Epitope mapping and characterization of antigenic determinants of heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli by using monoclonal antibodies.

A panel of monoclonal antibodies (MAbs) specific for the heat-stable enterotoxin (STh) of enterotoxigenic Escherichia coli was produced. All four MAbs (8G7, 53-4, 11C, and SH1) bound to native STh in an enzyme-linked immunosorbent assay to various degrees, with clone SH1 showing the best affinity. The MAbs were screened for neutralizing and guanylate cyclase-inhibiting activities by the suckling mouse assay and the cyclic GMP assay using T84 cells, respectively. The contact amino acid residues governing the reactivity of the four MAbs were precisely determined by using several chemically synthesized analogs of the various heat-stable enterotoxins (STa's). Three distinct antigenic sites of STh sufficiently removed from each other, one near the N terminus, another in the core functional region of the toxin, and the third in the C-terminal region, were recognized by the different MAbs. MAb SH1, which recognized Asn at position 4 and Tyr at position 5 from the N terminus was 100 times more potent in neutralizing the bioactivity of STh in the suckling mouse assay than was MAb 11C, which recognized Thr at position 16 and Tyr at position 19 from the N terminus of the STh molecule. The MAbs which recognized Leu at position 9 from the N terminus (MAb 53-4) and Tyr at position 19 from the N terminus (MAb 8G7) showed intermediate activities in the neutralization assay. The guanylate cyclase-inhibiting activities of SH1 and 11C essentially paralleled the results for the neutralization of bioactivity, while MAbs 53-4 and 8G7 exhibited reverse activity. These results indicate that MAbs that recognize the N-terminal residues which have been shown not to be essential for toxic activity have a potent protective capacity. None of the MAbs reacted with reduced and carboxy-methylated native STh. This suggests that all of the MAbs mediate their effect by reacting with conformation-dependent antigenic determinants.

Nosocomial outbreak of Campylobacter jejuni meningitis in newborn infants.

In a nosocomial outbreak of Campylobacter jejuni infection 11 newborn infants (7 female, 4 male) had meningitis. The outbreak was caused by a single strain of C jejuni, as demonstrated by biotyping (biotype I), serotyping (LAU 7/PEN 18 on heat-stable antigens, a new serotype on heat-labile antigens), and the identical susceptibility pattern and outer-membrane-protein profile on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Specific antibodies against the outbreak strain (enzyme-linked immunosorbent assay and Western blot) developed in all the babies. They were treated with gentamicin and ampicillin. All but one baby, who had a moderately dilated left lateral ventricle after the meningitis, recovered well. The source of infection could not be clearly determined. Thus, C jejuni can cause serious nosocomial infection; it should be considered as a possible agent of meningitis of unknown origin, particularly in newborn infants and other compromised hosts.

Evaluation of a new method for routine in vitro cultivation of Giardia lamblia from human duodenal fluid.

Although a method for in vitro cultivation of Giardia lamblia was described as early as 1927, only a few clinical isolates were cultured in vitro due to the complexity of the techniques. We developed a method which allows for the routine isolation of Giardia trophozoites from human duodenal fluid and maintenance of the organisms in axenic culture. This study evaluates the method in 198 patients. Seventeen strains of Giardia were isolated and cultivated axenically. The method was more sensitive than the microscopic examination of aspirated fluid and examination of an impression of mucosal biopsy. Five patients, however, excreted cysts in the stool, although no trophozoites could be demonstrated in the duodenal fluid. G. lamblia were cultivated from one patient who did not excrete cysts. The method will enable the collection of G. lamblia strains from clinical material in large numbers and can offer an important advance in epidemiological, biochemical, immunological, and therapeutic investigations of giardiasis.

In vitro susceptibilities of 25 Giardia lamblia isolates of human origin to six commonly used antiprotozoal agents.

The role drug resistance plays in the occurrence of chronic and recurrent giardiasis has not been established. Extensive data on the susceptibility to antimicrobial agents of living Giardia spp. trophozoites from human origin are lacking. We have determined with a macrodilution method in semisolid medium the in vitro susceptibility of 25 Giardia lamblia isolates, all obtained by routine cultivation of the duodenal fluid of children to six commonly used antiprotozoal drugs. The results showed tinidazole to be the most active drug (all isolates have MICs of less than or equal to 0.5 micrograms/ml). Metronidazole was equally active on all but one isolate, for which an MIC between 0.5 and 1 micrograms/ml was found. Furazolidone was the most active nonimidazole compound tested. More than 50% of the isolates were very susceptible to paromomycin, pyrimethamine, and chloroquine. Two of the strains presented an MIC for paromomycin higher than 10 micrograms/ml, and six strains needed more than 50 micrograms of pyrimethamine per ml to be inhibited. Decreased susceptibility of several of the isolates to different agents appears to be linked.

A competitive immunosorbent assay for the detection of heat-stable enterotoxin of Escherichia coli.

A competitive ELISA procedure for the detection of Escherichia coli heat stable enterotoxin (ST) with monoclonal antibody has been developed. This test is 10 times more sensitive than the suckling-mouse bioassay and it is specific, simple and cheap. A set of 882 strains of E. coli isolated from man were tested both by ST-ELISA and suckling-mouse bioassay, the latter serving as the reference method. Positive results in both tests were obtained with 152 strains. The remaining strains gave negative results in both tests, with the exception of two strains, known to be ST producers, that gave negative results in the suckling-mouse assay, but gave positive results by the ELISA method.

Enteropathogenic agents in children with diarrhoea in rural Zaire.

A systematic study of enteropathogenic agents in the stools of children was carried out in a rural area of Kivu Province in Zaire in June, 1979. 84 inpatients and 271 outpatients with diarrhoea were investigated together with 117 inpatient and 203 outpatient controls without diarrhoea. Campylobacter jejuni was the most frequently recovered pathogen in both inpatients (24%) and outpatients (13.7%). Enterotoxigenic Escherichia coli was the next most common among children presenting as outpatients with diarrhoea (10.3%). Yersinia enterocolitica was isolated for the first time in this region. Rotaviruses were found only in outpatients with diarrhoea, and usually in children under two years of age. C. jejuni and Vibrio cholerae could also be isolated from the faeces of domestic animals living in close contact with these village families. Analysis of the clinical features did not lead to the recognition of a pattern typical of any particular pathogen. Salmonella, Shigella, and enteropathogenic E. coli did not play a significant role in the cases studied.