Claudin-6 enhances cell invasiveness through claudin-1 in AGS human adenocarcinoma gastric cancer cells.

Claudins participate in tissue barrier function. The loss of this barrier is associated to metalloproteases-related extracellular matrix and basal membranes degradation. Claudin-1 is a pro-MMP-2 activator and claudin-6 transfected AGS (AGS-Cld6) cells are highly invasive. Our aim was to determine if claudin-6 was direct or indirectly associated with MMP-2 activation and cell invasiveness. Cytofluorometry, cell fractioning, immunoprecipitation, gelatin-zymography, cell migration and invasiveness assays were performed, claudin-2, -6, -7 and -9 transfected AGS cells, anti-MMP-2, -9 and -14, anti-claudins specific antibodies and claudin-1 small interfering RNA were used. The results showed a significant (p<0.001) overexpression of claudin-1 in AGS-Cld6 cell membranes. A strong MMP-2 activity was identified in culture supernatants of AGS-Cld6. Claudin-1 co-localized with MMP-2 and MMP-14; interestingly a significant increase in cell membrane and cytosol MMP-14 expression was detected in AGS-Cld6 cells (p<0.05). Silencing of claudin-1 in AGS-Cld6 cells showed a 60% MMP-2 activity decrease in culture supernatants and a significant decrease (p<0.05) in cell migration and invasiveness. Our results suggest that claudin-6 induces MMP-2 activation through claudin-1 membrane expression, which in turn promotes cell migration and invasiveness.

HIV-1 drug resistance prevalence, drug susceptibility and variant characterization in the Jacobi Medical Center paediatric cohort, Bronx, NY, USA.

OBJECTIVES: With the advent of combined antiretroviral therapy (cART), perinatally HIV-infected children are surviving into adolescence and beyond. However, drug resistance mutations (DRMs) compromise viral control, affecting the long-term effectiveness of ART. The aims of this study were to detect and identify DRMs in a HIV-1 infected paediatric cohort. METHODS: Paired plasma and dried blood spots (DBSs) specimens were obtained from HIV-1 perinatally infected patients attending the Jacobi Medical Center, New York, USA. Clinical, virological and immunological data for these patients were analysed. HIV-1 pol sequences were generated from samples to identify DRMs according to the International AIDS Society (IAS) 2011 list. RESULTS: Forty-seven perinatally infected patients were selected, with a median age of 17.7 years, of whom 97.4% were carrying subtype B. They had a mean viral load of 3143 HIV-1 RNA copies/mL and a mean CD4 count of 486 cells/µL at the time of sampling. Nineteen patients (40.4%) had achieved undetectable viraemia (< 50 copies/mL) and 40.5% had a CD4 count of > 500 cells/µL. Most of the patients (97.9%) had received cART, including protease inhibitor (PI)-based regimens in 59.6% of cases. The DRM prevalence was 54.1, 27.6 and 27.0% for nucleoside reverse transcriptase inhibitors (NRTIs), PIs and nonnucleoside reverse transcriptase inhibitors (NNRTIs), respectively. Almost two-thirds (64.9%) of the patients harboured DRMs to at least one drug class and 5.4% were triple resistant. The mean nucleotide similarity between plasma and DBS sequences was 97.9%. Identical DRM profiles were present in 60% of plasma-DBS paired sequences. A total of 30 DRMs were detected in plasma and 26 in DBSs, with 23 present in both. CONCLUSIONS: Although more perinatally HIV-1-infected children are reaching adulthood as a result of advances in cART, our study cohort presented a high prevalence of resistant viruses, especially viruses resistant to NRTIs. DBS specimens can be used for DRM detection.

Immune surveillance for six vaccinable pathogens using paired plasma and dried blood spots in HIV infected and uninfected children in Kinshasa.

Child vaccination reduces infant mortality rates. HIV-infected children present higher risk of diseases than non-infected. We report the protection coverage rates for 6 vaccine-preventable diseases in a paediatric population from the Democratic Republic of the Congo (DRC) and the impact of HIV infection, providing the first data on the validity of dried blood samples (DBS) to monitor the immune protection. During 2016-2018 DBS from 143 children/adolescents were collected in Kinshasa (DRC), being 52 HIV-infected. Forty-two had a paired plasma sample. Protective IgG was quantified (VirClia-IgG,VIRCELL) to obtain the optimal cut-off in IgG detection in DBS. ROC curves were generated with R software and statistical analyses with Stata. Protective IgG levels varied across pathogens, not reaching herd immunity. HIV-infected presented lower vaccine protection than uninfected for all analyzed pathogens, except rubella, with statistically significant differences for measles (30.8% vs. 53.8%; p = 0.008) and tetanus (3.8% vs. 22%; p = 0.0034). New cut-offs were calculated when using DBS to improve test performance. We reinforce the necessity to increase pediatric vaccination coverage in Kinshasa, especially in HIV seropositive, with less capacity to maintain adequate antibody levels. DBS were useful to monitor vaccination coverage in seroprevalence studies in resource-limited settings, after optimizing the cut-off value for each pathogen.

Two novel recessive mutations in KRT14 identified in a cohort of 21 Spanish families with epidermolysis bullosa simplex.

BACKGROUND: Basal epidermolysis bullosa simplex (EBS) is a group of blistering genodermatoses mostly caused by mutations in the keratin genes, KRT5 and KRT14. Recessive mutations represent about 5% of all EBS mutations, being common and specific in populations with high consanguinity, where affected patients show severe phenotypes. OBJECTIVES: To accomplish the first mutational analysis in patients of Spanish origin with EBS and to delineate a comprehensive genotype-phenotype correlation. METHODS: Twenty-one EBS families were analysed. Immunofluorescence mapping at the dermoepidermal junction level was performed on skin biopsies from patients. Mutation screening of the entire coding sequences of KRT5 and KRT14 in genomic DNA was assessed by polymerase chain reaction and direct sequencing. RESULTS: KRT5 or KRT14 causative mutations were identified in 18 of the 21 EBS families. A total of 14 different mutations were disclosed, of which 12 were dominant missense mutations and two truncating recessive mutations. Five of the 14 mutations were novel including three dominant in KRT5 (p.V186E, p.T321P and p.A428T) and two recessive in KRT14 (p.K116X and p.K250RfsX8). The two patients with EBS carrying homozygous recessive mutations were affected by severe phenotypes and belonged to consanguineous families. All five families with the EBS Dowling-Meara subtype carried recurrent mutations affecting the highly conserved ends of the α-helical rod domain of K5 and K14. The seven mutations associated with the localized EBS subtype were widely distributed along the KRT5 and KRT14 genes. Two families with mottled pigmentation carried the P25L mutation in KRT5, commonly associated with this subtype. CONCLUSIONS: This study further confirms the genotype-phenotype correlation established for EBS in other ethnic groups, and is the first in a Mediterranean country (excluding Israel). This study adds two novel recessive mutations to the worldwide record to date, which includes a total of 14 mutations. As in previous reports, the recessive mutations resulted in a lack of keratin K14, giving rise to a generalized and severe presentation.

Use of CT-scan in pediatric patients with thoraco-abdominal trauma: experience at a latin-american trauma facility. / Uso de la TAC en pacientes pediátricos con trauma toraco-abdominal: experiencia de un centro de trauma latinoamericano.

INTRODUCTION: CT-scan is the method of choice for major trauma assessment. However, it significantly increases radiation exposure in the pediatric population. The objective of this study was to analyze differences in clinical outcomes according to the preoperative use of CT-scan. MATERIALS AND METHODS: A retrospective observational study of pediatric patients admitted for trauma and requiring surgery was carried out. Patients were classified according to the previous use of CT-scan. ICU stay, re-admissions, and deaths were assessed. RESULTS: From 2011 to 2017, 737 patients under 18 years of age with external lesions were treated, 174 of whom required surgery. 48 patients (27.6%) underwent CT-scan prior to the procedure (Group 1), while the remaining 126 patients (72.4%) were directly scheduled for surgery (Group 2). Penetrating trauma occurred in 81% of patients, the proportion being significantly higher in Group 2 (p= 0.001). Median age was 15 years (interquartile range: 12-17), with no differences between groups. No significant differences were found in terms of hemodynamic instability at admission between groups (p= 0.596). At surgery, 3 out of 48 patients (6.3%) had no evident lesion. No significant differences were found in terms of re-admissions (p= 0.476), mortality (0.994), and ICU stay (0.466). CONCLUSION: The use of CT-scan as a diagnostic tool in pediatric trauma does not reduce mortality, ICU stay, or number of re-admissions. The use of tools such as ultrasound examination and simple X-ray should be protocolized to avoid unnecessary exposure to higher radiation doses. Prospective studies confirming this hypothesis are required.

INTRODUCCION: La tomografía axial computarizada (TAC) es el método de elección en la evaluación del trauma mayor, sin embargo, aumenta significativamente la exposición a radiación en la población pediátrica. El objetivo de este estudio es determinar diferencias en los desenlaces clínicos de acuerdo con el uso preoperatorio de la TAC. METODOS: Estudio observacional retrospectivo. Se incluyeron pacientes pediátricos ingresados por trauma que necesitaron manejo quirúrgico, y se clasificaron de acuerdo con el uso previo de TAC. Se evaluó tiempo en Unidad de Cuidados Intensivos (UCI), readmisiones, y muerte. RESULTADOS: Durante 2011 a 2017, 737 pacientes menores de 18 años consultaron por lesiones de causa externa, 174 requirieron intervención quirúrgica. A 48 (27,6%) se les realizó TAC previo al manejo quirúrgico (Grupo 1); los restantes 126 pacientes (72,4%) fueron llevados directamente a cirugía (Grupo 2). El trauma penetrante se presentó en un 81% de los pacientes, siendo significativamente mayor en el grupo 2 (p= 0,001). La mediana de edad fue 15 años (rango intercuartílico 12-17) sin diferencia entre los grupos. No hubo diferencias significativas en inestabilidad hemodinámica al ingreso entre los grupos (p= 0,596). Al momento de la cirugía, tres de 48 pacientes (6,3%) no presentaron ninguna lesión evidente. No hubo diferencias significativas en las readmisiones (p= 0,476), la mortalidad (0.994) y estancia en UCI (0,466). CONCLUSION: El uso de TAC como herramienta diagnóstica en trauma pediátrico no disminuye la mortalidad, días de estancia en UCI, ni el número de readmisiones. Debe protocolizarse el uso de herramientas como la ecografía y radiografía simple para evitar exposición innecesaria a dosis más altas de radiación. Se requieren estudios prospectivos que confirmen esta hipótesis.

The first COL7A1 mutation survey in a large Spanish dystrophic epidermolysis bullosa cohort: c.6527insC disclosed as an unusually recurrent mutation.

BACKGROUND: Dystrophic epidermolysis bullosa (DEB) is a genodermatosis caused by mutations in COL7A1. The clinical manifestations are highly variable from nail dystrophy to life-threatening blistering, making early molecular diagnosis and prognosis of utmost importance for the affected families. Mutation identification is mandatory for prenatal testing. OBJECTIVES: To conduct the first mutational analysis of COL7A1 in a Spanish cohort, to assess mutation consequences at protein/mRNA level and to establish genotype-phenotype correlations. METHODS: Forty-nine Spanish patients with DEB were studied. Antigen mapping was performed on patient skin biopsies. COL7A1 mutation screening in genomic DNA was performed by polymerase chain reaction (PCR) and direct sequencing. Mutation consequences were determined by reverse transcriptase-PCR. RESULTS: Eight patients belonged to three unrelated families with dominant DEB. Forty-one were affected with recessive DEB (RDEB). Specifically, 27 displayed the severe generalized subtype, eight the other generalized subtype and six a localized phenotype (two pretibial, three acral and one inversa). Thirty-five mutations were identified, 20 of which are novel. The pathogenic mutation c.6527insC accounted for 46.3% of Spanish RDEB alleles. A consistent genotype-phenotype correlation was established. CONCLUSIONS: Although the COL7A1 database indicates that most DEB mutations are family specific, the pathogenic mutation c.6527insC was highly recurrent in our cohort. This level of recurrence for a single genetic defect has never previously been reported for COL7A1. Our findings are essential to the clinicians caring for patients with DEB in Spain and in the large population of Spanish descendants in Latin America. They also provide geneticists a molecular clue for a priority mutation screening strategy.

[HIV-1 genetic variability in non Spaniard infected children]. / Identificación de las diferentes variantes geneticas del VIH-1 en niños de procedencia no española.

INTRODUCTION: The prevalence of HIV-1 non-B subtypes (HIV-NBS) is increasing in Europe, because of emigration from countries where genetic variants are endemic. Although HIV-NBS could have a different clinical evolution and could respond differently to antiretrovirals (AR) than B-subtypes, these variant's response remain undocumented. AIMS: To identify HIV-1 genetic variants and to determine clinical evolution in a non-Spaniard children infected with HIV-1. PATIENTS AND METHOD: Children with HIV-1 infection from endemic countries were tested for HIV-1 subtypes between 1-1-1988 and 31-12-2006. Twelve children less than 18 years old and born abroad were selected. RESULTS: HIV-NBS were isolated in 5 children (42%): CRF2_AG recombinant in 3 cases (Equatorial Guinea), Subtype C in one (Equatorial Guinea) and CRF13_cpx in last one (India). DISCUSSION: Because of the increasing frequency of patients with HIV-NBS and their unknown long-term evolution, all children from endemic countries should be tested for HIV subtypes. We believe new studies with more patients during longer times could reveal differences in these patient's clinical, immunological and virological evolution.

Tumor Biology as Predictor of Mortality in Liver Transplantation for Hepatocellular Carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most frequent primary malignant liver tumor, with the Milan criteria considered to be the gold standard for patient selection for liver transplantation (LT). MATERIALS AND METHODS: We performed a descriptive observational study, reviewing 20 years of experience of LT in patients with HCC in the Fundacion Valle del Lilí in Cali, Colombia. Subgroup analysis was undertaken for periods 1999 to 2007 and 2008 to 2015. RESULTS: Fifty-seven cases with a pretransplant HCC diagnosis were reviewed. In the first period patients within the Milan criteria had a recurrence-free survival at 5 years of 66.6%, and in those who exceeded the Milan criteria, recurrence-free survival was 75%. In the second period, patients within the Milan criteria, recurrence-free survival at 5 years was 93.5%, and in those who exceeded the Milan criteria, recurrence-free survival was 75.7%. No statistically significant difference was found in either period. For patients with mild and moderate tumor differentiation, the relapse survival rate at 5 years was 69.4% (95% confidence interval [CI] 35.8-87.8) and 74.7% (95% CI 44.5-90), respectively. All patients with poor tumor differentiation relapsed and died within 3 years. CONCLUSION: Global and recurrence-free survival among patients who met and patients who exceeded the Milan criteria was not significantly different, suggesting an expansion of the Milan criteria to include potential recipients who were previously excluded. Obtaining histologic differentiation and identifying vascular invasion will provide a more worthwhile contribution to LT decision making.

Bayesian network analysis of resistance pathways against HIV-1 protease inhibitors.

Interpretation of Human Immunodeficiency Virus 1 (HIV-1) genotypic drug resistance is still a major challenge in the follow-up of antiviral therapy in infected patients. Because of the high degree of HIV-1 natural variation, complex interactions and stochastic behaviour of evolution, the role of resistance mutations is in many cases not well understood. Using Bayesian network learning of HIV-1 sequence data from diverse subtypes (A, B, C, F and G), we could determine the specific role of many resistance mutations against the protease inhibitors (PIs) nelfinavir (NFV), indinavir (IDV), and saquinavir (SQV). Such networks visualize relationships between treatment, selection of resistance mutations and presence of polymorphisms in a graphical way. The analysis identified 30N, 88S, and 90M for nelfinavir, 90M for saquinavir, and 82A/T and 46I/L for indinavir as most probable major resistance mutations. Moreover we found striking similarities for the role of many mutations against all of these drugs. For example, for all three inhibitors, we found that the novel mutation 89I was minor and associated with mutations at positions 90 and 71. Bayesian network learning provides an autonomous method to gain insight in the role of resistance mutations and the influence of HIV-1 natural variation. We successfully applied the method to three protease inhibitors. The analysis shows differences with current knowledge especially concerning resistance development in several non-B subtypes.

Reacciones adversas a betalactámicos: una revisión de tema / Adverse reactions to beta-lactams: a topic review / Reações adversas a beta-lactâmicos: uma revisão de tópico

Las reacciones adversas a medicamentos son una de las principales causas de muerte en el mundo, producen muchos ingresos hospitalarios y aumentan los costos de atención. Dentro de los medicamentos que más se asocian con estas reacciones están los antibióticos y de estos los más comunes son los betalactámicos, ampliamente utilizados en las instituciones de salud. Las manifestaciones más frecuentes de las reacciones adversas a betalactámicos son alérgicas, dermatológicas, gastrointestinales, renales, hepáticas y neurológicas. Se realiza una revisión general de las reacciones adversas de estos medicamentos, se mencionan los distintos antibióticos betalactámicos con su clasificación y espectro de acción y más precisamente se explican las distintas reacciones adversas por uso de betalactámicos según el sistema comprometido.

Adverse drug reactions are one of the leading causes of death in the world. They are also responsible for an increase in hospital admissions and higher care costs. Among the most associated drugs with these reactions are antibiotics and of these the most common are beta-lactams, which are widely used in health institutions. The most fre-quent manifestations of adverse reactions to beta-lactams are allergic, dermatological, gastrointestinal, renal, hepatic and neurological reactions. A general review of the adverse reactions to these drugs is carried out. Also, the different beta-lactam antibiotics are described along with their classification and spectrum of action, and an accurate explanation of the different adverse reactions due to the use of beta-lactams according to the compromised system is made.

As reações adversas a medicamentos são uma das principais causas de morte no mundo, resultam em muitas admissões hospitalares e aumentam os custos do atendimento. Entre os medicamentos que mais se associam a essas reações estão os antibióticos e, destes, os mais comuns são os beta-lactâmicos, amplamente utilizados em instituições de saúde. As manifestações mais frequentes de reações adversas aos beta-lactâmicos são alérgicas, dermatológicas, gastrointestinais, renais, hepáticas e neurológicas. Faz-se uma revisão geral das reações adversas desses medicamentos, são mencionados os diferentes antibióticos beta-lactâmicos com sua classificação e espectro de ação, e mais precisamente explicam as diferentes reações adversas devidas ao uso de beta-lactâmicos de acordo com o sistema comprometido

Uso de la TAC en pacientes pediátricos con trauma toraco-abdominal: experiencia de un centro de trauma latinoamericano / Use of CT-scan in pediatric patients with thoraco- abdominal trauma: experience at a latin-american trauma facility

Introducción: La tomografía axial computarizada (TAC) es el método de elección en la evaluación del trauma mayor, sin embargo, aumentasignificativamente la exposición a radiación en la población pediátrica.El objetivo de este estudio es determinar diferencias en los desenlacesclínicos de acuerdo con el uso preoperatorio de la TAC. Métodos: Estudio observacional retrospectivo. Se incluyeron pacientes pediátricos ingresados por trauma que necesitaron manejo quirúrgico, y se clasificaron de acuerdo con el uso previo de TAC. Se evaluótiempo en Unidad de Cuidados Intensivos (UCI), readmisiones, y muerte.Resultados: Durante 2011 a 2017, 737 pacientes menores de 18años consultaron por lesiones de causa externa, 174 requirieron intervención quirúrgica. A 48 (27,6%) se les realizó TAC previo al manejoquirúrgico (Grupo 1); los restantes 126 pacientes (72,4%) fueron llevadosdirectamente a cirugía (Grupo 2). El trauma penetrante se presentó enun 81% de los pacientes, siendo significativamente mayor en el grupo2 (p= 0,001). La mediana de edad fue 15 años (rango intercuartílico 12-17) sin diferencia entre los grupos. No hubo diferencias significativasen inestabilidad hemodinámica al ingreso entre los grupos (p= 0,596).Al momento de la cirugía, tres de 48 pacientes (6,3%) no presentaronninguna lesión evidente. No hubo diferencias significativas en las read-misiones (p= 0,476), la mortalidad (0,994) y estancia en UCI (0,466). Conclusión: El uso de TAC como herramienta diagnóstica en trau-ma pediátrico no disminuye la mortalidad, días de estancia en UCI, niel número de readmisiones. Debe protocolizarse el uso de herramientascomo la ecografía y radiografía simple para evitar exposición innecesariaa dosis más altas de radiación. Se requieren estudios prospectivos queconfirmen esta hipótesis.(AU)

Introduction: CT-scan is the method of choice for major traumaassessment. However, it significantly increases radiation exposure in thepediatric population. The objective of this study was to analyze differ-ences in clinical outcomes according to the preoperative use of CT-scan. Materials and methods: A retrospective observational study ofpediatric patients admitted for trauma and requiring surgery was carriedout. Patients were classified according to the previous use of CT-scan.ICU stay, re-admissions, and deaths were assessed. Results: From 2011 to 2017, 737 patients under 18 years of agewith external lesions were treated, 174 of whom required surgery. 48patients (27.6%) underwent CT-scan prior to the procedure (Group 1),while the remaining 126 patients (72.4%) were directly scheduled forsurgery (Group 2). Penetrating trauma occurred in 81% of patients, theproportion being significantly higher in Group 2 (p= 0.001). Median agewas 15 years (interquartile range: 12-17), with no differences betweengroups. No significant differences were found in terms of hemodynamicinstability at admission between groups (p= 0.596). At surgery, 3 outof 48 patients (6.3%) had no evident lesion. No significant differenceswere found in terms of re-admissions (p= 0.476), mortality (0.994),and ICU stay (0.466). Conclusion: The use of CT-scan as a diagnostic tool in pediatrictrauma does not reduce mortality, ICU stay, or number of re-admissions.The use of tools such as ultrasound examination and simple X-ray shouldbe protocolized to avoid unnecessary exposure to higher radiation doses.Prospective studies confirming this hypothesis are required.(AU)

Intrathecal HIV-1 envelope glycoprotein gp120 induces enhanced pain states mediated by spinal cord proinflammatory cytokines.

Perispinal (intrathecal) injection of the human immunodeficiency virus-1 (HIV-1) envelope glycoprotein gp120 creates exaggerated pain states. Decreases in response thresholds to both heat stimuli (thermal hyperalgesia) and light tactile stimuli (mechanical allodynia) are rapidly induced after gp120 administration. gp120 is the portion of HIV-1 that binds to and activates microglia and astrocytes. These glial cells have been proposed to be key mediators of gp120-induced hyperalgesia and allodynia because these pain changes are blocked by drugs thought to affect glial function preferentially. The aim of the present series of studies was to determine whether gp120-induced pain changes involve proinflammatory cytokines [interleukin-1beta (IL-1) and tumor necrosis factor-alpha (TNF-alpha)], substances released from activated glia. IL-1 and TNF antagonists each prevented gp120-induced pain changes. Intrathecal gp120 produced time-dependent, site-specific increases in TNF and IL-1 protein release into lumbosacral CSF; parallel cytokine increases in lumbar dorsal spinal cord were also observed. Intrathecal administration of fluorocitrate (a glial metabolic inhibitor), TNF antagonist, and IL-1 antagonist each blocked gp120-induced increases in spinal IL-1 protein. These results support the concept that activated glia in dorsal spinal cord can create exaggerated pain states via the release of proinflammatory cytokines.

Posibles factores protectores de la infección por SARS-CoV-2 en una paciente vulnerable: a propósito de un caso / Possible protective factors of SARS-CoV-2 infection in a vulnerable patient: report of a case

No disponible

Clinical and virologic follow-up in perinatally HIV-1-infected children and adolescents in Madrid with triple-class antiretroviral drug-resistant viruses.

Drug resistance mutations compromise the success of antiretroviral treatment in human immunodeficiency virus type 1 (HIV-1)-infected children. We report the virologic and clinical follow-up of the Madrid cohort of perinatally HIV-infected children and adolescents after the selection of triple-class drug-resistant mutations (TC-DRM). We identified patients from the cohort carrying HIV-1 variants with TC-DRM to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors and protease inhibitors according to IAS-USA-2013. We recovered pol sequences or resistance profiles from 2000 to 2011 and clinical-immunologic-virologic data from the moment of TC-DRM detection until December 2013. Viruses harbouring TC-DRM were observed in 48 (9%) of the 534 children and adolescents from 2000 to 2011, rising to 24.4% among those 197 with resistance data. Among them, 95.8% were diagnosed before 2003, 91.7% were Spaniards, 89.6% carried HIV-1-subtype B and 75% received mono/dual therapy as first regimen. The most common TC-DRM present in ≥50% of them were D67NME, T215FVY, M41L and K103N (retrotranscriptase) and L90M (protease). The susceptibility to darunavir, tipranavir, etravirine and rilpivirine was 67.7%, 43.7%, 33.3% and 33.3%, respectively, and all reported high resistance to didanosine, abacavir and nelfinavir. Despite the presence of HIV-1 resistance mutations to the three main antiretroviral families in our paediatric cohort, some drugs maintained their susceptibility, mainly the new protease inhibitors (tipranavir and darunavir) and nonnucleoside reverse transcriptase inhibitors (etravirine and rilpivirine). These data will help to improve the clinical management of HIV-infected children with triple resistance in Spain.

Genotypic and phenotypic resistance to stavudine after long-term monotherapy. BMS-020 Spanish Study Group.

Protocol BMS 020 was a double-blind, prospective clinical trial in which two different doses of stavudine (20 and 40 mg twice daily) were compared in human immunodeficiency virus (HIV)-infected patients with previous exposure to zidovudine for longer than 16 weeks. Genotypic and phenotypic resistance to both zidovudine and stavudine were examined after at least 2 years of stavudine monotherapy. None of 35 tested individuals harboured the codon 50 and/or 75 mutations previously described to be associated with stavudine resistance. However, more than 80% of the individuals carried mutations associated with zidovudine resistance, despite all these patients having stopped zidovudine at least 2 years earlier. Significant phenotypic resistance to stavudine was observed only in 2 of 5 tested individuals, although IC50 values were increased only 6.6- and 9.2-fold, respectively. These two patients had suffered a decline in their CD4 count, and one of them had high levels of plasma viraemia. The sequence analysis of the reverse transcriptase (RT) gene (aa 30 to 240) in these five patients revealed no changes that could be involved in stavudine resistance. In contrast, and despite having stopped treatment with zidovudine more than 2 years before, phenotypic resistance to zidovudine was observed in all five subjects, with IC50 values raised by more than 75-fold in all of them. Moreover, all harboured codon substitutions within the RT gene associated with zidovudine resistance, and these mutations remained in viral genomes examined after virus co-culture, suggesting that they provided some biological advantage to mutants, even in the absence of drug pressure. In conclusion, both genotypic and phenotypic resistance to stavudine seem to be a rare event in patients exposed to the drug, even after long periods of exposure.

Influence of time and storage conditions on plasma HIV viral load measurements.

Quantification of human immunodeficiency virus (HIV) RNA in plasma from HIV-infected patients is now widely used as a clinical indicator of disease prognosis and of response to antiretroviral therapy. However, controversy exists as to whether values obtained under different testing conditions could vary significantly, thus jeopardizing the appropriate interpretation of data. Herein, we demonstrate that results obtained after testing plasma versus whole blood, or immediate versus deferred processing, do not appear to influence viral load measurements significantly. Thirty blood samples from HIV-infected patients were analysed. The second generation branched-DNA assay was used for quantification of plasma viral load. HIV RNA remained stable for at least 24 h at room temperature, either in plasma or in whole blood, in 72.4% of the samples (< 0.2 log difference in viral load values) although lower levels of HIV RNA tend to be seen in samples after being stored as whole blood at room temperature. Only 3.4% of samples showed a decline > 0.5 log when they were left as whole blood at room temperature for 24 h in comparison with testing after immediate plasma separation. Although immediate separation and refrigeration of plasma samples may reduce the chance of significant falls in viral load measurements, this level of processing can be limited in regions where clinical blood samples cannot be processed rapidly. Our data provide confidence in the results obtained when testing specimens, either plasma or whole blood, 24 h after venepuncture and storage at room temperature, mimicking the conditions in the transport of blood samples to reference centres.

Recombinant human immunodeficiency viruses type 1 circulating in Spain.

The protease, reverse transcriptase (RT), and envelope (env) genes of human immunodeficiency virus type 1 (HIV-1) clinical isolates from 13 immigrants (mainly of African origin) living in Spain were examined. Phylogenetic analyses were performed, taking as reference a panel of 25 HIV-1 sequences representing various subtypes. A discrepant topology was recognized in comparing the protease, RT, and/or env phylogenetic trees in 10 isolates, in which sequences clustering in 2 or 3 different HIV-1 subtypes were found. In eight of these strains, the discrepant region was env with respect to concordant pol genes. Five of nine patients harboring subtype G sequences at the protease and RT genes showed discrepant env sequences, being subtype A (three) or B (two). In addition, one recombinant H/A strain, one recombinant H/B isolate, and a triple recombinant, A/D/C, variant were found. This work represents the first phylogenetic characterization of HIV-1 recombinant clinical isolates in Spain.

Protease gene analysis of HIV type 1 non-B subtypes in Spain.

The protease gene of human immunodeficiency virus type 1 (HIV-1) clinical isolates found in 15 immigrants (most of African origin) living in Spain was examined. Phylogenetic analyses were performed, taking as reference a panel of 26 HIV protease gene sequences deposited with GenBank. All specimens belonged to four distinct HIV-1 non-B subtypes: C (three cases), F (one), G (nine), and H (two). Five patients harboring subtype G strains were further classified within the IbNg recombinant clade. A high degree of genetic polymorphism at the protease gene was seen in all subtypes. Moreover, changes at positions associated with drug resistance were seen in subtype G viruses carried by patients who had not been exposed to protease inhibitors. Plasma viremia was lower than expected for some samples, according to the clinical features and the CD4+ cell count, suggesting that viral load titers were underestimated by all three commercially available techniques. This work represents the first genetic characterization of subtypes C, F, G, and H in Spain.