This is a secondary analysis of data from an office-based, multicenter, prospective study that took place in France between October 2009 and June 2011 (unpublished data). The aims of the princeps study were to evaluate the frequency of GAS carriage in healthy children and to compare the performance of a RADT between children with pharyngitis and healthy children, with throat culture in a microbiology laboratory as the reference test (intermediate results presented at the 28^th Annual Meeting of The European Society for Paediatric Infectious Diseases, Nice, France, 2010; abstract A-229-0021-00920). This ancillary analysis was restricted to patients with pharyngitis from the first year of inclusion. The STARD statement was followed for reporting the results of the study [24].

Eligible patients were 3 to 15 years old who were evaluated by their paediatrician between October 2009 and May 2010, who had a diagnosis of pharyngitis and did not receive antibiotics for 7 days before inclusion. Pharyngitis was defined by inflammation of the pharynx and/or tonsils. In total, 17 French paediatricians who are part of a research and teaching network (ACTIV) participated in the study [25].

Throat samples were obtained by use of a double–swab collection–transportation system (Venturi Transystem Amies agar, COPAN Diagnostics, Corona, CA, USA). The RADT (StreptAtest, Dectrapharm, France), a GAS-specific immuno-chromatographic strip assay, was performed immediately with swab #1 collected in the paediatrician's office. Swab #2 was held at ambient temperature and sent within 72 hr to the Robert Debré Hospital laboratory by an express messenger service. On receipt, the swab was rolled over one-quarter of a trypticase soy agar plate with 5% sheep blood, and the inoculum was further distributed on the plate by streaking with a sterile wire loop. The plates were incubated anaerobically at 37°C and read after 18 to 24 hr. Negative cultures were reexamined after an additional 24 hr of incubation (48 hr total). ß-haemolytic colonies were further investigated by latex agglutination (Prolex, Pro-Lab Diagnostics, Richmond Hill, ON, Canada). With GAS positivity, inoculum size was estimated as follows: 1+, <10 colonies; 2+, 11–50 colonies; and 3+, >50 colonies per plate. Data for patients with 1+ and 2+ cultures were combined to produce a dichotomized variable (light or heavy GAS growth). Microbiologists were blinded to individual clinical data and RADT results.

We collected clinical data needed to calculate the McIsaac score for each patient. The McIsaac score predicts GAS pharyngitis and ranges from 0 to 5 [20]–[22]. McIsaac criteria include age 3 to 15 years, history of fever, tonsillar swelling or exudate, tender anterior cervical adenopathy, and absence of cough. As for other studies [13], [26], the adenopathy criterion was modified to include any anterior cervical adenopathy ≥1 cm and/or presence of tenderness because tender lymph nodes can be difficult to assess in children. As for other authors [13]–[15], extreme McIsaac scores were combined (scores 1 and 2, and 4 and 5), and for analysis of clinical components of the score, age was re-coded into 2 categories of equal range (3–8 years; 9–14 years).

In the original study, sample size was estimated so that the 95% confidence interval (95CI) for RADT sensitivity would be a +/−5% estimation. Assuming sensitivity to vary between 75% and 95% and a GAS prevalence of 35%, a sample of 714 children with pharyngitis was needed. No other sample size calculation was performed for this secondary analysis.

The study protocol was approved by the Institutional Review Board Comité de Protection des Personnes Ile-de-France XI (n°09016) and the French administrative authorities (CNIL, n°1354254; Afssaps, n°2009-A00086-51). Parent and patient approval for participation was obtained before inclusion. Data were double entered into 4D software version 6.4 (4D) and the database was fully anonymized.

Hospital laboratory throat culture was considered the reference test. First, we described patient demographic features and overall prevalence of groups A, C and G β-haemolytic streptococci. Patients with group C or G β-haemolytic streptococci were considered GAS-negative in the analysis. Then, GAS prevalence by McIsaac score and each McIsaac criterion were described. Second, we used chi-square tests to investigate the clinical spectrum effect on RADT diagnostic accuracy (sensitivity, specificity, likelihood ratios and predictive values) by McIsaac score and each McIsaac criterion. Third, chi-square tests were used to compare the distribution of GAS inoculum size by office-to-laboratory delay (≤48 vs. >48 hr) and to study the effect of inoculum size (light or heavy GAS growth) on RADT sensitivity. Fourth, the distribution of heavy inocula by McIsaac score was explored by a chi-square test for trend, and the association of clinical signs and inoculum size was evaluated by comparing the frequency of heavy inoculum according to each clinical criterion by chi-square tests. Fifth, a binomial model with an identity link was used to evaluate the combination of clinical spectrum effect and inoculum size on RADT sensitivity [27]. Differences in RADT sensitivity were estimated by restricting the analysis to patients with a positive throat culture and using the result of the RADT as the outcome in the model. RADT sensitivity was investigated by univariate modeling according to each McIsaac criterion and then, multivariate modeling by all McIsaac criteria and inoculum size. Potential interaction between inoculum size and each McIsaac criterion was assessed by use of a Wald test. For multivariate modeling, variables were added one by one following the crescent univariate statistical significance order until non-convergence of the model. Significant interactions were managed by introducing corresponding interaction terms in the multivariate model and, in case of non-convergence, by stratification on inoculum size. Sixth, the accuracy of the following McIsaac-score–based decision rule was studied in terms of sensitivity and specificity: (a) scores <2, no further testing or antibiotic; (b) scores 2–3, culture all, antibiotics only with positive culture; (c) scores ≥4, treat empirically with antibiotics [22]. Statistical analysis involved use of Stata/SE 12 (StataCorp, College Station, TX, USA).

In total, 807 children met the inclusion criteria; 1 patient with unreadable RADT results, 2 with lost throat swabs and 19 with missing data for McIsaac score calculation were secondarily excluded. Therefore, analysis involved 785 children (351 girls [44.7%]), with mean (SD) age 6.1 (2.5) years. Overall, GAS prevalence was 36.3% (95CI, 32.9%–39.8%). In all, 4 (<1%) Group C and 7 (<1%) Group G β-haemolytic streptococci were identified on BAP. The mean age of secondarily excluded children was 5.9 (3.0) years and GAS was found in throat cultures for 4/20 (20.0% [7.9%–39.2%]).

GAS prevalence increased with increasing McIsaac score (p<0.001) (Tables 1 and 2), from 24.3% to 41.2% for children with ≤2 and ≥4 McIsaac score, respectively. Some clinical criteria were also associated with GAS: age <9 years, absence of cough, and presence of fever (Table 2).

Overall, the sensitivity and specificity of the RADT was 86.7% (82.2%–90.4%) and 94.6% (92.2%–96.4%), respectively; positive and negative likelihood ratios 16.1 (11.1–23.2) and 0.14 (0.10–0.19), respectively; and positive and negative predictive values 90.1% (86.0%–93.4%) and 92.6% (89.9%–94.7%), respectively.

We found significant variations in RADT sensitivity and specificity by McIsaac score (Table 2). Sensitivity increased from 75% (55.1%–89.3%) to 84.3% (75.0%–91.1%) and to 89.9% (84.3%–94.0%; p  = 0.02), and specificity decreased from 97.7% (91.9%–99.7%) to 96.0% (91.8%–98.4%) and to 92.5% (88.4%–95.5%; p  = 0.04) with McIsaac scores ≤2, 3 and ≥4, respectively (Table 2). RADT likelihood ratios and predictive values varied, but not significantly, by McIsaac score.

The RADT performance varied significantly by McIsaac criteria. For example, sensitivity was significantly lower in children ≥9 years old than in younger children (66.7% [47.2%–82.7%] vs. 89.0% [84.5%–92.6%], p<0.01). The negative predictive value varied, but not significantly, by McIsaac score or clinical criteria, ranging from 88.8% (80.8%–94.3%) for children without tonsillar swelling or exudate to 94.6% (90.0%–97.5%) for children without fever.

Among 285 GAS-positive throat cultures, 243 (85% [80.6%–89.2%]) showed heavy inoculum. The distribution of inoculum size did not differ by office-to-laboratory delay (≤48 vs. >48 hr, p  = 0.97). RADT sensitivity varied widely and significantly between patients with light and heavy inoculum (40.5% [25.6%–56.7%] vs. 94.7% [91.0%–97.1%], p<0.001). Light inoculum was found in 65.8% (50.0%–81.6%) of false-negative RADT cases versus 6.9% (3.7%–10.1%) of true-positive cases (p<0.001).

Frequency of heavy inocula increased with increasing McIsaac score, although not significantly (p  = 0.09, table 1). Heavy inoculum was significantly more frequent in children <9 years old than in older children (87.1% [82.9%–91.2%] vs. 70.0% [52.6%–87.4%], p<0.05).

The results of univariate binomial modeling for RADT sensitivity were almost identical to those of stratified analysis (Tables 2 and 3): age ≥9 years and presence of cough were significantly associated with decreased RADT sensitivity (p<0.05). After stratification on inoculum size, wide and statistically significant variations in RADT sensitivity were found in patients with light inoculum (e.g., 37.4% variation in sensitivity by age, p<0.01), but not widely (<6%) nor significantly in patients with heavy inoculum. Interactions were found between inoculum size and age (p  = 0.03), anterior cervical adenopathy (p  = 0.01), and tonsillar swelling or exudate (p  = 0.07).

Because of these multiple interactions and because of no convergence in the multivariate model after introducing interaction terms, 2 multivariate models of RADT sensitivity were fit by stratifying on inoculum size (Table 3). Results of multivariate modeling were close to those of univariate modeling. Light inoculum was associated with wide and statistically significant adjusted variation in RADT sensitivity (e.g., from 23.6% to 40.9%), but heavy inoculum was not widely (≤6%) or significantly associated.

Among 785 children, 522 (66%) would have received antibiotics according to the decision rule. The rule had 98.9% (97.0%–99.8%) sensitivity and 52.0% (47.5%–56.5%) specificity.