<?xml version="1.0" encoding="UTF-8"?><!DOCTYPE article  PUBLIC "-//NLM//DTD Journal Publishing DTD v3.0 20080202//EN" "http://dtd.nlm.nih.gov/publishing/3.0/journalpublishing3.dtd"><article xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink" dtd-version="3.0" xml:lang="en" article-type="research article"><front><journal-meta><journal-id journal-id-type="publisher-id">AiM</journal-id><journal-title-group><journal-title>Advances in Microbiology</journal-title></journal-title-group><issn pub-type="epub">2165-3402</issn><publisher><publisher-name>Scientific Research Publishing</publisher-name></publisher></journal-meta><article-meta><article-id pub-id-type="doi">10.4236/aim.2023.135015</article-id><article-id pub-id-type="publisher-id">AiM-125275</article-id><article-categories><subj-group subj-group-type="heading"><subject>Articles</subject></subj-group><subj-group subj-group-type="Discipline-v2"><subject>Biomedical&amp;Life Sciences</subject></subj-group></article-categories><title-group><article-title>
 
 
  ST of &lt;i&gt;Streptococcus pneumonia&lt;/i&gt; Circulating in Burkina Faso before the Introduction of PCV-13, 2013
 
</article-title></title-group><contrib-group><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Kambiré</surname><given-names>Dinanibè</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref><xref ref-type="corresp" rid="cor1"><sup>*</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Tamboura</surname><given-names>Mamadou</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Ouédraogo</surname><given-names>Oumarou</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Tondé</surname><given-names>Issa</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Ouédraogo</surname><given-names>W. H. Gautier</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sanou</surname><given-names>Mahamoudou</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Zouré</surname><given-names>Abdou Azaque</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Compaoré</surname><given-names>Rebeca</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sagna</surname><given-names>Tani</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Soubeiga</surname><given-names>R. S. Théophile</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Zida</surname><given-names>Sylvie</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Aké</surname><given-names>Flavien</given-names></name><xref ref-type="aff" rid="aff3"><sup>3</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Abdoul-Salam</surname><given-names>Ouédraogo</given-names></name><xref ref-type="aff" rid="aff4"><sup>4</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Sangaré</surname><given-names>Lassana</given-names></name><xref ref-type="aff" rid="aff5"><sup>5</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Méda</surname><given-names>Isaïe</given-names></name><xref ref-type="aff" rid="aff6"><sup>6</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Kouanda</surname><given-names>Séni</given-names></name><xref ref-type="aff" rid="aff2"><sup>2</sup></xref></contrib><contrib contrib-type="author" xlink:type="simple"><name name-style="western"><surname>Ouédraogo-Traoré</surname><given-names>Rasmata</given-names></name><xref ref-type="aff" rid="aff1"><sup>1</sup></xref></contrib></contrib-group><aff id="aff3"><addr-line>Davycas International, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff6"><addr-line>Direction G&amp;amp;eacute;n&amp;amp;eacute;rale de la Sant&amp;amp;eacute; Publique, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff5"><addr-line>Centre Hospitalier Universitaire Sanon Sour&amp;amp;ocirc;, Bobo-Dioulasso, Burkina Faso</addr-line></aff><aff id="aff2"><addr-line>Institut de Recherche en Sciences de la Sant&amp;amp;eacute;, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff4"><addr-line>Centre Hospitalier Universitaire Yalgado Ou&amp;amp;eacute;draogo, Ouagadougou, Burkina Faso</addr-line></aff><aff id="aff1"><addr-line>Centre Hospitalier Universitaire P&amp;amp;eacute;diatrique Charles De Gaulle, Ouagadougou, Burkina Faso</addr-line></aff><pub-date pub-type="epub"><day>12</day><month>05</month><year>2023</year></pub-date><volume>13</volume><issue>05</issue><fpage>237</fpage><lpage>248</lpage><history><date date-type="received"><day>5,</day>	<month>April</month>	<year>2023</year></date><date date-type="rev-recd"><day>27,</day>	<month>May</month>	<year>2023</year>	</date><date date-type="accepted"><day>30,</day>	<month>May</month>	<year>2023</year></date></history><permissions><copyright-statement>&#169; Copyright  2014 by authors and Scientific Research Publishing Inc. </copyright-statement><copyright-year>2014</copyright-year><license><license-p>This work is licensed under the Creative Commons Attribution-NonCommercial International License (CC BY-NC).http://creativecommons.org/licenses/by-nc/4.0/</license-p></license></permissions><abstract><p>
 
 
  <b>Introduction:</b> Burkina Faso experiences regular cases of 
  Streptococcus pneumoniae meningitis. As part of the strategy to reduce cases of meningitis, the 13-valent pneumococcal conjugate vaccine (PCV-13) has been introduced in the Expanded Programme on Immunisation (EPI). Despite these efforts, there are some cases of pneumococcal meningitis including both vaccine and non-vaccine serotypes. The objective of this study was to describe the pneumococcal sequence types (ST) circulating in Burkina Faso before the introduction of the 13-valent pneumococcal conjugate vaccine (PCV-13). 
  <b>Methods:</b> It was a descriptive cross-sectional study that took place from 27
  <sup>th</sup> October 2013 to 7
  <sup>th</sup> January 2014. 
  S. pneumoniae strains were collected in Burkina Faso and Multi Locus Sequence Typing (MLST) was performed at the Pneumococcal Laboratory at the Centers for Disease Control and Prevention (CDC) in the USA (United States of America). MLST consists of 4 steps: amplification, purification, sequencing and interpretative reading of the results. The amplification used 7 primers consisting of sequences of 
  aroe, gdh, gki, recP, spi, xpt, ddl genes. 
  <b>Results:</b> Of 37 strains tested, 10 serotypes were identified. Serotype 1 was prevalent in 48.7% (18/37) followed by serotype 25F in 10.8% (4/37). Serotypes 5 and 12F/12A/12B/44/46 were 8.1% (3/37) each. Serotype 1 contained 5 STs including ST303 24.3% (9/37), ST217 8.1% (3/37) and ST618 8.1% (3/37); followed by serotype 25F with ST105 10.8% (4/37), serotype 5 with ST289 8.1% (3/37) and serogroup 12F/12A/12B/44/46 with ST 989 8.1% (3/37). 
  <b>Conclusion:</b> Pneumococci are characterised by their great variability both in number of serotypes and in ST within the same serotype. Thus, 10 serotypes have been identified. Also, within serotype 1, 5 different STs have been described. These data indicate the complexity of the pneumococcus which is strongly involved in purulent bacterial meningitis at national level. This requires continuous surveillance of pneumococcal meningitis through laboratory capacity building.
 
</p></abstract><kwd-group><kwd>&lt;i&gt;Streptococcus pneumoniae&lt;/i&gt;</kwd><kwd> Burkina Faso</kwd><kwd> PCV-13</kwd><kwd> Sequence Types</kwd></kwd-group></article-meta></front><body><sec id="s1"><title>1. Introduction</title><p>The African meningitis belt is the area of Africa south of the Sahara that extends from Senegal in the west to Ethiopia in the east that periodically experiences acute or purulent bacterial meningitis [<xref ref-type="bibr" rid="scirp.125275-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref2">2</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref3">3</xref>] . These purulent meningitises are mainly caused by three etiological agents, Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis, and are characterised by a predominantly neutrophilic leukocytosis [<xref ref-type="bibr" rid="scirp.125275-ref4">4</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] . Therefore, these pathogens cause meningitis epidemics especially in the meningitis belt of Lapeyssonia [<xref ref-type="bibr" rid="scirp.125275-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref6">6</xref>] . Furthermore, these meningitis epidemics are always accompanied by high mortality and morbidity rates [<xref ref-type="bibr" rid="scirp.125275-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref4">4</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref7">7</xref>] .</p><p>Burkina Faso, a country located in the heart of West Africa, regularly records cases of acute bacterial meningitis [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref7">7</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] . As part of its strategy to reduce meningitis cases, Burkina Faso introduced vaccines such as the Haemophilus influenzae type b vaccine in January 2006, MenAfriVac<sup>&#174;</sup> in December 2010 and PCV-13 in October 2013 [<xref ref-type="bibr" rid="scirp.125275-ref7">7</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref9">9</xref>] . The recently introduced PCV-13 significantly reduces the occurrence of meningitis and other pneumococcal infections in relevant targets [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref9">9</xref>] . As part of an overall strategy to monitor the actions of this vaccination campaign, the World Health Organization and Burkina Faso’s partners have supported the relevant departments of the Ministry of Health in moving from enhanced surveillance to case-by-case surveillance. Indeed, case-by-case surveillance is fundamentally different from enhanced surveillance because any suspected case of meningitis must be subject to a cerebrospinal fluid (CSF) sample that is analysed to identify the microorganisms involved, including pneumococci [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref9">9</xref>] .</p><p>S. pneumoniae occurs as gram-positive, candle-flame lanceolate diplococci and is strongly implicated in purulent and fatal meningitis due to its capsule [<xref ref-type="bibr" rid="scirp.125275-ref1">1</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref10">10</xref>] . This pneumococcal capsule is the source of a wide variety of serotypes that may or may not be vaccine serotypes [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref9">9</xref>] . The vaccine serotypes included in the pneumococcal conjugate vaccine (PCV13) are serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F or 23F [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] . Non-vaccine serotypes include serogroup 12F/12A/12B/44/46, serotypes 35B, 25F and 2. The serotypes also contain several standard sequences that are important for epidemiological surveillance [<xref ref-type="bibr" rid="scirp.125275-ref11">11</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref12">12</xref>] . Indeed, the determination of the different serotypes before the introduction of PCV-13 provided the government of Burkina Faso with scientific data to defend to World health organization (WHO) and its technical partners the interest of introducing PCV-13 and not pneumococcal conjugate vaccine seven valent (PCV7) or PCV10 [<xref ref-type="bibr" rid="scirp.125275-ref5">5</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref8">8</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref9">9</xref>] . Therefore, it is also important to have information on S. pneumoniae standard sequences (ST) from the same period. It is in this context that we propose to describe the type sequences of pneumococcal strains circulating in Burkina Faso before the introduction of PCV-13 in Burkina Faso.</p></sec><sec id="s2"><title>2. Methods</title><sec id="s2_1"><title>2.1. Setting, Type and Period of Study</title><p>This study was carried out concurrently in the meningitis reference laboratory (Charles De Gaulle Paediatric University Hospital), the national level laboratories (Yalgado Ou&#233;draogo University Hospital and the National Public Health Laboratory) in Burkina Faso and in the pneumococcal laboratory of the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia, United States of America. This was a descriptive cross-sectional study that took place from 27 October 2013 to 7 January 2014.</p></sec><sec id="s2_2"><title>2.2. Samples</title><p>The samples consisted of thirty-seven (37) strains of S. pneumoniae isolated from the cerebrospinal fluid (CSF) of patients with meningitis and came from the Central, Central West, Central South, Central North, Eastern and Northern regions.</p></sec><sec id="s2_3"><title>2.3. Laboratory Tests</title><p>S. pneumoniae strains isolated from the above laboratories in Burkina Faso were aliquoted in 0.5 mL of Greaves solution in a cryotube and transported to the CDC laboratory for testing. Once at the CDC, the strains were plated in fresh blood agar (FBA) and re-tested in the optochine with an inhibition diameter ≥ 14 mm. Next, the strains were sequenced in MLST which involves four main steps of amplification, purification, sequencing and interpretative reading of the results. The primer amplification used seven primers that carried the corresponding genes. The genes used in the sequencing of S. pneumoniae were aroe (shikimate dehydrogenase), gdh (glucose-6-phosphate dehydrogenase), gki (glucose kinase), recP (transketolase), spi (signal peptidase I), xpt (xanthine phosphoribosyltransferase), ddl (D-alanine-D-alanine ligase) (See <xref ref-type="table" rid="table">Table </xref>A4). The preparation of the aroe, gdh, gki, recp and xpt gene mix for the amplification of the 7 regions for MLST was done according to <xref ref-type="table" rid="table">Table </xref>A1. For the ddl gene, the preparation of the mix was done according to <xref ref-type="table" rid="table">Table </xref>A2. After the various preparations and distributions, the plate was placed in a thermocycler. <xref ref-type="table" rid="table">Table </xref>1 below gives the primers used for amplification of each of the seven genes.</p><p>Polymerase Chain Reaction (PCR) amplification for MLST sequencing of pneumococci followed the following conditions for the aroe, gdh, gki, recp, spi and xpt genes: [1X (94˚C, 1 min)]; [30X (94˚C, 15 sec); (54˚C, 30 sec); (72˚C, 45 sec)]; [1X (72˚C, 10 min)]; and [1X (4˚C, ∞]. For the ddl gene, the amplification conditions were as follows: [1X (94˚C, 1 min)]; [30X (94˚C, 15 sec); (50˚C, 1 min); (72˚C, 45 sec)]; [1X (72˚C, 10 min)] and [1X (4˚C, ∞]. Once the PCR was completed, the success of the amplification had to be verified. To do this, 2 μL of the amplicons underwent rapid migration on 1% agarose gel. Also, purification of the PCR product was performed. For this purpose, the ExoSAP-IT<sup>TM</sup> protocol was used according to the manufacturer’s instructions. For this purpose, in a clean 0.2 mL tube, 4 μL of amplicon + 3 μL of ExoSAP-IT<sup>TM</sup> was placed. This mixture was incubated at 37˚C for 15 minutes to degrade the primers and residual deoxyribonucleotide triphosphate (dNTP). Finally, incubate at 80˚C for 15 minutes to inactivate residual ExoSAP-IT<sup>TM</sup> reagents. The resulting PCR products are ready for amplification. For pneumococcal Deoxyribonucleic acid (DNA) sequencing by MLST, the composition of the reagents used in the preparation of the sequencing mix is given in <xref ref-type="table" rid="table">Table </xref>A4. Plates are introduced into the thermocycler under these conditions: [1X (96˚C, 1 min)]; [25X (96˚C, 10 sec); (55˚C, 5 sec); (60˚C, 4 min)]; [1X (4˚C, 7 min)] and [1X (4˚C, ∞)]. <xref ref-type="table" rid="table">Table </xref>A3 gives the AB sequencer used for sequencing the samples.</p><table-wrap id="table1" ><label><xref ref-type="table" rid="table">Table </xref>1</label><caption><title> The primer amplification used seven primers that carried the corresponding genes [<xref ref-type="bibr" rid="scirp.125275-ref13">13</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref14">14</xref>] </title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Genes</th><th align="center" valign="middle" >Primers sequences</th></tr></thead><tr><td align="center" valign="middle"  rowspan="2"  >aroe</td><td align="center" valign="middle" >aroe-F: TCC TAT TAA GCA TTC TAT TTC TCC CTT C (26)</td></tr><tr><td align="center" valign="middle" >aroe-R: ACA GGA GAG GAT TGG CCA TCC ATG CCC ACA CTG (33)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >gdh</td><td align="center" valign="middle" >gdh-F: ATG GAC AAA CCA GC(G/A/T/C) AG(C/T) TTC ACA AAA G (28)</td></tr><tr><td align="center" valign="middle" >gdh-R: ACG ATA GGT GAT ATC TGG TTG CCA AGT CCA TTT G (34)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >gki</td><td align="center" valign="middle" >gki-F: TCG TTT GGA CTT GCT TGG ATT GGC AG (26)</td></tr><tr><td align="center" valign="middle" >gki-R: AGA TGT GCG TAC TTG TGG GAA ACT ATT TTC ATC G (34)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >recp</td><td align="center" valign="middle" >recp-F: GAA TGT GTG ATT CAA TAA TCA CCT CAA ATA GAA GG (35)</td></tr><tr><td align="center" valign="middle" >recp-R: TGC TGT TTC GAT AGC AGC ATG GAT GGC TTC C (31)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >spi</td><td align="center" valign="middle" >spi-F: CGC TTA GAA AGG TAA GTT ATG AAT TT (26)</td></tr><tr><td align="center" valign="middle" >spi-R: GAA GAG GCT GAG ATT GGT GAT TCT CGG CC (29)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >xpt</td><td align="center" valign="middle" >xpt-F: TTA ACT TTT AGA CTT TAG GAG GTC TTA TG (29)</td></tr><tr><td align="center" valign="middle" >xpt-R: CGG CTG CTT GCG AGT GTT TTT CTT GAG (27)</td></tr><tr><td align="center" valign="middle"  rowspan="2"  >ddl</td><td align="center" valign="middle" >ddl-F: TAA AAT CAC GAC TAA GCG TGT TCT GG (26)</td></tr><tr><td align="center" valign="middle" >ddl-R: AAG TAG TGG GTA CAT AGA CCA CTG GG (26)</td></tr></tbody></table></table-wrap><p>Legend: number in front of each gene is the number of bases.</p></sec><sec id="s2_4"><title>2.4. Ethical Considerations</title><p>The study received approval from the ethical committee for health research of the Ministry of Health of Burkina Faso (Deliberation N˚2014-10-116).</p></sec><sec id="s2_5"><title>2.5. Data Analysis</title><p>For the descriptive statistics of the patients, the Stata 14.0 software was used to describe the different serotypes identified as well as the corresponding standard sequences (ST). Also, the STs were analysed with the CDC Fret programme using EditSeq/Seqman software and Codon Code Aligner software.</p></sec></sec><sec id="s3"><title>3. Results</title><p>The 37 S. pneumoniae strains were from the Centre (31.3%), East (28.1%), Centre-West (18.8%), North (12.5%), Centre-North (6.3%) and Centre-South (3.1%) regions. The youngest patient was 1 month old while the oldest was 55 years old. Of all the S. pneumoniae strains tested, 10 different serotypes were identified. Serotype 1 was the most common at 48.6% (18/37), followed by serotype 25F at 10.8% (4/37). Serotypes 5 and 12F/12A/12B/44/46 were 8.1% (3/37) each. Serotypes 4, 6B and 23F were 5.4% (2/37) each while serotypes 2, 14 and 35B were 2.7% (1/37) each. <xref ref-type="table" rid="table">Table </xref>2 gives the description of the different STs of the identified serotypes according to the 7 genes tested.</p><p>After the description of pneumococcal STs according the 7 genes used in MLST sequencing, it’s suitable to have more details of the proportion each ST described. Thus, the main ST described is ST303 of serotype 1 for 24.3% followed by ST105 of serotype 25F for 10.8%. <xref ref-type="table" rid="table">Table </xref>3 below gives for each serotype, the corresponding ST.</p><table-wrap id="table2" ><label><xref ref-type="table" rid="table">Table </xref>2</label><caption><title> Description of the different STs of the identified serotypes according to the 7 genes tested</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >S&#233;rotype</th><th align="center" valign="middle" >ST</th><th align="center" valign="middle" >aroe</th><th align="center" valign="middle" >gdh</th><th align="center" valign="middle" >gki</th><th align="center" valign="middle" >recp</th><th align="center" valign="middle" >spi</th><th align="center" valign="middle" >xpt</th><th align="center" valign="middle" >ddl</th></tr></thead><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >6B</td><td align="center" valign="middle" >12,693</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >134</td><td align="center" valign="middle" >367</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >6</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >217</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >18</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >618</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >5</td><td align="center" valign="middle" >289</td><td align="center" valign="middle" >16</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >9</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >41</td><td align="center" valign="middle" >33</td><td align="center" valign="middle" >33</td></tr><tr><td align="center" valign="middle" >5</td><td align="center" valign="middle" >289</td><td align="center" valign="middle" >16</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >9</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >41</td><td align="center" valign="middle" >33</td><td align="center" valign="middle" >33</td></tr><tr><td align="center" valign="middle" >35B</td><td align="center" valign="middle" >12,694</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >22</td><td align="center" valign="middle" >18</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >12,695</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >723</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >23F</td><td align="center" valign="middle" >802</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >53</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >72</td><td align="center" valign="middle" >38</td><td align="center" valign="middle" >31</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >23F</td><td align="center" valign="middle" >802</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >53</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >72</td><td align="center" valign="middle" >38</td><td align="center" valign="middle" >31</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >217</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >18</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >12F</td><td align="center" valign="middle" >989</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >89</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >112</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >4</td><td align="center" valign="middle" >5504</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >88</td><td align="center" valign="middle" >103</td><td align="center" valign="middle" >18</td><td align="center" valign="middle" >36</td><td align="center" valign="middle" >142</td><td align="center" valign="middle" >161</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >25F</td><td align="center" valign="middle" >105</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >15</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >12F</td><td align="center" valign="middle" >989</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >89</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >112</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >1316</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >25F</td><td align="center" valign="middle" >105</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >15</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td></tr><tr><td align="center" valign="middle" >5</td><td align="center" valign="middle" >289</td><td align="center" valign="middle" >16</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >9</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >41</td><td align="center" valign="middle" >33</td><td align="center" valign="middle" >33</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2830</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >155</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >4</td><td align="center" valign="middle" >12,696</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >103</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >9</td><td align="center" valign="middle" >14</td><td align="center" valign="middle" >161</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >618</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >6B</td><td align="center" valign="middle" >8052</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >20</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >17</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >618</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >25F</td><td align="center" valign="middle" >105</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >15</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td></tr><tr><td align="center" valign="middle" >12F</td><td align="center" valign="middle" >989</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >89</td><td align="center" valign="middle" >8</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >112</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr><tr><td align="center" valign="middle" >25F</td><td align="center" valign="middle" >105</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >15</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >6</td></tr><tr><td align="center" valign="middle" >2</td><td align="center" valign="middle" >12,697</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >13</td><td align="center" valign="middle" >36</td><td align="center" valign="middle" >12</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >6</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >14</td><td align="center" valign="middle" >12,698</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >17</td><td align="center" valign="middle" >21</td><td align="center" valign="middle" >14</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >217</td><td align="center" valign="middle" >10</td><td align="center" valign="middle" >18</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >7</td><td align="center" valign="middle" >19</td><td align="center" valign="middle" >9</td></tr></tbody></table></table-wrap><table-wrap id="table3" ><label><xref ref-type="table" rid="table">Table </xref>3</label><caption><title> Proportion of STs according the serotypes determined</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Serotypes</th><th align="center" valign="middle" >ST</th><th align="center" valign="middle" >Frequencies</th><th align="center" valign="middle" >Proportion</th></tr></thead><tr><td align="center" valign="middle" >25F</td><td align="center" valign="middle" >105</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >10.8</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >217</td><td align="center" valign="middle" >3</td><td align="center" valign="middle" >8.1</td></tr><tr><td align="center" valign="middle" >5</td><td align="center" valign="middle" >289</td><td align="center" valign="middle" >3</td><td align="center" valign="middle" >8.1</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >303</td><td align="center" valign="middle" >9</td><td align="center" valign="middle" >24.3</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >618</td><td align="center" valign="middle" >3</td><td align="center" valign="middle" >8.1</td></tr><tr><td align="center" valign="middle" >23F</td><td align="center" valign="middle" >802</td><td align="center" valign="middle" >2</td><td align="center" valign="middle" >5.4</td></tr><tr><td align="center" valign="middle" >12F</td><td align="center" valign="middle" >989</td><td align="center" valign="middle" >3</td><td align="center" valign="middle" >8.1</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >1316</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2830</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >4</td><td align="center" valign="middle" >5504</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >6B</td><td align="center" valign="middle" >8052</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >6B</td><td align="center" valign="middle" >12,693</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >35B</td><td align="center" valign="middle" >12,694</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >1</td><td align="center" valign="middle" >12,695</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >4</td><td align="center" valign="middle" >12,696</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >2</td><td align="center" valign="middle" >12,697</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >14</td><td align="center" valign="middle" >12,698</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >2.7</td></tr><tr><td align="center" valign="middle" >Total</td><td align="center" valign="middle" ></td><td align="center" valign="middle" >37</td><td align="center" valign="middle" >100</td></tr></tbody></table></table-wrap></sec><sec id="s4"><title>4. Discussion</title><p>The cases of S. pneumoniae meningitis that occurred in Burkina Faso before the introduction of PCV13 were due to a variety of serotypes. Thus, from <xref ref-type="table" rid="table">Table </xref>2, we were able to determine 10 different serotypes in the present study. From <xref ref-type="table" rid="table">Table </xref>3, we notice a single serotype may consist of one or more STs. This is the case for serotype 1, which is made up of 5 STs, with ST303 (9/17), ST217 (3/17) and ST618 (3/17) in the foreground. Results from other work in West Africa show a high prevalence of ST303 (7/9) within serotype 1 (1). This differs from previous work in the West African region which reported ST618 as 72.7% (92/127) [<xref ref-type="bibr" rid="scirp.125275-ref11">11</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref15">15</xref>] . In contrast, in South Africa, serotype 1 ST217 is reported at 96% (872/912) with three subclasses ST217 C1 at 92% (353/382), ST217C2 at 4% (15/382) and ST217C3 at 4% (14/382) [<xref ref-type="bibr" rid="scirp.125275-ref16">16</xref>] . Moreover, within this same serotype, there are various STs such as ST1316, ST2830, ST12695. These data show that there is great genetic diversity of pneumococcal strains circulating within the same country, from one country to another in the West African sub-region; and this, in time and space. This great genetic diversity could be explained by mutations in pneumococcal specie [<xref ref-type="bibr" rid="scirp.125275-ref16">16</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref17">17</xref>] [<xref ref-type="bibr" rid="scirp.125275-ref18">18</xref>] .</p><p>Nevertheless, a certain homogeneity is observed in our work for non-dominant serotypes such as 25F which contains only ST105 (4/4), serotype 5 with ST289 (3/3) and serogroup 12F/12A/12B/44/46 with ST 989 (3/3). In contrast, in North America, within serogroup 12F/12A/12B/44/46, the ST218 clonal complex is reported to be the most involved in meningitis epidemics [<xref ref-type="bibr" rid="scirp.125275-ref12">12</xref>] . Thus, the diversity of STs of the same serotype from one continent to another could also be seen. Also, many other serotypes, such as 6B, 4, 2 and 14, yielded only one ST due to the limited number of strains. Such results do not allow any consistent conclusions to be drawn. In this context, it seems very difficult to fight against pneumococcal meningitis worldwide without laboratories diagnosis. Thus, the surveillance of these serotypes and STs could be an opportunity in the use of data for the introduction of new vaccines.</p><p>At the end of this work, we note the following limitations. Firstly, the limited number of sequenced pneumococcal strains per region does not allow us to have a distribution of ST. Secondly, it should be remembered that Burkina Faso has 13 regions, whereas we did this work with strains from 6 regions. Thirdly, the data date from 2013-2014, and may not correspond to the current situation.</p><p>However, the data reported in this study provide a basis for future work on molecular surveillance of meningitis and the impact of vaccination on the distribution of different serotypes of S. pneumoniae. Also, they reinforce the need to increase and extend the possibilities of culture of pneumococcal strains in bacteriology laboratories in our country for a better surveillance of the epidemiological profile of meningitis.</p></sec><sec id="s5"><title>5. Conclusions</title><p>The aim of this work was to describe the typical sequences of S. pneumoniae strains in Burkina Faso. Indeed, pneumococci are characterised by their great variability in terms of both the number of serotypes and the typical sequences within the same serotype. Thus, out of all the strains, ten serotypes have been identified. Also, within serotype 1, five different standard sequences have been described.</p><p>This information gives an idea of the complexity of this pathogen which is greatly involved in purulent bacterial meningitis at both national and sub-regional levels. Preventive measures such as vaccination should be encouraged to protect the vulnerable segments of our society such as children under five years of age as well as people over sixty years of age.</p><p>This also requires continuous surveillance of pneumococcal meningitis, which requires laboratory capacity building and staff training.</p></sec><sec id="s6"><title>Acknowledgements</title><p>At the end of this work, we thank the Ministry of Health through its technical structures such as the Directorate of Population Health Protection (DPSP). We also thank our partners at CDC-Atlanta in Georgia, USA, particularly Dr. McGee Lesley, Dr. Srinivassan and Dr. Mahamoudou Ouattara.</p></sec><sec id="s7"><title>Authors Contributions</title><p>Conceptualization: KD, OTR, SL</p><p>Data curation: KD, TM, TI</p><p>Formal analysis: KD, OTR, ZAA, OO</p><p>Investigation: KD, SM, OTR</p><p>Resources: OTR, AF, MI</p><p>Supervision: OTR, KS, MI</p><p>Validation: OWHG, ZAA,</p><p>Visualization: OTR, KD, ASO</p><p>Writing &#177; original draft: KD, ASO, KBA</p><p>Writing &#177; review &amp; editing: OO, CR, ST, ZS, SRST</p></sec><sec id="s8"><title>Conflicts of Interest</title><p>The authors declare no conflicts of interest regarding the publication of this paper.</p></sec><sec id="s9"><title>Cite this paper</title><p>Dinanib&#232;, K., Mamadou, T., Oumarou, O., Issa, T., Gautier, O.W.H., Mahamoudou, S., Azaque, Z.A., Rebeca, C., Tani, S., Th&#233;ophile, S.R.S., Sylvie, Z., Flavien, A., Ou&#233;draogo, A.-S., Lassana, S., Isa&#239;e, M., S&#233;ni, K. and Rasmata, O.-T. (2023) ST of Streptococcus pneumoniae Circulating in Burkina Faso before the Introduction of PCV-13, 2013. Advances in Microbiology, 13, 237-248. https://doi.org/10.4236/aim.2023.135015</p></sec><sec id="s10"><title>Appendix</title><table-wrap id="table4" ><label><xref ref-type="table" rid="table">Table </xref>A1</label><caption><title> Preparation of mix for genes aroe, gdh, gki, recp and xpt for MLST amplification</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >PCR Master Mix</th><th align="center" valign="middle" >x1 (Volum/&#181;L)</th><th align="center" valign="middle" >x100</th></tr></thead><tr><td align="center" valign="middle" >PCR Water</td><td align="center" valign="middle" >15.8</td><td align="center" valign="middle" >1580</td></tr><tr><td align="center" valign="middle" >Buffer10X Tampon</td><td align="center" valign="middle" >2.5</td><td align="center" valign="middle" >250</td></tr><tr><td align="center" valign="middle" >10 mM dNTPs</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" >50</td></tr><tr><td align="center" valign="middle" >20 mM amorce sens</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" >50</td></tr><tr><td align="center" valign="middle" >20 mM amorce anti-sens</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" >50</td></tr><tr><td align="center" valign="middle" >Taq 5 U/&#181;L</td><td align="center" valign="middle" >0.2</td><td align="center" valign="middle" >20</td></tr><tr><td align="center" valign="middle" >Total</td><td align="center" valign="middle" >20</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >ADN</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >Final volum</td><td align="center" valign="middle" >25.0</td><td align="center" valign="middle" ></td></tr></tbody></table></table-wrap><table-wrap id="table5" ><label><xref ref-type="table" rid="table">Table </xref>A2</label><caption><title> Mix Preparation for ddl gene amplification</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >PCR Master Mix</th><th align="center" valign="middle" >x1 (Volum/&#181;L)</th><th align="center" valign="middle" >x100</th></tr></thead><tr><td align="center" valign="middle" >PCR Water</td><td align="center" valign="middle" >14.8</td><td align="center" valign="middle" >1480</td></tr><tr><td align="center" valign="middle" >Buffer10X</td><td align="center" valign="middle" >2.5</td><td align="center" valign="middle" >250</td></tr><tr><td align="center" valign="middle" >10 mM dNTPs</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" >50</td></tr><tr><td align="center" valign="middle" >20 mM Primer Forward</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >20 mM Primer Reverse</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >Taq 5 U/&#181;L</td><td align="center" valign="middle" >0.2</td><td align="center" valign="middle" >20</td></tr><tr><td align="center" valign="middle" >Total</td><td align="center" valign="middle" >20</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >DNA</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >Final Volum</td><td align="center" valign="middle" >25.0</td><td align="center" valign="middle" ></td></tr></tbody></table></table-wrap><table-wrap id="table6" ><label><xref ref-type="table" rid="table">Table </xref>A3</label><caption><title> Preparation of mix for gene spi amplification</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >PCR Master Mix</th><th align="center" valign="middle" >x1 (Volum/&#181;L)</th><th align="center" valign="middle" >x100</th></tr></thead><tr><td align="center" valign="middle" >PCR water</td><td align="center" valign="middle" >14.8</td><td align="center" valign="middle" >1480</td></tr><tr><td align="center" valign="middle" >10X Buffer</td><td align="center" valign="middle" >2.5</td><td align="center" valign="middle" >250</td></tr><tr><td align="center" valign="middle" >10 mM dNTPs</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" >50</td></tr><tr><td align="center" valign="middle" >20 mM Primer Forward</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >20 mM Primer Reverse</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >Taq 5 U/&#181;L</td><td align="center" valign="middle" >0.2</td><td align="center" valign="middle" >20</td></tr><tr><td align="center" valign="middle" >Total</td><td align="center" valign="middle" >20</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >DNA (Diluted 1:5)</td><td align="center" valign="middle" >5</td><td align="center" valign="middle" >From chelex extraction</td></tr><tr><td align="center" valign="middle" >Final volum</td><td align="center" valign="middle" >25.0</td><td align="center" valign="middle" ></td></tr></tbody></table></table-wrap><table-wrap id="table7" ><label><xref ref-type="table" rid="table">Table </xref>A4</label><caption><title> Mix preparation for MLST sequencing</title></caption><table><tbody><thead><tr><th align="center" valign="middle" >Sequencing Mix</th><th align="center" valign="middle" >x1 volum (&#181;L)</th><th align="center" valign="middle" >x100</th></tr></thead><tr><td align="center" valign="middle" >5X Sequencing Buffer</td><td align="center" valign="middle" >4</td><td align="center" valign="middle" >400</td></tr><tr><td align="center" valign="middle" >PCR Water</td><td align="center" valign="middle" >14.75</td><td align="center" valign="middle" >1475</td></tr><tr><td align="center" valign="middle" >Terminator nucleotides + Polymerase</td><td align="center" valign="middle" >1</td><td align="center" valign="middle" >100</td></tr><tr><td align="center" valign="middle" >Primer Forward (20 &#181;M/pmol)</td><td align="center" valign="middle" >0.25</td><td align="center" valign="middle" >25</td></tr><tr><td align="center" valign="middle" >Primer Reverse (20 &#181;M/pmol)</td><td align="center" valign="middle" >0.25</td><td align="center" valign="middle" >25</td></tr><tr><td align="center" valign="middle" >Purified DNA</td><td align="center" valign="middle" >0.5</td><td align="center" valign="middle" ></td></tr><tr><td align="center" valign="middle" >Final volum</td><td align="center" valign="middle" >20.5</td><td align="center" valign="middle" ></td></tr></tbody></table></table-wrap></sec></body><back><ref-list><title>References</title><ref 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