Serum antibodies to purified pneumolysin were determined by enzyme-linked immunosorbent assay (ELISA) in paired samples from 406 adult patients with community-acquired pneumonia and in samples from 184 healthy controls. A high sensitivity (83%) was obtained in patients with blood culture-confirmed pneumococcal pneumonia. In patients with a tentative pneumococcal diagnosis based on culture of samples from the sputum or the nasopharynx, 45% were positive by ELISA. The difference likely reflected the different relevance of cultural findings for the diagnosis of pneumococcal pneumonia. A significant rise in ELISA titer was found in 17% of the patients. When the diagnosis was also based on high titers, 25% were positive. Pneumococcal pneumonia diagnosed by the pneumolysin ELISA was significantly more common in the patients with a more severe disease and who required hospitalization (21 versus 5% for outpatients). Younger patients were more often positive for pneumococci as determined by high titers, while older patients showed titer rises. Mixed infections with other infectious agents were not uncommon. The finding of low titers in acute-phase samples from positive patients and in the youngest and oldest age groups of healthy controls were unexpected, indicating that further studies on the role of pneumolysin in pneumococcal disease are warranted.
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.
A pneumolysin-specific enzyme-linked immunosorbent assay (PLY-ELISA) for the detection of pneumolysin in urine was developed and evaluated in comparison with the commercially available Binax Now Streptococcus pneumoniae test (Binax, Portland, ME) for the diagnosis of pneumococcal infections. Assay sensitivity was evaluated using urine from 108 patients with culture-confirmed pneumococcal infections. In adults, the sensitivity and specificity of the PLY-ELISA were 56.6% and 92.2%, respectively. In children with nasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 62.5% and 87.5%, respectively, while specificities were 94.4% and 27.8%, respectively. In children with nonnasopharyngeal pneumococcal carriage, PLY-ELISA and Binax Now S. pneumoniae test sensitivities were 68.7% and 93.7%, respectively, and test specificities were 94.1% and 41.2%, respectively. The persistence of pneumolysin in urine of pneumococcal pneumonia patients decreased significantly after 4 to 6 days of treatment. Our data suggest that combining the high specificity of the PLY-ELISA with the high sensitivity of the Binax Now S. pneumoniae test would enable pneumococcal infections to be accurately diagnosed in children.
Blood-based pneumolysin PCR was compared to blood culture and detection of pneumolysin immune complexes, as well as to detection of antibodies to pneumolysin and to C polysaccharide, in the diagnosis of pneumococcal infection in 75 febrile children. Invasive pneumococcal infection was suspected on clinical grounds in 67 of the febrile children, and viral infection was suspected on clinical grounds in 8 of the febrile children. In addition, 15 healthy persons were examined to test the specificity of the PCR assay. Plasma, serum, and leukocyte fractions were analyzed by PCR. The combination of all test results led to the diagnosis of pneumococcal infection in 25 patients. Pneumolysin PCR was positive in 44% of these children, an increase occurred in the pneumolysin antibodies in 39% and in the C polysaccharide antibodies in 30% of the patients; pneumolysin immune complexes were found in convalescent serum in 30%, pneumolysin immune complexes occurred in acute-phase serum samples in 16%, and a positive blood culture was found in 20% of the patients. None of the healthy controls had positive results by PCR. The results suggest that the diagnosis of Streptococcus pneumoniae infection from blood samples necessitates the use of several different assays. Pneumolysin PCR was the most sensitive assay, but its clinical value is reduced by the fact that three blood fractions are needed.
Streptococcus pneumoniae is the primary etiological agent of community-acquired pneumonia and a major cause of meningitis and bacteremia. Three conserved pneumococcal proteins—pneumolysin, pneumococcal surface adhesin A (PsaA), and pneumococcal surface protein A (PspA)—are currently being investigated as vaccine candidates. Such protein-based vaccines, if proven effective, could provide a cheaper alternative to conjugate vaccine formulae. Few data from sub-Saharan Africa exist concerning the development of natural antibody to these antigens, however. To investigate the age-specific development of antiprotein immunoglobulin G (IgG) and IgA antibody responses, the sera of 220 persons 2 weeks to 84 years of age from coastal Kenya were assayed using enzyme-linked immunosorbent assays. IgG and IgA antibody responses to each antigen were observed in all age groups. Serum concentrations of IgG and IgA antibody responses to PspA and PdB (a recombinant toxoid derivative of pneumolysin), but not to PsaA, increased significantly with age (P < 0.001). No decline was observed in the sera of the elderly. Anti-protein IgG concentrations were only weakly correlated (0.30 < r < 0.56; P < 0.0001), as were IgA concentrations (0.24 < r < 0.54; P < 0.0001).
We have studied etiological diagnosis of acute otitis media (AOM) by comparing a newly developed pneumococcal PCR for Streptococcus pneumoniae to bacterial culture with 180 middle ear fluid (MEF) samples of 125 children with 125 episodes of AOM. For pneumococcal PCR assay, DNA from MEF samples was extracted by phenol-chloroform. The outer primers used amplified a 348-bp region of the pneumolysin gene, and the inner primers amplified a 208-bp region. S. pneumoniae was cultured in 33 (18%) samples, and pneumolysin PCR was positive for 51 (28%) of 180 MEF samples. Only 2 of 21 PCR-positive, S. pneumoniae culture-negative samples were positive for other otitis pathogens. By combining MEF culture and PCR results, 54 (30%) of 180 MEF samples had evidence of pneumococcal etiology. In conclusion, pneumolysin PCR is a sensitive and specific new method to study pneumococcal involvement in MEF samples of children with AOM.
Strep. pneumoniae was diagnosed as the cause of pneumonia in 40 (50%) of 80 consecutive adults admitted to St Stephen's Hospital with community-acquired pneumonia. None of the patients had evidence of Mycoplasma pneumoniae infection, and Legionella pneumophila serology was positive on only one occasion. In 29 patients (36%) no causative organism was demonstrated. The diagnosis of pneumococcal infection was obtained in 15 cases by isolating Strep. pneumoniae from the sputum, in 13 further cases by demonstrating pneumococcal capsular antigen in sputum, and in 12 other cases by detecting pneumococcal antigen in serum only. Only 2 cases with pneumococcal pneumonia were bacteraemic and 3 patients (7%), all aged more than 75 years, died. The relatively low bacteraemic and mortality rates suggest that community-acquired pneumococcal pneumonia currently seen in patients admitted to hospital in central London may not be so severe as in some other areas.
The serum antibody responses of splenectomized patients with non-Hodgkin's lymphoma (NHL) who had been immunized with a polyvalent pneumococcal vaccine (Pneumovax 23) were evaluated by an enzyme-linked immunosorbent assay with the 23-valent pneumococcal vaccine as the antigen. A response to immunization, defined as a twofold-or-higher rise of the prevaccination titer of antibodies against Streptococcus pneumoniae polysaccharide, was elicited in 5 of 11 patients with NHL. No significant difference in the level of antibodies against S. pneumoniae polysaccharide between lymphoma patients and patients who had undergone splenectomy for other reasons was detected (P = 0.83 and 0.87 before and after vaccination, respectively). NHL patients who did not respond to the first immunization received a booster dose of the polysaccharide vaccine. This injection did not increase the pneumococcal-antibody titer significantly (P = 0.7). We conclude that vaccination with pneumococcal polysaccharides in splenectomized patients with NHL elicits an adequate antibody response in 45.4% of the cases and should therefore be administered. Revaccination of the nonresponders does not further increase the pneumococcal-antibody levels.
Pneumococcal surface adhesin A (PsaA) is a 37-kDa common protein antigen of Streptococcus pneumoniae. In the present study, the protein was purified so that its immunoreactivity could be determined. PsaA was released and purified from cells by lysis in the presence of n-laurylsarcosine; this was followed by ammonium sulfate precipitation and subsequent preparative isoelectric focusing. A capture antibody enzyme-linked immunosorbent assay was used to determine the immunoreactivity of purified PsaA. The assay had a 67% sensitivity for sera from patients with bacteremic pneumococcal pneumonia. A specificity of 97% was estimated on the basis of a lack of reactivity with sera from patients with pneumonia caused by other organisms. PsaA is a potential vaccine candidate and may be useful as an antigen in a diagnostic assay for pneumococcal disease.
We have detected a cholesterol-dependent cytolysin, which we have named mitilysin, in a small number of Streptococcus mitis isolates. We have sequenced the mitilysin gene from seven isolates of S. mitis. Comparisons with the pneumococcal pneumolysin gene show 15 amino acid substitutions. S. mitis appear to release mitilysin extracellularly. Certain alleles of mitilysin are not recognized by a monoclonal antibody raised to the related toxin pneumolysin. Based on enzyme-linked immunosorbent assay and neutralization assay results, one isolate of S. mitis may produce a further hemolytic toxin in addition to mitilysin. As genetic exchange is known to occur between S. mitis and Streptococcus pneumoniae, this finding may have implications for the development of vaccines or therapies for pneumococcal disease that are based on pneumolysin.
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.
BACKGROUND--Transthoracic needle aspiration (TNA) with an ultrathin needle is a safe and highly specific procedure for obtaining a diagnosis in bacterial pneumonias, but its sensitivity is at best 70%. A study was performed to assess whether Streptococcus pneumoniae and Haemophilus influenzae type b antigen detection by latex agglutination from the TNA sample enhanced the diagnostic yield. METHODS--Blood cultures, TNA with an ultrathin needle (culture, Gram stain, and latex agglutination), serological tests, and pneumococcal antigen detection in the urine by counterimmunoelectrophoresis were performed in samples from 18 of 23 consecutive patients with severe community acquired pneumonia. RESULTS--The causative organism was identified in 16 cases (88%): S pneumoniae (10 cases), S pneumoniae plus H influenzae (two cases), Legionella pneumophila (three cases), and Mycoplasma pneumoniae (one case). The investigation of antigens by latex agglutination in the pulmonary aspirate increased the diagnostic yield of TNA from 50% to 78% and provided a rapid diagnosis (in less than two hours) with therapeutic implications in seven cases. Its effectiveness was not modified by prior antibiotic therapy. CONCLUSIONS--A latex agglutination test on the pulmonary aspirate enhances the diagnostic yield of TNA in severe community acquired pneumonia.
The pneumococcal C polysaccharide (PnC) is species specific and believed to be a cell wall component of all pneumococcal types. A sandwich enzyme-linked immunosorbent assay (ELISA) for detection of PnC in sputa has been developed by using a monoclonal antiphosphorylcholine antibody and a polyclonal rabbit anti-PnC antiserum in the test system. A 1-year study of adult hospitalized patients with community-acquired pneumonia was performed. A total of 147 patients with clinical and radiological evidence for pneumonia were accepted for the study. Of these, 105 patients provided a sputum sample upon admission to the ward. The sputa were cultured semiquantitatively as well as tested for the presence of antigen. Of the sputum samples from patients with Streptococcus pneumoniae, 27 of 33 (accounting for a sensitivity of 82%) were positive in the ELISA test. Of the sputum samples from patients with pneumonia of some other known or suspected etiology, 32 of 34 (accounting for a specificity of 94%) were negative. In addition, 7 sputum samples from 31 patients with pneumonia of unknown etiology were positive. The ELISA test described here is in our opinion a sensitive and specific test for detecting PnC from S. pneumoniae in sputa from patients with untreated pneumonia.
Serum samples from patients with documented influenza A virus infections were examined for antibodies to Legionella pneumophila and Mycoplasma pneumoniae to determine whether simultaneous or sequential infections with L. pneumophila and M. pneumoniae were complicating factors in influenza. When the frequency of copositivity of sera to influenza A virus and L. pneumophila was compared with the expected frequency for each infection alone, the difference was not statistically significant. However, when the frequency of copositivity of sera to influenza A virus and M. pneumoniae was compared with the expected frequency for each infection alone, there was a statistically significant (P less than 0.005) absence of coincident titers. Seasonal variations and differences in relative age frequencies for the two infections may partially explain the absence of coinfections. These data also suggest that in patients with either M. pneumoniae or influenza A virus infection, some type of protective mechanism which prevents coinfections with these organisms is present.
We have recently shown that a substantial proportion of antibody to pneumococcal polysaccharide as measured by enzyme-linked immunosorbent assay (ELISA) or radioimmunoassay is removed by adsorption with pneumococcal cell wall polysaccharide (CWPS). The present study was undertaken to validate the hypothesis that only serotype-specific antibody that remains after adsorption with CWPS provides protection against pneumococcal infection. Serum samples were obtained from human subjects before and after they had been vaccinated with pneumococcal polysaccharide vaccine. Antibody to Streptococcus pneumoniae serotype 4 was measured by ELISA without adsorption or after adsorption of serum with CWPS. Groups of mice were injected with graded doses of serum and then challenged intraperitoneally with 10, 100, or 1,000 50% lethal doses (LD50) of S. pneumoniae serotype 4. Without adsorption, prevaccination sera from five healthy adults appeared to contain up to 33 micrograms of antibody to S. pneumoniae serotype 4 antigen per ml; adsorption with CWPS removed all detectable antibody, and pretreating mice with up to 0.1 ml of these sera (less than or equal to 3.3 micrograms of antibody) failed to protect them against challenge with 100 LD50. In contrast, postvaccination sera contained 2.9 to 30 micrograms of antibody per ml that was not removed by adsorption. Diluting sera to administer desired amounts of serotype-specific immunoglobulin G showed a significant relationship between protection and antibody remaining after adsorption (P less than 0.05 by linear regression analysis); 150 ng was uniformly protective against 1,000 LD50, and 50 ng was protective against 100 LD50. These studies have, for the first time, quantitated the amount of serotype-specific antibody that protects mice against challenge with S. pneumoniae type 4. In light of these observations, it is necessary to reassess current concepts regarding the presence of antipneumococcal antibody in the unvaccinated population, responses to pneumococcal vaccination, and protective levels of immunoglobulin G.
The pneumococcal C polysaccharide (PnC) is species specific and believed to be a cell wall component of all capsular types. Antibodies against PnC in human sera have been demonstrated previously, but the question of whether a rise in these antibodies occurs during pneumococcal infections has not been investigated. We used an indirect enzyme-linked immunosorbent assay (ELISA) for the estimation of PnC antibodies in 124 hospital-treated patients with pneumonia. In 3 of 6 patients with pneumococcal bacteremia and in 17 of 44 patients with S. pneumoniae isolated in the blood, sputum, or nasopharynx, a significant rise in antibody levels was recorded, accounting for a sensitivity of 38.6%. Of 35 patients with pneumonia of other known or suspected etiology, 1 gave a positive result, corresponding to a specificity of 97.1%. In addition, 3 of 8 patients with PnC antigen in the sputum as the only etiological finding and 5 of 37 patients with unknown etiology gave positive results. The PnC antibodies did not seem to have any protective capacity against pneumonia caused by pneumococci. The ELISA, in which only one antigen preparation was used, was more simple than other tests in which traditional capsular antigen preparations are used. It might therefore be used as a supplemental method in the diagnosis of pneumococcal pneumonia. The problems involved in expressing serum titers obtained with the ELISA are discussed.
The most common infections in primary immune deficiency disease (PIDD) patients involve encapsulated bacteria, mainly Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (pneumococcus). Thus, it is important to know the titers of Hib- and pneumococcus-specific antibodies that are present in immune globulin (Ig) intravenous (IGIV) preparations used to treat PIDD. In this study, seven IGIV preparations were tested by enzyme-linked immunosorbent assay and opsonophagocytic activity for antibody titers to the capsular polysaccharides of Hib and five pneumococcal serotypes. Differences in Hib- and pneumococcus-specific antibody titer were observed among various IGIV preparations, with some products having higher- or lower-than-average titers. Opsonic activity also varied among preparations. As expected, IgG2 was the most active subclass of both binding and opsonic activity except against pneumococcal serotype 6B where IgG3 was the most active. This study determines antibody titers against capsular polysaccharides of Hib and pneumococcus in seven IGIV products that have been shown to be effective in reducing infections in PIDD patients. As donor antibody levels and manufacturing methods continue to change, it may prove useful from a regulatory point of view to reassess IGIV products periodically, to ensure that products maintain antibody levels that are important for the health of IGIV recipients.
Streptococcus pneumoniae is the most common cause of community-acquired pneumonia, but it is undoubtedly underdiagnosed. We used a nested PCR assay (targeting the pneumolysin gene) to detect S. pneumoniae DNA in multiple sample types from 474 adults with community-acquired pneumonia and 183 control patients who did not have pneumonia. Plasma or buffy coat samples were PCR positive in only 6 of the 21 patients with positive blood cultures for S. pneumoniae and in 12 other patients (4 of whom had no other laboratory evidence of S. pneumoniae infection). Buffy coat samples from two control patients (neither having evidence of S. pneumoniae infection), but no control plasma samples, were PCR positive. Although pneumococcal antigen was detected in the urine from 120 of 420 (29%) patients, only 4 of 227 (2%) urine samples tested were PCR positive. Overall, 256 of 318 (81%) patients had PCR-positive sputum samples, including 58 of 59 samples from which S. pneumoniae was cultured. Throat swab samples from 229 of 417 (55%) patients were PCR positive and, in those who produced sputum, 96% also had positive PCR results from sputum. Throat swabs from 73 of 126 (58%) control patients were also PCR positive. We conclude that the pneumolysin PCR assay adds little to existing diagnostic tests for S. pneumoniae and is unable to distinguish colonization from infection when respiratory samples are tested.
Sera from six outbreaks of legionellosis and four outbreaks of pneumonia of other etiologies were tested with the indirect immunofluorescence assay (IFA) as currently performed. The current IFA is at least as sensitive as the original test in detecting cases of Legionnaires disease (78 to 91%). By using Center for Disease Control criteria for a positive (fourfold increase in titer during convalescence to greater than or equal to 128) or presumptive (single titer greater than or equal to 256) serological test, the specificity exceeded 99%. No cross-reactions against Legionella pneumophila antigens were observed among sera from epidemic cases of Q fever, tularemia, and psittacosis; the only positive L. pneumophila IFA titer among the epidemic Mycoplasma pneumonia sera was reduced to a negative titer with an immunosorbent extracted from Escherichia coli strain O13:K92:H4. The slight increase in specificity (to 100%), however, was offset by a slight decrease in sensitivity. The sensitivity of the IFA was maximal when a conjugate that detected immunoglobulins G, M, and A was used. IFA titers were not significantly altered by replacing the monovalent serogroup 1 antigen with a polyvalent antigen (serogroups 1 through 4) nor by the presence of rheumatoid factor or heat-labile serum factors.
To improve the clinical diagnosis of pneumococcal infection in bacteremic and nonbacteremic community-acquired pneumonia (CAP), a Luminex technology-based multiplex urinary antigen detection (UAD) diagnostic assay was developed and validated. The UAD assay can simultaneously detect 13 different serotypes of Streptococcus pneumoniae by capturing serotype-specific S. pneumoniae polysaccharides (PnPSs) secreted in human urine. Assay specificity is achieved by capturing the polysaccharides with serotype-specific monoclonal antibodies (MAbs) on spectrally unique microspheres. Positivity for each serotype was based on positivity cutoff values calculated from a standard curve run on each assay plate together with positive- and negative-control urine samples. The assay is highly specific, since significant signals are detected only when each PnPS was paired with its homologous MAb-coated microspheres. Validation experiments demonstrated excellent accuracy and precision. The UAD assay and corresponding positivity cutoff values were clinically validated by assessing 776 urine specimens obtained from patients with X-ray-confirmed CAP. The UAD assay demonstrated 97% sensitivity and 100% specificity using samples obtained from patients with bacteremic, blood culture-positive CAP. Importantly, the UAD assay identified Streptococcus pneumoniae (13 serotypes) in a proportion of individuals with nonbacteremic CAP, a patient population for which the pneumococcal etiology of CAP was previously difficult to assess. Therefore, the UAD assay provides a specific, noninvasive, sensitive, and reproducible tool to support vaccine efficacy as well as epidemiological evaluation of pneumococcal disease, including CAP, in adults.
Antibodies against Mycoplasma pneumoniae antigen obtained by Tween-ether treatment from purified M. pneumoniae were measured by means of enzyme-linked immunosorbent assay (ELISA). Paired sera from 19 patients with pneumonia and from 13 patients with acute pancreatitis with a significant rise in complement fixing antibodies against M. pneumoniae were studied. Single sera from healthy 1-year-old children were used as controls. High levels of IgG and IgM class antibodies were seen in sera from patients with pneumonia while most patients with acute pancreatitis and all the children showed low levels of antibodies. The results indicate that ELISA using Tween-ether treated M. pneumoniae antigen could be used successfully in the specific laboratory diagnosis of M. pneumoniae infection.
Streptococcus pneumoniae is a common worldwide potential pathogen causing pneumonia among children and the detection of pneumococcal infections by conventional culturing techniques is cumbersome. The present study describes a comparative analysis of sensitive nested-PCR and bacterial culture in pediatric patients with clinical and radiological indication of S. pneumoniae infection.
PCR was performed using outer primers to amplify a 348-bp region and inner primers a 208-bp region of the pneumolysin gene. For pneumolysin PCR assay, DNA from peripheral blood and middle ear fluid (MEF) samples was extracted by salting out method. The sensitivity of the assay was evaluated with about 0.06 pg of purified S. pneumoniae genomic DNA.
Among 90 MEF culture negative samples from acute otitis media pediatric patients, 8.8% pneumolysin-PCR positivity was detected, demonstrating the sensitivity and reliability of PCR for rapid pneumonia evaluation. Binomial test of proportionality performed on (SPSS 17) gives P< 0.05 indicating that PCR technique is statistically significant and sensitive in the diagnosis of S. pneumoniae infection.
The research work evaluated the effectiveness and efficacy of nested-PCR for detecting S. pneumoniae in pediatric patients with clinical and radiological confirmation of bacterial infection. This simplified method permitted quick selection of the patients and played a significant role in preliminary management of pneumococcal infections.
Streptococcus pneumoniae; PCR; Screening; Pneumolysin; Gene; Pneumococcal Infections
Streptococcus pneumoniae is one of the most common etiologic agents of community-acquired pneumonia, particularly bacteremic pneumonia. Pneumolysin, a multifunctional cytotoxin, is a putative virulence factor for S. pneumoniae; however, a direct role for pneumolysin in the early pathogenesis of pneumococcal pneumonia has not been confirmed in vivo. We compared the growth of a pneumolysin-deficient (PLY[-]) type 2 S. pneumoniae strain with its isogenic wild-type strain (PLY[+]) after direct endotracheal instillation of bacteria into murine lungs. Compared with PLY(-) bacteria, infection with PLY(+) bacteria produced greater injury to the alveolar-capillary barrier, as assayed by albumin concentrations in alveolar lavage, and substantially greater numbers of PLY(+) bacteria were recovered in alveolar lavages and lung homogenates at 3 and 6 h after infection. The presence of pneumolysin also contributed to the development of bacteremia, which was detected at 3 h after intratracheal instillation of PLY(+) bacteria. The direct effects of pneumolysin on lung injury and on the ability of pneumococci to evade local lung defenses was confirmed by addition of purified recombinant pneumolysin to inocula of PLY(-) pneumococci, which promoted growth of PLY(-) bacteria in the lung to levels comparable to those seen with the PLY(+) strain. We further demonstrated the contributions of both the cytolytic and the complement-activating properties of pneumolysin on enhanced bacterial growth in murine lungs using genetically modified pneumolysin congeners and genetically complement-deficient mice. Thus, pneumolysin facilitates intraalveolar replication of pneumococci, penetration of bacteria from alveoli into the interstitium of the lung, and dissemination of pneumococci into the bloodstream during experimental pneumonia. Moreover, both the cytotoxic and the complement-activating activities of pneumolysin may contribute independently to the acute pulmonary injury and the high rates of bacteremia which characterize pneumococcal pneumonia.
Recently, there has been an increase in invasive pneumococcal disease (IPD) caused by serotype 1 Streptococcus pneumoniae throughout Europe. Serotype 1 IPD is associated with bacteremia and pneumonia in Europe and North America, especially in neonates, and is ranked among the top five most prevalent pneumococcal serotypes in at least 10 countries. The currently licensed pediatric pneumococcal vaccine does not afford protection to this serotype. Upon screening of 252 clinical isolates of S. pneumoniae, we discovered mutations in the pneumolysin gene of two out of the four serotype 1 strains present in the study group. Analysis of an additional 28 serotype 1 isolates from patients with IPD from various Scottish Health Boards, revealed that >50% had mutations in their pneumolysin genes. This resulted in the expression of nonhemolytic forms of pneumolysin. All of the strains producing nonhemolytic pneumolysin were sequence type 306 (ST306), whereas those producing “wild-type” pneumolysin were ST227. The mutations were in a region of pneumolysin involved in pore formation. These mutations can be made in vitro to give the nonhemolytic phenotype. Pneumolysin is generally conserved throughout all serotypes of S. pneumoniae and is essential for full invasive disease; however, it appears that serotype 1 ST306 does not require hemolytically active pneumolysin to cause IPD.
Antibody responses to a 23-valent pneumococcal vaccine for Streptococcus pneumoniae serotypes 6B, 14, 19F, and 23F in 84 patients with chronic pulmonary diseases over a 2-year period after vaccination were examined by using a third-generation enzyme-linked immunosorbent assay. Of these patients, 28 (31%) were low responders who had developed increases of at least twofold in the levels of serotype-specific immunoglobulin G (IgG) in sera for none of the four serotypes at 1 month after vaccination. Although no specific clinical features of low responders were evident, their prevaccination levels of IgG for all serotypes were higher than those of responders. In responders, the levels of IgG specific for serotypes 14 and 23F in sera were greatly increased 1 month after vaccination and those specific for serotypes 6B and 19F were moderately increased. In contrast, no significant increases in the levels of IgG specific for serotypes 6B, 19F, and 23F in the low responders during the same period were found, but the levels of IgG specific for serotype 14 did increase. Although a rapid decline in the levels of IgG for all serotypes in responders between 1 month and 6 months after vaccination was found, the levels of IgG specific for serotypes 14 and 23F in sera remained higher than the prevaccination levels for at least 2 years after vaccination. These data suggest the need for the revaccination of responders but not low responders among patients with chronic pulmonary diseases. Revaccination as early as 3 years postvaccination is recommended for responders to increase the reduced levels of IgG in sera, especially those specific for the weak vaccine antigens.