24 of follow-up. Discussion The results of this study show that a clinical approach analysing the virological response to short-term maraviroc exposure could 0 –1.7 –1.5 –1.3 –1.1 –0.9 –0.7 –0.5 –0.3 –0.1 0.1 0.3 25 Time on MCT (da y s) 8 P = 0.06 P = 0.09 P < 0.001 P < 0.001 Controls (n = 6) D/M (n = 11) R5 (n = 20) P = NS Δ Viral load (log10 copies/mL) Figure 1. Mean viral load evolution in the control group and MCT group patients during MCT according to the Trofile w result, not including patients with a non-reportable result of Trofile w . Viral load evolution during MCT is expressed as mean and 95% CI. No significant viral load reduction was observed in control group patients during MCT; similar viral load evolution was observed in patients with a dual/mixed (D/M) Trofile w result. However, a progressive viral load reduction during MCT was observed in patients with an R5 Trofile w result. On day 2 of MCT, differences in viral load reduction between R5 and D/M patients were almost significant (P¼0.06; Mann–Whitney U-test) and differences in viral load reduction between R5 and the control group were also almost significant (P¼0.09; Mann–Whitney U-test). Significant differences in viral load reduction on days 5 and 8 during MCT were achieved between R5 patients and the other groups (control and D/M, P,0.001; Mann–Whitney U-test). Table 1. Baseline characteristics of MCT group patients (n¼34) Age, years 37 (8–50) Male sex, n(%) 24 (70.6) HCV co-infection, a n(%) 13 (38.2) Viral load, log 10 copies/mL 4.13 (1.94–6.03) CD4þ, cells/mm 3 264 (2–871) Sexual transmission, n(%) 8 (23.5) IDU transmission, n(%) 19 (55.9) Vertical transmission, n(%) 5 (14.7) Blood transfusion, n(%) 2 (5.9) Stage C, CDC, n(%) 9 (26.5) Patients with CD4 ,200 cell/mm 3 ,n(%) 15 (44.1) Previous STI, n(%) 19 (55.9) Malignancies, n(%) 4 (11.8) IDU, intravenous drug user. Values other than n(%) are expressed as mean (minimum–maximum). a Positive PCR for HCV. Maraviroc exposure to assay HIV tropism 847 exposure. An ES-Trofile® was performed from blood samples the same day starting MCT (90%) or, if not possible, in samples obtained not more than 12 weeks before starting MCT. The result of MCT was compared with the tropism assay reported by ES-Trofile®. Baseline characteristics of the patients were: 36 (76.6%) were males, 18 (38.3%) showed hepatitis C virus coinfection and 13 (27.7%) had developed an AIDS-defining event (CDC stage C). Baseline median [interquartile range (IQR)] age was 43 [37 – 47] years, time since HIV diagnosis was 17 [12 – 20] years, viral load 4.71 log10 HIV-RNA copies/mL [4.19 – 5.07] and CD4+ cell count 221 [92 – 431] cell/mm3. Adherence to therapy during MCT was self-reported by patients and estimated through Pharmacy registers, being 100% in all the patients. Results of ES-Trofile® were: 29/47 (61.7%) patients were reported to be infected by R5-tropic HIV variants and 17/47 (36.2%) by dual/mixed (D/M) tropic virus; non-reportable result was reported in 1/47 patients (2.1%) and this patient achieved undetectability after MCT. As shown in figure 1a, mean viral load reduction > 1.6 log10 HIV-RNA copies/mL was observed during MCT in 26/29 patients with R5-tropic virus according to ES-Trofile®. On the other side, as shown in figure 1b, no viral load modification was observed in 13/17 patients with an ESTrofile® reported as D/M. However, 3/29 (10.3%) patients with R5-tropic virus showed no viral load modification after MCT (figure 1a), while 4/17 (23.5%) patients with D/M virus experienced a viral reduction > 1.5 log10 HIV-RNA copies/mL (figure 1b); hence, global discordance rate between MCT and ESTrofile® was 7/46 (15.2%). Discordance rate was higher in patients reported as D/M compared with patients with tropism reported as R5, although no statistically significant (23.5% vs 10.3%, respectively; p = 0.22, Chi-square test). Three patients with R5-tropic virus and no viral load reduction after MCT started a MRVsparing cART, according to MCT result. Immunovirological evolution of these patients after MCT is shown in figure 2a; two of them remain with undetectable viral load after 36 and 48 weeks, respectively, while the other patient achieved a viral load reduction > 3 log10 HIV-RNA copies/mL after 24 weeks. Three patients with D/M tropic virus and positive MCT started a MRV-containing regimen after MCT, associated to lamivudine plus abacavir. Immunovirological evolution of these patients is shown in figure 2b; viral load was undetectable after 12, 36 and 48 weeks, respectively. The other patient with positive MCT and an ES-Trofile® reported as D/M refused starting cART after MCT. Results presented herein show unexpected rates of discordance between the short-term virological response to MRV and the results reported by ES-Trofile®. Although a high global concordance was achieved (nearly 85%), rates of discordance around 15% was observed. Hence, clinicians are currently taking decisions based on an assay that might not ensure the virological success, attending to our results. Discordance observed in R5 patients could be explained because, despite maintaining the viral tropism as R5, MRV could be no effective due to changes mainly in the V3 loop that could be related with CCR5-antagonist resistance (Soulié et al, 2008); hence, despite an adequate tropism reported by ES-Trofile®, virological efficacy could be impaired. On the other side, discordance observed in patients with D/M tropic virus attending to ES-Trofile® was 23.5%, higher than in R5 patients; discordance in D/M patients could be explained due to the greater sensitivity of ES-Trofile® to detect minor X4-tropic variants, that could lead to a D/M result when R5-tropic variants are predominant enough to exert a virological response. Higher sensitivity to detect minor X4-tropic variants makes less likely to offer treatment with a CCR5-antagonist to patients with a minor representation of X4 virus. Moreover, ES-Trofile® is a qualitative test and clinicians only receive a categorical result (i.e. R5, X4, D/M), but percentage of X4 variants is not reported. Thus, our results show that establishing the clinically significant cut-off of X4-tropic variants is required to accurately consider some patients candidate to be treated with CCR5-antagonists. Alternatives to ES-Trofile® have been suggested, like other phenotypic or genotypic assays (Chueca et al, 2009; Poveda et al, 2009; Trouplin et al, 2001). Genotypic methods are being used to assay HIV tropism with a greater frequency due to their availability, good correlation with phenotypic methods and simplicity (Recordon-Pinson et al, 2010). Discordance between MCT and genotypic approaches is unknown and should be evaluated in future studies. The use of the ultra-deep sequencing (Rozera et al, 2009) could quantify minor variants, but this assay is not available in routine clinical practice and has not been clinically validated to consider a patient candidate to be treated with a CCR5-antagonist. In conclusion, our results show that ES-Trofile® does not completely correlate with the virological response to MRV after a short-term exposure, especially when D/M results are reported. These provocative results may have important clinical implications in routine clinical practice in order to accurately prescribe CCR5-antagonists treatment. Potential discordance between MCT and genotypic methods should be determined in future studies. ACKNOWLEDGEMENTS This work was supported by Redes Telemáticas de Investigación Cooperativa en Salud (RETICS; 2006, Red de SIDA RD06/0006/0021, 2007–2010) and by Fondo de Investigación Sanitaria grant PI06/0915. S.F.-M. has a grant from Fondo de Investigaciones Sanitarias (FIS06/00176). E.R.-M. has a grant from Fondo de Investigaciones Sanitarias (CP08/00172). A.G.-S. has a grant from Fundación Reina Mercedes. We are grateful to the patients that participated in this study, to José Manuel Lara Ruiz from the Immunology Service because of his technical support, to Marien Gutiérrez Sancho, Francisca Cano and Magdalena Rodríguez from the Hospital de Día (Infectious Diseases Service) and Dra Amparo Lluch (Pharmacy Service) for their support to the patients. Conflict of interest: none to declare. REFERENCES 1. Chueca N, Garrido C, Alvarez M, Poveda E, de Dios Luna J, Zahonero N, Hernández-Quero J, Soriano V, Maroto C, de Mendoza C, García F. 2009. Improvement in the Determination of HIV-1 Tropism Using the V3 Gene Sequence and a Combination of Bioinformatic Tools. J. Med. Virol. 81:763767. 2. Genebat M, Ruiz-Mateos E, León JA, González-Serna A, Pulido I, Rivas I, Ferrando-Martínez S, Sánchez B, Muñoz-Fernández MA, Leal M. 2009. Correlation between the Trofile test and virological response to a short-term maraviroc exposure in HIV-infected patients. J. Antimicrob. Chemother. 64:845-9. 3. Poveda E, Seclén E, González Mdel M, García F, Chueca N, Aguilera A, Rodríguez JJ, González-Lahoz J, Soriano V. 2009. Design and validation of new genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 antagonists. J. Antimicrob. Chemother. 63:1006-10. 4. Recondon-Pinson P, Soulié C, Flandre P, Descamps D, Lazrek M, Charpentier C, Montes B, Trabaud M-A, Cottalorda J, Schneider V, MorandJoubert L, Tamalet C, Desbois D, Macé M, Ferré V, Vabret A, Ruffault A, Pallier C, Raymond S, Izopet J, Reynes J, Marcelin A-G, Masquelier B and the ANRS AC11 Resistance Study Group. Evaluation of the genotypic prediction of HIV-1 coreceptor use versus a phenotypic assay and correlation with the virological response to maraviroc: the ANRS Geno Tropism Study. Antimicrob. Agents Chemother. doi: 10.1128/AAC.00148-10. 5. Reeves JD, Coakley E, Petropoulos CJ, Whitcomb JM. 2009. An enhancedsensitivity Trofile HIV coreceptor tropism assay for selecting patients for therapy with entry inhibitors targeting CCR5: A review of analytical and clinical studies. J. Viral Entry 3:94-102. 6. Rozera G, Abbate I, Bruselles A, Vlassi C, D'Offizi G, Narciso P, Chillemi G, Prosperi M, Ippolito G, Capobianchi MR. 2009. Massively parallel pyrosequencing highlights minority variants in the HIV-1 env quasispecies deriving from lymphomonocyte sub-populations. Retrovirology 6:15. 7. Soulié C, Malet I, Lambert-Niclot S, Tubiana R, Thévenin M, Simon A, Murphy M, Katlama C, Calvez V, Marcelin AG. 2008. Primary genotypic resistance of HIV-1 to CCR5 antagonists in CCR5 antagonist treatmentnaïve patients. AIDS 22:2212-4. 8. Trouplin V, Salvatori F, Cappello F, Obry V, Brelot A, Heveker N, Alizon M, Scarlatti G, Clavel F, Mammano F. 2001. Determination of Coreceptor Usage of Human Immunodeficiency Virus Type 1 from Patient Plasma Samples by Using a Recombinant Phenotypic Assay. J. Virol. 75: 251-259. 9. Whitcomb JM, Huang W, Fransen S, Limoli K, Toma J, Wrin T, Chappey C, Kiss LD, Paxinos EE, Petropoulos CJ. 2007. Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 co-receptor tropism. Antimicrob. Agents Chemother. 51:556-575. FIGURE CAPTIONS Figure 1a. Viral load evolution in patients reported as R5 by ES-Trofile® (N = 29). Mean viral load evolution and confidence interval 95% (CI 95%) is shown in 26/29 patients that achieved a viral load reduction > 1 log10 HIV-RNA copies/mL during MCT. Three patients (Discord 1, Discord 2 and Discord 3) showed no viral load modification during MCT despite ES-Trofile® was reported as R5 (3/29 = 10.3%). Figure 1b. Viral load evolution in patients reported as D/M by ES-Trofile® (N = 17). Mean viral load evolution and confidence interval 95% (CI 95%) is shown in 13/17 patients that showed no viral load modification during MCT. Four patients (Discord 4, Discord 5, Discord 6 and Discord 7) experienced a viral load reduction > 1.5 log10 HIV-RNA copies/mL despite ES-Trofile® was reported as D/M (4/17 = 23.5%). Figure 2a. Immunovirological evolution of patients with R5 tropism according to ES-Trofile® and negative MCT, once the new cART was started after MCT. Total CD4+T-cell increase and viral load reduction once cART was started after MCT, in patients with negative MCT and R5-tropic virus according to ES-Trofile®. A progressive CD4+T-cell increase and viral load reduction is observed. Two of them achieved undetectability (< 40 HIV-RNA copies/ml) after 36 and 48 weeks under cART after MCT, respectively; the other patient remain with low-level detectable viral load after 12 weeks under cART, and a viral load reduction > 3 HIV-RNA copies/ml was achieved at this timepoint. Figure 2b. Immunovirological evolution of patients with D/M tropism according to ES-Trofile® and positive MCT, once the new cART was started after MCT. Total CD4+T-cell increase and viral load reduction once cART was started after MCT, in patients with positive MCT and D/M-tropic virus according to ES-Trofile®. A progressive CD4+T-cell increase and viral load reduction is observed. All of them achieved undetectability (< 40 HIV-RNA copies/ml) after 12, 36 and 48 weeks under cART after MCT, respectively. 484 Current HIV Research, 2010, Vol. 8, No. 6 Genebat et al. cART. The first one developed fever and rash probably related to ABV, despite HLAB*5701 was negative, and treatment was modified to tenofovir/emtricitabine (TDF/FTC) plus MRV. The second one developed an acute hepatitis and cART was suspended; this patient recognized a huge alcohol intake, but the cause of the hepatitis could not be established. Once transaminases returned to baseline values, a new cART not including MRV was started. In summary, only 1/46 patient (2.2%) suspended MRV along the observational period due to an acute hepatitis probably not related to MRV. Clinical events were developed in 4/46 patients (8.7%). One patient died after 24 weeks follow up due to heroin overdose. One HCV-coinfected patient with previous Child C stage liver cirrhosis developed liver-related encephalopathy after 4 weeks with the new regimen, experiencing a good evolution with standard treatment. One patient developed a monometameric herpes-zoster after 24 weeks, with an excellent clinical response to acyclovir. Finally, one patient developed a non-Hodgkin lymphoma after 36 weeks of follow up; this patient had been in persistent virologic failure for many years and experienced a CD4+ gain above 200 cell/mm3 from baseline once MRVFig. (1). Virological response: percentage of patients with an undetectable viral load (grey, < 50 HIV-RNA copies/mL, black <200 HIVRNA copies/mL) in every timepoint, once MRV-containing cART was started. Notice that at baseline 17% of the patients had an undetectable viral load; these patients achieved undetectability during MCT and viral load was < 50 HIV-RNA copies/ml once cART was started. After 24 weeks of follow up (n=39), 38/39 patients (97%) had achieved undetectability and only 1/39 patients (3%) had a viral > 200 HIV-RNA copies/mL. Up to week 48 (n=27), 96.3% of patients had achieved < 50 HIV-RNA copies/mL and 100% a viral load < 200 HIVRNA copies/mL. Fig. (2). Immunological response: mean [CI 95%] CD4+ gain once MRV-containing cART was started, up to week 48. A progressive CD4+ increase is observed along the follow up. Maraviroc Efficacy in Clinical Practice Current HIV Research, 2010, Vol. 8, No. 6 485 containing cART was started. Viral load was undetectable when the clinical event occurred in all of them and MRVcontaining cART was maintained in every patient. Concomitant drugs were used together with MRVcontaining cART in 5/46 patients (10.9%): two patients were taking tuberculostatic drugs, two other patients were on chemotherapy and one patient was taking antipsychotic drugs. No increased toxicity or clinically relevant drug-drug interactions were observed. DISCUSSION Our results show that a MRV-containing cART combined with different ARV (including novel ARV) and in different clinical scenarios is long-term effective and well tolerated in an HIV-infected cohort with a high prevalence of HCV-coinfection. Despite limitations related to its observational design, our study adds relevant novelties: different clinical scenarios were considered and different ARV were added to MRV (conventional vs novel ARV-based cART); 50% of the patients showed chronic HCV or HBV coinfection and nearly 15% had developed malignancies at baseline; finally, results presented herein show the routine clinical practice avoiding Clinical Trials restrictions. As previously mentioned, MOTIVATE studies showed the immunovirological benefit of MRV in the context of a salvage therapy. In addition, in these studies the simultaneous use of other novel ARV was not permitted [1]. However, attending to current guidelines MRV should be considered from the first virologic failure [8] and not only once multidrug resistant virus have emerged, a context with a greater probability of X4-tropic virus emergence [9] in which MRV will not be useful. In Spain, it has been reported that CCR5-tropic HIV-1 virus is prevalent in nearly 70% of treatment-experienced patients [10], showing that MRV is active in most of these patients. Besides, novel cART combinations need to be explored in routine clinical practice, as shown when TDF plus didanosine were used together and unexpected high toxicity and immunovirological impairment were observed [11, 12]. Efficacy and tolerability of MRV in routine clinical practice and in a population with a high prevalence of HCV-coinfection has not been previously explored, as we do in the present study. Nozza et al. have recently shown the efficacy of MRV plus RGV and ETV as a salvage therapy in 28 HIV-infected patients, but only 8 patients showed HCV-coinfection [6]. In contrast, our study shows different scenarios in which a MRV-containing cART was prescribed: patients on persistent virologic failure, in whom MRV was combined with novel ARV (RGV plus a boosted PI, mainly DRV, instead of ETV) because therapeutic options were limited; and patients not on persistent virologic failure, in whom MRV was combined with more simplified regimens, an scenario not considered by Nozza et al. Global long-term immunovirological outcome obtained in our study is excellent, achieving nearly 97% of undetectability after 48 weeks and a progressive CD4+ gain. The mechanism of action of MRV, not acting against cellular enzymes, could reduce the possibility of developing serious adverse events and drug-drug interactions. On the other hand, blocking the CCR5 coreceptor has been reported to be associated with an increased susceptibility to certain infections [13]. Besides, other CCR5-antagonists have not been commercialized due to serious adverse events: vicriviroc was associated with the development of malignancies [14], while aplaviroc was related to severe hepatotoxicity [15]. In our study, only one patient suspended MRV and most treatment modifications were due to treatment simplifications. The only infectious event was a Fig. (3). Hepatotoxicity: mean liver enzymes (AST and ALT) evolution was analyzed to assess potential increased hepatotoxicity of MRVcontaining cART in a population with a high prevalence of HCV-coinfection. No significant modification in liver enzymes was observed during the observational period. 486 Current HIV Research, 2010, Vol. 8, No. 6 Genebat et al. monometameric herpes zoster and despite 15% of the patients had developed malignancies before starting MRVcontaining cART and 50% of patients showed chronic hepatitis coinfection, no increased hepatotoxicity or malignancies relapse were observed. The only AIDSdefining event observed in our study was a non-Hodgkin lymphoma; this patient had been on persistent virologic failure for many years before starting MRV-containing cART, and viral load was undetectable at the moment in which the neoplasm was developed. The excellent immunovirological data obtained with this MRV-based cART could be explained because: 1) patients were strictly followed because of the novelty of the new cART; hence, standard of care was improved in these patients; 2) apart from MRV, other novel ARV with a great antiviral activity were added in patients with multidrug resistant virus. Most of the patients included in this study started MRV therapy being classified as R5 through MCT, an attractive method recently reported [7]. Although it was not an objective of this study, it is worthy to note that the immunovirological evolution of patients classified as R5 by MCT was similar than patients classified as R5 by first generation Trofile® (data not shown). CONCLUSION We conclude that a MRV-containing cART is long-term effective and well tolerated in an HIV-infected population with a high prevalence of HCV-coinfection and using different combinations of ARV. ACKNOWLEDGEMENTS We would like to acknowledge the patients for their uninterested participation. We would like to thank Marien Gutiérrez Sancho, Francisca Cano and Magdalena Rodríguez from the Hospital de Día (Servicio de Enfermedades Infecciosas) and Dra Amparo Lluch from the Pharmacy Department for their support to the patients. FUNDING This work was supported by Redes Telemáticas de Investigación Cooperativa en Salud (RETICS; 2006, Red de SIDA RD06/0006/0021, 2007-2010) and a grant from Fondo de Investigaciones Sanitarias PS09/01595. E. R.-M. has a grant from Fondo de Investigaciones Sanitarias (CP08/00172). MC. R-S. was supported by Fundación para la Investigación y Prevención del SIDA en España (FIPSE) grant 36624/06 and by Fondo de Investigación Sanitaria grant PI06/0915. A. G-S. has a grant from Fundación Reina Mercedes. S.F.-M. has a grant from the Fondo de Investigaciones Sanitarias (FIS06/00176). A.G.-P. was supported by Proyecto de Excelencia, Consejería de Innovación, Ciencia y Empresa (P06-CTS-01579). G.M. was supported by Fundación para la Investigación y Prevención del SIDA en España (FIPSE) grant 366884/07. TRANSPARENCY DECLARATION Conflict of interests: none to declare. REFERENCES [1] Gulick RM, Lalezari J, Goodrich J, et al. Maraviroc for previously treated patients with R5 HIV-1 infection. N Engl J Med 2008; 359: 1429-41. [2] Steigbigel RT, Cooper DA, Teppler H, et al. Long-term efficacy and safety of Raltegravir combined with optimized background therapy in treatment-experienced patients with drug-resistant HIV infection: week 96 results of the BENCHMRK 1 and 2 phase iii trials. Clin Infect Dis 2010; 50: 605-12. [3] Arastéh K, Yeni P, Pozniak A, et al. Efficacy and safety of darunavir/ritonavir in treatment-experienced HIV type-1 patients in the POWER 1, 2 and 3 trials at week 96. Antivir Ther 2009; 14: 859-64. [4] Katlama C, Haubrich R, Lalezari J, et at. DUET-1, DUET-2 study groups.. Efficacy and safety of etravirine in treatment-experienced, HIV-1 patients: pooled 48 week analysis of two randomized, controlled trials. AIDS 2009; 23: 2289-300. [5] Whitcomb JM, Huang W, Fransen S, et al. Development and characterization of a novel single-cycle recombinant-virus assay to determine human immunodeficiency virus type 1 co-receptor tropism. Antimicrob Agents Chemother 2007; 51: 556-75. [6] Nozza S, Galli L, Visco F, et al. Raltegravir, maraviroc, etravirine: an effective protease inhibitor and nucleoside reverse transcriptase inhibitor-sparing regimen for salvage therapy in HIV-infected patients with triple-class experience. AIDS 2010; 24: 924-8. [7] Genebat M, Ruiz-Mateos E, León JA, et al. Correlation between Trofile® test and virological response to a short-term maraviroc exposure in HIV-infected patients. J Antimicrob Chemother 2009; 64: 845-9. [8] Hammer SM, Eron JJ Jr, Reiss P, et al. International AIDS SocietyUSA.. Antiretroviral treatment of adult HIV infection: 2008 recommendations of the International AIDS Society-USA panel. JAMA 2008; 300: 555-70. [9] Moore JP, Kitchen SG, Pugach P, Zack JA. The CCR5 and CXCR4 coreceptors-central to understanding the transmission and pathogenesis of human immunodeficiency virus type-1 infection. AIDS Res Hum Retroviruses 2004; 20: 111-26. [10] Moreno S, Clotet B, Sarría C, et al. Prevalence of CCR5-tropic HIV-1 Among Treatment-Experienced Individuals in Spain. HIV Clin Trials 2009; 10: 394-402. [11] Barreiro P, Soriano V. Suboptimal CD4 gains in HIV-infected patients receiving didanosine plus tenofovir. J Antimicrob Chemother 2006; 57: 806-9. [12] Martínez E, Milinkovic A, de Lazzari E, et al. Pancreatic toxic effects associated with co-administration of didanosine and tenofovir in HIV-infected adults. Lancet 2004; 364: 65-7. [13] Glass WG, McDermott DH, Lim JK, et al. CCR5 deficiency increases risk of symptomatic West Nile virus infection. J Exp Med 2006; 203: 35-40. [14] Gulick RM, Su Z, Flexner C, et al. AIDS Clinical Trials Group 5211 Team.. Phase 2 study of the safety and efficacy of vicriviroc, a CCR5 inhibitor, in HIV-1-Infected, treatment-experienced patients: AIDS clinical trials group 5211. J Infect Dis 2007; 196: 304-12. [15] Crabb C. GlaxoSmithKline ends aplaviroc trials. AIDS 2006; 20: 641. Received: April 14, 2010 Revised: July 16, 2010 Accepted: July 20, 2010 PMID: 20642436 DISCUSIÓN La comercialización simultánea de cuatro nuevos fármacos antirretrovirales, unos con alta barrera genética y otros con novedosos mecanismos de acción, ha permitido que desde principios de 2008 estemos disfrutando de lo que se ha llamado una segunda era de TARGA, facilitando por un lado el rescate de pacientes en fracaso virológico y por otro la simplificación de regímenes antirretrovirales previos con importantes efectos secundarios. Especialmente interesante por su mecanismo de acción extracelular es MRV, el único fármaco comercializado de la familia de los inhibidores de la entrada. En concreto, este fármaco ejerce su acción antirretroviral bloqueando el correceptor CCR5, un correceptor de quimiocinas fundamental para que se complete el proceso de entrada y fusión entre la proteína gp120 del VIH y el receptor CD4 del linfocito T, lo que permitirá finalmente que el material genómico del VIH penetre en la célula hospedadora. Sin embargo, además del CCR5, este proceso de entrada puede llevarse a cabo también a través del correceptor CXCR4, caso en el que MRV no sería eficaz. El uso de uno u otro correceptor varía a lo largo de la historia natural de la infección por VIH, siendo habitualmente cepas virales con tropismo R5 las que predominan en la infección aguda y durante los primeros años de infección crónica, para posteriormente emerger las cepas con tropismo X4. Por tanto, antes de indicar tratamiento con MRV es necesario conocer cuál es el correceptor empleado para completar este proceso de entrada, es decir, qué tropismo tienen las cepas virales de nuestro paciente. Para ello existen diferentes ensayos de tropismo: los métodos fenotípicos (Trofile® es el estándar de oro en la actualidad) son costosos, complejos y lentos, mientras que los genotípicos tienen alta sensibilidad para detectar cepas X4 pero tienen baja especificidad. Todo ello limita el uso de MRV en la práctica clínica. Como alternativa a estos ensayos ya conocidos, en el primer objetivo de la presente tesis se planteaba una herramienta clínica (MCT) que fuese sencilla y rápida. Los resultados de esta aproximación clínica se correlacionaron con el ensayo fenotípico clínicamente validado y de referencia (Trofile®), observándose una concordancia global del 93.5%. Además, MCT permitió determinar la sensibilidad clínica a MRV de pacientes en fracaso virológico pero con cargas virales por debajo de las 1000 copias/ml, una situación en la que Trofile® no puede determinar el tropismo viral. Por tanto, en este estudio se demostró que la respuesta virológica a una exposición a corto plazo a MRV podría ser una alternativa poco costosa y rápida para seleccionar a pacientes candidatos a tratamiento con antagonistas del CCR5. Desde entonces, la indicación clínica de MRV en nuestros pacientes se ha basado en el resultado de MCT, si bien antes de iniciar la misma se solicitaba el ensayo fenotípico. A tenor de nuestros resultados, y pese a una concordancia alta entre MCT y Trofile® en una aproximación inicial, las tasas de discordancia se vieron incrementadas a medida que el número de pacientes fue aumentando. La relevancia clínica de estas discrepancias es evidente, ya que podríamos estar indicando MRV en pacientes que no deberían recibirlo y viceversa. Por ello, nos planteamos analizar las tasas de discordancia entre ambos métodos, observando una discrepancia global mayor al 15%. Una posible explicación a esta elevada discrepancia es que los métodos fenotípicos tienden a aumentar la sensibilidad para detectar cepas con tropismo X4, de tal manera que Trofile® informa un resultado de tropismo como D/M si detecta al menos un 0.3% de cepas con tropismo X4. De esta manera, pacientes potencialmente candidatos a recibir MRV, no lo recibirían por la presencia de cepas minoritarias con tropismo X4, cuya relevancia clínica es desconocida hasta ahora. Por último, la indicación de MRV está basada en los resultados de los estudios MOTIVATE, es decir, en el contexto de un ensayo clínico y en los que se desconocía la tasa de coinfección por VHC de los pacientes, además de tener limitado el uso de otros antirretrovirales de reciente comercialización. El tercer estudio abordado en esta tesis demuestra que el uso de MRV en la rutina clínica diaria, en un grupo de pacientes con elevada tasa de coinfección por VHC y asociado a diferentes antirretrovirales es eficaz desde un punto de vista inmunovirológico y seguro. En resumen, los resultados presentados en esta tesis, de inmediata aplicación clínica, abren una puerta más para la determinación de la sensibilidad clínica a MRV, más allá de un mero resultado categórico de tropismo, y demuestran la eficacia y seguridad de MRV en su uso en la rutina clínica asistencial. Como consecuencia de los resultados aquí presentados, recomendamos el uso de MCT antes de indicar tratamiento con antagonistas de CCR5. CONCLUSIONES Como resultado de las publicaciones presentadas en la presente tesis doctoral, se desprenden las siguientes conclusiones:  Una aproximación clínica que analiza la respuesta virológica a una exposición a corto plazo a MRV es una herramienta sencilla y efectiva para determinar la indicación de antagonistas de CCR5.  Las tasas de discrepancia observadas entre MCT y Trofile® pueden tener relevancia clínica a la hora de prescribir con seguridad un TARGA que incluya antagonistas de CCR5.   Un régimen antirretroviral con MRV es eficaz y seguro a largo plazo asociado a diferentes regímenes antirretrovirales, en diferentes escenarios clínicos y en una población con elevada tasa de coinfección por VHC.  ANEXO: OTRAS PUBLICACIONES GENERADAS DURANTE EL PROGRAMA MIR Y DESARROLLO DE LA TESIS ORIGINAL RESEARCH Non-medically supervised treatment interruptions among participants in a universally accessible antiretroviral therapy programme DM Moore, 1,2 W Zhang, 1 B Yip, 1 M Genebat, 4 VD Lima, 1 JSG Montaner 1,2 and RS Hogg 1,3 1 British Columbia Centre for Excellence in HIV/AIDS, Vancouver, Canada, 2 Department of Medicine, Faculty of Medicine, University of British Columbia, Vancouver, Canada, 3 Faculty of Health Sciences, Simon Fraser University, Burnaby, Canada and 4 Department of Internal Medicine, Virgen del Rocio University Hospital, Seville, Spain Background We examined clinical outcomes, patient characteristics and trends over time of non-medically supervised treatment interruptions (TIs) from a free-of-charge antiretroviral therapy (ART) programme in British Columbia (BC), Canada. Methods Data from ART-naı ¨ve individuals 18 years old who initiated triple combination highly active antiretroviral therapy (HAART) between January 2000 and June 2006 were analysed. Participants having 3 month gap in HAART coverage were defined as having a TI. Cox proportional hazards modelling was used to examine factors associated with TIs and to examine factors associated with resumption of treatment. Results A total of 1707 participants were study eligible and 643 (37.7%) experienced TIs. TIs within 1 year of ART initiation decreased from 29% of individuals in 2000 to 19% in 2006 (Po0.001). TIs were independently associated with a history of injection drug use (IDU) (P50.02), higher baseline CD4 cell counts (Po0.001), hepatitis C co-infection (Po0.001) and the use of nelfinavir (NFV) (P50.04) or zidovudine (ZDV)/lamivudine (3TC) (P50.009) in the primary HAART regimen. Male gender (Po0.001), older age (Po0.001), AIDS at baseline (P50.008) and having a physician who had prescribed HAART to fewer patients (P50.03) were protective against TIs. Four hundred and eightyeight (71.9%) participants eventually restarted ART with male patients and those who developed an AIDS-defining illness prior to their TI more likely to restart therapy. Higher CD4 cell counts at the time of TI and unknown hepatitis C status were associated with a reduced likelihood of restarting ART. Conclusion Treatment interruptions were associated with younger, less ill, female and IDU participants. Most participants with interruptions eventually restarted therapy. Interruptions occurred less frequently in recent years. Keywords: access to therapy, adherence, antiretroviral therapy, treatment interruptions Accepted 11 August 2009 Introduction Improving access to highly active antiretroviral therapy (HAART) is an important public health objective in all regions of the globe. Not only is HAART associated with markedly improved survival among HIV-infected individuals [1,2], but it can also contribute to reducing the number of new HIV infections at the population level [3,4]. Continued access to HAART is often limited by patientincurred costs, especially in lowor middle-income countries [5] or in industrialized countries without universal health care insurance programmes [6]. However, other factors associated with poor access or continuation Correspondence: Dr David M. Moore, British Columbia Centre for Excellence in HIV/AIDS, 608 - 1081 Burrard St., Vancouver, BC, Canada V6Z 1Y6. Tel: 11 604 806 8781; fax: 11 604 806 9044; e-mail: [email protected] DOI: 10.1111/j.1468-1293.2009.00779.x r2009 British HIV Association HIV Medicine (2010), 11 ,299–307 299 Effect of the Substitution of One Nucleoside Analogue by One Non-nucleoside Reverse Transcriptase Inhibitor over Mitochondrial DNA Levels B. De Felipe, A. Blanco, N. Soriano-Sarabia, M. Á. Muñoz-Fernández, M. Genebat, A. Vallejo, M. Leal Abstract Background: Long-term antiretroviral therapy is associated with several side effects, like mitochondrial toxicity related to nucleoside reverse transcriptase inhibitors (NRTIs). Our objective was to analyze the effect of the substitution of one NRTI by one non-nucleoside reverse transcriptase inhibitor (NNRTI) in the antiretroviral regime of HIV-1-infected patients who were on a regime containing either two NRTIs and one NNRTI, or one NRTI, one NNRTI and one protease inhibitor (PI), over mtDNA level. Decreasing NRTIs could increase mtDNA level. Methods: Fifteen HIV-1-infected patients were included in the study. As controls, 17 healthy individuals and 15 HIV-1-infected patients naïve for antiretroviral treatment were also analyzed. mtDNA level was quantified at baseline and after 48 weeks of treatment. Results: Control groups showed higher levels of mtDNA than the study group (p < 0.001). Among this latter group, no statistical differences between baseline and after 48 weeks were found. Naïve HIV-infected patients had lower mtDNA than healthy volunteers (p < 0.001). Two patients had two consecutive blips (low viral load increases) but they did not show NNRTI-related resistance mutations. Conclusions: This study shows that although this treatment was immunovirologically effective, mtDNA level did not increase at least after 48 weeks. Infection 2008; 36: 266–270 DOI 10.1007/s15010-008-7312-z Introduction The introduction of highly active antiretroviral therapy (HAART) based on the combination of two nucleoside reverse transcriptase inhibitors (NRTIs) plus one protease inhibitor (PI) has led to a dramatic reduction in mortality and morbidity among HIV-infected patients [1, 2]. However, long-term use of PIs is associated with several side effects like lipodystrophy syndrome, severe hepatic damage and other metabolic disturbances, such as dyslipidemia or diabetes mellitus [3–6]. An effective alternative to reduce these side effects has been the substitution of PIs by non-nucleoside reverse transcriptase inhibitors (NNRTIs) in the antiretroviral regimen [7]. Nevertheless, NRTIs may inhibit human mitochondrial DNA (mtDNA) polymerase cdriving to mitochondrial toxicity and depletion [8, 9]. This inhibition leads to the development of different adverse effects [10, 11]. Changes in mitochondrial DNA in the peripheral blood cells have been reported to be a good marker of treatment toxicity [12]. Reported data have associated mitochondrial toxicity with the NRTIs included in the treatment [13], long-term antiretroviral treatment [14], different combinations among them [15], and/or genetic host factors. Others reported no association between mitochondrial toxicity and the type of NRTI used [16]. Furthermore, HIV infection with no antiretroviral treatment induces mtDNA depletion [17]. We hypothesized that mtDNA level could increase among those patients whose HAART regimen (either two NRTIs and one NNRTI, or one NRTIs, one PI and one NNRTI) is replaced by one NRTI and two NNRTIs. Hence, the objective of our study was to analyze the effect B. De Felipe, N. Soriano-Sarabia, M. Leal Service of Infectious Diseases, Virgen del Rocío University Hospital, Seville, Spain A. Blanco, M. Á. Muñoz-Fernández Laboratory of Immunobiology, Gregorio Marañón General University Hospital, Madrid, Spain M. Genebat Laboratory of Immunovirology, Service of Internal Medicine, Virgen del Rocío University Hospital, Seville, Spain B. De Felipe, A. Vallejo Laboratory of Molecular Virology, Virgen del Rocío University Hospital, Seville, Spain M. Leal (Corresponding author) Laboratory of Immunovirology, Service of Infectious Diseases, Hospital Universitario Virgen del Rocio, Manuel Siurot s/n, 41013 Seville, Spain; Phone: (+34/955) 012-396; Fax: -013-292 e-mail: [email protected] Received: July 25, 2007 ÆRevision accepted: January 8, 2008 Published online: May 3, 2008 Infection Brief Report 266 Infection 36 Æ2008 ÆNo. 3 URBAN &VOGEL Journal of Clinical Virology 40 (2007) 64–67 Short communication Control of HIV-1 RNA load after HAART interruption: Relationship with CCR5 co-receptor density and proviral DNA load in HIV-infected patients Natalia Soriano-Sarabiaa, Alejandro Vallejob,∗, Ger´ onimo Fern´ andezc, Miguel Genebatd, Sonia Guti´ errezd, Maria ´ Angeles Mu˜ noz-Fern´ andezc, Manuel Leala aService of Infectious Diseases, Virgen del Rocio University Hospital, Seville, Spain bLaboratory of Molecular Virology, Virgen del Roc´ıo University Hospital, Seville, Spain cLaboratory of Immunobiology, Gregorio Mara˜n´on University Hospital, Madrid, Spain dService of Internal Medicine, Virgen del Roc´ıo University Hospital, Seville, Spain Received 23 March 2007; accepted 18 June 2007 Abstract Background: CCR5 co-receptor density has been reported to play a role in the level of HIV production. In addition, reports about the relationship between proviral DNA load and plasma HIV load are controversial. Objectives: To analyse the role of CCR5 co-receptor density and proviral DNA load in the control of plasma HIV-viral load after HAART interruption, comparing patients whose plasma HIV load was persistently below 4 log10 RNA copies/mL, defined as “HIV controllers”, with patients who showed a viral load higher than 4 log10 RNA copies/mL, defined as “non-controllers”. Study design: Proviral DNA load quantification (N= 55) and CCR5 co-receptor density (N= 29) were determined in HIV-infected patients on prolonged HAART interruption. Results: Twenty-three percent of our HAART interruption cohort were classified as HIV controllers, while 77% were classified as noncontrollers. CCR5 co-receptor density was statistically higher in HIV controllers than in non-controllers, while proviral DNA load was not different between them. CCR5 co-receptor density in activated CD4 cells was independently associated with HIV plasma load after interruption. Conclusions: The observation of a higher CCR5 co-receptor expression in HIV controllers suggests that HIV infection leads to the selection of CD4 cells with low CCR5 co-receptor density after HAART interruption. © 2007 Elsevier B.V. All rights reserved. Keywords: HIV controllers; CCR5 density; Proviral DNA load; HAART interruption 1. Introduction In the absence of antiretroviral treatment some HIVinfected patients are able to maintain low to moderate plasma HIV-viral load (i.e., 75–10,000 RNA copies/mL) (Deeks et al., 2004) or even persistently undetectable, named as HIV controllers (Lambotte et al., 2005; Madeca et al., 2005). On the other hand, in our prolonged HAART interruption cohort ∗Corresponding author. E-mail address: av[email protected] (A. Vallejo). we described a group of patients whose viral load was persistently below 4 log10 RNA copies/mL (Vallejo et al., 2005). Understanding the mechanisms involved in HIV-viral load control is critical for the knowledge of HIV pathogenesis, i.e. the involvement of HIV-specific response has already been well documented (Betts et al., 1999; Emu et al., 2005; Pantaleo and Koup, 2004). However, other potential factors have not been deeply studied or reports are controversial. CCR5 co-receptor density has been reported to play a role in HIV production (Reynes et al., 2000). In addition, some studies suggest that proviral load may be an indicator of spread 1386-6532/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2007.06.010 Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited. 9. Klein MR, van Baalen CA, Holwerda AM, Kerkhof G Sr, Bende RJ, Keet IP, et al.Kinetics of Gag-specific cytotoxic T lymphocyte responses during the clinical course of HIV-1 infection: a longitudinal analysis of rapid progressors and long-term asymptomatics. J Exp Med 1995; 181:1365–1372. 10. Kuroda MJ, Schmitz JE, Charini WA, Nickerson CE, Lord CI, Forman MA, et al.Comparative analysis of cytotoxic T lymphocytes in lymph nodes and peripheral blood of simian immunodeficiency virus-infected rhesus monkeys. J Virol 1999; 73:1573–1579. 11. Borrow P, Lewicki H, Wei X, Horwitz MS, Peffer N, Meyers H, et al.Antiviral pressure exerted by HIV-1-specific cytotoxic T lymphocytes (CTLs) during primary infection demonstrated by rapid selection of CTL escape virus. Nat Med 1997; 3:205–211. 12. Sallusto F, Geginat J, Lanzavecchia A. Central memory and effector memory T cell subsets: function, generation, and maintenance. Annu Rev Immunol 2004; 22:745–763. 13. Wills MR, Okecha G, Weekes MP, Gandhi MK, Sissons PJ, Carmichael AJ. Identification of naive or antigen-experienced human CD8(R) T cells by expression of costimulation and chemokine receptors: analysis of the human cytomegalovirus-specific CD8(R) T cell response. J Immunol 2002; 168: 5455–5464. HIV–hepatitis C virus co-infection is associated with decreased plasmatic IL-7 levels Natalia Soriano-Sarabia a , Alejandro Vallejo a , Sonia Molina-Pinelo a ,MiguelGenebat a ,Marı´adelMar Rodrı´guez a , Armando Sa ´nchez-Quijano b ,Manuel Martı´nez-Moya c , Jorge Vivancos c and Manuel Leal d We analysed the potential influence of hepatitis C virus (HCV) co-infection over IL-7 levels and thymic function in naive HIV-infected patients and after effective HAART. HIV–HCV-co-infected patients had lower plasmatic IL-7 levels compared with HIV-monoinfected patients. This effect may not be associated either with HCV monoinfection or with the rate of liver injury. These lower levels may explain, at least partly, the lower CD4 cell repopulation of HIV–HCV-co-infected patients after HAART. HIV-infected patients co-infected by hepatitis C virus (HCV) may have an increased risk of progression to AIDS and lower CD4 cell repopulation after HAART [1]. Although these results have not been confirmed by other authors [2], a recent meta-analysis supported these findings [3]. This lower CD4 cell repopulation may be explained partly by the impairment of at least one of the main mechanisms involved in T-cell homeostasis, thymic function and plasmatic IL-7 levels [4,5]. A recent work has reported lower T-cell rearrangement excision circle (TREC) levels in HCV-monoinfected patients compared with non-infected controls, suggesting an immune impairment [6]. Whether HCV co-infection influences this CD4 cell homeostatic system has not been studied. In this work, we performed a cross-sectional study to analyse the potential influence of HCV co-infection over IL-7 levels and thymic function in both naive HIV-infected patients and after effective HAART. The CD4 cell count and IL-7 levels (Quantikine HS IL-7 immunoassay kit; R&D Systems, Minneapolis, Minnesota, USA) were measured in 97 naive HIV-infected patients, including 40 HCV-co-infected patients. Among these patients, 25 HIV-monoinfected and 20 HIV– HCV-co-infected patients had previously participated in other studies and had their thymic volume measurements recorded [7]. In addition, 92 HIV-infected patients on HAART, with undetectable HIV plasma viraemia, including 49 HCV-co-infected patients, were also analysed. Among them, 29 HIV-monoinfected and 24 HIV– HCV-co-infected patients had their thymic volume recorded [8]. A comparison of thymic volume between these two study populations was not possible because of technical changes. However, the analysis of the effect of HCV co-infection could be performed within each single group (naive patients and HAART-treated patients). The only limitation for the selection of the patients was the availability of frozen plasma samples. None of the HCVinfected patients had received treatment for HCV infection. Control populations for IL-7 level quantification included 28 healthy volunteers and 31 HCV-monoinfected patients who had never received treatment. In addition, in order to analyse the potential influence of liver injury on IL-7 levels, an additional group of 34 HIV–HCV-co-infected HAART-treated patients were studied. Subjects’written informed consent had been obtained and the ethical committee approved the study. Real-time polymerase chain reaction (LightCycler; Roche Diagnostics, Branchburg, New Jersey, USA) was used for the quantification of both the characteristic signaljoint sequences harboured in the generated TREC, and b-globin gene [7]. TREC levels were measured in peripheral blood mononuclear cells, yielding the number of TREC per 10 6 peripheral blood mononuclear cells in the same patients whose thymic volume had previously been measured. As shown in Fig. 1, HIV–HCV-co-infected patients, both naive and HAART-treated patients, showed statistically lower IL-7 levels than HIV-monoinfected patients. In addition, healthy volunteers showed statistically lower levels than naive HIV-infected patients, although no statistical differences with either HAART-treated patients or HCV-monoinfected patients were found. In order to determine which factors were associated with IL-7 levels, HIV-infected populations under study were analysed together (97 naive plus 92 HAART-treated patients). The CD4 cell count, HCV co-infection, TREC levels and thymic volume had P<0.1 in the univariate analysis and were introduced in the stepwise Research Letters 253 Journal of Clinical Virology 36 (2006) 13–16 Disseminate and fatal cytomegalovirus disease with thymitis in a naive HIV-patient after early initiation of HAART: Immune restoration disease? Sonia Guti´ erreza,1, Silvia Alconchelb, Ezequiel Ruiz-Mateosc,1, Miguel Genebata,1, Alejandro Vallejoc,1, Eduardo Lissena,1, Jorge Fern´ andez-Alonsob, Manuel Leala,∗,1 aDepartment of Internal Medicine, Virgen del Rocio University Hospital, Seville, Spain bDepartment of Anatomy Pathology, Virgen del Rocio University Hospital, Seville, Spain cDepartment of Biochemistry, Virgen del Rocio University Hospital, Seville, Spain Received 5 October 2005; received in revised form 10 December 2005; accepted 16 December 2005 Abstract We describe a na¨ ıve HIV-infected patient who developed a Pneumocystis carinii pneumonia and disseminate and fatal cytomegalovirus disease within 3 months after initiation of HAART, suggesting due to coincidence in time, an immune restoration disease. We propose an alternative hypothesis. © 2005 Elsevier B.V. All rights reserved. Keywords: Immune restoration disease; Thymitis cytomegalovirus; AIDS 1. Introduction HIV-patients under HAART may experience severe systemic inflammatory reactions that have been defined as immune restoration disease (IRD). It has been communicated that IRD has two different patterns; an earlier pattern during the first 3 months of HAART as an immune response against viable opportunistic pathogens, and a later pattern as an immune response against non-viable opportunistic pathogens months to years after HAART (French et al., 2004). During the IRD, a baseline CD4 cell count below 100 cells/mm3 has been reported among patients, while after HAART an increase above 200 cells/mm3is reached (Price et al., 2001). Outcomes range from minimal morbidity to fatal progression (French et al., 2004; Hirsch et al., 2004). In this way, atypical presentations of mycobacterial, cytomegalovirus (CMV), ∗Corresponding author at: Viral Hepatitis and AIDS Unit, Department of Internal Medicine, Virgen del Rocio University Hospital, Seville, PC 41013, Spain. Tel.: +34 955012396; fax: +34 955012390. E-mail address: [email protected] (M. Leal). 1Viral Hepatitis and AIDS Unit. hepatitis B virus, hepatitis C virus and JC virus have been described after initiating HAART (French et al., 2004; Safdar et al., 2002). We here report the case of a na¨ ıve HIV-infected patient who developed Pneumocystis carinii pneumonia as well as disseminated and fatal CMV infection coinciding with the initiation of HAART. 2. Case report In 1993, a 32-year-old woman was diagnosed of HIV infection in our unit. Then, her CD4 T cell count was 840 cells/mm3, and HIV plasma viral load (pVL) was above 75,000 copies/mL. The moment that primoinfection occurred in the past was unknown because it was asymptomatic. Since she declined to receive antiretroviral therapy, a progressive CD4+T cell count decrease was taking place during the following 9 years. In July 2002, she began HAART with zidovudine, lamivudine, and abacavir, having HIV pVL above 75,000 copies/mL, CD4+T cell count of 90 cells/mm3, and a thymic volume (measured by mediastinic computed 1386-6532/$ – see front matter © 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.jcv.2005.12.007 Journal of Antimicrobial Chemotherapy (2004) 53, 95–101 DOI: 10.1093/jac/dkh012 Advance Access publication 4 December 2003 95 ................................................................................................................................................................................................................................................................... JAC vol.53 no.1 © The British Society for Antimicrobial Chemotherapy 2003; all rights reserved. Long-term virological outcome and resistance mutations at virological rebound in HIV-infected adults on protease inhibitor-sparing highly active antiretroviral therapy Rafael de la Rosa 1 , Ezequiel Ruíz-Mateos 2 , Amalia Rubio 3 , María Antonia Abad 2 , Alejandro Vallejo 2 , Laura Rivero 4 , Miguel Genebat 4 , Armando Sánchez-Quijano 4 , Eduardo Lissen 4 and Manuel Leal 4 * Viral Hepatitis and AIDS Study Group, 1 Department of Medicine, San Sebastian Hospital, Ecija, Seville; 2 Department of Biochemistry, Virgen del Rocío University Hospital, Seville; 3 Department of Medical Biochemistry and Molecular Biology, University of Seville, Seville; 4 Department of Internal Medicine, Virgen del Rocío University Hospital, Avda. Manuel Siurot s/n, 41013 Seville, Spain Received 1 July 2003; returned 31 July 2003; revised 6 September 2003; accepted 3 October 2003 Objective : To assess the durability of the undetectability of HIV plasma viraemia (pV) and to determine the factors associated with virological rebound (VR) in HIV-infected adults on protease inhibitor (PI)-sparing highly active antiretroviral therapy (HAART). The development of resistance mutations during virologically successful therapy and VR was also analysed. Materials and methods : One hundred and twenty-six HIV-infected adults on PI-sparing HAART were prospectively followed from April 1998 to December 2002: Group 1 , naive for antiretroviral drugs ( n = 26); Group 2, previously PI-HAART-exposed patients ( n = 19); Group 3 , previously exposed to suboptimal therapy ( n = 81). Genotypic resistance tests on peripheral blood mononuclear cells or on plasma RNA (when feasible) were carried out when undetectable HIV pV was demonstrated for at least 48 weeks. Additionally, patients showing a therapy adherence >95% developing VR were also tested at rebound, at simplification and during previous suboptimal therapy exposure. Results : The median follow-up time was 630 [329–903] days. VR was considered as two consecutive pV levels >50 copies/mL. Twenty-two (17.5%) patients developed VR. Only therapy adherence <95% was independently associated with VR (adjusted hazard ratio: 8.42; 95% CI: 3.33–21.27). Twenty (40%) of the 50 patients with pV < 50 copies/mL for at least 48 weeks showed at least one thymidine-associated mutation (TAM) but none had NNRTI-resistance mutations. Ten (83.3%) of 12 available adherent patients showing VR harboured NNRTI-resistance-associated mutations; 50% of them were considered as wild-type strains at simplification time. However, the TAM number and resistance mutations profile found on suboptimal exposure were very similar to those found at VR on simplification therapy. Conclusions : PI-sparing HAART allows maintenance of successful long-term control of HIV replication, adherence to therapy being the main factor associated with VR. However, a small proportion of patients on simplification regimen may develop VR regardless of therapy compliance. VR on PI-sparing HAART is characterized by the emergence of NNRTI cross-resistance mutations. Finally, TAMs ‘archived’ during previous suboptimal exposures are partially involved in subsequent VR on simplification HAART. Keywords: PI-sparing HAART, simplification therapy, virological rebound, resistance mutations Introduction The introduction of highly active antiretroviral therapy (HAART) including protease inhibitor (PI) drugs and nucleoside reverse transcriptase inhibitors (NRTIs) has dramatically changed the course of human immunodeficiency virus (HIV) infection, reducing mortality and morbidity events associated with this disease. 1,2 These regimens have allowed successful control of HIV replication. However, the burden of toxicity resulting from the use of PI drugs is of concern as it constitutes a threat to the sustained success of HIV treatment. 3 Thus, .................................................................................................................................................................................................................................................................. *Corresponding author. Tel: +34-95-5012391; Fax: +34-95-5012390; E-mail: [email protected] by guest on October 1, 2010jac.oxfordjournals.orgDownloaded from “En las adversidades sale a la luz la virtud” Aristóteles …a Elena, virtuosa en las adversidades