Pneumococcal Serotypes in Urban Ugandan Children Vaccinated with the 10-Valent Pneumococcal Conjugate Vaccine

Pneumococcal Serotypes in Urban Ugandan Children Vaccinated with the 10-Valent Pneumococcal Conjugate Vaccine | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Pneumococcal Serotypes in Urban Ugandan Children Vaccinated with the 10-Valent Pneumococcal Conjugate Vaccine Alex Nakibinge, Thaddée Nshimiyimana, Amy E Steadman, Alfred Andama, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9423476/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract Objective Uganda introduced the 10-valent pneumococcal conjugate vaccine (PCV10) into routine infant immunization in 2014, but post-introduction molecular data on residual vaccine-type carriage remain limited. We assessed nasopharyngeal pneumococcal carriage and vaccine-type serotype distribution among urban Ugandan children aged ≤ 5 years, five years after PCV10 introduction, using real-time quantitative PCR (RT-qPCR). Results In this cross-sectional follow-up study, 156 stored nasopharyngeal bacterial isolates from 196 children were re-cultured and re-characterized; 64/90 phenotypically identified pneumococcal isolates were confirmed by lytA RT-qPCR, corresponding to an overall pneumococcal carriage prevalence of 33% (64/195). Molecular serotyping of all 64 pneumococcal isolates for PCV10/PCV13-associated capsular targets identified 23/64 (35.9%) vaccine-type isolates, while 41/64 (64.1%) were non-vaccine or non-typeable by the assay panel. Serotype 14 predominated (6/64, 9.4%), followed by 19A (5/64, 7.8%), and serotypes 3, 7F, and 9V (each 4/64, 6.3%); serotype 18C was detected in 1/64 (1.6%). Serotypes 1, 4, 5, 6A/6B, 19F, and 23F were not detected. Persistent carriage of PCV10 serotypes 7F, 9V, 14, and 18C indicates residual vaccine-type circulation despite an overall reduction in carriage prevalence. This is concerning, since a low universal carriage prevalence is necessary for effective pneumococcal disease control. Streptococcus pneumoniae Pneumococcus Carriage prevalence Invasive pneumococcal disease IPD Pneumococcal capsular serotypes PCV10 PCV13 RT-qPCR Children under 5 years Kampala Uganda Figures Figure 1 Introduction Streptococcus pneumoniae is a major global pathogen and the commonest bacterial cause of childhood pneumonia, otitis media, meningitis, and bacteremia [ 1 ]. WHO recognizes pneumococcal disease as a major public-health problem worldwide. In 2015, pneumococcal disease caused an estimated 294,000 deaths among children aged 1–59 months globally, despite vaccine availability and use [ 1 , 2 ]. Africa experiences the highest rates of pneumococcal illness and death [ 3 , 4 ], especially among children, the elderly, vulnerable populations, and those infected with HIV [ 4 – 6 ]. S. pneumoniae naturally lives in the human nasopharynx, with no known reservoirs in other animals [ 4 – 6 ]. Although carriage in the nasopharynx is typically asymptomatic, it is a prerequisite for the development of invasive pneumococcal disease (IPD) in carriers; carriage is crucial for the spread of the bacteria within the community [ 7 – 9 ]. Hence, studies on pneumococcal carriage are essential [ 10 , 11 ]. The capsular polysaccharide is the most important virulence factor in pneumococci because it surrounds the bacterium and shields it from the host’s immune response. Because of this, pneumococcal capsules have been extensively studied – research on pneumococcal capsules led to the discovery of deoxyribonucleic acid (DNA) as the genetic material in living organisms [ 4 ], the identification of various capsular serotypes, and the understanding of the serotype-specific nature of immune protection [ 4 ]. In summary, more than 100 distinct capsular serotypes have been identified so far [ 5 ], but only a few are clinically relevant worldwide [ 5 , 11 ]. Serotypes 1, 4, 5, 7F, 8, 12F, 14, 18C, and 19A are considered highly invasive and account for over 80% of IPD cases globally, whereas serotypes 6A, 6B, 11A, 15B/C, and 23F are generally thought to be less invasive [ 5 ]. Covalent coupling of the capsular polysaccharide to a protein carrier effectively converts the T-cell-independent Type 2 polysaccharide into a T-cell-dependent antigen [ 4 , 5 ]; carrier proteins used to date include a non-toxic mutant of the Corynebacterium diphtheriae toxin, ‘cross-reacting material 197’ (CRM197), and protein D of a non-typeable Haemophilus influenzae strain [ 4 ]. The use of the latter led to the development of the first 7-valent pneumococcal conjugate vaccine (PCV7), which was licensed in 2,000 and included capsular polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F, providing up to 90% protection against IPD in children [ 5 ]. However, due to serotype shifts caused by vaccine pressure, the protective coverage of PCV7 was reduced, and it did not include serotypes 1, 3, and 5, which are common in Africa, Asia, and Europe [ 5 , 12 ]. Later, a 10-valent conjugate vaccine (PCV10, which includes the PCV7 serotypes plus capsular polysaccharides of serotypes 1, 5, 7F) and a 13-valent conjugate vaccine (PCV13, which includes PCV10 plus serotypes 3, 6A, and 19A) were introduced for children in 2010 and licensed for adults in 2012 [ 5 ]. Furthermore, to cover a significant portion of IPD caused by other invasive serotypes, PCV20 (Prevnar 20) has been introduced; it offers broad protection against 20 pneumococcal serotypes, including the 13 in PCV13 (i.e., 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) plus seven additional serotypes (i.e., 8, 10A, 11A, 12F, 15B, 22F, 33F), Fig. 1 . PCV10 was introduced in Uganda in 2013 and added to the immunization schedule in 2014. In 2019, a review of the vaccine's impact in low- and upper-middle-income countries concluded that PCVs have a significant effect [ 13 ]; however, the level of impact varies widely [ 13 ] [ 14 ]. Furthermore, since the epidemiology of pneumococcal capsular types varies geographically and over time [ 14 ], understanding serotype distribution both spatially and temporally is essential for guiding vaccine use. Typically, sero-epidemiological data are lacking in many African settings. Therefore, this follow-up study aimed to examine pneumococcal carriage after PCV10 introduction in Uganda among children aged 5 years or younger, with a focus on carriage levels of vaccine-type (VT) serotypes. Thanks to notable advances in molecular technologies and our understanding of the pneumococcal genome [ 4 ], a real-time quantitative PCR (RT-qPCR) assay was employed to identify pneumococcal serotypes. Methods Setting : The study was conducted in the Genomics, Molecular, and Immunology (GMI) laboratories at the Department of Immunology and Molecular Biology, Makerere University College of Health Sciences in Kampala, Uganda, between March 2018 and May 2019. It was a follow-up study to previous works in Uganda [ 9 , 15 , 16 ] on carriage rates for S. pneumoniae , Haemophilus influenzae , Moraxella catarrhalis , and Staphylococcus aureus in Ugandan children pre- and post-PCV10 introduction. Note that these prior urban studies [ 9 , 16 ] did not genotype and/or serotype the pneumococci or other isolated bacterial species of interest. The Makerere GMI laboratories are accredited by the South African National Accreditation System (SANAS) https://www.sanas.co.za/Pages/index.aspx Pneumococcal isolates and antibiotic sensitivity testing : A total of 156 phenotypically characterized samples (90 S. pneumoniae , 40 H. influenzae , 14 M. catarrhalis , and 12 S. aureus isolated from children aged 5 years or younger [n = 194]) were retrieved from the − 80°C freezer and sub-cultured. Sample collection, culturing, and identification of isolates to the species level were described in the study by Nshimiyimana et al. [ 9 ]. However, all isolates were reidentified to the species level using biochemical and microbiological methods in this study. S. pneumoniae isolates were reidentified according to the WHO guidelines [ 17 , 18 ], i.e., colony characteristics (small, gray, alpha-hemolytic, glistening colonies tending to dip down in the center), Gram staining properties (positive diplococci), and biochemical characteristics (bile solubility and catalase reaction). An optochin sensitivity test was also performed: a disc impregnated with optochin was placed in the first streak area, and plates were incubated for 24–48 hours at 37°C under 5%-10% CO 2 . A zone of inhibition greater than 14 mm around the optochin disc allowed us to identify isolates as S. pneumoniae presumptively. For inhibition less than 14 mm, we performed the bile solubility test; if the turbid suspension was cleared in 2% sodium deoxycholate after 10–15 minutes at 35–37°C, the isolate was identified as S. pneumoniae . Phenotypic antibiotic susceptibility testing of pneumococci and other isolates was also described previously [ 9 ]; however, for S. pneumoniae , repeat testing was performed to verify the susceptibility patterns. Briefly, bacterial colonies from a 24-hour-old subculture were suspended in sterile normal saline to reach a turbidity equivalent to the 0.5 McFarland standard. Isolates were prepared for antibiotic susceptibility testing using the disk diffusion method on Mueller-Hinton Agar (MHA) medium supplemented with 5% sheep blood. After inoculating on MHA, antibiotic disks and/or E-test strips were placed on the streaked plates, which were then incubated for 24 hours at 35–37°C in 5%-10% CO 2 . S. pneumoniae isolates were tested for susceptibility to oxacillin (1 µg), erythromycin (15 µg), clindamycin (2 µg), tetracycline (30 µg), chloramphenicol (30 µg), and trimethoprim/sulfamethoxazole (co-trimoxazole) (1.25/23.5 µg). Additionally, E-tests for penicillin G (P 32 µg/ml to 0.002 µg/ml) and ceftriaxone (CRO 32 µg/ml to 0.002 µg/ml) were performed on isolates with ≤ 19 mm inhibition zone diameters around the oxacillin disc. Zone diameters and minimum inhibitory concentrations (MICs) for various antibiotics were read and interpreted according to CLSI guidelines [ 19 ]. For quality control, the reference S. pneumoniae strain ATCC 49619 was used. Molecular Assays : Chromosomal DNA extraction, molecular identification, and characterization were performed at GMI Labs using subcultured and re-identified isolates. Briefly, bacterial cells from overnight cultures in Brain Heart Infusion (BHI) broth were washed and centrifuged at 16,000g for 10 minutes. After discarding the supernatant, the bacterial pellet was washed with 2 mL of TE buffer (10 mM Tris, pH 8, 1 mM EDTA) and centrifuged at 14,000 g for 5 minutes. The supernatant was discarded, and 100 µL of lysozyme (40 mg/µL) was added, followed by incubation overnight at 37°C. Next, 150 µL of a fresh 10% SDS/200µg/mL Proteinase K mixture was added, and incubation continued at 65°C for 1 hour. Subsequently, 200 µL of 5 M NaCl and 200 µL of CTAB/NaCl solution were added, the mixture was vortexed, and incubated at 65°C for another hour. An equal volume of chloroform/isoamyl alcohol (24:1) was added, the mixture was briefly vortexed, and centrifuged at 14,000 g for 5 minutes, resulting in three distinct phases. Approximately 750 µL of the upper aqueous phase was transferred to a new labeled tube. To the precipitate, an equal volume of absolute ethanol was added, and the mixture was left at room temperature for 1 hour. After centrifugation at 14000g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 500 µL of 70% ethanol, then centrifuged again. The pellets were air-dried for 10 minutes, eluted in 100 µL of TE buffer, and stored at -20°C until PCR analysis. RT-qPCR assays were used to re-identify pneumococci, Haemophilus influenzae , and Moraxella catarrhalis . For pneumococci, we targeted the lytA gene, which encodes the major pneumococcal autolysin, serving as the "gold standard" target for RT-qPCR detection. For Haemophilus influenzae , we focused on the P6 gene (Protein D), while for Moraxella catarrhalis , the assay targeted the copB gene, which encodes the outer membrane protein. A ‘triplex RT-qPCR test’ was used to identify all three organisms in a single, rapid reaction utilizing specific fluorescent channels; see Table 1 . For S. aureus , we employed our previously published in-house conventional PCR assay targeting the nuc gene [ 8 ], on an Applied Biosystems Veriti 96-Well Thermal Cycler, Model 9902 (Connecticut, United States). Pneumococcal Serotyping : All pneumococcal isolates underwent RT-qPCR testing to identify capsular serotypes using primers and probes described in Table 1 . The assay aimed to detect serotypes included in the PCV10 vaccine currently used in Uganda, as well as those covered by PCV13 (see above). A multiplex RT-qPCR was performed in a 25 µl reaction containing 12.5 µl Platinum™ Quantitative PCR SuperMix-UDG (ThermoFisher Scientific, Waltham, Massachusetts, United States), 1.5 µl MgCl2 (50 mM), 2.5 µl primer-probe mix (50 picomoles), 3.5 µl nuclease-free water, and 50 ng pneumococcal template DNA. Five triplex reactions (each 25 µl) were prepared for different serogroups following a previously described protocol [ 20 ]. Primers and probes were grouped based on melting temperature (Tm) and expected amplicon size, with RT-qPCRs set up as follows: reaction 1 for serotypes 1, 5, 23F; reaction 2 for serotypes 4, 6A/6B/6C/6D, 9V/9A; reaction 3 for serotypes 14, 18C/18F/18B/18A, 19F; reaction 4 for serotypes 3, 19A, 7F/7A; reaction 5 for serotypes 6C/6D and 23A. An internal positive control targeting the cpsA gene of pneumococcal capsular polysaccharide synthesis, along with a no-template negative control, was included. The positive control was S. pneumoniae strain ATCC 49619. Thermocycling was performed on an ABI 7500 PCR system (Applied Biosystems / Thermo Fisher Scientific, Waltham, Massachusetts, United States) following the manufacturer’s instructions, using the program listed in Table 2 . A sample was considered positive only if its cycle-threshold (Ct) value matched the expected value for the target gene. Table 1 Primer/probe sequences used to genotype bacterial isolates and/or determine the pneumococcal capsular serotypes Target gene / serotype Primer and Probe sequence Role Reference lytA 5'-ACG CAA TCT AGC AGA TGA TGA AGC-3' (Forward) Identifies pneumococcus Carvalho et al. [ 21 ] 5'-TCG TGC GTT TTA ATT CGA GCT-3' (Reverse) 5'-/5 YakYel /TGC CGA AAA/ZEN/CGC TTG ATA (Probe) P6 5′-CCA GCT GCT AAA GTA TTA GTA GAA G-3′ (Forward) Identifies Haemophilus influenzae Obasi et al. [ 22 ] 5′-TTC ACC GTA AGA TAC TGT GCC-3′ (Reverse) VIC 5′-CAG ATG CAG TTG AAG GTT ATT TAG- MGB-3’ (Probe) copB 5′-GTG AGT GCC GCT TTT ACA ACC-3′ (Forward) Identifies Moraxella catarrhalis Obasi et al. [ 22 ] 5′-TGT ATC GCC TGC CAA GAC AA-3′ (Reverse) NED 5′-TGC TTT TGC AGC TGT TAG CCA GCC TAA-3′-MGB (Probe) nuc 5'-GCGATTGATGGTGATACGGTT-3' (Forward) Identifies Staphylococcus aureus Kateete et al. [ 8 ] 5'-AGCCAAGCCTTGACGAACTAAAGC-3' (Reverse) 1 5'-TT CAT CCC TAT GTG TGG TAT AG-3' (Forward) Identifies pneumococcal serotype 1 Carvalho et al. [ 23 ]; CDC [ 24 ]; Yun et al. [ 20 ] 5'-GCT TTA GAA GGT AGA GTT AAC ACC-3' (Reverse) 5'-/56- FAM /TGC CAA AGC CAG CCA T/31A (Probe) 3 5'-CCA CTA AAG CTT TGG CAA AAG AAA-3' (Forward) Identifies pneumococcal serotype 3 5'-CCC GAA CGT AAA GCT TCT TCA-3' (Reverse) 5'-/5 HEX /TTG TAG ACC/ZEN/GCC CCA CAA T (Probe) 4 5`-GCT TCT GCT GTA ACT GTT GTC C-3' (Forward) Identifies pneumococcal serotype 4 5'-CAC CAC CAT AGT AAC CAA AGT TCC-3' (Reverse) 5'-/5 Cy5 /TTC CAC AAA/TAO/AGA AGA GCC T (Probe) 5 5'-CAT GAT TTA TGC CCT CTT GCA A-3' (Forward) Identifies pneumococcal serotype 5 5'-GAC AGT ATA AGA AAA AGC AAG GGCTA (Reverse) 5'-/5 HEX /TCT TCT TCT/ZEN/CAT CGT TTC CG (Probe) 6A/B/C/D 5'-GTT TGC ACT AGA GTA TGG GAA GG-3' (Forward) Identifies pneumococcal serotype 6 5'-TAG CCT TTC TGA AAA CAT TTA GCG-3' (Reverse) 5'-/5 HEX /TGT TCT GCC/ZEN/CTG AGC AAC T (Probe) 7F/7A 5'-ATG AAG GCT TTG GTT TGA CAG-3' (Forward) Identifies pneumococcal serotype 7 5'-ATT CTC GCC ATC AAT TGC ATA TTC-3' (Reverse) 5'-/5 Cy5 /ACA CCA CTA/TAO/TAG GCT GTT GA (Probe) 9V/9A 5'-AGG TAT CCT ATA TAC TGC TTT AGG-3' (Forward) Identifies pneumococcal serotype 9 5'-ACGA ATC TGC CAA TAT CTG AAA G-3' (Reverse) 5'-/5 HEX /ACA + ACA+CAT + TGA+CAA + CCG CT/31A (Probe) 14 5'-AGA GTG TAT GAG GAA TCC-3' (Forward) Identifies pneumococcal serotype 14 5'-ATA TAT CTA CTG TAG AGG GAA T-3' (Reverse) 5'-/56- FAM /C QC CAA GTA/ZEN/AAT CAA (Probe) 19A 5'-CGC CTA GTC TAA ATA CCA-3 (Forward) Identifies pneumococcal serotype 19A 5'-GAG GTC ACC TAT AAT AGT AAGAG-3 (Reverse) 5'-/56- FAM /TAT CAA TGA/ZEN/GCC GAT CCG (Probe) 19F 5'-TGA GGT TAA GAT TGC TGA TCG-3' (Forward) Identifies pneumococcal serotype 19F 5'-CAC GAA TGA GAA CTC GAA TAA AAG-3' (Reverse) 5'- Cy5 /CGC + ACT+GTC + AAT+TCA CCT TC (Probe) 18C/18F/18A 5'-TCG ATG GCT AGA ACA GAT TTA TGG-3' (Forward) Identifies pneumococcal serotype 18 5'-CCA TTG TCC CTG TAA GAC CAT TG-3' (Reverse) 5'-5 HEX /AGG GAG TTG/ZEN/AAT CAA CCT A (Probe) 23 5'-GAC AGC ACC GAC AAT AGT CAT CTC-3' (Forward) Identifies pneumococcal serotype 23 5'-TCC ATC CCA ACC TAA CAC ACT TC-3' (Reverse) 5'-/5 Cy5 /ATT GTG TCC/TAO/ATA ACC CTT CG (Probe) The primers and probes were synthesized by Integrated DNA Technologies Inc (IDT), Coralville, Iowa, United States. Table 2 RT-qPCR program for identification of the pneumococcal serotypes Steps Temperature Time Acquisition Stage Cycles Initial denaturation 95°C 10 min No Hold 1 Denaturation 95°C 15 sec No Cycling 40 Combined annealing and extension 60°C 1 min Yes Results and Discussion All 156 stored isolates (90 pneumococci, 40 H. influenzae , 14 M. catarrhalis , 12 S. aureus ) from a previous study [ 9 ] were successfully sub-cultured and genotyped with a focus on molecular serotyping of S. pneumoniae . RT-qPCR genotyping confirmed 64 (71%) isolates as pneumococci, 16 (40%) H. influenzae , 9 (64%) M. catarrhalis , and 12 (100%) S. aureus , Table 3 . Table 3 Species-level confirmation of isolates by RT-qPCR Species # isolates tested # positive on RT-qPCR Frequency (%) Streptococcus pneumoniae 90 64 71 Haemophilus influenzae 40 16 40 Moraxella catarrhalis 14 9 64 Staphylococcus aureus 12 12 100 Total 156 86 Overall, based on RT-qPCR genotyping, the pneumococcal carriage prevalence in this urban pediatric population (n = 194, [ 9 ]) five years after introducing PCV10 is 33% (64/194), a significant significantly lower than the carriage rates reported in the pre-PCV era, which ranged from 56% to 77% [ 8 , 9 , 12 , 25 , 26 ]. Because the majority of isolates, i.e., 41/64 (64.1%), were not serotyped by our assay panel, they may represent non-vaccine serotypes or non-typeable variants. Furthermore, a decline in pneumococcal carriage after PCV vaccination is expected, given the documented reduction in carriage of VT serotypes following vaccination [ 27 ]. However, vaccination may not affect the overall pneumococcal carriage rate, since VT serotypes are replaced by non-VT serotypes [ 27 ]. The carriage rate observed in this study (33%) is consistent with recent reports of low serotype coverage for PCV10 in Ugandan children, estimated at 33%-42% [ 12 , 25 ]. Nevertheless, studies have generally noted higher carriage of VT serotypes years after PCV introduction in African settings compared to high-income countries [ 13 ], suggesting that PCVs may be less effective at reducing serotypes linked to IPD in low-income countries. Pneumococcal Serotypes: All 64 lytA -positive pneumococcal isolates were serotyped molecularly to investigate carriage of serotypes covered by the PCV10 and PCV13 vaccines, namely 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Note, since we serotyped only 23 (36%) of 64 lytA -positive isolates (Table 3 ), the rest were perhaps non-vaccine serotypes or non-typeable by the assay panel. Except for serotypes 1, 4, 5, 6A, 6B, 23F, and 19F, all other serotypes were found in at least one sample, but at very low frequencies (less than 10%), as shown in Table 4 . Serotype 14 was the most common, detected in six samples (9.4%), followed by serotype 19A in five samples (8%). Serotypes 3, 7F, and 9V were present in 4 samples each (6.3%), while serotype 18C was the least frequent, found in 1 sample (2%), as shown in Table 4 . Note that serotypes 3 and 7F coexisted in a sample (Table 4 ), and serotypes 3 and 19A are not covered by PCV10, which is currently used in Uganda (Fig. 1 ). Table 4 Frequency of VT* serotypes in children 5 years post PCV10 roll-out Isolate# Serotype Frequency (%) Chloramphenicol Tetracycline SXT Clindamycin Erythromycin Penicillin (≥ 0.06 mg/l)** Ceftriaxone (≤ 1mg/l) Ceftriaxone (≤ 0.5 mg/l)** SP121 14 6 (9.4) R R R S R R S S SP122 14 S I R S I R S S SP014 14 R I R S S R S S SP028 14 R R R S S R S S SP286 14 S I R R R R S S SP053 14 I S R I R R S I SP055 19A 5 (8) R S R S R R S S SP085 19A S S R R R R S I SP090 19A R S R S R R S S SP094 19A S R R R S R S S SP111 19A S R R R R R S S SP117 3 4 (6.3) R S R S I R S S SP118 3 S I R R R R S S SP119 3 R R R S R R R S SP126 3 R R R S R R S R SP126 7F 4 (6.3) S I R R R R S S SP130 7F R R R S R R S R SP133 7F R S R S I R S S SP134 7F R R R S R R R S SP136 9V 4 (6.3) S R R S R R S S SP143 9V S I R R R R S S SP148 9V S R R S R R S S SP149 9V R R R S R R S S SP160 18 1 (2) I S R I R R S I SP013 1 0 - - - - - - - - SP0144 4 0 - - - - - - - - SP0184 5 0 - - - - - - - - SP0177 6A 0 - - - - - - - - SP0164 6B 0 - - - - - - - - SP0196 19F 0 - - - - - - - - SP0104 23F 0 - - - - - - - - S, Susceptible; I, Intermediate; R, Resistant; -, Not applicable; SXT, trimethoprim-sulfamethoxazole; MDR, multidrug resistant. *With respect to the PCV10 and PCV13 vaccines. **CLSI interpretation guidelines for S. pneumoniae by minimum inhibitory concentration (MIC) susceptibility testing. In summary, four of the ten serotypes covered by the PCV10 vaccine (4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F)—specifically 14, 7F, 9V, and 18C—remain carried by children, though at low frequencies. This is concerning. These serotypes were also detected in studies in Eastern and Southwestern Uganda before the introduction of PCV10 [ 12 , 24 , 25 ], and Hammit et al. found serotypes 14 and 19A persisting in Kenyan children vaccinated with PCV10 [ 27 ]. Before PCV10 was introduced in Uganda, the most common serotypes reported were 19F, 6A/6B, and 23F [ 24 ], which appear to be suppressed by the vaccine. Overall, while vaccine trials and post-introduction studies in Africa show significant benefits of PCV immunization against IPD and all-cause mortality in children, African countries consistently report high residual carriage of VT serotypes [ 27 ]. This cannot be solely attributed to low vaccine coverage, as studies report high uptake rates—up to 90–95% in Kenya [ 28 ] and Malawi [ 29 ]. Since low overall carriage prevalence is necessary for effective control of pneumococcal carriage and, ultimately, IPD, persistent VT carriage in Africa might be high enough to sustain pneumococcal transmission among younger children and other vulnerable groups [ 30 ]. Additionally, pneumococcal serotypes in this study were highly resistant to common antibiotics, i.e., tetracycline, chloramphenicol, erythromycin, and clindamycin (Table 4 ). Moreover, additional serotypes, notably 29, are common in Uganda but are not covered by the current PCVs (Fig. 1 ); serotype 29 is reported to be associated with infection, antibiotic resistance, and invasive disease [ 25 ]. This is a concern in that, while IPD and pneumococcal infections are treatable with antibiotics, and immunization provides protection, shifts in serotypes and the growing antibiotic resistance in clinically relevant serotypes reduce the efficacy of both approaches to control [ 28 ]. Worldwide, ~ 600,000 deaths every year are attributed to antibiotic-resistant S. pneumoniae , and serotypes resistant to common antibiotics include 6B, 9V, 14, 19A, 19F, and 23F, which, unfortunately, are predominant in Uganda and generally in Africa [ 29 , 30 ], Table 4 . Conclusions Serotypes covered by PCV10 currently in use in Uganda can still be carried by vaccinated children years after vaccination. Since a low universal carriage prevalence is required for effective control of pneumococcal carriage and, eventually, IPD, residual VT carriage could be high enough to maintain pneumococcal transmission in the community. Limitations One limitation of this study is that the single-center, cross-sectional design and relatively small sample size could limit generalizability; hence, caution is necessary when interpreting our results. The study also lacked a control or comparison group, e.g., unvaccinated children; however, to assess the vaccine's impact, we compared our findings with pre-vaccine cohorts from other studies in the same setting before PCV10 was rolled out. For deeper insight into this setting, we recommend more robust approaches, such as whole-genome sequencing of the pneumococcal isolates. Another limitation is that we were unable to reconfirm 30 (33%) of the 90 stored pneumococci, suggesting i) degradation during storage or freeze/thaw effects, ii) an incorrect original biochemical/microbiological identification, or iii) effects of subculture on RT-qPCR. Abbreviations IPD Invasive pneumococcal disease PCV Pneumococcal conjugate vaccine VT Vaccine-type serotype CDC Centers for Disease Control MHA Mueller-Hinton Agar RT-qPCR Real-time quantitative polymerase chain reaction GMI Genomics, Molecular, and Immunology Laboratories BHI Brain Heart Infusion Tm Melting temperature TE Tris-EDTA buffer CTAB Cetyltrimethylammonium bromide buffer MIC Minimum inhibitory concentration Ct Cycle-threshold value CLSI The Clinical & Laboratory Standards Institute ATCC The American Type Culture Collection WHO The World Health Organization Declarations Ethics approval and consent to participate: The study obtained ethical approval from the Makerere University School of Biomedical Sciences Research and Ethics Committee (#SBS-620) and from the Mulago Hospital Research and Ethics Committee (#MHREC1574). Written informed consent was obtained from the parents and/or guardians of the recruited children, and consent for sample storage and use of stored samples in further studies was also obtained from the parents. The data were anonymized, and the authors did not have access to information that could identify individual participants during or after data collection. Consent for publication: Not applicable Competing interests: The authors declare that they have no competing interests Funding: None Author Contribution DPK conceived and designed the study, analyzed and interpreted the data, and drafted the manuscript. Alex N and TN performed the data collection and were major contributors in writing the manuscript. AS and POO provided research support and funding. DPK, Ann N, and AA supervised the project. All authors read and approved the final manuscript. Acknowledgements We thank the Global Health Labs (GHL), Bellevue, WA, United States, for ordering primers and probes from IDT Inc. and sending them to us. We also thank Ms. Praiscilla Kia and Ms. Esther Nakafu for their support rendered to this work. Data Availability All data supporting the findings of this study are available within the paper. References Loughran AJ, Orihuela CJ, Tuomanen EI. Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 2019;7(2). Epub 2019/03/16. 10.1128 /microbiolspec.GPP3-0004-2018. PubMed PMID: 30873934; PubMed Central PMCID: PMCPMC6422050. Koenraads M, Swarthout TD, Bar-Zeev N, Brown C, Msefula J, Denis B et al. Changing Incidence of Invasive Pneumococcal Disease in Infants Less Than 90 Days of Age Before and After Introduction of the 13-Valent Pneumococcal Conjugate Vaccine in Blantyre, Malawi: A 14-Year Hospital Based Surveillance Study. Pediatr Infect Dis J. 2022;41(9):764-8. Epub 2022/06/16. doi: 10.1097/inf.0000000000003606. PubMed PMID: 35703302; PubMed Central PMCID: PMCPMC9359774 Biologicals and from Takeda Pharmaceuticals outside the submitted work. No other competing interests were reported by authors. Other authors have no conflicts of interest to disclose. Wahl B, O'Brien KL, Greenbaum A, Majumder A, Liu L, Chu Y, et al. Burden of Streptococcus pneumoniae and Haemophilus influenzae type b disease in children in the era of conjugate vaccines: global, regional, and national estimates for 2000-15. Lancet Glob Health. 2018;6(7):e744–57. 10.1016/s2214-109x(18)30247-x . PubMed PMID: 29903376; PubMed Central PMCID: PMCPMC6005122. Geno KA, Gilbert GL, Song JY, Skovsted IC, Klugman KP, Jones C, et al. Pneumococcal Capsules and Their Types: Past, Present, and Future. Clin Microbiol Rev. 2015;28(3):871–99. 10.1128/cmr.00024-15 . PubMed PMID: 26085553; PubMed Central PMCID: PMCPMC4475641. Song JY, Nahm MH, Moseley MA. Clinical implications of pneumococcal serotypes: invasive disease potential, clinical presentations, and antibiotic resistance. J Korean Med Sci. 2013;28(1):4–15. 10.3346/jkms.2013.28.1.4 . Epub 20130108. O'Brien KL, Wolfson LJ, Watt JP, Henkle E, Deloria-Knoll M, McCall N, et al. Burden of disease caused by Streptococcus pneumoniae in children younger than 5 years: global estimates. Lancet. 2009;374(9693):893–902. 10.1016/s0140-6736(09)61204-6 . PubMed PMID: 19748398. Bogaert D, De Groot R, Hermans PW. Streptococcus pneumoniae colonisation: the key to pneumococcal disease. Lancet Infect Dis. 2004;4(3):144–54. 10.1016/s1473-3099(04)00938-7 . PubMed PMID: 14998500. Kateete DP, Kimani CN, Katabazi FA, Okeng A, Okee MS, Nanteza A et al. Identification of Staphylococcus aureus: DNase and Mannitol salt agar improve the efficiency of the tube coagulase test. 2010;9(1):23. Nshimiyimana T, Najjuka CF, Nalwanga W, Katende G, Kateete DP. Nasopharyngeal carriage and antibiotic susceptibility patterns of streptococcus pneumoniae, haemophilus influenzae, moraxella catarrhalis and staphylococcus aureus among urban Ugandan children post-PCV10 introduction: a cross-sectional study. Afr Health Sci. 2023;23(4):216–29. 10.4314/ahs.v23i4.24 . PubMed PMID: 38974281; PubMed Central PMCID: PMCPMC11225456. Simell B, Auranen K, Käyhty H, Goldblatt D, Dagan R, O'Brien KL. The fundamental link between pneumococcal carriage and disease. Expert Rev Vaccines. 2012;11(7):841–55. 10.1586/erv.12.53 . PubMed PMID: 22913260. Lin T-Y, Cheng-Hsun C, WPC Y, Abdul RM, Raja D, Leong CK, et al. Pneumococcal serotype prevalence and antibiotic resistance in children in South and Southeast Asia, 2012–2024. Hum Vaccines Immunotherapeutics. 2024;20(1):2417554. 10.1080/21645515.2024.2417554 . Nackers F, Cohuet S, de Waroux OlP, Langendorf C, Nyehangane D, Ndazima D et al. Carriage prevalence and serotype distribution of Streptococcus pneumoniae prior to 10-valent pneumococcal vaccine introduction: A population-based cross-sectional study in South Western Uganda, 2014. vaccine. 2017;35(39):5271-7. Swarthout TD, Fronterre C, Lourenço J, Obolski U, Gori A, Bar-Zeev N, et al. High residual carriage of vaccine-serotype Streptococcus pneumoniae after introduction of pneumococcal conjugate vaccine in Malawi. Nat Commun. 2020;11(1):2222. 10.1038/s41467-020-15786-9 . Epub 20200506. Hausdorff WP, Siber G, Paradiso PR. Geographical differences in invasive pneumococcal disease rates and serotype frequency in young children. Lancet. 2001;357(9260):950–2. 10.1016/s0140-6736(00)04222-7 . PubMed PMID: 11289365. Joloba M, Bajaksouzian S, Palavecino E, Whalen C, Jacobs MJI. High prevalence of carriage of antibiotic-resistant Streptococcus pneumoniae in children in Kampala Uganda. 2001;17(5):395–400. Kateete DP, Kajumbula H, Kaddu-Mulindwa DH, Ssevviri AK. Nasopharyngeal carriage rate of Streptococcus pneumoniae in Ugandan children with sickle cell disease. BMC Res Notes. 2012;5(1):28. 10.1186/1756-0500-5-28 . Satzke C, Turner P, Virolainen-Julkunen A, Adrian PV, Antonio M, Hare KM, et al. Standard method for detecting upper respiratory carriage of Streptococcus pneumoniae: updated recommendations from the World Health Organization Pneumococcal Carriage Working Group. Vaccine. 2013;32(1):165–79. World Health Organization. %J Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis Sp, manual. HiW. Laboratory methods for the diagnosis of meningitis caused by Neisseria meningitidis, Streptococcus pneumoniae and Haemophilus influenzae. WHO Man. 2011;(Ed. 2). CLSI. Performance Standards for Antimicrobial Susceptibility Testing. 26th edition. 2016 ed. CLSI Suppliment M100S. Wayne, PA: Clinical Laboratory Standards Institute; 2016. 2016;. Yun KW, Cho EY, Hong KB, Choi EH, Lee HJ. Streptococcus pneumoniae type determination by multiplex polymerase chain reaction. J Korean Med Sci. 2011;26(8):971–8. 10.3346/jkms.2011.26.8.971 . Epub 20110727. Carvalho Mda G, Tondella ML, McCaustland K, Weidlich L, McGee L, Mayer LW, et al. Evaluation and improvement of real-time PCR assays targeting lytA, ply, and psaA genes for detection of pneumococcal DNA. J Clin Microbiol. 2007;45(8):2460–6. 10.1128/jcm.02498-06 . Epub 20070530. Obasi CN, Barrett B, Brown R, Vrtis R, Barlow S, Muller D, et al. Detection of viral and bacterial pathogens in acute respiratory infections. J Infect. 2014;68(2):125–30. 10.1016/j.jinf.2013.10.013 . Epub 20131107. da Gloria Carvalho M, Pimenta FC, Jackson D, Roundtree A, Ahmad Y, Millar EV, et al. Revisiting pneumococcal carriage by use of broth enrichment and PCR techniques for enhanced detection of carriage and serotypes. J Clin Microbiol. 2010;48(5):1611–8. 10.1128/jcm.02243-09 . Epub 20100310. CDC. List of oligonucleotides used in pneumococcal serotype deduction by quadriplex real time PCR 2024 [cited 2024 12-12-2024]. Available from: https://www.cdc.gov/strep-lab/php/pneumococcus/serotyping-using-pcr.html#cdc_generic_section_2-real-time-pcr Lindstrand A, Kalyango J, Alfvén T, Darenberg J, Kadobera D, Bwanga F, et al. Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines. Be Appropriate? 2016;11(11):e0166018. Rutebemberwa E, Mpeka B, Pariyo G, Peterson S, Mworozi E, Bwanga F et al. High prevalence of antibiotic resistance in nasopharyngeal bacterial isolates from healthy children in rural Uganda: A cross-sectional study. 2015;120(4):249–56. Spijkerman J, Prevaes SM, van Gils EJ, Veenhoven RH, Bruin JP, Bogaert D, et al. Long-term effects of pneumococcal conjugate vaccine on nasopharyngeal carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis. PLoS ONE. 2012;7(6):e39730. Global burden of bacterial antimicrobial resistance. in 2019: a systematic analysis. Lancet. 2022;399(10325):629–55. Epub 20220119. doi: 10.1016/s0140-6736(21)02724-0. PubMed PMID: 35065702; PubMed Central PMCID: PMCPMC8841637. Kisakye A, Makumbi I, Nansera D, Lewis R, Braka F, Wobudeya E, et al. Surveillance for Streptococcus pneumoniae meningitis in children aged < 5 years: implications for immunization in Uganda. Clin Infect Dis. 2009;48(Suppl 2):S153–61. 10.1086/596495. PubMed PMID: 19191611. Nackers F, Cohuet S, le Polain de Waroux O, Langendorf C, Nyehangane D, Ndazima D, et al. Carriage prevalence and serotype distribution of Streptococcus pneumoniae prior to 10-valent pneumococcal vaccine introduction: A population-based cross-sectional study in South Western Uganda, 2014. Vaccine. 2017;35(39):5271–7. PubMed PMID: 28784282; PubMed Central PMCID: PMCPMC6616034. Additional Declarations No competing interests reported. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9423476","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":630823328,"identity":"21ca5319-01a3-4101-8620-c3344e5dcfb9","order_by":0,"name":"Alex Nakibinge","email":"","orcid":"","institution":"Makerere University College of Health Sciences (MakCHS)","correspondingAuthor":false,"prefix":"","firstName":"Alex","middleName":"","lastName":"Nakibinge","suffix":""},{"id":630823329,"identity":"89d889fc-b387-41ff-9147-2f5b3d87d8f2","order_by":1,"name":"Thaddée Nshimiyimana","email":"","orcid":"","institution":"University of Rwanda","correspondingAuthor":false,"prefix":"","firstName":"Thaddée","middleName":"","lastName":"Nshimiyimana","suffix":""},{"id":630823332,"identity":"1ce94b14-5189-4ec4-8ce7-908ffca8f94a","order_by":2,"name":"Amy E Steadman","email":"","orcid":"","institution":"Global Health Labs, Inc","correspondingAuthor":false,"prefix":"","firstName":"Amy","middleName":"E","lastName":"Steadman","suffix":""},{"id":630823333,"identity":"6201d0e4-0038-4813-aefa-b4f2f65997cb","order_by":3,"name":"Alfred Andama","email":"","orcid":"","institution":"Makerere University College of Health Sciences (MakCHS)","correspondingAuthor":false,"prefix":"","firstName":"Alfred","middleName":"","lastName":"Andama","suffix":""},{"id":630823334,"identity":"c991222e-a30f-4dee-a206-659daa3b8a5e","order_by":4,"name":"Peter Olupot-Olupot","email":"","orcid":"","institution":"Busitema University","correspondingAuthor":false,"prefix":"","firstName":"Peter","middleName":"","lastName":"Olupot-Olupot","suffix":""},{"id":630823338,"identity":"bf62d73e-3904-4a02-a04c-57924a418a2c","order_by":5,"name":"Ann Nanteza","email":"","orcid":"","institution":"Makerere University","correspondingAuthor":false,"prefix":"","firstName":"Ann","middleName":"","lastName":"Nanteza","suffix":""},{"id":630823339,"identity":"4d2ce474-24c9-45a0-a418-58533cc4bfbd","order_by":6,"name":"David Patrick Kateete","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABB0lEQVRIie3PMWrDMBSA4ScCyaJmViZfwaLQLfgqNgZP3grFQ1pcBPaiA8TE+A5dPCsY7MU0a0BLSqFToMpSuhTqlhAyRInHQvUjDW/4eBKAyfQXE93B4O6nCID0JhgAxdD2JHAgKOlBxk1LxRYCyxmxZrMrSmsSD14USu61ZNKG9jKHkHJce49ZKekChtcEJY2W2CJwKwwR4iSk7KqUqAC4AZTUerJ6+yUOt9537CuXTgGjj/Nk7YuOhB4nGDEUS28B+GfLTP+X9atY5nbg8zagGa+lnzF8S9xnoSXjlcfUNvKnaVpt1OdMTudN+qTU3YOW7J93PAy660J1gZzo0haTyWT6R30D1K1cHDUKaRQAAAAASUVORK5CYII=","orcid":"","institution":"Makerere University College of Health Sciences (MakCHS)","correspondingAuthor":true,"prefix":"","firstName":"David","middleName":"Patrick","lastName":"Kateete","suffix":""}],"badges":[],"createdAt":"2026-04-15 07:53:14","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9423476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9423476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108409166,"identity":"7f23a941-958f-452a-8659-0034e2ff47d3","added_by":"auto","created_at":"2026-05-04 09:58:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1515284,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe pneumococcal conjugate vaccines (PCVs) currently available and the serotypes covered by the vaccines. \u003c/strong\u003e*Available in Uganda. **Serotypes 6A and 29 are common in Uganda and associated with IPD and antibiotic resistance, but are not included in PCV10, which is currently administered to children in Uganda\u003c/p\u003e","description":"","filename":"Fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-9423476/v1/74cb9fce58f1c7dead7a9b12.png"},{"id":108492967,"identity":"f7d68cd4-1f3f-44c8-8b95-da2f4f07f64d","added_by":"auto","created_at":"2026-05-05 09:59:09","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2233360,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9423476/v1/50f6e5f1-2b53-4c11-aa27-dcec1d9c012f.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Pneumococcal Serotypes in Urban Ugandan Children Vaccinated with the 10-Valent Pneumococcal Conjugate Vaccine","fulltext":[{"header":"Introduction","content":"\u003cp\u003e \u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e is a major global pathogen and the commonest bacterial cause of childhood pneumonia, otitis media, meningitis, and bacteremia [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. WHO recognizes pneumococcal disease as a major public-health problem worldwide. In 2015, pneumococcal disease caused an estimated 294,000 deaths among children aged 1\u0026ndash;59 months globally, despite vaccine availability and use [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAfrica experiences the highest rates of pneumococcal illness and death [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], especially among children, the elderly, vulnerable populations, and those infected with HIV [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. \u003cem\u003eS. pneumoniae\u003c/em\u003e naturally lives in the human nasopharynx, with no known reservoirs in other animals [\u003cspan additionalcitationids=\"CR5\" citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Although carriage in the nasopharynx is typically asymptomatic, it is a prerequisite for the development of invasive pneumococcal disease (IPD) in carriers; carriage is crucial for the spread of the bacteria within the community [\u003cspan additionalcitationids=\"CR8\" citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Hence, studies on pneumococcal carriage are essential [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe capsular polysaccharide is the most important virulence factor in pneumococci because it surrounds the bacterium and shields it from the host\u0026rsquo;s immune response. Because of this, pneumococcal capsules have been extensively studied \u0026ndash; research on pneumococcal capsules led to the discovery of deoxyribonucleic acid (DNA) as the genetic material in living organisms [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], the identification of various capsular serotypes, and the understanding of the serotype-specific nature of immune protection [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In summary, more than 100 distinct capsular serotypes have been identified so far [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], but only a few are clinically relevant worldwide [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Serotypes 1, 4, 5, 7F, 8, 12F, 14, 18C, and 19A are considered highly invasive and account for over 80% of IPD cases globally, whereas serotypes 6A, 6B, 11A, 15B/C, and 23F are generally thought to be less invasive [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCovalent coupling of the capsular polysaccharide to a protein carrier effectively converts the T-cell-independent Type 2 polysaccharide into a T-cell-dependent antigen [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]; carrier proteins used to date include a non-toxic mutant of the \u003cem\u003eCorynebacterium diphtheriae\u003c/em\u003e toxin, \u0026lsquo;cross-reacting material 197\u0026rsquo; (CRM197), and protein D of a non-typeable \u003cem\u003eHaemophilus influenzae\u003c/em\u003e strain [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The use of the latter led to the development of the first 7-valent pneumococcal conjugate vaccine (PCV7), which was licensed in 2,000 and included capsular polysaccharides of serotypes 4, 6B, 9V, 14, 18C, 19F, and 23F, providing up to 90% protection against IPD in children [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. However, due to serotype shifts caused by vaccine pressure, the protective coverage of PCV7 was reduced, and it did not include serotypes 1, 3, and 5, which are common in Africa, Asia, and Europe [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Later, a 10-valent conjugate vaccine (PCV10, which includes the PCV7 serotypes plus capsular polysaccharides of serotypes 1, 5, 7F) and a 13-valent conjugate vaccine (PCV13, which includes PCV10 plus serotypes 3, 6A, and 19A) were introduced for children in 2010 and licensed for adults in 2012 [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Furthermore, to cover a significant portion of IPD caused by other invasive serotypes, PCV20 (Prevnar 20) has been introduced; it offers broad protection against 20 pneumococcal serotypes, including the 13 in PCV13 (i.e., 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, 23F) plus seven additional serotypes (i.e., 8, 10A, 11A, 12F, 15B, 22F, 33F), Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003ePCV10 was introduced in Uganda in 2013 and added to the immunization schedule in 2014. In 2019, a review of the vaccine's impact in low- and upper-middle-income countries concluded that PCVs have a significant effect [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]; however, the level of impact varies widely [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e] [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Furthermore, since the epidemiology of pneumococcal capsular types varies geographically and over time [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], understanding serotype distribution both spatially and temporally is essential for guiding vaccine use. Typically, sero-epidemiological data are lacking in many African settings. Therefore, this follow-up study aimed to examine pneumococcal carriage after PCV10 introduction in Uganda among children aged 5 years or younger, with a focus on carriage levels of vaccine-type (VT) serotypes. Thanks to notable advances in molecular technologies and our understanding of the pneumococcal genome [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], a real-time quantitative PCR (RT-qPCR) assay was employed to identify pneumococcal serotypes.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cp\u003e \u003cb\u003eSetting\u003c/b\u003e: The study was conducted in the Genomics, Molecular, and Immunology (GMI) laboratories at the Department of Immunology and Molecular Biology, Makerere University College of Health Sciences in Kampala, Uganda, between March 2018 and May 2019. It was a follow-up study to previous works in Uganda [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] on carriage rates for \u003cem\u003eS. pneumoniae\u003c/em\u003e, \u003cem\u003eHaemophilus influenzae\u003c/em\u003e, \u003cem\u003eMoraxella catarrhalis\u003c/em\u003e, and \u003cem\u003eStaphylococcus aureus\u003c/em\u003e in Ugandan children pre- and post-PCV10 introduction. Note that these prior urban studies [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e] did not genotype and/or serotype the pneumococci or other isolated bacterial species of interest. The Makerere GMI laboratories are accredited by the South African National Accreditation System (SANAS) \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.sanas.co.za/Pages/index.aspx\u003c/span\u003e\u003cspan address=\"https://www.sanas.co.za/Pages/index.aspx\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/p\u003e \u003cp\u003e \u003cb\u003ePneumococcal isolates and antibiotic sensitivity testing\u003c/b\u003e: A total of 156 phenotypically characterized samples (90 \u003cem\u003eS. pneumoniae\u003c/em\u003e, 40 \u003cem\u003eH. influenzae\u003c/em\u003e, 14 \u003cem\u003eM. catarrhalis\u003c/em\u003e, and 12 \u003cem\u003eS. aureus\u003c/em\u003e isolated from children aged 5 years or younger [n\u0026thinsp;=\u0026thinsp;194]) were retrieved from the \u0026minus;\u0026thinsp;80\u0026deg;C freezer and sub-cultured. Sample collection, culturing, and identification of isolates to the species level were described in the study by Nshimiyimana et al. [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. However, all isolates were reidentified to the species level using biochemical and microbiological methods in this study. \u003cem\u003eS. pneumoniae\u003c/em\u003e isolates were reidentified according to the WHO guidelines [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], i.e., colony characteristics (small, gray, alpha-hemolytic, glistening colonies tending to dip down in the center), Gram staining properties (positive diplococci), and biochemical characteristics (bile solubility and catalase reaction). An optochin sensitivity test was also performed: a disc impregnated with optochin was placed in the first streak area, and plates were incubated for 24\u0026ndash;48 hours at 37\u0026deg;C under 5%-10% CO\u003csub\u003e2\u003c/sub\u003e. A zone of inhibition greater than 14 mm around the optochin disc allowed us to identify isolates as \u003cem\u003eS. pneumoniae\u003c/em\u003e presumptively. For inhibition less than 14 mm, we performed the bile solubility test; if the turbid suspension was cleared in 2% sodium deoxycholate after 10\u0026ndash;15 minutes at 35\u0026ndash;37\u0026deg;C, the isolate was identified as \u003cem\u003eS. pneumoniae\u003c/em\u003e.\u003c/p\u003e \u003cp\u003ePhenotypic antibiotic susceptibility testing of pneumococci and other isolates was also described previously [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]; however, for \u003cem\u003eS. pneumoniae\u003c/em\u003e, repeat testing was performed to verify the susceptibility patterns. Briefly, bacterial colonies from a 24-hour-old subculture were suspended in sterile normal saline to reach a turbidity equivalent to the 0.5 McFarland standard. Isolates were prepared for antibiotic susceptibility testing using the disk diffusion method on Mueller-Hinton Agar (MHA) medium supplemented with 5% sheep blood. After inoculating on MHA, antibiotic disks and/or E-test strips were placed on the streaked plates, which were then incubated for 24 hours at 35\u0026ndash;37\u0026deg;C in 5%-10% CO\u003csub\u003e2\u003c/sub\u003e. \u003cem\u003eS. pneumoniae\u003c/em\u003e isolates were tested for susceptibility to oxacillin (1 \u0026micro;g), erythromycin (15 \u0026micro;g), clindamycin (2 \u0026micro;g), tetracycline (30 \u0026micro;g), chloramphenicol (30 \u0026micro;g), and trimethoprim/sulfamethoxazole (co-trimoxazole) (1.25/23.5 \u0026micro;g). Additionally, E-tests for penicillin G (P 32 \u0026micro;g/ml to 0.002 \u0026micro;g/ml) and ceftriaxone (CRO 32 \u0026micro;g/ml to 0.002 \u0026micro;g/ml) were performed on isolates with \u0026le;\u0026thinsp;19 mm inhibition zone diameters around the oxacillin disc. Zone diameters and minimum inhibitory concentrations (MICs) for various antibiotics were read and interpreted according to CLSI guidelines [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. For quality control, the reference \u003cem\u003eS. pneumoniae\u003c/em\u003e strain ATCC 49619 was used.\u003c/p\u003e \u003cp\u003e \u003cb\u003eMolecular Assays\u003c/b\u003e: Chromosomal DNA extraction, molecular identification, and characterization were performed at GMI Labs using subcultured and re-identified isolates. Briefly, bacterial cells from overnight cultures in Brain Heart Infusion (BHI) broth were washed and centrifuged at 16,000g for 10 minutes. After discarding the supernatant, the bacterial pellet was washed with 2 mL of TE buffer (10 mM Tris, pH 8, 1 mM EDTA) and centrifuged at 14,000 g for 5 minutes. The supernatant was discarded, and 100 \u0026micro;L of lysozyme (40 mg/\u0026micro;L) was added, followed by incubation overnight at 37\u0026deg;C. Next, 150 \u0026micro;L of a fresh 10% SDS/200\u0026micro;g/mL Proteinase K mixture was added, and incubation continued at 65\u0026deg;C for 1 hour. Subsequently, 200 \u0026micro;L of 5 M NaCl and 200 \u0026micro;L of CTAB/NaCl solution were added, the mixture was vortexed, and incubated at 65\u0026deg;C for another hour. An equal volume of chloroform/isoamyl alcohol (24:1) was added, the mixture was briefly vortexed, and centrifuged at 14,000 g for 5 minutes, resulting in three distinct phases. Approximately 750 \u0026micro;L of the upper aqueous phase was transferred to a new labeled tube. To the precipitate, an equal volume of absolute ethanol was added, and the mixture was left at room temperature for 1 hour. After centrifugation at 14000g for 5 minutes, the supernatant was discarded, and the pellet was resuspended in 500 \u0026micro;L of 70% ethanol, then centrifuged again. The pellets were air-dried for 10 minutes, eluted in 100 \u0026micro;L of TE buffer, and stored at -20\u0026deg;C until PCR analysis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eRT-qPCR assays\u003c/b\u003e were used to re-identify pneumococci, \u003cem\u003eHaemophilus influenzae\u003c/em\u003e, and \u003cem\u003eMoraxella catarrhalis\u003c/em\u003e. For pneumococci, we targeted the \u003cem\u003elytA\u003c/em\u003e gene, which encodes the major pneumococcal autolysin, serving as the \"gold standard\" target for RT-qPCR detection. For \u003cem\u003eHaemophilus influenzae\u003c/em\u003e, we focused on the \u003cem\u003eP6\u003c/em\u003e gene (Protein D), while for \u003cem\u003eMoraxella catarrhalis\u003c/em\u003e, the assay targeted the \u003cem\u003ecopB\u003c/em\u003e gene, which encodes the outer membrane protein. A \u0026lsquo;triplex RT-qPCR test\u0026rsquo; was used to identify all three organisms in a single, rapid reaction utilizing specific fluorescent channels; see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. For \u003cem\u003eS. aureus\u003c/em\u003e, we employed our previously published in-house conventional PCR assay targeting the \u003cem\u003enuc\u003c/em\u003e gene [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], on an Applied Biosystems Veriti 96-Well Thermal Cycler, Model 9902 (Connecticut, United States).\u003c/p\u003e \u003cp\u003e \u003cb\u003ePneumococcal Serotyping\u003c/b\u003e: All pneumococcal isolates underwent RT-qPCR testing to identify capsular serotypes using primers and probes described in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The assay aimed to detect serotypes included in the PCV10 vaccine currently used in Uganda, as well as those covered by PCV13 (see above). A multiplex RT-qPCR was performed in a 25 \u0026micro;l reaction containing 12.5 \u0026micro;l Platinum\u0026trade; Quantitative PCR SuperMix-UDG (ThermoFisher Scientific, Waltham, Massachusetts, United States), 1.5 \u0026micro;l MgCl2 (50 mM), 2.5 \u0026micro;l primer-probe mix (50 picomoles), 3.5 \u0026micro;l nuclease-free water, and 50 ng pneumococcal template DNA. Five triplex reactions (each 25 \u0026micro;l) were prepared for different serogroups following a previously described protocol [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Primers and probes were grouped based on melting temperature (Tm) and expected amplicon size, with RT-qPCRs set up as follows: reaction 1 for serotypes 1, 5, 23F; reaction 2 for serotypes 4, 6A/6B/6C/6D, 9V/9A; reaction 3 for serotypes 14, 18C/18F/18B/18A, 19F; reaction 4 for serotypes 3, 19A, 7F/7A; reaction 5 for serotypes 6C/6D and 23A. An internal positive control targeting the \u003cem\u003ecpsA\u003c/em\u003e gene of pneumococcal capsular polysaccharide synthesis, along with a no-template negative control, was included. The positive control was \u003cem\u003eS. pneumoniae\u003c/em\u003e strain ATCC 49619. Thermocycling was performed on an ABI 7500 PCR system (Applied Biosystems / Thermo Fisher Scientific, Waltham, Massachusetts, United States) following the manufacturer\u0026rsquo;s instructions, using the program listed in Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. A sample was considered positive only if its cycle-threshold (Ct) value matched the expected value for the target gene.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003ePrimer/probe sequences used to genotype bacterial isolates and/or determine the pneumococcal capsular serotypes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eTarget gene / serotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePrimer and Probe sequence\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eRole\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eReference\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003elytA\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ACG CAA TCT AGC AGA TGA TGA AGC-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcus\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eCarvalho et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TCG TGC GTT TTA ATT CGA GCT-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eYakYel\u003c/b\u003e/TGC CGA AAA/ZEN/CGC TTG ATA (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003eP6\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-CCA GCT GCT AAA GTA TTA GTA GAA G-3\u0026prime; (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies \u003cem\u003eHaemophilus influenzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eObasi et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TTC ACC GTA AGA TAC TGT GCC-3\u0026prime; (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eVIC\u003c/b\u003e 5\u0026prime;-CAG ATG CAG TTG AAG GTT ATT TAG- MGB-3\u0026rsquo; (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e\u003cem\u003ecopB\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-GTG AGT GCC GCT TTT ACA ACC-3\u0026prime; (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies \u003cem\u003eMoraxella catarrhalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eObasi et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u0026prime;-TGT ATC GCC TGC CAA GAC AA-3\u0026prime; (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e\u003cb\u003eNED\u003c/b\u003e 5\u0026prime;-TGC TTT TGC AGC TGT TAG CCA GCC TAA-3\u0026prime;-MGB (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e\u003cem\u003enuc\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCGATTGATGGTGATACGGTT-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eIdentifies \u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eKateete et al. [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AGCCAAGCCTTGACGAACTAAAGC-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TT CAT CCC TAT GTG TGG TAT AG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\" morerows=\"35\" rowspan=\"36\"\u003e \u003cp\u003eCarvalho et al. [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]; CDC [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]; Yun et al. [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GCT TTA GAA GGT AGA GTT AAC ACC-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/56-\u003cb\u003eFAM\u003c/b\u003e/TGC CAA AGC CAG CCA T/31A (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCA CTA AAG CTT TGG CAA AAG AAA-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 3\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCC GAA CGT AAA GCT TCT TCA-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eHEX\u003c/b\u003e/TTG TAG ACC/ZEN/GCC CCA CAA T (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5`-GCT TCT GCT GTA ACT GTT GTC C-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 4\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAC CAC CAT AGT AAC CAA AGT TCC-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eCy5\u003c/b\u003e/TTC CAC AAA/TAO/AGA AGA GCC T (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAT GAT TTA TGC CCT CTT GCA A-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 5\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GAC AGT ATA AGA AAA AGC AAG GGCTA (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eHEX\u003c/b\u003e/TCT TCT TCT/ZEN/CAT CGT TTC CG (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e6A/B/C/D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GTT TGC ACT AGA GTA TGG GAA GG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 6\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TAG CCT TTC TGA AAA CAT TTA GCG-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eHEX\u003c/b\u003e/TGT TCT GCC/ZEN/CTG AGC AAC T (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e7F/7A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ATG AAG GCT TTG GTT TGA CAG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 7\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ATT CTC GCC ATC AAT TGC ATA TTC-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eCy5\u003c/b\u003e/ACA CCA CTA/TAO/TAG GCT GTT GA (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e9V/9A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AGG TAT CCT ATA TAC TGC TTT AGG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 9\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ACGA ATC TGC CAA TAT CTG AAA G-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eHEX\u003c/b\u003e/ACA\u0026thinsp;+\u0026thinsp;ACA+CAT\u0026thinsp;+\u0026thinsp;TGA+CAA\u0026thinsp;+\u0026thinsp;CCG CT/31A (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-AGA GTG TAT GAG GAA TCC-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 14\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-ATA TAT CTA CTG TAG AGG GAA T-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/56-\u003cb\u003eFAM\u003c/b\u003e/C QC CAA GTA/ZEN/AAT CAA (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CGC CTA GTC TAA ATA CCA-3 (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 19A\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GAG GTC ACC TAT AAT AGT AAGAG-3 (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/56-\u003cb\u003eFAM\u003c/b\u003e/TAT CAA TGA/ZEN/GCC GAT CCG (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e19F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TGA GGT TAA GAT TGC TGA TCG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 19F\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CAC GAA TGA GAA CTC GAA TAA AAG-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-\u003cb\u003eCy5\u003c/b\u003e/CGC\u0026thinsp;+\u0026thinsp;ACT+GTC\u0026thinsp;+\u0026thinsp;AAT+TCA CCT TC (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e18C/18F/18A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TCG ATG GCT AGA ACA GAT TTA TGG-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-CCA TTG TCC CTG TAA GAC CAT TG-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-5\u003cb\u003eHEX\u003c/b\u003e/AGG GAG TTG/ZEN/AAT CAA CCT A (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003e23\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-GAC AGC ACC GAC AAT AGT CAT CTC-3' (Forward)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"2\" rowspan=\"3\"\u003e \u003cp\u003eIdentifies pneumococcal serotype 23\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-TCC ATC CCA ACC TAA CAC ACT TC-3' (Reverse)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5'-/5\u003cb\u003eCy5\u003c/b\u003e/ATT GTG TCC/TAO/ATA ACC CTT CG (Probe)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe primers and probes were synthesized by Integrated DNA Technologies Inc (IDT), Coralville, Iowa, United States.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eRT-qPCR program for identification of the pneumococcal serotypes\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSteps\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTemperature\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTime\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eAcquisition\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eStage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCycles\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eInitial denaturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e10 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eHold\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eDenaturation\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e95\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15 sec\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eNo\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003eCycling\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c6\" morerows=\"1\" rowspan=\"2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCombined annealing and extension\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e60\u0026deg;C\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 min\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eYes\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eAll 156 stored isolates (90 pneumococci, 40 \u003cem\u003eH. influenzae\u003c/em\u003e, 14 \u003cem\u003eM. catarrhalis\u003c/em\u003e, 12 \u003cem\u003eS. aureus\u003c/em\u003e) from a previous study [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e] were successfully sub-cultured and genotyped with a focus on molecular serotyping of \u003cem\u003eS. pneumoniae\u003c/em\u003e. RT-qPCR genotyping confirmed 64 (71%) isolates as pneumococci, 16 (40%) \u003cem\u003eH. influenzae\u003c/em\u003e, 9 (64%) \u003cem\u003eM. catarrhalis\u003c/em\u003e, and 12 (100%) \u003cem\u003eS. aureus\u003c/em\u003e, Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpecies-level confirmation of isolates by RT-qPCR\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSpecies\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003e# isolates tested\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e# positive on RT-qPCR\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eFrequency (%)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStreptococcus pneumoniae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e71\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eHaemophilus influenzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e16\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e40\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eMoraxella catarrhalis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e9\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e64\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cem\u003eStaphylococcus aureus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e12\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e100\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTotal\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e156\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e \u003cp\u003e86\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e\u0026nbsp;\u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eOverall, based on RT-qPCR genotyping, the pneumococcal carriage prevalence in this urban pediatric population (n\u0026thinsp;=\u0026thinsp;194, [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]) five years after introducing PCV10 is 33% (64/194), a significant significantly lower than the carriage rates reported in the pre-PCV era, which ranged from 56% to 77% [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Because the majority of isolates, i.e., 41/64 (64.1%), were not serotyped by our assay panel, they may represent non-vaccine serotypes or non-typeable variants. Furthermore, a decline in pneumococcal carriage after PCV vaccination is expected, given the documented reduction in carriage of VT serotypes following vaccination [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. However, vaccination may not affect the overall pneumococcal carriage rate, since VT serotypes are replaced by non-VT serotypes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. The carriage rate observed in this study (33%) is consistent with recent reports of low serotype coverage for PCV10 in Ugandan children, estimated at 33%-42% [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Nevertheless, studies have generally noted higher carriage of VT serotypes years after PCV introduction in African settings compared to high-income countries [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], suggesting that PCVs may be less effective at reducing serotypes linked to IPD in low-income countries.\u003c/p\u003e\n\u003ch3\u003ePneumococcal Serotypes:\u003c/h3\u003e\n\u003cp\u003eAll 64 \u003cem\u003elytA\u003c/em\u003e-positive pneumococcal isolates were serotyped molecularly to investigate carriage of serotypes covered by the PCV10 and PCV13 vaccines, namely 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. Note, since we serotyped only 23 (36%) of 64 \u003cem\u003elytA\u003c/em\u003e-positive isolates (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e), the rest were perhaps non-vaccine serotypes or non-typeable by the assay panel. Except for serotypes 1, 4, 5, 6A, 6B, 23F, and 19F, all other serotypes were found in at least one sample, but at very low frequencies (less than 10%), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Serotype 14 was the most common, detected in six samples (9.4%), followed by serotype 19A in five samples (8%). Serotypes 3, 7F, and 9V were present in 4 samples each (6.3%), while serotype 18C was the least frequent, found in 1 sample (2%), as shown in Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. Note that serotypes 3 and 7F coexisted in a sample (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e), and serotypes 3 and 19A are not covered by PCV10, which is currently used in Uganda (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab4\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 4\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eFrequency of VT* serotypes in children 5 years post PCV10 roll-out\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"11\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c9\" colnum=\"9\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c10\" colnum=\"10\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c11\" colnum=\"11\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eIsolate#\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSerotype\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eFrequency (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eChloramphenicol\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eTetracycline\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eSXT\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003eClindamycin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eErythromycin\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c9\"\u003e \u003cp\u003ePenicillin (\u0026ge;\u0026thinsp;0.06 mg/l)**\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c10\"\u003e \u003cp\u003eCeftriaxone (\u0026le;\u0026thinsp;1mg/l)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c11\"\u003e \u003cp\u003eCeftriaxone (\u0026le;\u0026thinsp;0.5 mg/l)**\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP121\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"5\" rowspan=\"6\"\u003e \u003cp\u003e6 (9.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP122\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP014\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP028\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP286\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP053\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP055\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" morerows=\"4\" rowspan=\"5\"\u003e \u003cp\u003e5 (8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP085\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP090\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP094\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e 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colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP119\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP126\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\" 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colname=\"c1\"\u003e \u003cp\u003eSP130\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e7F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e 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colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP148\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP149\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e9V\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP160\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e18\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1 (2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003eR\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003eS\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003eI\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP013\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0144\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0184\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e5\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0177\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6A\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0164\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e6B\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0196\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e19F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSP0104\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e23F\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c8\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c9\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c10\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c11\"\u003e \u003cp\u003e\u003cb\u003e-\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eS, Susceptible; I, Intermediate; R, Resistant; -, Not applicable; SXT, trimethoprim-sulfamethoxazole; MDR, multidrug resistant. *With respect to the PCV10 and PCV13 vaccines. **CLSI interpretation guidelines for \u003cem\u003eS. pneumoniae\u003c/em\u003e by minimum inhibitory concentration (MIC) susceptibility testing.\u003c/p\u003e \u003cp\u003eIn summary, four of the ten serotypes covered by the PCV10 vaccine (4, 6B, 9V, 14, 18C, 19F, 23F, 1, 5, 7F)\u0026mdash;specifically 14, 7F, 9V, and 18C\u0026mdash;remain carried by children, though at low frequencies. This is concerning. These serotypes were also detected in studies in Eastern and Southwestern Uganda before the introduction of PCV10 [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and Hammit et al. found serotypes 14 and 19A persisting in Kenyan children vaccinated with PCV10 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Before PCV10 was introduced in Uganda, the most common serotypes reported were 19F, 6A/6B, and 23F [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which appear to be suppressed by the vaccine. Overall, while vaccine trials and post-introduction studies in Africa show significant benefits of PCV immunization against IPD and all-cause mortality in children, African countries consistently report high residual carriage of VT serotypes [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. This cannot be solely attributed to low vaccine coverage, as studies report high uptake rates\u0026mdash;up to 90\u0026ndash;95% in Kenya [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] and Malawi [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Since low overall carriage prevalence is necessary for effective control of pneumococcal carriage and, ultimately, IPD, persistent VT carriage in Africa might be high enough to sustain pneumococcal transmission among younger children and other vulnerable groups [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAdditionally, pneumococcal serotypes in this study were highly resistant to common antibiotics, i.e., tetracycline, chloramphenicol, erythromycin, and clindamycin (Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e). Moreover, additional serotypes, notably 29, are common in Uganda but are not covered by the current PCVs (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e); serotype 29 is reported to be associated with infection, antibiotic resistance, and invasive disease [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. This is a concern in that, while IPD and pneumococcal infections are treatable with antibiotics, and immunization provides protection, shifts in serotypes and the growing antibiotic resistance in clinically relevant serotypes reduce the efficacy of both approaches to control [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. Worldwide, ~\u0026thinsp;600,000 deaths every year are attributed to antibiotic-resistant \u003cem\u003eS. pneumoniae\u003c/em\u003e, and serotypes resistant to common antibiotics include 6B, 9V, 14, 19A, 19F, and 23F, which, unfortunately, are predominant in Uganda and generally in Africa [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], Table\u0026nbsp;\u003cspan refid=\"Tab4\" class=\"InternalRef\"\u003e4\u003c/span\u003e.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eSerotypes covered by PCV10 currently in use in Uganda can still be carried by vaccinated children years after vaccination. Since a low universal carriage prevalence is required for effective control of pneumococcal carriage and, eventually, IPD, residual VT carriage could be high enough to maintain pneumococcal transmission in the community.\u003c/p\u003e\n\u003ch3\u003eLimitations\u003c/h3\u003e\n\u003cp\u003eOne limitation of this study is that the single-center, cross-sectional design and relatively small sample size could limit generalizability; hence, caution is necessary when interpreting our results. The study also lacked a control or comparison group, e.g., unvaccinated children; however, to assess the vaccine's impact, we compared our findings with pre-vaccine cohorts from other studies in the same setting before PCV10 was rolled out. For deeper insight into this setting, we recommend more robust approaches, such as whole-genome sequencing of the pneumococcal isolates. Another limitation is that we were unable to reconfirm 30 (33%) of the 90 stored pneumococci, suggesting i) degradation during storage or freeze/thaw effects, ii) an incorrect original biochemical/microbiological identification, or iii) effects of subculture on RT-qPCR.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIPD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eInvasive pneumococcal disease\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePCV\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePneumococcal conjugate vaccine\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eVT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eVaccine-type serotype\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCDC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCenters for Disease Control\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMHA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMueller-Hinton Agar\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eRT-qPCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eReal-time quantitative polymerase chain reaction\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGenomics, Molecular, and Immunology Laboratories\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eBHI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eBrain Heart Infusion\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTm\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMelting temperature\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eTE\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eTris-EDTA buffer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCTAB\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCetyltrimethylammonium bromide buffer\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMinimum inhibitory concentration\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCt\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eCycle-threshold value\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCLSI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe Clinical \u0026amp; Laboratory Standards Institute\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eATCC\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe American Type Culture Collection\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eWHO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eThe World Health Organization\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e \u003cstrong\u003eEthics approval and consent to participate:\u003c/strong\u003e \u003cp\u003eThe study obtained ethical approval from the Makerere University School of Biomedical Sciences Research and Ethics Committee (#SBS-620) and from the Mulago Hospital Research and Ethics Committee (#MHREC1574). Written informed consent was obtained from the parents and/or guardians of the recruited children, and consent for sample storage and use of stored samples in further studies was also obtained from the parents. The data were anonymized, and the authors did not have access to information that could identify individual participants during or after data collection.\u003c/p\u003e \u003c/p\u003e \u003cp\u003e \u003cstrong\u003eConsent for publication:\u003c/strong\u003e \u003cp\u003eNot applicable\u003c/p\u003e \u003c/p\u003e\u003cp\u003e \u003ch2\u003eCompeting interests:\u003c/h2\u003e \u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e \u003c/p\u003e\u003ch2\u003eFunding:\u003c/h2\u003e \u003cp\u003eNone\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eDPK conceived and designed the study, analyzed and interpreted the data, and drafted the manuscript. Alex N and TN performed the data collection and were major contributors in writing the manuscript. AS and POO provided research support and funding. DPK, Ann N, and AA supervised the project. All authors read and approved the final manuscript.\u003c/p\u003e\u003ch2\u003eAcknowledgements\u003c/h2\u003e \u003cp\u003eWe thank the Global Health Labs (GHL), Bellevue, WA, United States, for ordering primers and probes from IDT Inc. and sending them to us. We also thank Ms. Praiscilla Kia and Ms. Esther Nakafu for their support rendered to this work.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eAll data supporting the findings of this study are available within the paper.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLoughran AJ, Orihuela CJ, Tuomanen EI. Streptococcus pneumoniae: Invasion and Inflammation. Microbiol Spectr. 2019;7(2). 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Available from: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.cdc.gov/strep-lab/php/pneumococcus/serotyping-using-pcr.html#cdc_generic_section_2-real-time-pcr\u003c/span\u003e\u003cspan address=\"https://www.cdc.gov/strep-lab/php/pneumococcus/serotyping-using-pcr.html#cdc_generic_section_2-real-time-pcr\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLindstrand A, Kalyango J, Alfv\u0026eacute;n T, Darenberg J, Kadobera D, Bwanga F, et al. Pneumococcal Carriage in Children under Five Years in Uganda-Will Present Pneumococcal Conjugate Vaccines. Be Appropriate? 2016;11(11):e0166018.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRutebemberwa E, Mpeka B, Pariyo G, Peterson S, Mworozi E, Bwanga F et al. High prevalence of antibiotic resistance in nasopharyngeal bacterial isolates from healthy children in rural Uganda: A cross-sectional study. 2015;120(4):249\u0026ndash;56.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSpijkerman J, Prevaes SM, van Gils EJ, Veenhoven RH, Bruin JP, Bogaert D, et al. Long-term effects of pneumococcal conjugate vaccine on nasopharyngeal carriage of S. pneumoniae, S. aureus, H. influenzae and M. catarrhalis. PLoS ONE. 2012;7(6):e39730.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlobal burden of bacterial antimicrobial resistance. in 2019: a systematic analysis. Lancet. 2022;399(10325):629\u0026ndash;55. Epub 20220119. doi: 10.1016/s0140-6736(21)02724-0. PubMed PMID: 35065702; PubMed Central PMCID: PMCPMC8841637.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKisakye A, Makumbi I, Nansera D, Lewis R, Braka F, Wobudeya E, et al. Surveillance for Streptococcus pneumoniae meningitis in children aged\u0026thinsp;\u0026lt;\u0026thinsp;5 years: implications for immunization in Uganda. Clin Infect Dis. 2009;48(Suppl 2):S153\u0026ndash;61. 10.1086/596495. PubMed PMID: 19191611.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNackers F, Cohuet S, le Polain de Waroux O, Langendorf C, Nyehangane D, Ndazima D, et al. Carriage prevalence and serotype distribution of Streptococcus pneumoniae prior to 10-valent pneumococcal vaccine introduction: A population-based cross-sectional study in South Western Uganda, 2014. Vaccine. 2017;35(39):5271\u0026ndash;7. PubMed PMID: 28784282; PubMed Central PMCID: PMCPMC6616034.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Streptococcus pneumoniae, Pneumococcus, Carriage prevalence, Invasive pneumococcal disease, IPD, Pneumococcal capsular serotypes, PCV10, PCV13, RT-qPCR, Children under 5 years, Kampala, Uganda","lastPublishedDoi":"10.21203/rs.3.rs-9423476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9423476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eUganda introduced the 10-valent pneumococcal conjugate vaccine (PCV10) into routine infant immunization in 2014, but post-introduction molecular data on residual vaccine-type carriage remain limited. We assessed nasopharyngeal pneumococcal carriage and vaccine-type serotype distribution among urban Ugandan children aged\u0026thinsp;\u0026le;\u0026thinsp;5 years, five years after PCV10 introduction, using real-time quantitative PCR (RT-qPCR).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn this cross-sectional follow-up study, 156 stored nasopharyngeal bacterial isolates from 196 children were re-cultured and re-characterized; 64/90 phenotypically identified pneumococcal isolates were confirmed by \u003cem\u003elytA\u003c/em\u003e RT-qPCR, corresponding to an overall pneumococcal carriage prevalence of 33% (64/195). Molecular serotyping of all 64 pneumococcal isolates for PCV10/PCV13-associated capsular targets identified 23/64 (35.9%) vaccine-type isolates, while 41/64 (64.1%) were non-vaccine or non-typeable by the assay panel. Serotype 14 predominated (6/64, 9.4%), followed by 19A (5/64, 7.8%), and serotypes 3, 7F, and 9V (each 4/64, 6.3%); serotype 18C was detected in 1/64 (1.6%). Serotypes 1, 4, 5, 6A/6B, 19F, and 23F were not detected. Persistent carriage of PCV10 serotypes 7F, 9V, 14, and 18C indicates residual vaccine-type circulation despite an overall reduction in carriage prevalence. This is concerning, since a low universal carriage prevalence is necessary for effective pneumococcal disease control.\u003c/p\u003e","manuscriptTitle":"Pneumococcal Serotypes in Urban Ugandan Children Vaccinated with the 10-Valent Pneumococcal Conjugate Vaccine","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-04 09:58:51","doi":"10.21203/rs.3.rs-9423476/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"295846505788804648159459915842239772723","date":"2026-05-14T08:36:21+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T10:46:59+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"69482115444968803606052132195956759876","date":"2026-05-05T09:33:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"157048877758596368488116809291719620122","date":"2026-04-28T06:53:04+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"55484698199608574371983569580467005876","date":"2026-04-27T18:54:02+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-04-22T18:42:47+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2026-04-20T10:40:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-20T06:52:00+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-20T06:51:05+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2026-04-15T07:39:48+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"89c591e2-8ad7-4daa-bed3-3c0d09c81411","owner":[],"postedDate":"May 4th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewerAgreed","content":"295846505788804648159459915842239772723","date":"2026-05-14T08:36:21+00:00","index":38,"fulltext":""},{"type":"editorInvitedReview","content":"","date":"2026-05-05T10:46:59+00:00","index":30,"fulltext":""},{"type":"reviewerAgreed","content":"69482115444968803606052132195956759876","date":"2026-05-05T09:33:04+00:00","index":29,"fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-04T09:58:51+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-04 09:58:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9423476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9423476","identity":"rs-9423476","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}