RESUMO
OBJECTIVE: To develop and evaluate serological methods for chikungunya diagnosis and research in Nicaragua. METHODS: Two IgM ELISA capture systems (MAC-ELISA) for diagnosis of acute chikungunya virus (CHIKV) infections, and two Inhibition ELISA Methods (IEM) to measure total antibodies against CHIKV were developed using monoclonal antibodies (mAbs) and hyperimmune serum at the National Virology Laboratory of Nicaragua in 2014-2015. The sensitivity, specificity, predictive values, and agreement of the MAC-ELISAs were obtained by comparing the results of 198 samples (116 positive; 82 negative) with the Centers for Disease Control and Prevention's IgM ELISA (Atlanta, Georgia, United States; CDC-MAC-ELISA). For clinical evaluation of the four serological techniques, 260 paired acute and convalescent phase serum samples of suspected chikungunya cases were used. RESULTS: All four assays were standardized by determining the optimal concentrations of the different reagents. Processing times were substantially reduced compared to the CDC-MAC-ELISA. For the MAC-ELISA systems, a sensitivity of 96.6% and 97.4%, and a specificity of 98.8% and 91.5% were obtained using mAb and hyperimmune serum, respectively, compared with the CDC method. Clinical evaluation of the four serological techniques versus the CDC real-time RT-PCR assay resulted in a sensitivity of 95.7% and a specificity of 88.8%-95.9%. CONCLUSION: Two MAC-ELISA and two IEM systems were standardized, demonstrating very good quality for chikungunya diagnosis and research demands. This will achieve more efficient epidemiological surveillance in Nicaragua, the first country in Central America to produce its own reagents for serological diagnosis of CHIKV. The methods evaluated here can be applied in other countries and will contribute to sustainable diagnostic systems to combat the disease.
Assuntos
Anticorpos Antivirais/sangue , Febre de Chikungunya/diagnóstico , Vírus Chikungunya/imunologia , Febre de Chikungunya/sangue , Febre de Chikungunya/epidemiologia , Ensaio de Imunoadsorção Enzimática , Humanos , Nicarágua/epidemiologia , Vigilância da População , Testes SorológicosRESUMO
BACKGROUND: Zika virus (ZIKV), chikungunya virus (CHIKV), and dengue virus (DENV) cocirculate in Nicaragua. In this study, we sought to compare the quantified viremia and clinical presentation of patients infected with 1 or more of these viruses. METHODS: Acute-phase serum samples from 346 patients with a suspected arboviral illness were tested using a multiplex real-time reverse-transcription polymerase chain reaction for ZIKV, CHIKV, and DENV. Viremia was quantitated for each detected virus, and clinical information from request forms submitted with each sample was recorded. RESULTS: A total of 263 patients tested positive for 1 or more viruses: 192 patients tested positive for a single virus (monoinfections) and 71 patients tested positive for 2 or all 3 viruses (coinfections). Quantifiable viremia was lower in ZIKV infections compared with CHIKV or DENV (mean 4.70 vs 6.42 and 5.84 log10 copies/mL serum, respectively; P < .001 for both comparisons), and for each virus, mean viremia was significantly lower in coinfections than in monoinfections. Compared with patients with CHIKV or DENV, ZIKV patients were more likely to have a rash (P < .001) and less likely to be febrile (P < .05) or require hospitalization (P < .001). Among all patients, hospitalized cases had higher viremia than those who did not require hospitalization (7.1 vs 4.1 log10 copies/mL serum, respectively; P < .001). CONCLUSIONS: ZIKV, CHIKV, and DENV result in similar clinical presentations, and coinfections may be relatively common. Our findings illustrate the need for accurate, multiplex diagnostics for patient care and epidemiologic surveillance.
Assuntos
Febre de Chikungunya/virologia , Dengue/virologia , Viremia , Infecção por Zika virus/virologia , Adulto , Febre de Chikungunya/complicações , Febre de Chikungunya/fisiopatologia , Coinfecção , Dengue/complicações , Dengue/fisiopatologia , Feminino , Humanos , Masculino , Nicarágua , Viremia/fisiopatologia , Viremia/virologia , Adulto Jovem , Infecção por Zika virus/complicações , Infecção por Zika virus/fisiopatologiaRESUMO
Clinical manifestations of Zika virus, chikungunya virus, and dengue virus infections can be similar. To improve virus detection, streamline molecular workflow, and decrease test costs, we developed and evaluated a multiplex real-time reverse transcription PCR for these viruses.
Assuntos
Vírus Chikungunya/isolamento & purificação , Vírus da Dengue/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Zika virus/isolamento & purificação , Genoma Viral , Humanos , RNA Viral/genética , Sensibilidade e EspecificidadeRESUMO
Dengue virus (DENV) is the agent of the most common vector-borne disease worldwide. Using 199 clinical samples collected from Nicaragua and Sri Lanka, a laboratory-developed DENV multiplex real-time reverse transcription-PCR (rRT-PCR) proved more clinically sensitive than the FDA-approved CDC assay for DENV serotypes 1 to 4 when measured against a composite reference standard, with sensitivities of 97.4% versus 87.1%, respectively.
Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Dengue/virologia , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Virologia/métodos , Humanos , Nicarágua , Sensibilidade e Especificidade , Sorotipagem , Sri LankaRESUMO
A number of diagnostic tests are available for dengue virus (DENV) detection, including a variety of nucleic acid amplification tests (NAATs). However, reports describing a direct comparison of different NAATs have been limited. In this study, we report the design of an internally controlled real-time reverse transcriptase PCR (rRT-PCR) that detects all four DENV serotypes but does not distinguish between them (the pan-DENV assay). Two hundred clinical samples were then tested using four different DENV RT-PCR assays: the pan-DENV assay, a commercially produced, internally controlled DENV rRT-PCR (the Altona assay), a widely used heminested RT-PCR, and a serotype-specific multiplex rRT-PCR assay. The pan-DENV assay had a linear range extending from 1.0 to 7.0 log10 cDNA equivalents/µl and a lower limit of 95% detection ranging from 1.7 to 7.6 cDNA equivalents/µl, depending on the serotype. When measured against a composite reference standard, the pan-DENV assay proved to be more clinically sensitive than either the Altona or heminested assays, with a sensitivity of 98.0% compared to 72.3% and 78.8%, respectively (P ≤ 0.0001 for both comparisons). The pan-DENV assay detected DENV in significantly more samples collected on or after day 5 of illness and in a subgroup of patients with detectable anti-DENV IgM at presentation. No significant difference in sensitivity was observed between the pan-DENV assay and the multiplex rRT-PCR, despite the presence of an internal control in the former. The detection of DENV RNA late in the course of clinical illness should serve to lengthen the period during which a confirmed molecular diagnosis of DENV infection can be provided.
Assuntos
Vírus da Dengue/isolamento & purificação , Dengue/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Estudos de Coortes , Dengue/virologia , Vírus da Dengue/genética , Humanos , Técnicas de Diagnóstico Molecular/normas , Nicarágua , Reação em Cadeia da Polimerase em Tempo Real/normas , Reação em Cadeia da Polimerase Via Transcriptase Reversa/normas , Sensibilidade e Especificidade , Sri LankaRESUMO
Analogous to observations in RNA viruses such as human immunodeficiency virus, genetic variation associated with intrahost dengue virus (DENV) populations has been postulated to influence viral fitness and disease pathogenesis. Previous attempts to investigate intrahost genetic variation in DENV characterized only a few viral genes or a limited number of full-length genomes. We developed a whole-genome amplification approach coupled with deep sequencing to capture intrahost diversity across the entire coding region of DENV-2. Using this approach, we sequenced DENV-2 genomes from the serum of 22 Nicaraguan individuals with secondary DENV infection and captured â¼75% of the DENV genome in each sample (range, 40 to 98%). We identified and quantified variants using a highly sensitive and specific method and determined that the extent of diversity was considerably lower than previous estimates. Significant differences in intrahost diversity were detected between genes and also between antigenically distinct domains of the Envelope gene. Interestingly, a strong association was discerned between the extent of intrahost diversity in a few genes and viral clade identity. Additionally, the abundance of viral variants within a host, as well as the impact of viral mutations on amino acid encoding and predicted protein function, determined whether intrahost variants were observed at the interhost level in circulating Nicaraguan DENV-2 populations, strongly suggestive of purifying selection across transmission events. Our data illustrate the value of high-coverage genome-wide analysis of intrahost diversity for high-resolution mapping of the relationship between intrahost diversity and clinical, epidemiological, and virological parameters of viral infection.
Assuntos
Vírus da Dengue/classificação , Vírus da Dengue/genética , Dengue/virologia , Variação Genética , Genoma Viral , Adolescente , Criança , Análise por Conglomerados , Estudos de Coortes , Vírus da Dengue/isolamento & purificação , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Dados de Sequência Molecular , Nicarágua , Filogenia , Estudos Prospectivos , RNA Viral/genéticaRESUMO
BACKGROUND: Dengue is the most prevalent mosquito-borne viral disease in humans and a major urban public health problem worldwide. METHODS: A prospective cohort study of approximately 3800 children initially aged 2-9 years was established in Managua, Nicaragua, in 2004 to study the natural history of dengue transmission in an urban pediatric population. Blood samples from healthy subjects were collected annually prior to the dengue season, and identification of dengue cases occurred via enhanced passive surveillance at the study health center. RESULTS: Over the first four years of the study, seroprevalence of anti-dengue virus (DENV) antibodies increased from 22%-40% in the 2-year-old cohort and 90%-95% in the 9-year-old cohort. The incidence of symptomatic dengue cases and the ratio of inapparent to symptomatic DENV infection varied substantially from year to year. The switch in dominant transmission from DENV-1 to DENV-2 was accompanied by an increase in disease severity but, paradoxically, a decrease in transmission. Phylogeographic analysis of full-length DENV-2 sequences revealed strong geographic clustering of dengue cases. CONCLUSIONS: This large-scale cohort study of dengue in the Americas demonstrates year-to-year variation of dengue within a pediatric population, revealing expected patterns in transmission while highlighting the impact of interventions, climate, and viral evolution.
Assuntos
Dengue/epidemiologia , Dengue/transmissão , Vigilância da População , Distribuição por Idade , Criança , Pré-Escolar , Análise por Conglomerados , Vírus da Dengue/classificação , Feminino , Humanos , Incidência , Masculino , Nicarágua/epidemiologia , Estudos Prospectivos , Estudos Soroepidemiológicos , SorotipagemRESUMO
BACKGROUND: Numerous immunological approaches exist to diagnose dengue or detect dengue virus (DENV) infections. OBJECTIVES: To determine the best immunological markers and specimen types for dengue diagnosis and for measuring incidence of DENV infection in community-based studies. STUDY DESIGN: In one study, acute- and convalescent-phase samples were collected from hospitalized suspected pediatric dengue cases in Managua, Nicaragua, from September 2003 to February 2004. A second study examined specimens collected in a community setting in Managua before and after the 2003-2004 dengue season to measure incidence of DENV infection. In both studies, detection of anti-DENV IgM, IgA, and IgG in serum, filter-paper blood spots, and saliva was compared to a gold standard performed on serum samples. RESULTS: For dengue diagnosis, the highest sensitivity and specificity was obtained by measuring IgM or IgA in serum or filter-paper blood spots; intermediate and poor results were obtained in saliva for IgM and IgA, respectively. Detection of IgG alone in serum, filter-paper blood spots, or saliva functioned best for measuring DENV infection. CONCLUSIONS: Detection of IgM and IgA in serum and filter-paper blood spots yielded optimal results for diagnosis of dengue cases, whereas IgG was the best marker for measuring incidence of DENV infection.
Assuntos
Anticorpos Antivirais/análise , Anticorpos Antivirais/sangue , Sangue/imunologia , Dengue/diagnóstico , Saliva/imunologia , Soro/imunologia , Adolescente , Criança , Pré-Escolar , Dengue/epidemiologia , Feminino , Humanos , Imunoglobulina A/análise , Imunoglobulina A/sangue , Imunoglobulina G/análise , Imunoglobulina G/sangue , Imunoglobulina M/análise , Imunoglobulina M/sangue , Lactente , Masculino , Nicarágua/epidemiologia , Sensibilidade e Especificidade , Estudos Soroepidemiológicos , Testes SorológicosRESUMO
Dengue, the most prevalent arthropod-borne viral disease of humans, is caused by four serotypes of dengue virus (DENV 1-4). Although all four DENV serotypes cause a range of illness, defining precisely which clinical characteristics are associated with the distinct serotypes has been elusive. A cross-sectional study was conducted on 984 and 313 hospitalized children with confirmed DENV infections during two time periods, respectively, in the same hospitals in Nicaragua: a 3-year period (1999-2001) when DENV-2 accounted for 96% of the viruses identified, and the 2003 dengue season when DENV-1 predominated (87% of identified serotypes). When the two periods were compared, more shock (OR 1.91, 95% CI 1.35-2.71) and internal hemorrhage (OR 2.05, CI 1.16-3.78) were observed in the period when DENV-2 predominated, whereas increased vascular permeability was associated to a greater degree with the DENV-1 period (OR 2.36, CI 1.80-3.09). Compared with the DENV-2 period, the DENV-1 season was associated with more hospitalized primary dengue cases (OR 3.86, CI 2.72-5.48) and more primary DENV infections with severe manifestations (OR 2.93, CI 2.00-4.28). These findings provide new data to characterize the pathogenic potential of distinct DENV serotypes in human populations.
Assuntos
Vírus da Dengue/crescimento & desenvolvimento , Dengue/classificação , Adolescente , Anticorpos Antivirais/sangue , Criança , Pré-Escolar , Estudos Transversais , Dengue/sangue , Dengue/patologia , Dengue/virologia , Feminino , Hematócrito , Hemorragia/patologia , Hemorragia/virologia , Humanos , Lactente , Masculino , Contagem de Plaquetas , Sorotipagem , Choque/patologia , Choque/virologia , Trombocitopenia/patologia , Trombocitopenia/virologiaRESUMO
BACKGROUND: Dengue virus (DENV) and chikungunya virus (CHIKV) now co-circulate throughout tropical regions of the world, with billions of people living at risk of infection. The differentiation of these infections is important for epidemiologic surveillance as well as clinical care, though widely-used molecular diagnostics for DENV and CHIKV require the performance of two to four separate PCR reactions for detection. OBJECTIVES: In the current study, we sought to develop and evaluate a single-reaction, multiplex real-time RT-PCR (rRT-PCR) for the detection and differentiation of DENV and CHIKV (the pan-DENV-CHIKV rRT-PCR). STUDY DESIGN: From an alignment of all available CHIKV complete genome sequences in GenBank, a new CHIKV rRT-PCR was designed for use in multiplex with a previously described assay for pan-DENV detection. Analytical evaluation was performed in accordance with published recommendations, and the pan-DENV-CHIKV rRT-PCR was clinically compared to reference molecular diagnostics for DENV and CHIKV using 182 serum samples from suspected cases in Managua, Nicaragua. RESULTS: The pan-DENV-CHIKV rRT-PCR had a dynamic range extending from 7.0 to 2.0 log10copies/µL for each DENV serotype and CHIKV, and the lower limits of 95% detection were 7.9-37.4copies/µL. The pan-DENV-CHIKV rRT-PCR detected DENV in 81 patients compared to 75 using a reference, hemi-nested DENV RT-PCR, and it demonstrated perfect agreement with a reference CHIKV rRT-PCR (54 positive samples). CONCLUSIONS: The single-reaction, multiplex format of the pan-DENV-CHIKV rRT-PCR, combined with sensitive detection of both viruses, has the potential to improve detection while decreasing testing costs and streamlining molecular workflow.
Assuntos
Febre de Chikungunya/diagnóstico , Dengue/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Criança , Pré-Escolar , Humanos , NicaráguaRESUMO
Chikungunya virus (CHIKV) was recently introduced into the Americas. In Nicaragua, the first endogenous transmission of CHIKV was recognized in September 2014. We used an ongoing dengue cohort study of children aged 2-14 years in Managua, Nicaragua, to document the attack rate of symptomatic chikungunya in a presumably naive population. From September 2014 through March 2015, the overall clinical attack rate of laboratory-confirmed CHIKV infection was 2.9% (95% confidence interval [CI]: 2.3%, 3.4%). The attack rate was greater in children ≥ 8 years of age (4.1%; 95% CI: 3.2%, 5.1%) than in those < 8 years of age (1.5%; 95% CI: 0.9%, 2.1%). The mean age of CHIKV cases presenting with typical chikungunya symptoms was 9.8 years, compared with 7.8 years for cases presenting with undifferentiated fever (P = 0.04). Our data suggest that the clinical attack rate in children may underestimate the true burden of disease as some children, especially young children, may experience more atypical symptoms (e.g., undifferentiated fever).
Assuntos
Febre de Chikungunya/epidemiologia , Adolescente , Criança , Pré-Escolar , Feminino , Habitação , Humanos , Masculino , Nicarágua/epidemiologia , Fatores de Risco , Fatores SocioeconômicosRESUMO
To investigate age-related differences in dengue severity, 114 infants, 1,211 children, and 346 adults with laboratory-confirmed dengue virus (DEN) infections presenting to three hospitals in major urban centers in Nicaragua were recruited from 1999 to 2001. The age distribution of dengue cases and the circulating serotype (predominantly DEN2) were representative of national data. Similar results were obtained when either dengue hemorrhagic fever/dengue shock syndrome or its principal manifestations (vascular permeability, internal hemorrhage, marked thrombocytopenia, and/or shock) were analyzed in relation to age and immune status. The burden of disease and of severe dengue was found predominantly in infants 4-9 months of age and in children 5-9 years old, and secondary DEN infection was a risk factor for severity in children. Age-related differences were identified in the prevalence of specific clinical manifestations as well as in their association with a confirmed DEN diagnosis. This represents one of the few comprehensive studies to analyze characteristics of dengue in infants, children, and adults in the same population and highlights age-related differences in dengue severity.
Assuntos
Dengue/epidemiologia , Adolescente , Adulto , Distribuição por Idade , Fatores Etários , Criança , Pré-Escolar , Efeitos Psicossociais da Doença , Dengue/etiologia , Dengue/patologia , Feminino , Hospitais Urbanos , Humanos , Lactente , Recém-Nascido , Masculino , Pessoa de Meia-Idade , Nicarágua/epidemiologia , Prevalência , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
BACKGROUND: Dengue fever results from infection with one or more of four different serotypes of dengue virus (DENV). Despite the widespread nature of this infection, available molecular diagnostics have significant limitations. The aim of this study was to develop a multiplex, real-time, reverse transcriptase-PCR (rRT-PCR) for the detection, quantitation, and serotyping of dengue viruses in a single reaction. METHODOLOGY/PRINCIPAL FINDINGS: An rRT-PCR assay targeting the 5' untranslated region and capsid gene of the DENV genome was designed using molecular beacons to provide serotype specificity. Using reference DENV strains, the assay was linear from 7.0 to 1.0 log10 cDNA equivalents/µL for each serotype. The lower limit of detection using genomic RNA was 0.3, 13.8, 0.8, and 12.4 cDNA equivalents/µL for serotypes 1-4, respectively, which was 6- to 275-fold more analytically sensitive than a widely used hemi-nested RT-PCR. Using samples from Nicaragua collected within the first five days of illness, the multiplex rRT-PCR was positive in 100% (69/69) of specimens that were positive by the hemi-nested assay, with full serotype agreement. Furthermore, the multiplex rRT-PCR detected DENV RNA in 97.2% (35/36) of specimens from Sri Lanka positive for anti-DENV IgM antibodies compared to just 44.4% (16/36) by the hemi-nested RT-PCR. No amplification was observed in 80 clinical samples sent for routine quantitative hepatitis C virus testing or when genomic RNA from other flaviviruses was tested. CONCLUSIONS/SIGNIFICANCE: This single-reaction, quantitative, multiplex rRT-PCR for DENV serotyping demonstrates superior analytical and clinical performance, as well as simpler workflow compared to the hemi-nested RT-PCR reference. In particular, this multiplex rRT-PCR detects viral RNA and provides serotype information in specimens collected more than five days after fever onset and from patients who had already developed anti-DENV IgM antibodies. The implementation of this assay in dengue-endemic areas has the potential to improve both dengue diagnosis and epidemiologic surveillance.
Assuntos
Vírus da Dengue/genética , Reação em Cadeia da Polimerase Multiplex/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sorotipagem/métodos , Anticorpos Antivirais/genética , Vírus da Dengue/classificação , Feminino , Humanos , Masculino , RNA Viral/genética , Reprodutibilidade dos TestesRESUMO
ABSTRACT Objective To develop and evaluate serological methods for chikungunya diagnosis and research in Nicaragua. Methods Two IgM ELISA capture systems (MAC-ELISA) for diagnosis of acute chikungunya virus (CHIKV) infections, and two Inhibition ELISA Methods (IEM) to measure total antibodies against CHIKV were developed using monoclonal antibodies (mAbs) and hyperimmune serum at the National Virology Laboratory of Nicaragua in 2014-2015. The sensitivity, specificity, predictive values, and agreement of the MAC-ELISAs were obtained by comparing the results of 198 samples (116 positive; 82 negative) with the Centers for Disease Control and Prevention's IgM ELISA (Atlanta, Georgia, United States; CDC-MAC-ELISA). For clinical evaluation of the four serological techniques, 260 paired acute and convalescent phase serum samples of suspected chikungunya cases were used. Results All four assays were standardized by determining the optimal concentrations of the different reagents. Processing times were substantially reduced compared to the CDC-MAC-ELISA. For the MAC-ELISA systems, a sensitivity of 96.6% and 97.4%, and a specificity of 98.8% and 91.5% were obtained using mAb and hyperimmune serum, respectively, compared with the CDC method. Clinical evaluation of the four serological techniques versus the CDC real-time RT-PCR assay resulted in a sensitivity of 95.7% and a specificity of 88.8%-95.9%. Conclusion Two MAC-ELISA and two IEM systems were standardized, demonstrating very good quality for chikungunya diagnosis and research demands. This will achieve more efficient epidemiological surveillance in Nicaragua, the first country in Central America to produce its own reagents for serological diagnosis of CHIKV. The methods evaluated here can be applied in other countries and will contribute to sustainable diagnostic systems to combat the disease.
RESUMEN Objetivo Elaborar y evaluar métodos serológicos para el diagnóstico y la investigación del chikungunya. Métodos Se elaboraron dos sistemas de ELISA de captura de IgM (MAC-ELISA por sus siglas en inglés) para el diagnóstico de la infección aguda por el virus de (CHIKV) y dos métodos de ELISA de inhibición (MEI) para determinar el valor cuantitativo de los anticuerpos totales contra el CHIKV, en el Laboratorio Nacional de Virología de Nicaragua en 2014-2015, para lo cual se utilizaron anticuerpos monoclonales (AcMo) y sueros hiperinmunes. Se determinó la sensibilidad, la especificidad y los valores predictivos, así como la concordancia de los MAC-ELISA, comparando los resultados de 198 muestras (116 positivas y 82 negativas) con el ELISA de los Centros para el Control y la Prevención de Enfermedades de los Estados Unidos (Atlanta; MAC-ELISA-CDC). Para la evaluación clínica de las cuatro técnicas serológicas, se emplearon 260 muestras de suero obtenidas en la fase aguda y en la fase de convalecencia de presuntos casos de chikungunya. Resultados Se estandarizaron los cuatro métodos analíticos determinando las concentraciones óptimas de los diferentes reactivos. La duración del procesamiento se redujo sustancialmente en comparación con el MAC-ELISA-CDC. Con los sistemas de MAC-ELISA, se obtuvo una sensibilidad del 96,6% y del 97,4% y una especificidad del 98,8% y del 91,5% al utilizar AcMo y suero hiperinmune, respectivamente, en comparación con el método de los CDC. La evaluación clínica de las cuatro técnicas serológicas, en comparación con la PCR en tiempo real de los CDC, arrojó una sensibilidad del 95,7% y una especificidad del 88,8%-95,9%. Conclusiones Se estandarizaron dos sistemas de ELISA-MAC y dos de MEI y se comprobó que poseen la calidad adecuada para el diagnóstico y las investigaciones del chikungunya, con lo cual mejorará la eficiencia de la vigilancia epidemiológica en Nicaragua, el primer país centroamericano que produce sus propios reactivos para el diagnóstico serológico del CHIKV. Los métodos estudiados en este trabajo pueden aplicarse en otros países y contribuyen al desarrollo de sistemas de diagnóstico sostenibles para combatir la enfermedad.
Assuntos
Vírus Chikungunya/imunologia , Febre de Chikungunya/diagnóstico , Febre de Chikungunya/epidemiologia , Anticorpos Antivirais/sangue , Ensaio de Imunoadsorção Enzimática/métodos , Nicarágua/epidemiologiaRESUMO
The rapid spread of dengue is a worldwide public health problem. In two clinical studies of dengue in Managua, Nicaragua, we observed an abrupt increase in disease severity across several epidemic seasons of dengue virus serotype 2 (DENV-2) transmission. Waning DENV-1 immunity appeared to increase the risk of severe disease in subsequent DENV-2 infections after a period of cross-protection. The increase in severity coincided with replacement of the Asian/American DENV-2 NI-1 clade with a new virus clade, NI-2B. In vitro analyses of viral isolates from the two clades and analysis of viremia in patient blood samples support the emergence of a fitter virus in later, relative to earlier, epidemic seasons. In addition, the NI-1 clade of viruses was more virulent specifically in children who were immune to DENV-1, whereas DENV-3 immunity was associated with more severe disease among NI-2B infections. Our data demonstrate that the complex interaction between viral genetics and population dynamics of serotype-specific immunity contributes to the risk of severe dengue disease. Furthermore, this work provides insights into viral evolution and the interaction between viral and immunological determinants of viral fitness and virulence.
Assuntos
Vírus da Dengue/genética , Vírus da Dengue/imunologia , Dengue/patologia , Índice de Gravidade de Doença , Humanos , Dados de Sequência MolecularRESUMO
The purpose of this research was to evaluate the internal consistency, the test-retest reliability and the construct validity of the Frost Multidimensional Perfectionism Scale (FMPS) in women. The total sample was made up of 325 university women, with an average age of 20.75 years (SD = 2.81). The scale was administered twice (n = 189) with an interval of one or two months between the first and second time of administration. Results showed that Cronbach's Alpha for the total score of instrument was .87 and for the factors ranged from .66 to .80. The one-month test-retest reliability was .80 and for the two-months was .67. People with symptomatology of eating disorders showed significantly higher scores than the control group in the total score of the FMPS and three of its factors, Concerns about Mistakes, Doubts about Actions and Parental Expectations. These findings provide favorable evidence for internal consistency, test-retest reliability and construct validity of the FMPS.
El propósito de esta investigación fue evaluar la consistencia interna, la confiabilidad test-retest y la validez de constructo de la Frost Multidimensional Perfectionism Scale (FMPS) en mujeres. Se trabajó con una muestra de 325 mujeres universitarias, con una edad promedio de 20.75 años (DE = 2.81). Una muestra (n = 189) de participantes contestó la FMPS en dos ocasiones para el test-retest, con una diferencia de un mes o dos meses entre la primera y la segunda aplicación. Los resultados mostraron que el Alpha de Cronbach para el total del instrumento fue de .87 y para los factores el rango fue de .66 a .80. La confiabilidad test-retest a un mes fue de .80 y a dos meses fue de .67. Las personas con sintomatología de trastornos del comportamiento alimentario presentaron puntuaciones significativamente mayores que el grupo control en el total de la escala y tres de sus factores, Preocupación por los Errores, Indecisión de Acción y Expectativas Paternas. Se concluye que existe evidencia favorable sobre la consistencia interna, confiabilidad test retest y validez de constructo de la FMPS.
RESUMO
To investigate the incidence of dengue virus (DENV) infection in Nicaragua, a 2-year prospective study was conducted in schoolchildren 4-16 years old in the capital city of Managua. Blood samples were collected before the rainy season in 2001, 2002 and 2003, and were assayed for DENV-specific antibodies. Participants were monitored for dengue-like illness, and acute and convalescent blood samples were collected from suspected dengue cases. In 2001 and 2002, 602 and 397 students were recruited, respectively, and paired annual serum samples were available from 467 and 719 participants in 2001-2002 and 2002-2003, respectively. The overall seroprevalence of anti-DENV antibodies was 91%, increasing from 75% at age 4 to 100% at age 16. The incidence of DENV infection was 12% in Year 1 and 6% in Year 2 (P < 0.001). During Year 1, four laboratory-confirmed dengue cases were detected, with one DENV2 isolate; during Year 2, there were six confirmed dengue cases, with one DENV1 isolate. These and additional circulating serotypes were confirmed by plaque reduction neutralisation test. This study demonstrates surprisingly high transmission of DENV in urban Nicaragua.
Assuntos
Anticorpos Antivirais/sangue , Vírus da Dengue/imunologia , Dengue/epidemiologia , Adolescente , Distribuição por Idade , Criança , Pré-Escolar , Dengue/diagnóstico , Dengue/imunologia , Feminino , Humanos , Incidência , Masculino , Nicarágua/epidemiologia , Estudos Prospectivos , Sensibilidade e Especificidade , Estudos SoroepidemiológicosRESUMO
To evaluate alternative approaches to the serological diagnosis of dengue virus (DEN) infection, the detection of DEN-specific immunoglobulin M (IgM) and IgA antibodies in serum and saliva specimens was assessed in 147 patients with symptoms of DEN infection seen at the Ministry of Health in Nicaragua. Seventy-two serum samples were determined to be positive for anti-DEN antibodies by IgM capture enzyme-linked immunosorbent assay, the routine diagnostic procedure. Serum and saliva specimens were obtained from 50 healthy adults as additional controls. IgM was detected in the saliva of 65 of the 72 serum IgM-positive cases, 6 of the 75 serum IgM-negative cases, and none of the control group, resulting in a sensitivity of 90.3% and a specificity of 92.0% and demonstrating that salivary IgM is a useful diagnostic marker for DEN infection. Detection of IgA in serum may be another feasible alternative for the diagnosis of DEN infection, with serum IgA found in 68 (94.4%) of the IgM-positive cases. In contrast, detection of IgA in saliva was not found to be a useful tool for DEN diagnosis in the present study. Further studies of the kinetics of antibody detection in another set of 151 paired acute- and convalescent-phase serum samples showed that DEN-specific IgA antibodies were detected in more acute-phase samples than were IgM antibodies. Thus, we conclude that DEN-specific IgA in serum is a potential diagnostic target. Furthermore, given that saliva is a readily obtainable, noninvasive specimen, detection of DEN-specific salivary IgM should be considered a useful, cheaper diagnostic modality with similar sensitivity and specificity to IgM detection in serum.