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fecal specimens ( n = 40 , from 40 patients 127 days after symptom onset ) and urine specimens ( n = 133 , from 101 patients 257 days after symptom onset ) were collected from sars patients hospitalized in hong kong from march to may 2003 .
sars was confirmed by the presence of serum immunoglobulin ( ig ) g against sars - cov by an immunofluorescence assay ( 4 ) .
specimens were tested with polyclonal and monoclonal antibody  based capture elisas for sars - cov nucleocapsid protein and real - time qrt - pcr .
control urine ( n = 100 ) and fecal ( n = 100 ) specimens were obtained from hospitalized patients without sars . sars - cov nucleocapsid protein was detected by polyclonal antibody  based elisa according to published protocols ( 7,11 ) .
sars - cov nucleocapsid protein was detected by monoclonal antibody  based elisa using a modified protocol for serum samples ( 10 ) .
briefly , fecal and urine specimens were inactivated with 2% and 0.5% phenol , respectively , for 15 min before centrifugation and dilution in phosphate - buffered saline with 2% skim milk .
one hundred microliters of 1:10 diluted fecal specimens or 1:2 diluted urine specimens was added to wells previously coated with antinucleocapsid monoclonal antibodies .
plates were incubated , washed , treated with antinucleocapsid rabbit monoclonal antibodies , and analyzed as described previously ( 10,11 ) .
rna extraction and real - time qpcr assay specific for the 1b region of sars - cov were conducted as described previously ( 3,9 ) .
we compared the detection rates of 2 elisas and real - time qrt - pcr using the mcnemar test and studied the correlation between the optical density values at 450 nm ( od450 ) of the 2 elisas and log10 viral concentrations , as determined by real - time qrt - pcr , by linear regression ( spss version 11.0 , spss inc . ,
the cutoffs of the polyclonal antibody  based elisa have been determined previously , with specificities of 96% and 99% for fecal and urine specimens , respectively ( 11 ) .
the baselines of the monoclonal antibody  based elisa were determined by using 100 control fecal and urine specimens , with mean od450 values of 0.089 and 0.05 and standard deviation ( sd ) values of 0.074 and 0.03 , respectively .
the specificities of the monoclonal antibody  based elisa were 93% for fecal specimens and 98% for urine specimens , as determined using cutoffs defined as the mean + 2 sd .
of 40 fecal samples obtained from sars patients , 20 ( 50% ) obtained on days 9 to 23 after onset of symptoms were positive by the polyclonal antibody  based elisa , and 14 ( 35% ) obtained on days 2 to 21 were positive by the monoclonal antibody  based elisa .
of 133 urine samples , 6 ( 5% ) obtained on days 16 to 32 after onset of symptoms were positive by the polyclonal antibody  based elisa , and 11 ( 8% ) obtained on days 6 to 45 were positive by the monoclonal antibody  based elisa .
results of the polyclonal antibody  based elisa were comparable with our previous data on different specimens ( 11 ) .
the od450 values of both fecal ( pearson correlation 0.610 , p<0.0005 ) and urine specimens ( pearson correlation 0.475 , p<0.0005 ) detected by the 2 elisas were significantly correlated .
evaluation of polyclonal and monoclonal antibody  based enzyme - linked immunosorbent assays ( elisas ) for detecting nucleocapsid protein in fecal and urine specimens .
the dashed horizontal lines represent the corresponding cutoff optical density values at 450 nm ( od450 ) .
* sars - cov , severe acute respiratory syndrome coronavirus ; qrt - pcr , quantitative reverse transcription  polymerase chain reaction ; elisa , enzyme - linked immunosorbent assay .
the method of choice for early diagnosis of sars - cov infection should be the qrt - pcr .
moreover , qrt - pcr can detect sars - cov earlier in fecal specimens ( tables 1 and 2 ) . among the 40 fecal samples from sars patients , 32 ( 80% )
were positive by qrt - pcr , which was significantly higher than that of the polyclonal ( 50% ) and monoclonal ( 35% ) antibody - based elisas ( mcnemar test , p<0.005 and p<0.001 , respectively ) . of the 133 urine samples from sars patients , 33 ( 25% ) were positive by qrt - pcr , which was also significantly higher than that of the polyclonal ( 5% ) and monoclonal ( 8% ) antibody - based elisas ( mcnemar test , p<0.001 for both comparisons ) .
when qrt - pcr was used as a standard , the sensitivities of the polyclonal and monoclonal antibody  based elisas were 53.1% ( 17/32 ) and 43.8% ( 14/32 ) in fecal specimens , and 12.1% ( 4/33 ) and 15.2% ( 5/33 ) in urine specimens , respectively . the qrt - pcr can detect sars - cov in fecal specimens obtained on days 1 to 27 after onset of symptoms and in urine specimens obtained on days 9 to 45 .
moreover , 6 ( 75% ) of the 8 fecal specimens obtained on days 1 to 10 were positive by qrt - pcr .
all 3 tests had the highest detection rates in fecal specimens collected on days 16 to 20 , which suggested that this was the period of peak viral shedding in stool .
the detection rates in urine specimens were much lower than those in fecal specimens in all 3 assays . *
sars - cov , severe acute respiratory syndrome coronavirus ; qrt - pcr , quantitative reverse transcription  polymerase chain reaction ; elisa , enzyme - linked immunosorbent assay .
sars - cov can be detected during the late phase of illness . since sars - cov
can not be readily isolated from sars patients after week 3 of illness ( 14 ) , the detection of sars - cov beyond this time may be due to prolonged shedding of nonviable viruses in these patients or the presence of neutralizing immunoglobulins in clinical specimens , which has prevented viral replication in cell cultures .
higher detection rates by both elisas were found in specimens with higher viral concentrations ( table 2 ) .
there was also a significant correlation between viral load and elisa od450 values in fecal specimens tested with the monoclonal antibody  based elisa ( pearson correlation 0.424 , p = 0.003 ) , and in urine specimens tested with both the polyclonal and monoclonal antibody  based elisas ( pearson correlation 0.386 and 0.331 , respectively , p<0.0005 in both analysis ) . although the correlation between viral load and elisa od450 values in fecal specimens tested with the polyclonal antibody  based elisa was not significant , there was a trend for such a correlation ( pearson correlation 0.229 , p = 0.078 ) . in this study ,
fecal and urine samples were used because they are easier and safer to obtain and more readily available . in our previous reports ,
nucleocapsid protein was detected by the polyclonal antibody  based elisa in 83% of nasopharyngeal aspirates collected on days 11 to 15 after symptom onset and by the monoclonal antibody  based elisa in 85% of serum obtained during the first 10 days ( 10,11 ) .
these findings suggest that elisa may be more useful when used with nasopharyngeal aspirate and serum specimens .
however , these specimens were not included in the current study because only small amounts were available .
this study was supported by the research grant council grant ( hku 7532/03 m ) ; vice - chancellor sars research fund ( 21395035/39839/20700/420/01 and 21395061/27944/20700/420/01 ) , the university of hong kong ; and suen chi sun charitable foundation sars research fund .
using clinical samples from patients with severe acute respiratory syndrome , we showed that the sensitivities of a quantitative reverse transcription  polymerase chain reaction ( 80% for fecal samples and 
 25% for urine samples ) were higher than those of the polyclonal ( 50% and 5% ) and monoclonal ( 35% and 8% ) antibody - based nucleocapsid antigen capture enzyme - linked immunosorbent assays .