The pneumococcus is a diverse pathogen whose primary niche is the nasopharynx. Over 90 different serotypes exist, and nasopharyngeal carriage of multiple serotypes is common. Understanding pneumococcal carriage is essential for evaluating the impact of pneumococcal vaccines. Traditional serotyping methods are cumbersome and insufficient for detecting multiple serotype carriage, and there are few data comparing the new methods that have been developed over the past decade. We established the PneuCarriage project, a large, international multi-centre study dedicated to the identification of the best pneumococcal serotyping methods for carriage studies.
Methods and Findings
Reference sample sets were distributed to 15 research groups for blinded testing. Twenty pneumococcal serotyping methods were used to test 81 laboratory-prepared (spiked) samples. The five top-performing methods were used to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. Sensitivity and positive predictive value (PPV) were determined for the test methods and the reference method (traditional serotyping of >100 colonies from each sample).
For the alternate serotyping methods, the overall sensitivity ranged from 1% to 99% (reference method 98%), and PPV from 8% to 100% (reference method 100%), when testing the spiked samples. Fifteen methods had ≥70% sensitivity to detect the dominant (major) serotype, whilst only eight methods had ≥70% sensitivity to detect minor serotypes. For the field samples, the overall sensitivity ranged from 74.2% to 95.8% (reference method 93.8%), and PPV from 82.2% to 96.4% (reference method 99.6%). The microarray had the highest sensitivity (95.8%) and high PPV (93.7%). The major limitation of this study is that not all of the available alternative serotyping methods were included.
Most methods were able to detect the dominant serotype in a sample, but many performed poorly in detecting the minor serotype populations. Microarray with a culture amplification step was the top-performing method. Results from this comprehensive evaluation will inform future vaccine evaluation and impact studies, particularly in low-income settings, where pneumococcal disease burden remains high.
Catherine Satzke and collaborators exploit a panel of spiked and field pneumococcal samples to compare serotyping methods used for nasopharyngeal carriage studies.
About 800,000 young children, mostly living in low-income countries, die annually from pneumococcal diseases, illnesses caused by the Streptococcus pneumoniae bacterium. S. pneumoniae is transmitted through contact with infected respiratory secretions and harmlessly colonizes the nose and throat of many healthy children (nasopharyngeal and oropharyngeal carriage). Occasionally, however, S. pneumoniae spreads into the lungs, the blood stream, or the covering of the brain, where it causes pneumonia, septicemia, and meningitis, respectively. These potentially fatal invasive pneumococcal diseases can be treated with antibiotics but can also be prevented by vaccination. Vaccination primes the immune system to attack disease-causing organisms (pathogens) by exposing it to weakened or dead pathogens or to pathogen molecules that it recognizes as foreign (antigens). Because there are more than 90 S. pneumoniae variants, or “serotypes,” each characterized by an immunologically distinct complex sugar coat, S. pneumoniae vaccines have to include multiple serotypes. “Pneumococcal conjugate vaccines” (PCVs) effectively prevent invasive pneumococcal diseases caused by common serotypes in resource-rich countries and are now being introduced into resource-poor countries.
Why Was This Study Done?
Although vaccination with PCVs reduces the carriage of the pneumococcal serotypes contained in them (vaccine serotypes), in the absence of these serotypes, non-vaccine serotypes can rapidly colonize the nasopharynx and become more common in both carriage and disease. Thus, “serotype replacement,” which may be more pronounced in low-income settings, threatens the global control of pneumococcal disease through vaccination; thus, when evaluating the impact of pneumococcal vaccines, it is important to analyze the carriage of multiple serotypes. Unfortunately, the traditional serotyping method—in which bacterial colonies are grown and a small number of colonies are typed using tests called the Quellung reaction and latex agglutination—is cumbersome and frequently misses the carriage of multiple serotypes. Several new serotyping methods have been developed over the past decade, and, here, in a multi-center comparative study (the PneuCarriage project), the researchers investigate which of these new methods is best for the examination of pneumococcal carriage in vaccine evaluation studies.
What Did the Researchers Do and Find?
The researchers used 15 clinical isolates containing a mixture of pneumococcal serotypes to prepare 81 “spiked” samples, which they distributed to 15 laboratories for testing with 20 serotyping methods. They determined the sensitivity (the percentage of serotypes in the samples that were correctly identified) and the positive predictive value (PPV; the proportion of identified positives that were true positives) for each method and used the five top-performing methods (those with the highest sensitivity and PPV; a perfect test has a sensitivity and a PPV of 100%) to test 260 nasopharyngeal (field) samples collected from children in six high-burden countries. When testing the spiked samples, traditional serotyping of over 100 colonies per sample had a sensitivity of 98% and a PPV of 100% overall, whereas the sensitivity of the alternative methods ranged from 1% to 99%, and their PPV ranged from 8% to 100%. Fifteen methods detected the major serotype in the spiked samples with ≥70% sensitivity, but only eight detected the minor serotypes with the same sensitivity. For the field samples, the sensitivity and PPV of the top-performing tests ranged from 74.2% to 95.8% and from 82.2% to 96.4%, respectively (the sensitivity and PPV of the traditional method were 93.8% and 99.6%, respectively); a culture microarray method had the best overall performance (95.8% sensitivity and 93.7% PPV).
What Do These Findings Mean?
A pneumococcal serotyping method for use in carriage studies needs to have high sensitivity, to detect multiple serotypes in individual samples, and to detect most or all serotypes. These findings show that although most of the recently developed serotyping methods detected the dominant serotype in a sample, many failed to detect minor serotypes. Moreover, the performance of similar methods varied markedly, and methods optimized for testing pure isolates did not necessarily work well when testing more complex samples. These findings identified microarray with a culture amplification step as the top-performing method, but, importantly, this study did not test all the available serotyping methods. Also, because it assessed each method in only a single laboratory, no conclusions can be reached about the reproducibility of these methods or their suitability for use in less-experienced laboratories or in resource-limited settings. Nevertheless, these findings should help to guide future vaccine evaluation and impact studies, particularly in low-income settings, where the burden of pneumococcal disease remains high.
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at http://dx.doi.org/10.1371/journal.pmed.1001903.
The US Centers for Disease Control and Prevention provides information on all aspects of pneumococcal disease and pneumococcal vaccination, including personal stories (including some information in Spanish)
The UK National Health Service Choices website provides information about pneumococcal disease
Kidshealth, a website provided by the US-based not-for-profit Nemours Foundation, includes information on pneumococcal vaccination (in English and Spanish)
Public Health England provides guidance on pneumococcal disease and vaccination
The not-for-profit Immunization Action Coalition has information on pneumococcal disease, including personal stories
Gavi, a not-for-profit global vaccine alliance, is helping to roll out pneumococcal vaccination in resource-poor countries
More information about the PneuCarriage project is available
MedlinePlus provides links to other resources about pneumococcal infections (in English and Spanish)
The World Health Organization provides information about pneumococcal disease and pneumococcal vaccines.