RESUMEN
Real-time reverse transcriptase PCR (rRT-PCR) is the most accurate method for the detection of dengue virus (DENV) and yellow fever virus (YFV) in acute illness. However, performing rRT-PCR is not feasible for many laboratories in regions of endemicity. The current study compared new reverse transcription-insulated isothermal PCRs (the POCKIT DENV and YFV reagent sets) with laboratory-developed rRT-PCRs for both viruses using clinical samples and viral strains from different endemic regions. Sensitivity and specificity of the POCKIT DENV Reagent Set were 87.2% (68/78 samples) and 98.2% of samples (54/55), respectively. The YFV reagent set demonstrated sensitive detection of YFV RNA from six viral strains down to an estimated concentration of 2.5 log10 copies/mL and proved to be specific for YFV. Although the POCKIT assays require RNA extraction, they may provide accurate and less-complex options for molecular testing in laboratory settings where rRT-PCR is not practical.
Asunto(s)
Virus del Dengue/genética , Dengue/diagnóstico , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Fiebre Amarilla/diagnóstico , Virus de la Fiebre Amarilla/genética , Dengue/epidemiología , Dengue/virología , Enfermedades Endémicas/estadística & datos numéricos , Guatemala/epidemiología , Humanos , Paraguay/epidemiología , Juego de Reactivos para Diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/normas , Sensibilidad y Especificidad , Sri Lanka/epidemiología , Carga Viral/genética , Fiebre Amarilla/epidemiología , Fiebre Amarilla/virologíaRESUMEN
Background: We sought to characterize dengue virus (DENV) infections among febrile children enrolled in a pediatric cohort study who were clinically diagnosed with a non-dengue illness ("C cases"). Methods: DENV infections were detected and viral load quantitated by real-time reverse transcription-polymerase chain reaction in C cases presenting between January 2007 and January 2013. Results: One hundred forty-one of 2892 C cases (4.88%) tested positive for DENV. Of all febrile cases in the study, DENV-positive C cases accounted for an estimated 52.0% of patients with DENV viremia at presentation. Compared with previously detected, symptomatic dengue cases, DENV-positive C cases were significantly less likely to develop long-lasting humoral immune responses to DENV, as measured in healthy annual serum samples (79.7% vs 47.8%; P < .001). Humoral immunity was associated with viral load at presentation: 40 of 43 patients (93.0%) with a viral load ≥7.0 log10 copies/mL serum developed the expected rise in anti-DENV antibodies in annual samples versus 13 of 68 (19.1%) patients with a viral load below this level (P < .001). Conclusions: Antibody responses to DENV-positive C cases differ from responses to classic symptomatic dengue. These findings have important implications for DENV transmission modeling, immunology, and epidemiologic surveillance.
Asunto(s)
Formación de Anticuerpos/inmunología , Virus del Dengue/inmunología , Dengue/diagnóstico , Anticuerpos Antivirales/sangre , Estudios de Casos y Controles , Niño , Preescolar , Estudios de Cohortes , Dengue/epidemiología , Dengue/virología , Ensayo de Inmunoadsorción Enzimática , Femenino , Fiebre/etiología , Humanos , Incidencia , Masculino , Nicaragua/epidemiología , ARN Viral/sangre , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Viremia/genéticaRESUMEN
BACKGROUND: Infection with any of the 4 related dengue virus serotypes (DENV-1-4) is thought to result in lifelong immunity to homotypic reinfection (ie, reinfection with the same serotype). METHODS: Archived serum samples collected as part of an ongoing pediatric dengue cohort study in Nicaragua were tested for DENV by real-time reverse transcription polymerase chain reaction. Samples were collected from 2892 children who presented with an acute febrile illness clinically attributed to a non-DENV cause (hereafter, "C cases"). Test results were added to a database of previously identified symptomatic dengue cases in the cohort to identify repeat infections. RESULTS: Four patients with homotypic DENV reinfections were identified and confirmed among 29 repeat DENV infections (13.8%) with serotype confirmation. Homotypic reinfections with DENV-1, DENV-2, and DENV-3 occurred 325-621 days after the initial infection. Each patient experienced 1 symptomatic dengue case and 1 DENV-positive C case, and 2 patients presented with symptomatic dengue during their second infection. These DENV-positive C cases did not elicit long-lived humoral immune responses, despite viremia levels of up to 6.44 log10 copies per mL of serum. CONCLUSIONS: We describe the first set of virologically confirmed homotypic DENV reinfections. Such cases challenge the current understanding of DENV immunity and have important implications for modeling DENV transmission.
Asunto(s)
Virus del Dengue/clasificación , Virus del Dengue/aislamiento & purificación , Dengue/epidemiología , Dengue/virología , Serogrupo , Adolescente , Niño , Preescolar , Dengue/inmunología , Virus del Dengue/inmunología , Femenino , Genotipo , Humanos , Masculino , Nicaragua/epidemiología , Estudios Prospectivos , ARN Viral/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Recurrencia , Estudios Retrospectivos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Suero/virologíaRESUMEN
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.
Asunto(s)
Fiebre Chikungunya/diagnóstico , Dengue/diagnóstico , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Niño , Preescolar , Humanos , NicaraguaRESUMEN
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.
Asunto(s)
Virus del Dengue/clasificación , Virus del Dengue/aislamiento & purificación , Dengue/diagnóstico , Dengue/virología , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Virología/métodos , Humanos , Nicaragua , Sensibilidad y Especificidad , Serotipificación , Sri LankaRESUMEN
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.