Comparison of Quantitative Reverse Transcription-PCR to Viral Culture for Assessment of Respiratory Syncytial Virus Shedding

Abstract

Respiratory syncytial virus (RSV) has recently been recognized as a serious pathogen in elderly and immunocompromised adults. Diagnosis of acute infection in adults is often difficult due to the insensitivity of viral culture, and reverse transcription-PCR (RT-PCR) is a more sensitive alternative. The relationship of quantitative RT-PCR to viable virus has never been studied for RSV. Therefore, we compared a quantitative real-time RT-PCR with viral culture to assess viral load in adult volunteers challenged with the RSV A2 strain. Twelve of 13 volunteers were infected, and there was a high correlation (r ‫؍‬ 0.84) between quantitative RT-PCR and viral titer by cell culture. However, RT-PCR was more sensitive, with 73 of 169 (43%) samples positive compared to 58 (34%) samples positive by culture. The correlation between the two tests was highest early in the course of viral shedding (r ‫؍‬ 0.91, days 0 to 6), whereas during days 7 to 13, there was more variability (r ‫؍‬ 0.70). All subjects were culture negative by day 11, whereas one subject remained RT-PCR positive on day 12. All subjects were RT-PCR negative at day 28 postinfection. Quantitative RT-PCR has an excellent correlation with viral titers, as measured by culture, and should be a useful tool for future studies addressing viral load and disease pathogenesis.Respiratory syncytial virus (RSV) is a frequent cause of serious lower respiratory tract disease in young children (13). Reinfections occur throughout life and are generally mild in healthy adults. However, elderly or immunocompromised adults may develop severe RSV infection (4,5,23). In contrast to children, diagnosis in adults is often difficult due to the insensitivity of viral culture (7). This is likely due to thermolability of the virus and low titers of virus shed by adults and may also be due to the presence of preexisting nasal antibody in clinical specimens resulting in in vitro neutralization. Thus, investigators have increasingly relied on new sensitive molecular techniques such as reverse transcription PCR (RT-PCR) for the immediate diagnosis of RSV infection in adults (6, 9-11, 14, 30). In addition, the recent development of real-time RT-PCR, in which the concentration of amplification products is monitored as they accumulate during thermal cycling, allows quantification of RNA in specimens (18). The ability to assess the relationship of viral load to severity of illness and determination of the location of viral replication in the respiratory tract would improve the understanding of disease pathogenesis in adults as well as children. Quantitative RT-PCR has been used successfully to monitor disease progression and assess response to therapy for malignant diseases and other viral infections that are difficult to cultivate, such as human immunodeficiency virus and hepatitis C virus (2,12,15,20,29). However, the relationship of RNA copy to viable organisms and clinical illness is less well defined for respiratory pathogens, including RSV (9,16,17,19,22). Therefore, we developed a quanti...

Detection of Respiratory Syncytial Virus in Adults with Chronic Obstructive Pulmonary Disease

“…Group-specific real-time assay was performed by a one-tube nested RT-PCR method as previously described (19,21). The lower limit of detection is 0.01-0.05 pfu/ml for RSV group A or B or for one copy of RNA.…”

Utilidad de la reacción de polimerasa en cadena en tiempo real en el diagnóstico de infecciones por virus respiratorio sincicial en adultos

“…Si duplicamos la cifra de infecciones detectadas por TR-RPC en tiempo real, obtenemos cerca de 25% de adultos hospitalizados, con sospecha de IRV, en quienes se documenta un VRS. Las publicaciones en este tema, describen que el VRS está presente hasta en 20% de las IRV en adultos, con porcentajes variables en poblaciones específicas de estudio 4,[8][9][10] . Este sub-diagnóstico, que se obtendría por métodos convencionales, se debería a que en adultos la excreción viral es de menor cuantía que en la población pediátrica 13 , y que los métodos de diagnóstico convencionales tienen sensibilidades ampliamente variables 6 .…”

“…La dificultad en el diagnóstico se debe, en gran medida, a www.sochinf.cl Artículo Original la baja cuantía de partículas virales excretadas en adultos, en especial, en el grupo de adultos mayores 5 . Las técnicas de biología molecular, específicamente la RPC, aumenta la detección de VRS, con sensibilidad y especificidad cercanas a 98% 9,10 . El objetivo primario de nuestro trabajo fue determinar la utilidad de la técnica de TR-RPC en tiempo real, para identificar la presencia de VRS en pacientes adultos hospitalizados por infección respiratoria y en quienes la pesquisa de virus respiratorios por IFD fuera negativa; y como objetivo secundario, caracterizar clínicamente las infecciones por VRS detectadas por esta técnica.…”

RSV Associated Disease in Hospitalized Children in Southeast Brazil: Higher Frequency in Older Children Than in Infants

“…and HRV was performed in a final volume of 25µl, with 3µl of cDNA, 5µl of buffer (75mM Tris-HCl pH 9.0, 50mM KCl, 20mM (NH 4 ) 2 SO 4 ), 3µl of 50 mM MgCl 2 , 1µl of 10 mM dNTPs, 2.5µl of each primer (Table 1) at 10 pmol (Falsey et al 2003, van Woensel et al 2003, 0.5µl (2 U) of DNA polymerase (Biotools, Jupter, USA), and MilliQ water treated with DEPC (diethylpyrocarbonate, Sigma, St Louis, USA). The reaction consisted of 40 amplification cycles with denaturation at 94°C for 45 seconds, annealing at 54°C for 45 seconds, extension at 72°C for 45 seconds and final extension at 72°C for 7 minutes.…”