Search

In support of the surveillance of pneumococcal infections in the era of conjugate vaccines, a sensitive and specific multiplex immunoassay using xMAP beads has been developed for direct detection of pneumococcal serotype-specific polysaccharides in clinical samples, particularly urine. The assay was tested on panels of spiked urine specimens, clinical urine specimens and bacterial isolates. Each of the 14 serotypes in the multiplex assay can be detected to 0.1 ng purified polysaccharide ml(-1), or less. Testing of a panel of urine specimens from patients with culture-confirmed pneumococcal or non-pneumococcal disease indicated that the multiplex assay is both sensitive and specific. The correct pneumococcal serotype was identified directly from urine in 46/58 (79.3 %) patients who had a contemporaneous blood culture isolate of a multiplex assay serotype. Furthermore, the specificity of the assay on this panel of samples was 99.3 % (145/146). This multiplex assay could be useful, in conjunction with the pneumococcal screening test Binax NOW, in urine for diagnosis of pneumococcal disease and the identification of the aetiological serotype, and potentially be of benefit in culture-negative patients.

The diagnosis of severe Streptococcus pneumoniae infection relies heavily on insensitive culture techniques. To improve the usefulness of PCR assays, we developed a dual-PCR protocol (targeted at pneumolysin and autolysin) for EDTA blood samples. This was compared to the Binax NOW S. pneumoniae urine antigen test in patients with bacteremic pneumococcal infections. Patients with nonbacteremic community-acquired pneumonia also were tested by these methods to determine what proportion could be confirmed as pneumococcal infections. A direct comparison was made in a group of patients who each had both tests performed. The Binax NOW S. pneumoniae urine antigen test was positive in 51 of 58 bacteremic pneumococcal cases (sensitivity, 88%; 95% confidence interval [CI], 77 to 95%), whereas the dual PCR was positive in 31 cases (sensitivity, 53.5%; 95% CI, 40 to 67%; P < 0.0001), and all of these had detectable urinary antigens. Both tests gave positive results in 2 of 51 control patients (referred to as other-organism septicemia), giving a specificity of 96% (95% CI, 86.5 to 99.5%). In 77 patients with nonbacteremic community-acquired pneumonia, urinary antigen was detected significantly more often (in 21 patients [27%]) than a positive result by the dual-PCR protocol (6 [8%]) (P = 0.002). The development of a dual-PCR protocol enhanced the sensitivity compared to that of the individual assays, but it is still significantly less sensitive than the Binax NOW urine antigen test, as well as being more time-consuming and expensive. Urinary antigen detection is the nonculture diagnostic method of choice for patients with possible severe pneumococcal infection.

The clinical utility of Legionella urinary antigen assays for the diagnosis of Legionnaires' disease was assessed by using samples from 317 culture-proven cases. The sensitivities of the Binax enzyme immunoassay (EIA) and Biotest EIA were found to be 93.7 and 94.4% for travel-associated infection and 86.5 and 76.0% for community-acquired infection but only 44.2 and 45.7% for nosocomially acquired infection, respectively.

The Bartels Legionella Urinary Antigen enzyme immunoassay (Intracel, USA) is intended for the presumptive diagnosis of past or current Legionnaires' disease by qualitative detection of Legionella pneumophila serogroup 1 antigen in human urine. This test was evaluated using single urine samples collected from 349 patients with lower respiratory tract infection of known aetiology. Specificity was estimated as 100% (181 samples, 95% CI: 98%-100%); sensitivity for Legionella pneumophila serogroup 1 was 98.8% (167 samples, 95% CI: 95.7%-99.9%). Assessing assay results using a Visual Interpretation Card provided by the manufacturer in place of a photometer gave rise to one false-positive result among the 78 control samples examined. Providing the endpoint of this assay is determined photometrically, the Bartels Legionella Urinary Antigen enzyme immunoassay appears to be a highly specific and sensitive kit for the diagnosis of infection caused by Legionella pneumophila serogroup 1.

The new BinaxNOW Immunochromatographic (ICT) Assay for the detection of Legionella pneumophila antigens was used to test 535 urine specimens from patients with and without Legionnaires' disease. The specificity, calculated by testing 112 samples from patients with pneumonia of aetiologies other than Legionella infection, and 167 urine specimens from urinary tract infections, was found to be 97.1% if the manufacturer's guidelines were followed. However, it was determined that the 'false positive' results characterised by very weak bands could be discounted by re-examination of the results at 60 min, yielding a specificity of 100%. With this minor modification of the procedure applied to examination of urine samples from 117 patients with legionellosis confirmed by isolation of L. pneumophila and 70 patients who had seroconverted to L. pneumophila serogroup 1, sensitivity was calculated to be 79.7%. In comparison, the sensitivities of the Binax Urinary Antigen Enzyme Immunoassay (EIA) and Biotest Urin Antigen EIA were estimated to be 79.1 and 83.4%, respectively. Eleven cases (5.9%) were positive by BinaxNOW assay but negative by Binax or Biotest EIA, or both. The sensitivities of all assays increased to c. 94% if only diagnosis of cases confirmed by isolation of serogroup 1 L. pneumophila was considered, although the sensitivity for infections caused by L. pneumophila serogroup 1 monoclonal antibody (MAb) subgroup Bellingham was significantly lower than for other MAb subgroups. The Biotest EIA recognised 10 (45%) of the 22 cases not caused by L. pneumophila serogroup 1, whereas the two Binax kits detected only three each. The ICT assay BinaxNOW can be recommended as a rapid specific test for the diagnosis of Legionnaires' diseases caused by L. pneumophila serogroup 1, although very weak bands should be interpreted cautiously.

The relationship between serogroup and genotype of Legionella pneumophila strains was investigated by restriction fragment length polymorphism (RFLP) typing with a previously standardised method. Of the 51 RFLP types identified, 19 comprised strains of more than one serogroup. Several RFLP types included strains of five or more serogroups. To determine if sharing the same RFLP type indicates that strains are genotypically indistinguishable or merely that they are superficially similar, 31 strains were selected for further analysis with an extended range of restriction endonucleases and nucleic acid probes. In some cases, strains of a particular RFLP type were indistinguishable, while in others the restriction fragment patterns showed minor differences. It is possible that in the latter case the strains are diverging representatives of a parent clone. We conclude that analysis of restriction fragment patterns, either probed or unprobed, provides a more accurate measure of the ancestral relationship between strains than can be obtained with serological methods.

The enzyme linked immunosorbent assay (ELISA) described was developed to detect a soluble antigen in the urine of patients with Legionnaires' disease caused by Legionella pneumophila serogroup 1 (L.pn 1). The assay was evaluated and showed good specificity (100%) and intra-assay reproducibility. Antigen was detected in the urine of 93 (77%) of 120 patients, overall, and in 86% of patients from whom a specimen obtained within seven days of onset of illness was available. On all but one occasion the first urine sample taken from a patient for whom a positive ELISA result was obtained, was itself positive. In one case antigen was not detected at four days but was present on the fifth day after onset of symptoms. In two patients urinary antigen was detectable as early as two days after onset of symptoms. In another the antigen persisted for at least 60 days. More than half the patients, however, had stopped producing detectable antigen within 14 days of onset of symptoms. It is therefore important that where Legionnaires' disease is suspected urine is collected as early as possible in the course of the disease.

An enhanced chemiluminescence enzyme-linked immunosorbent assay has been developed for the detection of soluble antigen in the urine of patients with Legionnaires' disease (LD). In the assay antigen(s) in the urine samples are captured by a rabbit anti-L. pneumophila antibody coated onto microtitre strips. A fluorescein-isothiocyanate (FITC) conjugate of the same antibody is then added which binds to the captured antigen. Any immobilized FITC-labelled antibody is then detected with a horseradish peroxidase (HRP) conjugate of a monoclonal anti-FITC antibody. HRP activity is monitored after oxidation of luminol in the presence of H2O2 and iodophenol. The resulting luminescence is recorded using a camera luminometer. Urine specimens were available for testing from 31 patients with evidence of ongoing L. pneumophila serogroup 1 infection. A positive result was obtained in the cases of 12/12 specimens from culture-proven LD patients, and 16/19 specimens from patients with serological evidence of LD. Thus the sensitivity is estimated to be 28/31 (90%). The specificity was estimated using urine specimens from eight patients with non-L. pneumophila pneumonias of known aetiology. All eight specimens gave a negative result.

Formolised yolk sac antigens of Legionella pneumophila serogroups 1-6 were used to test 1792 serum specimens from 1431 patients with respiratory illness of serological evidence of Legionnaires' disease (LD). Thirty-five patients showed titres against the serogroup 1 antigen diagnostic for LD. Only two further cases were considered to have non-serogroup I infections (both serogroup 4) indicating that such infections are rare. Titres of greater than 1/16 against the serogroup 1 antigen occur in only 3% of subjects without LD and thus the demonstration of such a titre in patients with pneumonia during the early phase of illness can alert the clinician to the likelihood of LD. The supply of serogroup 1 antigen from the Division of Microbiological Reagents and Quality Control to routine diagnostic laboratories will be continued and monovalent serogroup 2-6 antigens will continue to be made available to reference laboratories.

The indirect fluorescent antibody (FA) technique for the diagnosis of Legionnaires' disease was used to investigate an outbreak of respiratory disease in a military population. The outbreak was later shown to be caused by an adenovirus. High titres were obtained using the ether-killed antigen supplied by the Center for Disease Control (CDC), Atlanta, but not with a formolised yolk-sac antigen prepared in out laboratory. The reactivity of these sera with the CDC antigen was removed by absorption with a partly identified gram-positive bacterium, whereas sera from persons with true Legionnaires' disease were unaffected by such treatment, suggesting that such reactivity is not specific for Legionnaires' disease. The lack of reactivity with negative control sera and strong reactivity from a small group of patients in whom seroconversion was demonstrated has led us to conclude that formolised yolk-sac antigen is a reliable antigen for the diagnosis of Legionnaires' disease by the FA method. Measurement of the sensitivity and specificity of the formolised yolk-sac antigen is still in progress.