The immunising effect of pneumococcal nasopharyngeal colonisation; protection against future colonisation and fatal invasive disease.

The human nasopharynx is an important ecological niche for Streptococcus pneumoniae, and asymptomatic nasopharyngeal carriage is a common precursor to invasive disease. However, knowledge of the immunological events, which occur during carriage, both on a cellular and humoral level, remains limited. Here, we present a long-term stable model of asymptomatic nasopharyngeal carriage using outbred naïve mice, in which we have investigated the effect of previous nasopharyngeal exposure to pneumococci, in the prevention of subsequent carriage and invasive disease. Carriage of D39 wildtype pneumococci restricted to the nasopharynx could be detected for at least 28 days post-infection, whereas nasopharyngeal carriage of a pneumolysin negative isogenic mutant (PLN-A) was cleared in 7-14 days. Both carriage events induced total and capsule specific IgA mucosal antibodies and increased levels of systemic antibodies (IgG against pneumococcal surface protein A (PspA) and IgM capsular polysaccharide), which increased over time and correlated to reduced nasopharyngeal pneumococcal numbers. Prior nasopharyngeal colonisation with PLN-A significantly reduced the duration of subsequent D39 wildtype carriage, and significantly increased survival following invasive pneumococcal challenge. In this case systemic anti-PspA and anti-capsular antibody IgM concentrations showed a strong correlation with reduced bacterial numbers in the lungs and nasopharynx, respectively and also with increased levels of IL17A and CD4+ T cells in lungs of pre-colonised mice. Prior nasopharyngeal colonisation with PLN-A also resulted in significant cross-serotype protection with mice protected from invasive disease with serotype 3 strain (A66) after pre-colonisation with a serotype 2 strain (D39). Our results suggest that both mucosal and systemic antibody as well as cellular host factors have a role in long-term protection against both colonisation and invasive pneumococcal challenge.

Identification of a surrogate marker for infection in the African green monkey model of inhalation anthrax.

In 2001, a bioterrorism attack involving Bacillus anthracis spore-laced letters resulted in 22 cases of inhalation anthrax, with five fatalities. This incident identified gaps in our health care system and precipitated a renewed interest in identifying both therapeutics and rapid diagnostic assays. To address those gaps, well-characterized animal models that resemble the human disease are needed. In addition, a rapid assay for a reliable diagnostic marker is key to the success of these efforts. In this study, we exposed African green monkeys to B. anthracis spores; examined clinical signs and physiological parameters, including fever, heart rate, complete blood count, and bacteremia; and evaluated the PCR assay and electrochemiluminescence (ECL) immunoassay for the biomarkers protective antigen and capsule. The results demonstrated that although there were neither objective clinical nor physiological signs that consistently identified either infection or the onset of clinical anthrax disease, the African green monkey is a suitable animal model exhibiting a disease course similar to that observed in the rhesus model and humans. We also demonstrated that detection of the biomarkers protective antigen and capsule correlated with bacterial loads in the blood of these nonhuman primates. The ECL immunoassay described here is simple and sensitive enough to provide results in one to two hours, making this assay a viable option for use in the diagnosis of anthrax, leading to timely initiation of treatment, which is a key component of B. anthracis therapeutic development.

Rapid detection of pneumococcal antigen in serum samples for diagnosing pneumococcal pneumonia.

OBJECTIVES: The aim of the study is to assess the usefulness of C polysaccharide and polysaccharide capsular antigen detection by immunochromatography (ICT) and enzyme immunoassay (EIA), respectively, in serum samples for diagnosing pneumococcal pneumonia. METHODS: Adult patients included in the study were classified in the following groups: In group 1 we studied 101 serum samples from patients with pneumonia due to Streptococcus pneumoniae. In 53 cases the pneumonia was bacteremic. The second group contained 113 serum samples from patients with no pneumococcal pneumonia. Group 3 was made up of 40 serum samples from healthy subjects with no clinical or radiological signs of pneumonia. RESULTS: Using ICT, antigen was detected in 50% of patients with pneumococcal pneumonia, in 64.3 and 40.9% of patients with bacteremic and non-bacteremic pneumococcal pneumonia, respectively. Using EIA, antigens were detected in 35.8% of patients with pneumococcal pneumonia, in 45 and 22.2% of patients with bacteremic and non-bacteremic pneumococcal pneumonia, respectively. CONCLUSIONS: In conclusion, the sensitivity of the tests is low. However, in special situations, where obtaining large volume of urine is difficult, they could be a complementary method in the rapid diagnosis of pneumococcal pneumonia.

Naturally-acquired immunity to Neisseria meningitidis group A.

Group A meningococcal disease is epidemic in Sudan, less common in Uganda, a country bordering the "meningitis belt," and rare in North America. The basis of naturally-acquired group A immunity is unknown but in North America protection has been attributed to a high prevalence of serum anticapsular antibodies elicited by cross-reacting bacteria. We measured group A anticapsular antibody concentrations and bactericidal titers in sera from 236 adults (47 from the Sudan obtained at the height of a group A epidemic, 57 from Uganda, and 132 from North America). Anticapsular antibody concentrations were higher in Sudanese sera than in North American or Ugandan sera (geometric mean of 31.5 versus 5.4 and 5.3 microg/ml, respectively, P < 0.0001). Bactericidal titers of > or =1:4 (presumed to be a protective titer when measured with human complement) were detected in 66% of Sudanese sera as compared with 27 and 23%, respectively, of North American and Ugandan sera (P < 0.0001). Bactericidal activity was inhibited by group A polysaccharide in 58% of the Sudanese bactericidal sera as compared to 17 and 6% of North America and Ugandan bactericidal sera (P < 0.0005). Approximately 50% of non-bactericidal Sudanese sera had high IgA anticapsular antibody concentrations, which were rare in bactericidal Sudanese sera. Thus, serum anticapsular antibodies and bactericidal activity are prevalent in Sudanese exposed to a group A epidemic. Cross-reacting group A anticapsular antibodies are prevalent in North American and Ugandan sera, but bactericidal activity is infrequent and when present is largely directed at non-capsular antigens.

Evaluation of a standardized F1 capsular antigen capture ELISA test kit for the rapid diagnosis of plague.

Rapid detection of soluble F1 capsular antigen in serum, bubo fluid or urine of patients proved to be a valuable tool in the presumptive diagnosis of plague. We evaluated a F1 capsular antigen capture ELISA resembling a commercially available test kit. The minimal detectable concentration was 4 ng/ml. The specificity was 100% when investigating 47 sera from healthy Malagasy subjects and 98.4% when 365 sera from German blood donors were studied. Sensitivity was determined on sera (n=11) and buboes (n=18) from bacteriologically confirmed Malagasy plague patients. Sensitivity was 90.1% for serum and 100% for buboes. A standardized F1 capsular antigen capture ELISA test kit might be well suited for the early detection of plague particularly in non-endemic areas where clinical microbiological laboratories have only limited access to alternative techniques for rapid identification of Yersinia pestis.

Rapid detection of contagious caprine pleuropneumonia using a Mycoplasma capricolum subsp. capripneumoniae capsular polysaccharide-specific antigen detection latex agglutination test.

Latex microspheres (diameter, 8 microm) were coated with anti-Mycoplasma capricolum subsp. capripneumoniae polyclonal immunoglobulin G (IgG) antiserum (anti-F38 biotype). The coated microspheres, when used in a latex agglutination test (LAT), detected M. capricolum subsp. capripneumoniae antigen in the serum of goats with contagious caprine pleuropneumoniae (CCPP). Beads also agglutinated strongly in the presence of purified M. capricolum subsp. capripneumoniae capsular polysaccharide (CPS). Preabsorption of CPS-specific antibodies prior to coating of the beads removed agglutinating activity in the presence of M. capricolum subsp. capripneumoniae, strongly suggesting that CPS is the likely soluble antigen recognized by the test. In addition, the specificity of the LAT exactly mirrored that of an M. capricolum subsp. capripneumoniae CPS-specific monoclonal antibody (WM25): of the 8 other mycoplasma species tested, agglutination was observed only with bovine serogroup 7. The LAT detected all 11 strains of M. capricolum subsp. capripneumoniae examined in this study, with a sensitivity level of 2 ng of CPS, or the equivalent of 1.7 x 10(4) CFU, in a reaction volume of 0.03 ml of serum. With field sera from goats with CCPP, the results of the LAT exhibited a 67% correlation with the results of the currently used complement fixation test (CFT), with the main discrepancy in diagnosis resulting from the increased sensitivity of the LAT compared to that of CFT. This antigen-detection LAT should prove particularly useful in identifying animals in the earliest stages of CCPP and combines sensitivity and low cost with ease of application in the field, without the need for any specialist training or equipment.

Immunogenicity of alpha-toxin, capsular polysaccharide (CPS) and recombinant fibronectin-binding protein (r-FnBP) of Staphylococcus aureus in rabbit.

This study was conducted to evaluate the antibody levels of alpha-toxin, capsular polysaccharides (CPS) and fibronectin-binding protein (FnBP) in rabbits immunized with an experimental vaccine against Staphylococcus aureus and to develop the bovine mastitis subunit vaccine in the future. Enzyme immunoassay was used for detection of IgG antibodies against staphylococcal CPS, alpha-toxin and FnBP. The levels of specific antibodies against CPS, alpha-toxin and FnBP in immunized rabbits were significantly increased after first immunization compared with control animals (p<0.05). Of three antigen used in vaccine, immunogenicity of CPS was relatively lower, compared with those of alpha toxin and fibronectin binding protein. Numbers of S. aureus in blood of immunized groups were lower than those of control group after bacterial challenge. But the bacterial numbers among immunized groups were not significantly different. S. aureus counts in excised organs were significantly lower in all immunized rabbits than in PBS-control group (p<0.05). The present study showed that alpha-toxin, capsular polysaccharide and fibronectin binding protein included in a subunit vaccine were protective.

Capsular types of Streptococcus pneumoniae isolated from blood and CSF during 1982-1987.

Knowledge about the type distribution of Streptococcus pneumoniae is fundamental to ensure an effective formulation of pneumococcal vaccine, especially with the possibility of producing a polysaccharide-protein-conjugated vaccine for the prevention of invasive disease in children. During the 6-year period 1982-1987, we received and typed 10,298 isolates from patients with invasive pneumococcal disease: 7,812 (76%) from blood and 2,486 (24%) from CSF. Of all isolates, 81% were recovered from individuals in Europe and 23% were from children. In order of frequency, S. pneumoniae types 6A + 6B, 14, 18C, 19F, 1, 7F, 23F, 19A, 4, and 5 were most commonly isolated from children, and types 3, 1, 14, 7F, 4, 6A + 6B, 8, 23F, 9V, and 19F, from adults. The pneumococcal types in the currently available 23-valent vaccine represented 87% of all isolates in this study, but the proportion of vaccine types varied somewhat with age and source. In all pneumococcal groups included in the vaccine, the vaccine types represented > 80% of the isolates, except in groups 6, 15, and 18.