Uptake of ciprofloxacin by macrophages.

Ciprofloxacin was concentrated within mouse peritoneal macrophages to between two and three times extracellular values. Uptake was rapid, occurred equally well with dead cells, and was not affected by lowering the pH or by prior ingestion of Staphylococcus aureus. Intracellular staphylococci were killed by extracellular concentrations of ciprofloxacin as low as 0.5 mg/l.

Neisseria gonorrhoeae: a versatile pathogen.

Neisseria gonorrhoeae is one of the most important causes of sexually transmitted disease. We do not fully understand the pathogenesis of infection with this organism, although recent improvements in immunological and molecular techniques have brought us closer to an answer. These techniques are now also being used to detect and identify N gonorrhoeae and to analyse the epidemiology of gonorrhoea. Plasmid and chromosomal mediated antibiotic resistance increases the difficulty of controlling gonorrhoea. Resistant strains occur all over the world and new patterns of resistance are still emerging. A better understanding of gonococcal pathogenicity is necessary for the development of an effective vaccine. Despite work on pili and outer membrane proteins no vaccine yet exists. The control of gonorrhoea still depends on diagnosis, treatment, and epidemiological control, facilities that are not widely available in many of those parts of the world where gonorrhoea is a major problem.

Opsonic activity of intravenous immunoglobulin preparations against Staphylococcus epidermidis.

Peritoneal dialysis effluent collected from patients undergoing continuous ambulatory peritoneal dialysis had no opsonic activity against Staphylococcus epidermidis and contained low concentrations of IgG and C3 (roughly equal to those found in 0.5% normal human serum). An intravenous immunoglobulin preparation that showed no opsonic activity against the same organism, on its own or when added to balanced salt solution or peritoneal dialysis fluid, showed good activity when combined with peritoneal dialysis effluent. This was probably due to the presence of low concentrations of C3 in the effluent as prior heat inactivation at 56 degrees C for 30 minutes eliminated any opsonic activity in the immunoglobulin-dialysis effluent mixture. Examination of a range of immunoglobulin preparations showed that their opsonic activity for S epidermidis in the absence of complement varied considerably. Luminol dependent chemiluminescence was unsatisfactory as a method for detecting complement independent immunoglobulin mediated opsonisation. Intravenous immunoglobulin preparations may be useful in boosting the peritoneal defences of those patients undergoing continuous ambulatory peritoneal dialysis (CAPD) who suffer from repeated intraperitoneal infections.

Grouping of beta-haemolytic streptococci by agglutination.

Streptococcal grouping sera, diluted and absorbed to remove cross-reactions, were bound to staphylococci and used to group trypsinised beta-haemolytic streptococci by coagglutination. The results compared well with those obtained using the Phadebact streptococcal grouping kit. The same sera bound to staphylococci without prior dilution and absorption could be used to group enzyme extracts of haemolytic streptococci by slide agglutination, the results again comparing favourably with those of the Phadebact kit.

Evaluation of an improved Streptex kit for the grouping of beta-haemolytic streptococci by agglutination.

A modified Streptex kit in which the extraction procedure had been simplified was used to group 200 streptococcal strains. Positive reactions could be obtained with live colonies and over 90% of isolates were correctly grouped from primary isolation plates. Some minor cross-reactions were seen with Streptex, but these were not strong enough to cause any confusion and no isolates were incorrectly grouped. The extraction enzyme in the Streptex kit was relatively poor at extracting the group-specific teichoic acid of group D strains but worked well with groups A, B, C, F and G.

Grouping and detection of group B streptococci by immunofluorescence.

Forty-nine clinical isolates of group B streptococci (GBS) were correctly grouped from broth culture by the Fluoro-Kit immunofluorescence test. A further 82 beta-haemolytic streptococci of groups A, C, D, F, and G were tested and gave no cross-reactions. The test was simple to perform and gave clear results. The Fluoro-Kit reagents, however, failed to detect GBS in 21 (51%) of 41 smears of rectal or vaginal swabs from pregnant women from which GBS were subsequently grown. Thirty-two (20%) of 159 culture-negative swabs gave positive immunofluorescent reactions.

Group B streptococci--gastrointestinal organisms?

Matched perianal swabs, rectal swabs, and faecal samples from a group of male homosexual patients attending a clinic for sexually transmitted disease were examined for the presence of group B streptococci (GBS). GBS recovery rates were as follows: perianal skin 31/115 (27%), rectal mucosa 18/72 (25%) and faeces 7/115 (6%). The recovery of GBS from faeces was similar to that obtained from faecal samples sent to the laboratory for routine investigation (5%). Although there was no difference in GBS recovery rates from rectal and perianal swabs, the latter did show heavier colonisation. These results suggest that gastrointestinal GBS carriage is mainly limited to the rectum and anal canal and that this may represent contamination from perianal skin.

Phagocytosis and killing of Gardnerella vaginalis by human neutrophils.

Gardnerella vaginalis was ingested and killed by neutrophils in the presence of normal human serum. Heat inactivation of the serum inhibited these processes. The opsonisation of some but not all G vaginalis strains was enhanced by immune rabbit serum. Immune serum did not, however, enhance intracellular killing. Blockade of the classical pathway of complement activation had no effect on the opsonic activity of human serum. These results suggest that the opsonisation, phagocytosis, and killing of G vaginalis by human serum and neutrophils is primarily mediated by complement activated by the alternative pathway. Serum without neutrophils has little effect on the viability of G vaginalis.

Evaluation of the Lumac kit for the detection of bacteriuria by bioluminescence.

Four hundred and twenty-two urine samples were screened for significant bacteriuria using bioluminescence and microscopy of uncentrifuged urine. A smaller number of false-negatives were seen with bioluminescence (10%) than with microscopy (40%) while both techniques gave a similar number of false-positives (18%). The kit required a large amount of manual preparation, largely pipetting. With this and the short shelf-life of the reconstituted reagents, it is not suitable for small numbers of urines. At 45p per urine, the cost of bioluminescence is too high.

Non-volatile fatty acids in the diagnosis of non-specific vaginitis.

In the vaginal washings of 100 women with symptomatic non-specific vaginitis a succinate/lactate ratio of greater than or equal to 0.4 had a diagnostic sensitivity of 80%, a specificity of 83% for this condition. The predictive value of a positive test was 94%, but that of a negative test was only 55%. A strong association between the presence of Gardnerella vaginalis, anaerobes, a vaginal pH of above 4.5, and amines was found not only in non-specific vaginitis, but also in trichomonal and gonococcal infection. A variety of primary changes may encourage the multiplication of both gardnerellae and anaerobes and their presence in non-specific vaginitis may be a secondary rather than a primary event.

Comparison of culture and microscopy in the diagnosis of Gardnerella vaginalis infection.

A comparison was made between human blood agar containing amphotericin B, nalidixic acid and either gentamicin or colistin for the isolation of Gardnerella vaginalis from cases of non-specific vaginitis seen in a clinic for sexually transmitted diseases. The medium containing gentamicin was more inhibitory for non-Gardnerella species, but not sufficiently inhibitory to allow direct plating in the clinic without spreading for single colonies. The diffuse beta haemolysis produced by G vaginalis on human, but not on horse blood agar, proved very useful in differentiating it from other vaginal organisms and was not affected by the antibiotics used. This characteristic, together with Gram stain morphology, oxidase and catalase, provides a simple, reliable methods of identifying G vaginalis. Sixty women with symptoms of vaginitis, in whom no other pathogen was isolated, were examined by culture and microscopy. Gardnerella vaginalis was grown from 45 whereas only 31 had positive microscopy (clue cells or Gram-variable bacilli). There was no significant difference between the rate of isolation of G vaginalis in the group with positive microscopy (25/31) and that with negative microscopy (20/31).

Evaluation of a direct immunofluorescence test for diagnosing gonorrhoea.

A new direct immunofluorescence reagent (Syva and Genetic Systems Inc) was evaluated for its ability to detect Neisseria gonorrhoeae in specimens from populations with a high prevalence of the infection. Gonorrhoea was diagnosed by culture in 45 of 105 (43%) urethral specimens from men and 17 of 90 (28%) urethral and 25 of 60 (42%) cervical specimens from women. In men the immunofluorescence test had a sensitivity of 84.4% and a specificity of 100%; Gram staining gave values of 94% and 100%, respectively. The sensitivity of the immunofluorescence test could be increased to 89% by testing duplicate smears. In women the immunofluorescence test had a sensitivity of 65% and a specificity of 98% for urethral samples and values of 72% and 94%, respectively for cervical samples. At both sites the sensitivity of the Gram stain was 40% and the specificity 100%. The testing of duplicate immunofluorescence smears increased the sensitivity to 76% for urethral and 88% for cervical samples.

Separation of Pneumocystis carinii from the lung of the steroid-suppressed rat.

Pneumocystis carinii pneumonia was induced in rats by chronic corticosteroid immunosuppression. The parasite was separated from virtually all contaminating host cells using the technique of unit gravity sedimentation. Cellular contamination was routinely below 0.02%. The same technique allowed partial separation of the cyst from the trophozoite stage.

Changes in penicillinase-producing Neisseria gonorrhoeae isolated in London.

To investigate changes in the gonococcal population over time, the plasmid content, serotype and auxotype have been determined for strains of penicillinase-producing Neisseria gonorrhoeae (PPNG) isolated from patients attending the Praed Street Clinic for Sexually Transmitted Diseases between 1978 and 1987. Three distinct changes have been identified: (i) in the main plasmid type, from PPNG with the 3.2-Mda plasmid before 1982 to strains with the 4.4-Mda plasmid between 1982 and 1985; (ii) the decline during this time of strains also carrying the 24.5-Mda conjugal plasmid; (iii) the re-emergence of PPNG with the 3.2-Mda plasmid in 1986. The three typing methods used have identified eight clusters of strains which have been prevalent in different years between 1978 and 1987.

Opsonic requirements of Staphylococcus epidermidis.

The opsonic requirements of 18 strains of Staphylococcus epidermidis were compared in pooled normal human serum and in peritoneal dialysate from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A serum concentration of 2.5% gave optimal opsonisation. The opsonisation of all strains was antibody- and complement-dependent, and there were no significant differences in the pattern of their opsonic requirements. Peritoneal-dialysis effluent from uninfected patients was a poor source of opsonins because of the low levels of immunoglobulin G and of the C3 component of complement it contained. Growth of S. epidermidis in peritoneal-dialysis effluent rather than in broth did not alter its opsonic requirements. Strains from patients undergoing CAPD and suffering from peritonitis were not more resistant to opsonisation and phagocytic killing than those from other sources.

Evaluation of a fluorescent monoclonal antibody reagent for identification of cultured Neisseria gonorrhoeae.

We evaluated a new fluorescent monoclonal antibody reagent for confirmation of identity of Neisseria gonorrhoeae. The reagent correctly identified all 161 fresh clinical isolates of N. gonorrhoeae, which included 11 penicillinase producing strains (PPNG). The reagent also correctly identified 21 stored PPNG strains. No cross reactions were seen with 58 fresh clinical isolates of N. meningitidis, 12 stored strains of N. lactamica, or with strains of Gardnerella vaginalis, lactobacilli, Candida spp., Staphylococcus epidermidis or Enterobacteriaceae. Some cross reaction was noted with strains of S. aureus, probably related to cell-wall protein. A. However, this reagent was highly sensitive and specific for use against oxidase positive, gram-negative cocci isolated in London.

Opsonophagocytosis of Pneumocystis carinii.

The interaction of Pneumocystis carinii purified from rat lungs with rat peritoneal macrophages and human circulating polymorphonuclear leucocytes was studied by amplified chemiluminescence and examination of stained cytospin preparations. A polyclonal rat antiserum to P. carinii was opsonic with both types of phagocyte. Complement had no opsonic properties alone but produced a synergic effect in combination with antibody.

Opsonophagocytosis of Campylobacter pylori.

The opsonic activity of human serum from various sources against Campylobacter pylori was compared. All sera, whether from control subjects with no symptoms of gastritis or peptic ulceration, or from symptomatic patients from whom C. pylori had or had not been isolated, opsonised C. pylori equally well. Opsonisation depended on the alternative pathway of complement activation but not on antibody. These findings suggest that antibody plays no role in protection against C. pylori and that the presence of antibody in patients' sera is mainly of diagnostic value.

The production and characterisation of rabbit antiserum and murine monoclonal antibodies to Haemophilus ducreyi.

Rabbit antiserum and murine monoclonal antibodies were raised against a strain of Haemophilus ducreyi. The antiserum gave high immunofluorescence titres and strong dot blot reactions with all H. ducreyi strains tested and the only cross reaction was with Bordetella pertussis. Three monoclonal antibodies, all of isotype IgG2a, also gave high immunofluorescence titres with H. ducreyi but did not cross react with any other species tested. Immunoblotting showed the monoclonal antibodies to react with a single polypeptide band of mol. wt 29,000 in the outer-membrane fraction of H. ducreyi. These antibodies have potential for use as diagnostic reagents and for investigating the pathogenicity of H. ducreyi.

Characterisation of monoclonal antibodies for detection of Mobiluncus spp. in genital specimens.

Monoclonal antibodies were raised to the anaerobic curved rods, Mobiluncus curtisii subsp. curtisii NCTC 11656, M. curtisii subsp. holmesii NCTC 11657 and M. mulieris NCTC 11658. Three antibodies reacted with the two subspecies of M. curtisii and, when used in combination against clinical isolates, showed 100% sensitivity and specificity in immunofluorescence studies. Immunoblotting showed that two of these antibodies reacted with an epitope on a protein which had an electrophoretic mobility corresponding to a Mr of 75 Kda in the absence of a reducing agent and 82 Kda in its presence in both type strains and in clinical isolates. The third antibody reacted with an epitope in type strains which had a mobility corresponding to 75 Kda and was unaffected by a reducing agent. However, in clinical isolates the epitope was present on a protein of 75 Kda or 71 Kda, or on both. A fourth antibody showed reactivity with M. mulieris NCTC 11658 alone, but only 6 (24%) of 25 clinical isolates gave positive results by immunofluorescence. The epitope is believed to be present on a protein of greater than 90 Kda. All four antibodies were shown by immunogold staining to be directed against epitopes exposed on the cell surface.