Confirmation of rubella within 4 days of rash onset: comparison of rubella virus RNA detection in oral fluid with immunoglobulin M detection in serum or oral fluid

Abstract

Rubella virus infection is typically diagnosed by the identification of rubella virus-specific immunoglobulin M (IgM) antibodies in serum, but approximately 50% of serum samples from rubella cases collected on the day of rash onset are negative for rubella virus-specific IgM. The ability to detect IgM in sera and oral fluids was compared with the ability to detect rubella virus RNA in oral fluids by reverse transcription-PCR (RT-PCR) by using paired samples taken within the first 4 days after rash onset from suspected rubella cases during an outbreak in Perú. Sera were tested for IgM by both indirect and capture enzyme immunoassays (EIAs), and oral fluids were tested for IgM by a capture EIA. Tests for IgM in serum were more sensitive for the confirmation of rubella than the test for IgM in oral fluid during the 4 days after rash onset. RT-PCR confirmed more suspected cases than serum IgM tests on days 1 and 2 after rash onset. The methods confirmed approximately the same number of cases on days 3 and 4 after rash onset. However, a few cases were detected by serum IgM tests but not by RT-PCR even on the day of rash onset. Nine RT-PCR-positive oral fluid specimens were shown to contain rubella virus sequences of genotype 1C. In summary, RT-PCR testing of oral fluid confirmed more rubella cases than IgM testing of either serum or oral fluid samples collected in the first 2 days after rash onset; the maximum number of confirmations of rubella cases was obtained by combining RT-PCR and serology testing.

FIG. 1.

Agarose gel of products obtained by conventional RT-PCR of representative samples from controls and suspected rubella cases. (Top panel) Products obtained by RT-PCR for the 185-bp amplicon using transport medium (negative laboratory controls) (lanes 1 to 8), samples from healthy blood donors (negative patient controls) (lanes 9 to 12), or samples from suspected rubella cases (lanes 13 to 20). The samples in lanes 13, 15, and 20 are positive for rubella RNA. Lane PC, positive control amplified from the RV RNA transcript containing a 30-nt insertion; lane M, DNA marker. (Bottom panel) Results for the controls and suspected rubella cases in which the samples were amplified with β-actin primers that produce a 150-bp fragment. The results for all blood donors and suspected rubella cases shown were positive for this control for RNA integrity. The eight negative laboratory control extracts from transport medium (lanes 1 to 8) were also positive for β-actin because the medium contained fetal bovine serum.

FIG. 2.

Percentage of suspected rubella cases, as confirmed by four tests. The results obtained by RT-PCR (a combination of conventional and real-time RT-PCR results) of OF (•), analysis of OF for IgM antibodies by the MI EIA (Δ), analysis of serum for IgM antibodies by the MI EIA (▸), and analysis of serum for IgM by the DBE EIA (○) during the first 4 days after rash onset are shown. (A) Percent positive when the results for samples with equivocal results are not included; (B) percent positive when the results for samples with equivocal results are included as positive results.

FIG. 3.

Phylogenetic analysis of 739-nt sequences from the E1-coding region of wild-type RVs. The unrooted tree was made by using the maximum-parsimony criterion of the PAUP search program (version 10.3; Genetics Computer Group). The tree was constructed by using the 739-nt sequences from positions 8731 to 9469 and contains the 32 WHO accepted reference strains and the 9 sequences from Perú (in boldface and italics). All reference viruses grouped as expected, which is the primary criterion for a valid RV genotyping analysis result.