{'fraction_non_alphanumeric': 0.026779133568094343, 'fraction_numerical': 0.011055605601506839, 'mean_word_length': 5.063555114200596, 'pattern_counts': {'":': 0, '<': 0, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination reagents are widely used in microbial diagnosis, identification and serotyping. Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality world-wide. Current vaccines target the pneumococcal capsule, and there are over 90 capsular serotypes. Serotyping pneumococcal isolates is therefore important for assessing the impact of vaccination programs and for epidemiological purposes. The World Health Organization has recommended latex agglutination as an alternative method to the ‘gold standard’ Quellung test for serotyping pneumococci. Latex agglutination is a relatively simple, quick and inexpensive method; and is therefore suitable for resource-poor settings as well as laboratories with high-volume workloads. Latex agglutination reagents can be prepared in-house utilizing commercially-sourced antibodies that are passively attached to latex particles. This manuscript describes a method of production and quality control of latex agglutination reagents, and details a sequential testing approach which is time- and cost-effective. This method of production and quality control may also be suitable for other testing purposes.


Introduction
Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality in children under the age of five years old worldwide, particularly in resource-poor settings 1,2 . Pneumococcal disease ranges from localized infections to life threatening conditions such as pneumonia, sepsis and meningitis 1,2 .
More than 90 serotypes of pneumococci have been identified based on differences in their capsular polysaccharide 3 . Current vaccines target the capsular polysaccharide and provide protection from the major serotypes causing invasive disease 4 . Serotyping pneumococcal isolates is important for assessing the impact of vaccination on carriage and disease as well as providing broader epidemiological information 5,6 . The current 'gold standard' method for pneumococcal serotyping is the Quellung test, but it is time consuming and requires some skill to perform 7 .
Latex agglutination is an alternative serotyping method recommended by the World Health Organization 7 . Latex agglutination is quick and simple to perform, and is employed in many laboratories worldwide [8][9][10][11] . Importantly, latex agglutination has shown comparable accuracy to the Quellung test for pneumococcal serotyping 10,[12][13][14] . Overall, this method is ideal for resource-poor settings as well as high throughput laboratories.
Latex agglutination reagents ('latex reagents') are created by the attachment of antibodies to latex particles 15 . In a positive reaction, these labeled particles agglutinate in the presence of specific antigen. Commercial latex reagents are available for a restricted range of serotypes. Latex reagents can also be prepared in-house using commercially available antisera 10 . Mixtures of antisera ('pools') as well as specific antisera to pneumococcal serogroups (e.g., group 19), individual serotypes (e.g., serotype 5), and to specific antigens ('factors', e.g., factor 19c recognizing serotype 19A) for further defining serotypes within groups are available 16 . This manuscript outlines the major steps in the production of latex reagents using passive attachment of commercially available antisera to latex particles. The quality control (QC) aspects of this method are described, and a protocol for the use of latex reagents for pneumococcal serotyping is included.

Conducting Latex Agglutination Serotyping
1. Remove all latex reagents from refrigerator and allow them to warm to RT for approximately 30 min. Immediately before use, gently invert the tubes to mix the reagents. 2. Label a microscope slide. 3. Place 15 µl of negative control latex reagent onto the microscope slide. 4. Using a sterile disposable 1 µl loop take a sweep of the pneumococcal culture so that the loop is approximately half full. 5. Gently smear the inoculum onto the surface of the slide close to the drop of negative control latex reagent. The inoculum should be visible on the glass slide. 6. Quickly and thoroughly mix the inoculum into the negative control latex reagent using the loop. Ensure that the drop does not spread further than its initial size. 7. Pick up the slide, holding it at both ends. Rock the slide back and forth for 1 min. Take care to keep the liquid moving slowly, and ensure that the size of the drop does not increase. NOTE: Alternatively use a plate rocker. 8. Observe for macroscopic agglutination (i.e., by naked eye) and clearing of the suspension against a black background. 9. If the negative control latex reagent shows no agglutination or clearing continue testing the culture, replacing the test latex reagents for the negative control latex reagent in steps 4.1 to 4.8 above. A fresh loop must be used each time. Proceed with testing of the latex reagents starting with the pools. 10. Test each of the 'pool' latex reagents in succession until a positive test is obtained.
NOTE: The order of testing of the pools may be done in 'rounds' to reflect the local incidence of particular serotypes (e.g., by testing for the three most likely pools on the first slide. If a positive reaction is not obtained, then test the next three most likely of the remaining pools on the second slide; and so on until a positive reaction is obtained). This ensures that the most common serotypes are tested first, minimizing the time and reagents needed. 11. Using the antisera manufacturer's key determine which individual antisera are represented in the positive pool. 12. Test each of the groups or types contained in the positive pool individually to determine the 'group' or 'type' as in steps 4.1 to 4.8 above. 13. Determine the result by reference to the antisera manufacturer's key. If a 'type' is determined (e.g., serotype 5), then no further testing is needed and this result is recorded. 14. If a 'group' is determined (e.g., group 19), then refer to the manufacturers key to determine the factors to be tested. Continue testing with the individual 'factor' latex reagents and determine the final serotype (e.g., serotype 19B).

Representative Results
A positive latex reaction occurs when type-specific antibody attached to the latex particle binds to the capsule of the pneumococcus, and agglutination of the antibody labeled particles ensues 15 . Figure 1A shows a positive reaction which is characterized by visible agglutination and clearing of the background suspension. A negative reaction is characterized by the latex agglutination reagent suspension remaining smooth and white ( Figure 1B). The reagents are optimized so that a positive reaction should be observed before the end of the one min time interval (usually detectable in approximately 20 sec). We do not recommend reading reactions after the 1 min time interval. Very occasionally, reactions display weak agglutination around the edge of the drop that is not accompanied by clearing of the background suspension, or appear 'stringy' with no background clearing (data not shown). These are most likely negative reactions. In such cases, we recommend retesting and/or further investigation using another serotyping method (for example the Quellung reaction 17 ).

Discussion
Latex agglutination is a simple, quick and inexpensive method for pneumococcal serotyping. Commercial pneumococcal latex agglutination reagents are available, but they currently do not differentiate all known pneumococcal serotypes A positive latex reaction occurs when specific antibodies bound to the latex particles attach to antigens on the polysaccharide capsule of the pneumococcal isolate 18,19 . The latex particles attached to the antibodies allow the specific antibody-antigen reaction to be visualized without magnification.
Latex agglutination is a suitable method for high throughput laboratories and also for resource-poor settings. Key advantages of the latex agglutination method are that no specialized equipment is required to perform the test, reagents have a shelf life of at least 2 years when stored at 4 °C 8 , and that it is inexpensive, simple and quick to perform. Serotyping a pneumococcal isolate by latex agglutination takes approximately 10 min, and up to four latex reagents can be tested in parallel on the one slide. Furthermore, if the prevalence of serotypes is known for the relevant epidemiological setting, testing can be performed in rounds starting with the pools containing the most common serotypes similarly to the Quellung test 17 . This approach minimizes the number of tests needed to determine the serotype. The method is also highly reproducible between different operators (data not shown). A disadvantage of latex agglutination serotyping is that producing the reagents is time consuming, taking approximately 4 hr; although multiple reagents can easily be produced in parallel. Furthermore, some antigens are common across different pneumococcal serotypes, resulting in cross-reactions with some antisera (e.g., serotype 29, 42 and Group 35). However, these cross reactions are well characterized when using commercial antisera (which is usually preabsorbed to remove the more problematic cross-reacting antibodies) and additional reaction tables are provided by the manufacturer in order to distinguish which serotype is present. Latex reagents may also cross react if the antibodies are not aligned properly on the latex particles; these are detected as QC failures. In our experience, rigorous QC is central to the success of this method, even when commercially available antisera are used for production. QC takes approximately 15 min per reagent and relies on a set of appropriate QC isolates as described above.
The method described here results in reagents that give a positive reaction well within the test period. In our experience, false positives are rare in practice (data not shown). Occasional false negative reactions are observed; usually these relate to known issues with a particular antiserum (e.g., serogroup 6 isolates may not react with pool B antiserum), or down regulation of capsule expression by the isolate. Using the serotyping algrorithm provided by the manufacturer is also important, as in most cases a false positive or negative reaction will lead to a 'blind end' result. In these cases, and when reactions are difficult to interpret, we recommend repeat-testing and/or testing by an alternative method such as the Quellung reaction.
Some troubleshooting is occasionally required to make satisfactory latex reagents. In the method described here, the antibody is attached to latex particles via passive adsorption 15,19 , so a critical variable is the concentration of antibody used, which determines how well the surface of the latex particles is covered and the subsequent alignment of the antibody active sites 15,20 . Consequently, if insufficient or excess antibody is attached to the latex particles, the antibody-antigen reactions will not be optimal, and unsatisfactory reagents may be produced 15,20,21 . As antibody titers vary between different lots of antisera, a standard set of dilutions may not produce reagents that perform well 8 . A useful starting point is to use a 1:40 dilution of antiserum, and if this is not successful, dilute the antiserum further. In our experience this usually resolves the problem 8 . In a small number of cases reagents may show weak agglutination that is not accompanied by clearing of the suspension when tested with target isolates. In these cases diluting the antiserum does not improve the quality of the reagent, but satisfactory reagents can usually be produced by omitting the centrifugation and wash steps 8,22 . Antibody coated latex particles are used commonly in diagnostic microbiology for detection, identification or serotyping of many different microbes 15,20 . As such, the method described here may be suitable for preparing latex reagents for other purposes, provided suitable antisera is available and adequate QC procedures are adopted.

Disclosures
The authors have nothing to disclose.