Development of a Respiratory Virus Panel Test for Detection of Twenty Human Respiratory Viruses by Use of Multiplex PCR and a Fluid Microbead-Based Assay

Abstract

Virology laboratories historically have used direct fluorescent-antibody assay (DFA) and culture to detect six or seven respiratory viruses. Following the discovery of five new human respiratory viruses since 2000, there is an increasing need for diagnostic tests to detect these emerging viruses. We have developed a new test that can detect 20 different respiratory virus types/subtypes in a single 5-h test. The assay employs multiplex PCR using 14 virus-specific primer pairs, followed by a multiplexed target-specific primer extension (TSPE) reaction using 21 primers for specific respiratory virus types and subtypes. TSPE products were sorted and identified by using a fluid microsphere-based array (Universal Array; TmBioscience Corporation, Toronto, Canada) and the Luminex x-MAP system. The assay detected influenza A and B viruses; influenza A virus subtypes H1, H3, and H5 (including subtype H5N1 of the Asian lineage); parainfluenza virus types 1, 2, 3, and 4; respiratory syncytial virus types A and B; adenovirus; metapneumovirus; rhinovirus; enterovirus; and coronaviruses OC43, 229E, severe acute respiratory syndrome coronavirus, NL63, and HKU1. In a prospective evaluation using 294 nasopharyngeal swab specimens, DFA/culture detected 119 positives and the respiratory virus panel (RVP) test detected 112 positives, for a sensitivity of 97%. The RVP test detected an additional 61 positive specimens that either were not detected by DFA/culture or were positive for viruses not tested for by DFA/culture. After resolution of discordant results by using a second unique PCR assay and by using a combined reference standard of positivity, the RVP test detected 180 of 183 true positives, for a sensitivity of 98.5%, whereas DFA and culture detected only 126 of 183 true positives, for a sensitivity of 68.8%. The RVP test should improve the capabilities of hospital and public health laboratories for diagnosing viral respiratory tract infections and should assist public health agencies in identifying etiologic agents in respiratory tract infection outbreaks.

Development and implementation of a molecular diagnostic platform for daily rapid detection of 15 respiratory viruses

“…Comparing analysis results with other diagnostic methods on clinical samples was considered the most relevant performance parameter in this case. The total positive diagnostic outcome of 57% of the 2004-2005 study is comparable to other prevalence studies of respiratory viruses in non-selected patient materials [Bellau-Pujol et al, 2005;Mahony et al, 2007]. Because the material is not clinically well defined, a large proportion of the diagnostic gap may be due to reasons such as patients sampled late in the course of the disease, patients tested for reasons other than viral respiratory tract disease, or inadequate sampling technique.…”

“…High-throughput systems combining PCR with fluid microbead multiplex product detection have also been recently described [Lee et al, 2007;Mahony et al, 2007]. However, most published assays have not been adapted for large-scale diagnostic use, or lack one or more important virus targets.…”

Bacteriemia oculta en niños atendidos en el Complejo Asistencial Dr. Sótero del Río: Experiencia post vacuna neumocóccica conjugada

“…Si el paciente no tiene foco y está en buenas condiciones generales, Respecto al diagnóstico de infección respiratoria viral, es importante mencionar que el examen se realizó en pacientes sin sintomatología respiratoria evidente y eso explica, probablemente, el alto número de pacientes con IFD positiva para ADV, causante a veces, de cuadros úni-camente febriles. Existe bastante evidencia en la literatura médica, que demuestra la sensibilidad y especificidad de los métodos moleculares muy por sobre la IFD (88 y 98% vs 68 y 70%, respectivamente) [19][20][21][22][23][24][25] . Gharabaghi y cols., compararon el rendimiento de cuatro ensayos comerciales de reacción de polimerasa en cadena-RPC con IFD y aislamiento viral en 750 hisopados nasofarín-geos de pacientes pediátricos, obteniendo un porcentaje de positividad adicional de 28,5%, lo que se traduce en un incremento de la sensibilidad de detección de virus respiratorios en niños, de 74,3% sobre la IFD y el cultivo viral 20 .…”

“…El microorganismo recuperado con mayor frecuencia desde hemocultivos de niños con BO probable fue S. pneumoniae y como era de esperar, fue menos frecuente, aunque estadísticamente no significativo, en el grupo de niños protegidos. Estudios como el de Kaiser Permanente describe el gran impacto de la PCV7 en niños entre 3 y 36 meses ambulatorios con bacteriemia por S. pneumoniae, con marcado descenso de su incidencia en Carolina del Norte [8][9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24][25][26][27] . En nuestro estudio es destacable que los pacientes protegidos y parcialmente protegidos, presentaron ENI por serotipos no vaccinales, mientras que los no protegidos tuvieron infecciones tanto por serotipos vaccinales como no vaccinales.…”

Diagnosis of influenza viruses with special reference to novel H1N1 2009 influenza virus

“…One such assay the xTAGTM RVP test was developed in 2005 immediately following SARS and H5N1 infl uenza and was designed to detect and type the three infl uenza A subtypes circulating at that time viz. H1, H3 and H5 [21,22].…”