Comparison of four methods using throat swabs to confirm rubella virus infection

Abstract

Laboratory tests are essential for confirming sporadic cases and outbreaks of rubella. Detection of rubella virus is often necessary to confirm rubella cases and to identify specimens to be used to characterize wild-type rubella viruses. The sensitivities of four methods for detecting rubella virus infection using throat swabs, which had been collected in Henan and Anhui provinces in China, were evaluated. The methods used were reverse transcription (RT)-PCR followed by Southern hybridization using RNA extracted directly from clinical specimens, virus growth in tissue culture followed by virus detection by RT-PCR, low-background immunofluorescence in infected tissue culture cells using monoclonal antibodies to the structural proteins of rubella virus, and a replicon-based method of detecting infectious virus. Among these four methods, direct RT-PCR followed by hybridization was the most sensitive method; the replicon-based method was the least difficult to perform.

FIG. 1.

Representative results for the protocol of RT-PCR plus hybridization. A shows the RT-PCR products from four throat swabs after electrophoresis on an agarose gel and staining with ethidium bromide, and B shows results after hybridization. The samples in each lane are as follows: lane 1, marker; lane 2, DIG marker; lane 3, H₂O; lane 4, RVTS25; lane 5, RVTS36; lane 6, RVTS37; lane 7, RVTS38; lane 8, empty; lane 9, positive control (rubella virus [Therien] RNA). Additional laboratory controls run with throat swabs were all negative (data not shown).

FIG. 2.

Representative results from IFA protocol and replicon protocol. (A) Vero cells infected with the supernatant from the first passage of clinical specimens were fixed at 3 days postinfection. The presence of virus was determined by the detection of viral structural proteins using rubella virus-specific mouse monoclonal antibodies and AlexaFluor 488-conjugated secondary antibody (green). The nuclei were stained using propidium iodide (red) as a counterstain. (B) GFP expressed from replicons was detected in Vero cells only when the infectious virus was present in the specimen (RVTS20) but not in the absence of infectious virus (RVTS35).