Serological diagnosis of HIV infection: practice and performance in western Europe.

Current laboratory practice in testing for HIV antibodies in western Europe was investigated by means of a questionnaire addressed to the 12 EC countries and Finland, Norway, Sweden and Switzerland. Despite inevitable regional differences there was a fair degree of homogeneity in broad laboratory organisation and in the types of tests and confirmatory strategies used. The primary test is always some form of enzyme linked immunosorbent assay (ELISA), though a number of laboratories also use agglutination tests. Confirmation is by an ELISA of a different type, or by Western blot, or both. The size and workload of laboratories covered a wide range. It is suggested that laboratories doing only a small number of tests at infrequent intervals should take extra care in validating their results and should be closely monitored. Twelve of the 16 countries studied have a quality assurance scheme for monitoring laboratory performance. Participation is voluntary but is invaluable even for the largest laboratories. The results suggest that the standard of laboratory diagnosis is reasonably uniform throughout the region, which is not only important for the individual patient but means that epidemiological comparisons of data from different areas have at least a sound technical base.

Human antibody response to lipopolysaccharides from Neisseria gonorrhoeae.

Red cells coated with lipopolysaccharides from three different strains of Neisseria gonorhoeae have been used as antigens in a haemagglutination test for gonococcal antibodies. For each strain the geometric mean titre in sera from 50 male and 25 female patients was significantly higher than that in 50 normal controls. The most useful smooth strain, G1, picked out 84% of females and 46% of males from a group of patients known to have gonorrhoea, but only gave 2% positives among controls. The rough strain, G2, gave 10% positives in controls and 31% in patients. The results suggest that the method is worth developing further as a diagnostic test and that strain differences are important. False positives were probably due to cross-reacting antibodies.

Identification of clinical isolates of Neisseria gonorrhoeae by a coagglutination test.

Ninety-eight per cent of laboratory isolates of Neisseria gonorrhoeae from a veneral disease clinic gave positive reactions with a gonococcal coagglutination test. The prototype reagent, however, was poor at distinguishing between different species of the genus Neisseria: 75% of strains of Neisseria meningitidis and 40% of other Neisseria species tested gave positive reactions. None of the origanisms other than Neisseria growing on the diagnostic cultures from the clinic gave positive reactions. We therefore suggest that the present reagent is unsuitable for testing isolates from the upper respiratory tract. The technique is simple, rapid, and convenient and with a more specific antibody could be useful. Results of coagglutination reactions of 126 strains of Neisseria grown on serum-containing and serum-free media were very similar and there is no need to use special serum-free media.

Acquisition of new genes by oral Neisseria.

It is suggested that part of the increased pharyngeal carriage of meningococci reported in patients with gonorrhoea is due to misidentification of gonococci which have been transformed to maltose fermenters by DNA from normal throat flora. The distribution of specific aminopeptidases in strains of gonococci, meningococci isolated from the throat and meningococci from systemic infections is consistent with this view. Gonococci oxidising maltose and gonococci with gamma-L-glutamyl aminopeptidase activity, both factors regarded as typical of Neisseria meningitidis, can be produced in vitro by transformation with DNA from N lactamica and N meningitidis. The clinical and theoretical implications of such changes are discussed.

Enzyme-linked immunosorbent assay (ELISA) to detect antibodies in gonorrhea using whole cells.

The choice of an antigen that will adequately differentiate between infected and non-infected patients has been a problem in detecting gonococcal antibodies for diagnosis. We have used the sensitive technique of ELISA to test various serotypes of Neisseria gonorrhoeae for their suitability as antigens. Whole cells of each serotype were attached to polystyrene plates using poly-L-lysine, N gonorrhoeae, strain H1 type 1 was used to detect antibodies in patients with known clinical history and then as a standard to evaluate the ability of different serotypes to differentiate between infected and non-infected groups.

Plasmids in throat and genital isolates of meningococci.

Plasmids 1.6, 2.8, or greater than 40 megadaltons in size were found in one urethral and nine throat strains of meningococci. Throat meningococci are known to be heterogeneous in their aminopeptidase profiles. Their unexpected content of plasmids is further evidence of their difference from classic systemic strains. Although the 2.8 megadalton plasmid has some resemblance to the well known 2.6 megadalton gonococcal plasmid, restriction enzyme studies gave no evidence of identity. Possible sources of the plasmids are discussed.

Immunity in acute bacterial infections.

A brief survey of the ways in which humoral and cellular immune responses deal with bacteria and their products shows that a variety of effector mechanisms is involved. Their efficiency varies, partly in relation to the kind of bacterial attack with which they have to deal. Some bacterial factors though not crudely toxic can interfere with specific host defences.

Resistance to infection: looking for new genes.

Genetic factors in resistance to infection, somewhat neglected with the development of microbiology, are again receiving careful attention. Although there are numerous reports of infection resulting from deficiencies in, for example, immunoglobulins or complement components, specific abnormalities in resistance associated with particular HLA groups have been unexpectedly rare. In mice, the Ity gene on chromosome 1, which is important in resistance to Salmonella typhimurium infection, may well be identical with the Lsh and Bcg genes concerned with resistance to Leishmania donovani and Mycobacterium bovis BCG. The most likely common factor determining resistance to three such disparate organisms is the macrophage, but direct evidence of its role is lacking. The high and low antibody-producing strains of mice selected by BIOZZI [2] are respectively sensitive and resistant to many intracellular infections including S. thyphimurium. Experiments with hybrids between Biozzi mice and other inbred strains suggest that the high line carries the Itys (salmonella susceptibility gene) plus another gene increasing susceptibility. Low line mice carry the Ityr gene plus another gene increasing resistance. Inbred mice are invaluable for analogizing gene interactions which, once understood, can be looked for in man.

Release of 14C label and complement killing of Escherichia coli.

When Escherichia coli labelled with 14C were killed by complement, characteristic 14C compounds were released even when complete cell disintegration was prevented or delayed by removal of lysozyme. Treatment with heated serum only resulted in the loss of small molecular weight compounds. Separation of the products was made easier if whole serum was replaced by a salt-precipitated fraction which contained no albumin or lysozyme but retained antibody and complement. Fractionation of the bacterial products on Sephadex G-200 showed two radioactive peaks containing lipids and proteins of which a preliminary examination was made. The release of these compounds was related to complement action, since they were not found when bacteria were killed by streptomycin or ultra-violet light in the absence of complement. Nor were they found when resistant bacteria were treated with complement and survived. The possible modes of action of complement on bacterial cell walls are discussed.

Classes of antibodies in acute gonorrhoea.

In patients with acute gonorrhoea the antibody responses to envelope antigens of Neisseria gonorrhoea were investigated with a sensitive enzyme-linked immunosorbent assay. IgG antibodies predominated even early in the disease but IgM and IgA antibodies were present. Surprisingly there were circulating antibodies linked to secretory piece. These were largely but not entirely IgA and were presumed to have been absorbed from sites of local production.

The cellular control of delayed hypersensitivity to Staphylococcus aureus in mice.

The cells which suppressed delayed hypersensitivity (DH) to Staphylococcus aureus in the mice were non-phagocytic, adhered to nylon wool and possessed surface immunoglobulin but no detectable Thy 1. antigen. They were probably B lymphocytes and the suppression which they mediated appeared to be specific. The mature suppressor cells were radiosensitive. Their precursors were sensitive to large doses of cyclophosphamide. Suppressor-cell activity in the spleen reached a peak after two subcutaneous S. aureus infections and declined thereafter. This decline might be related to the appearance of DH to S. aureus in mice after four or more staphylococcal infections.

Nature and Heterogeneity of the Antigens of Neisseria gonorrhoeae Involved in the Serum Bactericidal Reaction.

Sixty strains of Neisseria gonorrhoeae have been classified into four main groups according to their resistance to killing by human complement together with either normal human or immune rabbit antibodies. The rabbit antisera had been raised against 10 of the strains tested. The normal human antibodies had probably been formed against cross-reacting organisms since they could be removed by absorption with N. pharyngis sicca, N. pharyngis flavus, N. catarrhalis, or Escherichia coli. Bactericidal antibodies could be absorbed from both normal and immune sera by N. gonorrhoeae which had been autoclaved or trypsinized, and by red cells coated with gonococcal lipopolysaccharides. The results suggest that the antigens involved in the bactericidal reaction are lipopolysaccharides of several distinct specificities. Since individual sera always reacted more widely in hemagglutination than in bactericidal tests, it is postulated that surface-blocking antigens may restrict access to the lipopolysaccharides in the intact organisms.

The Ity/Lsh/Bcg gene significantly affects mouse resistance to Mycobacterium lepraemurium.

Mouse resistance to infection with Mycobacterium lepraemurium was measured by counting the total number of intact acid-fast bacilli in the spleen 8 weeks after i.v. injection of a standard inoculation. The effect of Ityr on resistance to M. lepraemurium was confirmed and the results extended to two Ityr strains of mice, A and C57L, not previously tested. Resistance to M. lepraemurium was also examined in the F1, backcross and F2 generations of BALB/c X CBA crosses, and in the congenic strain B10.LLshr that is Ityr. In all experiments the results were consistent with the view that resistance to M. lepraemurium is significantly affected by a gene close to or identical to the Ity/Lsh/Bcg gene on mouse chromosome 1. Sex had a marked effect on resistance to M. lepraemurium, so that the males of some genetically resistant strains were almost as susceptible as some genetically susceptible females.

Some physical properties of K antigens of Escherichia coli related to their biological activity.

The ability of K antigens from Escherichia coli to inhibit the agglutination of sheep red cells by rabbit antibody has been taken as a measure of the ability of such antigens to render strains of E. coli O-inagglutinable, resistant to complement and to phagocytosis, and to increase their virulence for mice. Analysis of K antigens by chromatography on Sepharose and diethylaminoethyl cellulose columns and by immunoelectrophoresis suggested that agglutination-inhibiting activity was directly related to molecular size rather than charge. The effect of alkali treatment showed inhibitory activity to be independent of K-specific antigenic determinants and confirmed its relation to molecular weight.

Effect of cyclophosphamide on delayed hypersensitivity to Staphylococcus aureus in mice.

Mice given cyclophosphamide 2-3 days before a single subcutaneous infection with Staphylococcus aures on cotton dust develop much more delayed type hypersensitivity (DTH) than normal. The enhancement is due to removal of rapidly dividing cells from the spleen. Passive transfer experiments before infection or before challenge show that immune serum suppresses the induction but not the expression but do not prevent induction. There may therefore be 2 suppressor or regulating systems involved. Cell commitment to suppressor function may be self-limiting. The results explain why DTH to staphylococci is only fully established after repeated infections and support the view that the suppressor system may function as a check on the excessive and potentially harmful development of DTH.