Point: is the era of viral culture over in the clinical microbiology laboratory?

Abstract

Conventional tube culture systems have long been the mainstay in clinical virology for the growth and identification of viruses from clinical specimens. Innovations such as centrifugation-enhanced shell vial and multiwell plate cultures and the use of genetically engineered and mixed cell lines, coupled with faster detection of viral replication, have allowed for reasonable turnaround times for even some of the most slowly growing clinically important human viruses. However, molecular methods, in particular, the PCR, have usurped the role of viral culture in many laboratories, limiting the use of this traditional method of virus detection or replacing it altogether. Advances and improvements in molecular technology over time have also resulted in newer generations of more rapid and accurate molecular assays for the detection, quantification, and genetic characterization of viruses. For this point-counterpoint, we have asked two individuals, Richard L. Hodinka of the Children's Hospital of Philadelphia, a clinical virologist whose laboratory has completely eliminated viral culture in favor of molecular methods, and Laurent Kaiser, head of the Virology Laboratory at the University of Geneva Hospital, who continues to be a strong advocate of viral culture, to discuss the relevance of viral culture in the molecular age.

Fig 1

Identification of influenza A (H1N1) virus of porcine origin in a pig farm employee by RT-PCR versus culture (see Fig. 2). A nasopharyngeal specimen from a farm employee with an influenza-like illness was screened for influenza virus by using a panel of specific RT-PCR assays. A generic RT-PCR used to screen animal and human matrix gene sequences of influenza A viruses (5) provided a signal (shown are the amplification and multicomponent plots) considered to represent ambiguous or even negative results, and all other combinations targeting the hemagglutinin genes of human viruses (seasonal H1, H1 2009, and H3) (5, 6) remained negative (data not shown). ΔRn, delta of the normalized reporter value.

Fig 2

(Left) In contrast to what was observed by RT-PCR, influenza A virus was easily cultivated on SIAT-1 MDCK cells, and a strong and specific cytopathic effect could be observed after 96 h. (Right) Immunofluorescence analysis using monoclonal antibodies directed against influenza virus nucleoprotein confirmed the presence of viral antigens in cells. The final hemagglutinin sequence obtained from the cell supernatant and subsequent phylogenetic analysis revealed a virus similar to avian-like swine influenza viruses, which predominate in European pigs (GenBank accession no. CY100132). One mismatch, which was probably the cause of the ambiguous amplification, was observed in the reverse primer.