This work was part of the PneuCarriage project, funded by the Bill and Melinda Gates Foundation with contribution by the Victorian Government&#8217;s Operational Infrastructure Support Program.

1. Preparation of in-house Latex Reagents
<ol>
	<li>Preparation of glycine buffered saline (GBS)
	<ol>
		<li>Add 1.5 g glycine, 11.7 g sodium chloride and 100 ml of distilled water to a 200 ml glass beaker.</li>
		<li>Mix to dissolve using a magnetic stirrer.</li>
		<li>Transfer mixture to a 500 ml measuring cylinder and add distilled water to make a total of 200 ml.</li>
		<li>Transfer the solution back to the 200 ml beaker, check the pH and adjust to 8.2 with 5 M sodium hydroxide. NOTE: Sodium hydroxide is corrosive, wear personal protective equipment.</li>
		<li>Filter sterilize the solution using 0.22 &#181;M filters and several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.</li>
		<li>Store at RT.</li>
	</ol>
	</li>
	<li>Preparation of GBS with 0.2% bovine serum albumin
	<ol>
		<li>Add 0.2 g of bovine serum albumin (BSA) powder and 100 ml of GBS to a 200 ml glass beaker and mix to dissolve using a magnetic stirrer.</li>
		<li>Filter sterilize the solution through 0.22 &#181;M filters using several 60 ml syringes into a 250 ml preautoclaved screw capped glass bottle.</li>
		<li>Store at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Latex reagent preparation
	<ol>
		<li>Label a 2 ml round bottom microfuge tube. 
		NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.</li>
		<li>Using a P1000 pipette with sterile tips add 975 &#181;l of GBS into the microfuge tube, then add 25 &#181;l of appropriate pneumococcal antiserum for the latex reagent being prepared (i.e., pool, group, type or factor). Invert five times to mix.</li>
		<li>Dilute polystyrene latex particles 1:10 by adding 120 &#181;l of latex particles to 1,080 &#181;l of sterile physiological saline. 
		NOTE: This can be made in larger volumes, but should be made fresh each time the latex reagents are produced.</li>
		<li>Add 1,000 &#181;l of the 1:10 latex suspension (prepared in step 1.3.3) to the 1,000 &#181;l 1:40 antiserum suspension (prepared in step 1.3.2). Invert five times to mix.</li>
		<li>Incubate at 37 &#176;C on a slowly rotating wheel (e.g., 4 rpm for a wheel of 38.5 cm diameter) for 2 hr.</li>
		<li>Centrifuge for 15 min at 1,100 x g. Discard the supernatant and gently resuspend the pellet in 2 ml of sterile saline.</li>
		<li>Centrifuge for 15 min at 1,100 x g. Discard the supernatant and add 1 ml of GBS and gently resuspend the pellet.</li>
		<li>Pipette the latex suspension into a labeled 5 ml screw capped tube. Add another 1 ml of GBS.</li>
		<li>Add 2 ml of GBS containing 0.2% BSA (w/v) (pH 8.2) (prepared in step 1.2). Add 40 &#181;l of 10% (w/v) solution of sodium azide as a preservative. 
		NOTE: Sodium azide is hazardous if inhaled, and is a skin and eye irritant. It is desirable to purchase a prepared 10% solution rather than the powdered form, as this minimizes the risk of inhalation exposure. Personal protective equipment (including gloves and splash goggles) should be worn when handling this reagent. Refer to the Material safety data sheet for details.</li>
		<li>Store latex reagents at 4 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of negative control latex reagent
	<ol>
		<li>Label a 2 ml round bottom microfuge tube. 
		NOTE: round bottom tubes are used to minimize particle settling which may affect antibody attachment.</li>
		<li>Using a P1000 pipette with sterile tips add 975 &#181;l of GBS into the microfuge tube, then add 25 &#181;l normal rabbit antiserum. Invert five times to mix.</li>
		<li>Proceed as in steps 1.3.3 to 1.3.10 above</li>
	</ol>
	</li>
</ol>
2. Quality Control (QC) of Latex Reagents
<ol>
	<li>Bring latex reagent to RT. Immediately before use, gently mix the latex reagent by inverting the tube several times.</li>
	<li>Check reagent for autoagglutination by pipetting 15 &#181;l onto a glass microscope slide and proceed as in steps 4.7 and 4.8 below. There should be no agglutination of the latex particles. The reagent should appear white and smooth.</li>
	<li>Test the reagent against a panel of low passage pneumococcal isolates. Use freshly grown cultures prepared as in step 3. The panel of isolates should include isolates of the &#8216;target&#8217; serotype (the specific serotype for the reagent being tested) and &#8216;non-target&#8217; serotypes (isolates of other serotypes that should not normally cross react with the reagent). Include at least one isolate of each target and non-target serotype for each latex reagent undergoing QC. If there is only one target or non-target serotype, test two different isolates of the particular serotype. 
	NOTE: The testing panel should include several isolates of each serotype, some of which are uncommon in invasive disease and/or type culture collections. It is preferable to include some carriage isolates, as they are often more demanding to serotype (data not shown), thereby further testing the capacity of the reagents and ensuring that the reagents are likely capable of serotyping both invasive and carriage isolates.</li>
	<li>Proceed with testing of latex reagents with target and non-target pneumococcal isolates as per the method described in steps 3 and 4 below.</li>
	<li>Quality control reagents at time of production and then at least annually thereafter. 
	NOTE: If a reagent does not pass QC, the batch must be discarded and a new preparation made. In the event of repeat QC failure we recommend the reagents be produced using the alternate methods described in Ortika et al., 20138 and in the discussion below.</li>
</ol>
3. Preparation of Pneumococcal Cultures for Serotyping
<ol>
	<li>Using a sterile loop select a single colony of the pneumococcal isolate and streak this out onto a solid nonselective blood agar so that the primary inoculum covers approximately one third of the plate surface. Streak for single colonies. 
	NOTE: Plates prepared from defibrinated horse or sheep blood are suitable; but blood plates prepared with human blood or citrated animal blood are not.</li>
	<li>Incubate plate O/N&#160;at 37 &#176;C in an atmosphere of 5% CO2.</li>
	<li>Observe the growth on the plate for purity and assess that there is sufficient growth for serotyping. 
	NOTE: A pure culture of the pneumococcal isolate is required for serotyping. In our experience, confluent, or near confluent growth covering approximately one third of the plate surface is usually sufficient to complete serotyping. As pneumococcal cultures may be prone to autolysis, cultures prepared for serotyping should be tested within 24 hr of subculture. Do not take the culture plate out of the incubator more than 15 to 20 min before serotyping commences.</li>
</ol>
4. Conducting Latex Agglutination Serotyping
<ol>
	<li>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.</li>
	<li>Label a microscope slide.</li>
	<li>Place 15 &#181;l of negative control latex reagent onto the microscope slide.</li>
	<li>Using a sterile disposable 1 &#181;l loop take a sweep of the pneumococcal culture so that the loop is approximately half full.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>Observe for macroscopic agglutination (i.e., by naked eye) and clearing of the suspension against a black background.</li>
	<li>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.</li>
	<li>Test each of the &#39;pool&#39; latex reagents in succession until a positive test is obtained. 
	NOTE: The order of testing of the pools may be done in &#8216;rounds&#8217; to reflect the local incidence of particular serotypes (e.g.,&#160;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.</li>
	<li>Using the antisera manufacturer&#8217;s key determine which individual antisera are represented in the positive pool.</li>
	<li>Test each of the groups or types contained in the positive pool individually to determine the &#8216;group&#8217; or &#8216;type&#8217; as in steps 4.1 to 4.8 above.</li>
	<li>Determine the result by reference to the antisera manufacturer&#8217;s key. If a &#8216;type&#8217; is determined (e.g., serotype 5), then no further testing is needed and this result is recorded.</li>
	<li>If a &#8216;group&#8217; is determined (e.g., group 19), then refer to the manufacturers key to determine the factors to be tested. Continue testing with the individual &#8216;factor&#8217; latex reagents and determine the final serotype (e.g., serotype 19B).</li>
</ol>

<ol>
	<li>Rudan, I., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epidemiology+and+etiology+of+childhood+pneumonia+in+2010:+estimates+of+incidence,+severe+morbidity,+mortality,+underlying+risk+factors+and+causative+pathogens+for+192+countries.">Epidemiology and etiology of childhood pneumonia in 2010: estimates of incidence, severe morbidity, mortality, underlying risk factors and causative pathogens for 192 countries.</a> J Glob Health. 3 (10401), (2013).</li><li>O'Brien, K. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Burden+of+disease+caused+by+Streptococcus+pneumoniae+in+children+younger+than+5+years:+global+estimates.">Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates.</a> Lancet. 374, 893-902 (2009).</li><li>Henrichsen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Six+newly+recognized+types+of+Streptococcus+pneumoniae.">Six newly recognized types of Streptococcus pneumoniae.</a> J Clin Microbiol. 33, 2759-2762 (1995).</li><li>Hausdorff, W. P., Bryant, J., Paradiso, P. R., Siber, G. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Which+pneumococcal+serogroups+cause+the+most+invasive+disease:+implications+for+conjugate+vaccine+formulation+and+use,+part+I.">Which pneumococcal serogroups cause the most invasive disease: implications for conjugate vaccine formulation and use, part I.</a> Clin Infect Dis. 30, 100-121 (2000).</li><li>Mulholland, K., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+after+pneumococcal+vaccination.">Serotype replacement after pneumococcal vaccination.</a> Lancet. 379, 1388-1389 (2012).</li><li>Weinberger, D. M., Malley, R., Lipsitch, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+replacement+in+disease+after+pneumococcal+vaccination.">Serotype replacement in disease after pneumococcal vaccination.</a> Lancet. 378, 1962-1973 (2011).</li><li>Satzke, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Standard+method+for+detecting+upper+respiratory+carriage+of+Streptococcus+pneumoniae:+Updated+recommendations+from+the+World+Health+Organization+Pneumococcal+Carriage+Working+Group.">Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: Updated recommendations from the World Health Organization Pneumococcal Carriage Working Group.</a> Vaccine. , (2013).</li><li>Ortika, B. D., Habib, M., Dunne, E. M., Porter, B. D., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Production+of+latex+agglutination+reagents+for+pneumococcal+serotyping.">Production of latex agglutination reagents for pneumococcal serotyping.</a> BMC Res Notes. 6 (49), (2013).</li><li>Adegbola, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Serotype+and+antimicrobial+susceptibility+patterns+of+isolates+of+Streptococcus+pneumoniae+causing+invasive+disease+in+The+Gambia+1996-2003.">Serotype and antimicrobial susceptibility patterns of isolates of Streptococcus pneumoniae causing invasive disease in The Gambia 1996-2003.</a> Trop Med Int Health. 11, 1128-1135 (2006).</li><li>Slotved, H. C., Kaltoft, M., Skovsted, I. C., Kerrn, M. 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D., Satzke, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capsular+serotyping+of+Streptococcus+pneumoniae+using+the+Quellung+reaction.">Capsular serotyping of Streptococcus pneumoniae using the Quellung reaction.</a> J. Vis. Exp. (84), (2014).</li><li>Arai, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+antiserum-coated+latex+particles+for+serotyping+Streptococcus+pneumoniae.">Use of antiserum-coated latex particles for serotyping Streptococcus pneumoniae.</a> Microbiol Immunol. 45, 159-162 (2001).</li><li>Lafong, A. C., Crothers, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simple+latex+agglutination+method+for+typing+pneumococci.">Simple latex agglutination method for typing pneumococci.</a> J Clin Pathol. 41, 230-231 (1988).</li><li>Ortega-Vinuesa, J. L., Bastos-Gonzalez, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+factors+affecting+the+performances+of+latex+agglutination+tests.">A review of factors affecting the performances of latex agglutination tests.</a> J Biomater Sci Polym Ed. 12, 379-408 (2001).</li><li>Yap, K. 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The authors have nothing to disclose.

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 serotypes10,12. However, latex agglutination reagents can be easily produced in-house using purchased antisera raised against specific pneumococcal capsular antigens. In the method described here, antibodies are passively attached to latex particles to make a set of latex agglutination reagents.
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 &#176;C8, 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 test17. 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 &#8216;blind end&#8217; 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 adsorption15,19, 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 sites15,20. 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 produced15,20,21. As antibody titers vary between different lots of antisera, a standard set of dilutions may not produce reagents that perform well8. 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 problem8. 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 steps8,22.
Antibody coated latex particles are used commonly in diagnostic microbiology for detection, identification or serotyping of many different microbes15,20. 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.

Streptococcus pneumoniae (the pneumococcus) is a major cause of morbidity and mortality in children under the age of five years old world-wide, particularly in resource-poor settings1,2. Pneumococcal disease ranges from localized infections to life threatening conditions such as pneumonia, sepsis and meningitis1,2.
More than 90 serotypes of pneumococci have been identified based on differences in their capsular polysaccharide3. Current vaccines target the capsular polysaccharide and provide protection from the major serotypes causing invasive disease4. Serotyping pneumococcal isolates is important for assessing the impact of vaccination on carriage and disease as well as providing broader epidemiological information5,6. The current &#8216;gold standard&#8217; method for pneumococcal serotyping is the Quellung test, but it is time consuming and requires some skill to perform7. Latex agglutination is an alternative serotyping method recommended by the World Health Organization7. Latex agglutination is quick and simple to perform, and is employed in many laboratories worldwide8-11. Importantly, latex agglutination has shown comparable accuracy to the Quellung test for pneumococcal serotyping10,12-14. Overall, this method is ideal for resource-poor settings as well as high throughput laboratories.
Latex agglutination reagents (&#8216;latex reagents&#8217;) are created by the attachment of antibodies to latex particles15. 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 antisera10. Mixtures of antisera (&#8216;pools&#8217;) as well as specific antisera to pneumococcal serogroups (e.g., group 19), individual serotypes (e.g., serotype 5), and to specific antigens (&#8216;factors&#8217;, e.g., factor 19c recognizing serotype 19A) for further defining serotypes within groups are available16.
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.

<table><tbody><tr><td>Microparticles based on polystyrene, 800 nm</td><td>Sigma</td><td>65984-10 ml-F</td><td>Or other volumes as required</td></tr><tr><td>Normal rabbit serum</td><td>Antibodies Australia&amp;nbsp;</td><td>NRS-1ml</td><td>Or other volumes as required, bring to RT before use</td></tr><tr><td>Pneumococcus (Neufeld) antisera</td><td>SSI Diagnostica</td><td>Various</td><td>Bring to RT before use&lt;br/&gt; &lt;a href=&quot;http://www.ssi.dk/ssidiagnostica&quot;&gt;http://www.ssi.dk/ssidiagnostica&lt;/a&gt;</td></tr><tr><td>Sterile Bovine Albumin&amp;nbsp;</td><td>Sigma</td><td>A-4503</td><td>Bring to rRT before use</td></tr><tr><td>Sodium azide 10% (v/v) solution&amp;nbsp;</td><td>VWR International</td><td>ROAC3902/100ml</td><td>Caution: hazardous if inhaled; skin and eye irritant</td></tr><tr><td>Eppendorf Safe-Lock tubes, 2 ml</td><td>Eppendorf</td><td>0030 120.094</td><td>Round bottom</td></tr><tr><td>Sodium chloride</td><td>Merck</td><td>10241.AP</td><td></td></tr><tr><td>Calibrated disposable inoculating loops 1 &amp;micro;l</td><td>Copan</td><td>CD175SO1</td><td></td></tr><tr><td>Glass microscope slides 76 mm x 26 mm</td><td>Thermo Fisher Scientific</td><td>LBS 2950RC</td><td></td></tr><tr><td>5 M NaOH</td><td>BDH</td><td>10252</td><td>Caution: highly corrosive</td></tr><tr><td>Glycine</td><td>Merck</td><td>10119.05</td><td></td></tr><tr><td>Horse Blood Agar (HBA) plates</td><td>Thermo Fisher Scientific</td><td>PP2001</td><td>Bring to RT before use&lt;br/&gt; &lt;a href=&quot;http://www.thermofisher.com.au/&quot;&gt;http://www.thermofisher.com.au&lt;/a&gt;</td></tr><tr><td>Rotating wheel</td><td>Wyble Engineering Development Corporation</td><td></td><td></td></tr><tr><td>Polystyrene flat bottom screw cap tube, 5 ml</td><td>Technoplas</td><td>S5016SU</td><td></td></tr><tr><td>60 ml syringes without needles</td><td>Terumo</td><td>SS-60L</td><td></td></tr><tr><td>Millex-GP syringe filter unit, 0.22 &amp;micro;m</td><td>Merck Millipore</td><td>SLGP033RS</td><td></td></tr><tr><td>Brochure on Neufeld antisera</td><td>Statens Serum Institut</td><td></td><td>Key to determining serotypes&lt;br/&gt; &lt;a href=&quot;http://www.ssi.dk/ssidiagnostica&quot;&gt;http://www.ssi.dk/ssidiagnostica&lt;/a&gt;</td></tr><tr><td>Key to pneumococcal factor serum</td><td>Statens Serum Institut</td><td>18058</td><td>For determining serotype within Groups&lt;br/&gt; &lt;a href=&quot;http://www.ssi.dk/ssidiagnostica&quot;&gt;http://www.ssi.dk/ssidiagnostica&lt;/a&gt;</td></tr><tr><td>*alternative sources are available for most materials</td><td></td><td></td><td></td></tr></tbody></table>

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 &#8216;gold standard&#8217; 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.

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 ensues15. 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&#160;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 &#8216;stringy&#8217; 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 reaction17).
<img alt="Figure 1" fo:content-width="5in" src="/files/ftp_upload/51747/51747fig1highres.jpg" width="500" /> 
Figure 1.&#160;Positive and negative latex agglutination reactions. Preparations of a pneumococcal isolate mixed with latex agglutination reagent showing a positive reaction (A) or a negative reaction (B). Agglutination accompanied by clearing of the background is seen in the positive reaction (A).There is no visible agglutination with the negative control latex reagent or in a negative test reaction (B). Photographs8 used with permission. <a href="https://www.jove.com/files/ftp_upload/51747/51747fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination at JoVE.com

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Latex agglutination testing is a simple, rapid and inexpensive method for serotyping Streptococcus pneumoniae, and has also been widely applied in diagnostic microbiology. This manuscript describes the in-house production of latex agglutination reagents, quality control procedures and the application of this technique to pneumococcal serotyping.

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 ...

capsular-serotyping-of-streptococcus-pneumoniae-by-latex-agglutination

Research

JoVE Journal

Immunology and Infection

21.8K Views.  Murdoch Childrens Research Institute.  Latex agglutinatie test is een eenvoudige, snelle en goedkope werkwijze voor serotypering Streptococcus pneumoniae en ook grote schaal toegepast in diagnostische microbiologie. Dit manuscript beschrijft de in-house productie van latex agglutinatie reagentia, procedures voor kwaliteitscontrole en de toepassing van deze techniek om pneumokokken serotypering. 

Video: Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

Particle Agglutination Method for Poliovirus Identification

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute flaccid paralysis (AFP) cases. In the World Health Organization (WHO) Global Polio Laboratory Network, PV isolation and identification are currently being performed by using cell culture system and real-time RT-PCR, respectively. In the post-eradication era of PV, simple and rapid identification procedures would be helpful for rapid confirmation of polio cases at the national laboratories. In the present study, we will show the procedure of novel PA assay developed for PV identification. This PA assay utilizes interaction of PV receptor (PVR) molecule and virion that is specific and uniform affinity to all the serotypes of PV. The procedure is simple (one step procedure in reaction plates) and rapid (results can be obtained within 2 h of reaction), and the result is visually observed (observation of agglutination of gelatin particles).

A recently developed novel particle agglutination (PA) assay utilizing virus receptor molecule allowed a rapid and easy identification of poliovirus (PV). In this article, we will show the procedure for the PA assay for PV identification.

In the Global Polio Eradication Initiative, laboratory diagnosis plays a critical role by isolating and identifying PV from the stool samples of acute ...

Biochemical and Structural Characterization of the Carbohydrate Transport Substrate-binding-protein SP0092

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant strains. Carbohydrate substrate binding proteins (SBPs) represent viable targets for the development of protein-based vaccines and new antimicrobials because of their extracellular localization and the centrality of carbohydrate import for pneumococcal metabolism, respectively. Described here is a rationalized integrated protocol to carry out a comprehensive characterization of SP0092, which can be extended to other carbohydrate SBPs from the pneumococcus and other bacteria. This procedure can aid the structure-based design of inhibitors for this class of proteins. Presented in the first part of this manuscript are protocols for biochemical analysis by thermal shift assay, multi angle light scattering (MALS), and size exclusion chromatography (SEC), which optimize the stability and homogeneity of the sample directed to crystallization trials and so enhance the probability of success. The second part of this procedure describes the characterization of the SBP crystals using a tunable wavelength anomalous diffraction synchrotron beamline, and data collection protocols for measuring data that can be used to resolve the crystallized protein structure.

A streamlined protocol for performing an extensive biochemical and structural characterization of a carbohydrate substrate binding protein from Streptococcus pneumoniae is presented.

Development of new antimicrobials and vaccines for Streptococcus pneumoniae (pneumococcus) are necessary to halt the rapid rise in multiple resistant ...

Biochemistry

Experimental Human Pneumococcal Carriage

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major source of transmission and the prerequisite of invasive disease. A model of carriage will allow accurate determination of the immunological correlates of protection, the immunizing effect of carriage and the effect of host pressure on the pathogen in the nasopharyngeal niche. Further, methods of carriage detection useful in epidemiologic studies, including vaccine studies, can be compared.
Aim
We aim to develop an EHPC platform that is a safe and useful reproducible method that could be used to down-select candidate novel pneumococcal vaccines with prevention of carriage as a surrogate of vaccine induced immunity. It will work towards testing of candidate vaccines and descriptions of the mechanisms underlying EHPC and vaccine protection from carriage1. Current conjugate vaccines against pneumococcus protect children from invasive disease although new vaccines are urgently needed as the current vaccine does not confer optimal protection against non-bacteraemic pneumonia and there has been evidence of serotype replacement with non-vaccine serotypes2-4.
Method
We inoculate with S. pneumoniae suspended in 100 &mu;l of saline. Safety is a major factor in the development of the EHPC model and is achieved through intensive volunteer screening and monitoring. A safety committee consisting of clinicians and scientists that are independent from the study provides objective feedback on a weekly basis.
The bacterial inoculum is standardized and requires that no animal products are inoculated into volunteers (vegetable-based media and saline). The doses required for colonization (104-105) are much lower than those used in animal models (107)5. Detecting pneumococcal carriage is enhanced by a high volume (ideally &gt;10 ml) nasal wash that is relatively mucus free. This protocol will deal with the most important parts of the protocol in turn. These are (a) volunteer selection, (b) pneumococcal inoculum preparation, (c) inoculation, (d) follow-up and (e) carriage detection.
Results
Our current protocol has been safe in over 100 volunteers at a range of doses using two different bacterial serotypes6. A dose ranging study using S. pneumoniae 6B and 23F is currently being conducted to determine the optimal inoculation dose for 50% carriage. A predicted 50% rate of carriage will allow the EHPC model to have high sensitivity for vaccine efficacy with small study numbers.

Experimental human pneumococcal carriage offers a natural model of carriage and a potential model for use in vaccine development. This technique is valuable yet complex and involves clinical risk by introducing a pathogen into a human. We have developed a detailed protocol.

Experimental human pneumococcal carriage (EHPC) is scientifically important because nasopharyngeal carriage of Streptococcus pneumoniae is both the major ...

Medicine

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing the mucosal surface of the nasopharynx, where it can reside, multiply and eventually overcome host defences to invade to other tissues of the host. Establishment of an infection in the normally lower respiratory tract results in pneumonia. Alternatively, the bacteria can disseminate into the bloodstream causing bacteraemia, which is associated with high mortality rates2, or else lead directly to the development of pneumococcal meningitis. Understanding the kinetics of, and immune responses to, nasopharyngeal colonization is an important aspect of S. pneumoniae infection models.
Our mouse model of intranasal colonization is adapted from human models3 and has been used by multiple research groups in the study of host-pathogen responses in the nasopharynx4-7. In the first part of the model, we use a clinical isolate of S. pneumoniae to establish a self-limiting bacterial colonization that is similar to carriage events in human adults. The procedure detailed herein involves preparation of a bacterial inoculum, followed by the establishment of a colonization event through delivery of the inoculum via an intranasal route of administration. Resident macrophages are the predominant cell type in the nasopharynx during the steady state. Typically, there are few lymphocytes present in uninfected mice8, however mucosal colonization will lead to low- to high-grade inflammation (depending on the virulence of the bacterial species and strain) that will result in an immune response and the subsequent recruitment of host immune cells. These cells can be isolated by a lavage of the tracheal contents through the nares, and correlated to the density of colonization bacteria to better understand the kinetics of the infection.

Characterization of Inflammatory Responses During Intranasal Colonization with Streptococcus pneumoniae

Colonization of the murine nasopharynx with Streptococcus pneumoniae and the subsequent extraction of adherent or recruited cells is described. This technique involves flushing the nasopharynx and collection of the fluid through the nares and is adaptable for various readouts, including differential cell quantification and analysis of mRNA expression in situ.

Nasopharyngeal colonization by Streptococcus pneumoniae is a prerequisite to invasion to the lungs or bloodstream1. This organism is capable of colonizing ...

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal epidemiology, capsular serotyping can provide useful information for vaccine efficacy and impact studies.&#160;The Quellung reaction is the gold standard method for pneumococcal capsular serotyping. The method involves testing a pneumococcal cell suspension with pooled and specific antisera directed against the capsular polysaccharide. The antigen-antibody reactions are observed microscopically. The protocol has three main steps: 1) preparation of a bacterial cell suspension, 2) mixing of cells and antisera on a glass slide, and 3) reading the Quellung reaction using a microscope. The Quellung reaction is reasonably simple to perform and can be applied wherever a suitable microscope and antisera are available.

Capsular Serotyping of Streptococcus pneumoniae Using the Quellung Reaction

The Quellung reaction is the gold standard technique for serotyping Streptococcus pneumoniae. This technique utilizes a microscope and specific pneumococcal antisera and is commonly used in reference and research laboratories worldwide.

There are over 90 different capsular serotypes of Streptococcus pneumoniae (the pneumococcus). As well as being a tool for understanding pneumococcal ...

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled microscopic lesions (microlesions) that are remarkable due to the absence of infiltrating immune cells. Due to their release of cardiotoxic products, S. pneumoniae within microlesions are thought to contribute to the heart failure that is frequently observed during fulminate invasive pneumococcal disease in adults. Herein is demonstrated a protocol for experimental mouse infection that leads to reproducible cardiac microlesion formation within 30 hr. Instruction is provided on microlesion identification in hematoxylin &#38; eosin stained heart sections and the morphological distinctions between early and late microlesions are highlighted. Instruction is provided on a protocol for verification of S. pneumoniae within microlesions using antibodies against pneumococcal capsular polysaccharide and immunofluorescent microscopy. Last, a protocol for antibiotic intervention that rescues infected mice and for the detection and assessment of scar formation in the hearts of convalescent mice is provided. Together, these protocols will facilitate the investigation of the molecular mechanisms underlying pneumococcal cardiac invasion, cardiomyocyte death, cardiac remodeling as a result of exposure to S. pneumoniae, and the immune response to the pneumococci in the heart.

Visualization of Streptococcus pneumoniae within Cardiac Microlesions and Subsequent Cardiac Remodeling

Streptococcus pneumoniae forms discrete non-purulent microscopic lesions in the heart. Outlined is the protocol for a murine model of cardiac microlesion formation. Instruction is provided on microlesion visualization using microscopy, discrimination between early and late microlesions, and methods to detect cardiac remodeling in hearts of convalescent animals.

During bacteremia Streptococcus pneumoniae can translocate across the vascular endothelium into the myocardium and form discrete bacteria-filled ...

Homogeneous Glycoconjugate Produced by Combined Unnatural Amino Acid Incorporation and Click-Chemistry for Vaccine Purposes

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new protein functions or to have access to protein conjugates. Stop codon suppression, in particular amber codon suppression, has emerged as the most popular method to genetically introduce UAAs at defined positions. This methodology is herein applied to the preparation of a carrier protein containing an UAA harboring a bioorthogonal functional group. This reactive handle can next be used to specifically and efficiently graft a synthetic oligosaccharide hapten to provide a homogeneous glycoconjugate vaccine. The protocol is limited to the synthesis of glycoconjugates in a 1:1 carbohydrate hapten/carrier protein ratio but amenable to numerous pairs of biorthogonal functional groups. Glycococonjugate vaccine homogeneity is an important criterion to ensure complete physico-chemical characterization, thereby, satisfying more and more demanding drug regulatory agency recommendations, a criterion which is unmet by classical conjugation strategies. Moreover, this protocol makes it possible to finely tune the structure of the actual conjugate vaccine, giving rise to tools to address structure-immunogenicity relationships.

Genetic code expansion is applied for the introduction of an unnatural amino acid bearing a biorthogonal functional group on a carrier protein at a defined site. The biorthogonal function is further used for the site-selective coupling of a carbohydrate antigen to provide a homogeneous glycoconjugate vaccine.

Genetic code expansion is a powerful tool to introduce unnatural amino acids (UAAs) into proteins to modify their characteristics, to study or create new ...

Bioengineering

Opsonophagocytic Killing Assay to Assess Immunological Responses Against Bacterial Pathogens

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental procedure in which phagocytic cells are co-cultured with bacterial units. The immune cells will phagocytose and kill the bacterial cultures in a complement-dependent manner. The efficiency of the immune-mediated cell killing is dependent on a number of factors and can be used to determine how different bacterial cultures compare with regard to resistance to cell death. In this way, the efficacy of potential immune-based therapeutics can be assessed against specific bacterial strains and/or serotypes. In this protocol, we describe a simplified OPKA that utilizes basic culture conditions and cell counting to determine bacterial cell viability after co-culture with treatment conditions and HL-60 immune cells. This method has been successfully utilized with a number of different pneumococcal serotypes, capsular and acapsular strains, and other bacterial species. The advantages of this OPKA protocol are its simplicity, versatility (as this assay is not limited to antibody treatments as opsonins), and minimization of time and reagents to assess basic experimental groups.

This opsonophagocytic killing assay is used to compare the ability of phagocytic immune cells to respond to and kill bacteria based on different treatments and/or conditions. Classically, this assay serves as the gold&#160;standard for assessing effector functions of antibodies raised against a bacterium as opsonin.

A key aspect of the immune response to bacterial colonization of the host is phagocytosis. An opsonophagocytic killing assay (OPKA) is an experimental ...

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is requisite to phagocytosis and killing, the assay is an alternative method to&#160;opsono-phagocytic killing assays. An advantage of the opsono-adherence&#160;assay is the option of using inactivated pathogens and mammalian cell lines, which allows standardization across multiple experiments. The use of an inactivated pathogen in the assay also facilitates work with biosafety level 3 infectious agents and other virulent pathogens. In our work, the opsono-adherence assay was used to assess the functional ability of antibodies, from sera of animals immunized with an anthrax capsule-based vaccine, to induce adherence of fixed Bacillus anthracis to a mouse macrophage cell line, RAW 264.7. Automated fluorescence microscopy was used to capture images of bacilli adhering to macrophages. Increased adherence was correlated with the presence of anti-capsule antibodies in the serum. Non-human primates that exhibited high serum anti-capsule antibody concentrations were protected from anthrax challenge. Thus, the opsono-adherence assay can be used to elucidate the biological functions of antigen specific antibodies in sera, to evaluate the efficacy of vaccine candidates and other therapeutics, and to serve as a possible correlate of immunity.&#160;&#160;&#160;&#160;&#160;&#160;

Opsono-Adherence Assay to Evaluate Functional Antibodies in Vaccine Development Against Bacillus anthracis and Other Encapsulated Pathogens

The opsono-adherence assay is an alternative method to the opsono-phagocytic killing assay to evaluate the opsonic functions of antibodies in vaccine development.

The opsono-adherence assay is a functional assay that enumerates the attachment of bacterial pathogens to professional phagocytes. Because adherence is ...

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, studies in this serotype have been hindered by the lack of genetic tools to modify it. In this study, we describe a method to genetically modify a serotype 1 clinical isolate (strain 519/43). Interestingly, this was achieved by exploiting the Pneumococcus&#8217; ability to naturally acquire DNA. However, unlike most pneumococci, the use of linear DNA was not successful; to mutate this important strain, a suicide plasmid had to be used. This methodology has provided the means for a deeper understanding of this elusive serotype, both in terms of its biology and pathogenicity. To validate the method, the major known pneumococcal toxin, pneumolysin, was mutated because it has a well-known and easy to follow phenotype. We showed that the mutant, as expected, lost its ability to lyse red blood cells. By being able to mutate an important gene in the serotype of interest, we were able to observe different phenotypes for loss of function mutants upon intraperitoneal and intranasal infections from the ones observed for other serotypes. In summary, this study proves that strain 519/43 (serotype 1) can be genetically modified.

Constructing Mutants in Serotype 1 Streptococcus pneumoniae strain 519/43

Here, we describe a S. pneumoniae serotype 1 strain 519/43 that can be genetically modified by using its ability to naturally acquire DNA and a suicide-plasmid. As proof of principle, an isogenic mutant in the pneumolysin (ply) gene was made.

Streptococcus pneumoniae serotype 1 remains a huge problem in low-and-middle income countries, particularly in sub-Saharan Africa. Despite its importance, ...

Capsular Serotyping of Streptococcus pneumoniae by Latex Agglutination

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