- Volume 39, Issue 1, 1993

A simple and economic method for the detection and identification of human papillomaviruses (HPV) is described. The method has been developed with cloned HPV DNA and DNA from clinical samples. Genomic fragments were obtained from several different HPV types, including the ones most frequently encountered in the genital tract by polymerase chain reaction (PCR) amplification directed by degenerate general primers. The amplification fragments were identified by a form of miniature fingerprinting, with a set of restriction enzymes that gave a unique digestion pattern for each HPV type. Different strategies are proposed, based on PCR and restriction analysis, and this approach to identification was compared with more classic methods such as Southern hybridisation.

The immunological response of cystic fibrosis (CF) patients to lipopolysaccharide (LPS) antigens of Pseudomonas cepacia was investigated. Enzyme-linked immunosorbent assays (ELISA) with either P. cepacia whole cells or extracted core LPS from a clinical isolate of P. cepacia as antigen were used to measure serum IgG and sputum IgA anti-P. cepacia antibodies. The ELISA with core LPS distinguished nine CF patients colonised by P. cepacia from nine age- and sex-matched non-colonised CF patients. The rate of increase of anti-P. cepacia IgG antibodies after bacteriologically proven P. cepacia colonisation varied in individual patients: In some patients the first isolation of P. cepacia was preceded or accompanied by a two-to-four-fold rise in anti-P. cepacia LPS IgG titres. Absorption studies and immunoblot analysis of serum from patients colonised with P. cepacia demonstrated that a significant component of the anti-P. cepacia core LPS antibodies was specific for P. cepacia and did not react with the core LPS of P. aeruginosa. Immunoblotting also illustrated that there may be a degree of core heterogeneity between different isolates of P. cepacia. Detection of P. cepacia LPS specific antibodies in serum (IgG) and sputum (IgA) from CF patients is recommended to assist the identification of P. cepacia colonisation in CF patients.

The routine use of ELISA and complement fixation tests in the diagnosis of suspected clinical cases of hydatid disease was evaluated. In the ELISA test, dialysed and filtered sheep cyst fluid was used as antigen and two positive cut-off points—+3SD and + 2SD of the mean absorbance values of the control sera—were evaluated. The predictive values of ELISA tests were 82% and 90% for positive tests, and 86% and 82% for negative tests, respectively with the two cut-off points. In a population survey of blood donors and veterinary workers in Powys, 4% and 8%, respectively, had ELISA values above the lower cut-off point. However, it would not be appropriate to use the same test for diagnostic population screening in Wales since the predictive value of the test is likely to be very low in this setting. Serological surveys with the ELISA may be of use in monitoring the progress of the South Powys Hydatid Control Programme. The use of cumulative percentages was found to be a useful method of comparing whole distributions of results in different populations.

Adherence to buccal epithelial cells (BEC) and the role played in the binding by lipoteichoic acid (LTA) and other superficial components have been studied in reference and clinical strains of Streptococcus bovis either glucan-positive biotype I or glucan-negative biotype II. To avoid the synthesis of glucan by biotype I strains, adherence was studied in bacteria grown in Todd-Hewitt broth, a sucrose deficient medium. Both biotypes were shown to bind to BEC and clinical isolates, irrespective of biotype attached to the same degree but in greater numbers than reference strains, Inhibition studies suggest that at least two mechanisms,“LTA and protein-mediate” are responsible for the adherence of both glucan-positive and negative strains of S. bovis. Moreover, in glucan-positive strains capsular polysaccharides may be also involved.

Three Staphylococcus aureus strains (303, 18Z and TG), exhibiting various patterns of survival within abscesses, were significantly more sensitive to the bactericidal activity of oleic acid during the log phase of growth than at other stages of the growth cycle. Cells entering the stationary phase showed diminished sensitivity to the fatty acid. These changes were reflected by changes in the LD50 and also by differences in the rate of killing by oleic acid. Additional changes were noted: The rate of killing by oleic acid declined over a 4-day period; a progressively greater proportion of the staphylococcal population became resistant to even high concentrations of oleic acid; from the fourth day onwards c. 50—55% of the cocci were totally resistant to the fatty acid. Strains 303 and 18Z became more sensitive to mono-olein during the log phase of growth, but strain TG was very resistant to mono-olein throughout the growth cycle. Growth in the presence of glycine 6% to reduce cross-links in the peptidoglycan did not alter bacterial sensitivity to oleic acid. However, all three S. aureus strains exhibited significant increases in membrane fluidity during the log phase of growth, but upon entering the stationary phase membrane fluidity again decreased. Concomitant changes in carotenoid content occurred during the growth cycle, but these changes did not appear to be solely responsible for the changes in sensitivity to the lipids.

To establish an experimental model to study gastric spiral non-cultivable bacteria, 30 4-week-old female CFW (LOB) mice were inoculated with porcine gastric mucus containing “Gastrospirillum suis” and 25 mice were inoculated with mucus without “G. suis”. Mice were examined 3, 7, 14, 21, 28 and 60 days after inoculation. Fragments from the membranous, oxyntic and antral gastric mucosa and from the duodenal mucosa were obtained for histological and microbiological analysis. Tightly spiralled bacteria were seen in smears and in histological sections of the antral and oxyntic mucosa from all G. suis-infected mice. The pre-formed urease test also gave positive results in both tissues. In control mice, no tightly spiralled bacteria were seen. By 7 days after inoculation, the test animals had developed an inflammatory infiltrate of mononuclear cells, some neutrophils and a few eosinophils, mainly in the lower third of the antral and oxyntic mucosa, which persisted for the remainder of the observation period. This model can assist in the understanding of several clinical, pathological and immunological aspects of infection with spiral gastric bacteria, particularly those associated with non-cultivable spiral bacteria.

Experiments were performed to determine the effects of products of bacterial growth (including endotoxin) on phagocytosis and intracellular killing by polymorphonuclear leucocytes (PMNL) in urine. Bacteriologically filtered supernates of two strains of Escherichia coli grown in urine were added in varying amounts to mixtures of PMNL and E. coli, also in urine. Phagocytosis of the two strains was reduced from > 90% in controls to 66% and 48%, respectively, in the presence of undiluted culture filtrate (containing endotoxin 2—2.5 μg/ml). Intracellular killing was also decreased and was abolished by dilutions corresponding to endotoxin concentrations of 0.6 and 0.75 μg/ml. When PMNL exposed to these inhibitory dilutions were resuspended in fresh urine, their phagocytic ability was fully restored and 13—24% of their killing activity was regained. A minimum concentration of commercially purified E. coli endotoxin of 200 μg/ml was required to abolished PMNL killing, with phagocytosis uninhibited. The results strongly suggest that bacterial growth metabolites, not endotoxin, are responsible for the depression of phagocytosis and intracellular killing in infected urine. A moderate dilution of the bacterial products in urine permits good PMNL function. Extrapolating this to the clinical situation, diluting the urine by water loading (as recommended for patients with urinary infections) should ensure efficient activity of PMNL under in-vivo conditions providing urinary pH and osmolality are not adversely affected.

Detection of Salmonella typhi infection by a co-agglutination assay for specific O, H and Vi antigens and by blood culture were compared for 110 patients with suspected typhoid fever. Blood cultures were positive for S. typhi in 25.5% of patients. Co-agglutination tests with patients’ serum and with blood culture supernates gave positive results in 70.9% and 67.3% of cases respectively. S. typhi antigens Hd and O9 were detected in patients’ serum by co-agglutination in 96.4% of blood culture-positive, and 62.2% of blood culture-negative patients. Co-agglutination results were uniformly negative with serum samples from a control group of 50 healthy individuals, 20 patients with febrile non-typhoid infectious disease and 20 patients with non-infectious febrile disease. Of the 25 patients with suspected typhoid fever who had not received prior antibiotic treatment, 88% yielded positive blood cultures and 96% gave positive results in serum co-agglutination tests. By contrast, of the 95 patients who had received prior antibiotics, only 7% yielded positive blood cultures, but 63.5% gave positive results in serum co-agglutination tests. Co-agglutination tests with serum offer a simple, rapid, sensitive, specific and economical method for the early diagnosis of typhoid fever.