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A micromethod for the lymphoproliferative assay has been evaluated statistically with non-stimulated human lymphocyte combinations. Different variance components were estimated by means of a hierarchical analysis of variance. Instead of using an arbitrary value for a significant difference between stimulated and non-stimulated cultures, the value can be calculated on the basis of the components of variance in the experimental design. The method disclosed systematic differences between the values of control cultures depending on the lymphocyte combinations and groups of persons examined, which underlines that every experiment should always contain non-stimulated cultures. Further, cell-cell cooperation and differences in spontaneous DNA synthesis within individual lymphocyte populations could be demonstrated.

In 1985, a total of 1,140 patients (907 men and 503 women) were treated for gonorrhoea at the Copenhagen County Outpatients Clinic at Rudolph Berghs Hospital. Failure of the standard treatment regimen (pivampicillin plus probenecid; patients stating penicillin allergy received a Sulfotrim-regimen) occurred in 79 (5.6%). A significant association between the in vitro susceptibility to penicillin and the efficacy of the standard treatment regimen was found; among 1,139 patients with uncomplicated urogenital gonorrhoea caused by penicillin susceptible gonococci, the failure rate was low (0.4-1.9%); the failure rate was high in patients infected with penicillin resistant gonococci. Continued surveillance of the in vitro antimicrobial susceptibility of gonococci is recommended as a basis for the choice of standard treatment regimen.

The results of two different in vitro investigations are presented: a comparison between gentamicin and netilmicin and a presentation of a regression line for netilmicin. The first investigation comprises 335 bacterial strains. The method used is a plate dilution technique and the susceptibility of the strains is given as IC50 values, i.e. 50% inhibiting concentrations. The conclusion of the study is that netilmicin offers no advantage over gentamicin. In the second investigation a regression line is presented giving the relation between IC50 values for netilmicin and the size of the inhibition zone, based upon 109 bacterial strains. For the disc diffusion method a 20-hour prediffusion technique is used. The disc content is 15 micrograms netilmicin base. The results are discussed and a grouping as to susceptibility is proposed based on the IC50 values found and the blood level obtained after scheduled dose of netilmicin.

The results of blood cultures from a large number of Danish hospitals examined at Statens Seruminstitut in 1977 and 1978 have been analysed. Out of a total of 17, 518 cultures in 1977 and 17,820 in 1978, 1,626 from 830 patients and 1,544 from 832 patients yielded growth of pathogenic microorganisms. Cases of bacteremia with Enterobacteriaceae constituted approximately 50% of cases both in 1977 and 1978. Polymicrobial bacteremia occurred in 8.1% of bacteremic cases in 1977 and 7.5% of those in 1978. The microorganisms isolated and their frequencies were surprisingly similar to those found in 1965 and 1971 in an earlier investigation from this department. No change in antibiotic susceptibility was observed between 1977 and 1978. Retesting of 50 E. coli strains from 1965, 1971 and 1978 for susceptibility towards ampicillin, cephalotin, streptomycin and tetracycline indicated that no significant changes had occurred, contrary to the results of the earlier investigation. This fact stresses the importance of exploring the possibility of unnoticed methodological changes having occurred in the sensitivity testing systems before any conclusions can be drawn concerning apparent differences in antibiotic susceptibilities.

In 1982, 2 of 14 Plasmodium falciparum infections acquired in East Africa and diagnosed in Copenhagen were resistant to treatment with sulfadoxine plus pyrimethamine (Fansidar), while in 1983, 6 of 18 were so. The in vivo tests were supplemented by determinations of drug concentrations in serum, and 4 isolates from in vivo-sensitive cases and 6 from in vivo-resistant cases were selected for in vivo tests. These were performed in ordinary RPMI 1640 medium and in a medium with physiological p-aminobenzoic acid and folic acid concentrations. Pharmacokinetic aberrations were found to be of possible importance in only 2 of the in vivo-resistant cases. In vitro susceptibility to sulfadoxine was found to be uniformly low in all isolates. Testing with a combination of sulfadoxine and pyrimethamine in the medium with physiological concentrations of cofactors probably reflects the in vivo situation most accurately, but in all but 1 of the isolates studied in vitro the in vivo susceptibility to Fansidar would be predicted by in vitro susceptibility to pyrimethamine in either medium. The concentration of p-aminobenzoic acid in serum, quantitated by high performance liquid chromatography, was found to be subject to wide variation, and this may have implications for in vitro testing.

In the period 1975-1981, 4060 cases of Staphylococcus aureus bacteremia were recorded in Denmark, and the corresponding strains were examined. The percentage of strains, resistant to penicillin only, rose to 82, and the percentage of multiply-resistant strains fell to five. Newer phage types (94, 96 and 95) increased from 10% to 27% of the material. These strains were usually resistant only to penicillin, but produced large amounts of penicillinase. The ample penicillinase production has also been characteristic for previous epidemic strains, and it is furthermore correlated to mortality. The overall mortality of 34.6% was lower than that of the preceding period. Mortality rates were highest in elderly patients, nosocomial cases, patients with serious primary diseases and endocarditis cases.

A number of laboratory variables, including Hb., ESR and several phase proteins, fluctuated in concord with the clinical signs of synovitis activity in patients with rheumatoid arthritis during a controlled study of 3 disease-modifying anti-rheumatic drugs (DMARD). The correlation between laboratory variables and clinical synovitis was significant in a large patient population but the correlation coefficients were not of such magnitude that any of the laboratory variables reflected clinical synovitis activity in a reliable manner in the individual patients. In patients treated with azathioprine, the response of the Hb, (and consequently of the ESR), was reduced compared to patients given other DMARD. This phenomenon was caused by the bone marrow suppressing effect of azathioprine. However, the effect of azathioprine on the clinical synovitis activity did not differ from that of the 2 other drugs. Similar results were found by reviewing the literature about controlled trials of DMARD. In the present trial the clinical evaluation was performed under optimal conditions. In daily clinical practice the evaluations of the joints may be less than optimal since they may be performed by different rheumatologists with varying experience. Consequently, it may be difficult to do without the unreliable laboratory variables mentioned in the routine assessments of disease activity, unless the quality of routine evaluations of synovitis activity is improved considerably.

Optimum conditions for the production and assay of guinea pig interleukin-2 (IL-2) have been established. The mitogenic activities of serial dilutions of guinea pig IL-2 preparations were compared in cultures of guinea pig peripheral blood lymphocytes prestimulated for 7 days with phytohemagglutinin (PHA) used at 1 microgram/ml. Parallel log dose-log response curves were used for quantitative comparisons. Optimum IL-2 yields were obtained from cultures of lymph node lymphocytes stimulated for 20 h with Concanavalin A (ConA) at 5 micrograms/ml. Guinea pig T cell lines reactive to mycobacterial antigens were propagated for several months using our IL-2 preparations. The molecular weight of guinea pig IL-2 was estimated to be 30,000 using S-200 gel filtration. The species specificities of guinea pig, human, mouse and rat IL-2s were examined. It was shown that guinea pig T lymphocyte blasts were stimulated only weakly with human IL-2 and not at all with mouse and rat IL-2.

In 499 patients operated on for cataract, the clinical postoperative signs of extraocular inflammation (conjunctival hyperaemia, chemosis, discharge and oedema of the lids), the number of infiltrates around the corneoscleral sutures, and the severity of intraocular inflammation in the anterior chamber (aqueous-flare) were assessed on the fourth postoperative day and correlated with the bacterial conjunctival flora examined both qualitatively and quantitatively on the same day. Patients with potential pathogens (Staphylococcus aureus, gram-negative bacilli and streptococci) on the conjunctiva following operation did not show any increased inflammatory reactions when compared with those without such pathogen. The quantity of bacteria, i.e. number of colonies, did not appear to play a role. The reasons are discussed. The clinical postoperative inflammatory signs were further correlated with the following factors: surgical complications, quality of suturing technique, use of alpha chymotrypsin, systemic disease, sex and age. A positive correlation was found between the severity of extraocular inflammation and retained lens material and hyphaema. Furthermore, extraocular reactions were more severe in males than in females. The incidence of infiltrates around corneoscleral sutures was found to vary with age, i.e. occurred more frequently in patients less than 60 years. No relationship was found between the severity of aqueous-flare and the above mentioned factors.

Forty-nine clinical strains of coagulase-negative staphylococci were investigated for susceptibility to methicillin, oxacillin, cloxacillin, dicloxacillin, flucloxacillin and cephalothin. The highest level of resistance was found to methicillin and the lowest to cephalothin. The resistance-level of the isoxazolyl penicillins showed a high degree of uniformity. However more strains were resistant to cloxacillin and oxacillin than to dicloxacillin and flucloxacillin. Only a weak correlation was found between beta-lactamase production, and resistance to the six antibiotics. Methicillin was the most stable. The inactivation of cephalothin differed from that of the other antibiotics.

The variations in the incidence of Mycoplasma pneumoniae infections in Denmark over a period of 17 years could be demonstrated in the central serological laboratory which serves the whole of the population. This observation was made possible for the first and major part of this study by testing cold agglutinin (CA) positive sera, which had been kept frozen sine 1958, for antibodies to M.pneumoniae. The second part of the study is based upon results from routine tests for CA and M.pneumoniae antibodies on all samples which we receive. A statistical analysis of the total material indicates that four epidemics of M.pneumoniae antibodies on all samples which we receive. A statistical analysis of the total material indicates that four epidemics of M.pneumoniae infection had taken place from January 1958 to December 1974 and that these epidemics occurred at regular four and a half year intervals. By a follow-up of the study a fifth epidemic was demonstrated during the first eight months of 1975 which broke the regular periodicity by appearing two years earlier than expected. The consequences of including only CA positive sera in this study was investigated. Antibodies to M.pneumoniae were measured by either an indirect immunofluorescence test, an indirect haemagglutination test or a complement fixation test. The observed difference in sensitivity of these tests is discussed in relationship to a possible influence on the overall incidence.

On the basis of a study from De Gamles By, Copenhagen, concerning the mortality and changes in the patients' need for nursing care over a 2-year period, calculation is made of the time-related changes in the care status, using a special model for homes for elderly persons. This model is based on a policy that only independent persons are received into such homes and that they remain there until they die. It is found that the need for care becomes stabilized after a period of 5-6 years and remains at a level corresponding to about 45% independent, 36% slightly dependent, and 17% heavily dependent patients (the "minimum need for care"). Using the model, a few examples are given to illustrate the changes which would arise in the need for care in De Gamles By if, on the basis of the primary care status on January 15, 1969, (A) only independent patients were admitted, and (B) the current admittance policy was continued.

A quantitative assay method for determination of thiomersal in biological products is described and evaluated. A microbiological method using agar diffusion from filter paper discs is used. The advantages of the method in terms of low cost and reliability for determining the actual antimicrobial activity in the preparation are discussed. The assay method is in routine use in the production control of vaccines at our Institute.

Based on an account of the need for nursing care in the entire patient population in De Gamles By, Copenhagen, on January 15, 1967, and again on January 15, 1969, it is shown that the need for care varies considerably, despite a policy of admitting only independent persons. The consequences of this variability for the size of the nursing staff required are discussed, together with some aspects of admittance policy.

During the years 1966-1974, 167,297 strains isolated from 167,297 patients or staff members in Danish hospitals were registered at Staten Seruminstitu. All the strains were phage-typed and examined for production of a 'Tween'-80-splitting enzyme and resistance to mercuric chloride. 158,236 strains were examined for resistance to antibiotics. Since 1968, a steep decrease in the number of strains resistant to three or more antibiotics (multiple-resistant) and in strains of the 83A complex was noticed. In recent years an increase in strains belonging to phage-group I and in those referred to as miscellaneous and non-identified was registered. The increase in the non-typable strains might be explained by the shift of the concentration of the typing phages from 1000 X RTD to 100 X RTD. It is concluded that at least two factors may have contributed to the reduction of the multiple-resistant strains: an altered antibiotic policy, restricting the use of streptomycin and tetracyclines, and an improved hospital hygiene, diminishing the spread of identical strains within the various departments. However, it is emphasized that the consumption of methicillin is still increasing.

The relative potencies of a number of rabies immunoglobulin preparations were estimated in an international collaborative study comprising eight laboratories in five countries. Two assay methods were used: a virus neutralization test in mice (MNT) and a virus neutralization test in cell culture (RFFIT). Differences between the results obtained by the two methods, which have been reported, could not be generally corroborated. The results indicate that in some laboratories the MNT cause difficulties and give results different from those obtained by RFFIT. In other laboratories such difficulties are not encountered. The results seem to indicate that the RFFIT is a more reliable method than the MNT.

A seroepidemiological survey has shown a remarkable shift in the previously reported regular cyclic pattern of Mycoplasma pneumoniae epidemics which culminated every 4.5 years during the period 1958-74. The last of four regular epidemics occurred in 1972. It was followed by 'premature' epidemics in 1975 and 1977/8 which inaugurated a change from an epidemic to an endemic pattern of the infection in Denmark. Over the following 9 years (1978-86) there has been an irregular but significant decrease in the annual number of seropositive samples with the usual high incidence during winter seasons. This endemic period terminated in the fourth trimester of 1987 with the development of a new epidemic. The hypothesis is advanced that a sixfold increase of children in day care may have influenced the change from an epidemic to an endemic situation which after 9 years led to a new epidemic of M. pneumoniae infection.

A micromethod for the lymphocyte proliferative assay and multifactorial analysis of stimulation indices classified according to subjects, lymphocyte combinations and antigens was used to elucidate deficient Mantoux (Mx) reactions in BCG-vaccinated subjects. It was possible to group each of the factors so that interactions between the two latter factors were not present within but only between groups. Comparisons between the subject groups showed a clear association between results of this in vitro test with PPD antigen and the Mx test carried out with 1 TU. The in vitro test had the advantage of being more sensitive and of not introducing the immune reactions occasionally seen with the Mx test. Thymus-processed lymphocytes were the cells responsible with functions both as effector and regulator cells, B lymphocytes had only regulatory properties.

On the basis of an account of the mortality and changes in the need for care over a 2-year period among independent persons and patients requiring care in De Gamles By, Copenhagen, an assessment is made of the movements of patients and the need for care in a model of a geriatric institution. This model includes both purpose-built residential apartments for independent persons and places for dependent patients requiring nursing care. It is shown that the accommodation for the latter patients rapidly becomes occupied by the independent persons from the residential apartments who subsequently need nursing care. Thus, the places in the nursing section are blocked for the admittance of dependent persons living in the community outside. This situation arises when there are equal numbers of both types of accomodation. Where the number of residential apartments is greater than the number of places in the nursing section the care function must be transferred to the residential apartments, and this is regarded as impracticable.

Ten patients with cystic fibrosis (CF) and chronic broncho-pulmonary Pseudomonas aeruginosa infection were given imipenem/cilastatin (100 mg/kg/day) in combination with tobramycin (15 mg/kg/day). Forced vital capacity and forced expiratory volume in the first second improved significantly in nine out of ten patients, and most of the patients improved clinically. P. aeruginosa was not eradicated in any patient and resistance against imipenem developed in all patients during treatment. A concomitant increase in MIC of piperacillin and ceftazidime occurred during treatment. In-vitro bactericidal synergy of imipenem and tobramycin was noted in 57% of pretreatment isolates. Seven patients complained of adverse reactions, mainly gastrointestinal, and treatment of three patients was discontinued after 5, 8, and 12 days of therapy, because of rash or nausea and vomiting. The side effects were considered to be due to imipenem/cilastatin. Because of the rapid development of imipenem resistance despite combination therapy, the high proportion of side effects, and the risk of induction of beta-lactam resistance, imipenem/cilastatin cannot be recommended for routine treatment of CF-patients with P. aeruginosa infection.

The influence of the volume of blood cultured on the rate of detection of bacteremia was evaluated in a routine 12-tube blood culture system using 1693 samples from 1502 patients. Blood samples were drawn simultaneously into two transport tubes. The blood volume cultured was the only varying parameter. Generally, 17% more cultures with clinically significant microorganisms (both Enterobacteriaceae and gram-positive cocci) were found when blood from two instead of one tube was used (in most cases comparing 13-16 ml of blood with 6.5-8 ml). Of the most prevalent species, the maximum average extra yield was observed for Staphylococcus aureus (26%) followed by Escherichia coli (16%) and Streptococcus pneumoniae (12%). In adults most cases of bacteremia are low-grade. The grade of bacteremia in our patient population was on average as low as 0.25 CFU/ml blood. Therefore, all patients suspected of having bacteremia should have the benefit of a sufficient volume of blood cultured. Since the volume of blood cultured seems to be the single most important factor in the detection of bacteremia, it is imperative that the volume is the same in comparative studies of different blood culture systems.

A mouse model using intraperitoneal inoculation of Streptococcus pneumoniae type 3 was used to compare in vitro and in vivo effects of 14 cephalosporins, selected to encompass a wide range of minimal inhibitory concentrations (MICs) against the organism. Antibiotics were subcutaneously administered as single doses 1 hr after inoculation of pneumococci, and the effect was measured as the 50% effective dose (ED50). The correlation between log ED50 and log MIC was highly significant (r = .87, P less than .001). Pharmacokinetic properties of the cephalosporins were estimated after a fixed dose of 5 mg per mouse (167 mg/kg) for all drugs. The only correlation that was significant was between log ED50 and the time the serum concentration remained above the MIC for each drug (r = -.90, P less than .001). Ceftriaxone was the most-effective cephalosporin in vivo because of a combination of high in vitro activity and prolonged serum elimination half-life.

In the period 1969-1974 a decreasing number of Danish hospital departments recorded epidemic occurrences of Staphylococcus aureus strains, and the local spread of strains was less extensive. Multiple-resistant strains of the 83 A complex were succeeded by type 94 strains, resistant to penicillin only. The level of methicillin resistance fell from 19 to 6%. 12% of methicillin resistant strains are now sensitive to streptomycin and/or tetracyclines; they do not represent a few clones, but belong to a wide spectrum of phage types. The changes follow a reduction in the consumption of streptomycin and tetracyclines not of methicillin or other penicillins. As a contrast to the general reduction of combined resistance to streptomycin and tetracyclines, the strains in dermatological departments, where tetracyclines are commonly used, maintain a high degree of resistance to tetracyclines alone.

The development of tuberculin sensitivity in groups of guinea pigs immunized with living BCG vaccine or with heat-killed Mycobacterium tuberculosis suspended in oil (TB) was measured by skin tests and lymphocyte transformation (LT) tests with tuberculin PPD. LT tests on lymphocytes from peripheral blood (PBL) and lymph nodes (LNL) and skin tests were carried out on different groups of guinea pigs for 1 year following immunization. Three ways of expressing the LT results were considered, i.e. as stimulation index, delta cpm and uncorrected cpm values. Since neither of the first two methods were equally applicable to all of the data, we decided to express the data as geometric means of cpm values for stimulated and control cultures, respectively. For both immunogens the sensitivity measured by PBL LT and skin tests showed a closely parallel development; LNL reactivity appeared earlier and remained at a higher level than PBL reactivity. Antibody levels in the guinea pigs were measured using a radioimmunoassay technique, were found to be dependent on the immunization period and not correlated to the cellular immunity.

We followed the cases of 26 consecutive postmenopausal patients operated on for primary breast cancer. Serum specimens were obtained each month for 1.5 years and stored at -80 degrees C until assayed for carcinoembryonic antigen (CEA) and other quantities. Ten patients developed recurrence, while 16 qualified as controls (no clinical recurrence for at least 1.7 years after the last venipuncture). Using the homeostatic autoregressive time series model, modified by us to be particularly sensitive to sustained deviations from the mean, we detected four recurrences by CEA without having any falsely positive alarms. Group-based reference limits and application of the unmodified homeostatic model were less effective (fewer detected and shorter lead time). Simulation studies, involving use of a mathematical model relating CEA concentration to tumor growth and using parameters estimated from patient data, verified this and indicated that at least five stable baseline values are needed to detect 100% of recurrences before they are detected by the group-based limit.

The homology percentages between DNA from M. smegmatis ATCC 607 and DNA from nine various species of mycobacteria have been determined. DNA-DNA hybridization was measured in a spectrophotometer. The technique, calculation of results and the uncertainty of the method are described.

Ten patients with cystic fibrosis and chronic broncho-pulmonary Pseudomonas aeruginosa infection received 45 mg imipenem/cilastatin per kg body weight/day, intravenously for two weeks. The treatment was safe with only minor side effects and clinical parameters improved considerably during therapy. In all patients resistance of Ps. aeruginosa to imipenem developed in the second week of treatment; in seven patients the therapy selected for a resistant strain and in three resistance developed in the original strain. The resistance persisted after cessation of treatment and thus the clinical usefulness of imipenem/cilastatin as monotherapy in CF-patients with Ps. aeruginosa seems to be limited.

As part of a cooperative inter-laboratory WHO supported project for the fractionation of Mycobacterium tuberculosis skin test preparations, four fractions (designated 15, 7, 4.75 and 3.5%) were evaluated by comparative skin tests on sensitized guinea-pigs. The 7% fraction was the most potent in both homologously and heterologously sensitized animals, and the 4.75% and 3.5% gel fractions showed the lowest activity. Significant levels of cross-reactivity in guinea-pigs immunized with M. bovis BCG, M. kansasii, M. avium and M. intracellulare were demonstrated for all fractions examined, thus reflecting the antigenic relationships among these mycobacteria. These four fractions may qualify as starting material for further studies aiming at a reduction of skin test cross-reactivity.

In a double blind trial of 36 patients with rheumatoid arthritis a new scintimetric method was applied to three comparable patient groups before and after eight months' treatment with levamisole, penicillamine, or azathioprine. Technetium-99m pyrophosphate scintigraphy of both hands was performed on a gammacamera with a computer attached, immediately after the administration of the tracer. The uptake ratio of each proximal interphalangeal (PIP) joint and the mid-half of the adjacent proximal phalanx was calculated from the activity counted for one minute. A scintimetric index was expressed as the sum of eight uptake ratios. This index was higher for each of the three patient groups before start of treatment than that for 10 age matched controls. After eight months of treatment the index, the number of PIP joints with clinical signs of synovitis, and the total circumference of the PIP joints decreased in the penicillamine and azathioprine groups. The scintimetric method reliably reflected local synovitis activity and its changes but, like grip strength and PIP circumference, was not a representative measure of the overall activity of the disease.

The study is a retrospective and prospective serological investigation of the incidence of Mycoplasma (M.) pneumoniae infection in Denmark during a 17-year period. By least square multiple regression analysis it was shown that four major outbreaks or epidemics had occurred which culminated at regular intervals of four and a half years. The study was based on cold agglutinin (CA) positive sera tested for antibodies to M. pneumoniae by an indirect immunofluorescence (IF) test, an indirect haemagglutination (IHA) test or a complement fixation (CF) test. By the criteria chosen for a positive test, the CF test was found to detect more cases than the IF or IHA tests. A shift during the study from the latter two tests to the CF test influenced the incidence, but not the periodicity of epidemics. The consequence of including only CA positive sera in the study was investigated.

Treponema pallidum organisms suspended in equal parts of Nelson's survival medium alone and with addition of various sera were counted in 5 microliters samples before and after 18 hours' anaerobic incubation at 35 degrees C. Samples in which the treponemes were not Poisson-distributed were excluded from the study. A loss of greater than 30% occurred in 230 out of 391 samples. The disappearance of treponemes did not seem to be caused by treponemal agglutination during incubation. There was no adherence of treponemes to the formed elements remaining in the suspension. The loss was not dependent on the concentration of syphilitic serum, nor was it influenced by the presence of immobilizing antibody in the serum. Lack of rigorous anaerobiosis during the manipulations did not influence the treponemal loss. The loss was independent of the density of the treponemal suspensions which did not contain unheated guinea-pig serum (GPS). The relative frequency of the loss exceeding 30% was highest in suspensions containing unheated GPS. However, the extent of loss in individual samples was haphazardly distributed among suspensions of all the compositions examined. The loss of treponemes seems to depend on at least two factors: a) a factor which is eluted together with the treponemes from the syphilitic orchitis and b) the presence of active guinea-pig complement. It is conjectured that the complement is non-immunologically activated and acts through the alternative pathway.

The conjunctival flora of 499 patients was studied the day before cataract extraction, no antibiotics or chemotherapeutical agents had been used before admission. Staphylococcus albus was by far the most common micro-organism (95.4%), followed by corynebacteria (44%), Staphylococcus aureus (14.9%), gram-negative bacilli (7.8%) and pneumo-streptococci (4.4%). Corynebacteria was isolated more frequently in the presence of S. albus, while S. aureus and gram-negative bacilli were found more frequently in the absence of S. albus. No relationship could be demonstrated between the occurrence of pneumo-streptococci and S. albus. The flora of the nose and skin of the face were studied and compared with the conjunctival flora, and a similarity could be observed. Furthermore, strains of S. aureus isolated at the same time from the two or three regions, in most cases, showed the same bacterio-phage type complex. The conjunctival flora was further correlated with sex, age, season, and number of polymorphonuclear neutrophils recovered from the conjunctival fluid. The incidence of corynebacteria and gram-negative bacilli was found to be higher in males, while corynebacteria was the only organism to show seasonal variation, i.e. was isolated more frequently in the second and third quarters of the year. No correlation could be found between age or number of neutrophils.

A number of studies have reported hyperoestrogenaemia in men surviving an acute myocardial infarction (AMI). This has led to speculations that hyperoestrogenaemia might be a coronary risk factor. It is not clear why plasma levels of oestrogens should be elevated in coronary heart disease. To ascertain whether hyperoestrogenaemia might be present before the onset of a myocardial infarction, we performed a prospective study in 252 men above the age of forty who within the following 5 years suffered an AMI and, as controls, 526 men matched for age and coronary risk factors and 329 randomly selected men. Mean plasma concentrations of oestradiol and oestrone did not differ significantly between the groups. This suggests that hyperoestrogenaemia is not a coronary risk factor and thus, if present after the AMI, is more likely develop concurrently with the myocardial infarction.

Skin reactivity to tuberculin has been studied during the course of experimental leptospirosis in guinea pigs. A depression of the delayed hypersensitivity to tuberculin was demonstrated in the infected animals. The depression was most pronounced when icterus had developed. The depression was not correlated with the amount of infectious units administered or with the demonstration of live leptospirae in the peritoneal cavity. In the infected animals there was no correlation between the initial and the final skin tests which is in contrast to findings in the control group.

To investigate minimal requirements for tetanus revaccination to secure continuous protection, recently recommended by WHO, 637 subjects with documented vaccination history were studied. Antitoxin concentration in serum relative to time corresponded to a steep decline in the first years after vaccination continuing exponentially. By multiple regression analyses duration of immunity after three-dose primary vaccination was calculated to be 5 years (upper 95% confidence limit of estimated risk of serum antitoxin concentration below 0.01 IU ml-1 still less than 0.1%). Serum antitoxin concentration relative to time after revaccination depended upon age at revaccination and interval from primary vaccination. When given in childhood 5 years after primary vaccination revaccination was calculated to offer protection for approximately equal to 21 years, but protection was considerably shorter when given to the elderly. Cross-sectional and longitudinal analyses were compared and statistical approaches were introduced which may be generally applicable for evaluation of vaccination programmes. It was concluded that a vaccination programme consisting of primary vaccination in infancy and one revaccination 5 years later will secure continuous protection to about the age of 25 years. This is considerably simpler than programmes recommended in many countries, in which risk of hyperimmunization is apparent.

MPB 70 is a protein found in large quantities in the culture filtrate (CF) of the Tokyo and some other strains of Mycobacterium bovis BCG, and it has a remarkable degree of specificity for these strains. We estimated the molecular weight of MPB 70 to 22,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). SDS-PAGE and immunoblotting showed that MPB 70 was present in high quantities in CF from BCG Tokyo, that it could be also demonstrated in BCG Copenhagen, and that it was absent in CF from M. tuberculosis H37Rv. When the purified MPB 70 preparation used in the present study was run in SDS-PAGE, blotted and stained with a polyclonal rabbit or a monoclonal mouse anti-MPB 70 antibody, several bands in addition to the main 22 kDa band were seen, indicating a tendency of the MPB 70 molecules and/or fragments thereof to form very stable aggregates with themselves. The biological activity of MPB 70 was studied in groups of guinea pigs sensitized with live BCG of the Tokyo and Copenhagen strains. Guinea pigs from both groups developed reactivity to tuberculin PPD as assessed by skin tests and lymphocyte stimulation tests with peripheral blood or lymph node lymphocytes. In addition, a strong and persistent reactivity to MPB 70 was demonstrated in the BCG Tokyo group with both methods. Guinea pigs sensitized with the Copenhagen strain were only weakly reactive to MPB 70. Skin reactions in guinea pigs that had been repeatedly tested with MPB 70 and tuberculin were compared with reactions in animals tested only once. Reactions to MPB 70 in BCG Tokyo sensitized guinea pigs were suppressed by repeated tests, whereas tuberculin reactions were boosted by the interim tests. The levels of specific anti-MPB 70 antibodies were higher in BCG Tokyo- than in BCG Copenhagen-sensitized guinea pigs. MPB 70 has a high degree of specificity and is a strongly immunogenic protein, which may prove useful in studies of mycobacterial immunology.

A lysis-centrifugation blood culture system (Isolator) and a conventional system (4 tubes of nutrient broth, 4 tubes of semisolid agar, and 4 tubes of thioglycollate agar) were compared after different lengths of incubation by cumulative scoring of differences in detection power. After the first half day of incubation, the Isolator system was already significantly faster in detecting isolates of clinical significance (15 vs. 4, P = 0.02). Maximum difference in first or only detection system was seen after two days of incubation and was based on an overall superior detection of Staphylococcus aureus (11 vs. 0, P = 0.001), and an earlier detection of Enterobacteriaceae (30 vs. 13, P = 0.01) in the Isolator system. On the contrary, the detection of Streptococcus pneumoniae was significantly inferior in the Isolator system (0 vs. 10, P = 0.002). The earlier finding of clinically significant microorganisms in the Isolator system certainly contributes to good patient-care. A drawback of the Isolator system was the finding of clinically insignificant coagulase-negative staphylococci in 11%, compared with 1% in the conventional system. This led to a considerable waste of time and materials in the laboratory. The comparison of the two blood culture systems, based on statistical analysis of cumulative differences in detection power, expressed as the earliest or only findings, gives the optimal information, and is in our opinion the clinically most relevant comparison.

The antibacterial activity of four new fluoroquinolone carboxylic acids, pefloxacin, ofloxacin, enoxacin and ciprofloxacin, against 256 clinical isolates was investigated by means of an agar dilution method. Generally, all quinolones tested had a high activity against Gram-negative bacteria. More than 90% of Enterobacteriaceae strains were inhibited by a quinolone concentration of 0.4 microgram/ml. Also strains usually resistant to conventional beta-lactam antibiotics, and sometimes to third-generation cephalosporins, like Enterobacter spp., Serratia spp, and Yersinia spp. were susceptible to the tested quinolones. Ciprofloxacin was 5 to 25-fold more potent on a weight basis against Enterobacteriaceae than the other quinolones. Neisseria meningitidis, Neisseria gonorrhoeae, and Haemophilus influenzae were extremely susceptible to the new quinolones. Ciprofloxacin was about 10 times more potent against Pseudomonas aeruginosa than the other quinolones, and was the only quinolone that was sufficiently active against all tested P. aeruginosa strains (MIC less than or equal to 0.4 microgram/ml). The activity against Gram-positive bacteria was considerably lower. All the quinolones investigated had an acceptable activity against many of the methicillin-sensitive and methicillin-resistant Staphylococcus aureus and coagulase-negative staphylococci. The majority of the Streptococcus spp. tested was quinolone-resistant, and was Listeria monocytogenes. Generally, it was evident that ciprofloxacin was more potent on a weight basis than the other quinolones, but this difference was counterbalanced by a higher achievable serum concentration for ofloxacin. Some of the investigated fluoroquinolones might constitute valid therapeutical alternatives to beta-lactam antibodies and aminoglycosides in the treatment of serious bacterial infections.

The stimulating effects of 2-mercaptoethanol (2-ME) reported in murine cell cultures were confirmed for guinea pig lymphocyte transformation (LT). 2-ME was mitogenic for peripheral blood lymphocytes (PBL) and lymph node lymphocytes (LNL) and enhanced PPD-induced LT. The effect on LNL was dependent on the magnitude of the response of non-2-ME treated cultures. The 2-ME effect on PPD-stimulated LNL was reversed when stimulation index (SI) replaced the delta cpm estimate. Mycostatin (MYC) inhibited the blastogenic response of PBL control cultures, whereas its effect on LNL control cultures varied. The interpretation of the effect of MYC on PPD-stimulated cultures was dependent on the use of the delta cpm or SI estimates. The interpretation of LT experiments is therefore highly dependent on whether delta cpm or SI is used for expression of results. Analysis of effects on control and stimulated cultures should precede the addition of 2-ME or MYC to cultures for LT.

The methods used for obtaining bacterial cultures from the normal conjunctiva were reviewed. A comparison was made between four methods; a) platinum loop, b)calcium alginate swab c) dry, and d) wet cotton wool swab, using agar (all methods) and serum-bouillon (methods b, c, d) as culture media. The comparisons were based on the isolation rate of Staphylococcus albus and corynebacteria. At the same time, four topical anaesthetics (benoxinate, tetracaine, proparacaine, cocaine) and one vital stain (tetrazolium - alcian blue mixture) instilled into the conjunctiva immediately before taking samples, were studied for their effects on the isolation rate of Staphylococcus albus and corynebacteria. For isolating Staphylococcus albus, method d was found to be superior to the others when the methods were used in the above mentioned order, and when agar was the only culture medium used. On the other hand, after instillation of the above mentioned eyedrops, causing the so-called "washing-out" effect (Fahmy et al. 1974), method a was found better than the others, providing the methods were used in the a-b-c-d order and when agar only was used. When considering the growth of Staphylococcus albus in serum boullion as well, methods b, c, and d were found equal in effect, but somewhat superior to a. Corynebacteria showed nearly the same isolation rate with all the methods before as well as after the instillation of eye drops.

The biological activities of tuberculin PPD RT 23 and the International Standard for Purified Protein Derivative of Mammalian Tuberculin (PPD-M) were compared in sensitized and unsensitized guinea-pigs by skin tests and lymphocyte stimulation (LS) tests. Estimates of relative potency (RP) from skin test results were dependent on the dose level, on the immunogen used, and, in guinea-pigs immunized with killed tubercle bacilli in oil, also on the immunization time. Relative potency estimates from LS results were dependent on the source of the lymphocytes and were different from estimates obtained from skin tests. Lymphocyte stimulation dose-response curves for the tuberculins were qualitatively different. In contrast to RT 23, PPD-M gave rise to non-specific skin reaction in unsensitized guinea-pigs. Both tuberculins were mitogenic to lymph node lymphocytes isolated from unsensitized guinea-pigs, PPD-M being the more mitogenic of the two tuberculins. The present results confirm that qualitatively different tuberculins cannot be unambiguously calibrated in identical terms and thus emphasize that the uncritical use of (international) standards should be avoided in tuberculin calibration.