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)

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)

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)

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)

Regulation of porcine endogenous retrovirus by dual LTR1+2 (Long Terminal Region) miRNA in primary porcine kidney cells

Porcine endogenous retroviruses (PERVs) integrate into germline DNA as proviral genome that enables vertical transmission from parents to their offspring. The provirus usually survives as part of the host genome rather than as an infectious agent, but may become pathogenic if it crosses species barriers. Therefore, replication-competent PERV should be controlled through selective breeding or knockout technologies. Two microRNAs (miRNAs), dual LTR1 and LTR2, were selected to inhibit the expression of PERV in primary porcine kidney cells. The inhibition efficiency of the miRNAs was compared based on their inhibition of different PERV regions, specifically long terminal repeats (LTRs), gag, pol, and env. Gene expression was quantified using real-time polymerase chain reaction and the C-type reverse transcriptase (RT) activity was determined. The messenger RNA (mRNA) expression of the PERV LTR and env regions was determined in HeLa cells co-cultured with primary porcine kidney cells. The mRNA expression of the LTR, gag, pol, and env regions of PERV was dramatically inhibited by dual miRNA from 24 to 144 h after transfection, with the highest inhibition observed for the LTR and pol regions at 120 h. Additionally, the RT activity of PERV in the co-culture experiment of porcine and human cells was reduced by 84.4% at the sixth passage. The dual LTR 1+2 miRNA efficiently silences PERV in primary porcine kidney cells.

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.

Comparison of R-mix Virus Culture and Multiplex Reverse Transcriptase Polymerase Chain Reaction for Assessment of Neonatal Respiratory Viral Infection

OBJECTIVE: Respiratory viral infection of the neonatal period is highly contagious. Rapid and accurate diagnosis is important for proper treatment and prevention. However, the existing diagnostic method, respiratory virus cell culture, takes a long time to diagnose. Recent development of rapid diagnostic methods such as multiplex reverse transcriptase polymerase chain reaction (RT-PCR) enable early detection and effective treatment of respiratory viral infections. We compared the efficiency of multiplex RT-PCR and R-mix virus culture for rapid detection of respiratory viruses. METHODS: We retrospectively analyzed the clinical features and results of R-mix virus culture and multiplex RT-PCR with nasopharyngeal aspiration specimens in 117 newborns admitted to neonatal intensive care unit suspected of infectious diseases. RESULTS: R-mix virus culture was positive in 29 cases (24.8%) and RT-PCR in 86 cases (73.5%). R-mix virus culture and multiplex RTPCR were identical in 54 cases (positive 26 cases, negative 28 cases). Among 75 cases that showed different results, 60 showed negative result in R-mix virus culture and positive result in multiplex RT-PCR, and three showed positive result in R-mix virus culture and negative result in multiplex RT-PCR. Different viruses were detected in the remaining 12 cases by both methods. CONCLUSION: Multiplex RT-PCR is faster than R-mix virus culture and has the advantage of identifying new respiratory viruses. On the other hand, Multiplex RT-PCR is more susceptible to false positives and mixed infections than R-mix virus culture, so more attention is required when interpreting test results.

Sequential Bilateral Rapid Destructive Inflammatory Coxarthrosis in a Patient with Human Immunodeficiency Virus / 대한고관절학회지

The diagnostic criteria for sequential rapidly destructive coxarthrosis remain unclear and this condition is rarely reported in patients with human immunodeficiency virus (HIV). Here, we report a case of an HIV-infected 73-year old female who experienced hip joint destruction. The patient was diagnosed with HIV in 2012 (at age 68 years) and began continuous treatment with nucleoside reverse transcriptase and protease inhibitors. Twenty-nine months after her HIV diagnosis, the patient experienced osteonecrosis of the right hip and underwent a total hip arthroplasty (THA). Twelve months post right-hip THA, X-ray results showed good outcomes. Eight months later (20 months post THA), however, osteolysis of the left femoral head was detected upon radiological exam and THA of the left hip was performed; chronic inflammation and fibrosis were identified in the resultant biopsy. Favorable results were obtained at 3 months after the second surgery.

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.

Application of CRISPR/Cas9 in Treating Hepatitis B Virus

The advent of oral antiviral agents has revolutionized hepatitis B treatment. It has led to decreased incidence and mortality related to hepatocellular carcinoma. However, although nucleos(t)ide analogs (NA) are fast and potent in inhibiting hepatitis B virus (HBV) polymerase and reverse transcriptase activity, complete cure of the virus is not possible. The complete eradication of HBV requires the covalently-closed-circular DNA (cccDNA) to be eliminated. Novel gene editing methods, such as zing finger nucleases, transcription activator-like effector nucleases, and the clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) system, designed to target specific DNA sequences has great potential for therapeutic application. Among these, the CRISPR/Cas9 system may be the most feasible approach to eradicate HBV cccDNA. Further studies are needed to develop a more efficient and safer method of delivery using the CRISPR/Cas9 system to achieve complete cure of chronic hepatitis B.

Duplex nested reverse transcriptase polymerase chain reaction for simultaneous detection of type 2 porcine reproductive and respiratory syndrome virus and porcine circovirus type 2 from tissue samples

There are high levels of co-incidence of porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) in porcine tissue. This study established a duplex nested reverse transcriptase polymerase chain reaction (RT-PCR) method that targets the genomic RNA of type 2 PRRSV and the mRNA of PCV2 in infected tissues. The method amplified discriminative bands of 347 bp and 265 bp specific for type 2 PRRSV and PCV2, respectively. The limits of detection of the duplex nested RT-PCR were 10(1.5) TCID₅₀/mL for type 2 PRRSV and 10² infected cells/mL for PCV2. The kappa statistic, which measures agreement between methods, was 0.867, indicating a good level of agreement. This RNA-based duplex RT-PCR approach can be another way to detect type 2 PRRSV and PCV2 simultaneously and with improved convenience.

Safety and Efficacy of Ziagen (Abacavir Sulfate) in HIV-Infected Korean Patients

BACKGROUND: Abacavir is a widely-used nucleoside reverse transcriptase inhibitor for the treatment of human immunodeficiency virus (HIV) infection. Mandatory postmarketing surveillance was conducted in Korea to monitor the safety and evaluate the effectiveness of Ziagen® (abacavir sulfate 300 mg; ViiV Healthcare, Middlesex, UK). MATERIALS AND METHODS: An open-label, multi-center, non-interventional postmarketing surveillance study was conducted from June 2010 to June 2016 to monitor the safety and effectiveness of Ziagen across 12 hospitals in Korea. Subjects older than 18 years taking Ziagen according to prescribing information were enrolled. The primary outcome was defined as the occurrence of any adverse events after Ziagen administration. Secondary outcomes included the occurrence of adverse drug reactions, occurrence of serious adverse events, and effectiveness of Ziagen administration. RESULTS: A total of 669 patients were enrolled in this study, with a total observation period of 1047.8 person-years. Of these, 90.7% of patients were male. The mean age of patients was 45.8±11.9 years. One-hundred ninety-six (29.3%) patients reported 315 adverse events, and four patients reported seven serious adverse events, without any fatal events. There was one potential case of an abacavir hypersensitivity reaction. Among the 97 adverse drug reactions that were reported from 75 patients, the most frequent adverse drug reactions included diarrhea (12 events), dyspepsia (10 events), and rash (9 events). No ischemic heart disease was observed. In the effectiveness analysis, 91% of patients achieved HIV-1 RNA under 50 copies/mL after 24 months of observation with abacavir administration. CONCLUSION: Our data showed the safety and effectiveness of Ziagen in a real-world setting. During the study period, Ziagen was well-tolerated, with one incident of a clinically suspected abacavir hypersensitivity reaction. The postmarketing surveillance of Ziagen did not highlight any new safety information. These data may be helpful in understanding abacavir and the HIV treatment practices in Korea.

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.

Compiling Multicopy Single-Stranded DNA Sequences from Bacterial Genome Sequences

A retron is a bacterial retroelement that encodes an RNA gene and a reverse transcriptase (RT). The former, once transcribed, works as a template primer for reverse transcription by the latter. The resulting DNA is covalently linked to the upstream part of the RNA; this chimera is called multicopy single-stranded DNA (msDNA), which is extrachromosomal DNA found in many bacterial species. Based on the conserved features in the eight known msDNA sequences, we developed a detection method and applied it to scan National Center for Biotechnology Information (NCBI) RefSeq bacterial genome sequences. Among 16,844 bacterial sequences possessing a retron-type RT domain, we identified 48 unique types of msDNA. Currently, the biological role of msDNA is not well understood. Our work will be a useful tool in studying the distribution, evolution, and physiological role of msDNA.

Distinctive Drug-resistant Mutation Profiles and Interpretations of HIV-1 Proviral DNA Revealed by Deep Sequencing in Reverse Transcriptase / 生物医学与环境科学（英文）

<p><b>OBJECTIVE</b>To investigate distinctive features in drug-resistant mutations (DRMs) and interpretations for reverse transcriptase inhibitors (RTIs) between proviral DNA and paired viral RNA in HIV-1-infected patients.</p><p><b>METHODS</b>Forty-three HIV-1-infected individuals receiving first-line antiretroviral therapy were recruited to participate in a multicenter AIDS Cohort Study in Anhui and Henan Provinces in China in 2004. Drug resistance genotyping was performed by bulk sequencing and deep sequencing on the plasma and whole blood of 77 samples, respectively. Drug-resistance interpretation was compared between viral RNA and paired proviral DNA.</p><p><b>RESULTS</b>Compared with bulk sequencing, deep sequencing could detect more DRMs and samples with DRMs in both viral RNA and proviral DNA. The mutations M184I and M230I were more prevalent in proviral DNA than in viral RNA (Fisher's exact test, P<0.05). Considering 'majority resistant variants', 15 samples (19.48%) showed differences in drug resistance interpretation between viral RNA and proviral DNA, and 5 of these samples with different DRMs between proviral DNA and paired viral RNA showed a higher level of drug resistance to the first-line drugs. Considering 'minority resistant variants', 22 samples (28.57%) were associated with a higher level of drug resistance to the tested RTIs for proviral DNA when compared with paired viral RNA.</p><p><b>CONCLUSION</b>Compared with viral RNA, the distinctive information of DRMs and drug resistance interpretations for proviral DNA could be obtained by deep sequencing, which could provide more detailed and precise information for drug resistance monitoring and the rational design of optimal antiretroviral therapy regimens.</p>

Receptor for Hepatitis B and D Virus / 대한내과학회지

Chronic hepatitis B affects 400 million people worldwide and is one of the leading causes of liver-related morbidity and mortality. All clinically available hepatitis B virus (HBV) drugs are nucleoside or nucleotide analogs that inhibit viral reverse transcriptase (RT) activity. Resistance to these HBV drugs has been widely reported, and is due to specific mutations in the viral RT domain. Therefore, the development of new, non-polymerase targeting anti-HBV agents is urgently needed. A potential drug target, the HBV receptor that mediates the viral entry process, has been recently identified using human primary hepatocytes, northern tree shrew (Tupaia belangeri) hepatocytes, and HepaRG cells. A transporter of bile acids, sodium taurocholate cotransporting polypeptide (NTCP), was identified as the receptor for HBV and hepatitis D virus, and the transport function of NTCP was correlated with HBV entry. Therefore, functional inhibitors of NTCP may inhibit HBV infection, and viral entry was blocked by several NTCP receptor-targeting compounds. The HBV receptor is an attractive target for development of entry inhibitors, and serves as a model for the mechanistic study of HBV entry and infection. This review will summarize the characteristics and clinical importance of NTCP, and will discuss the potential therapeutic use of NTCP inhibitors to prevent HBV entry.

Variability of Reverse Transcriptase Gene and S Gene in Lamivudine-treated Chronic Hepatitis B Patients / 病毒学报

We wished to undertake molecular characterization of the reverse transcriptase (RT) gene and overlapping surface (S) gene in lamivudine-treated patients with chronic infection with the hepatitis B virus (HBV). Sequencing analyses of the HBV RT/S gene of isolates from 25 chronic hepatitis B (CHB) patients with the YMDD mutation and 30 treatment-naïve CHB patients were undertaken. In patients with the YMDD mutation, rtM2041 was the major type of mutation (20/25, 80%). rtL80I was present in most of the patients with rtM204I (14/20, 70%). rtL180M coexisted with rtM204V (5/5, 100%). Patients with the YMDD mutation had a significantly higher prevalence of mutation of the RT gene than treatment-naïve CHB patients (P < 0.05). Classical primary resistance and secondary/compensatory mutations were detected at only five sites (rtL80, rtV173, rtL180, rtM204, rtM250) in CHB patients with the YMDD mutation. The frequency of nucleos(t)ide analog resistance (NAr) mutation within the RT gene in patients with the YMDD mutation was significantly higher than that in treatment-naïve patients (P < 0.05). Amino-acid mutations within the RT gene were also associated with other types of NAr in patients with the YMDD mutation. The rate of amino-acid variants within the S gene region was significantly higher in patients with the YMDD mutation than that in treatment-naïve patients (P < 0.05). sM133L and sG145R variants were also present in patients with the YMDD mutation. These observations suggest that CHB patients with the YMDD mutation also have NAr mutations related to other NA drugs, which might lead to cross-resistance in CHB patients. Variants present in the S gene region could cause changes in the antigenicity of HBsAg, which could result in a false-negative diagnosis of HBsAg and immune in escape of the HBV.