Resistencia antirretroviral en pacientes con virus de inmuno- deficiencia humana en Hospital Nacional Mario Catarino Rivas / Resistance to antiretroviral drugs in patients with human immunodeficiency virus in Mario Catarino Rivas National Hospital

Introducción: La terapia antirretroviral de primera línea utilizada en el país desde hace varios años, consiste en un inhibidor de la transcriptasa reversa no nucleósido con dos inhibidores de la transcriptasa reversa nucleósidos, está asociada al fallo terapéutico y se ha reportado resis- tencia a la misma. Objetivo: conocer la resistencia a la terapia antirretroviral de primera línea en pacientes tratados por Virus de Inmunodeficiencia Humana en Hospital Nacional Mario Catarino Rivas desde octubre 2016 al 2017. Pacientes y métodos: Investigación observacio- nal, descriptiva; tipo cohorte transversal, retrospectiva. Población de 313 pacientes a quienes se le realizó prueba de genotipo, de los cuales se tomaron como muestra 291 pacientes distri- buidos por grupos: sin previa exposición a antirretrovirales, pediátricos, con 12 meses de trata- miento y 48 o más meses de tratamiento a quienes se les realizó prueba de genotipo. Resulta- dos: Hubo amplificación en el 52% (152) de los pacientes, de los cuales el 56% (85) presentó resistencia a tratamiento antirretroviral, con prevalencia de resistencia de inhibidores de trans- criptasa reversa análogo de nucleósidos del 75% (64), los inhibidores de transcriptasa reversa no nucleósidos (ITRNN) con 89% (76) y los inhibidores de proteasa del 15% (13). Se encontró una prevalencia de resistencia primaria del 19% en pacientes de diagnóstico nuevo. Conclu- sión: Se recomienda el cambio de primera línea de terapia antirretroviral ya que se identifica- ron mutaciones de resistencia a los ITRNN en un 91% en los pacientes con diagnóstico recien- te y sin exposición a ARV. La OMS recomienda retirar los ITRNN como primera línea e incluir fármacos con mejor barrera genética, cuando los niveles de resistencia para los ITRNN sean >10%...(AU)

Sensibilidad y especificidad de pruebas moleculares en odontología / Sensitivity and specificity of molecular tests in dentistry

La biología molecular tiene mayor afinidad en las áreas de la salud, en odontología su principal aplicación ha sido en la identificación de microorganismos orales patógenos mediante el uso de secuencias genéticas específicas (ácido desoxirribonucleico [DNA], ácido ribonucleico [RNA] y proteínas). Las pruebas a nivel molecular se caracterizan por su rapidez, reproductibilidad, sensibilidad y especificidad de los microorganismos diana. El presente artículo de revisión bibliográfica servirá como herramienta para comprender los principios de las técnicas más destacadas como son: PCR estándar y RT-PCR en tiempo real, PCR con transcriptasa inversa, microarreglos y ensayo por inmunoabsorción ligado a enzimas (ELISA), además de sus ventajas y desventajas respecto a las pruebas convencionales (AU)

Molecular biology has a greater affinity in the areas of health. In dentistry, its main application has been the identification of pathogenic oral microorganisms, through the use of specific genetic sequences (deoxyribonucleic acid [DNA], ribonucleic acid [RNA] and proteins). Molecular tests are characterized by their rapidity, reproducibility, sensitivity and specificity of target microorganisms. This literature review article will serve as a tool to understand the principles of the most prominent techniques such as: Standard PCR, Real-time RT-PCR, Reverse transcriptase PCR, microarrays and Enzyme-linked immunosorbent assay (ELISA), in addition to their advantages and disadvantages with respect to conventional tests (AU)

Pacientes asintomáticos positivos a la COVID-19 / COVID-19 positive asymptomatic patients

Introducción: La COVID-19 es una enfermedad emergente de categoría pandémica. Las formas clínicas son variables y existe una elevada frecuencia de la forma asintomática. Objetivo: Describir las características clínicas y epidemiológicas en los pacientes de la COVID-19 asintomáticos. Métodos: Se realizó un estudio descriptivo, con todos los pacientes asintomáticos al ingreso, con diagnóstico confirmado, por la prueba de reacción en cadena de la polimerasa-transcriptasa inversa en tiempo real, para el SARS-CoV-2 en el Hospital Militar Dr. Fermín Valdés Domínguez, desde marzo hasta julio de 2020. Fueron estudiadas las variables expresividad clínica, proporción de asintomáticos, edad, sexo, antecedentes epidemiológicos y clínicos. Se utilizaron medidas de frecuencias. Resultados: El 41,8 por ciento de los pacientes permanecieron asintomáticos, predominó el grupo etario entre 20 y 39 años (33,3 por ciento) y el sexo femenino (55,6 por ciento). La fuente de infección más frecuente fue el contacto con caso confirmado (88,9 por ciento) y los municipios de mayor frecuencia fueron Holguín, Gibara y Banes. Los factores de riesgo clínico preponderante fueron: la condición de adulto mayor (20 por ciento) y la hipertensión arterial (17,8 por ciento). El 57, 8 por ciento de los pacientes no tenían comorbilidades. Conclusiones: Predominaron los adultos jóvenes, del sexo femenino, pertenecientes a los municipios Holguín, Gibara y Banes, con antecedentes epidemiológicos de ser contactos de casos confirmados. La condición adulto mayor y la hipertensión arterial fueron los factores de riesgo más frecuentes y la mayoría de los casos no presentaron comorbilidades(AU)

Introduction: COVID-19 is an emerging disease of pandemic category. The clinical forms are variable and the asymptomatic form has high frequency. Objective: To describe the clinical and epidemiological characteristics of COVID-19 positive and asymptomatic patients. Methods: A descriptive study was carried out, including all asymptomatic patients on admission, whit a confirmed diagnosis, by the real-time reverse transcriptase-polymerase chain reaction test for SARS-CoV-2 infection at Military Hospital Dr. Fermín Valdés Domínguez from March to July 2020. The variables age, sex, clinical expressiveness, clinical and epidemiological history were studied. Frequencies and percentage were used. Results: The 41,8 percent of the patients remained asymptomatic, the age group between 20 and 39 years (33,3 percent) and female sex predominated (55,6 percent). The most frequent source of infection was contact with confirmed case (88,9 percent) and the most frequent municipalities were Holguín, Gibara and Banes. Older adults and hypertension were most frequent clinical risk factors (20,0 and 57,8 percent respectively) and 57,8 percent of the patients had no comorbidities. Conclusions: Young adults predominated, female sex, from Holguín, Gibara and Banes municipalities with a history of being a contact confirmed case. Elderly condition and hypertension were the most frequent risk factors and most of the cases did not have comorbidities(AU)

Desenvolvimento e caracterização de dispersões sólidas de efavirenz / Development and characterization of efavirenz solid dispersions

A baixa solubilidade aquosa dos insumos farmacêuticos ativos (IFA) é um grande desafio no desenvolvimento de formulações farmacêuticas, pois pode resultar em biodisponibilidade insuficiente e variável. Diversas estratégias de modificação do estado sólido dos compostos ativos, têm sido propostas para incrementar a solubilidade de fármacos pouco solúveis em água. Dentre as estratégias abordadas a ispersão sólida (DS) é uma das formas mais promissoras de aumentar a solubilidade, dissolução e a biodisponibilidade de IFAs com baixa solubilidade aquosa. O efavirenz (EFV) é um inibidor não nucleosídeo da transcriptase reversa (NNRTI) e um dos componentes da terapia antirretroviral de alta atividade (HAART), sendo parte da primeira linha de tratamento de infecções do vírus HIV tipo 1. O antirretroviral está classificado como pertencente à classe II do SCB, e exibe baixa solubilidade aquosa (solubilidade menor que 10 µg/mL) e alta permeabilidade com absorção dependente da taxa de dissolução, resultando em biodisponibilidade oral baixa e variável. A administração de fármacos pouco solúveis na forma de DS é um método atraente para aumentar a biodisponibilidade in vivo. Neste estudo, um método de triagem rápida por evaporação de solvente foi empregado para preparar DS de EFV, variando-se proporções em misturas compostas pelos carreadores, polivinilpirrolidona K-28/32 (PVP K-28/32), copovidona (CoPVP), hidroxipropilmetilcelulose ftalato (HPMCP-50, HPMCP-55 e HPMCP-55s), poloxâmero 188 (P188) e poloxâmero 407 (P407). A solubilidade das DS foi avaliada por meio do método do equilíbrio (shake-flask), onde selecionou-se os polímeros P188 e P407 que conduziram a uma elevada capacidade de saturação em meio aquoso, superior a 1.000 vezes ao fármaco puro. As propriedades físico-químicas e do estado sólido das amostras foram avaliadas por meio de calorimetria exploratória diferencial (DSC); termogravimetria (TG); espectroscopia do infravermelho com transformada de Fourier (FTIR), difratometria de raios X pelo método do pó (DRXP) e ensaios de dissolução com emprego do aparato IV USP. Os resultados de DRXP demonstraram que os carreadores P188 e P407 foram capazes de estabilizar o EFV na forma amorfa nas DS, fato esse evidenciado pela ausência de picos característicos do antirretroviral

he low aqueous solubility of the active pharmaceutical ingredient (API) is a major challenge in the development of pharmaceutical formulations as it may result in insufficient and variable bioavailability. Several strategies for modifying the solid-state of the active compounds have been proposed to increase solubility of drugs that are poorly soluble in water. Among the strategies approaches, solid dispersion (SD) is one of the most promising ways to increase solubility, dissolution and bioavailability of APIs with low aqueous solubility. Efavirenz (EFV) is a non-nucleoside reverse transcriptase inhibitor (NNRTI) and one of the components of highly active antiretroviral therapy (HAART), being part of the first line of treatment of type 1 HIV virus infections. The antiretroviral is classified as belonging to BCS class II, and exhibits low aqueous solubility (solubility less than 10 µg / mL) and high permeability with dissolution ratedependent absorption, resulting in low and variable oral bioavailability. Drug delivery of poorly aqueous soluble drugs in form SD is an appealing method to increase in vivo bioavailability. In this study, a fast screening method of solvent evaporation method was used to prepare EFV SD, varying the proportions in mixtures composed by the carriers polyvinylpyrrolidone K-28/32 (PVP K-28/32), copovidone (CoPVP), hydroxypropylmethylcellulose phthalate (HPMCP-50, HPMCP-55 e HPMCP-55s), poloxamer 188 (P188) e poloxamer 407 (P407). The solubility of the samples was evaluated by the method of equilibrium (shake-flask), wherein the polymers P188 and P407 were selected due to the capacity to promote high saturation in aqueous medium, 1,000 times superior to the pure drug. The physicochemical and solid-state properties of the samples were evaluated by differential scanning calorimetry (DSC); thermogravimetry (TG); Fourier transform infrared spectroscopy (FTIR), X-ray powder diffraction (XRPD) and dissolution assays using the IV USP apparatus. The results of XRPD demonstrated that the carriers P188 and P407 were able to stabilize the EFV in amorphous form in the SD, a fact evidenced by the absence of characteristic peaks of the antiretroviral

Precisión diagnóstica de pruebas basadas en amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) para SARS-CoV-2 / Diagnostic Accuracy of Tests Based on Loop-mediated Isothermal Amplification reverse transcriptase (RT-LAMP) for SARS-CoV-2

ANTECEDENTES: Los coronavirus son una familia de virus causantes de enfermedades respiratorias, digestivas y del sistema nervioso en humanos y animales. En diciembre de 2019, se identificó en la provincia de Wuhan, China una cepa de coronavirus nunca antes encontrada en humanos, la cual recibió el nombre de SARS-CoV-2. La infección por SARS-CoV-2 se ha extendido a más de 212 países y fue declarada como pandemia por la Organización Mundial de la Salud. En nuestro país, se ha reportado 65 015 casos y un total de 1 814 fallecidos. La técnica molecular estándar para detectar SARS-CoV-2 es la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR). Estudios en cepas diferentes de coronavirus, sugieren que las pruebas moleculares basadas en amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) podrían mostrar mayor sensibilidad y especificidad que las pruebas RT-PCR, además de ser más rápidas y no requerir reactivos o instrumentos costosos. OBJETIVO: Describir la evidencia científica disponible sobre la precisión diagnóstica de las pruebas RT-LAMP para SARS-CoV-2. MÉTODO: Búsqueda sistemática en Medline (Pubmed), Cochrane Central Register of Controlled Trials (CENTRAL), Medrxiv y Chinese Clinical Trial Registry (CCTR) de estudios en idioma español o inglés publicados entre el 01 de diciembre de 2019 y el 06 de mayo de 2020, complementada con una búsqueda en Google Scholar. La calidad metodológica se evaluó usando el instrumento QUADAS 2. RESULTADOS: Se identificaron 09 estudios publicados en el año 2020, procedentes de Australia, Corea del Sur, Israel, Pakistán, China y Reino Unido. El número de muestras analizadas varió entre 21 y 260. Un estudio fue desarrollado en pacientes de un asilo de ancianos, mientras que el resto estudios fue desarrollado en un ámbito hospitalario. La evaluación de calidad mostró una probabilidad alta de sesgo en las dimensiones de selección de individuos y prueba de referencia. En cuanto a la aplicabilidad de los resultados del estudio, existe una probabilidad incierta en la dimensión de selección de los pacientes y la clasificación de la condición según la prueba de referencia. La calidad global de la evidencia es muy baja. CONCLUSIONES: • Las pruebas RT-LAMP fueron desarrolladas para identificar con mayor frecuencia el gen N (04 estudios), seguido de los genes ORF1a y N (02 estudios), gen RdRp (01 estudio), gen ORF1a (01 estudio) y genes ORF1ab y S (01 estudio). Existió variabilidad en los protocolos seguidos en cada estudio. Comparado con la prueba de reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR), las pruebas basadas en amplificación isotérmica mediada en lazo de transcriptasa inversa (RT-LAMP) mostraron una sensibilidad entre 80% y 100%, y una especificidad entre 73% y 100% para el diagnóstico de SARS-CoV-2. El valor predictivo positivo de las pruebas RT-LAMP varió entre 73% y 100%, mientras que el valor predictivo negativo varió entre 75% y 100%. Ambos valores son influenciados por la prevalencia de la enfermedad, la cual varió en los estudios entre 9,1% y 70,8% (mediana: 62,7%). La calidad de la evidencia para la precisión diagnóstica de las pruebas RT-LAMP desarrolladas en los estudios identificados es muy baja, debido al alto riesgo de sesgo, imprecisión en los resultados y aplicabilidad incierta.(AU)

Variabilidad genética en regiones codificantes del antígeno de superficie y el dominio de la transcriptasa inversa de la polimerasa del virus de la hepatitis B, Colombia, 2002-2014 / Genetic variability in coding regions of the surface antigen and reverse transcriptase domain of hepatitis B virus polymerase, Colombia, 2002-2014

Introducción. Se estima que 240 millones de personas en el mundo tienen infección crónica con el virus de la hepatitis B (HBV). En Colombia, la endemia es variable y circulan diferentes genotipos virales. Las mutaciones a lo largo del genoma se han asociado con resistencia antiviral, el escape ante la reacción de anticuerpos neutralizadores tras la vacunación o a la infección natural, la infección oculta y la progresión a carcinoma hepatocelular. Objetivo. Identificar los genotipos y las mutaciones presentes en la región codificante del antígeno de superficie (S) y del dominio de la transcriptasa inversa (reverse transcriptase, RT) de la polimerasa del HBV en muestras de suero remitidas al Instituto Nacional de Salud de Colombia para el diagnóstico de hepatitis B, entre el 2002 y el 2014. Materiales y métodos. En 495 muestras de suero positivas para el antígeno de superficie de la hepatitis B (HBsAg) se buscó el ADN viral, se amplificó y secuenció un fragmento de 1.591 nucleótidos y, posteriormente, se hizo el análisis filogenético correspondiente. Resultados. En 66 de las muestras se logró detectar el genoma viral y 28 de ellas se secuenciaron exitosamente. El análisis filogenético permitió identificar los genotipos y subgenotipos F3 y A2. Una muestra presentó simultáneamente las sustituciones de resistencia L180M y M204V, otra presentó la sustitución I169L y en una se identificó la mutación P120Q, previamente asociada con variantes de escape. Dos muestras presentaron una deleción de 105 nucleótidos en la región preS1-preS2. Conclusiones. Se corroboró la circulación en Colombia de los genotipos y subgenotipos F3 y A2, así como la presencia de mutaciones de resistencia y escape. El presente estudio constituye un aporte a la epidemiologia molecular del HBV en Colombia.

Introduction: Despite the availability of an effective vaccine and treatment to reduce the viral load and progressive hepatocellular injury, approximately 240 million people worldwide are chronically infected with the hepatitis B virus (HBV). In Colombia, the circulation of different viral genotypes has been confirmed. Mutations in the genome have been associated to antiviral therapy resistance, viral escape to neutralizing antibodies, occult infection and progression to hepatocellular carcinoma. Objective: To identify the genotypes and the presence of mutations in the coding region of the surface (S) antigen and the reverse transcriptase (RT) domain of the polymerase of HBV obtained from serum samples for hepatitis B diagnosis received by the Instituto Nacional de Salud during the period 2002-2014. Materials and methods: A total of 495 serum samples with previous HBsAg reactive result were used for molecular detection. A fragment of 1,591 nucleotides was sequenced, and the corresponding phylogenetic analysis was performed. Results: We detected the viral genome of HBV in 66 samples and 28 were successfully sequenced. The phylogenetic analysis allowed the identification of subgenotypes F3 and A2. The L180M and M204V resistance mutations were simultaneously identified in one sample, while the I169L resistance mutation was identified in another one. A single escape mutation, P120Q, was identified in one more. Two samples showed a deletion of 105 nucleotides in the preS1-preS2 region. Conclusions: The circulation of genotypes/subgenotypes F3 and A2 of HBV in Colombia was corroborated, as well as the presence of some resistance and escape mutations. The present study constitutes a contribution to the molecular epidemiology of HBV in Colombia.

Resistencias transmitidas del VIH-1 en pacientes sin exposición previa a terapia antirretroviral (Naïve) Cali-Colombia 2008-2015 / Transmitted HIV-1 resistance in never exposed to antiretroviral therapy patients, Cali-Colombia 2008-2015

Objetivo: Determinar la frecuencia de resistencias transmitidas en pacientes VIH no expuestos a terapia antirretroviral. Metodología: Estudio transversal, entre 2008 y 2015 participaron 342 pacientes mayores de 18 años, con infección VIH-1, sin exposición a antirretrovirales, con genotipo de resistencias previo al inicio con antirretrovirales y consentimiento informado. Se incluyeron mutaciones de resistencias definidas por Organización Mundial de Salud (OMS)-2009 e internacional AIDS Society-USA 2015. Se recolectaron características sociodemográficas y clínicas relacionadas con VIH. Resultados: Edad promedio 32±10,5 años; 74% hombres. Al genotipo, mediana de carga viral 26.802 copias/mL, y 343 células T-CD4/mm3 . Según lista de Organización Mundial de Salud-2009 la frecuencia de mutaciones fue 3.2% y considerando mutaciones de internacional AIDS Society y base de datos VIH-Stanford, esta fue 7.9%. Mutaciones más comunes: K103N/S (2.1%), Q58E (2%), V108I y E138A (1.2%). Mutaciones en transcriptasa reversa 4.4% para inhibidores no nucleosídicos y 1.2% para nucleosídicos; para inhibidores de proteasa 2.3%. En pacientes con probable infección retroviral <1 año, la prevalencia de resistencias transmitidas, según la lista de OMS-2009, fue 7.3%, mientras que, con infección ≥1 año fue 1.6% (p=0.008). Conclusión: En pacientes con probable infección <1 año, la frecuencia de mutaciones transmitidas superó el umbral recomendado por la Organización Mundial de Salud para implementar genotipo de resistencias.

Introduction: This study determined the frequency of transmitted drug resistance in patients not exposed to ART, attended in Cali-Colombia. Methodology: A cross-sectional study between 2008 and 2015 involved 342 patients older than 18 years, with confirmed HIV infection, without exposure to antiretrovirals, with resistance genotype prior to initiation of antiretroviral and informed consent. Resistance mutations defined by WHO-2009 and international AIDS Society-USA 2015 were included. Sociodemographic and clinical characteristics related to HIV were also collected. Results: The mean age was 32 ± 10.5 years; 74% were men. At the time of genotype, median viral load was 26,802 copies / mL, and 343 T-CD4 / mm3 cells. According to the WHO-2009 list, the frequency of mutations was 3.2% and considering the AIDS Society-USA list and the Stanford HIV database mutations, this reached 7.9%. The most common mutations were K103N/S (2.1%), Q58E (2%), V108I and E138A (1.2%). Reverse transcriptase mutations were found in 4.4% for non-nucleoside inhibitors and 1.2% for nucleoside; for protease inhibitors were 2.3%. In patients with retroviral infection <1 year, the transmitted resistances prevalence, using only the WHO-2009 list, reach 7.3%, while in those with ≥1 year only was 1.6% (p=0.008). Conclusions: The frequency of transmitted mutations in naïve patients with retroviral infection <1 year exceeded the prevalence threshold recommended by the WHO to implement the resistance genotype.

Molecular detection of mixed infections with multiple dengue virus serotypes in suspected dengue samples in Tamaulipas, Mexico

This study aimed to detect dengue virus (DENV) serotypes in serum samples obtained in Matamoros Tamaulipas, Mexico, and to determine the concordance of conventional nested reverse transcriptase polymerase chain reaction (RT-PCR) and a serological test [enzyme-linked immunosorbent assay (ELISA NS1)]. Here, we detected mixed infections consisting of four serotypes of DENV. The most prevalent serotype was DENV-1, followed by DENV-4. This is the first report of DENV-4 in our region. Mixed infections were also detected in 21.5% of samples, and the predominant coinfection consisted of DENV-1 and DENV-2. Therefore, continuous epidemiological surveillance of DENV in this area is required to predict future forms of dengue heterologous infections and the effect of this on health care.

Mutations in the S gene and in the overlapping reverse transcriptase region in chronic hepatitis B Chinese patients with coexistence of HBsAg and anti-HBs

Abstract Background The mechanism underlying the coexistence of hepatitis B surface antigen and antibodies to HBsAg in chronic hepatitis B patients remains unknown. Aims This research aimed to determine the clinical and virological features of the rare pattern. Methods A total of 32 chronic hepatitis B patients infected by HBV genotype C were included: 15 carrying both HBsAg and anti-HBs (group I) and 17 solely positive for HBsAg (group II). S gene and reverse transcriptase region sequences were amplified, sequenced and compared with the reference sequences. Results The amino acid variability within major hydrophilic region, especially the “a” determinant region, and within reverse transcriptase for regions overlapping the major hydrophilic region in group I is significantly higher than those in group II. Mutation sI126S/T within the “a” determinant was the most frequent change, and only patients from group I had the sQ129R, sG130N, sF134I, sG145R amino acid changes, which are known to alter immunogenicity. Conclusions In chronic patients, the concurrent HBsAg/anti-HBs serological profile is associated with an increased aa variability in several key areas of HBV genome. Additional research on these genetic mutants are needed to clarify their biological significance for viral persistence.

Human coronavirus ocurrence in different populations of Sao Paulo: a comprehensive nine-year study using a pancoronavirus RT-PCR assay

Human coronaviruses (HCoVs) are considered one of the most common respiratory viruses associated with respiratory tract illnesses. An emergent human coronavirus was identified as the causal agent of an epidemic of severe acute respiratory syndrome (SARS) during 2002-2003. The severity of the disease combined with its rapid spread requires the continuous surveillance of coronaviruses in worldwide populations. Epidemiological and clinical data of HCoVs infectious in the Brazilian population are scarce and restricted to one or two groups of patients. Our study aimed to investigate retrospectively the presence of HCoVs in different populations of São Paulo presenting acute respiratory tract infections (ARIs) during the years of 2001-2010. A pancoronavirus RT-PCR was performed in this study. Coronaviruses were detected in 126 (11.5%) of 1,087 specimens. Peaks detection frequency was observed during 2002-2004 and 2008-2009, with the highest detection in 2008. The prevalence of HCoVs was higher among children with heart diseases (24.6%), patients under stem cell transplantation program (24.3%) and renal transplanted patients (20.2%). Coryza, cough and fever were the most common symptoms at presentation of positive cases and wheezing, a lower respiratory tract infection symptom was reported by 12% of the total, and 27% of high at-risk patients. HCoVs may have an important role among patients with underlying conditions and transplanted ones

Perfil Genotípico de Resistência do HIV em pacientes com falha virológica ao esquema antirretroviral de primeira linha na coorte de pacientes com HIV/AIDS do Instituto de Pesquisa Clinica Evandro Chagas-Fiocruz / Genotypic resistance profile of HIV in patients with virological failure to first-line antiretroviral regimen in a cohort of patients with HIV / AIDS Clinical Research Institute Evandro Chagas-Fiocruz

Introdução: Ao longo dos trinta anos de identificação do HIV como agente etiológico, muitas mudanças ocorreram, principalmente em relação ao tratamento dos pacientes. [...] Objetivos: Descrever o perfil genotípico por ocasião da primeira falha virológica em uso de um esquema inicial de cART; Avaliar o impacto do perfil mutacional no momento da primeira falha no uso potencial da etravirina em esquemas antirretrovirais subsequentes; Descrever o perfil da população baseando-se no escore de mutações da etravirina e analisar os fatores relacionados à redução na sensibilidade a esta droga; Avaliar a prevalência de genotipagens com de vírus selvagem e analisar os fatores associados à sua ocorrência; Descrever os fatores relacionados à mutação K65R na primeira falha à cART. Métodos: Estudo com coleta de dados a partir de prontuários médicos e exames de genotipagem de pacientes com HIV acompanhados no IPEC que tiveram a falha virológica ao esquema antirretroviral inicial no período de 2000 a 2012. [...] Resultados: Foram incluídos 166 pacientes nesse estudo. O subtipo viral predominante foi o B (65,3 por cento). Nos 113 pacientes que usaram esquemas baseados em ITRNN (66,5 por cento), a mutação mais frequente para esta classe foi a 103N. Entre os 17 pacientes que fizeram uso de IP sem booster, as mutações mais frequentes na protease foram a 30N, 32I e 46ILV e entre os 36 pacientes que fizeram uso de IP com booster as mutações mais frequentes na protease foram a 82ATF, 90M e 46ILV...

Introduction: Over thirty years of HIV epidemic, many changes have occurred, especially in term's of patients treatment. [...] Objectives: To describe the genotypic profile at the first virologic failure while using the firstline cART; evaluate the impact of the mutational profile at the time of first failure in the potential subsequent use of etravirine for salvage regimens; describe the profile of the population relying etravirine´s mutations and analyze the factors related to reduced sensitivityto this drug; evaluate the prevalence as well as the associated risk factors of genotyping withthe presence of wild-type virus; describe the factors related to the presence of K65R mutationat the first failure to cART.Methods: The study included data collection from medical records and genotyping of HIV patients followed at IPEC who had presented virologic failure for initial antiretroviral regimenin the period of 2000 to 2012. [...] Results: 166 patients were included in this study.The predominant virus subtype was B (65.3 percent). Among the 113 patients using NNRTI -based regimens, the most frequent mutation to NNRTI was 103N. Among the 17 patients who used an unboosted PI, the most prevalent mutations in protease were 30N, 32I and 46ILV. Among the 36 patients who used boosted PI the most frequent mutations in protease were 82ATF, 90M and 46ILV. The most frequent NRTI mutation into the three groups was 184VI...

Estudo de doença residual minima em leucemia linfoide aguda da criança e do adolescente / Minimal residual disease study in acute lymphoblastic leukemia of child and adolesce

A leucemia linfóide aguda (LLA) é o câncer mais comum da infância. Os atuais protocolos de tratamento da LLA levam à cura em 70 por cento dos casos e parte do sucesso se deve à aplicação de diferentes tratamentos para os pacientes estratificados em diferentes grupos de risco, segundo fatores prognóstico pré-tratamento (contagem leucocitária e idade ao diagnóstico). Contudo, pacientes considerados em remissão podem apresentar conteúdo substancial de células neoplásicas, chamada doença residual mínima (DRM), cuja proliferação está associada com a recaída da doença e que podem estar em níveis indetectáveis pelas técnicas convencionais de análise morfológica. Vários trabalhos têm mostrado que é possível prever a evolução clínica dos pacientes com base na DRM, porém, no protocolo do Grupo Brasileiro de Tratamento da Leucemia Infantil (GBTLI), a DRM não é utilizada como critério de realocação dos pacientes nos grupos de risco. Desta forma, o presente estudo objetivou (1) obter dados prospectivos de DRM com o uso de reação em cadeia da polimerase (PCR) para detecção de rearranjos de genes de imunoglobulina (Ig) e receptores de células T (TCR); (2) comparar resultados de DRM com fatores prognósticos ao diagnóstico e de resposta utilizados nos protocolos do GBTLI e (3) avaliar o valor preditivo da DRM em pacientes tratados pelo protocolo GBTLI-99. No total, foram estudadas 91 amostras de LLA pediátrica classificadas...

Acute lymphoblastic leukemia (ALL) is the most common pediatric cancer. The recent ALL treatment protocols can achieve the complete remission in 70 per cent of cases and this success is due to different treatments for patients stratified into different risk groups , according to pre-treatment prognostic factors (white count cells and diagnosis age). Therefore, patients considered in remission may have substantial contents of neoplasic cells, the minimal residual disease (MRD). The proliferation of such neoplasic cells is associated with disease relapse and they can be undeteced by conventional methods. It has been demonstrated that the clinical evolution of patients based on MRD can be forecasted. Therefore, in the Brazilian Group for Childhood Leukemia Treatment (GBTLI), MRD is not used as a reallocation criterion of patients in risk groups. The present study aimed to (1) obtain MRD data through the use of polymerase chain reaction (PCR) for the detection of immunoglobulin (Ig) and T cell receptor genes rearrangements (TCR); (2) compare the results of MRD with prognostic factors at diagnosis and response factors used in the GBTLI protocols and (3) estimate the predictive value of MRD in patients treated...

Do DNA extraction methods and Taq polimerase quality improve the double repetitive element (DRE) PCR typing method for Mycobacterium tuberculosis strains?

Double repetitive element (DRE) PCR amplification is a simple Mycobacterium tuberculosis typing method, however amplification failure or poor resolution of bands commit its efficacy. In order to verify if whether or not these features could be minimized by improving DNA extraction procedures or Taq polymerise quality, DRE-PCR was performed on 24 M. tuberculosis DNA samples extracted by heat-shock, mechanical and enzymatic methods applying conventional and hot start Taq pol. We demonstrated that when dealing with the Mycobacterium tuberculosis DRE-PCR typing method, Taq pol of better quality might be more important to improve amplification than the DNA extraction method.

Amplificação de duplo elemento repetido (DRE) por PCR é um método simples para tipagem de Mycobacterium tuberculosis, entretanto falha ou a baixa resolução das bandas na amplificação compromete a eficiência do método. Com o objetivo de verificar se estes problemas podem ou não ser minimizados pela utilização de diferentes procedimentos de extração de DNA ou de qualidades de Taq polimerase, DRE-PCR foi ensaiado em 24 amostras de DNA de M. tuberculosis extraídos pelos métodos de choque-térmico, - mecânico e enzimático utilizando Taq polimerase convencional e hot start Taq pol. Foi demonstrado que a qualidade da Taq pol utilizada talvez seja mais importante para uma melhor amplificação que o método de extração de DNA empregado.

Identificación molecular de mutaciones puntuales relacionadas con resistencia a drogas en VIH-1 de pacientes peruanos / Molecular identification of point mutations related to HIV-1 drug resistance in peruvian patients

Objetivo: Identificar mutaciones puntuales relacionadas con resistencia a drogas en VIH-1 de pacientes peruanos.Materiales y métodos: Se seleccionaron 11 muestras de VIH provenientes de sangre total de sujetos con tratamientoantirretroviral (ARV). Posteriormente se realizó la optimización de la amplificación de 337 pb del gen de la transcriptasareversa (tr) y 377 pb de todo el gen de la proteasa (prt). Los productos de PCR fueron secuenciados directamentepara el análisis de mutaciones de resistencia. Las secuencias finales fueron analizadas en programas de análisis demutaciones de la HIV Drug Resistance Database de la Universidad de Stanford. Resultados: La optimización de laconcentración de ADN (2,5 ng / μL) así como la concentración de magnesio (4 mM) fueron factores críticos para la amplificaciónde la tr y la prt respectivamente. De otro lado, el análisis de secuencia reveló la presencia de las mutacionesT215Y y la M184V en tr, implicadas en conferir resistencia a zidovudina (AZT) y estavudina (D4T) así como a lamivudina(3TC) y emtricitabina (FTC) respectivamente. En prt se observaron las mutaciones D30N y N88D implicadas enconferir resistencia a nelfinavir (NFV). Es importante señalar que sólo tres muestras de VIH-1 presentaron mutacionesde resistencia, las demás mostraron mutaciones compensatorias. Conclusiones: Se demuestra la existencia de mutacionesde resistencia a ARV a nivel de tr y prt de VIH-1 en sujetos peruanos que reciben terapia TARGA. Se requierende mayores estudios para establecer un perfil de resistencia a ARV en la población peruana.

Objective: Identify point mutations related to HIV-1 drug resistance in Peruvian patients. Materials and methods: A total of 11 HIV-1 positive samples were selected, all were obtained from the whole blood of subjects undergoing anti-retro viral (ARV) treatment. 337 gene base pairs (bp) from reverse transcriptase (rt) and 377 bp from the whole protease gene (prt) were optimized. PCR products were sequenced directly for the analysis of resistance mutations. Final sequences were analyzed in the University of Stanford HIV Drug Resistance Database programs. Results: Optimization of DNA concentration (2,5 ng / µL), as well as magnesium concentration (4mM) were critical factors for rt and prt amplification, respectively. On the other hand, the sequence analysis showed the presence of T215Y and M184V mutations in rt , implicated in zidovudine (AZT), stavudine (D4T), lamivudina (3TC) and emtricitabine (FTC) resistance, respectively. In prt, mutations D30N and N88D were observed. These mutations have been implicated in nelfinavir (NFV) resistance. It must be highlighted that only three HIV-1 positive samples showed resistance mutations. The remaining samples showed compensatory mutations. Conclusions: This study demonstrated the existence of ARV resistance mutations at the HIV-1 rt and prt levels in Peruvian patients undergoing HAART therapy. Further studies are required to establish an ARV resistance pattern in the Peruvian population.

La transcriptasa reversa del VIH: estudios estructurales y cinéticos / The reverse transcriptasa of the HIV: structural and kinetic studies

La transcriptasa reversa (TR) es una polimerasa de ADN, compuesta por dos subunidades 66 y 51 KDa. Sintetiza ADN usando un ácido nucleico complementario como templado. Esta enzima posee tres actividades distintas: una polimerasa de ADN dependiente de ARN que sintetiza la cadena negativa del ADN proviral, una actividad polimerasa de ADN dependiente de ADN que cataliza la construcción de la hebra positiva y una actividad de RNAsa H. La TR del VIH utiliza un ARN de transferencia (ARNt) específico para llevar acabo la fabricación del ADN complementario, lo cual induce un cambio de conformación que afecta las actividades de la polimerasa. Diversos investigadores han encontrado que la afinidad del sustrato por la TR del VIH es significativamente alta; la Kd para un dNTP de la TR viral es 4nM, una de las más bajas encontradas para las polimerasas de ADN y posee a su vez valores de procesividad y selectividad de gran magnitud. Distintas evidencias remarcan el hecho de que la TR sufre un cambio conformacional cuando se encuentra acomplejada de la forma E-ADN-dNTP, y que esta acción puede ser inhibida por el apareamiento de un nucleótido incorrecto. La enzima debe acomodar los duplex generados de cada reacción y debido a esto la TR podría no ser capaz de restringir la geometría del apareamiento, como sucede para el resto de las polimerasas, originando la aparición de los diferentes mutantes del VIH. Los inhibidores de la transcriptasa reversa del VIH se pueden agrupar en tres categorías: análogos de nucleósidos, no nucleósidos y oligonucleótidos. Los inhibidores análogos de nucleósidos más utilizados clínicamente son: Lamivudina, Zerit, Retrovir, Videz, AZT o Zidoduvina y Viramune. Los no nucleósidos ya empleados en terapias son: Viramune y rescriptor. Por todas sus características, la TR del VIH se encuentra catalogada como una de las enzimas de mayor funcionalidad y complejidad que se haya estudiado

Frequência das mutações que conferem resistência aos anti-retrovirais em um grupo de indivíduos infectados pelo HIV-1, em Salvador, Bahia / Frequency of mutations that confer resistance to antiretroviral drugs in a group of individuals infected by HIV-1 in Salvador, Bahia

A grande variabilidade genética do HIV é um importante obstáculo para a manutenção de terapêuticas de longa eficácia. Assim, o conhecimento sistemático das mutações que conferem resistência aos anti-retrovirais é de grande importância clínica e terapêutica. A partir de uma coleção de plasma de pacientes do Estado da Bahia, realizamos um estudo com o intuito de investigar a freqüência de mutações que conferem resistência aos anti-retrovirais, empregando o ensaio LIPA HIV-1 RT para analisar as mutações no gene da transcriptase reversa e a técnica de RFLP, utilizando a enzima de restrição Hinc lI, para identificar as mutações nas posições 82 e 90 no gene da protease. As amostras selecionadas foram provenientes de pacientes virgens de tratamento (VT; n=22) e de pacientes que estavam sob terapia antiretroviral, na forma de terapia dupla com INTR (TD; n=48) e terapia tripla com INTR e inibidores da protease (TT; n=41). Neste último grupo, realizamos também o teste fenotípico HIV-1 PR/RT Antivirogram TM VIRCO em 17 pacientes. No grupo de pacientes VT, apenas 1/20 (5%) apresentou a mutação M184V no gene da TR, enquanto que 3/22 (13,6%) apresentaram a mutação L90M no gene da protease. Já no grupo TD, encontramos mutações que conferem resistência ao AZT como T215Y/F, K70R e L41M em, respectivamente, 60%, 48% e 48% das amostras analisadas. Mutações que conferem resistência ao 3TC (M184V-44%) e ao ddI (L746%). Nos indivíduos sob TT, as freqüências de mutações no gene da protease foram de 34%-V82A, 15%-L90M e 39%- V82AJL90M. A análise fenotípica mostrou que as drogas menos ativas dos INTR foram AZT e 3TC e dos IP foram saquinavir, ritonavir, indinavir e nelfinavir. Estes resultados estão de acordo com a elevada freqüência de mutações encontradas no gene da protease V82A-60%, L90M-24% e V82AJL90M-65%. Nossos resultados mostram que as mutações genotípicas encontradas têm uma forte correlação com o tipo de drogas anti-retrovirais em uso pelos pacientes incluídos no estudo.

Prevalence of primary and secondary resistant mutations to antiretroviral drug in a population of Puerto Rican infected with HIV

INTRODUCTION: Several studies have reported increasing number of therapeutic failures with HAART in HIV-infected individuals. In order to assess the impact HIV antiretroviral resistance could have on treatment, we decided to determine the prevalence of primary and secondary antiretroviral resistant genotypes in a population of HIV-infected Puerto Ricans and compare the mutational distribution pattern with that reported in Europe and US. METHOD: In a total of 80 plasma samples from patients with detectable viral load of over 1,000 RNA copies/ml, the Trugene Visible Genetics HIV sequencing method was used to detect antiretroviral resistance mutations. RESULTS: We found 55 subjects (69 per cent) with high level of resistance to ZDV in the reverse transcriptase gene and 46 subjects (58 per cent) with high level of resistance to NFV in the protease gene. Mutation frequencies to the NRTI ranged in appearance from as high as 54 per cent (i.e., M184V) in the studied subjects to a low of less than 5 per cent (i.e., M184I and V75T). For the NNRTI the most common mutation was K103N in 40 per cent of the subjects and found to confer cross resistance to NVP, DLV and EFV. Another concerning finding is the increasing trend of the frequency of primary and secondary resistant mutations from year 2000 to 20001. Nine (23 per cent) of the total detected primary mutations, to either RTI or PI, showed an increase of at least 5 per cent from one year to the other. Similarly, there were 6 (11 per cent) secondary resistant mutations showing an increase of at least 5 per cent during the two years studied. CONCLUSIONS: In two year period we detected a tendency to increase in primary and secondary HIV-resistant mutation in a population of HIV-infected Puerto Ricans.