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Journal of Clinical Microbiology, December 2001, p. 4532-4534, Vol. 39, No. 12
Institute of Medical Microbiology, Clinical
Virology, University of Muenster,1 and
Institute of Public Health, North
Rhine-Westphalia,2 Muenster, Germany
Received 2 May 2001/Returned for modification 23 July 2001/Accepted 17 September 2001
The two assays evaluated in this study (the Ridascreen rotavirus
and the Pathfinder rotavirus) exhibited comparable sensitivities (100%) but highly divergent positive predictive values (93.74 and
57.7%, respectively) when compared on 393 specimens. This difference
should be considered when using these tests on collectives with an
unknown or low prevalence.
Enzyme immunoassays (EIAs) have
replaced electron micoscropy (EM) as the standard method for the
detection of rotaviruses in stool samples in the 1980s (1, 3-6,
8-13, 15-18). Typically, these assays are applied on stool
specimens from children younger than 2 years with diarrheal disease.
Since the past few years, however, we have observed an increasing
tendency in clinical practice to include antigen detection assays in
pretransplantation evaluation protocols. Therefore, in the present
study we examined the impact of this strategy on the predictive values
of two representative commercial test kits for rotavirus antigen
detection in stool samples: the Ridascreen rotavirus (R-Biopharm,
Darmstadt, Germany) as an automated test in microtiter plate format and
the Pathfinder rotavirus (Kallestadt, Austin, Tex.) as a manually
performed test designed for small series or single specimens. EM served
as a supportive method for defining a formal "gold standard."
As a high-risk collective for our study, 192 stool samples were
acquired from the pediatric unit of the university hospital of
Münster during the rotavirus season in 1997 to 1998, which were
either submitted with the clinical diagnosis of enteritis or
macroscopically liquid (mostly both). As a low-prevalence collective, 201 formed stools from healthy adults were randomly selected from a
series of routine examinations from the Institute of Public Health,
North Rhine-Westfalia. Both EIAs were performed exactly as specified by
the manufacturers. Specimens exhibiting borderline results were
retested once, and the latter result was used for evaluation. EM was
performed by standard methods with negative staining after
concentration of virus particles at 100,000 × g (7). Specimens without clearly recognizable virus
particles were considered negative after an examination time of 20 min
at ×50,000 magnification. To define a formal gold standard, all
samples which had positive results in at least two of the three methods were regarded as true positive.
An overview of the results is shown in Table
1. A total of 15 samples in this study
were considered true positive; 14 of these were recognized by EM
(sensitivity, 93.9%), and none of the negative samples was wrongly
considered positive (specificity, 100% [Table 1]). The Pathfinder
test recognized all positive samples as positive (sensitivity, 100%)
but gave 11 false-positive results (specificity, 79.1% [Table 1]), 1 of which emerged after retesting a primarily borderline specimen
(specimen 1302 [Table 2]). The
Ridascreen test also recognized all true-positive specimens, but it
gave one false positive result (sensitivity, 100%; specificity, 99.73%). A detailed analysis of the 11 "false-positive" pathfinder results (Fig. 1; Table 2) showed that the
majority of these (8 of 11) were not caused by optical densities (ODs)
near the cutoff; thus, specificity could not have been substantially
improved by increasing the cutoff. Eight of the false-positive
Pathfinder results were obtained in the adult group with formed stools.
The positive and negative predictive values (PPV and NPV, respectively) were 100 and 99.73% for EM, 93.74 and 100% for the Ridascreen rotavirus, and 57.7 and 100% for the Pathfinder rotavirus. These numbers were calculated using the rotavirus prevalence in the entire
specimen collective (3.82%; 15 of 393). If the evaluation was
restricted to the "classical" patient subset for rotavirus tests
(children younger than 2 years [n = 124]), the
rotavirus prevalence increased to 11.29% (14 positives of 124 samples)
and, consequently, the PPV of the Pathfinder rotavirus test improved to
82.35%. This was an effect not only of higher prevalence but also of
an improved specificity itself (now only 3 false positives in 110 true
negatives [97.27%]). Because the Pathfinder test is a manually
performed test, we suspected that the relatively large number of
false-positive results was due to a lack of care during the washing
steps. We therefore retested all false-positive samples, taking special
care with the washing steps. As shown in Table 2, retesting did not
improve the performance significantly (9 of 11 false positives still
remained positive).
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4532-4534.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Evaluation of Two Enzyme Immunoassays for Detection
of Human Rotaviruses in Fecal Specimens
ABSTRACT
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TABLE 1.
Summary of results
TABLE 2.
Raw data of the 11 discordant positive results obtained
with the Pathfinder testa
View larger version (16K):
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FIG. 1.
Plot of the relative ODs of the pathfinder rotavirus and
the ridascreen rotavirus EIA. The ODs are normalized by division with
the cutoff of the respective individual run.
In summary, it turned out that the EIAs were equivalent with respect to their sensitivities and were comparable or even slightly superior to EM. This is in concordance with the majority of publications covering this subject (4, 5, 8, 12, 13, 15, 17, 18). Some investigators have postulated that EIAs are probably a better standard method for detection of rotaviruses than is EM due to the low sensitivity of the latter (2, 5); however, in these studies EM was performed without ultracentrifugation of samples, which has been demonstrated to be an essential step in achieving maximum sensitivity (7). Rabenau et al. (14) have also evaluated the Ridascreen rotavirus test along with a panel of other assays and shown that this test, after adjustment of the cutoff value, exhibited superior sensitivity and specificity values for the detection of rotaviruses, which is in accordance with our study. In contrast, the false-positive results of the Pathfinder rotavirus assay could not be erased by increasing the cutoff value (Table 2; Fig. 1). Thus, in view of the broad interassay variability seen on retesting of the specimens giving false-positive results with the Pathfinder rotavirus assay, the most probable explanation for these false-positive results is the manual test format, which cannot guarantee highly reproducible performance compared with an automated test system.
We conclude that the two tests evaluated here exhibit comparable performance when used in a "classical" patient collective. However, if specimens are obtained from an "atypical" patient collective, PPVs can drop dramatically and the minor-looking differences in specificity may become vital. This study clarifies that tests originally designed for confirmation of a strong clinical suspect cannot be recommended for screening purposes. However, if testing of individual patients from "atypical" collectives is explicitly requested, it is crucial to use the more specific tests (e.g., the Ridascreen rotavirus in the setting of this study).
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ACKNOWLEDGMENTS |
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We thank Sabine Lima and Maria Seehusen for their excellent technical assistance, and we thank r-Biopharm and Sanofi Pasteur for supplying the EIA reagents.
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FOOTNOTES |
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* Corresponding author. Mailing address: Institute of Medical Microbiology, Clinical Virology, von-Stauffenberg-Str. 36, 48151 Muenster, Germany. Phone: (49) 251 7793 111. Fax: (49) 251 7793 104. E-mail: eingb{at}uni-muenster.de.
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Journal of Clinical Microbiology, December 2001, p. 4532-4534, Vol. 39, No. 12
0095-1137/01/$04.00+0 DOI: 10.1128/JCM.39.12.4532-4534.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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