Replacing traditional diagnostics of fecal viral pathogens by a comprehensive panel of real-time PCRs

Abstract

Molecular DNA-based diagnostics are increasingly being used for diagnosis of viral infections. For enteric viruses, PCR assays have also been developed. The aims of this study were to compile and evaluate a comprehensive panel of PCR assays for diagnosis of viruses causing diarrheal disease and to evaluate its use in a largely pediatric population in a 750-bed university medical center. The PCR panel was designed to include assays for detection of adenovirus, astrovirus, enterovirus, norovirus, parechovirus, rotavirus, and sapovirus. The results of the PCR panel were evaluated in relation to conventional viral diagnostics consisting of viral culture and/or rotavirus and adenovirus rapid antigen tests on samples that were taken for routine diagnostics. Comparing conventional with PCR-based testing, the number of viruses detected increased dramatically from 25 to 106 when PCR assays were used. This increase was due mainly to detection of previously undetected viruses, i.e., astrovirus, norovirus, and sapovirus. In 24% of the samples, norovirus was detected. Also, the lower detection limit of PCR-based adenovirus, enterovirus, parechovirus, and rotavirus diagnostics further increased the detection rate. By focusing on samples from patients with complaints of gastroenteritis, detection of a causative agent was increased from 49% by conventional tests to 97% by molecular diagnostics. However, many samples containing low viral loads were found in patients with complaints other than intestinal complaints. In conclusion, the proposed comprehensive PCR panel with appropriate cutoff values can be used for sensitive, rapid, and clinically relevant diagnosis of gastrointestinal viruses.

Fig. 1.

Overview of results from conventional testing compared to those of the panel of real-time PCRs. Conventional testing consisted of a rapid antigen test for adenovirus and rotavirus and/or viral culture.

Fig. 2.

Comparison of the number of viruses found by real-time PCR in samples where only one virus was detected (single) and in those samples where multiple viruses were found (multiple). On the second y axis, the average C_T values found are represented by diamonds, and the lines represent the range of C_T values found within each group.

Fig. 3.

Comparison of clinical symptoms of patients who were PCR positive for rotavirus and those who had either rotavirus RAT-positive detection or rotavirus RAT-negative detection. The latter group is furthermore divided into those with a low C_T value (higher rotavirus load) and high C_T values (lower rotavirus loads). In this figure, only data of patients in which rotavirus was the only virus detected by any method have been included.