A. Analytical performances of the PneumoResp RDT
Specificity of the PneumoResp RDT
The specificity of the test was evaluated on 52 bacterial strains belonging to 24 different species, including 30 strains of streptococci of the mitis group (Streptococcus mitis, Streptococcus oralis, Streptococcus gordonii, Streptococcus sanguinis, Streptococcus parasanguinis, Streptococcus peroris, Streptococcus pneumoniae and Streptococcus pseudopneumoniae) and 22 strains of bacteria belonging to other genera (Alloscardovia omnicolens, Enterococcus avium, Enterococcus faecalis, Enterococcus faecium, Haemophilus influenzae, Haemophilus parainfluenzae, Klebsiella pneumoniae, Parvimonas micra and Staphylococcus aureus) or to non-mitis streptococci (Streptococcus agalactiae, Streptococcus anginosus, Streptococcus constellatus, Streptococcus pyogenes, Streptococcus salivarius and Streptococcus vestibularis) in order to verify the absence of reactivity with the antigens used in the test with regard to the bacteria potentially present in the oral or respiratory microbiota. No cross-reactions were observed for strains belonging to the tested species, except for one of the 3 strains of P. micra and for the 3 strains of S. pseudopneumoniae. The PneumoResp RDT was positive for the 4 tested strains of S. pneumoniae (3 clinical strains and the ATCC reference strain 49619) that were used as positive controls.
Correlation between qPCR assays and semi-quantitative culture
Using dilutions of the ATCC strain 49619 of S. pneumoniae, a correlation was established between bacterial loads obtained by culture (expressed in CFU/ml) and PCR assays (expressed in Ct); the results are shown in Figure 1. The sensitivity of the two PCR assays was shown to be 103 CFU/ml for the ply gene and 104 CFU/ml for the lytA gene.
B. Clinical performances of the PneumoResp RDT
Population and study design
The population consisted of 196 children consulting for symptoms compatible with acute respiratory infection at the University Hospital of Saint-Etienne, France, between October 2017 and July 2018. The study was performed on 196 respiratory samples (sputum or nasopharyngeal secretions) sent to the Microbiology Department of this hospital.
For patients with clinical suspicion of lower respiratory infection, a chest X-ray was performed. The diagnosis of pneumonia was defined on the coexistence of evocative clinical criteria and an abnormal parenchymal image on chest X-ray [3]; S. pneumoniae was estimated responsible for pneumonia if the bacterial load was greater than or equal to 107 CFU/ml for S. pneumoniae by semi-quantitative culture of respiratory specimens (sputum or nasopharyngeal secretions), irrespectively of the presence of other pathogen(s) [4,5].
The demographic and clinical characteristics of included patients are listed in Table 1.
Overall microbiological results from clinical specimens
Table 2 summarizes the results obtained for the 196 collected respiratory specimens using semi-quantitative culture, qPCR and RDT. All the 70 strains of S. pneumoniae that were recovered from culture were susceptible to optochin and positive with the bile salt lysis test.
Performances of the RDT by comparison to culture, PCR assays and clinical data
The same 196 respiratory specimens were used to evaluate the performances of the PneumoResp RDT. The RDT was first tested on undiluted fluidized samples; in case of positive result, the test was repeated after a 1:100 dilution. Table 3 shows the performances of the RDT by comparison to the semi-quantitative culture and to the two PCR assays with high bacterial load (Ct corresponding to107 CFU/ml or more); it was also tested with regard to the criteria of S. pneumoniae pneumonia that were listed above.
The sensitivity and negative predictive value (NPV) of the RDT on undiluted specimens were shown to be both of 100% by comparison to all the tested conditions depicted in Table 3, except for the ply PCR assay, which were both of 98.4. This means that no sample exhibiting a high bacterial load was missed by the RDT and that a negative RDT result was highly predictive of a weak or negative pneumococcal load. Another illustration of the correlation between bacterial loads evaluated by the two PCR assays and the results of RDT is shown on Figure 2.
With regard to the criteria of S. pneumoniae pneumonia, the results of Table 3 show that, if the RDT was negative on an undiluted specimen, pneumococcal pneumonia could be ruled out at day 0; in addition, all the 23 presumed pneumococcal pneumonia were tested positive by using the RDT on diluted specimen at day 0 (Figure 3).