High multiple carriage and emergence of Streptococcus pneumoniae vaccine serotype variants in Malawian children

This study characterised the diversity of pneumococcal carriage in Malawian children by employing a sensitive microarray serotyping method. We observed a much higher diversity of pneumococcal serotypes in Malawian children than reported elsewhere in Africa [29], where serotyping was by a less sensitive latex agglutination assay. The higher serotype diversity in Malawian children could be attributed high rates of multiple carriage and by the use of a more sensitive method of identification. We also observed that carriage of NVT represented a high proportion (~40 %) of all serotypes circulating in Malawi. These NVTs may ultimately increase in the Malawian population due to serotype replacement following the recent introduction of PCV13 [11], potentially leading to an increase in NVT IPD cases [12, 30]. A recent study in Germany has reported an increase of IPD due to NVT serotypes 15A and 23B post PCV13 [31]. Serotype 15A was detected in our dataset (Fig. 1) and it would be important to monitor the prevalence of serotype 15A and other NVT serotypes in both carriage and IPD in Malawi, post PCV13. We have shown, statistically, that younger children (0–2 years) carried a significantly higher proportion of PCV13 serotypes compared to older children (3–13 years) (p = 0.028). This supports observations from a recent carriage study in Kathmandu, Nepal where 44.4 % (132/297) of the pneumococcal serotype positive swabs from children aged 0–24 months contained one or more PCV13 serotypes [32]. This finding suggests that children in this age group are a reservoir of vaccine serotypes. Targeting this group for pneumococcal conjugate vaccination would therefore prevent the spread of vaccine serotypes within the community, thereby ensuring herd immunity.

Carriage of NT strains represented <1 % of all the carried population detected in Malawian children. Three categories of NTs have been reported recently [33] based on (i) complete deletion of the cps gene cluster (NT1), (ii) the sole presence of novel surface protein nspA gene (NT2) at the CPS locus or (iii) the presence of a conserved aliB-cluster (NT3) at the CPS locus. NTs are usually associated with carriage rather than IPD [34], which could explain why they are not included in the current vaccine formulations. However, a recent study reported that the highest rates of genetic recombination occurred in NT pneumococcal strains, which suggests their potential importance in genetic exchange events as well as species adaptation [9]. Although children in Malawi carried only a small proportion of NTs (<1 %), the ability by NTs to recombine readily may be central to the spread of antibiotic resistance, which could have a negative impact on disease control efforts.

At 40 %, we have demonstrated that the rate of multiple carriage among Malawian children is as high as has been reported elsewhere [4, 35]. Multiple carriage is thought to promote the horizontal gene transfer of antibiotic resistance and virulence genes [8, 36‐38], which may contribute to the pathogen adaptation and increased risk of disease in the host. A recent report suggests that multiple carriage may promote co-infection with two or more pneumococcal serotypes [39]. It is therefore important to understand the dynamics of multiple carriage in a given setting in order to control pneumococcal spread and disease.

We did not find any statistically significant difference in the prevalence of multiple carriage between HIV negative and HIV positive children, which is similar to our recent report in adults [14]. To date, the effect of HIV infection on pneumococcal carriage is currently not fully understood; hence further studies on much larger datasets need to be conducted to address such questions. Although pneumococcal carriage rates have been shown to decrease with age [40], this study could not establish the association between age and the prevalence of multiple carriage in children and further studies are therefore recommended.

The polysaccharide capsule is essential for pneumococcal survival and transmission within the host by acting as a barrier to phagocytic killing [41]. The capsule is also a target for current conjugate vaccine formulations. One of the key mechanisms by which S. pneumoniae survives the host immune response and the effect of vaccination is to alter its CPS locus, through mutations and genetic recombination. We detected naturally occurring CPS locus variants in vaccine-associated serotypes 6B, 19A and 20. Although multiple carriage is reported to promote genetic recombination through horizontal gene transfer [42], it is not clear whether this played a role in altering the CPS genes of vaccine serotypes reported here.

Serotype 6B causes 10 % of IPD in young children globally and ranks second after serotype 14 [24]. In Malawi, serotype 6B is the second most isolated strain from invasive disease in children after serotype 1 [25]. In our data set, serotype 6B variants were genetically distinct from wildtype 6B serotypes. They demonstrated a significantly high SNPs density (Fig. 4a) and genetic recombination events (Fig. 4b), suggesting carriage of a different lineage of serotype 6B in Malawi. The 6B variants also had novel sequence types, and contained an insertion of the licD-family phosphotransferase gene (Fig. 5). To ascertain the potential behaviour of the 6B variants under vaccine pressure as the national programme expands, further work in mouse models is recommended.

The cps locus variant of serotype 19A showed an inversion in the rmlD gene (Fig. 6). Although inversions do not change the genetic composition of the sequence, recent findings suggest gene inversions may actually lower the expression level of the affected gene, resulting in abnormalities at phenotypic level [43]. In our setting, the rmlD gene inversion in the 19A variant may impair the functionality of the whole rml gene cluster necessary for rhamnose biosynthesis, a component of the polysaccharide capsule. This could lead to the production of an altered 19A capsule, which may not be recognised by the vaccine.

A structural difference of the polysaccharide capsule within serogroup 20 has previously been reported [44]. This structural difference was due to a truncation and loss of function of the whaF gene [44]. The truncation in the whaF gene correlated with the loss of an αGlc residue in the capsular polysaccharide repeat unit of serotype 20A [44]. The Malawian serotype 20 variants contained a 716 bp deletion within the whaF gene (Fig. 7). This deletion would render the whaF gene non-functional, leading to a loss of an αGlc residue in the capsular polysaccharide repeat unit of the variant. It is therefore likely that the serotype 20 variants circulating in Malawi belong to subtype 20A. However, it is unclear how this deletion affects the ability of the 20 variant to colonise and cause invasive disease, although harbouring an intact allele of the whaF gene has been associated with invasive strain strains of subtype 20B [44].

This study had some limitations, which made it impossible to draw some conclusions from the analysis. Because of the limited sample size, we were not able to characterise serotype-specific associations in multiple carriage. To address such limitations, a follow up study with additional samples would be recommended in this population. The microarray technique employed has limited ability to discriminate closely related serotypes, which are detected as a group, however this limitation is common to all known phenotypic and genotypic serotyping methods [45]. In addition, microarray cannot differentiate NTs from the Mitis-group Streptococci on pneumococcal positive samples [32], which may lead to an inaccurate estimation of NTs in carriage.