BACKGROUND Evolutionary changes in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) include indels in non-structural, structural, and accessory open reading frames (ORFs) or genes. OBJECTIVES We track indels in accessory ORFs to infer evolutionary gene patterns and epidemiological links between outbreaks. METHODS Genomes from Coronavirus disease 2019 (COVID-19) case-patients were Illumina sequenced using ARTIC_V3. The assembled genomes were analysed to detect substitutions and indels. FINDINGS We reported the emergence and spread of a unique 4-nucleotide deletion in the accessory ORF6, an interesting gene with immune modulation activity. The deletion in ORF6 removes one repeat unit of a two 4-nucleotide repeat, which shows that directly repeated sequences in the SARS-CoV-2 genome are associated with indels, even outside the context of extended repeat regions. The 4-nucleotide deletion produces a frameshifting change that results in a protein with two inserted amino acids, increasing the coding information of this accessory ORF. Epidemiological and genomic data indicate that the deletion variant has a single common ancestor and was initially detected in a health care outbreak and later in other COVID-19 cases, establishing a transmission cluster in the Uruguayan population. MAIN CONCLUSIONS Our findings provide evidence for the origin and spread of deletion variants and emphasise indels’ importance in epidemiological studies, including differentiating consecutive outbreaks occurring in the same health facility.

Los métodos tradicionales de identificación fenotípica del género Mycobacterium son lentos y poco sensibles, requiriéndose cuatro a seis semanas para lograr un diagnóstico apropiado a partir de un cultivo positivo. Los procedimientos moleculares han permitido acortar este período, obteniéndose resultados entre las 36 a 72 horas. En nuestro país la incidencia de M. tuberculosis es baja y no existen datos acerca de con qué frecuencia los casos diagnosticados como tuberculosis pulmonar son en realidad causados por Mycobacterium no tuberculosis (MNT), normalmente saprofitas. Desde el punto de vista terapéutico, el diagnóstico etiológico a través de la identificación precisa de la especie de Mycobacterium infectante resulta un aporte significativo, dado que el tratamiento y el manejo de sus contactos son diferentes según sea la especie involucrada. Por estas razones se introdujo en nuestro laboratorio el diagnóstico de Mycobacterium a través de su identificación genotípica. Para ello se eligieron dos marcadores moleculares de ADN: la secuencia de inserción IS6110, característica de los genomas del complejo M. tuberculosis, y la secuencia del gen ribosomal 16s (ADNr 16s) para estudiar la identidad específica dentro del género Mycobacterium. Una vez puestas a punto las técnicas moleculares seleccionadas, se procedió al estudio retrospectivo de una colección de 80 aislamientos, identificados como Mycobacterium por métodos fenotípicos. La mayoría de los aislamientos (75/80) resultaron cepas del complejo M. tuberculosis. Los restantes cinco fueron identificados como cepas MNT, tres de ellas causantes de infecciones pulmonares.

Résumé Les méthodes traditionnelles d'identification phénotypi-que du genre Mycobacterium sont lentes et peu sensibles, quatre à six semaines étant nécessaires pour avoir un diagnostic approprié à partir d'une culture positive. Les procédés moléculaires ont permis de raccourcir cette période: on obtient des résultants au bout de 36-72 heures. Dans notre pays, l'incidence de M. tuberculosis est basse et il n'y a pas de registres pour savoir la fréquence avec laquelle les cas diagnostiqués comme tuberculose pulmonaire sont en réalité causés par Mycobacterium non tuberculose (MNT), normalement saprophytes. Du point de vue thérapeutique, le diagnostic étiolo-gique à travers l'identification précise de l'espèce de mycobacterium infectante est un apport important, étant donné que le traitement varie selon l'espèce en question. Voilà pourquoi on a introduit dans notre laboratoire le diagnostic de Mycobacterium à travers son identification génotypique. Pour ce faire, on a choisi deux marqueurs moléculaires d'ADN: la séquence d'insertion IS6110, caractéristique des génomes du complexe M.tuberculose, et la séquence du gène ribosome 16s (ADN 16s) pour étudier l'identité spécifique dans le genre Mycobacterium. Une fois mises à point les techniques moléculaires sélectionnées, on fait une étude rétrospective d'une collec-tion de 80 isolements, identifiés comme Mycobacterium par des méthodes phénotypiques. La plupart des isolements (75/80) étaient des cèpes du complexe M. tuberculose. Les autres cinq ont été identifiés comme des cèpes MNT, dont trois étant la cause d'infections pulmonaires.

Introducción: las hepatitis causadas por el virus de la hepatitis C (VHC) se han transformado en uno de los principales problemas asociados a las infecciones emergentes. El VHC posee una alta variabilidad genética identificándose desde su descubrimiento a la fecha seis tipos principales y un número creciente de subtipos virales asociados a diferente respuesta al tratamiento y a la evolución natural de la enfermedad. Objetivo: contribuir al diagnóstico, al seguimiento de los infectados y al conocimiento de la epidemiología a través del análisis molecular determinando el genotipo y la carga viral. Material y método: se analizaron las muestras de 175 pacientes referidos entre marzo de 1997 y junio de 2001 para el diagnóstico de infección por VHC. En todos los casos se realizó determinación de anticuerpos por enzima inmuno análisis (ELISA) e inmunoblot y amplificación del genoma de VHC mediante técnicas de transcripción reversa y reacción en cadena de la polimerasa (RT-PCR). A una submuestra de pacientes PCR positivos se determinó el genotipo y carga viral. Resultados: se estableció viremia en 125 pacientes de un total de 175 (173 con serología positiva y dos casos con serología negativa para VHC). El análisis de los polimorfismos de restricción (RFLP’s) y de la secuencia nucleotídica en 51 portadores crónicos mostró la siguiente distribución de genotipos y sus correspondientes subtipos: a) genotipo 1: 35 casos [subtipos 1a (17), 1b (16), variante (Mon1 y Mon2)]; b) genotipo 2: 3 casos [(subtipos 2a (1) y 2b (2)] y c) genotipo 3:13 casos [subtipos 3a (13)]. Conclusiones: en el presente trabajo se muestra la importancia de la implementación de técnicas moleculares aplicadas al diagnóstico y seguimiento de portadores de HVC, constituyendo una nueva herramienta, altamente precisa, sensible y específica. La gran capacidad analítica, a través de la secuenciación automática y el posterior análisis filogenético, nos ha permitido identificar en dos pacientes una nueva variante genética de HCV que no había sido descrita, y que plantea nuevas interrogantes. Estas fueron publicadas e ingresadas al Gen-Bank con los nombres Mon 1 y Mon 2. Con respecto a la determinación de la carga viral no se evidenció ningún tipo de correlación entre el genotipo infectante y el valor de carga obtenido, siendo por ello la viremia genotipo independiente.

Résumé Introduction: Les hépatites provoquées par le virus C (VHC) sont devenues un des principaux problèmes associés aux infections émergentes. Le VHC possède une haute variabilité génétique; jusqu’à nos jours, on a identifié six types principaux et un nombre croissant de sous-types viraux associés à une réponse différente au traitement et à l’évolution naturelle de la maladie. But: contribuer au diagnostic, au suivi des infectés et à la connaissance de l’épidémiologie au moyen de l’analyse moléculaire qui détermine le génotype et la charge virale. Matériel et méthodes: on analyse les échantillons de 175 patients entre mars 1997 et juin 2001 pour le diagnostic d’infection par VHC. â tous les cas, on réalise une détermination d’anti-corps par enzyme immune analyse (ELISA) et immunoblot et amplification du génome de VHC au moyen de techniques de transcription reverse et réaction en chaîne de la polymérase (RT-PCR). A un sous-échantillon de patients PCR positifs on a déterminé le génotype et la charge virale. Résultats: on a constaté virémie chez 125 patients d’un total de 175 (173 ayant sérologie positive et 2 cas à sérologie négative pour VHC). L’analyse des polymorphismes de restriction(RFLP’s) et de la séquence nucléotide chez 51 porteurs chroniques, a révélé la distribution de génotypes et leurs sous-types correspon-dants suivante: a) génotype 1:35 cas [sous-types 1a (17), 1b (16), variante (Mon1 et Mon2)]; b) génotype 2:3 cas [sous-types 2a (1) et 2b (2)] et c) génotype 3:13 cas [sous-types 3a (13)]. Conclusions: dans ce travail, on signale l’importance de l’emploi de techniques moléculaires appliquées au diagnostic et au suivi des porteurs de HVC, qui sont un nouvel instrument, très précis, sensible et spécifique. La grande capacité analytique, à travers le séquencement automatique et l’analyse phylogénétique postérieure, nous a permis d’identifier chez deux patients une nouvelle variante génétique de HVC qui n’avait pas été décrite et qui pose de nouveaux questionnements. Ils ont été publiés e inclus à la Gen-Bank sous le nom Mon 1 et Mon 2. En ce qui concerne la détermination de la charge virale, aucune relation entre le génotype infestant et la valeur de charge obtenue n’a été mise en évidence, restant alors la virémie génotype indépendante.

Summary Background. Hepatitis C virus infection appears to be as one of the main problems associated with emerging infectious diseases. VHC has considerably genetic plasticity; since its categorisation, six primary strains (and an increasingly high number of types) associated with treatment responses and the natural progress of the disease have been identified. Objectives. To improve diagnosis and follow up for patients with VHC, and to broad epidemiological knowledge using molecular analysis in determining genotype and viral charge. Methods. We analysed samples of 175 patients with VHC infectious disease referred from March 1997 to June 2001. All patients underwent ELISA, immunoblot and amplification of VHC through reverse transcription and polymerase chain reaction (RT-PCR). Genotype and viral charge were determined in a sub-sample of patients positive for PCR. Results. Viremia was found in 125 out of 175 patients (173 positive serology for VHC and 2 negative serology for VHC). Analysis of restriction polymorphism (RFLP’s) and nucleotide sequence in 51 chronic carriers showed the following distribution of genotypes and types: a) genotype 1: 35 cases [type 1a (17), 1b (16), variants (Mon1 and Mon2)]; b) genotype 2: 3 cases [(type 2ª (1) and 2b (2)], and c) genotype 3: 13 cases [type 3a (13)]. Conclusions. Molecular techniques applied to diagnosis and follow up of patients with HVC infectious disease showed to be a new, specific and highly precise tool. Trough automatic sequential ordering and the subsequent philological analysis two HCV variants have been described. They were been entered at the Gen-Bank as Mon 1 and Mon 2. As there was not been found any association between infectious genotype and viral charge, viremia was genotypically non-dependent.

O estudo de quatro anos de duração (1987-1990) abarcou as principais características clínico-epidemiológicas de pneumonia em crianças, tal como foi diagnosticado no serviço de emergência do Hospital Infantil, e também as etiologias e fatores implicados nos casos mais severos. Determinou-se a etiologia em 47.7% dos 541 casos de pneumonia, que incluíam 283 patógenos, dos quais 38.6% eram virus e 12.6% bactérias. Observou-se etiologia viral e mista mais freqüentemente nas crianças de menos de 12 meses. A etiologia bacteriana prevaleceu nas idades entre 6 e 23 meses. Com relação aos virus, a predominância foi de RSV com prevalência de 66% do total de virus. Os mais importantes agentes bacterianos foram S. pneumoniae (64%) e H. influenzae (19%). O H. influenzae e as infecções mistas incidiram significativamente durante 1988 assinalando variações anuais na severidade das doenças. Observou-se correlação entre a gravidade dos casos e a alta porcentagem de diagnóstico etiológico: pacientes com ritmos respiratórios mais altos que 70rpm, derrame pleural ou com comprometimento extenso de parênquima pulmonar, apresentaram maior índice de positividade laboratorial. Reconheceu-se vários fatores de risco individuais e familiares ao comparar crianças com pneumonia com controles sadios.

The 4-year study (1987-1990) covered the major clinical-epidemiological characteristics of pneumonia in children as diagnosed at the emergency service of the Children's Hospital, as well as etiologies, and factors involved in the most severe cases. Etiology was determined in 47.7% of the 541 pneumonia cases, involving 283 pathogens of which 38.6% were viruses and 12.6% bacteria. Viral and mixed etiologies were more frequent in children under 12 months of age. Bacteria predominated in ages between 6 and 23 months. Among the viruses, respiratory syncytial virus predominated (66%). The bacterial pneumonias accounted for 12.2% of the recognized etiologies. The most important bacterial agents were S. pneumoniae (64%) and H. influenzae (19%). H. influenzae and mixed infections had a relevant participation during the 1988 season, pointing to annual variations in the relative participation of pathogens and its possible implication in severity of diseases. Correlation of severity and increased percentage of etiological diagnosis was assessed: patients with respiratory rates over 70 rpm, or pleural effusion and/or extensive pulmonary parenchyma compromise yielded higher positive laboratory results. Various individual and family risk factors were recognized when comparing pneumonia children with healthy controls.

Para obter informação básica sobre a incidência, duração, características clínicas e etiologia das infecções respiratórias agudas (IRA), foram observadas 276 crianças de famílias pobres que moram em Montevidéo durante 32 meses. A população alvo foi dividida em dois grupos para análise dos resultados: Crianças com menos de 12 meses e acima dessa idade. Durante o período de seguimento foram registrados 1056 episódios de IRA. A incidência de IRA foi 5.2 por criança/ano. Foi 87% mais alta nos lactentes do que nos grupos de mais idade, com duração maior dos episódios. A maioria das infecções foi pouco grave. Observaram-se poucos casos com taquipnéia e retrações, mas 12 crianças foram hospitalizadas e dois lactentes morreram. Etiologia viral foi identificada em 15.3% dos episódios. RSV foi o agente predominante, produzindo epidemias anuais. Registrou-se durante os episódios de IRA a colonização moderada até alta no trato respiratório superior de S. pneumoniae (32.3%) e Hemophilus sp. (18.9%). Este estudo de comunidade forneceu informações originais sobre IRA no Uruguai. Permitiu avaliar o impacto destas infecções nas crianças.

To obtain base line data on incidence, duration, clinical characteristics and etiology of acute respiratory infections (ARI), 276 children from deprived families living in Montevideo were followed during 32 months. The target population was divided into two groups for the analysis of the results: children aged less than 12 months and those older than this age. During the follow-up period 1.056 ARI episodes were recorded. ARI incidence was 5.2 per child/year. It was 87% higher in infants than in the older group, as was the duration of the episodes. Most of the diseases were mild. Tachypnea and retractions were seldom observed, but 12 children were refered to the hospital, and 2 infants died. Viral etiology was identified in 15.3% of the episodes. RSV was the predominant agent producing annual outbreaks. Moderate to heavy colonization of the upper respiratory tract by Streptococcus pneumoniae (32.3%) and Hemophilus sp. (18.9%) was recorded during ARI episodes. This community-based study furnish original data on ARI in Uruguay. It enabled to asses the impact of these infections on childhood.