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Forum Londonescortadult Szh Watchdefault 0 London Escort Adult Bifidobacterial Lipoglycan as a New Cause for False-Positive Platelia Aspergillus Enzyme-Linked Immunosorbent Assay Reactivity--《微生物临床杂志》--医学期刊频道--首席医学网

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    DISCUSSION

    Most bifidobacteria showed significant reactivity with the PA ELISA in contrast to other gram-positive bacteria containing LTA or lipomannan or gram-negative bacteria containing LPS (Table 1; Fig. 3). ELISA reactivity was clearly shown to be associated with bacterial lipoglycans containing a -1,5-galactofuranosyl chain. Only a few bifidobacterial lipoglycans have been structurally analyzed, but also other bifidobacterial species seem to contain reactive residues in their cell wall (7, 18, 30). This specific epitope can be detected as a surface antigen, shown by the reactivity of the suspended cell pellet, but also as an exoantigen, shown by the reactivity of the cell-free supernatant of a liquid culture (Table 3). Secretion of lipoglycans during normal growth was already shown by Op den Camp et al. with B. bifidum subsp. pennsylvanicum (33). The complete removal of PA ELISA reactivity after deacylation of the supernatant, compared to the 50% decrease with GM, suggests that only the acylated micellar form reacts in the PA ELISA.

    When our results were extrapolated to the neonatal host, a calculation was made to estimate the PA ELISA reactivity of feces as potential source of cross-reactivity in the neonate. The bifidobacterial numbers found in feces (15, 29) are very high, and the EI per gram of feces is also high, ranging from 4,000 to 100,000. In order to confirm this calculation, FISH analysis was performed on neonatal fecal samples with a genus-specific probe for bifidobacteria. This 16S rRNA hybridization technique is a fast method to quantify bifidobacteria in the human gut (21). The presence of bifidobacteria was associated with PA ELISA reactivity of the fecal samples, but reactivity showed some variation, which probably depends on the bifidobacterial species present, as shown by the calculated examples. The results clearly show that the bifidobacterial community in the gut is a significant source of PA ELISA reactivity and that, considering the dilution in the blood volume, the serum concentration could become high enough after transmucosal passage to be detected with the PA ELISA. Furthermore, false-positive PA reactivity in fully breast-fed neonates could not come from galactomannan since breast milk reacts negatively in the PA ELISA (results not shown).

    E. lenta is, like Bifidobacterium species, a gram-positive, obligatory anaerobic, non-spore-forming rod and has been found in adult feces at concentrations of 108.5 cells/g, in 4 out of 12 subjects that were tested (detection limit, 107 cells/g) (41). Furthermore, the number increased in the intestinal microflora of healthy subjects after administration of antibacterial agents (32). Comparable species have also been found in feces of children at 109.6 cells/g, but the bacteria were not identified to species level (15, 29). So, in addition to Bifidobacterium, this species might also form an important source of PA ELISA reactivity in the intestine. Since E. lenta is present in the fecal flora of adults, it might also be a cause of false PA ELISA serum reactivity in adults. Recently developed species-specific oligonucleotide probes might be helpful with future studies of E. lenta in the human intestine (19, 41).

    Positivity of fecal samples has already been shown (1) but was always correlated with consumption of food containing fungal GM from contaminated sources (1, 23). In addition to the host's own microflora as a source of bifidobacteria, ELISA reactivity of certain food products is more likely to result from bifidobacterial lipoglycan than from fungal GM. Because of their health-promoting effects, bifidobacteria are widely used as probiotics and food additives and are present in fermented foods like milk, olives, sauerkraut, yogurt, butter, and cheese (3). Especially B. lactis and Bifidobacterium animalis are often used, and amounts of 107 to 108 of bifidobacteria per gram of food can be found (20, 40, 50). As can be seen in Fig. 3, B. lactis (recently reclassified as a subspecies of B. animalis [50]) and B. animalis show high reactivities and could act as a source of ELISA serum reactivity after consumption of food products. Furthermore, bifidobacteria are used as fecal indicator organisms and have been found on meat and meat products and in raw milk (2, 11). Especially meat products are often GM positive (23) but are more likely contaminated with bifidobacteria instead of fungal GM.

    The frequent isolation of bifidobacteria from clinical infections in recent years has raised debate whether the bacteria are actually infective (3, 17, 36). However, even if there's a lack of pathogenicity in immunocompromised patients (3), bifidobacteria can invade the host by bacterial translocation (17). This phenomenon is caused by a diminished intestinal barrier, resulting in the passage of bacteria or bacterial components or products across the mucous membrane and epithelium. Not only intestinal mucosal injury by for instance cytotoxic chemotherapy or an immature intestinal mucosa but also immunodeficiency in the host, overgrowth of intestinal bacteria, and treatment with antibiotics and/or immunosuppressive agents have been shown to promote translocation of intestinal bacteria (i.e., bifidobacteria) (6, 17). The actual translocation of reactive components across the intestinal wall remains to be proven but seems even more likely for an exocellular lipoglycan molecule of 10 kDa. Penicillin treatment of B. bifidum subsp. pennsylvanicum resulted in an increase of lipoglycan excretion (33). This was not the result of bacteriolysis, as was also observed for acylated or deacylated LTAs of other bacteria and for other inhibitors of cell wall synthesis (33, 35, 43, 49). Consequently, antibiotic treatment of immunocompromised patients will increase the free lipoglycan pool in the intestine which may easily translocate to the blood.

    In the blood, lipoglycan might bind to different components, resulting in a mixture of Galf-containing molecules that react in the PA ELISA, as is the case with GM (27). LTAs and lipoglycans spontaneously bind to mammalian cell membranes. In a study on B. bifidum subsp. pennsylvanicum it was shown that its lipoglycan bound reversibly to human colonocytes (8.3 x 108 binding sites/cell) and erythrocytes (2.1 x 106 binding sites/cell) (34). The lipid part of the molecule was shown to be responsible for binding. Furthermore, the lipoglycan of B. bifidum subsp. pennsylvanicum exhibited strong binding to the macrophage scavenger receptor, comparable to the LTA of Staphylococcus aureus (13). In order to analyze the nature and structure of the cross-reacting molecule(s) present in (false-) positive serum samples, a method is needed that specifically isolates reactive components because of their small amounts present. Several techniques (28) can then be used for further analysis to discriminate between false- and true-positive PA ELISA results and thereby prevent unnecessary preemptive treatment of patients.

    The Bifidobacterium lipoglycan offers an explanation for the occurrence of a high rate of false PA ELISA reactivity among neonates and infants. Furthermore, together with E. lenta, these bacteria might also be the cause of false-positive reactivity in adult patients.

    ACKNOWLEDGMENTS

    We thank Marc Tabouret, Bio-Rad (Steenvoorde, France), for kindly providing the purified GM. We also thank Arie Oosterhof (Dept. Biochemistry, Radboud University, Nijmegen, The Netherlands), Ger Bongaerts (Dept. Medical Microbiology, Radboud University Nijmegen Medical Center, Nijmegen, The Netherlands), and Winclove BioIndustries BV (Amsterdam, The Netherlands) for kindly providing some of the bifidobacterial species. We thank Gjalt Welling (Dept. Medical Microbiology, University of Groningen) for performing the FISH analysis.

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