Pneumococcal antigen persistence in sputum from patients with community-acquired pneumonia.

The purpose of this study was to establish the diagnostic value of pneumococcal capsular antigen by comparing this with the results of Gram stain and culture in representative and nonrepresentative sputa during follow-up in patients with community-acquired pneumonia. Antigen was detected by a latex particle agglutination test. At the time of hospital admission, antigen was detected in 17 representative sputum specimens from 30 patients with pneumococcal pneumonia, which was comparable to the results of Gram stain and culture. In five additional patients, antigen was demonstrated in nonrepresentative specimens. During follow-up under antibiotic treatment, this number increased by six: three patients with representative and three patients with nonrepresentative sputum, respectively. Two of the 22 patients with pneumonia of other known cause had an antigen-positive sputum on admission and in another two patients, sputum antigen was detected during follow-up. Ten of 34 patients with pneumonia of unknown cause had detectable antigen in representative or nonrepresentative sputum on admission. During follow-up, antigen was detected in sputa of an additional seven patients. There was no difference in duration of antigen persistence between patients with pneumococcal pneumonia and pneumonia of unknown cause. It was observed that the first antigen-positive sputum specimen was always detected within the first five days of the hospital stay. We conclude that antigen detection in both representative and nonrepresentative sputum specimens at the time of hospital admission and during follow-up is of additional value for the diagnosis of pneumococcal pneumonia. It markedly increases the number of patients with pneumococcal pneumonia detected, who would otherwise be considered to have pneumonia of unknown cause. However, antigen-positive results should be interpreted carefully, especially in those pneumonia patients with chronic bronchitis, because detectable antigen may be caused by pneumococcal carriership of the lower respiratory tract.

Anti-HBc titers and anti-HBc immunoglobulin (M/G) classes in acute, chronic and resolved hepatitis B.

IgM-anti-HBc and IgG-anti-HBc serum titers were determined by indirect immunofluorescence in a prospective longitudinal study of 50 patients with hepatitis B, 43 of whom recovered completely. 37 of the recovered patients and all 7 non-recovering patients were followed up for a median of 5 years. Five of the non-recovering patients were followed up from the initial acute stage of the disease. IgM-anti-HBc was present in the acute stage in 39/43 of the recovery patients. The median maximal titer, 1:1000, was reached during the week before peak SGPT. It always disappeared in recovering patients within a median period of 5 weeks after peak SGPT. IgG-anti-HBc was present in all 43 recovering patients in the acute stage of disease with a median maximal titer of 1:1000, maintained for at least 10 weeks. After 5 years, 28 of 37 recovered patients were still IgG-anti-HBc positive with a median titer of 1:200. All non-recovering patients showed persistent IgM as well as IgG-anti-HBc positivity. In the acute stage the medians of the maximal titers were 1:100 for IgM-anti-HBc and 1:1000 for Igg-anti-HBc. After 5 years they were 1:100 for IgM and 1:10000 for IgG-anti-HBc. The presence of IgM-anti-HBc in a preceding study was considered to be a marker of hepatitis B virus replication. From this study no evidence can be obtained to support the view that the titer level of anti-HBc is reliable in the differentiation between infectious anti-HBc positive blood, as there was no difference (p = 0.4) between the number of patients with an anti-HBc level of 1:1000 after at least five years, who had recovered (9/28) and who had not recovered (3/7).

Inexpensive 4-hour micro-agar dilution susceptibility determination method.

Using a micro-agar dilution (MAD) method in which microscope slides are covered with a thin film of agar, and MICs are read microscopically after a 4-h incubation, 18 antibiotics were tested against 29 to 32 microorganisms each. Identical MICs were obtained for microscopic MAD MICs performed in duplicate in 87.1% of the antibiotic-microorganism combinations, and 97.9% were identical within one dilution. When read macroscopically after an 18-h incubation, identical duplicate MICs were obtained in 86.8% of the cases, and 98.4% were identical within one dilution. Using agar dilution as the "gold standard," the correlation obtained with MAD slides read microscopically at 4 h was 94.3%, and macroscopic correlation at 18 h was 97.6%. The correlation of MAD slides with agar dilution for the groups of microorganisms most frequently used was as follows (microscopic/macroscopic): Staphylococcus aureus 96%/98%; Streptococcaceae 97%/98%; Enterobacteriaceae 98%/99%; and Pseudomonadaceae 95%/98%. At the present rate of exchange (fl 1.60 = $1.00f1p4he cost of a MAD slide, including labor, is $1.28 (20 microorganisms tested) or $0.06 per microorganism-antibiotic combination tested. This method is easy to perform, rapid, and inexpensive. It is suitable for use in routine and research laboratories.

Demonstration of circulating pneumococcal immunoglobulin G immune complexes in patients with community-acquired pneumonia by means of an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay was developed for quantitation of circulating immune complexes (CICs) containing specific antipneumococcal immunoglobulin G (IgG). These CICs were detected in 17 (85%) of 20 patients with bacteremic pneumococcal pneumonia, 4 (36.4%) of 11 patients with probable pneumococcal pneumonia, 3 (16.7%) of 18 patients with pneumonia of other (nonpneumococcal) etiology, and 13 (41.9%) of 31 patients with pneumonia of unknown etiology. There was no correlation between CICs and serum IgG antibody levels. Pneumococcal capsular antigen was demonstrated in dissociated CICs by latex agglutination.

Pneumococcal antibody levels in patients with acute lung infiltrates.

We assessed the diagnostic value of serial serum antibody titers (IgG, IgM) to a polyvalent pneumococcal antigen preparation containing capsular polysaccharides from 14 different serotypes in the differential diagnosis between infectious lung infiltrates and lung infarction. A two-fold or higher change in antibody level, measured by means of an enzyme-linked immunosorbent assay (ELISA) was considered significant. Of 30 patients with pneumococcal pneumonia, 13 were infected with a Streptococcus pneumoniae serotype included in the vaccine (group A), five with a non-vaccine type (group B), and in 12 patients the S. pneumoniae serotype was not identified (group C). The sensitivity was 62% (group A). A heterotypic antibody rise was observed in one patient (group B). There was no difference in antibody rises between groups A and C. In 13 patients the pulmonary infiltrates were associated with different etiological factors (group D). The specificity determined in this group was 85%. The positive predictive value of an antibody rise was 89% (SD = 0.07) in pneumococcal pneumonia and a negative result signified in only 46% of the patients (SD = 0.10) that the pulmonary infiltrates were not associated with pneumococcal infection. Four patients suffering from pulmonary infarction had no antibody rise. Preliminary data of a current similar study, using a 23-valent antigen of pneumococcal capsular polysaccharides supported the aforementioned results. It is noteworthy that ten additional patients with lung infarction showed no seroconversion. The results suggest that serum antibody changes to a polyvalent pneumococcal vaccine may be of value in the differential diagnosis between infectious lung infiltrates and lung infarction.

Minimum number of pneumococci required for capsular antigen to be detectable by latex agglutination.

Forty-eight strains of Streptococcus pneumoniae were tested in vitro to determine the minimum number required for pneumococcal capsular antigen to be detectable by latex agglutination. It was found that 10(6) to 10(7) microorganisms per ml were needed and that antigen remained detectable even when viable pneumococci could no longer be demonstrated.

Immunoglobulin classes of smooth muscle antibody in the course of acute hepatitis B: prognostic significance.

A prospective longitudinal study was performed in 48 patients with acute hepatitis B (AHB) of whom 38 were previously healthy (PH) and 10 drug addicted (DA). Smooth muscle antibody was present in 23/38 PH and in 8/10 DA patients for a median of 4 weeks; other autoantibodies were not found. In the PH patients SMA was of IgM class in 23/23 and 8/23 of the IgG class as well. In the 8 DA patients 2 had IgM-SMA only, 3 (IgM+IgG)-SMA and 3IgG-SMA only. IgG-SMA presence could not be related to the duration or titer height of SMA nor to the type of fluorescence patterns. In SMA-negative patients IgM-anti-HBc was cleared within 6 weeks and in IgM-SMA positive patients within 32 weeks (medians 4 and 5 weeks) after maximal S-GPT. IgM-anti-HBc persisted for years in 3/3 IgG-SMA positive and in 2/11 IgG-IgM positive patients. In the remaining 9 IgG-IgM SMA positive patients it disappeared within 15 (median 9) weeks after maximal S-GPT. All 34 patients without SMA or with IgM-SMA only recovered completely. The 3 patients with IgG-SMA and 2 of the 11 patients with IgG+IgM SMA developed chronicity. Determination of SMA and of its immunoglobulin classes, at maximal SGPT may in acute hepatitis B be of help in predicting the outcome of disease.

A longitudinal study on the occurrence of autoantibodies in the course of acute hepatitis B.

The presence or absence of autoantibodies in acute hepatitis B was investigated longitudinally in a prospective study of 38 patients, 37 of whom recovered completely. Antibodies to nuclei, bile canaliculi or mitochondria were not found in any of the 354 investigated sera. Smooth muscle antibody (SMA) was present in 23 patients for median 4 weeks and from (median) -1 to +3 weeks from peak SGPT. Titers reached from 1:10 to 1:400, with a median of 1:50. In the patient with persistent HBs-antigenemia, SMA - present in low titer (1:10) - persisted as well. Besides smooth muscle cells, other localisations were: glomerular (15 patients), around hepatocytes ('polygonal' 11 pts), around renal tubuli (10) and in the gastric mucosal layer (8). These fluorescence patterns, the presence of which was not correlated to the SMA titer height, disappeared either earlier than or simultaneously with smooth muscle cellular fluorescence. A maximal titer greater than 1:50 was associated with a thymol turbidity ten times the upper limit of normal or more (i.e. greater than 25 S-H U) and with the peak serumgammaglobulin about simultaneously with peak SGPT. The presence of SMA was not correlated with extrahepatic manifestations nor with the peak values attained for ESR, SGPT, alkaline phosphatase or bilirubin.

Effect of washing sputum on detection of pneumococcal capsular antigen.

The effect of washing of sputum on detection of pneumococcal capsular antigen was investigated. A total of 357 sputa from 104 patients was tested. Antigen could be detected in 164 (46%) of the sputa in both the washed and unwashed portions, and could not be detected in either portion in a further 180 (50%) sputa. Four (1%) of the sputa agglutinated in the negative control, and were considered to be auto-agglutinating. In 9 (3%) sputa antigen could be detected in the unwashed portion, but not in the washed portion. There were no specimens in which antigen could be detected in the washed portion only. These data indicate that pneumococcal capsular antigen can be detected as reliably in washed sputum as in unwashed sputum.

Detection of pneumococcal capsular antigen in the presence of penicillin in vitro.

Eight strains of Streptococcus pneumoniae were tested in vitro for their ability to produce capsular antigen in the presence of penicillin. It was found that, provided 10(6) to 10(7) pneumococci/ml were present, capsular antigen could be detected during the 72 h in which the experiment was conducted, irrespective of whether penicillin was added at 0 h or 8 h, and even when no viable pneumococci remained. When fewer pneumococci were present, capsular antigen could not be detected at any time in the presence of penicillin. Control cultures, without penicillin, yielded detectable capsular antigen only when the threshold value of 10(6)-10(7) pneumococci/ml was reached. It is concluded that the presence of penicillin does not influence the detection of pneumococcal capsular antigen, but demonstration of this antigen is totally dependent on the number of pneumococci present.

The role of antigen detection in pneumococcal carriers: a comparison between cultures and capsular antigen detection in upper respiratory tract secretions.

During the winter season upper respiratory tract secretions from 166 patients with stable chronic obstructive pulmonary disease (COPD) or asthma were simultaneously cultured for Streptococcus pneumoniae and tested for pneumococcal capsular antigen. Latex agglutination was employed to investigate the effect of pneumococcal carriership on pneumococcal capsular antigen detection in upper respiratory tract secretions. All specimens originating from the oropharynx, nasopharynx and saliva were both cultured and investigated in parallel for the presence of antigen. The recovery of pneumococci from the different areas was unequally distributed (oropharynx 29%, nasopharynx 8%, and saliva 16%), with the highest isolation rate from the oropharynx alone. Only 4 (3%) of the oropharyngeal swabs, 1 (1%) of the nasopharyngeal swabs and 14 (9%) of the saliva specimens yielded both pneumococcal antigen and a positive culture for S. pneumoniae. A further 9 (6%) of the oropharyngeal swabs, 5 (3%) of the nasopharyngeal swabs, and 50 (33%) of the saliva specimens were antigen positive only, with no pneumococci isolated on culture. It is speculated that these reactions were due to cross-reacting microorganisms (especially alpha-haemolytic streptococci) present in saliva and contaminating the oropharynx and the nasopharynx. Quantitative cultures of 9 oropharyngeal swabs yielded S. pneumoniae in concentrations too low to be detectable by latex agglutination. The study indicates that there is a poor relation between pneumococcal colonization and antigen detection in the oropharynx and nasopharynx. Antigen present in these secretions is probably not an important disrupting factor by contamination when detecting pneumococcal antigen in washed sputum. The false positive antigen results in saliva are probably due to cross-reactions with alpha-haemolytic streptococci.

The detection of platelet isoantibodies by membrane immunofluorescence.

A membrane ummunofluorescence test for the detection of platelet isoantibodies is described. Gel filtration of the incubation mixture was incorporated in the procedure and proved effective for the removal of serum proteins from the platelet suspension. With this technique isoantibodies were found in the serum of 13 out of a group of 16 patients who had received multiple transfusions. The results were checked by measuring the uptake of 125I-labeled anti-IgG fraction by gel-filtered platelets. Subsequently the membrane immunofluorescence method was also compared with established techniques described for the detection of isoantibodies such as the microtest for lymphocytotoxicity and a complement-fixation method and the procedures based on the release of labeled serotonin, the phagocytosis of chromium-tagged platelets, the increase of platelet factor 3 activity, and on platelet aggregation. We had the opportunity to investigate the serum of one patient for the presence of isoantibodies against platelets from HLA identical siblings both before and after the administration of their platelets. On the basis of this experience it is concluded that the membrane immunofluorescence test for platelet isoantibodies is a relatively simple method with a high degree of specificity and adequate sensitivity.

In vitro studies on stability and development of metronidazole resistance in Helicobacter pylori.

Seventy isolates of Helicobacter pylori from antral biopsy samples were tested for their susceptibilities to metronidazole by agar dilution. Seven (10%) of these clinical isolates appeared to be resistant to metronidazole. Sixty-three strains were susceptible. In 42 (67%) of the 63 susceptible isolates, resistant isolates were obtained by serial passage on plates containing subinhibitory concentrations of metronidazole. In 10 of these 42 strains, the acquired resistance appeared to be unstable. The difference between the stability of resistance that occurred after one or two passages and the stability of resistance that occurred after three passages was statistically significant (P < 0.006). Primary resistance in clinical isolates was a stable phenomenon. Whether the resistance that emerges during therapy in patients is stable or unstable needs to be established.

Pneumococcal capsular antigen detection and pneumococcal serology in patients with community acquired pneumonia.

BACKGROUND: Methods to determine the microbial cause of community acquired pneumonia include detection of pneumococcal antigen and measurement of pneumococcal capsular antibody response. Their usefulness compared with conventional microbiological techniques was investigated in patients with pneumonia, some of whom had been treated with antibiotics. METHODS: Pneumococcal capsular antigen was detected by latex agglutination in sputum and the results compared prospectively with results of conventional microbiological techniques in 90 patients with community acquired pneumonia. Serum, urine, and pleural fluid samples were also tested for antigen. Serum pneumococcal capsular antibody titres were measured. RESULTS: A diagnosis was established by conventional microbiological techniques in 53 patients, 30 of whom had pneumococcal pneumonia. The sensitivity of antigen detection in first day sputum specimens (n = 18) in those with pneumococcal pneumonia was 94%; antigen was present in 23 of the 27 patients who produced representative sputum on admission and during follow up. The specificity of antigen detection in sputum in patients with non-pneumococcal pneumonia and lung infarction was 87%. Antigen was present in 12 of 25 patients with pneumonia of unknown aetiology who produced representative sputum. Antigen was rarely detected in serum and urine, but was present in pleural fluid in three of four patients with pneumococcal pneumonia and in all four patients with pneumonia of unknown aetiology. Pneumococcal antigen remained detectable in patients treated with antibiotics. Pneumococcal capsular antibody detection was as specific (85%) as antigen detection, but had a lower sensitivity (50%). CONCLUSION: Pneumococcal antigen detection in sputum or pleural fluid is of value in making a rapid diagnosis and provides an additional diagnostic result in patients with pneumococcal pneumonia, especially those receiving antibiotic treatment.

Faecal endotoxin and activity of the gut-associated lymphoid tissue in patients with malignant (B-cell) lymphoma.

On the basis of previously obtained evidence that Gram-negative bacteria may influence the activity of leukaemia, a study of the composition of the flora, the immune stimulation by the Gram-negative bacteria and the endotoxin concentration in faeces was conducted in patients with low-grade malignant B-cell lymphoma as well as in patients with acute leukaemia. In these patients it was investigated whether the number of facultative anaerobic Gram-negative bacteria in the faeces correlated with endotoxin concentration. In addition, IgA coating of Gram-negative bacteria in the faeces was determined and the titre of circulating antibodies to endogenous Enterobacteriaceae species from the faeces of the corresponding patient was studied. No clear difference was found to exist in the percentage of IgA-coated Gram-negative bacteria in the faeces and in the circulating antibody titres to endogenous Enterobacteriaceae between healthy control persons, lymphoma patients and patients with acute leukaemia. Antimicrobial treatment to establish selective decontamination (SD) of Enterobacteriaceae species from the digestive tract did cause a significant decrease in the faecal endotoxin concentration in a subset of patients treated for SD with polymyxin.