Infecciones por Chlamydia trachomatis en una unidad de cuidados intensivos neonatales / Chlamydia trachomatis infections in a neonatal intensive care unit

Las infecciones por Chlamydia trachomatis han aumentado su prevalencia, especialmente en jóvenes embarazadas. Esto adquiere relevancia en pediatría por el elevado riesgo de transmisión vertical al neonato y su potencial gravedad en el lactante. Estas infecciones requieren de un alto índice de sospecha, por cuadro clínico atípico y signos radiológicos inespecíficos. Los métodos diagnósticos convencionales presentan limitaciones para su detección. Las técnicas moleculares son las recomendadas por su elevada sensibilidad, especificidad y rapidez, lo cual permite una terapéutica adecuada y oportuna. En este estudio, desarrollado en una unidad de cuidados intensivos neonatales de un hospital de alta complejidad durante 12 años, se describieron las características de la población, su presentación clínica y evolución. La detección microbiológica se realizó por métodos moleculares. Se incluyeron 29 pacientes (p) con infección por C. trachomatis (3,9% del total de muestras enviadas),13 p con infección respiratoria y 16 p con compromiso ocular. La mediana de edad fue de 19 días al momento del diagnóstico y el 65% de las gestantes tenía <25 años. Veinticuatro p (83%) eran recién nacidos a término y 23 p (79%) previamente sanos. Nueve p (31%) presentaron fiebre al momento del ingreso y 12 (41%) eosinofilia. De los 13 p con enfermedad respiratoria, 9 (69%) consultaron por tos y 11 (85%) con hipoxemia, con requerimientos de oxígeno en 8 (61%), asistencia respiratoria mecánica en 3 (23%) y uno (16%) requirió ECMO. Los hallazgos radiológicos mostraron un patrón intersticial inespecífico. Nueve p (31%) presentaron coinfección y uno falleció asociado a influenza A (AU)

The prevalence of Chlamydia trachomatis infections has increased, especially among young pregnant women. This is of particular relevance in pediatrics due to the high risk of motherto-child transmission and the potential severity of the infection in infants. A high index of suspicion is required for these infections due to the atypical clinical features and non-specific radiological signs. The usefulness of conventional diagnostic methods is limited. Molecular techniques are recommended because of their high sensitivity, specificity, and speed, allowing for adequate and timely treatment. In this 12-year study conducted in a neonatal intensive care unit of a tertiary-care hospital, patient characteristics, clinical presentation, and outcome are described. Microbiological detection was performed using molecular methods. Twenty-nine patients with C. trachomatis infection (3.9% of the total samples submitted), of whom 13 had respiratory tract infection and 16 ocular involvement, were included. The median age at diagnosis was 19 days and 65% of the mothers were <25 years old. Twenty-four p (83%) were term newborns and 23 patients (79%) were previously healthy. On admission, 9 patients (31%) had fever and 12 (41%) had eosinophilia. Of the 13 patients with respiratory tract involvement, 9 (69%) consulted for cough and 11 (85%) had hypoxemia, requiring oxygen in 8 (61%), mechanical ventilation in 3 (23%), and ECMO in 1 (16%). Radiological findings showed a nonspecific interstitial pattern. Nine patients (31%) presented with coinfection, one of whom died due to an associated influenza A infection (AU)

Detección rápida de aneuploidías para la definición de incompatibilidad con la vida extrauterina independiente / Rapid detection of aneuploidies for definition of incompatibility with independent extrauterine life

OBJETIVO: Analizar la implementación de la prueba rápida de reacción en cadena de la polimerasa cuantitativa y fluorescente (QF-PCR) para la detección de aneuploidías. MÉTODO: Se incluyeron todas las pacientes que se realizaron una QF-PCR entre septiembre de 2017 y mayo de 2021. En todos los casos se consignaron los datos clínicos, ecográficos y de laboratorio, y se efectuó un seguimiento de quienes se realizaron además cariograma y su resultado fue normal. RESULTADOS: Se realizaron 213 procedimientos invasivos genéticos prenatales, siendo 72 para detección rápida de aneuploidía mediante QF-PCR. El promedio de edad de las madres con QF-PCR fue de 37 años y 48 pacientes (67%) tenían menos de 15 semanas de gestación. La QF-PCR demostró aneuploidía de los cromosomas 18, 13 y de triploidía en 21 de 49 casos informados como anormales. De los 22 casos sin sugerencia de alteración, 17 accedieron a proseguir el estudio con cariotipo, que resultó anormal en 6 casos. Hubo 4 casos de discordancia entre la QF-PCR y el cariotipo, que pudo afectar el manejo clínico de la gestación. En 25/72 casos (34,7%) la aneuploidía era letal. CONCLUSIONES: Considerando la necesidad de tener un diagnóstico rápido, pero también completo y que permita un consejo genético apropiado, debería integrarse la QF-PCR a un protocolo de diagnóstico que considere variables clínicas y ecográficas.

OBJECTIVE: To analyze the performance of QF-PCR test for the detection of aneuploidies. METHOD: All patients who underwent QF-PCR from September 2017 to May 2021, were included. Clinical, ultrasound and laboratory data were recorded in all cases, as well as follow-up of the cases, including those performing karyotype and the result was normal. RESULTS: 213 prenatal genetic invasive procedures were performed in the study period, 72 for rapid detection of aneuploidy by QF-PCR. 48 patients (67%) were less than 15 weeks at the time of ultrasound diagnosis. The QF-PCR test demonstrated aneuploidy of chromosomes 18, 13, and triploidy in 21/49 cases reported as abnormal. Of the cases without suggestion of alteration (22), 17 agreed to continue the study with a karyotype, which was abnormal in 6 cases. There were 4 cases of discrepancy between QF-PCR and karyotype, which could affect the clinical management of pregnancy. 25/72 cases (34. 7%) corresponded to lethal aneuploidy. CONCLUSIONS: Our results justify the use of QF-PCR. Considering the need to have a rapid diagnosis, but also complete and that allows appropriate genetic counseling, it is that QF-PCR should be integrated into a protocol that considers clinical and ultrasound variables.

Neumomediastino espontáneo en paciente con COVID-19 / Sponteneous pneumomediastinum in patient with COVID-19

Introducción: El neumomediastino espontáneo o síndrome Hamman es una complicación poco frecuente y rara. Se define como la presencia de aire o gas dentro del mediastino sin una causa identificada. Objetivo: Presentar un caso clínico con una complicación poco frecuente, neumomediastino espontáneo, en un paciente con enfermedad por COVID-2019. Caso clínico: Paciente de 86 años con cuadro clínico manifestado por fiebre de 38o C y síntomas respiratorios (tos con secreción blanquecina, disnea de moderados esfuerzos). Se realiza prueba de reacción en cadena de polimerasa para enfermedad por coronavirus 2019, esta fue positiva. Al cuarto día de su hospitalización concurre con empeoramiento clínico dentro que lo destaca tos y disnea progresiva acompañado con saturación de oxigeno menor a 91 por ciento. Se realizaron estudios imagenológicos de alta resolución (angiotomografía computarizada de tórax) en la cual se evidencia la presencia de neumomediastino. Desarrollo: La pandemia por enfermedad por coronavirus 2019 ha dado lugar a una emergencia de salud pública a nivel mundial, en la que es importante que el personal de la salud esté familiarizados con los síntomas, los resultados de imágenes y con las complicaciones de esta enfermedad, como el neumomediastino encontrado en este caso. Conclusión . El neumomediastino espontáneo es una complicación poco frecuente que se presenta en la fase inflamatoria de esta enfermedad(AU)

Introduction: Spontaneous pneumomediastinum or Hamman syndrome is a rare and infrequent complication. It is defined as the presence of air or gas within the mediastinum without an identified cause. Objective: To report a clinical case of spontaneous pneumomediastinum in a patient with COVID-2019, a disease with a rare complication. Clinical case report: We report the case of an 86-year-old patient with a clinical condicion of fever of 38o C and respiratory symptoms (cough with whitish secretions, dyspnea on moderate exertion). He underwent a polymerase chain reaction test for coronavirus disease 2019, which resulted positive. On the fourth day of his hospitalization, he his clinical condition worsened, including cough and progressive dyspnea accompanied by oxygen saturation less than 91 percent. The presence of pneumomediastinum was revealed by high-resolution imaging studies (computed tomography angiography of the chest). Discussion: The 2019 coronavirus disease pandemic has given rise to a global public health emergency, which requires health personnel to be familiar with symptoms, imaging results, and complications of this disease, such as pneumomediastinum found in this case. Conclution: Spontaneous pneumomediastinum is a rare complication that occurs in the inflammatory phase of this disease(AU)

First report of fibropapillomatosis (FP) and Chelonid alphaherpesvirus 5 (ChHV5) in a green sea turtle (Chelonia mydas) from the historically fibropapillomatosis-free Fernando de Noronha Archipelago, Northeastern Brazil / Primeiro caso de fibropapilomatose (FP) e de Chelonid alphaherpesvirus 5 (ChHV5) em tartaruga-verde (Chelonia mydas) no Arquipélago de Fernando de Noronha, Nordeste do Brasil

Fibropapillomatosis (FP) is an infectious disease caused by Chelonid alphaherpesvirus 5 (ChHV5). Nevertheless, its clinical manifestations are considered multifactorial. Due to its relevance, FP is currently monitored in sea turtle populations in the United States, Australia, Caribbean, and Brazil. Between 2000 and 2020, the TAMAR Project/ TAMAR Project Foundation analyzed the prevalence of FP in nine states and oceanic islands along the Brazilian coast, including Fernando de Noronha Archipelago (FNA), a historically FP-free area. A total of 4,435 green sea turtles (Chelonia mydas) were monitored from 2010 to 2016. Additionally, in 2012 and 2014, 43 FP-free skin samples were analyzed for ChHV5 using a qualitative PCR for the UL30 polymerase (pol) sequence. In 2015, a bilateral ocular nodule characterized as an FP tumor was reported in one of the monitored individuals undergoing rehabilitation. Tissue samples were collected following surgical removal of the tumor. Characterization of a 454 bp UL30 polymerase gene revealed a ChHV5 sequence previously reported in other areas of the Atlantic Brazilian coast. In the years following this finding from January 2017 to March 2020, a total of 360 C. mydas were monitored in the same area and no FP tumors were detected. This is the first report of FP and the first detection of ChHV5 in FNA, a finding of great concern considering this site's historical absence of FP occurrence. This study highlights the importance of monitoring this disease in historically FP-free areas of the Brazilian Atlantic coast.(AU)

A fibropapilomatose (FP) é uma doença infecciosa causada pelo Chelonid alphaherpesvirus 5 (ChHV5). No entanto, as manifestações clínicas da doença são consideradas multifatoriais. Esta doença é monitorada atualmente em populações de tartarugas marinhas nos EUA, Austrália, Caribe e Brasil. Desde 2000, o Projeto TAMAR/Fundação Projeto TAMAR analisa a presença de FP em nove estados da costa brasileira e ilhas oceânicas, incluindo o arquipélago de Fernando de Noronha, uma área historicamente livre de FP. Um total de 4.435 indivíduos de Chelonia mydas foram monitorados de 2010 a 2016 e 43 amostras de pele foram analisadas para detectar ChHV5 em 2012 e 2014 com o objetivo de avaliar a presença do vírus em tecidos sem FP, usando uma PCR qualitativa para detecção de sequências do gene da UL30 polimerase. Em 2015, uma tartaruga verde (C. mydas) foi relatada com um nódulo ocular bilateral caracterizado como FP. Amostras de tecido foram coletadas durante sua reabilitação e procedimento cirúrgico para remover o tumor. A caracterização parcial de uma sequência de 454 bp do gene UL30 polimerase detectou ChHV5 anteriormente relatado em outras áreas da costa atlântica brasileira. Após estes achados, de janeiro de 2017 a março de 2020, um total de 360 indivíduos de C. mydas foram monitorados e nenhum caso de FP foi registrado. Este é o primeiro relato de FP e a primeira caracterização de ChHV5 no arquipélago de Fernando de Noronha, uma questão preocupante e que ressalta a importância do monitoramento desta doença em áreas historicamente livres de FP na costa atlântica brasileira.(AU)

The Advances and Application of ABO Blood Group Genotyping Technology --Review / 中国实验血液学杂志

The ABO blood group system is the most important blood group system in clinical transfusion. Serological technology is a routine method for the identification of ABO blood groups, however, which have some limitations in the identification of complicated ABO samples with weakened antigens or antibodies, abnormal plasma proteins, polyagglutination, or cold agglutinin, etc. With the development of molecular biology technology, ABO blood group gene was cloned, and ABO blood group genotyping technology based on DNA was established. The genotyping technologies with different throughputs such as PCR-SSP, Droplet-AS-PCR, PCR-RFLP, PCR-SBT, SNaPshot, MALDI-TOF MS and NGS have emerged. Genotyping has overcome the limitations of serology, and has become an indispensable method to solve difficult blood type, providing strong support for the correct identification of ABO blood group, and providing guarantee for precision blood transfusion. This review summarizes the progress and application of ABO blood group genotyping methods.

Using multiple-fragment amplification combined with Gibson assembly to clone genes with site-directed mutations / 生物工程学报

In order to develop a simple and efficient site-directed mutagenesis solution, the Gibson assembly technique was used to clone the cyclin dependent kinase 4 gene with single or double site mutations, with the aim to simplify the overlap extension PCR. The gene fragments containing site mutations were amplified using a strategy similar to overlap extension PCR. Meanwhile, an empty plasmid was digested by double restriction endonucleases to generate a linearized vector with a short adaptor overlapping with the targeted gene fragments. The gene fragments were directly spliced with the linearized vector by Gibson assembly in an isothermal, single-reaction, creating a recombinant plasmid. After the recombinant plasmids were transformed into competent Escherichia coli DH5α, several clones were screened from each group. Through restriction analysis and DNA sequencing, it was found that the randomly selected clones were 100% target mutants. Since there was neither tedious multiple-round PCR amplification nor frequent DNA extraction operation, and there was no need to digest the original plasmid, this protocol circumvents many factors that may interfere with the conventional site-directed mutagenesis. Hence, genes with single or multiple mutations could be cloned easily and efficiently. In summary, the major defects associated with overlap extension PCR and rolling circle amplification were circumvented in this protocol, making it a good solution for site-directed mutagenesis.

Characterization of a family B DNA polymerase from Thermococcus eurythermalis A501 and its application in PCR / 生物工程学报

DNA polymerases are widely used in PCR and play important roles in life science research and related fields. Development of high-performance DNA polymerases is of great commercial interest as the current commercial DNA polymerases could not fully satisfy the requirements of scientific research. In this study, we cloned and expressed a family B DNA polymerase (NCBI accession number TEU_RS04875) from Thermococcus eurythermalis A501, characterized its enzymatic property and evaluated its application in PCR. The recombinant Teu-PolB was expressed in E. coli and purified with affinity chromatography and ion-exchange chromatography. The enzymatic properties of Teu-PolB were characterized using fluorescence-labeled oligonucleotides as substrates. The application potential of Teu-PolB in PCR was evaluated using the phage λ genomic DNA as a template. Teu-PolB has DNA polymerase and 3'→5' exonuclease activities, and is highly thermostable with a half-life of 2 h at 98 ℃. The most suitable PCR buffer is consisted of 50 mmol/L Tris-HCl pH 8.0, 2.5 mmol/L MgCl2, 60 mmol/L KCl, 10 mmol/L (NH4)2SO4, 0.015% Triton X-100 and 0.01% BSA, and the optimal extension temperature is 68 ℃. Under the optimized conditions, a 4 kb target fragment was successfully amplified with an extension rate of 2 kb/min. The yield of the Teu-PolB amplified-DNA was lower than that of Taq DNA polymerase, but its extension rate and fidelity was higher than that of Taq and Pfu DNA polymerases. The biochemical properties of Teu-PolB demonstrate that this enzyme can be used in PCR amplification with high thermostability, good salt tolerance, high extension rate and high fidelity.

Polimorfismo genético del correceptor CCR5 en pacientes cubanos VIH/sida de la tercera edad / Genetic polymorphism of the CCR5 coreceptor in Cuban elderly HIV/AIDS patients

Introducción: El polimorfismo en algunos genes de quimiocinas se asocia con resistencia a la infección por VIH-1, en este sentido la presencia de la mutación Δ32 del correceptor CCR5 en homocigosis, se relaciona con resistencia a la infección y la mutación heterocigótica con un retraso en la progresión de la enfermedad. Objetivos: Identificar la frecuencia del polimorfismo genético del correceptor CCR5 en los pacientes bajo estudio, así como su relación con los niveles de linfocitos T CD4+, la carga viral y las enfermedades oportunistas. Métodos: Se realizó un estudio de corte transversal en 45 pacientes VIH/sida de la tercera edad, cubanos atendidos en el Centro Hospitalario Universitario del IPK durante los meses de enero a mayo de 2019 en el servicio de Medicina del Centro Hospitalario Universitario del IPK, a los que se les realizó la reacción en cadena de la polimerasa (PCR) para determinar el polimorfismo genético del correceptor CCR5. Resultados: El polimorfismo genético del correceptor CCR5 que predominó fue el homocigótico salvaje con 87 por ciento seguido del heterocigótico Δ32 con 13 por ciento. El 80 por ciento de los pacientes presentaron carga viral no detectable y el 56 por ciento niveles de linfocitos T CD4+ por encima de 350 cél/µL. La enfermedad oportunista que predominó fue la neumonía por Pneumocystis jirovecii en 32 por ciento de los sujetos estudiados. No se observaron diferencias estadísticamente significativas entre el polimorfismo genético del correceptor CCR5 y los niveles de linfocitos T CD4+, la carga viral y las enfermedades oportunistas presentes en los pacientes estudiados. Conclusiones: Los polimorfismos genéticos del correceptor CCR5 hallados fueron el homocigótico salvaje y el heterocigótico-∆32. Fue limitado el polimorfismo del gen en los pacientes estudiados(AU)

Introduction: Polymorphism in some chemokine genes is associated to resistance to HIV-1 infection. Homozygous Δ32 mutation of the CCR5 coreceptor is related to resistance to infection, whereas heterozygous mutation is related to a delay in the progress of the disease. Objectives: Identify the frequency of genetic polymorphism of the CCR5 coreceptor in the patients studied, as well as its relationship to CD4+ T lymphocyte levels, viral load and opportunistic diseases. Methods: A cross-sectional study was conducted of 45 Cuban elderly HIV/AIDS patients attending the Medicine Service of the University Hospital Center at IPK from January to May 2019. These patients underwent polymerase chain reaction testing (PCR) to determine genetic polymorphism of the CCR5 coreceptor. Results: A predominance was found of wild homozygotous genetic polymorphism of the CCR5 coreceptor with 87 percent, followed by heterozygotous Δ32 genetic polymorphism with 13 percent. In 80 percent of the patients studied the viral load was undetectable, whereas in 56 percent CD4+ T lymphocyte levels were above 350 cel/µl. The prevailing opportunistic disease was Pneumocystis jirovecii pneumonia in 32 percent of the subjects. Statistically significant differences were not found between genetic polymorphism of the CCR5 coreceptor and CD4+ T lymphocyte levels, viral load and the opportunistic diseases present in the patients studied. Conclusions: The genetic polymorphisms of the CCR5 coreceptor found in the study were of the wild homozygotous and heterozygotous Δ32 types. Gene polymorphism was limited in the patients studied(AU)

Sistema para la Gestión de Personas Aisladas: solución informática para el Centro de Aislamiento UCI-MINSAP / Sistema para la Gestión de Personas Aisladas: IT solution for the Isolation Center UCI-MINSAP

La aplicación Sistema para la Gestión de Personas Aisladas (SGPA-UCI) fue desarrollada para informatizar el proceso de ingreso, pruebas de PCR y egreso de personas aisladas en el centro de aislamiento instalado en la Universidad de las Ciencias Informáticas (UCI), como parte del enfrentamiento a la Covid-19 en Cuba. Para el desarrollo de la aplicación se utilizó Symfony como framework de desarrollo y para el monitoreo de los datos, se diseñaron tableros con el empleo de Grafana. La solución informática facilitó la gestión de más de 10 mil personas aisladas en el centro de aislamiento, en un período de aproximadamente 6 meses, obteniendo los datos primarios de las personas y facilitando el proceso de ingreso, preparación de PCR, procesamiento de resultados recibidos y proceso de egreso. El sistema para la gestión de personas aisladas permitió el proceso de seguimiento a las personas en el centro de aislamiento UCI-MINSAP durante el enfrentamiento a la pandemia Covid-19(AU)

The System for the Management of Isolated Persons (SGPA-UCI) application was developed to computerize the process of admission, PCR tests and discharge of isolated people in the isolation center created in the University of Computer Sciences (UCI), as part of the confrontation with Covid-19 in Cuba. For the development of the application, Symfony was used as a development framework and for data monitoring, dashboards were designed using Grafana. The computer solution simplified the management of more than 10 thousand people isolated in the isolation center, in a period of approximately 6 months, obtaining the primary data of the people and facilitating the process of admission, preparation of PCR, processing of received results and exit process. The system for the management of isolated people allowed the process of monitoring people in the UCI-MINSAP isolation center during the confrontation with the Covid-19 pandemic(AU)

Técnicas de citogenética y biología molecular para el diagnóstico y seguimiento de la leucemia promielocítica / Cytogenetic and molecular biology techniques for the diagnosis and monitoring of promyelocytic leukemia

Introducción: La leucemia promielocítica es un subtipo de leucemia mieloide aguda que se presenta frecuentemente con una coagulopatía potencialmente mortal, por lo que representa una emergencia médica. En la gran mayoría de los pacientes ocurre la t(15;17)(q24;q21) que genera el gen aberrante PML-RARA. Mediante diferentes técnicas de citogenética y de la biología molecular que detectan dichas aberraciones es posible diagnosticar la entidad de manera inequívoca y estudiar la enfermedad mínima residual. Objetivo: Describir, comparar y analizar las técnicas de citogenética y de la biología molecular que son útiles para el diagnóstico y el seguimiento del paciente con leucemia promielocítica. Así como señalar sus ventajas y limitaciones. Métodos: Se realizó revisión de la bibliografía científica de los últimos cinco años relacionada con el tema a través de PUBMED. Se realizó análisis y resumen de la información. Análisis y síntesis de la información: Se describen dos técnicas de citogenética y tres moleculares basadas en la aplicación de la reacción en cadena de la polimerasa. Se comparan y analizan sus ventajas y limitaciones. Conclusiones: Algunas de estas técnicas son útiles únicamente para el diagnóstico, mientras que otras, por su alta sensibilidad, se recomiendan para el seguimiento del paciente con leucemia promielocítica(AU)

Introduction: Promyelocytic leukemia (PML) is a subtype of acute myeloid leukemia that frequently presents with a potentially fatal coagulopathy, therefore it represents a medical emergency. In the vast majority of patients, the t (15; 17) (q24; q21) occurs, which generates the aberrant gene PML-RARA. Using different cytogenetic and molecular biology techniques that detect these aberrations, it is possible to unequivocally diagnose the entity and study minimal residual disease. Objective: To describe, compare and analyze cytogenetics and molecular biology techniques that are useful for diagnosis and follow-up of the patient with Promyelocytic leukemia. As well as pointing out its advantages and limitations. Methods: A review of the scientific bibliography of the last five years related to the subject was carried out through PUBMED. An analysis and summary of the information was made. Analysis and synthesis of the information: Two cytogenetic and three molecular techniques are described based on the application of the polymerase chain reaction. Its advantages and limitations are compared and analyzed. Conclusions: Some of these techniques are only useful for diagnosis, while others, due to their high sensitivity, are recommended for monitoring the patient with Promyelocytic leukemia(AU)

Identification of genetic variants the CCKAR gene and based on body measurement and carcass quality characteristics in Qinchuan beef cattle (Bos taurus)

BACKGROUND: This study aimed to explore genetic polymorphisms of the CCKAR gene and their relationship with the growth and development of Qinchuan cattle which could be used as molecular markers for the improvement of the breeding of Qinchuan cattle. RESULTS: Here, we have identified seven single nucleotide polymorphisms (SNPs) at loci g. 1463 C>G; g. 1532 T>A; g. 1570 G>A; g. 1594 C>A; g. 1640 T>C; g. 1677 G>C; and g. 1735 C>T in the coding region of the bovine CCKAR gene. The frequencies identified on allelic and genotypic characteristics have shown that all seven SNPs diverged from the Hardy-Weinberg-Equilibrium. The SNP2, SNP3, SNP6 and SNP7 had the lowest polymorphism information content values, and remaining SNPs were found to be moderate (0.25 < PIC < 0.50). The genotype CG in SNP1 at loci g.1463 C>G had the greatest association with WH, HW, CD and CCF, while the genotype TA at the very same loci was associated with BFT, ULA and IMF content in Qinchuan cattle. The CCKAR gene expression level in adipose tissue, small intestine, liver and skeleton muscle was found to be higher, whereas, the expression level of mRNA in organs of other digestive system including reticulum, abomasum and omasum was moderate. Some expression of CCKAR mRNA was found in the large intestine, kidney and rumen. CONCLUSIONS: In summary, our finding suggested that the CCKAR gene could be used as a potential candidate for the improvement of carcass quality and body measurements of Qinchuan cattle.

Evolución de las infecciones por Pneumocystis Jirovecii en el Hospital de Pediatría Juan P. Garrahan / Evolution of Pneumocystis Jirovecii infections at Hospital de Pediatría Juan P. Garrahan

Pneumocystis jirovecii es un hongo oportunista, causante de neumonía en huéspedes inmunocomprometidos. Es una infección grave con elevada tasa de mortalidad en pacientes oncohematológicos y receptores de trasplante de células progenitoras hematopoyéticas. La administración de corticosteroides es el principal factor de riesgo para adquirir esta infección. Actualmente las infecciones ocurren en aquellos pacientes que no reciben adecuada profilaxis. Las técnicas de diagnóstico molecular son las recomendadas por su elevada sensibilidad, especificidad y rapidez. La frecuencia global de P. jirovecii en pacientes inmunocomprometidos de nuestro hospital, durante el período evaluado fue de 4,8%, con una mortalidad global del 20%. Como factores de mal pronóstico se reportan la presencia de coinfecciones y la necesidad de asistencia respiratoria mecánica. Es importante la sospecha precoz en pacientes de riesgo, confirmada con un diagnóstico preciso mediante métodos moleculares para una intervención adecuada y oportuna (AU)

Pneumocystis jirovecii is an opportunistic fungus, causing pneumonia in immunocompromised hosts. It is a severe infection with a high mortality rate in oncology/hematology patients and hematopoietic stem cell transplant recipients. The administration of corticosteroids is the main risk factor for acquiring this infection. Currently infections occur in patients who do not receive adequate prophylaxis. Molecular diagnostic techniques are recommended because of their high sensitivity, specificity, and speed. In the study period, the overall incidence of P. jirovecii in immunocompromised patients at our hospital was 4.8%, with an overall mortality rate of 20%. Factors of a poor prognosis are the presence of coinfections and the need for mechanical respiratory assistance. Early suspicion in high-risk patients is important to confirm the diagnosis through molecular studies and start adequate and early treatment (AU)

Optimización de unidades formadoras de colonias y detección del virus de papiloma humano en sangre de cordón umbilical / Colony-forming units optimization and human papillomavirus detection in umbilical cord blood

Resumen Introducción: Se requiere analizar diversos parámetros para el control de calidad adecuado de las unidades de sangre de cordón umbilical (USCU) cuando se utilizan con fines terapéuticos. Objetivo: Optimizar las unidades formadoras de colonias (UFC) de cultivos clonogénicos y detectar el genoma del virus del papiloma humano (VPH) en USCU. Métodos: Se incluyeron 141 muestras de sangre de cordón umbilical (SCU), de segmento y de UFC de cultivos clonogénicos de USCU. Se realizó extracción de ADN, cuantificación y amplificación por PCR del gen endógeno GAPDH. Se detectó el gen L1 del VPH con los oligonucleótidos MY09/MY11 y GP5/GP6+; los productos de PCR se migraron en electroforesis de agarosa. El ADN purificado de las UFC se analizó mediante electroforesis de agarosa y algunos ADN, con la técnica sequence specific priming. Resultados: La concentración de ADN extraído de UFC fue superior comparada con la de SCU (p = 0.0041) y la de segmento (p < 0.0001); así como la de SCU comparada con la de segmento (p < 0.0001). Todas las muestras fueron positivas para la amplificación de GAPDH y negativas para MY09/MY11 y GP5/GP6+. Conclusiones: Las USCU criopreservadas fueron VPH netativas; además, es factible obtener ADN en altas concentraciones y con alta pureza a partir de UFC de los cultivos clonogénicos.

Abstract Introduction: Analysis of several markers is required for adequate quality control in umbilical cord blood units (UCBU) when are used for therapeutic purposes. Objective: To optimize colony-forming units (CFU) from clonogenic cultures and to detect the human papillomavirus (HPV) genome in UCBU. Methods: One hundred and forty-one umbilical cord blood (UCB), segment or CFU samples from UCBU clonogenic cultures were included. DNA extraction, quantification and endogenous GAPDH gene PCR amplification were carried out. Subsequently, HPV L1 gene was detected using the MY09/MY11 and GP5/GP6+ oligonucleotides. PCR products were analyzed with electrophoresis in agarose gel. CFU-extracted purified DNA was analyzed by electrophoresis in agarose gel, as well as some DNAs, using the SSP technique. Results: CFU-extracted DNA concentration was higher in comparison with that of UCB (p = 0.0041) and that of the segment (p < 0.0001), as well as that of UCB in comparison with that of the segment (p < 0.0001). All samples were positive for GAPDH amplification and negative for MY09/MY11 and GP5/GP6+. Conclusions: Cryopreserved UCBUs were HPV-negative. Obtaining CFU DNA from clonogenic cultures with high concentrations and purity is feasible.

Development of Kompetitive Allele Specific PCR markers for identification of persimmon varieties using genotyping-by-sequencing

BACKGROUND: Persimmon (Diospyros kaki Thunb.) is the most widely cultivated species of the genus Diospyros. In this study, genetic diversity and variations in persimmon genotypes were investigated using single nucleotide polymorphism (SNP) markers identified by genotyping-by-sequencing (GBS) analysis. RESULTS: Ninety-five persimmon accessions grown in the Pear Research Institute, National Institute Horticultural and Herbal Science, were sequenced using the Illumina Hiseq2500 platform and polymorphic SNPs were detected to develop molecular markers. These reliable SNPs were analyzed using the Kompetitive Allele Specific PCR (KASP) assay to discriminate among persimmon genotypes. GBS generated a total of 447,495,724 trimmed reads, of which 89.7% were raw reads. After demultiplexing and sequence quality trimming, 108,876,644 clean reads were mapped to the reference transcriptome. An average of 1,146,070 genotype reads were mapped. Filtering of raw SNPs in each sample led to selection of a total of 1,725,401 high-quality SNPs. The number of homozygous and heterozygous SNPs ranged from 1,933 to 6,834 and from 846 to 5,927, respectively. CONCLUSIONS: Of the 49 SNPs selected for development of an identification system for persimmons, 15 SNPs were used in the KASP assay to analyze 32 persimmon accessions. These KASP markers discriminated among all accessions.

Development of a species-specific PCR assay for authentication of Agkistrodon acutus based on mitochondrial cytochrome b gene

BACKGROUND: Agkistrodon acutus, a traditional Chinese medicine, clinically used in the treatment of rheumatism, tumor, and cardiovascular and cerebrovascular diseases. Due to the unique medicinal value and the difficulty of artificial breeding of Agkistrodon acutus, the supply of Agkistrodon acutus on the market exceeds the demand, and a large number of its adulterants are found on the market. In this study, the cytb gene sequences of Agkistrodon acutus and 9 snakes were compared and analyzed, specific primers were designed, and specific PCR methods were established to detect Agkistrodon acutus medicinal samples on the market. RESULTS: This method was successfully applied to distinguish the snake from other adulterated species, and tested 18 Agkistrodon acutus samples randomly purchased from six cities. Twelve samples were counterfeit and six were genuine. The standard reference material of Agkistrodon acutus was cloned by molecular cloning and sequencing, and the gene sequence difference with other species was significant. It shows that the region could be used as the fingerprint region of the target species. CONCLUSIONS: The proposed method can be used as a species-specific marker and can be highly distinguished from other adulterated snake species, which is helpful to effectively avoid the problem of false sale of Agkistrodon acutus.

Isolation and identification of Salmonella spp. in raw milk from dairy herds in Colombia / Isolamento e identificação de Salmonella spp. no leite cru de rebanhos leiteiros na Colômbia

Salmonellosis is a foodborne disease (FBD) that affects public health and can cause death in people. Many outbreaks of Salmonellosis have been reported due to the contamination of raw milk and dairy products with the pathogen. To determine the prevalence of Salmonella spp. in milk samples from four dairy herds in the Sabana of Bogotá in 2017, 112 milk samples were taken directly from the mammary gland during milking. All milk samples were cultured and tested to isolate and identify Salmonella spp. using microbiological and molecular methods. Salmonella spp. prevalence of milk samples was found to be 20.5% (n=23). The main Salmonella serovars isolated were S. Newport (60.87%), S.Typhimurium (17.4%), S. Virchow, S. Bredeney, and S. Anatum (4.3% each one of the serovars). However, it was not possible to determine the Salmonella serotype in two isolates. The prevalence of Salmonella spp. in milk has not been studied extensively in Colombia. The 20.5% in the prevalence might be due to fact that the sample was taken directly from the mammary gland allowing a better chance of isolation by avoiding the dilutional effect of mixed milk from different cows in the buckets. This also suggests that the infection of the udder could have occurred by hematogenous dissemination or by milking machine contamination. This study highlights the need to implement measures to prevent contamination and reduce the problem in the herds, which will result in milk and dairy products with high standards of innocuity and quality and decrease the risk of foodborne illness(AU)

A salmonelose é uma doença transmitida por alimentos que afeta a saúde pública e pode causar a morte de pessoas. Muitos surtos de salmonelose têm sido relatados devido à contaminação de leite cru e produtos lácteos com o patógeno. Para determinar a prevalência de Salmonella spp. em amostras de leite de quatro rebanhos leiteiros na Sabana de Bogotá em 2017, cento e doze amostras de leite foram colhidas diretamente da glândula mamária durante a ordenha. Todas as amostras de leite foram cultivadas para isolar e identificar Salmonella spp. usando métodos microbiológicos e moleculares. A prevalência de Salmonella spp. nas amostras de leite foi de 20,5% (n = 23). Os principais sorovares de Salmonellaidentificados foram S. Newport (60,87%), S. Typhimurium (17,4%), S. Virchow, S. Bredeney e S. Anatum (4,3% cada um dos sorovares). No entanto, não foram determinados os sorovares de dois isolados. A prevalência de Salmonella spp. no leite ainda não foi extensivamente estudada na Colômbia. Os 20,5% na prevalência podem ser devidos ao fato de a amostra ter sido colhida diretamente da glândula mamária, permitindo uma melhor chance de isolamento, evitando o efeito de diluição do leite misto de diferentes vacas nos baldes, o que pode indicar infecção do úbere pela disseminação hematogênica ou por contaminação da ordenhadeira. Este estudo destaca a necessidade da implementação de medidas destinadas a prevenir a contaminação e reduzir o problema nos rebanhos, resultando em leite e produtos lácteos com altos padrões de inocuidade e qualidade, diminuindo o risco de doenças de origem alimentar.(AU)

Analysis of the qPCR Assay Efficacy as Molecular Diagnostic in Patients with Chemotherapy and/or Antibiotic Therapy

Abstract High sensitivity of qPCR assay can be compromised by the presence of PCR inhibitors in samples analyzed. The aim of this study was to analyze the RT-qPCR assay efficiency considering the RNA quality/quantity and the presence of PCR inhibitors in patients with chemotherapy and/or antibiotic therapy. We analyzed 60 samples using RT-qPCR from individuals suspected of leukemia and 44 samples were quantified by fluorimetry and spectrophotometry. The efficiency of the RT-qPCR assay was evaluated comparing the threshold cycle (Ct) from tested samples and the standard curve. The 260/280 and 260/230 ratios, the presence of PCR inhibitors and the amount of sample (ng) used in the RT-qPCR reaction can be associated with 56.8% (R²=0.56, p<0.05) in the Ct obtained. The decrease of the RT-qPCR efficiency can be explained in 42,8% due to the variation of the 260/280 ratio (R²=0.42,p<0.05). The presence of antibiotics in the blood sample can be associated in 11.3% with the variability of 260/280 ratio (R²=0.11,p<0.05). Presence of chemotherapeutic drugs in the blood sample was not correlated with Ct variation (p=0.17). The spectrophotometer determines a RNA quantification with 2.2 times higher than the fluorimeter (t=2.2, p=0,03) and this difference is correlated with the 260/280 ratio (R²=0.36, p<0.05). Samples with low purity had a reduction in the qPCR efficiency, although we did not observe false results.

La pandemia de COVID-19, estudio de casos: Australia, Nueva Zelandia y Cuba / COVID-19 pandemic; case studies: Australia, New Zealand and Cuba

Introducción: La COVID-19, enfermedad respiratoria viral, producida por el SARS- CoV- 2, los primeros casos aparecieron en Wuhan, China, en diciembre 2019, evolucionó a pandemia. La OMS declaró emergencia mundial el 30 de enero del 2020. Se presentan los datos generales de la epidemia en Cuba, Australia y Nueva Zelandia. Objetivo: Describir la evolución de la epidemia en dichos países, las medidas tomadas y sus resultados. Material y Métodos: Investigación descriptiva, cuanti - cualitativa, utilizó la revisión documental, para cotejar información publicada sobre la epidemia en los países seleccionados, en revistas médicas, prensa periódica, sitios web oficiales. Se analizó información hasta el 13 de junio. Resultados: Australia tuvo 7 283 casos, 6 888 (94,48 por ciento) recuperados. Realizaron 1 782 651 test diagnósticos (69,91 por 10 000 habitantes) y positividad de 0.4 por ciento. Reportaron 102 fallecidos, mayores tasas entre 70 - 89 años, letalidad de 1,39 por ciento. Nueva Zelandia totalizó 1 515 casos, con 1 483 recuperados (97,8 por ciento), fallecieron 22. Realizaron 310 297 (36 por 10 000 habitantes) pruebas de PCR, con 0,7 por ciento de positivos. La letalidad fue de 1,9 por ciento. Cuba, acumulaba 2 238 casos, recuperados 1 923 (86 por ciento). Fallecieron 84 pacientes, con letalidad de 3,75 por ciento. Realizaron PCR (12,16 x 10 000 hab.), con 1,7 por ciento positivas. Conclusiones: El control resultó de la voluntad política de enfrentar y contener la epidemia con drásticas medidas de distanciamiento social, cierre de fronteras y aislamiento de territorios, aplicación de test diagnósticos, y la existencia de sistemas de salud públicos robustos y gratuitos(AU)

Introduction: COVID-19 is a viral respiratory disease produced by SARS-CoV-2. The first cases were diagnosed in Wuhan, China in December 2019; then the disease became a pandemic. The WHO declared it a global emergency on January 30, 2020. General data on the epidemic in Cuba, Australia and New Zealand are presented. Objective: To present the evolution of the epidemic in these countries as well as the measures taken and their results. Material and Methods: A descriptive, quantitative and qualitative research used documentary review to compare information about the epidemic in the selected countries. The information was obtained from medical journals, periodical press, and official websites and it was analyzed before June 13. Results: Australia had 7,283 cases of which 6,888 (94.48 percent) patients recovered. They performed 1,782,651 diagnostic tests (69.91 per 10,000 inhabitants) and the positivity was 0.4 percent. They reported 102 deaths with higher rates in people aged 70 - 89 years, and a case fatality of 1.39 percent. New Zealand reported 1,515 cases, with 1,483 recovered (97.8 percent) and 22 deaths. They performed 310,297 (36 per 10,000 population) PCR tests, with 0.7 percent positive cases. The case fatality was 1.9 percent. Cuba accumulated 2,238 cases and 1,923 (86 percent) recovered. A total of 84 patients died, with a lethality of 3.75 percent. PCR tests (12.16 x 10,000 inhabitants) were performed reporting 1.7 percent of positive cases. Conclusions: The control resulted from the political will to confront and contain the epidemic with drastic measures of social distancing, closure of borders and isolation of territories, application of diagnostic tests, and the existence of robust and free public health systems(AU)

Eficacia protectora de una vacuna inactivada contra el virus de la enfermedad hemorrágica del conejo tipo 2 preparada a partir de un aislado local en Egipto / Protective efficacy of an inactivated vaccine against Rabbit hemorrhagic disease virus 2 prepared from a local isolate in Egypt

Rabbit hemorrhagic disease is a contagious viral disease of rabbits controlled by vaccination. The present study was aimed to diagnose rabbit hemorrhagic disease from 11 infected farms from Qalubia governorate during 2019 and to prepare homologous vaccine against rabbit hemorrhagic disease virus 2. For this purpose, 11 liver samples were collected from suspected cases and subjected to detection and identification of circulating rabbit hemorrhagic disease virus. Ten samples were confirmed to be rabbit hemorrhagic disease virus using hemagglutination test, animal inoculation and reverse transcriptase polymerase chain reaction. Sequencing and phylogenetic analysis of two isolates (R5&R6) revealed the presence of rabbit hemorrhagic disease virus 2 (A/Qalubia/2019 and B/Qalubia/2019) under accession number MT07629 and MT067630 respectively. The inactivated rabbit hemorrhagic disease virus vaccines were prepared using Montanide ISA 206 oil or aluminum hydroxide gel adjuvants. Prepared vaccines were inoculated subcutaneously in susceptible rabbits and submitted to sterility, safety and potency tests. Obtained results showed that mean hemagglutination inhibition titer for aluminum hydroxide gel vaccine was 6,7.7,8.9 and 9.1 log2 while, Montanide vaccine reached to 6.7,8.7,9.2 and 9.5 log2 at 1st, 2nd, 3rd, and 4th weeks post vaccination, respectively. Immunized rabbits with Montanide vaccine showed better protection reach to 70 percent, 90 percent percent, 100 percent and 100 percent when compared to aluminum hydroxide gel vaccine 60 percent, 70 percent, 90 percent and 90 percent at 1st, 2nd, 3rd and 4th weeks post vaccination respectively. It was concluded that newly emerged rabbit hemorrhagic disease virus 2 was isolated from suspected cases. The two prepared vaccines were sterile, safe and potent. The oily adjuvanted rabbit hemorrhagic disease virus 2 vaccine stimulated an earlier and higher humoral immune response than the aluminum hydroxide gel adjuvanted vaccine. This humoral immune response achieved significant level of protection(AU)

La enfermedad hemorrágica del conejo es una enfermedad viral contagiosa de los conejos que se controla mediante vacunación. El presente estudio tuvo como objetivo diagnosticar la enfermedad hemorrágica del conejo en 11 granjas infectadas de la provincia de Qalubia, durante 2019 y preparar una vacuna homóloga contra el virus de la enfermedad hemorrágica del conejo tipo 2. Para este propósito, se recolectaron 11 muestras de hígado de casos sospechosos y se sometieron a detección e identificación de virus circulante de la enfermedad hemorrágica del conejo. Se confirmó que diez muestras eran positivas al virus de la enfermedad hemorrágica del conejo, utilizando para ello la prueba de hemaglutinación, inoculación en animales y Reacción en cadena de la polimerasa con transcriptasa inversa. La secuenciación y el análisis filogenético de dos aislamientos (R5 y R6) revelaron la presencia del virus de la enfermedad hemorrágica del conejo tipo 2 (A/Qalubia/2019 y B/Qalubia/2019) con los números de acceso MT07629 y MT067630 respectivamente. Las vacunas inactivadas del virus de la enfermedad hemorrágica del conejo se prepararon usando adyuvantes de gel de hidróxido de aluminio o aceite Montanide ISA 206. Las vacunas preparadas se inocularon por vía subcutánea en conejos susceptibles y se sometieron a pruebas de esterilidad, seguridad y potencia. Los resultados obtenidos mostraron que el título medio de inhibición de la hemaglutinación para la vacuna en gel de hidróxido de aluminio fue de 6; 7,7; 8,9 y 9,1 log2, mientras que la vacuna de Montanide alcanzó 6,7; 8,7; 9,2 y 9,5 log2 en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Los conejos inmunizados con la vacuna Montanide tuvieron una mejor protección, alcanzándose niveles de 70 por ciento, 90 por ciento, 100 por ciento y 100 por ciento en comparación con la vacuna en gel de hidróxido de aluminio 60 por ciento, 70 por ciento, 90 por ciento y 90 por ciento en la 1ª, 2ª, 3ª y 4ª semanas después de la vacunación, respectivamente. Se concluyó que el virus de la enfermedad hemorrágica del conejo tipo 2 de reciente aparición se aisló de los casos sospechosos. Las dos vacunas preparadas fueron estériles, seguras y potentes. La vacuna contra el virus de la enfermedad hemorrágica del conejo tipo 2 con adyuvante oleoso estimuló una respuesta inmune humoral más temprana y mayor que la vacuna con adyuvante en gel de hidróxido de aluminio. Esta respuesta inmune humoral confirió un nivel significativo de protección(AU)