Detection of SARS-associated coronavirus in throat wash and saliva in early diagnosis

Abstract

The severe acute respiratory syndrome-associated coronavirus (SARS-CoV) is thought to be transmitted primarily through dispersal of droplets, but little is known about the load of SARS-CoV in oral droplets. We examined oral specimens, including throat wash and saliva, and found large amounts of SARS-CoV RNA in both throat wash (9.58 x 10(2) to 5.93 x 10(6) copies/mL) and saliva (7.08 x 10(3) to 6.38 x 10(8) copies/mL) from all specimens of 17 consecutive probable SARS case-patients, supporting the possibility of transmission through oral droplets. Immunofluorescence study showed replication of SARS-CoV in the cells derived from throat wash, demonstrating the possibility of developing a convenient antigen detection assay. This finding, with the high detection rate a median of 4 days after disease onset and before the development of lung lesions in four patients, suggests that throat wash and saliva should be included in sample collection guidelines for SARS diagnosis.

Figure 1

Quantification of the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) RNA by real-time reverse transcription–polymerase chain reaction (RT-PCR) assay. (A) Location of the forward and reverse primers and probe in the genome of SARS-CoV, with the genome positions shown according to the Urbani strain (20). (B) A schematic diagram of the construct, ORF1b/pCRII-TOPO, and the protocol for generating the in vitro transcribed RNA as the standard for the real-time RT-PCR assay is shown. The relationship between known input RNA copies to the threshold cycle (CT) is shown at the bottom.

Figure 2

Detection of the severe acute respiratory syndrome–associated coronavirus (SARS-CoV) in the epithelial cells in throat wash from SARS patients by an indirect immunofluorescence assay. (A,B) Spot slides of SARS-CoV–infected Vero E6 cells were incubated with the preimmune (A) or postimmune (B) serum from a rabbit immunized with the recombinant nucleocapsid protein of the SARS-CoV, followed by fluorescein isothiocyanate–conjugated goat anti-rabbit immunoglobulin G. Panels A and B demonstrate the specificity of the reagents. (C to G) Epithelial cells in throat wash from a healthy control (E) and two SARS patients, ID17 (C,D) and ID11 (F,G), were incubated with the preimmune (C,F) or postimmune (D,E,G) rabbit serum. (H) The light microscopic picture of (G), taken with the fluorescent light on.