Comparison of two different combined test strips with fluorescent microspheres or colored microspheres as tracers for rotavirus and adenovirus detection

Rotavirus (RV) and enteric adenovirus (AdV) infections mainly result in acute infantile gastroenteritis, transmitted via the fecal–oral route. The symptoms include watery diarrhea, vomiting, headaches, fever, and gastralgic abdominal spasms [1, 2]. Symptoms may appear 1–2 days after viral infection, and persist for 1–10 days after the development of gastroenteritis, but different viral infections have different durations (RV approximately 3 days; AdV 5–8 days) [3], and the clinical features differ. Therefore, differential diagnosis is very important, especially for early diagnosis and treatment. Symptoms may also manifest in adults [4, 5]. RV and AdV account for the first and second most common viral infections among children under 5 years, respectively [6‐8]. Therefore, RV and AdV are tested for simultaneously during the clinical diagnosis of viral gastroenteritis [4, 5].

Several methods for detecting RV and AdV are currently available: 1) viruses were cultured in cells, and immunological or molecular biological methods are used to identify the viruses [9]; 2) immunological methods are used directly to detect the presence of antigens or IgM and IgG antibodies in the specimens [10‐12]; and 3) molecular biological methods are used directly to detect antigens [10, 13]. Methods 1 and 3 are limited with respect to technology and equipment, especially in primary medical institutions and field tests. With regard to Method 2, tests based on gold immunochromatographic assays, in which colloidal gold is used as the tracer utilized in antigen–antibody reactions, are simple, fast, require no special equipment or professionals, and have been used extensively in several fields including medicine and food safety [14‐16]. However, this method has low sensitivity [17], and may fail to detect low titers of RV and AdV.

An emerging detection technique uses fluorescent microspheres [18, 19] instead of colloidal gold particles [20]. Reaction bands can be observed with the naked eye following illumination with an ordinary UV lamp or special equipment. This technique is comparatively sensitive and stable [21, 22]. High-resolution spectra that are recognizable by the naked eye can be obtained from stimulated microspheres, with sensitivities higher than those of colloidal gold particles [23].The relevant products are currently at the basic research and development stage, and are based on fluorescent microspheres (mainly for single-item detection) [24‐26]. Colored microspheres are also used as detection tracers. They can be produced in any color (e.g., black, blue, orange, gray, red, pink, green, violet, or yellow) and in various sizes (most from 10 μm to 1000 μm) from materials including polyethylene, silica, and glass [27, 28]. However, the sensitivity of colored microsphere tracers is not well known, and will be evaluated in the present study.

The objective of the present study was to compare the two different methods—using fluorescent microspheretracers (FMT) or colored microspheretracers (CMT)—and to demonstrate the efficiency of FMT in relation to CMT in combined RV–AdV detection. This will provide a more reliable mean of detecting RV and AdV infections, and will have a significant clinical effect on the proper diagnosis and therapy of viral gastrointestinal infections, especially in primary medical institutions.

The use of colloidal gold test strips for the detection of RV and AdV is gradually increasing, both in China and abroad. However, owing to technological limitations, there are very few combined detection products. The CerTest rotavirus–adenovirus blister test strip (CMT) is popular in clinical practice and scientific research [32, 33]. However, CMT strips do sometimes fail to detect viruses (false negative results) during clinical tests owing to deficiencies in the colored microsphere technique, especially when there are insufficient quantities of RV and AdV in the stool specimens. Thus, there is a huge demand for lateral flow immunochromatographic techniques with highly sensitive fluorescent microspheres as tracers. Currently, there are few manufacturers of combined RV–AdV fluorescent microspheredetection test strips in China and abroad.

In the present study, CMT and real-time RT-PCR were used as the reference products to evaluate the detection performance of combined RV–AdV fluorescent microsphere detection test strips. CMT uses colored microspheres (red and blue, etc.) as the tracers, which produce significant results when used to detect diagnostic reagents in vitro. The efficiency of the test trips can them be determined. Thus, colored latex microsphere immunoassays have gradually attracted attention in the industry [34‐36].

RV is the most common cause of severe acute gastroenteritis in children [1], and the most common enteropathogens have been extensively studied in China [37‐39]. In the present study, when a combined RV–AdV FMT strip was used to detect viruses in the clinical specimens, the rate of positive RV detection in the 350 clinical specimens was 37.4% (131/350), and the rate of positive AdV detection was 20.6% (72/350). Such high infection rates demonstrate that it is necessary to detect both RV and AdV in clinical specimens from patients with gastroenteritis in China.

The results show that the combined RV–AdV FMT detection strips were significantly more sensitive than the CMT strips. Although the FMT technique seems to be more sensitive, it has some limitations that must be considered, such as the risk of false positives. Therefore, caution should be exercised, especially during the detection of weakly positive specimens [40]. Low specimen numbers also constitute a limitation. Hence, there is a requirement for further development by medical institutions, and for large-scale clinical specimen verification. There was cross reaction with the other common viral intestinal infections with FMT. The test results should be obtained within 30 min of specimen collection to provide timely, accurate, and reliable diagnoses in clinical settings.

Both FMT and CMT were able to detect RV and AdV with high specificity, although FMT had higher detection sensitivity than CMT.