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.

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.

The specificity of serum and local antibodies in female gonorrhoea.

Immunoblotting has been used to compare the specificity of serum and local IgG and IgA antibodies in 13 women with gonorrhoea and in 13 controls. The technique allowed the simultaneous detection of antibodies to the major outer membrane proteins I, II, and III, pili and lipopolysaccharide; antibodies to another antigen which is probably a 'carbohydrate' were also detected. Serum and local IgG and IgA were found to be produced to several antigens during gonococcal infections, although the quantity of antibody was greater in serum. There was little change in the specificity of serum antibodies whereas the local response to LPS and pili increased over the two week study period. Serum antibody to LPS was more often IgG than IgA. Sera contained antibodies to 'carbohydrate', pili and lipopolysaccharide (LPS) whilst the local response was largely to the latter two antigens. Antibody to the outer membrane proteins was rarely detected. Control sera, but not vaginal washings, contained IgG and IgA to the major antigens but the staining of the immunoblots was less intense than those from patient's sera suggesting quantitative differences.

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.

Application of immuno-blotting to the differentiation of specific antibodies in gonorrhoea.

The technique of immuno-blotting was used to examine the specificity of serum antibodies in patients with gonorrhoea and to define the significant antigenic determinant present in the desoxycholate extracts of gonococcal cell walls used previously for serological diagnosis. IgG but not IgA antibody was directed predominantly against lipopolysaccharide. Antibodies to outer membrane proteins were not found as frequently as expected. There was a suggestion from the results that antibodies to antigen PI was more common in systemic than in local infections.

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.

Genetic control of resistance to Salmonella typhimurium infection in high and low antibody responder mice.

Mice selected by Biozzi for high and low responses to sheep erythrocytes were investigated for resistance to subcutaneous Salmonella typhimurium infection. The resistance was measured by LD50 values, viable bacterial counts in liver and spleen at 10 days, and the kinetics of infection over 4 weeks. High responder mice were susceptible to S. typhimurium injected subcutaneously (LD50 less than 10) and low line resistant (LD50 3 x 10(6)). Control of natural resistance to S. typhimurium in inbred mice is primarily by a single gene. Ity, on chromosome 1. Results with hybrid generations of Biozzi mice with either BALB/c (sensitive) or CBA (resistant) inbred mice indicated additional genetic control of resistance in Biozzi mice. Analysis of resistance data of backcrosses of (high x low)F1 with either parental strain showed this genetic control to be at least one other gene in the Biozzi mice, not linked to Ity. The antibody responses in the hybrid generations and inbred and Biozzi parental strains were tested by haemagglutination assays and ELISA. After specific stimulation of the mice there was an inverse relationship between resistance to S. typhimurium and antibody levels.

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.

Analysis of antibodies in local and disseminated Neisseria gonorrhoeae infections by means of gel electrophoresis-derived ELISA.

Major antigens in Neisseria gonorrhoeae were identified by surface labelling the organisms with 125I and electrophoresing extracts in polyacrylamide with sodium dodecyl sulphate. Horizontal slices of the gels were cut out and tested in individual wells against patients' sera using ELISA. Serum from local gonococcal infections reacted with Protein II and, probably, lipopolysaccharide, but not with Protein I in deoxycholate (DOC) extracts and gave no reaction with Triton X-100 extracts. Serum from disseminated gonococcal infections reacted with Protein I in the DOC extract and with pili and a number of undefined possibly cytoplasmic membrane antigens in the Triton X-100 extract. The significance of the results and the potential of the method are discussed.

Inbred mouse strain resistance to Mycobacterium lepraemurium follows the Ity/Lsh pattern.

Inbred mouse strains and their F1 hybrids infected intravenously with Mycobacterium lepraemurium showed different mean survival times (MST). BALB/c and C57BL mice were particularly susceptible, whereas C3H, CBA and DBA/2 mice were relatively resistant. Resistance as judged by MST was dominant in the F1 hybrids. A similar ranking order was obtained by comparing the doubling time of the bacillus in the bone marrow, the increase in spleen weight between 4 and 12 weeks after infection, and the pathology of the liver during infection. The general pattern suggests that mouse resistance to M. lepraemurium is, at least in part, controlled by a gene with the same strain distribution as the genes for resistance to Salmonella typhimurium (Ity') and Leishmania donovani (Lsh') and the gene controlling resistance to Mycobacterium bovis BCG (Bcg). Ity, Lsh and Bcg are all known to be on chromosome 1, suggesting a centre controlling reactions to intracellular infections.

The protein-lipopolysaccharide complex extracted with trichloracetic acid from Salmonella typhimurium effective in protection of mice against S. typhimurium infection.

A protein-lipopolysaccharide complex has previously been postulated as necessary to protect susceptible mice against Salmonella typhimurium infection. Lipopolysaccharide attached to non-specific proteins, bovine serum albumin or methylated BSA, gave a specific delayed-type hypersensitivity (DTH) reaction when injected into the footpad of mice sensitized with sublethal doses of S. typhimurium. However, immunization of BALB/c mice with the complex gave no survivors after challenge with 100 LD50 S. typhimurium. Trichloracetic acid extraction of bacterial cultures produced lipopolysaccharide with attached protein. This method gave simple and convenient production of an active factor, demonstrating few major protein bands after electrophoresis. The complex elicited specific DTH reactions in sensitized mice and protected 37% of BALB/c mice against 100 LD50 S. typhimurium. Combinations of protein:lipopolysaccharide were used in DTH experiments to determine the relative importance of the components. A minimum requirement for both was demonstrated, although the ratio was not critical. Use of O-antigenic mutant strains of Salmonella indicated a role for protein in the specificity of activity of the complex.