Group A Streptococcus pharyngitis and pharyngeal carriage: A meta-analysis

<div><p>Objective</p><p>Antibiotic treatment of Group A Streptococcus (GAS) pharyngitis is important in acute rheumatic fever (ARF) prevention, however clinical guidelines for prescription vary. GAS carriers with acute viral infections may receive antibiotics unnecessarily. This review assessed the prevalence of GAS pharyngitis and carriage in different settings.</p><p>Methods</p><p>A random-effects meta-analysis was performed. Prevalence estimates for GAS+ve pharyngitis, serologically-confirmed GAS pharyngitis and asymptomatic pharyngeal carriage were generated. Findings were stratified by age group, recruitment method and country income level. Medline and EMBASE databases were searched for relevant literature published between 1 January 1946 and 7 April 2017. Studies reporting prevalence data on GAS+ve or serologically-confirmed GAS pharyngitis that stated participants exhibited symptoms of pharyngitis or upper respiratory tract infection (URTI) were included. Included studies reporting the prevalence of asymptomatic GAS carriage needed to state participants were asymptomatic.</p><p>Results</p><p>285 eligible studies were identified. The prevalence of GAS+ve pharyngitis was 24.1% (95% CI: 22.6–25.6%) in clinical settings (which used ‘passive recruitment’ methods), but less in sore throat management programmes (which used ‘active recruitment’, 10.0%, 8.1–12.4%). GAS+ve pharyngitis was more prevalent in high-income countries (24.3%, 22.6–26.1%) compared with low/middle-income countries (17.6%, 14.9–20.7%). In clinical settings, approximately 10% of children swabbed with a sore throat have serologically-confirmed GAS pharyngitis, but this increases to around 50–60% when the child is GAS culture-positive. The prevalence of serologically-confirmed GAS pharyngitis was 10.3% (6.6–15.7%) in children from high-income countries and their asymptomatic GAS carriage prevalence was 10.5% (8.4–12.9%). A lower carriage prevalence was detected in children from low/middle income countries (5.9%, 4.3–8.1%).</p><p>Conclusions</p><p>In active sore throat management programmes, if the prevalence of GAS detection approaches the asymptomatic carriage rate (around 6–11%), there may be little benefit from antibiotic treatment as the majority of culture-positive patients are likely carriers.</p></div

Results of literature search and study selection.

<p>Results of literature search and study selection.</p

Prevalence of GAS culture-positive pharyngitis by age group, recruitment strategy and setting.

<p>Prevalence of GAS culture-positive pharyngitis by age group, recruitment strategy and setting.</p

The prevalence of GAS carriage by age group, recruitment strategy and setting.

<p>The prevalence of GAS carriage by age group, recruitment strategy and setting.</p

Total unequivocal serologically-confirmed GAS pharyngitis prevalence by age group, recruitment strategy and setting, including where GAS culture positive swabs were obtained.

<p>Total unequivocal serologically-confirmed GAS pharyngitis prevalence by age group, recruitment strategy and setting, including where GAS culture positive swabs were obtained.</p

Site-directed mutagenesis of predicted epitope on prM-E protein.

<p>(A) To confirm the binding epitope of D29 Fab-IgG, residues within the P3 and P9 predicted sequences were mutated to generate mutants 1–6 (M1-6); M3/4, M3/5, M4/5 and M5/6 contain combinational-mutations as stated. (B) Reactivity of antibodies with the mutants was tested by Western blot analysis. Cleared lysate of DENV2-infected Vero cells (DV2), pCMV-prM-E- (prM-E) or mutants-transfected HEK 293 T cells (M1-5/6) were separated on 12% SDS-PAGE in non-reducing condition, followed by detection with h4G2, m2H2 and D29 Fab-IgG.</p

Localization of P3 and P9 peptide sequence on 3D crystal structure of prM-E heterodimer (PDB 3C6E).

<p>P3 (Purple) and P9 (green) peptide sequences were aligned with the predicted clusters (Cluster 1 – Navy blue; Cluster 2 – Black) on the prM-E crystal structure. The path of peptide phage sequences was highlighted with the participating residues from the cluster and the peptide phage labeled. Matched residues were purple (P3) or green (P9) and numbered accordingly; mis-matched residues were grey. The respective proteins and domains were highlighted as above.</p

Localization of D29 Fab-IgG predicted epitopes in 3D crystal structures of DENV2 prM-E heterodimer (PDB 3C6E) and the binding specificity of peptide-phages.

<p>(A) Clusters of conformational epitopes predicted by Pepitope server are displayed on the prM-E heterodimer crystal structure at neutral pH with their solvent-accessible surfaces highlighted. The prM is pink, EDI is red, EDII is yellow, EDIII is blue, FP is cyan. (B) The binding specificity of peptide-phages (P1-P9) to D29 Fab-IgG was tested in a direct ELISA format with 10 µg/ml of D29 Fab-IgG, h3H5, m2H2 and non-DENV specific human antibody (Hu) immobilized on a Maxisorb plate. Ability of peptide-phages to inhibit D29 Fab-IgG binding to DENV2 was investigated by (C) ELISA and (D) Western blot analysis. For ELISA, peptide-phages (10¹² pfu/ml) were incubated with D29 Fab-IgG for 1 hr at RT before application to immobilized DENV2 for 5 min at RT. Bound D29 Fab-IgG was detected with HRP-conjugated anti-Human IgG-Fc. The percentage of inhibition of D29 Fab-IgG binding by the peptide-phages shown is the average of three experiments. Error bars represent the standard errors of the mean (***p-value <0.005). For Western blot analysis, 0.5 µg/ml of D29 Fab-IgG was incubated with 8×10⁶ pfu of purified DENV2, 5% SM or 4×10¹¹ pfu of peptide-phage clones for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT.</p

Competition Assays and Western Blots of D29 Fab-IgG and monoclonal anti-DENV antibodies with known epitope.

<p>(A) Serially diluted h3H5, h4G2 or m2H2 was incubated with immobilized DENV2 (2×10⁷ pfu/ml) for 1 hr at RT before addition of 2.5 µg/ml of D29 Fab-IgG for a further hour at RT. Bound D29 Fab-IgG was detected by HRP-conjugated anti-human IgG-Fc antibody. 2.5 µg/ml of HRP-conjugated m2H2 was used to compete against m2H2. Values displayed are the average of three independent experiments and error bars represent standard errors of the mean. For Western Blot analysis, DENV1-4 (D1-D4) infected Vero cell lysate and 0.5 µg of recombinant DENV2 E protein (ecto-domain) (rE) were separated on 12% SDS-PAGE in (B) non-reducing, (C) reducing conditions; followed by detection with 1 µg/ml of h3H5, h4G2, m2H2 and D29 Fab-IgG. (D) For competition Western blot analysis, 0.5 µg/ml of D29 Fab-IgG was incubated with 1 µg/ml of h4G2, h3H5, m2H2 for 1 hr at RT before applying to membrane transblotted with DENV2 viral lysate for 30 min at RT.</p

Immunoprecipitation of DENV2 proteins with anti-DENV antibodies.

<p>(A) For immunoprecipitation in native condition, DENV2-infected BHK cells were incubated with media supplemented with 250 µCi/ml ³⁵S-methionine before lysis. Viral proteins were precipitated with D29 Fab-IgG or h4G2. (B) For immunoprecipitation with 1.25% SDS, cleared lysate of DENV2-infected BHK cells were incubated with h3H5, h4G2, m2H2, D29 Fab-IgG or non-DENV specific human antibody (IgG) followed by analysis with silver staining and Western blotting. Precipitated prM and E proteins were verified by (anti-prM) HRP-conjugated m2H2 or (anti-E) h3H5 respectively. HC - antibody heavy chain; LC - antibody light chain.</p