A clinical validation of Bordetella pertussis and Bordetella parapertussis polymerase chain reaction: comparison with culture and serology using samples from patients with suspected whooping cough from a highly immunized population.

The aim of this study was to validate the performance of the polymerase chain reaction (PCR) assay for Bordetella pertussis and Bordetella parapertussis in comparison with both culture and serology. The number of samples positive in PCR was 2.4-fold higher than the number of samples positive in culture. In serologically confirmed cases, the sensitivity of PCR and culture depended on the duration of disease and the age of the patient, being less sensitive in older age and later in disease. The sensitivity of the PCR in patients with < 10 days of symptoms was 70%, 50%, and 10% in the age groups < 1 year, 1-4 years, and > or = 5 years, respectively. Evidence suggested that the effect of age on sensitivity may be due to differences in immune responses. The low IgA response in the < 1 year age group may be related to the high number of samples positive in PCR and culture, even late in disease.

Polymerase chain reaction assay for pertussis: simultaneous detection and discrimination of Bordetella pertussis and Bordetella parapertussis.

A polymerase chain reaction (PCR) assay which allows the simultaneous detection and discrimination of the two causative agents of pertussis, Bordetella pertussis and Bordetella parapertussis, was developed. Primer pairs were based on insertion sequence elements IS481 and IS1001. IS481 is specific for B. pertussis and is present in about 80 copies per cell, while IS1001 is specific for B. parapertussis and is found in 20 copies per cell. An internal control was included in the PCR assay to monitor the performance of the PCR and to identify possible inhibitory components in clinical samples. Discrimination of amplified DNA derived from the internal control, B. pertussis, or B. parapertussis was accomplished by differential spacing of the primers. The sensitivity of the combined PCR method was found to be very high and allowed the detection of one cell of either pathogen. The usefulness of the method was investigated by using a limited number of clinical samples derived from patients with serologically proven pertussis.

Isolation of a repeated sequence from the genome of Bordetella parapertussis with characteristics of an insertion sequence element.

A repeated DNA sequence in the genome of Bordetella parapertussis was identified by means of heteroduplex formation and S1 nuclease treatment. Cloning and sequencing of the repeated sequence, revealed that it comprised 1306 basepairs, contained inverted repeats at its termini, and two large open reading frames. This insertion sequence element was designated IS1001, and was found in about 20 copies in 10 B. parapertussis strains analyzed. Identical hybridization patterns were observed in B. parapertussis when IS1001 was used as a probe. B. pertussis strains did not hybridize to IS1001.

Characterization of IS1001, an insertion sequence element of Bordetella parapertussis.

By analysis of repetitive DNA in Bordetella parapertussis, an insertion sequence element, designated IS1001, was identified. Sequence analysis revealed that IS1001 comprised 1,306 bp and contained inverted repeats at its termini. Furthermore, several open reading frames that may code for transposition functions were identified. The largest open reading frame coded for a protein comprising 406 amino acid residues and showed homology to TnpA, which is encoded by an insertion sequence element (IS1096) found in Mycobacterium smegmatis. Examination of flanking sequences revealed that insertion of IS1001 occurs preferentially in stretches of T's or A's and results in a duplication of target sequences of 6 to 8 bases. IS1001 was found in about 20 copies in 10 B. parapertussis strains analyzed. No restriction fragment length polymorphism was observed in B. parapertussis when IS1001 was used as a probe. An insertion sequence element similar or identical to IS1001 was found in B. bronchiseptica strains isolated from pigs and a rabbit. In these strains, about five copies of the IS1001-like element were present at different positions in the bacterial chromosome. Neither B. pertussis nor B. bronchiseptica strains isolated from humans and dogs contained an IS1001-like element. Therefore, IS1001 may be used as a specific probe for the detection of B. parapertussis in human clinical samples.

Sensitivity and specificity of an enzyme-linked immunosorbent assay using the recombinant DNA-derived Treponema pallidum protein TmpA for serodiagnosis of syphilis and the potential use of TmpA for assessing the effect of antibiotic therapy.

The recombinant DNA-derived Treponema pallidum membrane protein TmpA, purified from Escherichia coli K-12, was used in an enzyme-linked immunosorbent assay (ELISA) to evaluate its suitability in a screening test for syphilis and to monitor the effect of antibiotic treatment. The sensitivity of the TmpA ELISA was 76% for primary syphilis, 100% for secondary syphilis, and 98% for early latent syphilis. All except 1 of 15 serum samples positive for yaws were positive in this test. A specificity of 99.6% was found by testing more than 938 donor samples. The sensitivity and specificity of the TmpA ELISA are comparable to that of the T. pallidum hemagglutination assay, and therefore the test may be useful for the diagnosis of untreated syphilis. After antibiotic treatment, the level of anti-TmpA antibodies in sera of syphilis patients dropped sharply within 1 year. Thus, TmpA might be a useful antigen for monitoring successful treatment of syphilis.

Serological identification of pig enterotoxigenic Escherichia coli strains not belonging to the classical serotypes.

Escherichia coli strains isolated from pigs suspected to have succumbed to E. coli enterotoxicosis and not belonging to the commonly incriminated (classical) serotypes (O8:K87:K88, O45:K88, O138:K81:K88, O141:K85:K88, O147:K89:K88, O149:K91;K88, and O157:K88) were tested for enterotoxigenicity in the ligated gut test (LGT) using pig intestine. Of 202 strains tested, 54 strains belonging to 13 different O groups were positive in the LGT. Four of these strains had K88 antigen and one possessed K99 antigen. The majority of the strains was not agglutinated by any of the standard OK antisera. Four new K antigens ("K200", "K442", "K2346" and "K2347") were provisionally designated. K200 was found in pig enterotoxigenic strains belonging to O group 8 and carrying flagellar antigen H31 and in non-enterotoxigenic non-motile strains of O group 8, as well as in O group 20 strains isolated from calves succumbing to E. coli septicemia in two countries. The provisional antigen K2346 was encountered in 18 enterotoxigenic strains with various O antigens from two countries. It is proposed to include these two K antigens into the international E. coli antigens scheme. Attempts to demonstrate a common antigen in the nonclassical enterotoxigenic strains lacking K88 and K99 antigens failed.

Detection of the K99 antigen by means of agglutination and immunoelectrophoresis in Escherichia coli isolates from calves and its correlation with entertoxigenicity.

The common antigen of calf enterotoxigenic strains of Escherichia coli, recently established as the K99 antigen, was studied by means of the slide agglutination test and immunoelectrophoresis. Specific antisera were obtained by absorption of crude antisera with ultrasonicates of autologous cells grown at 18C or by injection into rabbits of the purifies K99 antigen obtained by preparative electrophoresis. The K99 antigen was usually undetectable in calf enterotoxigenic E. coli cultures with capsular K antigens of the A variety grown at 37C on commercially available nutrient agar plates designed for the isolation of Enterobacteriaceae, but was rapidly detectable when grown on a buffered semi-synthetic medium at pH 7.5 (Minca medium). An alternative procedure for the isolation and identification of calf entertosigenic E. coli strains from feces, using the Minca medium, is proposed. K99 was found in 70 of 74 strains of E. coli, the enterotixigenicity of which was established in the ligated gut test in calves. None of the 20 cultures negative in the ligated gut test possessed K99 antigen. The K99 antigen is therefore probably a useful diagnostic tool for the identification of calf enterotixigenic E. coli strains, taking into account that K99 and enterotoxigenicity are controlled by different plasmids.

Serotyping and biotyping of 160 Escherichia coli strains: comparative study.

One hundred and sixty Escherichia coli strains were serotyped and biotyped. Serotyping revealed 68 different types, with 25 strains not typable. Biotyping was possible in all strains but revealed 55 different types. One biotype could be subdivided into 35 different serotypes, indicating that for this biotype the series of biochemical and fermentation reactions could be extended for further differentiation.

Escherichia coli O antigen typing by means of a mechanized microtechnique.

A mechanized microtechnique for the serological typing of Escherichia coli cultures is described.