- Volume 46, Issue 1, 1997

The effects of sample storage, target preparation and annealing temperature on a nested polymerase chain reaction (PCR) test for Toxoplasma gondii DNA were investigated with experimentally seeded amniotic fluids to simulate congenital infection. Aliquots of 17 amniotic fluid samples remaining after investigation for conditions other than toxoplasmosis and seeded to contain small numbers of T. gondii tachyzoites, were tested after storage for up to 2 weeks. There was no deterioration in test sensitivity on samples stored for 1–2 weeks at room temperature; thus, samples sent by post to reference laboratories are acceptable for examination. There was no significant difference in the results from two target preparation methods or two different annealing temperatures. One-to-ten parasites were detectable in 13 of 17 test samples. The significant feature in the remaining four samples was the use of washed stored parasites to seed the amniotic fluids rather than any feature of the amniotic fluids used or the test. False positive results were found in up to 15% of unseeded amniotic fluid samples, which contrasts with only 1.9% for the same PCR test in other routine or negative control samples tested as part of the laboratory’s reference diagnostic work. The difference illustrates the increased risk of cross-contamination in PCR when the majority of specimens tested are positive. The results suggest that PCR techniques are likely to be sensitive and effective tools for the routine diagnosis of congenital toxoplasma infection.

The surface properties of various Escherichia coli isolates associated with diarrhoeal illness were compared by aqueous partitioning between polyethylene glycol (PEG) and Dextran phases. Two well characterised strains of enteropathogenic E. coli (EPEC) were found to be very hydrophobic, based on the critical polymer concentration. EPEC strain E2348 cured of the EPEC adherence factor (EAF) plasmid had much reduced surface hydrophobicity. Partitioning of a series of diarrhoeagenic E. coli strains demonstrated that the majority of EAF+ EPEC strains were significantly more hydrophobic than EAF− EPEC strains. E. coli strains defined as enteroaggregative on the basis of hybridisation with a specific DNA probe showed much greater heterogeneity in their partitioning behaviour, possibly indicating that the AAF/I pili were not expressed in all strains. The E. coli K-12 strain used as a transformation host for adhesion studies had very low surface hydrophobicity but had a detectable negative charge. No alteration in these properties was observed when transformed with EPEC and recombinant plasmids known to specify adherence to tissue culture cells.

The Mast resistotyping scheme was assessed with 228 strains of Campylobacter jejuni and C. coli from enteric infections in man and from a diverse selection of other sources (livestock, chickens and river water). Most (153 of 158) C. jejuni examined were of the three most common Penner (heat stable, HS) serotypes, HS1, HS2 and HS4 complex. Fourteen resistotypes were identified in the 158 strains of C. jejuni and 16 in the 70 isolates of C. coli. The predominant codes were 00 (44% of C. jejuni; 33% of C. coli) and 40 (21% of both species). The scheme was simple to use but reproducibility and interpretation of sensitivity zones-notably for fluorouracil, triphenyltetrazolium chloride and metronidazole-was occasionally problematic. Overall, resistotypes did not correlate with Penner HS serotypes or with three key genomic markers (ribotype, PFGE macrorestriction-type and fla-type). Although resistotyping offers a rapid means for distinguishing between some strains of C. jejuni and C. coli, discrimination for common resistotypes can be achieved only in combination with other typing methods.

Lactulose exerts a beneficial effect on hepatic encephalopathy by decreasing toxic shortchain (iC4–nC6) fatty acid (isobutyrate, butyrate, isovalerate, valerate, isocaproate and caproate) production. However, the precise mechanism by which lactulose exerts this effect remains uncertain. This study investigated the effect of lactulose on faecal flora, particularly Clostridium difficile, which produces mostly iC4–nC6 fatty acids. An in-vitro faecal incubation system was used to estimate how lactulose influences production of short-chain (C2–nC6) fatty acids and lactate. Faecal specimens were collected from patients with liver cirrhosis, who carried C. difficile in the colon. Supplementation of lactulose along with blood in faecal specimens decreased iC4–nC6 fatty acids production and increased acetate and lactate production, resulting in increased faecal acidity. These changes were statistically significant when compared with supplementation by blood alone. Quantitative faecal culture demonstrated that lactulose supplementation suppressed the growth of C. difficile and Bacteroides spp. (B. fragilis group), iC4–nC6 fatty acids-producing organisms. These results suggest that decreased faecal levels of iC4–nC6 fatty acids after lactulose supplementation may be related to suppression of iC4–nC6 fatty acids-producing faecal organisms, especially C. difficile.

Evaluation of human papillomavirus (HPV) diversity in various grades of cervical lesions is helpful for understanding the characteristics of HPV infection in the pathogenesis of cervical neoplasia. A total of 227 women with normal cervices (n = 72), low-and high-grade cervical squamous intraepithelial lesions (SILs) (n = 55 and 53, respectively) and cervical carcinomas (n = 47) were screened for human papillomavirus (HPV) types 6, 11, 16 and 18 infection by the polymerase chain reaction. The prevalence of multiple HPV infections in patients with normal cervices, low-grade SILs, high-grade SILs and cervical carcinomas was 22.2%, 61.8%, 41.5% and 21.3%, respectively, while the prevalence of a single-type infection was 36.1%, 21.8%, 30.2% and 61.7%, respectively. HPV 16/11 and 16/18 were the most common combinations observed in multiple infections. Multiple HPV infections were seen most frequently in patients with low-grade SILs, and the prevalence decreased with increasing severity of cervical neoplasia. In contrast, infection with a single HPV type was most commonly observed in patients with cervical carcinoma, and the prevalence decreased with decreasing severity of cervical neoplasia. HPV 16 was the predominant single-type infection in patients with cervical carcinoma and this prevalence decreased steadily with decreasing severity of cervical neoplasia. Conversely, HPV 11 was the predominant single-type infection in patients with normal cervices. This prevalence decreased with increasing severity of cervical neoplasia. Patients with low-grade SILs had a higher prevalence of HPVs, regardless of single or multiple infection status, and larger copy numbers of virus genome were seen more frequently in patients with more severe lesions.