Dissemination of the Omicron Variant and Its Sub-Lineages among Residents and Travelers in Its First Year of Emergence in Venezuela.

The emergence of the SARS-CoV-2 Variant of Concern (VOC), Omicron, has been characterized by an explosive number of cases in almost every part of the world. The dissemination of different sub-lineages and recombinant genomes also led to several posterior waves in many countries. The circulation of this VOC and its major sub-lineages (BA.1 to BA.5) was monitored in community cases and in international travelers returning to Venezuela by a rapid partial sequencing method. The specific sub-lineage assignment was performed by complete genome sequencing. Epidemic waves of SARS-CoV-2 cases were observed among international travelers during 2022, a situation not seen before December 2021. The succession of the Omicron VOC sub-lineages BA.1 to BA.5 occurred sequentially, except for BA.3, which was almost not detected. However, the sub-lineages generally circulated two months earlier in international travelers than in community cases. The diversity of Omicron sub-lineages found in international travelers was related to the one found in the USA, consistent with the most frequent destination of international travel from Venezuela this year. These differences are compatible with the delay observed sometimes in Latin American countries in the circulation of the different lineages of the Omicron VOC. Once the sub-lineages were introduced in the country, community transmission was responsible for generating a characteristic distribution of them, with a predominance of sub-lineages not necessarily similar to the one observed in travelers or neighboring countries.

Description of a One-Year Succession of Variants of Interest and Concern of SARS-CoV-2 in Venezuela.

Some of the lineages of SARS-CoV-2, the new coronavirus responsible for COVID-19, exhibit higher transmissibility or partial resistance to antibody-mediated neutralization and were designated by WHO as Variants of Interests (VOIs) or Concern (VOCs). The aim of this study was to monitor the dissemination of VOIs and VOCs in Venezuela from March 2021 to February 2022. A 614 nt genomic fragment was sequenced for the detection of some relevant mutations of these variants. Their presence was confirmed by complete genome sequencing, with a correlation higher than 99% between both methodologies. After the introduction of the Gamma VOC since the beginning of the year 2021, the variants Alpha VOC and Lambda VOI were detected as early as March 2021, at a very low frequency. In contrast, the Mu VOI, detected in May 2021, was able to circulate throughout the country. After the detection of the Delta VOC in June 2021, it became the predominant circulating variant. With the arrival of the Omicron VOC in December, this variant was able to displace the Delta one in less than one month.

Detection of the Omicron variant of SARS-CoV-2 in international travelers returning to Venezuela.

BACKGROUND: By the end of 2021, the SARS-CoV-2 Variant of Concern (VOC) Delta was predominant in most of the world. At the end of November, the Omicron variant was first detected in South Africa. This variant was immediately classified as VOC, due to the explosive increase of cases in South Africa, and the great number of mutations exhibited by this new lineage. Since then, Omicron VOC displaced Delta one in almost every country. Venezuela implemented in May 2021 molecular testing of all the passengers arriving at Venezuelan airports. METHODS: In this study, we analyzed the presence of variants of SARS-CoV-2 in those positive samples, by sequencing a small fragment of the Spike genomic region. RESULTS: The Omicron variant was found in passengers arriving to Venezuela from the beginning of December. Complete genome analysis confirmed the presence of the Omicron VOC. The detection of this VOC coincided with an unprecedented increase in the frequency of passengers with positive nucleic acid testing. CONCLUSIONS: Genomic surveillance of samples for international travelers returning to Venezuela allowed us to rapidly detect the introduction of the Omicron variant in the country.

First Case of Monkeypox in Venezuela: Partial Complete Genome Sequence Allowed Its Grouping into the West African Clade II.

The ongoing epidemic of monkeypox virus (MPXV) infection has already reached more than 50,000 persons worldwide until the end of August 2022. We report the first case detected in Venezuela. The patient reported traveling from Spain and contact with friends tested positive for MPXV after his return. Partial complete genome phylogenetic analysis allowed to group the isolate within the clade II of MPXV, the major one circulating worldwide. No other case of MPXV has been detected until the end of August 2022 in the country, although the presence of undiagnosed cases due to the fear of stigmatization cannot be ruled out.

Introduction and rapid dissemination of SARS-CoV-2 Gamma Variant of Concern in Venezuela.

In less than two years since SARS-CoV-2 emerged, the new coronavirus responsible for COVID-19, has accumulated a great number of mutations. Many of these mutations are located in the Spike protein and some of them confer to the virus higher transmissibility or partial resistance to antibody mediated neutralization. Viral variants with such confirmed abilities are designated by WHO as Variants of Concern (VOCs). The aim of this study was to monitor the introduction of variants and VOCs in Venezuela. A small fragment of the viral genome was sequenced for the detection of the most relevant mutations found in VOCs. This approach allowed the detection of Gamma VOC. Its presence was confirmed by complete genome sequencing. The Gamma VOC was detected in Venezuela since January 2021, and in March 2021 was predominant in the East and Central side of the country, representing more than 95% of cases sequenced in all the country in April-May 2021. In addition to the Gamma VOC, other isolates carrying the mutation E484K were also detected. The frequency of this mutation has been increasing worldwide, as shown in a survey of sequences carrying E484K mutation in GISAID, and was detected in Venezuela in many probable cases of reinfection. Complete genome sequencing of these cases allowed us to identify E484K mutation in association with Gamma VOC and other lineages. In conclusion, the strategy adopted in this study is suitable for genomic surveillance of variants for countries lacking robust genome sequencing capacities. In the period studied, Gamma VOC seems to have rapidly become the dominant variant throughout the country.

SARS-CoV-2 genetic diversity in Venezuela: Predominance of D614G variants and analysis of one outbreak.

SARS-CoV-2 is the new coronavirus responsible for COVID-19 disease. The first two cases of COVID-19 were detected in Venezuela on March 13, 2020. The aim of this study was the genetic characterization of Venezuelan SARS-CoV-2 isolates. A total of 7 full SARS-CoV-2 genome sequences were obtained by Sanger sequencing, from patients of different regions of Venezuela, mainly from the beginning of the epidemic. Ten out of 11 isolates (6 complete genomes and 4 partial spike genomic regions) belonged to lineage B, bearing the D614G mutation in the Spike protein. Isolates from the first outbreak that occurred in the Margarita Island harbored an in-frame deletion in its sequence, without amino acids 83-85 of the NSP1 of the ORF1. The search for deletions in 48,635 sequences showed that the NSP1 gene exhibit the highest frequency of deletions along the whole genome. Structural analysis suggests a change in the N-terminal domain with the presence of this deletion. In contrast, isolates circulating later in this island lacked the deletion, suggesting new introductions to the island after this first outbreak. In conclusion, a high diversity of SARS-CoV-2 isolates were found circulating in Venezuela, with predominance of the D614G mutation. The first small outbreak in Margarita Island seemed to be associated with a strain carrying a small deletion in the NSP1 protein, but these isolates do not seem to be responsible for the larger outbreak which started in July.

Performance of molecular methods for identification of unusual subtypes of hepatitis C virus genotype 2

Introduction: Hepatitis C virus (HCV) displays high genetic variability, with seven genotypes and numerous subtypes. The determination of the viral type has been essential for the selection and timing of antiviral treatment. In Venezuela, HCV genotype 2 is relatively diverse, being particularly prevalent subtype 2j. Objective: To evaluate the performance of methodologies for genotyping HCV, particularly for identification of subtype 2j. Materials and methods: HCV genotype and subtype were determined by reverse hybridization technique (LiPA) and sequencing of the HCV 5'UTR and NS5B regions. Results: A total of 65 samples from HCV-infected patients were analyzed. PCR amplifications of the 5'UTR region exhibited the highest sensitivity (100% vs 91% for LiPA and 77% for NS5B). Genotype determination, taking as reference test NS5B, showed 100% concordance with the other methods, and 67% and 59% for subtypes with 5´NC and LiPA, respectively. NS5B sequencing allowed the identification of subtypes 2j and 2s, which were not detected by the other methods. A specific LiPA pattern was not observed for HCV subtype 2j. Conclusion: Although being the methodology with lowest sensitivity for amplification of HCV RNA, sequencing NS5B region remains a powerful tool for correct discrimination of the different HCV subtypes, which is of epidemiological relevance.

Use of Synthetic Peptides and Multiple Antigen Blot Assay in the Immunodiagnosis of Hepatitis C Virus Infection.

Acute hepatitis C virus (HCV) infection is usually asymptomatic, therefore, early diagnosis is rare. It may remain undiagnosed in individuals who progress to chronic infection, often until serious liver damage has developed. To incorporate the diagnosis of this viral disease in a multiple-diagnostic assay, we first analyzed by immunoinformatics the HCV subtype 1a polyprotein (specifically Core, E2, NS3, NS5A proteins) to select antigenic peptides to be tested initially by the Pepscan technique. Next, we performed the immunodiagnosis of HCV infection, using the Multiple Antigen Blot Assay (MABA). In 22 patients' sera included in this study, a 20-mer linear peptide belonging to the N-terminus of the worldwide conserved Core protein showed 100% sensitivity and specificity; other sequences showed different levels of antibody recognition. The use of MABA in combination with synthetic peptides as a source of multiple, specific, and nonexpensive antigens for other infectious diseases could represent a rapid, integrated, and inexpensive diagnostic methodology.

Desempeño de métodos moleculares para la identificación de subtipos poco comunes del virus de la hepatitis C, genotipo 2 / Performance of molecular methods for identification of unusual subtypes of hepatitis C virus genotype 2

Resumen Introducción. El virus de la hepatitis C (HCV) presenta una gran variabilidad genética, con siete genotipos y numerosos subtipos. La determinación del tipo viral ha sido fundamental para la escogencia y la duración del tratamiento antiviral adecuado. En Venezuela, el genotipo 2 del HCV es relativamente diverso, siendo particularmente prevalente el subtipo 2j. Objetivo. Evaluar el desempeño de las metodologías para la determinación del genotipo del HCV, particularmente para la identificación del subtipo 2j. Materiales y métodos. Se determinaron el genotipo y el subtipo del HCV mediante la técnica de hibridación inversa LiPA (Line Probe Assay) y secuenciación de las regiones genómicas 5'NC y NS5B del virus. Resultados. En 65 muestras analizadas, la metodología basada en la amplificación de la región 5'NC mostró mayor sensibilidad (100 %), en comparación con la técnica LiPA (91 %) y la secuenciación de la región NS5B (77 %). La determinación de genotipo, tomando como método de referencia la secuenciación de NS5B, mostró un alto grado de concordancia para la secuenciación de la región 5´NC y la hibridación inversa LiPA, con 100 % en la asignación de genotipos, comparado con 70 % y 66 % para los subtipos, respectivamente. La secuenciación de la región NS5B permitió identificar los subtipos 2j y 2s, los cuales no fueron detectados por las otras metodologías. No se observó un patrón característico para las muestras subtipo 2j en la hibridación inversa LiPA. Conclusión. Aunque es la metodología con menor sensibilidad, la secuenciación de la región NS5B es una herramienta poderosa para la correcta discriminación de los distintos subtipos circulantes del HCV, lo cual reviste importancia epidemiológica.

Abstract Introduction: Hepatitis C virus (HCV) displays high genetic variability, with seven genotypes and numerous subtypes. The determination of the viral type has been essential for the selection and timing of antiviral treatment. In Venezuela, HCV genotype 2 is relatively diverse, being particularly prevalent subtype 2j. Objective: To evaluate the performance of methodologies for genotyping HCV, particularly for identification of subtype 2j. Materials and methods: HCV genotype and subtype were determined by reverse hybridization technique (LiPA) and sequencing of the HCV 5'UTR and NS5B regions. Results: A total of 65 samples from HCV-infected patients were analyzed. PCR amplifications of the 5'UTR region exhibited the highest sensitivity (100% vs 91% for LiPA and 77% for NS5B). Genotype determination, taking as reference test NS5B, showed 100% concordance with the other methods, and 67% and 59% for subtypes with 5´NC and LiPA, respectively. NS5B sequencing allowed the identification of subtypes 2j and 2s, which were not detected by the other methods. A specific LiPA pattern was not observed for HCV subtype 2j. Conclusion: Although being the methodology with lowest sensitivity for amplification of HCV RNA, sequencing NS5B region remains a powerful tool for correct discrimination of the different HCV subtypes, which is of epidemiological relevance.

Gerencia de diagnóstico y vigilancia epidemiológica del Instituto Nacional de Higiene "Rafael Rangel": Una década de avances y logros / Diagnostic and epidemiological surveillance management The National Institute of Hygiene "Rafael Rangel": A decade of advances and achievements

Buscando en los registros de las principales actividades de la Gerencia de Diagnóstico y Vigilancia Epidemiológica ha sido difícil elegir entre tantas vivencias, aquellos elementos que marcaron pauta durante la década 2008 ­ 2018. No obstante, es de resaltar que los desafíos afrontados ante la aparición de brotes, epidemias y la primera pandemia del siglo XXI, trajeron consigo un cúmulo de experiencias que se presentan en este artículo. Como centro nacional de referencia en las áreas de Bacteriología, Micología y Virología, continuamos aportando soluciones a la salud pública nacional mediante la actualización profesional de nuestro personal y la formación de la generación de relevo, en la que participan profesionales de excelencia, altamente especializados y sensibilizados con la problemática y los requerimientos de nuestra población. Asimismo, a través de la coordinación, supervisión y evaluación de la Red de laboratorios de salud pública, se contribuye con el fortalecimiento del diagnóstico de enfermedades transmisibles y vigilancia epidemiológica en el país. El trabajo realizado en estos diez años ha sido excelente, crucial y prioritario para enfrentar las emergencias. Debemos seguir trabajando en dos aspectos claves: 1. Mayor integración del laboratorio con el componente epidemiológico y clínico del país para ser más útiles al sistema de salud, y 2. Consolidar la creación del edificio sede del Centro de Diagnóstico de Enfermedades Transmisibles del Instituto Nacional de Higiene "Rafael Rangel" (INHRR), proyecto en el que estamos trabajando con la asesoría de la OPS/OMS.

Looking at the records of the main activities of the Diagnostic and Epidemiological Surveillance Management, it has been difficult to choose between many experiences, those elements that set the standard during the 2008 ­ 2018 decade. However, it is noteworthy that the challenges faced with the emergence of outbreaks, epidemics and the first pandemic of the 21st century, brought with it a wealth of experiences that are presented in this article. As a national reference center in Bacteriology, Mycology and Virology areas, we continue to provide solutions to public health through the professional updating of our staff and formation of the relief generation, in which participate professionals of excellence, highly specialized and sensitized with the problems and requirements of our population. Likewise, through the coordination, supervision and evaluation of the public health laboratories network, it contributes to the strengthening of the communicable diseases diagnosis and epidemiological surveillance in the country. The work done in these ten years has been excellent, crucial and priority to face emergencies. We must continue working on two key aspects: 1. Greater laboratory integration with the epidemiological and clinical component of the country to be more useful to the health system, and 2. Consolidate headquarters building creation of the National Institute of Hygiene "Rafael Rangel" (INHRR) Diagnostic Center for Communicable Diseases, project in which we are working with the PAHO / WHO advice.

Optimización de la técnica de PCR reversa para la detección del VIH en plasma de pacientes infectados / Optimization of the reverse PCR technique for HIV detection in plasma of infected patients

Aunque la técnica convencional para el diagnóstico del VIH es la PCR a partir de ADN proviral, la técnica RT-PCR cualitativa podría constituir una herramienta de apoyo en el diagnóstico del VIH en pacientes infectados, ya que una de sus principales ventajas es el uso de muestras de plasma, en lugar de sangre total, lo cual facilitaría su transporte entre las distintas regiones del país. En este estudio se planteó como objetivo evaluar las condiciones óptimas, en cuanto al uso de cebadores y enzima transcriptasa reversa, para síntesis de ADN complementario (RT-PCR) de la región de envoltura del VIH, a partir de muestras de plasma de pacientes VIH positivos. Los resultados obtenidos en este trabajo sugieren que la utilización de cebadores azarosos, en una mezcla de reacción con la enzima RT “SuperScript” TM (200 U/mL), permite maximizar la eficiencia de amplificación del gen de la envoltura de VIH-1, en comparación con cebadores específicos. Esto permite ofrecer una alternativa metodológica a partir de muestras de plasma, para la detección del VIH en aquellas personas que por diversas razones no pueden trasladarse a la institución de referencia.

Even though PCR is the conventional technique for HIV diagnosis in proviral DNA, the qualitative RT-PCR technique could constitute a support tool for HIV diagnosis in infected patients since one of its main advantages is the use of plasma samples instead of whole blood, which facilitates its transportation from the various regions of the country. The main objective of this study was the evaluation of the optimal conditions regarding use of primers and reverse transcriptase enzyme, for the synthesis of complementary DNA (RT-PCR) of the HIV sheath in plasma samples of HIV positive patients. The results obtained in this study suggest that the use of random primers, in a reactive mixture with the RT “SuperScript” enzyme TM (200 U/mL), allows maximizing the amplifying efficiency of the HIV-1 sheath gene, as compared with specific primers. This offers a methodological alternative using plasma samples for HIV detection in those persons who due to various reasons cannot travel to the reference institution.

Coinfección de hepatitis B y/o hepatitis C en pacientes infectados por virus de inmunodeficiencia humana (VIH) / Hepatitis B and/or hepatitis C co-infection in patients infected by the human immunodefficiency virus (HIV)

La coinfección con los virus de hepatitis B (VHB) y/o hepatitis C (VHC) puede provocar complicaciones en el paciente VIH+. El objetivo de este estudio fue evaluar la frecuencia de marcadores serológicos en la coinfección del VHB y/o VHC en plasmas de pacientes infectados por VIH y su correlación con el estatus virológico del VIH e inmunológico del paciente. Se evaluaron 1.846 plasmas positivos para VIH, referidos al Instituto Nacional de Higiene “Rafael Rangel” para la determinación de marcadores serológicos del VHB y VHC. Se realizaron análisis de carga viral del VIH-1 y recuento de linfocitos T CD4+/CD8+ para evaluar el estatus virológico e inmunológico, respectivamente de la población estudiada. La frecuencia de coinfección por VHB ó VHC fue de 15% y 5%, respectivamente mientras que la coinfección VHB/VHC fue de 0,16% (3/1.846) en pacientes infectados por VIH. No se observó asociación entre presencia de marcadores serológicos del VHB ó el VHC y bajos ó elevados niveles de ARN genómico del VIH (p=0,81 y p=0,31, respectivamente) ni valores bajos ó normales del índice CD4/CD8 (p=0,75 y p=0,06, respectivamente). Estos resultados sugieren que la coinfección con VHB o VHC no parece influir en los estatus virológico e inmunológico de la población evaluada.

Co-infection with hepatitis B (HBV) virus and/or hepatitis C (HCV) virus can induce complications in HIV+ patients. The purpose of this study was to evaluate the frequency of serologic markers in HBV and/ or HCV in plasma of HIV infected patients, and its correlation with the HIV viral status and the immunological status of the patient. The study included the evaluation of 1,846 HIV positive plasmas referred to the Instituto Nacional de Higiene “Rafael Rangel” for the determination of HBV and HCV serologic markers. The evaluation of the viral and immunological status was done by the analysis of the HIV-1 viral load and CD4+/CD8+ T lymphocyte counts, respectively, in the population studied. The frequency of HBV or HCV co-infection was 15% and 5%, respectively, while HBV/HCV co-infection was 0.16% (3/1,846) in HIV infected patients. There was no association between the presence of HBV or HCV serologic markers and low or normal values of the HIV genomic RNA (p=0.81 and p=0.31, respectively) nor low or normal values of the CD4/CD8 index (p=0.75 and p=0.06, respectively). These results suggest that HBV or HCV co-infection does not seem to influence the viral and immunological status of the evaluated population.

Detección del virus de inmunodeficiencia humana tipo 1 mediante la PCR, en neonatos de madres seropositivas / Detection of type 1 human immunodefficiency virus through the PCR in newborns from seropositive mothers

Resumen El diagnóstico temprano de la infección por el VIH-1 en el recién nacido es importante para su manejo clínico oportuno. Los objetivos de este trabajo fueron la detección del ADN proviral del VIH-1 mediante la técnica de PCR en neonatos de madres seropositivas al VIH-1 y la determinación de los posibles factores de transmisión vertical por VIH-1 en la población estudiada. Se analizaron 214 muestras sanguíneas de niños entre 0 y 18 meses de edad, referidos al INHRR entre Septiembre 2005-Agosto 2006. Todas las muestras se colectaron de manera estéril con EDTA, las células mononucleares de sangre periférica se separaron mediante gradiente de HISTOPAQUE. Posteriormente el ADN proviral fue extraido mediante columnas de silica-gel (QIAGEN). La amplificación del material genético de cada muestra se efectuó por dos ensayos de PCR a dos rondas, utilizando iniciadores conservados de los genes env y gag del VIH-1. Se encontró ADN proviral del VIH-1 en un 8% (17/214 muestras) de la población infantil evaluada, de los cuales el 82,4% mostraron elevados niveles de carga viral (>5.0 log10). Se corroboró la utilidad de la PCR como herramienta molecular en el diagnóstico oportuno de infección perinatal por el VIH-1.

Abstract The early diagnosis of HIV-1 infection is important for the opportune management of these patients. Objectives of this work were detection of proviral HIV-1 DNA through the PCR technique in newborns from HVI-1 seropositive mothers and determination of the possible vertical HIV-1 transmission factors in the population studied. 214 blood samples taken from children aged between 0 and 18 months referred to the INHRR during the September 2005-August 2006 period. All the samples were mixed with EDTA under sterile conditions. Peripheral blood mononuclear cells were separated by an HISTOPAQUE gradient. Later, the proviral DNA was extracted through silica-gel columns (QIAGEN). The amplification of the genetic material of each sample was obtained through two PCR determinations in two stages, using initiators conserved from env and gag HIV-1 genes. Proviral HIV-1 DNA was found in 8% (17/214) of the samples of the child population evaluated, 82.4% of which showed elevated viral load levels (>5.0 log10). The usefulness of PCR as a molecular tool for the opportune diagnosis of perinatal HIV-1 infection is corroborated.

Estudio molecular de la infección por el virus papiloma humano (VPH) en pacientes atendidos en el Hospital J. M de Los Ríos: tipificación y correlación clínica / Molecular study of the papillomavirus human infectiony in patient attending in the J. M. de los Ríos Hospital: clinical typing and correlation

La infección por el virus de papiloma humano (VPH) constituye un problema de salud pública mundial, sobre todo los países en desarrollo. Esta entidad incluye población de ambos sexos, desde la niñez hasta la edad adulta. El mecanismo de transmisión puede ser sexual, pero no necesariamente. La presencia de VPH en niños puede producir lesiones muy severas y recurrentes de diferente localización: cutánea, anogenital, oral, esófago, vías respiratorias superiores, conjuntiva ocular, entre otras, llegando a ocasionar severas complicaciones y desenlaces fatales. Utilizando la reacción en cadena de la polimerasa se estudiaron un grupo de pacientes provenientes del Hospital "J.M. de Los Ríos" y de otras instituciones, quienes presentaban diversas lesiones causadas por el virus

Estudio de reacción en cadena de la polimerasa (PCR) para mycobacterium tuberculosis: Hospital de Niños "J.M. de Los Ríos" / Polimerase chain reaction study for the mycobacterium tuberculosis: J.M. de Los Ríos Hospital

Se analizan los resultados de la aplicación de la reacción en cadena de polimerasa (PCR) para mycrobacterium tuberculosis, realizados en el Hospital de Niños, entre marzo del 2000 hasta enero del 2002. Las muestras son de contenido gástrico, lavado bronquial, esputo, orina, líquido cefalorraquídeo, líquido pleural y biopsia de ganglio. Para la extracción y amplificación de ADN se utilizó Protocolo de Laboratorio Abbott. Como control negativo se utilizó esperma de salmón y como control positivo extracto de ADN inactivo de M. tuberculosis H37Ra