Infección congénita por citomegalovirus en neonatos muy bajo peso / Congenital cytomegalovirus infection in very low birth weight neonates

Introducción: La infección congénita por el citomegalovirus en neonatos menores de 1500 gramos puede ser causa de morbilidad, mortalidad y discapacidad. Objetivo: Describir el comportamiento de la infección congénita por citomegalovirus en un servicio de neonatología. Métodos: Se realizó un estudio descriptivo y transversal con 61 neonatos. Se les realizó detección de citomegalovirus en la primera semana de vida en suero y orina, mediante reacción en cadena de la polimerasa, para determinar infección congénita. Se evaluaron variables perinatales en todos los neonatos, así como elementos clínicos y resultados de exámenes complementarios en los infectados. Resultados: La incidencia de infección congénita fue de un 10 por ciento (6/61). El 5 por ciento de los estudios fueron positivos (6/122). Ninguna muestra de orina resultó positiva (0/61) y en el 10 por ciento de las muestras de suero (6/61) se detectó el genoma del virus. Se encontró asociación entre valoración nutricional al nacer e infección por citomegalovirus (p< 0,05). El 83 por ciento de los neonatos infectados presentaron algún signo clínico y el síndrome de dificultad respiratoria fue el más frecuente (67 por ciento). En todos los neonatos con infección congénita el ultrasonido cerebral fue normal y en el 33 por ciento se detectó retinopatía de la prematuridad en el fondo de ojo. Conclusiones: La incidencia de infección congénita por citomegalovirus es alta en este grupo de riesgo. Los signos clínicos encontrados y los resultados del fondo de ojo en neonatos con infección congénita se relacionaron con la prematuridad y la valoración nutricional de hipotrófico se asoció con esta infección(AU)

Introduction: Congenital cytomegalovirus infection in neonates weighing less than 1500 grams can be a cause of morbidity, mortality, and disability. Objective: To describe the behavior of congenital cytomegalovirus infection in a neonatal service. Methods: A descriptive and cross-sectional study was conducted with 61 neonates. Cytomegalovirus was detected in the first week of life in serum and urine, by polymerase chain reaction, to determine congenital infection. Perinatal variables were evaluated in all neonates, as well as clinical elements and results of complementary examinations in infected infants. Results: The incidence of congenital infection was 10 percent (6/61). 5 percent of the studies were positive (6/122). No urine samples were positive (0/61) and the virus genome was detected in 10 percent of serum samples (6/61). An association was found between nutritional assessment at birth and cytomegalovirus infection (p < 0.05). A total of 83 percent of infected neonates had some clinical sign, with respiratory distress syndrome being the most common (67 percent). In all neonates with congenital infection, brain ultrasound was normal, and retinopathy of prematurity was detected in 33 percent of patients with fundus retinopathy. Conclusions: The incidence of congenital cytomegalovirus infection is high in this risk group. The clinical signs found and the results of the fundus in neonates with congenital infection were related to prematurity and the nutritional assessment of hypotrophic was associated with this infection(AU)

On the Sensitivity of the Virion Envelope to Lipid Peroxidation.

Emerging viruses are a public health threat best managed with broad spectrum antivirals. Common viral structures, like capsids or virion envelopes, have been proposed as targets for broadly active antiviral drugs. For example, a number of lipoperoxidators have been proposed to preferentially affect viral infectivity by targeting metabolically inactive enveloped virions while sparing metabolically active cells. However, this presumed preferential virion sensitivity to lipoperoxidation remains untested. To test whether virions are indeed more sensitive to lipoperoxidation than are cells, we analyzed the effects of two classic generic lipoperoxidators: lipophilic 2,2'-azobis(2,4-dimethylvaleronitrile) (AMVN) and hydrophilic 2,2'-azobis(2-methylpropionamidine) dihydrochloride (AAPH) on Vero and human foreskin fibroblasts (HFF) cell viability, HSV-1 plaquing efficiency, and virion and cell lipoperoxidation. Cells or virions were incubated with the lipoperoxidators at 37°C for 2 h or incubated in atmospheric O2, and dose responses (half maximal cytotoxic and effective concentration [CC50 and EC50]) were evaluated by three or four parameter regression. The HSV-1 virions were slightly more sensitive to lipoperoxidators than were the cells (selectivity index [SI], 3.3 to 7.4). The effects of the lipophilic AMVN on both cell and virion viability directly correlated with the extent of membrane lipoperoxidation as evaluated by two different probes, C11-Bodipy and liperfluo. Moreover, the hydrophilic AAPH-induced virion inactivation at lower concentrations than did lipoperoxidation. Known lipoperoxidators inhibit infectivity via lipoperoxidation-independent mechanisms. Antioxidants protected against a loss of viral infectivity by less than 5-fold. Carrier bovine serum albumin (BSA) protected against both peroxidators to a similar extent when present together with the lipoperoxidating agents, suggesting that BSA quenches them as expected. Virions incubated in atmospheric oxidative conditions suffered losses of infectivity that were similar to those of chemically peroxidated virions, and they were protected by water soluble vitamin C and BSA with no evident lipoperoxidation, indicating predominant peroxidative damage to nonlipid virion components. Thus, lipoperoxidation is not a mechanism by which to specifically inhibit the infectivity of enveloped viruses, and the effects of known lipoperoxidators on virion infectivity are not solely mediated by lipoperoxidation. IMPORTANCE Small molecules that induce lipoperoxidation have been proposed repeatedly as potential antiviral drugs based on a presumed unique sensitivity of virions to this type of damage. Several small molecules that inactivate virions without affecting cells have been proposed to act primarily by inducing lipoperoxidation. However, the preferential sensitivity of virions to lipoperoxidators had not been experimentally evaluated. Using two of the best characterized small molecule lipoperoxidators, which are widely considered to be the prototypical water soluble and liposoluble lipoperoxidators, we show that lipoperoxidators have no preference for virions over cells. Moreover, they also inactivate virions by mechanisms other than the induction of lipoperoxidation. Therefore, the general induction of lipoperoxidation is not a path by which to develop antivirals. Moreover, molecules with specific antiviral activity which are not cytotoxic and have no preference to localize to virions over cells are unlikely to act primarily by inducing lipoperoxidation.

[Genetic analysis of the mutations in HIV-1 infected population in Ecuador]. / Análisis genético de las mutaciones presentes en las poblaciones virales en pacientes con infección por VIH-1 en Ecuador.

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

HIV-1 Antiretroviral Resistance in Cuba, 2009-2014.

INTRODUCTION By the end of 2017, there were more than 28,000 individuals living with HIV in Cuba, over 80% receiving antiretroviral therapy, which dramatically reduces viral replication, improves immune status and decreases risk of transmission. These results could be jeopardized by emergence of HIV-1 drug resistance. In 2009, a test for HIV-1 genotypic resistance was introduced in routine clinical practice in Cuba. OBJECTIVE Investigate antiretroviral resistance and its relation to subtype distribution in HIV-1 treatment-naïve and previously treated patients in Cuba. METHODS Resistance and HIV-1 subtype distribution were determined in 342 antiretroviral treatment-naïve patients and 584 previously treated for HIV-1 whose blood specimens were sent to the Pedro Kourí Tropical Medicine Institute during 2009-2014. Transmitted drug resistance was determined using the Calibrated Population Resistance Tool v.6. Drug resistance analysis was conducted using the algorithm Rega v9.1.0. RESULTS Prevalence of transmitted drug resistance was 11.4%, and 41% of mutated viruses exhibited dual-class resistance to nucleoside reverse transcriptase inhibitor and non-nucleoside reverse transcriptase inhibitor. Overall, 84.9% of patients had ≥1 resistance mutation, 80% had ≥1 nucleoside reverse transcriptase inhibitor mutation, 71.4% had ≥1 non-nucleoside reverse transcriptase inhibitor mutation and 31.7% had ≥1 protease inhibitor mutation. K65R and K101E mutations were significantly more frequent in subtype C, L210W in CRF19_cpx, and M47V/I in CRF BGs (20, 23, 24). Full class resistance to nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, protease inhibitors and multidrug resistance were detected in 21.2%, 32.4%, 8% and 4.1% of patients, respectively. Average percentage resistance to nucleoside reverse transcriptase inhibitor, protease inhibitor, full class resistance to nucleoside reverse transcriptase inhibitor, protease inhibitor and multidrug resistance increased in patients failing two or more regimens. Nevertheless, after 2011, a declining trend was observed in the frequency of multidrug resistance and full class resistance to nucleoside reverse transcriptase inhibitors and protease inhibitors. CONCLUSIONS Detected levels of transmitted drug resistance highlight the need for a national surveillance study in treatment-naïve patients. Resistance prevalence is high in previously treated patients but appears to be decreasing over time. The frequency of resistance mutations in recombinant forms of HIV in Cuba needs further study. KEYWORDS Antiretroviral therapy, highly active antiretroviral therapy, HIV, anti-HIV agents, drug resistance, multiple drug resistance, Cuba.

Análisis genético de las mutaciones presentes en las poblaciones virales en pacientes con infección por VIH-1 en Ecuador / Genetic analysis of the mutations in HIV-1 infected population in Ecuador

Resumen Introducción Las recomendaciones internacionales de tratamiento anti-retroviral incluyen pruebas de resistencia para orientar el régimen de tratamiento en cada paciente, lo que no está disponible de forma estable en Ecuador. Objetivo Describir las mutaciones que confieren resistencia a anti-retrovirales en una población de pacientes ecuatorianos. Metodología A partir de muestras de plasma de 101 pacientes con VIH-1 con fallo a la terapia anti-retroviral, 15 niños y 86 adultos, se realizó pirosecuenciación con el GS Junior (Roche) y se analizaron las secuencias con el programa DeepChek. Resultados Las mutaciones más frecuentes fueron M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L y L90M en adultos, y F77L, K103N/S, M46L/I, V82T/F/A/S/L y L90M en niños. Se encontró una elevada resistencia a los inhibidores de la transcriptasa reversa (TR) no análogos de nucleósidos en poblaciones minoritarias virales de adultos y niños (34,9 y 70%, respectivamente), en los niños, tanto las poblaciones virales mayoritarias como minoritarias, fueron resistente a inhibidores de proteasa (> 45%). Los pacientes que tuvieron un mayor número de esquemas terapéuticos presentaron mayores niveles de resistencia a los anti-retrovirales. La mayoría de las muestras fueron del subtipo B en la región de la TR y proteasa, y CRF25_cpx en integrasa. Conclusiones Se muestran las mutaciones y la resistencia a antiretrovirales en una población de pacientes ecuatorianos con infección por VIH-1, que permitirán realizar un llamado de alerta a las autoridades de salud sobre la necesidad de realizar estudios de resistencia.

Background The international recommendations of antiretroviral treatment include resistance tests to guide the treatment regimen in each patient, which is not available on a regular basis in Ecuador. Aim To describe mutations that confer resistance to antiretrovirals in a population of Ecuadorian patients. Methods Plasma samples from 101 HIV-1 patients with failure to antiretroviral therapy, divided into 15 children and 86 adults, were studied with the GS Junior (Roche) and the sequences were analyzed with the DeepChek program. Results The most frequent mutations were M184V/I, K101E/P/H, K103N/S, D30N, M46L/I, I54L/M, V82T/F/A/S/L and L90M in adults and F77L, K103N/S, M46L/I, V82T/F/A/S/L and L90M in children. High resistance to non-nucleoside reverse transcriptase (RT) inhibitors in minority viral populations of adults and children (34.9% and 70%) was detected; in children both viral populations (majority and minority viral populations) (> 45%) were protease inhibitor resistant. Patients who had a greater number of therapeutic regimens had higher levels of resistance to antiretrovirals. Most of the samples were subtype B in the TR and protease region, and CRF25_cpx in integrase. Conclusions Mutations and resistance to antiretrovirals are shown in a population of Ecuadorian patients with HIV-1. These results will make it possible to issue a warning to health authorities about the need for resistance studies.

Herpesviruses excretion in saliva of pediatric transplant recipients.

BACKGROUND: Saliva samples could be used for follow-up of herpesviruses infection in pediatric transplant recipients. OBJECTIVE: With the aim of determining the frequency of herpesviral infections in saliva samples after transplantation, and the association with viremia and complications, a pilot longitudinal follow-up of pediatric Cuban transplanted recipients (kidney and liver) was performed. METHODS: Quantitative real-time polymerase chain reaction of cytomegalovirus (CMV), Epstein-Barr virus, herpes simplex virus, human herpesevirus-6 (HHV6), varicella zoster virus, and human herpesvirus-8 were serially assayed in saliva and serum samples from 27 transplanted patients, during 32 weeks after the graft. Samples taken immediately after the graft were used as control samples. RESULTS: Herpesviruses were detected in 88.9% of saliva and in 37.0% of serum samples. HHV6 and CMV were the viruses more frequently detected (70.4%) in saliva and they were significantly more frequent during the follow-up in comparison with control samples (P < .05). Most patients (22/27) had more than one virus shedding concurrently. Patients with CMV in saliva were associated with CMV viremia (P = .009), particularly at the cutoff of 252.5 copies/mL, with a less accurate level of area under the curve. No association between CMV viral load in saliva and viral disease or response to the antiviral treatment was observed. CONCLUSIONS: The association found between CMV shedding in saliva and CMV viremia in this study opens the possibility of future studies of using viral load in saliva as a predictor of viremia. The implementation of herpesviral load in saliva samples for early clinical intervention in pediatric recipients needs a study with a large number of samples for further conclusions.

Detección de Chlamydia trachomatis en muestras de exudado endocervical mediante una prueba de diagnóstico rápido y dos técnicas de reacción en cadena de la polimerasa / Detection of Chlamydia trachomatis in endocervical swab using rapid test and two reaction techniques polymerase chain

Introducción: Chlamydia trachomatis es el principal agente bacteriano que produce infecciones de transmisión sexual.Objetivo: detectar la presencia de C. trachomatis utilizando una prueba de diagnóstico rápido y compararla con la reacción en cadena de la polimerasa (RCP).Métodos: se procesaron 50 muestras de exudado endocervical, de mujeres sintomáticas del municipio 10 de Octubre. A las muestras se les aplicó la prueba Chlamy-check-1, un ensayo de RCP del gen del plásmido críptico y una RCP en tiempo real (RCP-TR) de la proteína mayor de la membrana externa (MOMP) de C. trachomatis, que fue utilizada como referencia. Se calculó, sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN).Resultados: de las muestras estudiadas, 44 resultaron positivas por la prueba rápida, mientras que por la RCP del plásmido críptico solo 3 muestras (6 por ciento) amplificaron. Al aplicar la RCP-TR, 4 muestras (8 por ciento) se confirmaron como positivas, coincidiendo 3 por los tres métodos de diagnóstico. Al evaluar la prueba Chlamy-check-1 frente a la prueba de referencia se observó una sensibilidad de 100 por ciento, mientras que la especificidad fue de 13 por ciento, así como un VPP de 9,1 por ciento y VPN de 100 por ciento. Por el contrario, la RCP del plásmido críptico mostró una sensibilidad y especificidad de 75 y 100 por ciento, respectivamente; un VPP de 100 por ciento y VPN de 97,9 por ciento.Conclusiones: se obtuvo diferencia entre los porcentajes de positividad detectados con la prueba rápida, y las técnicas de RCP. La baja especificidad de la prueba rápida indica la necesidad de realizar estudios de evaluación de este estuche diagnóstico(AU)

Introduction: Chlamydia trachomatis is the leading bacterial agent that causes sexually transmitted infections.Objective: to detect the presence of C. trachomatis using a rapid test and compare it with the chain reaction (PCR).Methods: 50 endocervical exudates taken from symptomatic women were processed in Diez de October municipality. The samples were applied the Chlamy-check-1 test, a PCR assay of the cryptic plasmid gene and a real-time PCR (RT-PCR) of major outer membrane protein (MOMP) of C. trachomatis which was used as reference. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive value were calculated.Results: 44 samples were positive by the rapid test, whereas only three samples (6 percent) amplified by cryptic plasmid PCR. Applying RT-PCR, 4 samples (8 percent) were confirmed as positive, 3 samples matched with three diagnostic methods. In assessing the Chlamy-check-1 versus the reference test, 100 percent of sensitivity was observed, while the specificity was 13 percent> Also PPV was 9.1percent and NPV was 100 percent. On the contrary, the cryptic plasmid PCR had 75 and 100 percent of sensitivity and specificity respectively, 100 percent PPV and 97.9 percent NPV.Conclusions: the difference was obtained between the percentages of positivity detected with both the rapid test, and CPR techniques. The low specificity of the rapid test indicates the need for further studies to evaluate this diagnostic kit(AU)

Detección de Chlamydia trachomatis en muestras de exudado endocervical mediante una prueba de diagnóstico rápido y dos técnicas de reacción en cadena de la polimerasa / Detection of Chlamydia trachomatis in endocervical swab using rapid test and two reaction techniques polymerase chain

Introducción: Chlamydia trachomatis es el principal agente bacteriano que produce infecciones de transmisión sexual.Objetivo: detectar la presencia de C. trachomatis utilizando una prueba de diagnóstico rápido y compararla con la reacción en cadena de la polimerasa (RCP).Métodos: se procesaron 50 muestras de exudado endocervical, de mujeres sintomáticas del municipio 10 de Octubre. A las muestras se les aplicó la prueba Chlamy-check-1, un ensayo de RCP del gen del plásmido críptico y una RCP en tiempo real (RCP-TR) de la proteína mayor de la membrana externa (MOMP) de C. trachomatis, que fue utilizada como referencia. Se calculó, sensibilidad, especificidad, valor predictivo positivo (VPP) y negativo (VPN).Resultados: de las muestras estudiadas, 44 resultaron positivas por la prueba rápida, mientras que por la RCP del plásmido críptico solo 3 muestras (6 porciento) amplificaron. Al aplicar la RCP-TR, 4 muestras (8 porciento) se confirmaron como positivas, coincidiendo 3 por los tres métodos de diagnóstico. Al evaluar la prueba Chlamy-check-1 frente a la prueba de referencia se observó una sensibilidad de 100 porciento, mientras que la especificidad fue de 13 porciento, así como un VPP de 9,1 porciento y VPN de 100 porciento. Por el contrario, la RCP del plásmido críptico mostró una sensibilidad y especificidad de 75 y 100 porciento, respectivamente; un VPP de 100 porciento y VPN de 97,9 porciento.Conclusiones: se obtuvo diferencia entre los porcentajes de positividad detectados con la prueba rápida, y las técnicas de RCP. La baja especificidad de la prueba rápida indica la necesidad de realizar estudios de evaluación de este estuche diagnóstico.

Introduction: Chlamydia trachomatis is the leading bacterial agent that causes sexually transmitted infections.Objective: to detect the presence of C. trachomatis using a rapid test and compare it with the chain reaction (PCR).Methods: 50 endocervical exudates taken from symptomatic women were processed in Diez de October municipality. The samples were applied the Chlamy-check-1 test, a PCR assay of the cryptic plasmid gene and a real-time PCR (RT-PCR) of major outer membrane protein (MOMP) of C. trachomatis which was used as reference. Sensitivity, specificity, positive (PPV) and negative (NPV) predictive value were calculated.Results: 44 samples were positive by the rapid test, whereas only three samples (6 percent) amplified by cryptic plasmid PCR. Applying RT-PCR, 4 samples (8 percent) were confirmed as positive, 3 samples matched with three diagnostic methods. In assessing the Chlamy-check-1 versus the reference test, 100 percent of sensitivity was observed, while the specificity was 13 percent> Also PPV was 9.1percent and NPV was 100 percent. On the contrary, the cryptic plasmid PCR had 75 and 100 percent of sensitivity and specificity respectively, 100 percent PPV and 97.9 percent NPV.Conclusions: the difference was obtained between the percentages of positivity detected with both the rapid test, and CPR techniques. The low specificity of the rapid test indicates the need for further studies to evaluate this diagnostic kit.

Normalización de un sistema de reacción en cadena de la polimerasa en tiempo real para la cuantificación del herpesvirus humano 8 / Standardization of a real-time polymerase chain reaction system for quantification of human herpesvirus 8

OBJETIVO: normalizar un sistema de reacción en cadena de la polimerasa en tiempo real para determinar la carga viral del herpesvirus humano 8, en diferentes muestras clínicas de pacientes en los que se sospeche la infección por este agente. MÉTODOS: se evaluaron 3 de los métodos reportados internacionalmente para obtener ADN estándar en la construcción de curvas externas estándar, que permiten determinar el número de copias de ADN diana en la muestra problema. RESULTADOS: se obtuvieron 3 ADN estándar a partir del clonaje de un fragmento del gen ORF26 del herpesvirus humano 8 en un vector (ADN plasmídico), con la utilización de productos purificados de reacción en cadena de la polimerasa y el empleo de ADN genómico de la línea celular BCBL. Se pudieron construir las curvas patrón a partir de cada uno de los ADN estándar obtenidos, los que mostraron una fuerte correlación lineal (r= -1) y valores muy bajos de error a lo largo de 6 magnitudes de concentración de ADN diana. El límite inferior de detección a partir del ADN plasmídico y de los productos de reacción en cadena de la polimerasa fue de hasta 100 copias, mientras que con el ADN genómico fue de hasta 10 copias; este último sistema resultó el más sensible. CONCLUSIONES: la reacción en cadena de la polimerasa en tiempo real normalizada a partir de los 3 ADN estándar probó ser un sistema rápido, específico y altamente sensible que permitirá un mejor diagnóstico y además desarrollar estudios sobre la patogenia de la infección por el herpesvirus humano 8 en Cuba.

OBJECTIVE: to standardize a real-time polymerase chain reaction system to determine the human herpes virus 8 viral load in several samples from patients suspected of this type of infection. METHODS: three internationally known methods were evaluated to obtain standard DNA in standard external curve constructions, which allow determining the number of target DNA copies in the suspected samples. RESULTS: three standards DNA were obtained from cloning ORF26 gene fragment of human herpesvirus 8 in a vector (plasmid DNA), with the use of purified polymerase chain reaction products and of genomic DNA of BCBL cell lines. The pattern curves were constructed on the basis of each of the resulting standard DNA, which showed strong linear correlation (r= -1) and very low error values throughout 6 target DNA concentrations. The lower detection limit based on plasmid DNA and the polymerase chain reaction products was 100 copies, whereas that obtained with genomic DNA reached up to 10 copies; this last system turned to be the most susceptible. CONCLUSIONS: real-time polymerase chain reaction system, standardized for the three standard DNA proved to be a rapid, specific and highly sensitive system for better diagnosis, and for the development of studies on the pathogenesis of human herpesvirus 8 infection in Cuba.

Normalización de un sistema de reacción en cadena de la polimerasa en tiempo real para la cuantificación del herpesvirus humano 8 / Standardization of a real-time polymerase chain reaction system for quantification of human herpesvirus 8

OBJETIVO: normalizar un sistema de reacción en cadena de la polimerasa en tiempo real para determinar la carga viral del herpesvirus humano 8, en diferentes muestras clínicas de pacientes en los que se sospeche la infección por este agente. MÉTODOS: se evaluaron 3 de los métodos reportados internacionalmente para obtener ADN estándar en la construcción de curvas externas estándar, que permiten determinar el número de copias de ADN diana en la muestra problema. RESULTADOS: se obtuvieron 3 ADN estándar a partir del clonaje de un fragmento del gen ORF26 del herpesvirus humano 8 en un vector (ADN plasmídico), con la utilización de productos purificados de reacción en cadena de la polimerasa y el empleo de ADN genómico de la línea celular BCBL. Se pudieron construir las curvas patrón a partir de cada uno de los ADN estándar obtenidos, los que mostraron una fuerte correlación lineal (r= -1) y valores muy bajos de error a lo largo de 6 magnitudes de concentración de ADN diana. El límite inferior de detección a partir del ADN plasmídico y de los productos de reacción en cadena de la polimerasa fue de hasta 100 copias, mientras que con el ADN genómico fue de hasta 10 copias; este último sistema resultó el más sensible. CONCLUSIONES: la reacción en cadena de la polimerasa en tiempo real normalizada a partir de los 3 ADN estándar probó ser un sistema rápido, específico y altamente sensible que permitirá un mejor diagnóstico y además desarrollar estudios sobre la patogenia de la infección por el herpesvirus humano 8 en Cuba(AU)

OBJECTIVE: to standardize a real-time polymerase chain reaction system to determine the human herpes virus 8 viral load in several samples from patients suspected of this type of infection. METHODS: three internationally known methods were evaluated to obtain standard DNA in standard external curve constructions, which allow determining the number of target DNA copies in the suspected samples. RESULTS: three standards DNA were obtained from cloning ORF26 gene fragment of human herpesvirus 8 in a vector (plasmid DNA), with the use of purified polymerase chain reaction products and of genomic DNA of BCBL cell lines. The pattern curves were constructed on the basis of each of the resulting standard DNA, which showed strong linear correlation (r= -1) and very low error values throughout 6 target DNA concentrations. The lower detection limit based on plasmid DNA and the polymerase chain reaction products was 100 copies, whereas that obtained with genomic DNA reached up to 10 copies; this last system turned to be the most susceptible. CONCLUSIONS: real-time polymerase chain reaction system, standardized for the three standard DNA proved to be a rapid, specific and highly sensitive system for better diagnosis, and for the development of studies on the pathogenesis of human herpesvirus 8 infection in Cuba(AU)