Antonio Aguilera

Evaluation of Eight Different Bioinformatics Tools To Predict Viral Tropism in Different Human Immunodeficiency Virus Type 1 Subtypes

ABSTRACT Human immunodeficiency virus type 1 (HIV-1) tropism can be assessed using phenotypic assays, but this is quite laborious, expensive, and time-consuming and can be made only in sophisticated laboratories. More accessible albeit reliable tools for testing of HIV-1 tropism are needed in view of the prompt introduction of CCR5 antagonists in clinical practice. Bioinformatics tools based on V3 sequences might help to predict HIV-1 tropism; however, most of these methods have been designed by taking only genetic information derived from HIV-1 subtype B into consideration. The aim of this study was to evaluate the performances of several genotypic tools to predict HIV-1 tropism in non-B subtypes, as data on this issue are scarce. Plasma samples were tested using a new phenotypic tropism assay (Phenoscript-tropism; Eurofins), and results were compared with estimates of coreceptor usage using eight different genotypic predictor softwares (Support Vector Machine [SVM], C4.5, C4.5 with positions 8 to 12 only, PART, Charge Rule, geno2pheno coreceptor, Position-Specific Scoring Matrix X4R5 [PSSMX4R5], and PSSMsinsi). A total of 150 samples were tested, with 115 belonging to patients infected with non-B subtypes and 35 drawn from subtype B-infected patients, which were taken as controls. When non-B subtypes were tested, the concordances between the results obtained using the phenotypic assay and distinct genotypic tools were as follows: 78.8% for SVM, 77.5% for C4.5, 82.5% for C4.5 with positions 8 to 12 only, 82.5% for PART, 82.5% for Charge Rule, 82.5% for PSSMX4R5, 83.8% for PSSMsinsi, and 71.3% for geno2pheno. When clade B viruses were tested, the best concordances were seen for PSSMX4R5 (91.4%), PSSMsinsi (88.6%), and geno2pheno (88.6%). The sensitivity for detecting X4 variants was lower for non-B than for B viruses, especially in the case of PSSMsinsi (38.4% versus 100%, respectively), SVMwetcat (46% versus 100%, respectively), and PART (30% versus 90%, respectively). In summary, while inferences of HIV-1 coreceptor usage using genotypic tools seem to be reliable for clade B viruses, their performances are poor for non-B subtypes, in which they particularly fail to detect X4 variants.

Design and validation of new genotypic tools for easy and reliable estimation of HIV tropism before using CCR5 antagonists

BACKGROUND Genotypic tools may allow easier and less expensive estimation of HIV tropism before prescription of CCR5 antagonists compared with the Trofile assay (Monogram Biosciences, South San Francisco, CA, USA). METHODS Paired genotypic and Trofile results were compared in plasma samples derived from the maraviroc expanded access programme (EAP) in Europe. A new genotypic approach was built to improve the sensitivity to detect X4 variants based on an optimization of the webPSSM algorithm. Then, the new tool was validated in specimens from patients included in the ALLEGRO trial, a multicentre study conducted in Spain to assess the prevalence of R5 variants in treatment-experienced HIV patients. RESULTS A total of 266 specimens from the maraviroc EAP were tested. Overall geno/pheno concordance was above 72%. A high specificity was generally seen for the detection of X4 variants using genotypic tools (ranging from 58% to 95%), while sensitivity was low (ranging from 31% to 76%). The PSSM score was then optimized to enhance the sensitivity to detect X4 variants changing the original threshold for R5 categorization. The new PSSM algorithms, PSSM(X4R5-8) and PSSM(SINSI-6.4), considered as X4 all V3 scoring values above -8 or -6.4, respectively, increasing the sensitivity to detect X4 variants up to 80%. The new algorithms were then validated in 148 specimens derived from patients included in the ALLEGRO trial. The sensitivity/specificity to detect X4 variants was 93%/69% for PSSM(X4R5-8) and 93%/70% for PSSM(SINSI-6.4). CONCLUSIONS PSSM(X4R5-8) and PSSM(SINSI-6.4) may confidently assist therapeutic decisions for using CCR5 antagonists in HIV patients, providing an easier and rapid estimation of tropism in clinical samples.

Rilpivirine Resistance Mutations in HIV Patients Failing Non-Nucleoside Reverse Transcriptase Inhibitor-Based Therapies

Objective:Rilpivirine (RPV) is the latest approved nonnucleoside reverse transcriptase inhibitor (NNRTI). It displays in-vitro activity extending over other NNRTI-resistant HIV strains. There is scarce information about the rate of RPV resistance-associated mutations (RAMs) in patients failing other NNRTIs. Methods:RPV RAMs were examined in plasma samples collected from HIV patients that had recently failed NNRTI-based regimens at 22 clinics in Spain. Results:Resistance tests from a total of 1064 patients failing efavirenz (EFV) (54.5%), nevirapine (NVP) (40%) or etravirine (ETR) (5.5%) were examined. The prevalence of RPV RAMs was K101E (9.1%), K101P (1.4%), E138A (3.9%), E138G (0.3%), E138K (0.3%), E138Q (0.8%), V179L (0.2%), Y181C (21.8%), Y181I (0.5%), Y181V (0.2%), H221Y (8.3%), F227C (0.1%) and M230L (1.5%). K101E/M184I was seen in 1%. E138K/M184I were absent. Mutations L100I and V108I were significantly more frequent in patients failing EFV than NVP (7.9 vs. 0.2 and 12.2 vs. 7.3%, respectively). Conversely, Y181C, Y181I, V106A, H221Y and F227L were more prevalent following NVP than EFV failures. Using the Spanish resistance interpretation algorithm, 206 genotypes (19.3%) from patients failing NNRTI (NVP 52%, EFV 40.8% and ETR 7.8%) were considered as RPV resistant. In patients with ETR failure, cross-resistance to RPV was seen in 27.6%, mainly as result of Y181C (81.3%), V179I (43.8%), V90I (31.3%) and V108I (18.8%). Conclusion:RPV resistance is overall recognized in nearly 20% of patients failing other NNRTIs. It is more common following ETR (27.6%) or NVP (25%) failures than EFV (14.5%). E138 mutants are rarely seen in this context.

Diarrea por adenovirus y astrovirus en pacientes inmunodeficientes hospitalizados

Fundamento La diarrea, aguda o cronica, es una complicacion comun en los pacientes inmunodeprimidos tales como los infectados por el virus de la inmunodeficiencia humana (VIH), sometidos a trasplantes de medula osea o de organo solido y aquellos con leucemias u otras alteraciones del sistema inmunitario. Dada la importancia de reconocer las posibles etiologias de la diarrea a al ahora de administrar una terapia antimicrobiana especifica o evitar un diagnostico incorrecto de rechazo postrasplante, hemos investigado la presencia de antigeno de astrovirus y adenovirus en las heces de pacientes inmunodeprimidos. Pacientes Y METodos Se analizaron las heces de 258 pacientes inmunodeprimidos hospitalizados en el Complejo Hospitalaria Universitario de Santiago de Compostela y diagnosticados de diarrea durante 1997- 99. La deteccion de los antigenos viricos se realizo mediante enzimoinmunoanalisis (EIA). Tambien se analizaron para otros enteropatogenos comunes. Resultados El antigeno de adenovirus fue positivo en 5 casos (2%) y el astrovirus en 12 (5%). Los pacientes mas frecuentemente afectados fueron los prematuros y los hematologicos. Se detecto antigeno de astrovirus en tres pacientes infectados por VIH. La mayoria de los casos positivos presentaban una alteracion de la flora intestinal o presencia de toxina de Clostridium difficile, ambas situaciones relacionadas con tratamiento antibiotico prolongado. Conclusiones Los astrovirus y los adenovirus son enteropatogenos a considerar en individuos inmunocomprometidos hospitalizados. Es, pues, conveniente realizar un diagnostico certero de la etiologia de la diarrea de cara a la administracion de un tratamiento antimicrobiano, en los casos en que este sea necesario, o evitar un diagnostico incorrecto de rechazo postrasplante.

Human Immunodeficiency Virus Type 2 Infection in Spain

The first cases of human immunodeficiency virus type 2 (HIV-2) infection in Spain were identified in 1988, in 3 African immigrants living in Barcelona. Since then, up to December 1998, 92 individuals with HIV-2 infection have been reported in Spain. Most are adult men, infected through heterosexual contacts, originating from West African countries, and currently living in the largest urban Spanish cities. Fifteen individuals have developed AIDS, meanwhile the rest remain asymptomatic. For 22 subjects, HIV-2 subtyping was performed on proviral DNA, 16 being infected with subtype A (8 Spanish born and 8 African immigrants) and the remaining with subtype B (two Spanish born and 4 originating from Equatorial Guinea). Coinfection with HIV-1 was demonstrated in 9 individuals. In conclusion, HIV-2 is currently circulating in Spain with a low prevalence and without evidence for increase over time.

Prevalence of HTLV infection in pregnant women in Spain

Objective: To estimate the prevalence of HTLV infection among pregnant women in Spain. Methods: A commercial ELISA incorporating HTLV-I and HTLV-II antigens was used for HTLV antibody screening. Repeatedly reactive samples were further examined by western blot. Moreover, confirmation with PCR was performed when cells were available. Results: 20 366 pregnant women in 12 different Spanish cities were tested in a 3 year period (July 1996 to August 1999). 32 samples were repeatedly reactive by ELISA, and 10 of them were confirmed as positive by western blot (eight for HTLV-II and two for HTLV-I). In addition, three of 13 women who had an indeterminate western blot pattern yielded positive results for HTLV-II by PCR. All 11 HTLV-II infected women had been born in Spain, and all but one were former drug users. Seven of them were coinfected with HIV-1. One HTLV-I infected woman was from Peru, where HTLV is endemic and where she most probably was infected during sexual intercourse. Conclusion: The overall prevalence of HTLV infection among pregnant women in Spain is 0.064% (13/20 366), and HTLV-II instead of HTLV-I is the most commonly found variant. A strong relation was found among HTLV-II infection and specific epidemiological features, such as Spanish nationality and injecting drug use. Although HTLV-II can be vertically transmitted, mainly through breast feeding, both the low prevalence of infection and its lack of pathogenicity would not support the introduction of HTLV antenatal screening in Spain.

Trends in the prevalence and distribution of HTLV-1 and HTLV-2 infections in Spain

BackgroundAlthough most HTLV infections in Spain have been found in native intravenous drug users carrying HTLV-2, the large immigration flows from Latin America and Sub-Saharan Africa in recent years may have changed the prevalence and distribution of HTLV-1 and HTLV-2 infections, and hypothetically open the opportunity for introducing HTLV-3 or HTLV-4 in Spain. To assess the current seroprevalence of HTLV infection in Spain a national multicenter, cross-sectional, study was conducted in June 2009.ResultsA total of 6,460 consecutive outpatients attending 16 hospitals were examined. Overall, 12% were immigrants, and their main origin was Latin America (4.9%), Africa (3.6%) and other European countries (2.8%). Nine individuals were seroreactive for HTLV antibodies (overall prevalence, 0.14%). Evidence of HTLV-1 infection was confirmed by Western blot in 4 subjects (prevalence 0.06%) while HTLV-2 infection was found in 5 (prevalence 0.08%). Infection with HTLV types 1, 2, 3 and 4 was discarded by Western blot and specific PCR assays in another two specimens initially reactive in the enzyme immunoassay. All but one HTLV-1 cases were Latin-Americans while all persons with HTLV-2 infection were native Spaniards.ConclusionsThe overall prevalence of HTLV infections in Spain remains low, with no evidence of HTLV-3 or HTLV-4 infections so far.

Drug resistance mutations in patients infected with HIV-2 living in Spain

BACKGROUND In contrast with HIV-1, information about drug resistance in HIV-2 is scarce and mainly derived from small series of patients failing antiretroviral therapy. METHODS The spectrum of changes in the reverse transcriptase (RT), protease (PR) and integrase (INT) genes was examined in HIV-2 individuals enrolled in the HIV-2 Spanish register. RESULTS From a total of 236 HIV-2-infected individuals registered in Spain from 1989 to June 2010, 53 PR, 44 RT and 8 INT sequences were obtained. Low plasma viraemia precluded collection of this information from most of the remaining cases. No major mutations associated with drug resistance in HIV-1 were recognized in 29 PR, 20 RT and 5 INT sequences from antiretroviral-naive HIV-2 individuals, although natural polymorphisms with potential effects on susceptibility to PR inhibitors were recognized at 10 positions (L10V/I, V32I, M36I, M46I, I47V, Q58E, A71V/I, G73A, V82I and L89I/V) and for nucleoside reverse transcriptase inhibitors at three positions (T69N, V75I and K219E). In 24 antiretroviral-experienced patients with virological failure the most frequent major RT resistance mutations were M184V (58%), Q151M (33%) and K65R (21%), which are rarely seen thymidine analogue mutations. In PR the most frequent major changes were V47A (17%), I54M (17%), I82F (13%), L90M (29%) and L99F (29%). Two of the three patients who failed on raltegravir had N155H in the INT region. CONCLUSIONS Drug resistance mutations in HIV-2 are selected at the same positions as in HIV-1, although with different frequency. Polymorphisms in the RT and PR associated with drug resistance in HIV-1 as compensatory changes are common in untreated HIV-2 subjects. These findings highlight the need for specific guidelines for interpreting genotypic resistance patterns in HIV-2 infection.

Infecciones por VIH-2 y HTLV-I/II en España

Hasta diciembre de 2002, se han identificado en Espana un total de 56, 566 y 109 casos de infeccion por los virus de la leucemia humana T tipos I y II (HTLV-I, HTLV-II) y de la inmunodeficiencia humana tipo 2 (VIH-2), respectivamente. La mayor parte de los sujetos infectados por VIH-2 y HTLV-I corresponden a inmigrantes procedentes de zonas endemicas, o espanoles que han viajado a aquellas regiones o que han mantenido relaciones sexuales con oriundos de ellas. Por el contrario, la infeccion por el HTLV-II predomina entre espanoles adictos a drogas por via parenteral (ADVP) que con frecuencia estan coinfectados por el VIH-1. Entre los sujetos infectados por el HTLV-I, 12 pacientes han desarrollado mielopatia subaguda y cuatro leucemia de celulas T del adulto. Tan solo 20 (18,3%) de los pacientes infectados por el VIH-2 han desarrollado sida. No se ha observado un incremento en la incidencia de la infeccion por el VIH-2 y el HTLV-I en estos anos. Por el contrario, la infeccion por el HTLV-II se ha extendido rapidamente en el colectivo de pacientes infectados por el VIH-1 adictos a drogas por via parenteral (ADVP) en prisiones con una prevalencia del 18% en determinadas carceles espanolas. No obstante, la prevalencia de dicha infeccion sigue siendo baja fuera del ambito carcelario entre los pacientes infectados por el VIH-1 ADVP (4,7%).

Using NS5B Sequencing for Hepatitis C Virus Genotyping Reveals Discordances with Commercial Platforms

We aimed to evaluate the correct assignment of HCV genotypes by three commercial methods—Trugene HCV genotyping kit (Siemens), VERSANT HCV Genotype 2.0 assay (Siemens), and Real-Time HCV genotype II (Abbott)—compared to NS5B sequencing. We studied 327 clinical samples that carried representative HCV genotypes of the most frequent geno/subtypes in Spain. After commercial genotyping, the sequencing of a 367 bp fragment in the NS5B gene was used to assign genotypes. Major discrepancies were defined, e.g. differences in the assigned genotype by one of the three methods and NS5B sequencing, including misclassification of subtypes 1a and 1b. Minor discrepancies were considered when differences at subtype levels, other than 1a and 1b, were observed. The overall discordance with the reference method was 34% for Trugene and 15% for VERSANT HCV2.0. The Abbott assay correctly identified all 1a and 1b subtypes, but did not subtype all the 2, 3, 4 and 5 (34%) genotypes. Major discordances were found in 16% of cases for Trugene HCV, and the majority were 1b- to 1a-related discordances; major discordances were found for VERSANT HCV 2.0 in 6% of cases, which were all but one 1b to 1a cases. These results indicated that the Trugene assay especially, and to a lesser extent, Versant HCV 2.0, can fail to differentiate HCV subtypes 1a and 1b, and lead to critical errors in clinical practice for correctly using directly acting antiviral agents.