Immunodetection of pneumolysin in human urine by ELISA.

An ELISA test has been employed for the detection of pneumolysin (PLY) in urine from 14 pneumococcal pneumonia patients and from 11 healthy adult volunteers. The urines of all the 11 healthy adult volunteers developed signals around the mean of the blanks, whereas all the pneumococcal pneumonia patient urines rendered signals at least five times this mean. Chemiluminescent Western blot analyses of these urines, carried out with the PLY-specific rabbit polyclonal IgG preparation used in ELISA, were negative. The 30-kDa filtrates of three high-signal urines were ELISA negative, suggesting that this ELISA test mainly detected high molecular weight forms in urine rather than free PLY-derived antigenic fragments. The urine sample, which rendered the highest ELISA signal, was then concentrated by filtration through a 10-kDa filter. When this concentrate was subjected to Western blot with the ELISA-capture monoclonal antibody, a major band was developed. Its relative molecular mass was similar to that of recombinant PLY and its peptide mass fingerprinting showed peptides corresponding to amino acid stretches from the four domains of the PLY molecule. When the pool of PLY-negative urines was sham-contaminated with purified recombinant pneumolysin, a conspicuous matrix effect was observed; nevertheless, this ELISA test was still reproducible and highly sensitive, detecting pneumolysin in the order of picograms per milliliter. A comparison was also made between this PLY-ELISA and the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in analysing bacterial isolates. On the basis of the minimum number of pneumococci examined, both tests were shown to have similar potency, but strain-dependent discrepancies were observed. This ELISA could provide an alternative to the Binax NOW Streptococcus pneumoniae Urinary Antigen Test in the diagnosis of pneumococcal pneumonia.

[Community-acquired pneumonia: serum adenosine-deaminase activity in the aetiological diagnosis]. / Neumonía adquirida en la comunidad: adenosindesaminasa plasmática como marcador etiológico.

BACKGROUND: Adenosine deaminase (ADA) is a cytoplasmic enzyme which activity is increased in disorders that stimulate cells involved in the immune system. In community-acquired pneumonia (CAP), increased levels of serum ADA have been associated with the presence of atypical microorganisms as the source of the former. Previous studies have shown ADA increases in non-infectious diseases. We evaluated the factors that may influence plasmatic ADA (ADAp) levels in CAP patients. PATIENTS AND METHODS: A study with cases (245 episodes of CAP) and controls (49) was designed, and the differences in ADAp activity with regard to organisms, comorbidity factors and complications were analyzed. A logistic regression analysis was performed. RESULTS: CAP caused by atypical microorganisms were found to have increased ADAp values. Variables that independently increased ADAp levels were: atypical etiology (OR = 5.9), liver disease (OR = 5.8), diabetes mellitus (OR = 1.9), and prior antibiotic consumption (OR = 1.7). CONCLUSIONS: ADAp is an etiologic marker that could be useful in the empiric approach of the treatment of CAP.