Melissa Snyder, Ph.D.
Associate Professor of Laboratory Medicine and Pathology
Division Chair, Clinical Biochemistry
Mayo Clinic, Rochester, Minnesota
Hi everyone. My name is Melissa Snyder, and I’m an associate professor of laboratory medicine and pathology in the Department of Laboratory Medicine and Pathology at Mayo Clinic in Rochester, Minnesota. I am also co-director of the Antibody Immunology Laboratory.
Before beginning my presentation, I want to confirm that I have no disclosures relevant to this topic.
I have two learning objectives for this presentation. First, for participants to be able to identify clinical application of testing for Streptococcus pneumoniae polysaccharide antibodies and second, for participants to be able to describe tests available for measurement of Streptococcus pneumoniae polysaccharide antibodies.
Streptococcus pneumoniae is a gram-positive, encapsulated bacterium. Although generally thought of as a diplococci, this bacteria can also be found singly or in larger chains, as shown in this picture. The structure of the streptococcus pneumonia bacteria includes an inner cellular membrane, which is composed of a lipid bilayer, and a cell wall, which consists of cell wall C polysaccharide. This polysaccharide is invariant across the various serotypes, which we will discuss in a moment, and induces antibodies which are not protective in terms of an immune response against streptococcus pneumonia. Lastly, we have the outer capsule, which is composed largely of various polysaccharides.
A variety of infections can be caused by Streptococcus pneumoniae. Often these are respiratory tract infections, including pneumonia and otitis media. However, in some individuals, infection with Streptococcus pneumoniae can lead to invasive disease, such as septicemia or meningitis. Certain risk factors are associated with an increased risk of infection. These risk factors include age, especially in children less than 2 years of age and adults above 65 years old, cigarette smoking, certain chronic diseases, such as chronic obstructive pulmonary disease, or COPD, and congestive heart failure, and neurological diseases, including seizure disorders. Lastly, and probably not surprisingly, individuals with various primary or secondary immunodeficiencies are also at risk for infection with Streptococcus pneumoniae, including individuals with hypogammaglobulinemia, complement deficiency, or asplenia.
The polysaccharides present in the bacterial capsule of Streptococcus pneumoniae define various serotypes of the bacteria. To date, more than 90 serotypes have been identified, based on differences in the polysaccharides present in the bacterial capsule. The characterization and identification of these serotypes is important, as there are critical differences in the behavior of these serotypes. Some serotypes are found more frequently in carriers of streptococcus pneumonia, while other serotypes are known to have a higher potential for invasive disease or antibiotic resistance. Still, other serotypes have been found to be more virulent, with a higher potential for transmission between individuals.
There are several Streptococcus pneumoniae vaccines available for clinical use. The vaccines are divided into two categories: nonconjugated and conjugated vaccines. In the nonconjugated vaccines, free polysaccharides representing the various serotypes induce a T-cell-independent antibody response. These vaccines are referred to as PPSVs, which stands for “purified polysaccharide vaccines.” Pneumovax is a 23-valent nonconjugated vaccine, meaning that it contains polysaccharides present in the outer bacterial capsule from 23 different serotypes. While very effective, the primary issue with this type of non-conjugated polysaccharide vaccine is that it is generally not effective in children, especially in children under the age of 2 years. This is because a young child’s immune system is not mature and has only weak responses to pure polysaccharides. For this reason, the conjugated vaccines were developed. In the conjugated vaccines, polysaccharides are linked to a protein. By conjugating the polysaccharides to a protein carrier, a T-cell-dependent antibody response can be initiated, which is significantly more robust in children. These conjugated vaccines are identified as PCVs, or “polysaccharide conjugate vaccines.” There are three PCVs currently available for clinical use: Prevnar 13, Prevnar 15, and Prevnar 20, representing 13, 15, and 20 serotypes, respectively.
The serotypes present in each of the vaccines are shown in this table. The serotypes included in the vaccines were originally chosen based on associations between the serotypes and risks of respiratory and invasive infections. I won’t go through this table in detail, except to say that the serotypes in Prevnar 13, 15, and 20 are mostly subsets of the serotypes included in Pneumovax, except for the 6A serotype, which is only found in polysaccharide conjugate vaccines. Since introduction of routine pneumococcal vaccinations, the incidence of pneumococcal-related diseases has decreased, particularly those associated with serotypes included in the vaccines. However, as vaccines have been effective in preventing disease associated with certain serotypes, other serotypes have emerged as public health concerns. It is for this reason that pneumococcal vaccines will likely continue to evolve in terms of which serotypes are included both in the conjugated and non-conjugated versions.
Current recommendations include routine use of streptococcal pneumoniae vaccines in children and adults. In infants and children, Prevnar 13 or Prevnar 15 are indicated in a series of four doses occurring over ages 2 to 15 months. In adults over 65 years of age, a single dose of Prevnar 20 is recommended. Alternatively, a single dose of Prevnar 15 followed more than 1 year later by a single dose of Pneumovax is also appropriate. This vaccination strategy is also indicated for adults less than 65 years who have underlying medical conditions which increase their risk for streptococcal infection.
Approximately 4 to 6 weeks after vaccination, most healthy individuals will develop pneumococcal-specific antibodies, with the serotype specificities being dependent upon the specific vaccine. However, some individuals with primary or secondary immunodeficiencies may not demonstrate an adequate antibody response after vaccination. Measurement of antibodies against pneumococcal polysaccharides may be useful for diagnostic evaluation for suspected immunodeficiency. In fact, for some humoral immunodeficiencies, assessment of polysaccharide vaccine responses is included in the classification criteria. In addition, measurement of the antibodies can be used to characterize the vaccine response in terms of how many serotypes the patient responded to, which serotypes the patient responded to, and the magnitude of the response.
Mayo Clinic Laboratories offers 3 tests which can be used to measure pneumococcal-specific antibodies. The first test is “Streptococcus pneumoniae IgG antibodies, Total, Serum,” with the test code PNTO. The second test is “Streptococcus pneumoniae IgG Antibodies, 23 Serotypes, Serum” and has the test code PN23M. The last test is “Streptococcus pneumoniae IgG Antibodies, Total, with Reflex, Serum,” also identified as PNTOR. For the next part of this presentation, I will review each of these tests.
The “Streptococcus pneumoniae IgG Antibodies, Total, Serum” test is a semi-quantitative measurement of the total pneumococcal IgG response. The testing is performed by enzyme immunoassay in which the capture antigen is a mixture of the 23 polysaccharides present in Pneumovax. With this testing, a single result is obtained, which provides an overall assessment of the pneumococcal vaccine response.
For the total pneumococcal testing, we have established interpretive ranges, which were based on correlations with the serotype-specific testing, which we will review shortly. On the total pneumococcal antibody testing, a result <9.7 mcg/mL is indicative of a poor vaccine response. On the other end of the spectrum, results >270 mcg/mL correlate with a strong response to the vaccine. Results between 9.7 and 40 mcg/mL are consistent with a more modest vaccine response, while results from 180-270 mcg/mL likely indicate a moderate to robust antibody response. Results within the 40-180 mcg/mL range are more difficult to interpret. For patients who fall in this category, testing for serotype-specific antibodies may be useful.
And that brings us to the next test, which is “Streptococcus pneumoniae IgG Antibodies, 23 Serotypes, Serum.” This test is used to measure IgG antibodies that are specific for individual polysaccharides, again the 23 polysaccharides found in Pneumovax. This test is performed by multiplex immunoassay in which individual fluorescent bead sets are conjugated to specific polysaccharides. In this assay, multiple results are provided such that the antibody response against each individual polysaccharide can be assessed.
The serotype-specific testing has been available for over 10 years. However, recently, a method modification was implemented. The testing is still performed by bead-based multiplex immunoassay, but we have moved to magnetic beads and a new coupling chemistry, which is used for conjugation of the polysaccharides to the beads. We have realized several improvements with this method change, including better analytical specificity, reduced imprecision, and more consistency across reagent lots. In addition, it is important to note that a change to the reference range occurred with this method modification, such that a result ≥1.0 mcg/mL for each serotype is considered to be consistent with a response to the vaccine.
Guidelines are available to aid in the interpretation of serotype-specific antibody responses in the context of pneumococcal vaccination. When comparing pre- to post-vaccine antibody concentrations, which is the optimal approach, a ≥2-fold change in concentration for >50% of serotypes in children less than 6 years of age or for >70% of serotypes in individuals above 6 years is consistent with a normal response to the vaccine. If assessing only a single sample, concentrations ≥1.0 mcg/mL for >50% of serotypes in children less than 6 years of age or for >70% of serotypes in individuals above 6 years is consistent with a normal response to the vaccine. These guidelines have been published and I direct you to this practice parameter for more detailed information.
The final test we will review is “Streptococcus pneumoniae IgG antibodies, Total, with Reflex, Serum.” When this test is ordered, the total pneumococcal antibody concentration is measured by enzyme immunoassay. Depending upon the results, reflex testing for serotype-specific antibodies may be performed, either based on lab-defined criteria or healthcare provider request.
The reflex testing approach is based on the interpretive ranges I showed on an earlier slide for the total pneumococcal test. For results <9.7 mcg/mL or >270 mcg/mL, serotype-specific antibody testing is not indicated, as the total concentration clearly indicates either a very poor or very robust vaccine response, respectively. Results in the range of 40-180 mcg/mL are the most unclear in terms of a vaccine response, and further assessment with serotype-specific testing is suggested. For samples in this range, testing for serotype-specific antibodies is performed automatically. For the categories 9.7-40 mcg/mL and 180-270 mcg/mL, serotype-specific testing may be indicated for some patients. In these cases, the direct healthcare providers are in the best position to make that decision and reflex testing is available at their discretion.
Mayo Clinic Laboratories offers a group of tests which are useful for assessing antibodies responses to pneumococcal vaccination. The Streptococcus pneumoniae IgG Antibodies, Total, Serum” provides a global measurement of the antibody response, while “Streptococcus pneumoniae IgG Antibodies, 23 Serotypes, Serum” gives a more detailed, serotype-specific assessment. “Streptococcus pneumoniae IgG Antibodies, Total, with Reflex, Serum” incorporates both of the previous tests, taking a reflex approach.
Thank you for joining me for this presentation on Streptococcus pneumoniae antibody testing. I hope you find it useful in your daily routine practice.
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