Streptococcus pneumoniae Serotype Epidemiology among PCV-10 Vaccinated and Unvaccinated Children at Gertrude’s Children’s Hospital, Nairobi County: A Cross-Sectional Study

Background: Serotype replacement and emergence of multidrug resistant S. pneumoniae has exacerbated the need for continuous regional serotype surveillance especially in the developing world. We investigated S. pneumoniae serotypes circulating among vaccinated and unvaccinated children ≤5 years in Nairobi County post PCV10 era. Methods: A total of 206 vaccinated and unvaccinated children attending Gertrude’s Children’s Hospital (GCH) were recruited for this study. Nasopharyngeal swabs collected using Copan Flocked Swabs were the main study specimen. Culturing and isolation of S. pneumoniae was done on BA with gentamicin and BA plates respectively at the GCH main laboratory. Serotyping was done using the Quellung reaction at the KEMRI-Wellcome Trust, Kilifi. Results: Out of the 206 subjects sampled, 20.39% (42) were found to be carriers of S. pneumoniae. About 52% (n=22) of the S. pneumoniae carriers had received the recommended dose of PCV-10, while 48% (n=20) of the carriers had not. Almost all (n=41; 19.90% of subjects) isolates contained non-vaccine type S. pneumoniae serotypes, while n=1 of the serotypes (in 0.49% of subjects) were untypeable. Serotypes 28F, 6A, 11A, 3 and 7C were prevalent in both vaccinated and unvaccinated children, whereas serotypes 23A, 17F, 35F, 48, 13 and 35B, and 23B, 20, 19B, 21, untypeable, 15B and 39 were found among unvaccinated and vaccinated groups, respectively. Conclusions: All S. pneumoniae serotypes isolated from the subjects sampled were non PCV-10 vaccine type. These results therefore highlight the importance of monitoring and evaluation to provide epidemiological information to determine the effectiveness of PCV10 in Kenya’s Public health services.


Amendments from Version 1
After receiving reviews from the three proposed peer reviewers, I have made corrections to include their concerns as follows: 1. Changed description of Streptococcus pneumoniae from "highly invasive" to "friendly gram positive inhabitant of the human upper respiratory tract" in paragraph 1.
2. Edited the use of the abbreviation "SPn" to "S. pneumoniae" across the text.
3. Reviewed paragraphs 1 & 5 to remove noted repetitions and inconsistencies regarding the exact number of S. pneumoniae serotypes 4. I changed the previous wording of the conclusion in the abstract from "Kenyan children currently using PCV-10 vaccine are not protected" to "these results therefore highlight the importance of monitoring and evaluation to provide epidemiological information to determine the effectiveness of PCV10 in Kenya's Public health services" 5. Reviewed the paper to indicate that the evaluation was done after introduction of PCV-10 in 2010 and also reviewed the methodology section to make it a summarized version of what is in the main text.

See referee reports
REVISED not protect against these conditions if they are caused by agents other than S. pneumoniae or from strains not included in the vaccine (Moffitt & Malley, 2011 Pneumococcal conjugate (PCVs) and polysaccharide (PPVs) vaccines are designed according to their virulence mechanisms and how they generally interact with the human immune system (Castañeda-Orjuela et al., 2012). The WHO has advised that all children ≤5 years should be immunized against pneumococcal disease and continuous surveillance done to keep out the disease especially in the developing world (Vandenbos et al., 2013). The need for continuous surveillance has been exacerbated by the acute emergence of multi-drug resistant S. pneumoniae strains and escalated child mortality and morbidity due to pneumococcal disease, despite the availability of PCVs and PPVs (Väkeväinen et al., 2010). This study therefore sought to establish the S. pneumoniae serotypes among vaccinated and unvaccinated children ≤5 years of age in Nairobi County, Kenya.

Study Location
This study was conducted among children ≤5 years attending the outpatient department of Gertrude's Children's Hospital in Nairobi County between May 2017 and February 2018. Subjects were clinically assessed by a physician and those who presented with pneumococcal disease symptoms recommended to the study nurse for recruitment. Gertrude's Children's Hospital is the largest standalone health care facility specializing in pediatric care in East and Central Africa. The hospital is accredited by the Joint Commission on International Accreditation (JCIA). S. pneumoniae isolation and stocking was done at Gertrude's Children's Hospital Main Laboratory and capsular serotyping done at KEMRI Wellcome Trust, Kilifi, Kenya.

Study Design
This was a descriptive cross-sectional study. S. pneumoniae serotype epidemiology among PCV-10 vaccinated and unvaccinated children between 6 months and 5 years of age was measured. Children who had no history of any chronic disease and whose parents or legal guardians consented to the study were systematically recruited. Children whose parents or legal guardians declined to give consent and those with any known immunosuppressive conditions were excluded from the study.

Introduction
Streptococcus pneumoniae is a friendly gram positive inhabitant of the human upper respiratory tract but can be highly invasive in some conditions (Mitchell & Mitchell, 2010

− =
Where n= desired minimal sample size; z= standard normal deviation (1.96, from the tailed normal table); p = prevalence rate; and m= the desired degree of accuracy at a 95% confidence level of 0.05. This gave a sample size of 206.

Identification of S. pneumoniae
Nasopharyngeal swabs were per nasally collected using Copan flocked swabs and temporarily suspended in Armies medium for transportation to the main laboratory. Each swab was inoculated onto a selective gentamicin with 5% sheep blood agar (BA) plate. All swabs were plated within 24 h of collection. The plates were incubated at 37°C in a 5% CO 2 atmosphere and examined at 16-24 h and then again at 40-48 h for growth of S. pneumoniae. Isolates were identified as S. pneumoniae by colony morphology (Mucoid, draughtsman appearance, α-haemolysis) and susceptibility to optochin (positive, ≥14 mm zone of inhibition; negative, <14 mm zone of inhibition). Plates with colonies akin to S. pneumoniae morphological features but with optochin clearance zones below 14 mm were further subjected to solubility in bile salts (positive, bile soluble; negative, bile insoluble).
The isolation of a single colony indicated carriage. Single colonies were picked using sterile inoculating loops and evenly plated on BA. After 24-48 h, enough inoculum was stocked in brain heart infusion (BHI) agar with 5% sheep blood (Ultralab East Africa, Ltd), gently vortexed and stored at -70°C for serotyping.
Serotyping of S. pneumoniae Capsular serotyping was done using the Quellung reaction test. Frozen vials containing S. pneumoniae stocks stored at -70°C were thawed at room temperature for about 30 minutes. A loopful of the stored S. pneumoniae cells were suspended in 50 µl PBS and gently vortexed. Subsequently, 10 µl of the suspended cells were added on to a glass slide and mixed with 5 µl pooled antisera (Statens Serum Institute, cat. No.16744). The glass slide was swirled gently while observing for any agglutination reaction until a positive reaction was observed with various pooled antisera. The process was repeated with individual groups under various antisera pools.
After that, 10 µl of the suspended cells in PBS were added to a glass slide and mixed with various S. pneumoniae serotypespecific antisera included in the antisera pools that gave a positive reaction. This was done until a positive reaction with the particular serotype specific antisera was observed. Those serotypes that did not belong to any pool were typed directly until a positive agglutination reaction was observed. The cells/ PBS/serotype-specific antisera mixture on the glass slide were covered with a cover slip and observed under a phase contrast microscope with a ×100 objective lens with oil emulsion.

Discussion
This study found that 20.39% of all children studied, from both the PCV-10 vaccinated and unvaccinated groups, were carriers of S. pneumoniae. While this is a significant reduction from the pre-vaccine era, it is still high compared to malaria, diarrhea and HIV/AIDS (Feikin et al., 2010). In total, n=41 of the serotypes found were non-vaccine type (in 19.90% of the subjects), with one additional untypeable serotype. This is a very important finding as it explains the high level of child morbidity and mortality due to pneumococcal disease despite the availability of PCV-10.
While these findings agree partially agree with those of (Jacobs et al. (2008), where a significant decrease in the vaccine type S. pneumoniae serotypes found in isolates was observed, a 97.6% (n=41) decrease is, at the very least, surprising. This trend may be attributed to the increased level of antimicrobial misuse by a greater percent of the study population (Domenech de Cellès et al., 2011). 10-valent pneumococcal conjugate vaccine contains 10 different serotypes, which include: 1, 4, 5, 6B, 7F, 9V, 14, 18C, 19F, 23F (Slotved et al., 2016). None of these 10 serotypes was found in the study population yet this is the vaccine currently included in KEPI, targeting the same population.
S. pneumoniae carriage decreased with age as 11.65% (n=24) were obtained from children aged between 6-24 months and 8.74% (n=18) from children >24 months. The study results demonstrated a linear relationship between child age and S. pneumoniae carriage. A similar study done elsewhere reported findings that partly agree with this and partly disagrees (Hill et al., 2008).
The former being attributable to development of S. pneumoniaespecific IgG antibodies due to vaccination and during that window before most children start attending school (Corscadden et al., 2013). Unlike findings from a study by (de Paz et al. (2015), serotype 28F was the most prevalent and was present in all five age groups profiled. This is a likely scenario of serotype replacement as S. pneumoniae attempts to evade the action of the immune system and eventually shares the resistant genes within the microbial community, especially in the nasopharyngeal region (Donati et al., 2010).
Serotypes 28F, 6A, 11A, 3 and 7C were prevalent in both vaccinated and unvaccinated children, whereas serotypes 23A, 17F, 35F, 48, 13, 35B and 23B, 20, 19B, 21, untypeable, 15B, 39 were found among unvaccinated and vaccinated groups respectively. There exist different antigenic features between and within various strains of S. pneumoniae (Song et al., 2012). While the majority, if not all, S. pneumoniae serotypes are capable of causing disease, the frequency with which they are isolated varies (Kalin, 1998). In this case, vaccination would only be partially effective and, if so, due to inter-strain antigenic characteristics.
While trying to evade the action of the immune system, S. pneumoniae has a tendency to exchange resistant genes and other antigenic correlates at the nasopharyngeal region (Johnston et al., 2014). Resistance to antimicrobial agents is occasioned by among other factors, misuse of antibiotics (Dinsbach, 2012). This is largely due to lack of properly enforced antibiotic use regulations by the authorities.   The study reports pneumococcal carriage in a cohort of Kenyan children. This has significant implication when it comes to the evaluation of the impact of PCV10 on the population structure of pneumococcus.

Specific comments:
Pneumococcus is a normal commensal, not "highly invasive" as is reported in intro.
The authors must avoid the use of non-standard nomenclature such as SPn. Further, there is inconsistent use of S. pneumoniae and pneumococcus throughout the text. These cannot be used interchangeably.
The introduction needs to be reworked and repetitions avoided, i.e. in paragraph 1 and 5, the authors talk about 90 serotypes. The last sentence of paragraph 5 does not include references.
The study was conducted between 2017 and 2018, it would really have benefited from the WHO working group report on pneumococcal carriage studies . Most importantly, 79% (164/206) being non-viable seriously indicates problems in the experimental design. The authors don't say anything 1 4.

11.
non-viable seriously indicates problems in the experimental design. The authors don't say anything about broth enrichment in order to improve recovery of the pneumococcus. Amies media is not ideal for pneumococcus compared to STGG. A 10ul innoculum is very little, did the authors attempt to use bigger volumes especially for the samples with no growth? A 2% BA plate is used as primary culture then selective media, if possible, do this in parallel.
Need to be more consistent in reporting proportions.
What was the PCV10 vaccine coverage at each timepoint? Also authors need to report non-pcv as non-PCV10 because some of the serotypes the report as "non-vacccine" such as serotype 3 are included in PCV13.
Repetition of results. The authors do not report any metadata looking at risk factors for carriage, hence the "epi" in title must fall out.
I have read this submission. I believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.
Author Response 06 Jan 2019 , Kenyatta University, Kenya Michael Walekhwa Dear Dr. Ondigo, Many thanks for your review of this article.
I have read through and keenly updated the areas you highlighted during your review. Kindly revisit.

Many thanks
No competing interests were disclosed.

Competing Interests:
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