Figure 1.
Detection of Streptococcus pneumoniae in saliva samples from schoolchildren.
Results of qPCR-based detection of S. pneumoniae-specific genes lytA and piaA and of live S. pneumoniae isolation from raw (A) and culture-enriched (B) samples of saliva collected from 50 schoolchildren. Each dot or cross represents an individual sample. The position of symbols corresponds to CT values for lytA- and piaA-specific signals as marked on corresponding axes. Dots represent samples classified as positive for S. pneumoniae with qPCR. Red dots represent samples from which live pneumococci were isolated from primary cultures of raw saliva on gentamicin-supplemented plates inoculated on the day of sample collection (A and B). Blue dots represent additional samples from which live pneumococci were isolated from re-cultured gentamicin plate culture harvests (B). Crosses represent samples classified by qPCR as negative for S. pneumoniae. Dotted lines mark the threshold assigned to discriminate between positive (CT<40) and negative samples, and the total number of 45 cycles in each qPCR reaction. Numbers in square brackets depict (in red) the number cultures from which live S. pneumoniae was isolated and (in black) number of samples with CT values <40 for both targeted genes. The number in the upper right corner depicts the number of samples classified with qPCR as negative for S. pneumoniae. Spearman's rank correlation coefficient (rho) and the associated P value (p) for samples generating any lytA- or piaA-specific signal (CT<45) are shown.
Figure 2.
Impact of culture-enrichment on qPCR detection of Streptococcus pneumoniae gene lytA in saliva from schoolchildren.
Each dot or cross represents an individual sample. The position of symbols corresponds to CT values for lytA-specific signals in DNA extracted from raw and culture-enriched sample of saliva as marked on corresponding axes. Dots represent 44 samples classified as positive and crosses represent 6 samples classified as negative for S. pneumoniae with qPCR. Open dots represent 32 saliva samples classified as positive for S. pneumoniae in both raw and culture-enriched samples. Green dots represent 12 samples classified as positive only after culture-enrichment. Dotted lines mark the threshold assigned to discriminate between positive (CT <40) and negative samples, and the total number of 45 cycles in each qPCR reaction. There was a significant increase in quantity of both lytA and (not shown) piaA detected with qPCR in culture-enriched compared to raw saliva samples (Mann-Whitney, p = <0.0001).
Figure 3.
Presence of serotype-specific Streptococcus pneumoniae DNA sequences detected with qPCR in saliva samples from schoolchildren.
Results of capsular gene detection in DNA extracted from culture-enriched samples of saliva collected from 50 children in assays considered as reliable (A) in the study or as unreliable (B) due to lack of specificity as determined by being no more than 2 CT stronger than the matching signal for the pneumococcal gene lytA in the same sample (dashed line). Each symbol represents individual serotype-specific signal detected with qPCR. Position of the symbol corresponds to the CT value for lytA-specific signal considered to be representative of overall quantity of S. pneumoniae in a sample (X-axis) and to the CT value for a particular serotype-specific signal (Y-axis). Results are colour-coded per serotype as shown in the legend. In A, diamonds represent samples from which a strain of a particular serotype was cultured, coloured dots represent samples culture-negative but qPCR positive for a serotype and open dots depict samples from carriers but negative in qPCR for any serotype-specific signal thus considered qPCR-non-typeable (NT). Numbers in brackets next to a serotype symbol in the legend report the total number of samples classified as positive in serotype-specific qPCR. In B, crosses depict serotype-specific signals detected in assays classified as unreliable in the study. Note that in all five qPCR assays there were a number of samples with a signal specific for serotype considerably stronger than for lytA.
Table 1.
Summary of the specificity of molecular methods used in the study to determine the sample serotype composition.
Figure 4.
Streptococcus pneumoniae serotype carriage in saliva of schoolchildren.
Serotypes detected are ranked by decreasing number of samples classified as positive in the study (upper X-axis). Every y-axis interval tick represents an individual carrier. Carriers are ranked according to decreasing number of serotypes detected as stated in the figure legend. Open circles depict strains of S. pneumoniae cultured from a sample (serotypes 3, 6A, 6C, 15B, 11A and 16F). NT – samples classified as positive for S. pneumoniae but as negative for the presence of any of the serotypes tested for.