Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20ºC from one to fifteen months after collection

Abstract

Key words: respiratory syncytial virus -HEp-2 cell culture -nasopharyngeal aspirates Several diagnostic methods have been developed since respiratory syncytial virus (RSV) was defined as an important respiratory human pathogen. During the 1980s and early 1990s, viral isolation in tissue culture was widely used in the diagnosis of RSV infections (Arens et al. 1986, Waner et al. 1990. Although this method is considered the gold standard for laboratory diagnosis of RSV, results are not available quickly enough to be the basis for initiating antiviral therapy or infection control measures. Primary isolation of RSV in conventional cell culture generally takes three to seven days, but can range from two to ten days (Piedra et al. 2002).For faster diagnosis, the isolation of RSV in cell culture has been replaced by antigen detection-based assays and new sensitive molecular techniques such as reverse transcriptase-polymerase chain reaction (RT-PCR) (WHO 2000, Henrickson 2004, Perkins et al. 2005. However, the cell culture method continues to be valuable because it allows the amplification of small amounts of virus that are present in a specimen, providing isolates for subtyping and further analysis. Moreover, it is less likely to diagnose false epidemics and permits the recovery of several additional agents that may be present in a specimen (Halstead et al. 1990, Piedra et al. 2002, Hall & McCarthy 2004.RSV is a highly thermolabile virus requiring stringent adherence to transport and storage guidelines, and immediate inoculation of specimens in permissive cell lines for optimal recovery of the virus (Piedra et al. 2002). When this is not possible, specimens need be stored carefully. RSV does not tolerate slow freezing and thawing. Approximately 50% of RSV infectivity is lost when samples are submitted to a single freezer-thaw cycle, with a complete loss of viability when they are frozen slowly at -20 o C and then thawed. The residual viral titer can be maintained for several years when the clinical samples with RSV are stored at -70 o C (Tristram & Welliver 1995, Piedra et al. 2002.Since 2001, the identification of RSV infections at the Virology Laboratory of the Pathology and Legal Medicine Department of the Federal University of Ceará (UFC) has been performed using indirect fluorescence-antibody (IFA). Due to the lack of a -70 o C freezer, the respiratory specimens collected from children with acute respiratory infections (ARI) were stored at -20 o C.The purpose of this study was to establish the rate of recovery of RSV in cell culture from nasopharyngeal aspirates (NPA) stored at -20ºC from one to 15 months after collection.NPA were obtained from children with ARI within seven days of onset, treated at the Albert Sabin Pediatric Hospital, located in Fortaleza, Ceará, Brazil, from January 2003 to August 2004. They were obtained by manual suction through a nasal catheter as described previously (Gardner & McQuillin 1980). Samples were transported to the laboratory on wet ice. An aliquot of each sample was processed imm...

Acute Respiratory Illness in Rural Haiti

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A molecular epidemiological study of human respiratory syncytial virus in Croatia, 2011–2014

“…Negative PCR results can result from storage conditions because HRSV is a highly thermolabile virus that is known for its intolerance of freezing and thawing cycles (Nunes and Moura, 2006). In some samples, the viral load was insufficient to obtain sequences of good quality, therefore 362 sequences were acquired and analysed.…”

Development of a multiplex RT-PCR for simultaneous diagnosis of human metapneumovirus (HMPV) and human respiratory syncytial virus (HRSV) from clinical specimens

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