Comparison of direct electron microscopy, immune electron microscopy, and rotavirus enzyme-linked immunosorbent assay for detection of gastroenteritis viruses in children

Abstract

An approximate 10% suspension in water of the first available stool sample from 411 infants and young children with acute gastroenteritis was examined by electron microscopy (EM) after 2 min of negative staining. This procedure enabled the detection of 88% of the 199 rotavirus infections, all of the 22 adenovirus infections, and 47% of the 15 approximately 27-nm virus infections ultimately detected by a combination of techniques, including immune electron microscopy (IEM) and rotavirus enzyme-linked immunosorbent assay (ELISA). Of the 204 infections detected by direct EM of stools, 76% were detected within 2 min of viewing, and 94% were detected within 6 min of viewing. Type 1 and type 2 rotavirus particles were visualized with approximately equal efficiency, although type 2 rotavirus infections were more common. Rectal swab preparations were clearly inferior to stool preparations for the detection of virus infection by direct EM. IEM examination was required for efficient visualization of viruses in rectal swab specimens. ELISA was the most sensitive method for the detection of rotaviruses; with this method, all infections in which rotavirus particles were visualized by EM or IEM were detected. However, 73% of the 1,834 specimens which were presumptively positive for rotavirus by conventional indirect ELISA proved to be falsely positive on the basis of EM, IEM, blocking ELISA, confirmatory ELISA, or a combination of these methods. False-positive rotavirus ELISA reactions apparently were eliminated when fecal specimens were tested in a modified confirmatory ELISA with a lower dilution of rotavirus-negative (pre-immunization) than rotavirus-positive (post-immunization) capture antibody from the same animal.

Enzyme-linked immunosorbent assay for the detection of the coronavirus-like agent and its antibodies in pigs with porcine epidemic diarrhea

“…This was required to eliminate false-positive results that otherwise would have been encountered during this study in 15 of a total of 42 fecal specimens. Similar experiences have been reported with solid-phase immunoassays for detection of human rotavirus (Yolken et al, 1977;Cukor et al, 1978;Brandt et al, 1981) and Norwalk enteritis virus (Greenberg et al, 1978) in feces. As suggested by Brandt et al (1981), these false-positive reactions may be due to the presence in the test samples of intestinal bacteria or their products, which bind nonspecifically to the antibodies used as reagents in ELISA.…”

Analysis of Human Rotaviruses from a Single Location Over an 18-Year Time Span Suggests that Protein Coadaption Influences Gene Constellations

“…Sample collection, RNA extraction, and G/P typing. Fecal specimens were collected from infants and young children who were hospitalized with diarrhea at Children's Hospital National Medical Center, Washington, DC, as described previously (18)(19)(20)(21). The fecal specimens were tested for evidence of RV using electron microscopy and for viral antigens using an enzyme-linked immunosorbent assay (ELISA) (23).…”

“…During the years 1974 to 1991, fecal specimens were collected from infants and young children who were hospitalized with acute gastroenteritis at Children's Hospital National Medical Center in Washington, DC (18)(19)(20)(21). Total RNA was extracted from RV-positive specimens and classified into preliminary G/P types using a microtiter plate hybridization-based PCR-ELISA (23) ( Table 1).…”

“…To better understand the limitations of gene reassortment among human RVs, our laboratories have been systematically analyzing the sequences of genogroup 1 strains found in fecal specimens that were collected from children with RV gastroenteritis at Children's Hospital National Medical Center in Washington, DC (18)(19)(20)(21). This specimen collection is unique in that it includes samples from as early as 1974 -just a few years after the discovery of RVs-to as late as 1991 (22) (Table 1).…”

Comparison of enzyme-linked immunosorbent assay and RT-PCR for the detection of porcine epidemic diarrhoea virus

“…extremely autolysed tissues or those stored at room temperature for prolonged periods. In addition, false-positive reactions in the ELISA assay may be due to the non-specific binding of antibodies used as reagents to intestinal bacteria or their products (Brandt et al, 1981). However, this was probably Fig.…”