Evaluation of a commercial latex agglutination test for detecting rotavirus A and human adenovirus in children's stool specimens.

OBJECTIVES
Rotavirus A and human adenovirus are the two most common causes of infantile diarrhea; thus, it is of great importance to find out a rapid and accurate diagnostic method. This study aimed to evaluate the diagnostic significance of latex agglutination test for detection of rotavirus A and human adenovirus.


METHODS
A prospective study was conducted on 214 diarrhea children from September 2018 to March 2019 in our hospital. Fresh stool samples were collected for detection of rotavirus A and human adenovirus by latex agglutination test and quantitative reverse transcription polymerase chain reaction (RT-qPCR). Then, the consistency of results detected by these two methods was analyzed． RESULTS: With performing the latex agglutination test, it was revealed that positive rates for detecting rotavirus A virus and human adenovirus were 23.83% (51/214) and 25.24% (54/214), respectively. Meanwhile, results of RT-qPCR showed that positive rates for detecting rotavirus A virus and human adenovirus were 58 (27.10%) and 59 (27.57%), respectively. Using RT-qPCR as the gold standard, the sensitivity and specificity of the latex agglutination test for detecting rotavirus A were 81.03% and 97.44%, and the corresponding values for detecting human adenovirus were 76.27% and 94.19%, respectively.


CONCLUSION
This latex agglutination test showed a satisfactory consistency with RT-qPCR for detecting rotavirus A and human adenovirus. The mentioned commercial assay may be highly appropriate for rapid screening of rotavirus A and human adenovirus.

In humans, RVs, which were firstly discovered in 1973 in duodenal biopsy of nine children who suffered from acute diarrhea, are non-enveloped viruses of the Reoviridae family, 5,6 and group A (RVA) was found as the most common infectious variant in humans. AdV is a relatively large non-enveloped dsDNA virus possessing a molecular weight of ~150 MDa. 7 It has more than 80 different serotypes belonging to species A-G, 8 and they mainly infect humans through respiratory, ocular, and gastrointestinal. AdV has become the second principal reason for infantile diarrhea behind RV. 9 Rapid and accurate diagnosis of AGE in early stage is of great importance for the treatment of AGE. Hence, a rapid and accurate diagnostic method for AGE is urgently required.
Several techniques can be used to detect these two viruses, including scanning electron microscopy, polyacrylamide gel electrophoresis (PAGE), antigen detection assays, quantitative reverse transcription polymerase chain reaction (RT-qPCR), enzyme-linked immunoassay technology (ELISA), and virus isolation. However, it was previously noted that due complex operation, expensive equipment, etc, both scanning electron microscopy and PAGE are not highly appropriate to be used in the clinical practice. Moreover, the culture may lead to false-negative results when fastidious or slowly growing viruses are involved. [10][11][12][13] At present, antigen detection assays and RT-qPCR are the two most common detective methods worldwide.
In the present study, we utilized a commercial latex agglutination test kit, which is extensively used in numerous institutions across China. In clinical practice, RT-qPCR is taken as a main method for detection of RV and AdV; however, it is extremely costly, making it inappropriate for early diagnosis. Hence, in the current research, RT-qPCR was used as the gold standard to assess the capacity of the commercial LAT method for rapid detection of RV and AdV in outpatient children.

| RT-qPCR for detection of rotavirus A and human adenovirus
Stool samples were collected and mixed with 1 mL normal saline.
Then, 200 μL supernatant was separated from the mixture, which was centrifuged at 400 g at 20°C for 30 seconds, and DNA/RNA was extracted by NAE32 nucleic acid automatic extraction kit (DAAN Gene Co., Ltd.). The detection of rotavirus A and adenovirus was performed by SLAN 96P real-time PCR System (HONGSHI Co., Ltd.) with a total volume of 25 μL. For each assay, negative and positive controls were implemented. FAM channel was used to detect rotavirus A, and HEX channel was employed to detect adenovirus. The amplification of RT-qPCR products was conducted under the following conditions: for 20 minutes at 50°C; for 5 minutes at 95°C; five cycles for 10 seconds at 95°C, for 15 seconds at 55°C, and for 30 seconds at 72°C; and followed by 35 cycles for 10 seconds at 95°C and for 45 seconds at 60°C.

| Statistical analysis
The main purpose of the present study was to assess the sensitivity and specificity of the latex agglutination test compared with RT-qPCR, which was considered as a standard method. The results were analyzed by using SPSS 20.0 software. The kappa test and Student's t test were used to determine statistical significance. P < .05 was considered statistically significant. The kappa (κ) value between 0.61 and 0.8 represented good agreement and between 0.81 and 1 reflected very good agreement. 14,15

| RE SULTS
To investigate the performance of commercial latex agglutination test kit, a total of 214 samples were collected in this study. As shown in

| D ISCUSS I ON
Diarrhea is a common and frequent disease in infants and young children, 16  According to a previous study, virus is further prevalent in autumn and winter seasons, because viruses may be more stable at a low temperature. 18 In the present study, we successfully enrolled 214 patients who were admitted to our hospital in the aforementioned seasons. Our data showed that positive rates of RV were 23.83% and 27.10% for latex agglutination test and RT-qPCR, respectively. Moreover, the positive rates of AdV were 25.23% and 27.57% for latex agglutination test and RT-qPCR, respectively. Both of the positive rates were relatively remarkable, while previous studies reported different prevalence rates. [19][20][21] The results of the current research indicated that there was no significant difference (P > .05) between the latex agglutination test and RT-qPCR in the detection of RV and AdV. Meanwhile, it was noted that there was a good agreement in detection of RV and AdV between these two methods. To evaluate the performance of the commercial LAT assay kit, we used RT-qPCR as the gold standard. for confirming the viruses, while LAT is highly recommended for screening. This research was a case-control study, and the coincidence rate of the positive rate between the two assays was slightly more than daily monitoring. In the next study, we will expand the sample size to find out differences between the two methods. Moreover, we found that there were two cross-reactional samples, indicating that the test line of RV was red and the test line of AdV was blue. There were several reasons can cause cross-reaction, but the most probably reason may be the fecal samples with too much blood, which was caused by multiple bowel movements. In clinical practice, special attention is needed for these cases.

| CON CLUS IONS
The proposed commercial latex agglutination test is a low-cost screening method for identification of rotavirus and adenovirus with a middle level of sensitivity and specificity. However, the mentioned method is not highly appropriate for accurate diagnosis of rotavirus and adenovirus in hospitalized children. In the future study, we will further evaluate fast diagnostic methods with consideration of lager sample size.

ACK N OWLED G M ENTS
This study was supported by the Natural Science Foundation of Zhejiang Province (LY15H050001).

CO N FLI C T O F I NTE R E S T
The authors declare that there is no conflict of interest.