A new and efficient enrichment method for metagenomic sequencing of Monkeypox virus.

BACKGROUND: The methodology described in previous literature for Monkeypox virus (MPXV) sequencing shows low efficiency when using metagenomic approaches. The aim of the present study was to evaluate a new fine-tuned method for extraction and enrichment of genomic MPXV DNA using clinical samples and to compare it to a non-enrichment metagenomic approach. RESULTS: A new procedure that allows sample enrichment in MPXV DNA, avoiding wasting the sequencing capacity in human DNA, was designed. This procedure consisted of host DNA depletion using a saponin/NaCl combination treatment and DNase, together with high g-force centrifugations. After typical quality control, samples using the enrichment method contained around 96% of reads not classified as human DNA, while the non-enrichment protocol showed around 5-10%. When reads not belonging to Orthopoxvirus were removed, enriched samples kept about 50% of the original read counts, while non-enriched ones kept only 2-7%. CONCLUSIONS: Results showed a very significant improvement in sequencing efficiency, increasing the number of reads belonging to MPXV, the depth of coverage and the trustworthiness of the consensus sequences. This, in turn, allows for more samples to be included in a single cartridge, reducing costs and time to diagnosis, which can be very important factors when dealing with a contagious disease.

Implementación generalizada del diagnóstico en un solo paso (DUSP) de la hepatitis C en Galicia: resultados para la reflexión / Generalized implementation of reflex testing of hepatitis C in Galicia: Results for reflection

Introducción: La implementación del diagnóstico en un solo paso (DUSP) de la infección activa por virus de la hepatitis C (VHC) conjuntamente con la incorporación de alertas informativas ha demostrado que reduce de forma significativa, respecto al diagnóstico tradicional, la cifra de pacientes que no eran remitidos para valoración terapéutica. Métodos: A partir de la implementación en 2018 del DUSP en los servicios de microbiología de los hospitales del Servicio Gallego de Salud (SERGAS), se identifican y caracterizan de manera retrospectiva los nuevos diagnósticos de infección activa por VHC. Resultados: En 2018 se identificaron mediante DUSP un total de 258 pacientes con infección activa por VHC desconocida (70,2% hombres, mediana de edad de 52 años) procedentes de consultas de unidades de atención primaria y especializada en un 54,8% y 39,8%, respectivamente, así como de otras localizaciones en un 5,4%. De los 258 pacientes, el 81,0% fueron derivados para valoración terapéutica, con una mediana de 54 días desde su diagnóstico. En el 58,3% de los casos se determinó el DUSP mediante carga viral, el genotipo predominante fue el 1a (30,7%), un 52,1% fue tratado y se observó una respuesta viral sostenida en el 93,7% de estos. Conclusión: La implementación en toda Galicia del DUSP de la hepatitis C conjuntamente con alertas informativas ha permitido obtener, en conjunto, tasas de derivación para tratamiento similares a las obtenidas en otros estudios. Sin embargo, existe una amplia variabilidad entre los distintos centros, que exigen la incorporación de mejoras, como la formación o la utilización de medidas de rescate para su optimización.(AU)

Introduction: The implementation of reflex testing of active hepatitis C virus (HCV) infection, together with the incorporation of informative alerts in the reports, has shown that it significantly reduces the number of patients who were not referred for therapeutic evaluation. Methods: Since the implementation in 2018 of the DUSP in the microbiology services of the Galician Health Service hospitals (SERGAS), new diagnoses of active HCV infection have been retrospectively identified and characterized. Results: In 2018, a total of 258 patients with unknown active HCV infection (70,2% men, middle age 52 years) were identified through by reflex testing from consultations of primary and specialized care units in 54.8% and 39.8% respectively, as well as from other locations by 5.4%. Of the 258 patients, 81.0% were referred for therapeutic evaluation, with a median of 54 days from their diagnosis. In 58.3% of the cases the reflex testing was determined by viral load, the predominant genotype was 1a (30,7%) and 52,1% were treated, observing sustained viral response in 93.7% of these. Conclusion: The generalized implementation of the HCV reflex testing together with informative alerts in Galicia has allowed us to obtain referral rates for treatment similar to those obtained in other studies. However, there is a wide variability between the different centers that require the incorporation of improvements, such as training or the use of rescue measures for optimization.(AU)

Generalized implementation of reflex testing of hepatitis C in Galicia: Results for reflection.

INTRODUCTION: The implementation of reflex testing of active hepatitis C virus (HCV) infection, together with the incorporation of informative alerts in the reports, has shown that it significantly reduces the number of patients who were not referred for therapeutic evaluation. METHODS: Since the implementation in 2018 of the DUSP in the Microbiology Services of the Galician Health Service hospitals (SERGAS), new diagnoses of active HCV infection have been retrospectively identified and characterized. RESULTS: In 2018, a total of 258 patients with unknown active HCV infection (70,2% men, middle age 52 years) were identified through by reflex testing from consultations of primary and specialized care units in 54.8% and 39.8% respectively, as well as from other locations by 5.4%. Of the 258 patients, 81.0% were referred for therapeutic evaluation, with a median of 54 days from their diagnosis. In 58.3% of the cases the reflex testing was determined by viral load, the predominant genotype was 1a (30,7%) and 52,1% were treated, observing sustained viral response (SVR) in 93.7 % of these. CONCLUSION: The generalized implementation of the HCV reflex testing together with informative alerts in Galicia has allowed us to obtain referral rates for treatment similar to those obtained in other studies. However, there is a wide variability between the different centers that require the incorporation of improvements, such as training or the use of rescue measures for optimization.

Safety and Immunogenicity of SARS-CoV-2 vaccines in people with HIV.

OBJECTIVE: To evaluate the safety and the serological response after two doses of mRNA-based SARS-CoV-2 vaccination in people with HIV (PWH). METHODS: Participants were evaluated 4 weeks after the second dose of mRNA-1273 or BNT162b2 vaccine. Tolerability was evaluated with a specific adverse event questionnaire. Patient's sera were analysed using LIAISON SARS-CoV-2 TrimericS IgG (DiaSorin). RESULTS: One-hundred PWH were included, 75% of them men, with a mean age of 44 ±â€Š11 years old, all receiving antiretroviral treatment and mostly with controlled viral loads (98% with HIV RNA <50 copies/ml) and 96% had >200 CD4+/µl. All patients seroconverted after vaccination (antibody concentration ≥33.8 binding antibody units [BAU]/ml). Only 3% of the patients had a low antibody concentration (<520 BAU/ml), whereas 67% of them had concentrations above the assay's detection range (>2080 BAU/ml). Fifty-six patients had local or systemic symptoms, with mild arthromyalgia being the most common systemic symptom. No severe adverse events were reported. CONCLUSIONS: Vaccination with two doses of mRNA-1273 or BNT162b2 is well tolerated in PWH under effective antiretroviral treatment and it leads to a successful antibody response.

Resistance-associated substitutions after sofosbuvir/velpatasvir/voxilaprevir triple therapy failure.

Direct-acting antivirals (DAAs) resolve chronic HCV infection in >95% of patients, but a small percentage do not respond to DAA-based therapy. These may be difficult to treat because of resistance-associated substitutions (RAS) emerging after treatment failure. Triple therapy with sofosbuvir (SOF)/velpatasvir (VEL)/voxilaprevir (VOX) is the recommended retreatment after DAA-based failure. However, in rare cases, failure to triple therapy occurs, and there is little information characterizing the viruses that relapse. To determine the RAS profile after failing SOF/VEL/VOX, and seek suitable alternatives for retreatment, samples from 5 patients were analysed using MiSeq Illumina deep sequencing before and after triple therapy. All patients were men, aged 59-78 years, 2 HCV genotype (G) 1b and 3 G3a. The most prevalent NS3 substitutions after SOF/VEL/VOX failure were Y56F and A166T. Four patients had the NS5A RAS, Y93H, after triple failure, and Y93H was observed in both G1b patients before retreatment and after SOF/ledipasvir failure. In 2 G3a patients, Y93H appeared at triple failure, and on the other G3a, A30K persisted in 100% of viral genomes. Finally, G1b patients showed C316N in NS5B, associated with SOF failure, but G3a patients had no known NS5B substitutions. HCV RAS analysis identified the following substitutions present at higher rates after triple failure: Y56F in NS3 (G1b), A166T in NS3 (G3a), A30K or Y93H in NS5A, and C316N in NS5B (G1b). A RAS-based salvage treatment (SOF + glecaprevir/pibrentasvir + RBV) was successfully used in one G3a patient.

Generalized implementation of reflex testing of hepatitis C in Galicia: Results for reflection. / Implementación generalizada del diagnóstico en un solo paso (DUSP) de la hepatitis C en Galicia: resultados para la reflexión.

INTRODUCTION: The implementation of reflex testing of active hepatitis C virus (HCV) infection, together with the incorporation of informative alerts in the reports, has shown that it significantly reduces the number of patients who were not referred for therapeutic evaluation. METHODS: Since the implementation in 2018 of the DUSP in the microbiology services of the Galician Health Service hospitals (SERGAS), new diagnoses of active HCV infection have been retrospectively identified and characterized. RESULTS: In 2018, a total of 258 patients with unknown active HCV infection (70,2% men, middle age 52 years) were identified through by reflex testing from consultations of primary and specialized care units in 54.8% and 39.8% respectively, as well as from other locations by 5.4%. Of the 258 patients, 81.0% were referred for therapeutic evaluation, with a median of 54 days from their diagnosis. In 58.3% of the cases the reflex testing was determined by viral load, the predominant genotype was 1a (30,7%) and 52,1% were treated, observing sustained viral response in 93.7% of these. CONCLUSION: The generalized implementation of the HCV reflex testing together with informative alerts in Galicia has allowed us to obtain referral rates for treatment similar to those obtained in other studies. However, there is a wide variability between the different centers that require the incorporation of improvements, such as training or the use of rescue measures for optimization.

Direct, indirect and total effectiveness of bivalent HPV vaccine in women in Galicia, Spain.

Bivalent human papillomavirus (HPV) vaccine was incorporated into the childhood vaccination calendar in Galicia, Spain in 2008. The objectives of this study were to estimate direct, indirect and total effectiveness of HPV vaccine and to identify sexual habits changes in the post-vaccination period in Galicia, Spain.Endocervical scrapings of 745 women attending 7 Health Areas of the Galician Public Health Service were collected in the post-vaccination period, from 2014-2017. Two groups were studied: women born between 1989 and 1993 (n = 397) and women born in 1994 or later (n = 348). Twelve high-risk human papillomavirus (HR-HPV) genotypes were detected by Cobas® 4800 HPV test (Roche Diagnostics, Mannheim, Germany). The Linear Array® HPV Genotyping Test (Roche Diagnostics) was used for HR-HPV genotype detection other than HPV 16/18. Information about sexual habits was collected by a self-filled questionnaire. Post-vaccination data were compared to previously published pre-vaccination data obtained between 2008 and 2010 in Galicia from women of the same age (18-26 years old, n = 523). The Stata 14.2 software was employed for statistical analyses.Data from 392 unvaccinated and 353 vaccinated women were compared. For unvaccinated and vaccinated women, HPV 16/18 prevalence was 9.2% and 0.8%, respectively, and HPV 31/33/45 prevalence was 8.4% and 1.1%, respectively. Direct, indirect and total effectiveness of the HPV vaccine were (%, 95% CI): 94 (72-99), 30 (-11-56) and 95 (79-99), respectively, for HPV 16/18 and 83 (46-94), -10 (-88-33) and 84 (54-94), respectively, for HPV 31/33/45. The number of women with first intercourse before 17 years old and 3 or more sexual partners along life was higher in the post-vaccination period (p < 0.05). A positive impact of bivalent HPV vaccine was observed, both on direct and cross protection. Sexual habits could have changed in the post-vaccination period.

Any impact of blips and low-level viraemia episodes among HIV-infected patients with sustained virological suppression on ART?

OBJECTIVES: The objective of this study was to evaluate the prevalence of blips and risk of virological failure (VF) among HIV-infected patients with sustained virological suppression (HIV-RNA <50 copies/mL) on ART. METHODS: Newly diagnosed (2004-13) HIV-infected patients with sustained virological suppression on ART (minimum follow-up of 3 months) were identified. Risk of VF was evaluated according to different plasma HIV-RNA quantification values based on the limits of quantification/detection of current commercial assays (20 copies/mL). Kaplan-Meier and Cox proportional hazards models were used to compare the cumulative incidence of VF. RESULTS: A total of 565 newly diagnosed HIV-infected patients were identified: 453 started ART and 354 achieved virological suppression. Prevalence of blips (isolated HIV-RNA ranging from 50 to 200 copies/mL) and VF (HIV-RNA ≥50 copies/mL) was 22.7% and 8.8%, respectively (mean follow-up of 42 months). Multivariate analysis identified differences between HIV-RNA values as an independent predictor of VF (P = 0.008); risk of VF was higher for patients with blips [HR 2.500 (95% CI 0.524-11.926)] and for those with at least three consecutive detected, but not quantified, HIV-RNA determinations (HIV-RNA <20 copies/mL) [HR 3.813 (95% CI 0.675-21.535)]. Moreover, only HIV-infected patients with at least three consecutive detected, but not quantified, HIV-RNA determinations showed a higher probability of virological rebound with >200 copies/mL [33.7% at 24 and 60 months versus <5% for other HIV-RNA values; HR 6.943 (0.728-66.261), P = 0.092]. CONCLUSIONS: Blips are frequent (22.7%) among HIV-infected patients with sustained virological suppression on ART. HIV patients with blips and at least three consecutive detected, but not quantified, HIV-RNA determinations (<20 copies/mL) had a higher risk of VF. These findings highlight the relevance of maintaining HIV-RNA levels below the limits of quantification of current assays (<20 copies/mL).

Molecular characterization of HIV-1 infection in Northwest Spain (2009-2013): Investigation of the subtype F outbreak.

BACKGROUND: HIV-1 subtype B is the predominant one in European regions several, while other subtypes and recombinants are also circulating with high prevalence. A sub-epidemic of subtype F with specific characteristics and low response to treatment has been recently identified in Galicia. In this study we investigated the characteristics of the HIV-1 subtype F sub-epidemic in A Coruña and Santiago de Compostela in Northwest Spain. METHODS: 420 newly HIV-1 diagnosed patients during 2009-2013 were enrolled in this study. HIV-1 subtyping was carried out using automated subtyping tools and phylogenetic analysis. Molecular epidemiology investigation of subtypes B and F was performed by means of phylogenetic analysis using fast maximum likelihood. Phylodynamic analysis was performed using Bayesian method as implemented in BEAST v1.8. RESULTS: Subtype B found to be the predominant (61.2% and 70.4%) followed by subtype F (25.6% and 12.0%) in both areas (A Coruña and Santiago de Compostela, respectively). The latter found to mainly spread among men having sex with men (MSM). The vast majority of subtype F lineages from both areas clustered monophyletically, while subtype B sequences clustered in several tree branches. The exponential growth of subtype F sub-epidemic dated back in 2008 by means of phylodynamic analysis. Most of new infections during 2009-2013 occurred within the subtype F transmission cluster. CONCLUSIONS: Subtype F circulates at high prevalence in A Coruña and Santiago de Compostela in Northwest Spain, suggesting that the HIV-1 epidemic in this region has distinct characteristics to the rest of Spain. Subtype F has being spreading among MSM and is currently the most actively spreading network. The single cluster spread of this local sub-epidemic might provide an explanation for the distinct characteristics and the low response to antiretroviral treatment.

Seroprevalence of HCV and HIV infections by year of birth in Spain: impact of US CDC and USPSTF recommendations for HCV and HIV testing.

BACKGROUND: The US Centers for Disease Control and Prevention (CDC) recently add the advice of one-time testing of HCV infection in persons born during 1945-1965. Moreover, the US Preventive Services Task Force (USPSTF) newly recommended one-time HIV testing for persons aged 15-65. Herein, we evaluate the potential impact of these recommendations in a reference medical area of Spain. METHODS: All assays results entries for HCV and HIV serological markers ordered at a reference lab from primary care and specialized physicians between 2008 and 2012 were recorded in a medical area which covers 501,526 citizens in Northern Spain. The year of birth were also documented. RESULTS: A total of 108,159 anti-HCV-Ab results were generated during the study period. The global rate of anti-HCV-Ab+ was 7.7% (95% CI: 7.6%-7.9%), being more prevalent in men than women (8.6% vs. 4.5%). By year of birth, the highest prevalence was found in persons born between 1955 and 1970. HCV genotype 1 was the most prevalent (59.7%) followed by genotype 3 (22.7%). Regard HIV infection, among 65,279 anti-HIV results generated the prevalence of anti-HIV+ was 1.1% (95% CI: 1.0%-1.2%), being more frequent in men (2% vs 0.5%). The years of birth with highest rates of HIV infection exactly match with those for HCV infection. CONCLUSIONS: The highest rates of HCV and HIV infections are found between 1960 and 1965. Different historical and social circumstances such as the huge intravenous drug use epidemic in the eighties in Spain, might explain it. Therefore, each country needs to determine its own HCV and HIV seroprevalences by year of birth to establish the proper recommendations for the screening of both infections.

High prevalence of subtype F in newly diagnosed HIV-1 persons in northwest Spain and evidence for impaired treatment response.

HIV-1 non-B subtype variants were found in 37.8% of 296 newly diagnosed persons in northwest Spain over the past 5 years. Subtype F was the most prevalent non-B subtype (29.6%) and displayed preferential transmission among MSM. Virologic response rates to antiretroviral therapy were lower among F subtypes compared to B subtypes at weeks 24 (31% vs. 78.3%), 48 (51.7% vs. 85.2%), and 96 (61.1% vs. 94.3%) of therapy. Subtype F was independently associated with virological response at 24 weeks.

Resistance to the most recent protease and non-nucleoside reverse transcriptase inhibitors across HIV-1 non-B subtypes.

OBJECTIVES: Limited data are available on resistance to etravirine, rilpivirine, darunavir and tipranavir in patients infected with HIV-1 non-B subtypes, in which natural polymorphisms at certain positions could influence the barrier and/or pathways to drug resistance. METHODS: FASTA format sequences from the reverse transcriptase and protease genes recorded within the Spanish Drug Resistance database (ResRIS) were examined. RESULTS: From 8272 genotypes derived from 5930 different HIV-1 patients included in ResRIS, 5276 genotypes had complete treatment information. Overall, 85% were from antiretroviral-experienced subjects and 7.5% belonged to HIV-1 non-B subtypes: CRF02_AG, C, F and G being the most prevalent variants. For etravirine, only G190A was more prevalent in B than non-B subtypes, whereas V90I and V179E were more frequent in non-B than B subtypes. For rilpivirine, V108I and Y188I were more frequent in B than non-B subtypes, whereas V90I was more prevalent in non-B subtypes. Despite these differences, the overall prevalence of resistance did not differ significantly when comparing etravirine or rilpivirine in B versus non-B subtypes (11.3% versus 7.4%, P = 0.13, and 10.5% versus 7.4%, P = 0.23, respectively). Despite more frequent natural polymorphisms in non-B than B subtypes at tipranavir resistance positions, the prevalence of tipranavir resistance was greater in B than non-B subtypes (11% versus 4.3%, P = 0.004), reflecting a greater antiretroviral exposure in the former. Darunavir resistance did not differ significantly when comparing B and non-B subtypes (5.8% versus 5.5%, P = 0.998). CONCLUSIONS: The rate of resistance to the most recently approved protease and non-nucleoside reverse transcriptase inhibitors is low in antiretroviral-experienced patients, regardless of the HIV-1 subtype.

Clinical, virological and biochemical evidence supporting the association of HIV-1 reverse transcriptase polymorphism R284K and thymidine analogue resistance mutations M41L, L210W and T215Y in patients failing tenofovir/emtricitabine therapy.

BACKGROUND: Thymidine analogue resistance mutations (TAMs) selected under treatment with nucleoside analogues generate two distinct genotypic profiles in the HIV-1 reverse transcriptase (RT): (i) TAM1: M41L, L210W and T215Y, and (ii) TAM2: D67N, K70R and K219E/Q, and sometimes T215F. Secondary mutations, including thumb subdomain polymorphisms (e.g. R284K) have been identified in association with TAMs. We have identified mutational clusters associated with virological failure during salvage therapy with tenofovir/emtricitabine-based regimens. In this context, we have studied the role of R284K as a secondary mutation associated with mutations of the TAM1 complex. RESULTS: The cross-sectional study carried out with > 200 HIV-1 genotypes showed that virological failure to tenofovir/emtricitabine was strongly associated with the presence of M184V (P < 10-10) and TAMs (P < 10-3), while K65R was relatively uncommon in previously-treated patients failing antiretroviral therapy. Clusters of mutations were identified, and among them, the TAM1 complex showed the highest correlation coefficients. Covariation of TAM1 mutations and V118I, V179I, M184V and R284K was observed. Virological studies showed that the combination of R284K with TAM1 mutations confers a fitness advantage in the presence of zidovudine or tenofovir. Studies with recombinant HIV-1 RTs showed that when associated with TAM1 mutations, R284K had a minimal impact on zidovudine or tenofovir inhibition, and in their ability to excise the inhibitors from blocked DNA primers. However, the mutant RT M41L/L210W/T215Y/R284K showed an increased catalytic rate for nucleotide incorporation and a higher RNase H activity in comparison with WT and mutant M41L/L210W/T215Y RTs. These effects were consistent with its enhanced chain-terminated primer rescue on DNA/DNA template-primers, but not on RNA/DNA complexes, and can explain the higher fitness of HIV-1 having TAM1/R284K mutations. CONCLUSIONS: Our study shows the association of R284K and TAM1 mutations in individuals failing therapy with tenofovir/emtricitabine, and unveils a novel mechanism by which secondary mutations are selected in the context of drug-resistance mutations.

[Prevalence of antiretroviral drug resistance among previously untreated Spanish patients infected with HIV]. / Prevalencia de resistencia a los fármacos antirretrovirales en pacientes españoles infectados por el VIH y sin tratamiento previo.

INTRODUCTION: Genotypic resistance was tested to investigate changes in the rates of resistance to antiretroviral drugs in non-treated patients in Spain. MATERIAL AND METHOD: A total of 209 HIV-infected patients from different autonomous communities in Spain without prior antiretroviral (AR) drug treatment were studied from 1997 to 2001. Regions of the HIV pol gene coding for protease (PR) and retrotranscriptase (RT) were sequenced in plasma samples by RT PCR amplification and automated PCR sequencing. RESULTS: At least one primary RT or PR mutation was detected in 14 patients (6.7%); 11 of them were associated with resistance to RT inhibitors (5.3%) and 3 to PR inhibitors (1.4%). The changes in the resistance rate between March 1997-February 1999 and March 1999-February 2001 were as follows: resistance mutations were detected in 3 of the 111 patients studied in the first period, and in 10 of 98 patients in the second period (2.7% versus 10.2%, P = 0.025). The infection time was less than three months in 1.5% of cases, less than 1 year in 13.4%, more than 1 year in 45.9% and unknown in 39.2%. CONCLUSION: The rate of primary resistance in naive patients is low in Spain, although there may be a trend toward an increase. The rising prevalence of resistance is a cause for concern.

Prevalencia de resistencia a los fármacos antirretrovirales en pacientes españoles infectados por el VIH y sin tratamiento previo / Prevalence of antiretroviral drug resistance among naive Spanish patients infected with HIV

Introducción. Para conocer la evolución de la tasa de resistencia a los antirretrovirales en España en pacientes no tratados previamente, estudiamos la resistencia genotípica. Material y método. Se estudiaron muestras plasmáticas de 209 pacientes infectados por el virus de la inmunodeficiencia humana (VIH), procedentes de diversas comunidades autónomas, que no habían recibido tratamiento antirretroviral desde 1997 a 2001. Analizamos la secuencia del gen pol que codifica la proteasa (PR) y la retrotranscriptasa (RT) del VIH usando un sistema de secuenciación automático. Resultados. En 14 pacientes (6,7%) se detectó al menos una mutación en la RT o mutación primaria en la PR. En 11 pacientes, las mutaciones se asociaron a resistencia a los inhibidores de la RT (5,3%) y en tres a los inhibidores de la PR (1,4%). La evolución de la tasa de resistencia entre marzo de 1997 a febrero de 1999 y de marzo de 1999 a febrero de 2001 fue la siguiente: se detectaron mutaciones codificadoras de resistencias en 3 de los 111 pacientes estudiados en el primer período mientras que se identificaron en 10 casos de 98 pacientes en el segundo período (2,7% frente a 10,2%; p = 0,025). En el 1,5% de los casos, el tiempo de la infección fue inferior a 3 meses, en el 13,4% 1 año y en el 39,2% de duración desconocida. Conclusión. El porcentaje de pacientes no tratados previamente con resistencia primaria en España es bajo pero preocupa la probable tendencia a su incremento (AU)

Introduction. Genotypic resistance was tested to investigate changes in the rates of resistance to antiretroviral drugs in non-treated patients in Spain. Material and method. A total of 209 HIV-infected patients from different autonomous communities in Spain without prior antiretroviral (AR) drug treatment were studied from 1997 to 2001. Regions of the HIV pol gene coding for protease (PR) and retrotranscriptase (RT) were sequenced in plasma samples by RT PCR amplification and automated PCR sequencing. Results. At least one primary RT or PR mutation was detected in 14 patients (6.7%); 11 of them were associated with resistance to RT inhibitors (5.3%) and 3 to PR inhibitors (1.4%). The changes in the resistance rate between March 1997-February 1999 and March 1999-February 2001 were as follows: resistance mutations were detected in 3 of the 111 patients studied in the first period, and in 10 of 98 patients in the second period (2.7% versus 10.2%, P = 0.025). The infection time was less than three months in 1.5% of cases, less than 1 year in 13.4%, more than 1 year in 45.9% and unknown in 39.2%. Conclusion. The rate of primary resistance in naive patients is low in Spain, although there may be a trend toward an increase. The rising prevalence of resistance is a cause for concern (AU)