Sheffield medium for cultivation of Haemophilus ducreyi.

Our interest in the role of Haemophilus ducreyi in genital ulceration led us to examine the various media commonly used for the cultivation of the organism. We describe an improved medium for the routine isolation of H ducreyi. In comparative studies using 50 test strains originally isolated in the United Kingdom, Canada, the United States of America, and Kenya, the new medium proved superior to three standard media in requiring a shorter incubation period to first visible growth, giving larger colonies in the same period, and making possible a starch aggregation test which we have found helpful in the presumptive identification of H ducreyi from clinical material.

Haemophilus ducreyi infections--time for reappraisal.

As the literature on Haemophilus ducreyi and clinical chancroid is reviewed, it becomes obvious that many significant findings have been forgotten over the years. As a result, from the time of Ducrey's original description of the organism in 1890 until about 1977, both clinical and laboratory experts in the United Kingdom believed that H. ducreyi infections were rare, generally acquired abroad, and almost impossible to confirm in the routine laboratory! In consequence it was a common view that it was not worth looking for H. ducreyi until all other possible causes of genital ulceration had been excluded. Moreover, the search for such an infection stopped as soon as any other cause for the patient's lesions had been found. A decision to ignore this 'rule' in Sheffield led to our looking for H. ducreyi in specimens from an unselected series of patients with genital ulceration including a number with herpes genitalis infections. The surprise finding of H. ducreyi in circumstances suggesting that it was a secondary invader made us re-examine the whole question of H. ducreyi infections and chancroid and wonder if the same organism can act as a primary pathogen and as a secondary invader. An account of the media and methods we used and of the characteristics of the organism is presented. In an attempt to find out more about the characteristic coherent colonies of H. ducreyi we studied them with the scanning electron microscope. It is clear that the whole subject of H. ducreyi infections has been neglected in the United Kingdom, but we believe that interest has now been aroused and progress will surely follow. Some areas for further investigation are suggested.

Pathogenic microbial flora of genital ulcers in Sheffield with particular reference to herpes simplex virus and Haemophilus ducreyi.

The pathogenic microbial flora of genital ulcers in 161 (80 men and 81 women) unselected patients was studied prospectively. In only one case was Treponema pallidum responsible whereas herpes simplex virus was considered to be the cause of 130 (80.8%) genital ulcers. H ducreyi was isolated from 46 (28.6%) patients, most commonly as a secondary pathogen in herpetic lesions. Two or more pathogens were isolated from the ulcers in 67 (41.6%) patients, and in 21 (13%) patients no pathogens were isolated. Our results indicate an urgent need for antiviral treatment to reduce the local reservoir of genital herpes, challenge traditional concepts about the prevalence of H ducreyi in Britain, and call for a reappraisal of its role in the causation of genital ulcers.

Chancroid in Sheffield. A report of 22 cases diagnosed by isolating Haemophilus ducreyi in a modified medium.

The causative organism of chancroid, Haemophilus ducreyi, is generally considered to be very fastidious and its isolation, maintenance, and detailed study very demanding. In this study a modified medium was developed, which allowed the organism to be isolated more frequently than previously would have been expected. Twenty-two cases of chancroid were confirmed by the isolation of H ducreyi in 160 patients with genital ulceration examined over a one-year period. The cases were apparently unrelated, and in only five was there a history of recent sexual contact abroad. Concurrent infection with other sexually transmitted diseases was present in 18 (81.8%) patients, and in 14 (63.6%) both H ducreyi and herpes simplex virus were isolated form the same genital ulcers. Thus, these findings indicate that chancroid is underdiagnosed in England and that H ducreyi may frequently occur as a secondary invader of damaged genital skin and mucosa.

Induction of a cell wall variant of the gonococcus by human amylase.

Human amylase has been shown to be an effective and powerful inhibitor of the gonococcus in vitro. Its action appears to be on the bacterial cell wall. When tested in osmotically stabilised liquid media the organism was not inhibited; however the organisms which grew in such cultures appeared to be cell wall variants, which were less sensitive to penicillin than the parent strains. Studies on these variants suggest that they are "transitional-phase variants." Since cervical mucus has much in common with an alkaline osmotically stabilised liquid medium and also contains a high concentration of amylase, it seems possible that these variants may occur in vivo.

A contribution of cellular immunity to protection against influenza in man.

The degree of lymphocyte transformations and leukocyte migration inhibition (LMI) in the presence of inactivated A/Scotland/74 (H3N2) influenza virus vaccine was measured in blood samples collected from 56 medical student volunteers. At the same time the volunteers were skin tested, using the same vaccine. Using the antigenically similar WRL 105 (H3N2), recombinant influenza virus, the level of haemagglutination-inhibiting (HI) antibodies in serum, and neutralizing antibodies in nasal washings collected from the volunteers, were also determined. Each volunteer was then inoculated with live, attenuated WRL 105 influenza virus vaccine and infections demonstrated by virus isolations and serology. Correlations between the ability to infect the volunteers and the various parameters of humoral and cellular immunity were then determined. The results showed a good correlation between the level of serum HI antibody and infection. Thus 16 of 20 volunteers with serum HI antibody titres of 1:10, but only 6 of 20 volunteers with antibody levels of 1:30, showed evidence of infection. No direct correlation was observed between any of the other parameters measured and infection by WRL 105 virus. However, when the LMI and serum HI antibody levels were considered together, a contribution of cellular immunity, as measured by the LMI test, could be found. Of 19 volunteers with low serum HI antibody and low LMI levels, 16 were infected, whereas of 13 volunteers with low HI antibody, but with high LMI levels, only 6 showed evidence of infection with WRL 105 influenza virus.

A simple manganous chloride and Congo red disc method for differentiating Neisseria gonorrhoeae from Neisseria meningitidis.

Manganous chloride and Congo red incorporated into blotting paper discs have been used to differentiate gonococci from meningococci. The new technique is simple and reliable; the materials for the test are inexpensive. The method will increase the efficiency of distinguishing between the pathogenic Neisseria in any clinical bacteriology laboratory and especially in those in the tropical areas.

N. gonorrhoeae: pathogenicity of colonial type 5.

This paper records the results of the intravenous inoculation of chick embryos with type 5 gonococci and establishes that these strains are pathogenic.

Reversion of Kellogg's colonial types of Neisseria gonorrhoeae in liquid medium.

Colonial type variation of gonococci is well known, but change from type 4 to type 4 to type 1 is rare except in vivo. By observing quantitatively subcultures from a new liquid medium it was possible to follow the day-to-day changes in the ratio of colonial types present. The results obtained showed that type 1 colonies could be derived from type 5 inocula even in unsupplemented media. In unsupplemented liquid medium, type 4 inocula did not revert to other types and indeed colonial type 4 appeared to be the final form of the organism before it died out. If, however, iron in the form of ferric citrate was added to the medium, reversion occurred and type 1 colonies rapidly came to predominate.

The survival of gonococci and meningococci in subcutaneous diffusion chambers in mice.

Studies are reported on the survival of gonococci and meningococci in Millipore diffusion chambers implanted subcutaneously in normal mice and in pre-immunised mice. The chambers allowed the passage of nutrients and humoral factors but excluded host cells. After an initial fall in the viable count--attributed to lysis by non-specific serum factors--there was evidence of multiplication of Neisseria gonorrhoeae; the subsequent development of specific antibody led to the disappearance of gonococci 16 days after the chambers were implanted. N. meningitidis behaved differently in the implanted chambers. Meningococci did not appear to be lysed by non-specific humoral factors and so the viable count showed no initial fall. The meningococci survived for 49 days, at which time the entire chamber was rejected.

Prolonged survival of Neisseria gonorrhoeae in a new liquid medium.

Despite substantial improvements in solid culture media for gonococci and the development of liquid media for the short-term growth of the organism, we know of no study of the long-term survival of Neisseria gonorrhoeae in liquid medium. A new liquid medium for N. gonorrhoeae formulated in this laboratory is simple, efficient, and promising. It will support growth and sustain inoculated colony types for periods of up to 3 weeks. Moreover, it has been used successfully for the primary isolation of gonococci from patients. From the evidence at present available, we believe that this medium will find a useful place in gonococcal research.