RESUMEN
Polymerase chain reaction (PCR) is a widely used technique in the diagnosis of viral infections due to its low cost, high sensitivity, and specificity. Although the more advanced variations of PCR, such as real-time PCR and digital PCR are now available to researchers, conventional PCR is still used in many research studies. Here we describe the protocol for tri-primer diagnostic reverse transcription polymerase chain reaction for detection of rubella in throat swabs and further detailed protocol for a two fragment genotyping using two different sets of primers. In tri-primer diagnostic PCR, one forward and two reverse primers are used to detect clade I and clade II of the rubella virus. In the two fragments genotyping, each fragment of the genome is amplified, sequenced separately, and then the overlapping regions are aligned and full length sequence window is obtained.
Asunto(s)
Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Virus de la Rubéola , Genotipo , ARN Viral/genética , Virus de la Rubéola/genéticaRESUMEN
BACKGROUND: India has set a goal to eliminate measles and rubella/Congenital Rubella Syndrome (CRS) by 2023. Towards this goal, India conducted nationwide supplementary immunization activity (SIA) with measles-rubella containing vaccine (MRCV) targeting children aged between 9 months to <15 years and established a hospital-based sentinel surveillance for CRS. Reliable data about incidence of CRS is necessary to monitor progress towards the elimination goal. METHODS: We conducted serosurveys in 2019-20 among pregnant women attending antenatal clinics of 6 hospitals, which were also sentinel sites for CRS surveillance, to estimate the prevalence of IgG antibodies against rubella. We systematically sampled 1800 women attending antenatal clinics and tested their sera for IgG antibodies against rubella. We used rubella seroprevalence data from the current survey and the survey conducted in 2017 among antenatal women from another 6 CRS surveillance sites to construct a catalytic models to estimate the incidence and burden of CRS. RESULT: The seroprevalence of rubella antibodies was 82.3% (95% CI: 80.4-84.0). Rubella seropositivity did not differ by age group and educational status. Based on the constant and age-dependent force of infection models, we estimated that the annual incidence of CRS in India was 225.58 per 100,000 live births (95% CI: 217.49-232.41) and 65.47 per 100,000 live births (95% CI: 41.60-104.16) respectively. This translated to an estimated 14,520 (95% CI: 9,225-23,100) and 50,028 (95% CI: 48,234-51,543) infants with CRS every year based on age-dependent and constant force of infection models respectively. CONCLUSIONS: Our findings indicated that about one fifth of women in the reproductive age group in India were susceptible for rubella. The estimates of CRS incidence will serve as a baseline to monitor the impact of MRCV SIAs, as well progress towards the elimination goal of rubella/CRS.
Asunto(s)
Síndrome de Rubéola Congénita/epidemiología , Síndrome de Rubéola Congénita/patología , Adolescente , Anticuerpos Antivirales/sangre , Niño , Preescolar , Estudios Transversales , Femenino , Humanos , Inmunoglobulina G/sangre , Incidencia , India/epidemiología , Lactante , Masculino , Síndrome de Rubéola Congénita/sangre , Estudios SeroepidemiológicosRESUMEN
BACKGROUND: Government of India is committed to eliminate measles and control rubella/congenital rubella syndrome (CRS) by 2020. In 2016, CRS surveillance was established in five sentinel sites. We analyzed surveillance data to describe the epidemiology of CRS in India. METHODOLOGY/PRINCIPAL FINDINGS: We used case definitions adapted from the WHO-recommended standards for CRS surveillance. Suspected patients underwent complete clinical examination including cardiovascular system, ophthalmic examination and assessment for hearing impairment. Sera were tested for presence of IgM and IgG antibodies against rubella. Of the 645 suspected CRS patients enrolled during two years, 137 (21.2%) were classified as laboratory confirmed CRS and 8 (1.2%) as congenital rubella infection. The median age of laboratory confirmed CRS infants was 3 months. Common clinical features among laboratory confirmed CRS patients included structural heart defects in 108 (78.8%), one or more eye signs (cataract, glaucoma, pigmentary retinopathy) in 82 (59.9%) and hearing impairment in 51. (38.6%) Thirty-three (24.1%) laboratory confirmed CRS patients died over a period of 2 years. Surveillance met the quality indicators in terms of adequacy of investigation, adequacy of sample collection for serological diagnosis as well as virological confirmation. CONCLUSIONS/SIGNIFICANCE: About one fifth suspected CRS patients were laboratory confirmed, indicating significance of rubella as a persistent public health problem in India. Continued surveillance will generate data to monitor the progress made by the rubella control program in the country.
Asunto(s)
Síndrome de Rubéola Congénita/epidemiología , Adolescente , Adulto , Anticuerpos Antivirales/sangre , Femenino , Humanos , Inmunoglobulina M/sangre , India/epidemiología , Lactante , Recién Nacido , Masculino , Síndrome de Rubéola Congénita/sangre , Síndrome de Rubéola Congénita/diagnóstico , Síndrome de Rubéola Congénita/mortalidad , Vigilancia de Guardia , Adulto JovenRESUMEN
Rubella or German measles is an infection caused by rubella virus (RV). Infection of children and adults is usually characterized by a mild exanthematous febrile illness. However, RV is a major cause of birth defects and fetal death following infection in pregnant women. RV is a teratogen and is a major cause of public health concern as there are more than 100,000 cases of congenital rubella syndrome (CRS) estimated to occur every year. Several lines of evidence in the field of molecular biology of RV have provided deeper insights into the teratogenesis process. The damage to the growing fetus in infected mothers is multifactorial, arising from a combination of cellular damage, as well as its effect on the dividing cells. This review focuses on the findings in the molecular biology of RV, with special emphasis on the mitochondrial, cytoskeleton and the gene expression changes. Further, the review addresses in detail, the role of apoptosis in the teratogenesis process.
Asunto(s)
Anomalías Congénitas/virología , Complicaciones Infecciosas del Embarazo/virología , Síndrome de Rubéola Congénita/virología , Virus de la Rubéola/fisiología , Rubéola (Sarampión Alemán)/complicaciones , Teratogénesis , Apoptosis/fisiología , Femenino , Humanos , Mitocondrias/virología , Embarazo , Rubéola (Sarampión Alemán)/virología , Transducción de Señal , Replicación Viral/fisiologíaRESUMEN
Enzyme linked immunosorbent assay (ELISA) for antibody identification, is important for laboratory confirmation of rubella infection in different settings. The Enzygnost rubella ELISA, widely used in the World Health Organization (WHO) Global Measles and Rubella Laboratory Network, is expensive and often unavailable. Qualitative and quantitative performance of the Euroimmun ELISA was compared with the Enzygnost ELISA, for detection of rubella specific IgM, using 283 sera collected from suspected congenital rubella syndrome (CRS) patients and IgG antibodies using 435 sera from a serosurvey among pregnant women. Good qualitative agreement was observed for detection of both rubella specific IgM (94.7% agreement and κ of 0.86) and IgG (96.3% agreement and κ of 0.84). Bland-Altman analysis for IgG yielded a mean difference of 0.781â¯IU/ml with 97.1% values within ±2 SD of the mean difference. Our study findings suggest that Euroimmun ELISA may be considered for detection of rubella specific IgM in suspected CRS cases and rubella specific IgG in surveillance studies.
Asunto(s)
Anticuerpos Antivirales/sangre , Pruebas Diagnósticas de Rutina/métodos , Ensayo de Inmunoadsorción Enzimática/métodos , Complicaciones Infecciosas del Embarazo/diagnóstico , Juego de Reactivos para Diagnóstico , Virus de la Rubéola/inmunología , Rubéola (Sarampión Alemán)/diagnóstico , Femenino , Humanos , Inmunoglobulina G/sangre , Inmunoglobulina M/sangre , Lactante , Recién Nacido , EmbarazoRESUMEN
Rubella or German measles is an infection caused by rubella virus (RV). Infection of children and adults is usually characterized by a mild exanthematous febrile illness. However, RV is a major cause of birth defects and fetal death following infection in pregnant women. RV is a teratogen and is a major cause of public health concern as there are more than 100,000 cases of congenital rubella syndrome (CRS) estimated to occur every year. Several lines of evidence in the field of molecular biology of RV have provided deeper insights into the teratogenesis process. The damage to the growing fetus in infected mothers is multifactorial, arising from a combination of cellular damage, as well as its effect on the dividing cells. This review focuses on the findings in the molecular biology of RV, with special emphasis on the mitochondrial, cytoskeleton and the gene expression changes. Further, the review addresses in detail, the role of apoptosis in the teratogenesis process.
Asunto(s)
Humanos , Femenino , Embarazo , Complicaciones Infecciosas del Embarazo/virología , Rubéola (Sarampión Alemán)/complicaciones , Virus de la Rubéola/fisiología , Anomalías Congénitas/virología , Síndrome de Rubéola Congénita/virología , Teratogénesis , Rubéola (Sarampión Alemán)/virología , Replicación Viral/fisiología , Transducción de Señal , Apoptosis/fisiología , Mitocondrias/virologíaRESUMEN
BACKGROUND: We conducted a sero-survey among pregnant women attending antenatal clinics of six hospitals which also function as sentinel sites for CRS surveillance, to estimate the prevalence of IgG antibodies against rubella. METHODS: We systematically sampled 1800 pregnant women attending antenatal clinics and tested their sera for IgG antibodies against rubella. We classified sera as seropositive (titre ≥10â¯IU/ml), sero-negative (titre <8â¯IU/ml) or indeterminate (titre 8-9.9â¯IU/ml) per manufacturer's instructions. In a sub-sample, we estimated the titers of IgG antibodies against rubella. IgG titer of ≥10â¯IU/mL was considered protective. RESULTS: Of 1800 sera tested, 1502 (83.4%) were seropositive and 24 (1.3%) were indeterminate and 274 (15.2%) were sero-negative. Rubella sero-positivity did not differ by age group, educational status or place of residence. Three hundred and eighty three (87.8%) of the 436 sera had IgG concentrations ≥10â¯IU/mL. CONCLUSION: The results of the serosurvey indicate high levels of rubella sero-positivity in pregnant women. High sero-prevalence in the absence of routine childhood immunization indicates continued transmission of rubella virus in cities where sentinel sites are located.
Asunto(s)
Anticuerpos Antivirales/sangre , Complicaciones Infecciosas del Embarazo/epidemiología , Rubéola (Sarampión Alemán)/epidemiología , Adolescente , Adulto , Estudios Transversales , Femenino , Humanos , Inmunoglobulina G/sangre , India/epidemiología , Embarazo , Mujeres Embarazadas , Prevalencia , Virus de la Rubéola , Vigilancia de Guardia , Estudios Seroepidemiológicos , Centros de Atención Terciaria , Vacunación/estadística & datos numéricos , Adulto JovenRESUMEN
Rubella infection during pregnancy can result in miscarriage, fetal death, stillbirth, or a constellation of congenital malformations known as congenital rubella syndrome (CRS). The 11 countries in the World Health Organization (WHO) South-East Asia Region are committed to the elimination of measles and control of rubella and CRS by 2020. Until 2016, when the Government of India's Ministry of Health and Family Welfare and the Indian Council of Medical Research initiated surveillance for CRS in five sentinel sites, India did not conduct systematic surveillance for CRS. During the first 8 months of surveillance, 207 patients with suspected CRS were identified. Based on clinical details and serologic investigations, 72 (34.8%) cases were classified as laboratory-confirmed CRS, four (1.9%) as congenital rubella infection, 11 (5.3%) as clinically compatible cases, and 120 (58.0%) were excluded as noncases. The experience gained during the first phase of surveillance will be useful in expanding the surveillance network, and data from the surveillance network will be used to help monitor progress toward control of rubella and CRS in India.
Asunto(s)
Síndrome de Rubéola Congénita/diagnóstico , Síndrome de Rubéola Congénita/epidemiología , Virus de la Rubéola/aislamiento & purificación , Vigilancia de Guardia , Adolescente , Adulto , Femenino , Humanos , India/epidemiología , Lactante , Recién Nacido , Masculino , Embarazo , Virus de la Rubéola/genética , Adulto JovenRESUMEN
We report 3 atypical rubella cases in a family cluster in India. The index case-patient showed only mild febrile illness, whereas the other 2 patients showed acute encephalitis and died of the disease. We confirmed rubella in the index and third cases using next-generation sequencing and IgM.
Asunto(s)
Encefalitis Viral/diagnóstico , Fenotipo , Rubéola (Sarampión Alemán)/diagnóstico , Enfermedad Aguda , Anticuerpos Antivirales/inmunología , Biomarcadores , Niño , Preescolar , Encefalitis Viral/inmunología , Encefalitis Viral/virología , Familia , Resultado Fatal , Femenino , Genoma Viral , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Inmunoglobulina G/inmunología , Inmunoglobulina M/inmunología , India , Masculino , ARN Viral , Rubéola (Sarampión Alemán)/inmunología , Rubéola (Sarampión Alemán)/virología , Hermanos , Evaluación de SíntomasRESUMEN
Efficient coupling of cellular energy production to metabolic demand is crucial to maintain organismal homeostasis. Here, we report that the mitochondrial Sirtuin Sirt4 regulates mitochondrial ATP homeostasis. We find that Sirt4 affects mitochondrial uncoupling via the adenine nucleotide translocator 2 (ANT2). Loss of Sirt4 expression leads to decreased cellular ATP levelsin vitro and in vivo while Sirt4 overexpression is associated with increased ATP levels. Further, we provide evidence that lack of Sirt4 activates a retrograde signaling response from the mitochondria to the nucleus that includes AMPK, PGC1α, key regulators of ß-oxidation such as Acetyl-CoA carboxylase, and components of the mitochondrial respiratory machinery. This study highlights the ability of Sirt4 to regulate ATP levels via ANT2 and a feedback loop involving AMPK.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Translocador 2 del Nucleótido Adenina/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Mitocondriales/metabolismo , Sirtuinas/metabolismo , Animales , Respiración de la Célula , Metabolismo Energético , Ácidos Grasos/metabolismo , Regulación de la Expresión Génica , Células HEK293 , Células Hep G2 , Homeostasis , Humanos , Masculino , Ratones , Mitocondrias/metabolismo , Recambio Mitocondrial , Oxidación-Reducción , Transducción de SeñalRESUMEN
Cellular processes such as proliferation, differentiation and death are intrinsically dependent upon the redox status of a cell. Among other indicators of redox flux, cellular NAD(H) levels play a predominant role in transcriptional reprogramming. In addition to this, normal physiological functions of a cell are regulated in response to perturbations in NAD(H) levels (for example, due to alterations in diet/metabolism) to maintain homeostatic conditions. Cells achieve this homeostasis by reprogramming various components that include changes in chromatin structure and function (transcription). The interdependence of changes in gene expression and NAD(H) is evolutionarily conserved and is considered crucial for the survival of a species (by affecting reproductive capacity and longevity). Proteins that bind and/or use NAD(H) as a co-substrate (such as, CtBP and PARPs/Sirtuins respectively) are known to induce changes in chromatin structure and transcriptional profiles. In fact, their ability to sense perturbations in NAD(H) levels has been implicated in their roles in development, stress responses, metabolic homeostasis, reproduction and aging or age-related diseases. It is also becoming increasingly clear that both the levels/activities of these proteins and the availability of NAD(H) are equally important. Here we discuss the pivotal role of NAD(H) in controlling the functions of some of these proteins, the functional interplay between them and physiological implications during calorie restriction, energy homeostasis, circadian rhythm and aging.