Development and Evaluation of a Rapid, Simple, and Sensitive Immunochromatographic Assay To Detect Thermostable Direct Hemolysin Produced by Vibrio parahaemolyticus in Enrichment Cultures of Stool Specimens

Abstract

Thermostable direct hemolysin (TDH) is considered to be a major virulence factor in Vibrio parahaemolyticus, and most cases of V. parahaemolyticus diarrhea in humans are caused by tdh gene-positive strains. In the present study, we developed an immunochromatographic assay to detect TDH (TDH-ICA) and evaluated the utility of TDH-ICA for the diagnosis of V. parahaemolyticus diarrhea. TDH-ICA allowed the detection of 0.2 ng/ml of TDH within 10 min. Fecal homogenates were spiked with various numbers of tdh-positive V. parahaemolyticus organisms, and their enrichment cultures were tested with TDH-ICA. The results of detection of TDH in the enrichment cultures by TDH-ICA were in accord with the results of recovery of the spiked V. parahaemolyticus organisms from the enrichment cultures by plating onto thiosulfate-citrate-bile salts-sucrose agar. When enrichment cultures of 217 stool specimens from patients with diarrhea were tested with TDH-ICA, the TDH-ICA results showed 100% sensitivity and specificity compared to the results of isolation of V. parahaemolyticus from the stool specimens by a conventional bacterial culture test. Since TDH-ICA was able to detect TDH in a fecal enrichment culture within 10 min, TDH-ICA testing of a fecal enrichment culture could be completed rapidly and easily within approximately 16 h, including incubation time for the fecal enrichment culture. These results indicate that TDH-ICA is a rapid, simple, and sensitive TDH detection method and that TDH-ICA testing of a fecal enrichment culture is useful as an adjunct to facilitate the early diagnosis of V. parahaemolyticus diarrhea.Vibrio parahaemolyticus is a major food-borne pathogen that causes diarrhea in humans through seafood consumption, but not all strains are considered to be pathogenic. Thermostable direct hemolysin (TDH) has been found to be closely associated with the enteropathogenicity of V. parahaemolyticus (5). In the late 1980s, cases of diarrhea caused by Vibrio parahaemolyticus strains that did not produce TDH were found (8). The strains produced a new hemolysin that was immunologically similar but not identical to TDH. The hemolysin was referred to as TDH-related hemolysin (TRH). Currently, TDH and TRH are regarded as important virulence factors of V. parahaemolyticus (5, 13), and therefore, strains possessing the tdh gene encoding TDH and/or the trh gene encoding TRH are considered pathogenic strains (14). Since most cases (approximately 90%) of V. parahaemolyticus diarrhea in humans are caused by tdh-positive strains, including strains that possess both the tdh and trh genes (1, 2, 16, 17, 18), tdh-positive V. parahaemolyticus is a major causative organism of V. parahaemolyticus diarrhea.Cases of V. parahaemolyticus diarrhea in humans are commonly diagnosed by isolating this pathogen from a stool specimen by a bacterial culture test. The test procedure includes the plating of stool or its enrichment culture onto selective agar medium and the subsequent identification of suspected colonies on the agar medium. The identif...

Screening for a Single-Chain Variable-Fragment Antibody That Can Effectively Neutralize the Cytotoxicity of the Vibrio parahaemolyticus Thermolabile Hemolysin

“…ibrio parahaemolyticus, a Gram-negative motile bacterium that inhabits marine and estuarine environments throughout the world (7,23,36), is a major food-borne pathogen that causes life-threatening diseases in humans through the consumption of raw or undercooked seafood (5,21,34 It is well known that V. parahaemolyticus contains many different kinds of toxins, such as thermostable direct hemolysin (TDH), TDH-related hemolysin (TRH), and some noncharacterized proteins. TDH is considered one of the major virulence factors of V. parahaemolyticus, and its function has been well characterized and discussed (25).…”

A highly sensitive and flexible magnetic nanoprobe labeled immunochromatographic assay platform for pathogen Vibrio parahaemolyticus

“…Lateral flow immunochromatographic assay (LFIA) is an extended application of ELISA, it is rapid, simple, and appropriate for point-of-care test (Zhang et al, 2009;Dzantiev et al, 2014). The labels used in LFIA system mainly include colloidal gold nanoparticles (Kawatsu et al, 2006;Ju et al, 2010;Preechakasedkit et al, 2012;, magnetic nanoparticles Xu et al, 2009;Wang et al, 2015a), fluorescent microspheres (Xu et al, 2013), quantum dot and so on. Compared with other labels, magnetic nanoparticles can provide a higher sensitivity , because the magnetic signals from surface and internal membrane of LFIA can be detected by a Magnetic Assay Reader (MAR).…”

Development and Evaluation of Immunochromatographic Assay for Simple and Rapid Detection of Campylobacter jejuni and Campylobacter coli in Human Stool Specimens

“…Test strips and MAb-colloidal-gold conjugates were prepared according to the protocol described previously (19). Briefly, to prepare the test strips, an absorbent pad (Millipore, Bedford, MA) was attached to a laminated membrane card (HiFlow Plus 135; Millipore) so that the pad slightly overlapped the membrane, and the assembly was then cut into strips consisting of the membrane (5 by 25 mm) with the absorbent pad (5 by 17 mm).…”