Use of a Multiplex PCR Assay To Assess the Presence of Treponema pallidum in Mucocutaneous Ulcerations in Patients with Suspected Syphilis.

We evaluated the utility of the commercial Allplex genital ulcer real-time PCR multiplex assay for detecting Treponema pallidum, herpes simplex virus 1 (HSV-1) and 2 (HSV-2), and Chlamydia trachomatis serovar L (lymphogranuloma venereum [LGV]) DNA in mucosal and genital ulcers in the context of suspected syphilis. In total, 374 documented genital and mucosal ulcers from patients with and without syphilis presenting at several sexually transmitted infection (STI) centers in France from October 2010 to December 2016 were analyzed at the National Reference Center (CNR) for Bacterial STIs at Cochin Hospital in Paris. T. pallidum subsp. pallidum detection results were compared with the final diagnosis based on a combination of clinical examination, serological results, and in-house nested PCR (nPCR). Detections of HSV and LGV were validated against reference methods. We found that 44.6% of the 374 samples tested were positive for T. pallidum subsp. pallidum, 21% for HSV, and 0.8% for LGV. No positive results were obtained for 30.7% of samples, and 4.8% presented coinfections. For T. pallidum subsp. pallidum detection, the overall sensitivity was 80% (95% confidence interval [CI], 76.1 to 84.1%), specificity was 98.8% (95% CI, 97.7 to 99.9%), positive predictive value was 98.8% (95% CI, 97.7 to 99.9%) and negative predictive value was 80.2% (95% CI, 76.2 to 84.2%), with a rate of concordance with the reference method of 92.5% (k = 0.85). This PCR multiplex assay is suitable for T. pallidum subsp. pallidum detection in routine use and facilitates the simultaneous rapid detection of a broad panel of pathogens relevant in a context of suspected syphilis lesions.

Evaluation of a PCR test for detection of treponema pallidum in swabs and blood.

Syphilis diagnosis is based on clinical observation, serological analysis, and dark-field microscopy (DFM) detection of Treponema pallidum subsp. pallidum, the etiological agent of syphilis, in skin ulcers. We performed a nested PCR (nPCR) assay specifically amplifying the tpp47 gene of T. pallidum from swab and blood specimens. We studied a cohort of 294 patients with suspected syphilis and 35 healthy volunteers. Eighty-seven of the 294 patients had primary syphilis, 103 had secondary syphilis, 40 had latent syphilis, and 64 were found not to have syphilis. The T. pallidum nPCR results for swab specimens were highly concordant with syphilis diagnosis, with a sensitivity of 82% and a specificity of 95%. Reasonable agreement was observed between the results obtained with the nPCR and DFM methods (kappa = 0.53). No agreement was found between the nPCR detection of T. pallidum in blood and the diagnosis of syphilis, with sensitivities of 29, 18, 14.7, and 24% and specificities of 96, 92, 93, and 97% for peripheral blood mononuclear cell (PBMC), plasma, serum, and whole-blood fractions, respectively. HIV status did not affect the frequency of T. pallidum detection in any of the specimens tested. Swab specimens from mucosal or skin lesions seemed to be more useful than blood for the efficient detection of the T. pallidum genome and, thus, for the diagnosis of syphilis.

Remodeling of VE-cadherin junctions by the human herpes virus 8 G-protein coupled receptor.

Kaposi Sarcoma (KS) are opportunistic tumors, associated with human herpes virus 8 (HHV8) infection. KS development is highly favored by immune-depression and remains the second most frequent tumor in acquired immune deficiency syndrome patients. Although it has been shown that experimental expression of the HHV8 G-protein-coupled receptor (vGPCR) in the endothelial compartment is alone sufficient to recapitulate the formation and progression of KS-like lesions, its functional effects on endothelial homeostasis are not fully understood. Here we show that vGPCR expression in endothelial cells induces an increase in paracellular permeability both in vivo and in vitro. By using pharmacological inhibitors and small interference RNA-based knockdown, we demonstrate an essential role for the PI(3)Kinase-γ/Rac nexus in vGPCR-mediated permeability. This was further accompanied by dramatic remodeling of VE-cadherin-dependent cell-cell junctions. Importantly, this in vitro vGPCR-initiated signaling signature was observed in a large panel of human KS. Altogether, our results support the hypothesis that endothelial vGPCR signaling is co-opted in KS, and unveil new key cellular targets for therapeutic intervention.

Expression of putative Escherichia coli heat-labile enterotoxin (LT) receptors on intestinal brush borders from pigs of different ages.

Many strains of enterotoxigenic Escherichia coli (ETEC) that cause diarrhoea in young piglets secrete a heat-labile enterotoxin (LTp) that binds to specific glycoconjugates on porcine intestinal epithelial cells. Binding of LTp to an appropriate glycoconjugate facilitates the uptake and trafficking of the toxin into the cell, where it stimulates intracellular changes that promote fluid secretion and diarrhoea. The objective of the current study was to identify the LTp-binding glycoconjugates on porcine intestinal epithelial cells, the natural target cells for LTp. We found that LTp binds, in an age-correlated manner, to an acidic glycosphingolipid (GSL) that co-migrated with GM1 on thin-layer chromatography (TLC), a small acidic GSL that appears to be a sulphatide, a neutral GSL that co-migrated with neolactotetraglycosylceramide (nLc4) on TLC, and two glycoproteins (36 and 205 kDa). Of these potential LTp receptors, the GM1-co-migrating GSL was detected most intensely in young animals, while the other four LTp-binding glycoconjugates were detected most intensely in older pigs (> or= 4 weeks). Since ETEC primarily cause disease in young piglets, the GM1-co-migrating GSL is the most likely candidate for a functional LTp receptor.

Escherichia coli strain RDEC-1 AF/R1 endogenous fimbrial glycoconjugate receptor molecules in rabbit small intestine.

Escherichia coli strain RDEC-1 causes a diarrheagenic infection in rabbits with AF/R1 fimbriae, which have been identified as an important colonization factor in RDEC-1 adherence leading to disease. The AF/R1-mediated RDEC-1 adherence model has been used as a model systems for E. coli diarrheal diseases. In this study, RDEC-1 adhered specifically to small intestinal brush borders, with both sialic acid and beta-galactosyl residues apparently involved. The AF/R1-mediated adherence activity of [(14)C]-labeled RDEC-1 was analyzed quantitatively by using 24-well plates coated with purified brush borders and purified microvilli. Two microvillus membrane proteins (130 and 140 kDa) were individually isolated, and chicken antibody raised to each protein inhibited bacterial adherence. These same two proteins, previously shown to be recognized by AF/R1, were individually digested with trypsin, and the amino acid sequences of peptides were determined by reversed-phase capillary liquid chromatography-mass spectrometry tandem mass spectrometry (LC-MS). This LC-MS analysis indicated that these proteins are subunits of the rabbit sucrase-isomaltase protein (SI) complex. Guinea pig serum raised to purified rabbit SI complex inhibited bacterial adherence to microvilli. Additionally, as determined by high-performance thin-layer chromatography and autoradiography, RDEC-1 adhered selectively, via AF/R1 fimbriae, to a glycolipid tentatively identified as galactosylceramide (Gal beta 1-1Cer) in the lipid extract of rabbit small intestinal brush borders. RDEC-1 adherence to Gal beta 1-1Cer was partially inhibited in the presence of galactose. These combined results indicate that the endogenous receptor molecule for AF/R1 fimbriae of RDEC-1 is each individual component of the SI complex, although binding to glycolipid may be responsible for an additional adherence mechanism.

Identification of an intestinal neutral glycosphingolipid as a phenotype-specific receptor for the K88ad fimbrial adhesin of Escherichia coli.

In this study, we identified a receptor for the K88ad fimbrial adhesin of Escherichia coli in neutral glycosphingolipid preparations from intestinal epithelial cells of K88ad-adhesive pigs, which was absent in preparations from K88ad-nonadhesive pigs. Neither K88ab nor K88ac adhesin variants bound to this neutral glycosphingolipid. Because this receptor is an intestinal glycosphingolipid that binds K88ad adhesin, it has been designated IGLad. Carbohydrate compositional analysis of a partially purified preparation of IGLad identified galactose, glucose, and N-acetylglucosamine in a ratio of 1.5:1.0:0.5 as the major monosaccharides. Preliminary characterization experiments using lectins showed that IGLad contains the terminal glycanic structure Galbeta1-4GlcNAc. Removal of terminal beta-linked galactose residues from IGLad decreased the recognition of IGLad by the K88ad adhesin, indicating that terminal beta-linked galactose is an essential component of the K88ad adhesin recognition site on IGLad. Studies with purified glycosphingolipid standards demonstrated that K88ad adhesin binds to neolactotetraosylceramide (nLc4Cer) (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) , lactotriosylceramide (GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) and lactotetraosylceramide (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glcbeta1-1Cer) . Based on these studies, IGLad appears to be nLc4Cer.

K88 adhesins of enterotoxigenic Escherichia coli and their porcine enterocyte receptors.

The three antigenic variants of the K88 fimbrial adhesin (K88ab, K88ac, and K88ad) of enterotoxigenic Escherichia coli (ETEC) each exhibit unique specificity with regard to their hemagglutination characteristics. The variants are also unique in the specificity of their binding to the brush borders of enterocytes isolated from pigs with different genetic backgrounds. Diversity in enterocyte binding specificity suggests the existence of several K88 receptors, expressed individually or in various combinations on porcine enterocytes. Three candidate receptors have been identified that may explain the adhesion of K88 fimbrial variants to various porcine enterocytes. These receptors are an intestinal mucin-type sialoglycoprotein (IMTGP), an intestinal transferrin (GP74), and an intestinal neutral glycosphingolipid (IGLad). The IMTGP binds K88ab and K88ac, but not K88ad. The GP74 binds K88ab, but not K88ac or K88ad, and the IGLad binds K88ad, but not K88ab or K88ac. Each of the candidate receptors has been found in brush borders that are adhesive for the fimbriae that bind the respective receptor. They have not been found in brush borders that are not adhesive for those same fimbriae. The presence of IMTGP was highly correlated with susceptibility of neonatal gnotobiotic pigs to ETEC expressing K88ab or K88ac.

Expression of mucin-type glycoprotein K88 receptors strongly correlates with piglet susceptibility to K88(+) enterotoxigenic Escherichia coli, but adhesion of this bacterium to brush borders does not.

Three antigenic variants of the K88 fimbrial adhesin exist in nature, K88ab, K88ac, and K88ad. Enterotoxigenic Escherichia coli (ETEC) strains that produce these fimbriae cause life-threatening diarrhea in some but not all young pigs. The susceptibility of pigs to these organisms has been correlated with the adherence of bacteria to isolated enterocyte brush borders. Whether that correlation holds for multiple K88 variants and over a broad genetic base of pigs is unknown and was the impetus for this study. We also desired to examine the correlation of the expression of a porcine intestinal brush border mucin-type glycoprotein (IMTGP) which binds K88ab and K88ac with the susceptibility of piglets to K88(+) ETEC. Of 31 neonatal gnotobiotic pigs inoculated with K88ab+ or K88ac+ ETEC, 13 developed severe diarrhea, became dehydrated, and died or became moribund. Another pig became severely lethargic but not dehydrated. In vitro brush border adherence analysis was not possible for 10 of the severely ill pigs due to colonization by challenge strains. However, of the 17 pigs that did not become severely ill, 8 (47%) had brush borders that supported the adherence of K88ab+ and K88ac+ bacteria in vitro, suggesting a poor correlation between in vitro brush border adherence and piglet susceptibility to K88(+) ETEC. By contrast, the expression of IMTGP was highly correlated with susceptibility to K88(+) ETEC. Of the 12 pigs that produced IMTGP, 11 developed severe diarrhea. The other pig that produced IMTGP became lethargic but not severely diarrheic. Only 2 of 18 pigs that did not produce IMTGP became severely diarrheic. Colonizing bacteria were observed in histologic sections of intestines from all pigs that expressed IMTGP except for the one that did not develop severe diarrhea. However, colonizing bacteria were observed in histologic sections from only one pig that did not produce IMTGP. The bacterial concentration in the jejuna and ilea of pigs expressing IMTGP was significantly greater (P < 0.005) than that in pigs not expressing IMTGP. These observations suggest the IMTGP is a biologically relevant receptor for K88ab+ and K88ac+ E. coli or a correlate for expression for such a receptor.

Characterization of the carbohydrate moiety of intestinal mucin-type sialoglycoprotein receptors for the K88ac fimbrial adhesin of Escherichia coli.

We have previously identified two mucin-type sialoglycoproteins from porcine intestinal epithelial cells with approximate molecular masses of 210 (intestinal mucin-type glycoprotein IMTGP-1) and 240 kDa (IMTGP-2) as receptors for the K88ab and K88ac fimbrial adhesins of Escherichia coli. These receptors are detected in intestinal brush border membrane preparations from pigs with adhesive phenotypes but not from pigs with nonadhesive phenotypes and are postulated to be important determinants of the susceptibility of pigs to K88ab+ and K88ac+ enterotoxigenic E. coli infections. Using exoglycosidase digestion studies, we have now determined that beta-linked galactose is an important component in the recognition of IMTGP-1 and IMTGP-2 by the K88ac adhesin. In addition, we observed a differential distribution of the K88ac adhesin binding activity of IMTGP-1 and IMTGP-2 along the crypt-villus axis, suggesting that receptor activity is dependent on the maturation state of the intestinal epithelial cells. Brush borders from immature intestinal epithelial cells possessed the highest concentrations of IMTGP-1 and IMTGP-2 receptor activity, with a progressive decrease in receptor activity as the cells mature. To characterize the differences in the carbohydrate moieties of IMTGP-1 and IMTGP-2, we developed a procedure for purifying the receptors, using phenol extraction followed by serial lectin affinity chromatography. Carbohydrate compositional analysis of the purified receptors indicated that the carbohydrate moieties of IMTGP-1 and IMTGP-2 consist of both N- and O-glycans containing galactose, glucose, sialic acid, mannose, N-acetylgalactosamine, N-acetylglucosamine, and fucose. The major difference between the two receptors is that IMTGP-2 contains a higher percentage of monosaccharides (mannose and glucose) commonly found in N-glycans.

Transferrin associated with the porcine intestinal mucosa is a receptor specific for K88ab fimbriae of Escherichia coli.

Putative receptors of Escherichia coli K88 fimbriae are either tightly membrane bound or an integral part of membranes. Thus, proteins associated with piglet small intestinal mucosae were solubilized by a detergent (deoxycholate). A 74-kDa glycoprotein (GP74) purified from enterocyte and brush border membrane preparations was specifically detected in vitro by K88ab fimbriae. GP74 was recognized only in the mucosae of phenotypically adhesive animals. Metaperiodate treatment abolished the recognition, indicating that K88ab fimbriae-GP74 binding required the carbohydrate moiety. This glycoprotein belongs to the transferrin family and differed from the serum transferrin of the same adhesive-phenotype piglets. Unlike intestinal transferrin, serum transferrin was recognized independently of the adhesion phenotype. The glycan moieties of intestinal and serum transferrins differed in their molar compositions. Transferrin GP74 contained one monosialylated and monofucosylated glycan chain of the N-acetyllactosamine type. Intestinal holotransferrin exhibited pI values of 5.2, 5.3, 5.5, and 5.6, whereas serum holotransferrin pI values ranged between 5.4 and 6.2. Since mucosal transferrin was found intimately entrapped on membranes, we hypothesize that a K88ab fimbriae-transferrin-cell transferrin receptor complex might allow the bacteria to adhere to specific sites of the mucosa.

Susceptibility of infant mice to F5 (K99) E. coli infection: differences in glycosyltransferase activities in intestinal mucosa of inbred CBA and DBA/2 strains.

Enterotoxigenic Escherichia coli (ETEC) strains expressing F5 (K99) fimbriae cause diarrhoea in the young animal through adhesion to specific sialoglycolipids of the small intestine surface. We studied here an infant mouse diarrhoea model, as CBA infant mice are susceptible to F5-positive ETEC infection, whereas DBA/2 ones are resistant. In an attempt to determine an enzymatic basis for susceptibility and resistance, we investigated the intestine ganglioside pattern in relation to the activity of glycosyltransferases responsible for the globo- and ganglio-series. We observed that the intestine of susceptible CBA infant mice displayed a characteristic sialoglycolipid pattern containing mainly the F5 receptors. The two murine strains differed in the relative activities of galactosyltransferases (GbOse3Cer and GM1 synthases), N-acetylgalactosylaminyltransferases (GA2 and GM2 synthases) and sialytransferases (GM3 and GD3 synthases). An elevated GM3-synthase activity was observed in the intestine of susceptible CBA infant mice, at the age of high susceptibility. Hence, we conclude that the marked specificity of mouse type correlated with susceptibility and resistance to F5-positive ETEC infection which could be controlled through the regulation of glycosyltransferase activities.