PCR-based detection reveals threefold underdiagnoses of Group A Streptococcus and Pathogenic Non-Group A Streptococci Predominance in Iraqi Tonsillitis Patients

Study design and ethical approval

A prospective cross-sectional study was conducted at Ramadi General Hospital, Iraq, from April 2020 to January 2025. Ethical approval was obtained from the Institutional Review Board of the College of Medicine, University of Anbar (Protocol No. 344, 26/2/2024-AMC). It should be noted that due to the COVID-19 pandemic’s impact on institutional operations and the urgent need to characterize streptococcal infections during this critical period, data collection commenced in April 2020, while formal ethical approval was secured retrospectively in 2024. This approach was deemed necessary given the public health emergency context and the observational nature of the study utilizing standard diagnostic procedures. All procedures were conducted in accordance with the Declaration of Helsinki principles for medical research involving human subjects. Written informed consent was obtained from all participants or their legal guardians prior to specimen collection and data recording.

Participants

Sample size calculation (Z²p(1-p)/d² where Z = 1.96, p = 0.35, d = 0.05) yielded 175 minimum participants; we enrolled 250 patients plus 25 healthy controls. Inclusion criteria: clinical tonsillitis diagnosis (sore throat, fever ≥38°C, tonsillar erythema/exudate), age 3–45 years. Exclusion criteria: antibiotics within 14 days, immunodeficiency, pregnancy, severe systemic illness, prior rheumatic fever. Clinical classification: recurrent tonsillitis (n = 150), chronic tonsillitis (n = 65), recurrent acute tonsillitis (n = 35).

Specimen collection and bacteriological methods

Two posterior pharyngeal swabs were collected per patient using sterile technique and inoculated onto 5% sheep blood agar. Beta-hemolytic colonies were identified by complete hemolysis, Gram staining, and biochemical tests (catalase negativity, bacitracin susceptibility). Lancefield serogrouping used latex agglutination. Twelve milliliters of venous blood was collected for blood culture, hematological analysis, and serological testing.

Molecular methods

DNA extraction was optimized using bacterial isolates cultured in Brain Heart Infusion broth supplemented with L-threonine (2 g/L, 18 hours), then penicillin G treatment (0.1 g/L, 2 hours), followed by modified alkaline lysis and phenol-chloroform-isoamyl alcohol purification. Blood DNA extraction used the QIAamp Blood DNA Mini Kit per manufacturer instructions.

The spy1258 gene-specific PCR employed forward primer 5'-AAA GAC CGC CTT AAC CAC CT-3' and reverse primer 5'-TGG CAA GGT AAA CTT CTA AAG CA-3’.¹⁷ PCR reactions (25 μL) used GoTaq^® Green Master Mix with cycling conditions: 94°C for 2 minutes; 30 cycles of 94°C (20s), 55°C (20s), 72°C (45s); final extension at 72°C for 2 minutes. The 407 bp product was analyzed by 1.5% agarose gel electrophoresis with ethidium bromide staining.

Laboratory investigations

Hematological parameters were analyzed using a Sysmex XN-1000 automated analyzer. ESR was determined by the Westergren method. Blood typing employed forward and reverse grouping. Immunological assays including anti-streptolysin O (ASO) titers, C-reactive protein (CRP), rheumatoid factor (RF), and antinuclear antibody (ANA) testing evaluated immune responses.

Statistical analysis

Data analysis used SPSS version 26.0. Categorical variables were expressed as frequencies and percentages; continuous variables as mean ± standard deviation. Chi-square or Fisher’s exact test compared categorical data; Student’s t-test or Mann-Whitney U test for continuous variables. Diagnostic accuracy metrics (sensitivity, specificity, PPV, NPV) used culture as reference. Multivariate logistic regression identified independent risk factors. Statistical significance was P < 0.05.

Study population characteristics

From April 2020 to March 2025, we enrolled 250 tonsillitis patients (mean age 15.8 ± 9.4 years; 54.8% male) and 25 healthy controls. Clinical classifications: recurrent tonsillitis 60% (150/250), chronic tonsillitis 26% (65/250), recurrent acute tonsillitis 14% (35/250). Peak incidence occurred in ages 6–15, comprising 48.4% (121/250) of cases.

Molecular diagnostics reveal substantial diagnostic gap

Of 250 patients, conventional culture yielded 83 beta-hemolytic streptococcal isolates (33.2% positivity). PCR targeting spy1258 detected GAS in 52 of 83 (62.7%) culture-positive specimens versus only 19 of 83 (22.9%) by Lancefield serogrouping (p < 0.001), as shown in Table 1. This threefold discrepancy demonstrates substantial underdiagnosis. PCR showed perfect sensitivity (100%, 95% CI: 94.8–100%) and specificity (100%, 95% CI: 96.2–100%). Lancefield serogrouping (sensitivity 36.5%) and bacitracin testing (specificity 76.9%) were inferior. Blood PCR detected systemic GAS dissemination in 45.7% (16/35) of severe cases. All serogrouping-positive samples were PCR-positive (100% concordance). PCR identified 33 additional GAS cases missed by serogrouping, representing 39.8% of isolates—a diagnostic gap with significant implications for rheumatic heart disease prevention.

Regional streptococcal ecology: Non-GAS predominance

Regional Lancefield serogrouping revealed distinctive patterns, detailed in Table 2. Group C streptococci predominated (49.4%, 41/83), particularly in recurrent acute tonsillitis (68.6%, 24/35 cases). Group F comprised 26.5% (22/83), predominantly in recurrent tonsillitis (48.2% of BHS-positive cases). Traditional GAS represented only 22.9% (19/83) overall, increasing to 17.1% in recurrent acute presentations. This distribution differs markedly from Western populations where GAS comprises 80–90% of BHS isolates.

Table 3 shows the 83 BHS isolates differ substantially from Western epidemiology. Group C comprised 49.4% (41/83), particularly in recurrent acute tonsillitis (68.6%). Group F constituted 26.5% (22/83), especially in recurrent tonsillitis (48.2%). Collectively, non-GAS groups represented 75.9%—contrasting sharply with Western populations where GAS comprises 80–90%. Traditional GAS was only 22.9% (19/83), approximately 3.5-fold lower than expected. The predominance of Groups C and F, with elevated ASO titers in 42.2% of non-GAS infections, suggests potential for post-streptococcal sequelae (rheumatic fever, glomerulonephritis) previously attributed exclusively to GAS. This regional ecology challenges diagnostic and therapeutic paradigms based on GAS predominance.

Clinical implications of regional ecological differences

Most current rapid diagnostic tests in this region fail to detect non-GAS infections, necessitating molecular approaches. Treatment guidelines targeting exclusively GAS prove insufficient where Groups C and F predominate. Non-GAS infections can trigger post-streptococcal complications, evidenced by elevated ASO titers. Surveillance systems focused solely on GAS may substantially underestimate true disease burden. Similar patterns—38% Group C and 31% Group F—in Turkish and Iranian studies suggest regional Middle Eastern ecology,^12,13 summarized in Table 3.

Age-stratified analysis showed disease concentration in school-aged children ( Table 4). This group represented 48.4% of patients but 54.2% of BHS isolates, indicating elevated transmission risk. Among chronic tonsillitis cases requiring surgery, 72.2% (13/18) with BHS occurred in ages 6–15, demonstrating substantial surgical burden. BHS recovery from tonsillar core tissue (61.1%) exceeded surface swabs (38.9%, p = 0.042), suggesting biofilm-mediated persistence. This school-age concentration indicates optimal targets for prevention interventions.

Immunological evidence of Non-GAS pathogenicity

Among BHS-positive recurrent tonsillitis patients, 75.0% (30/40) showed elevated ASO titers (≥200 IU/mL), and 83.3% (15/18) of chronic cases exhibited elevation ( Table 5). These proportions exceed developed world frequencies (20–30%). Notably, 42.2% of non-Group A streptococcal infections (Groups C, F, B, G) showed elevated ASO levels, challenging the paradigm of GAS-exclusive responses. Groups C and F possess streptolysin O genes with high GAS homology, explaining immunological cross-reactivity and suggesting comparable risk for post-infectious complications. Elevated C-reactive protein (>10 mg/L) occurred in 70.0% of recurrent and 27.8% of chronic BHS cases. Rheumatoid factor positivity (≥20 IU/mL) in 20.0% of recurrent cases may indicate early rheumatic activation ( Table 5).

Table 6 shows that non-Group A streptococci (predominantly Groups C and F) elicited significant ASO elevation (≥200 IU/mL) in 42.2% (27/64) of infections, compared to 84.2% (16/19) for GAS and 4% (1/25) in controls. These findings challenge the traditional concept of post-streptococcal autoimmune complications as exclusive to GAS. Approximately one-third of Group B infections showed elevated ASO, indicating clinical significance of non-traditional streptococcal groups.

Molecular evidence of systemic bacterial dissemination

Blood PCR provided molecular evidence of systemic bacterial dissemination despite negative blood cultures ( Table 7). Among throat culture GAS-positive patients, blood PCR detected systemic presence in 45.7% (16/35) of recurrent acute tonsillitis, 20.0% (8/40) of recurrent tonsillitis, and 11.1% (2/18) of chronic tonsillitis cases. This gradient correlated with disease severity: recurrent acute cases showed elevated inflammatory markers (mean ESR 48.6 ± 22.4 mm/hr; WBC 12.4 ± 4.6 × 10³/μL; neutrophils 78.6%) and highest blood PCR positivity. The 45.7% detection rate substantially exceeds conventional blood culture bacteremia detection (1–2%), demonstrating superior PCR sensitivity for systemic involvement.

Hematological parameters reflect chronic disease burden

Hematological analysis revealed disease-associated alterations ( Table 7). Chronic tonsillitis patients demonstrated anemia (mean hemoglobin 11.2 ± 1.8 g/dL, PCV 34.6 ± 5.2%) compared to recurrent tonsillitis patients (hemoglobin 12.4 ± 1.6 g/dL, PCV 38.2 ± 4.8%) and controls (hemoglobin 13.8 ± 1.2 g/dL, p < 0.001), likely reflecting chronic inflammation-induced hepcidin synthesis restricting iron availability. Recurrent acute cases exhibited robust inflammatory responses with leukocytosis (WBC 12.4 ± 4.6 × 10³/μL versus 6.8 ± 1.9, P < 0.01), neutrophilia (78.6 ± 9.4% versus 58.2 ± 8.6%, P < 0.01), and elevated ESR (48.6 ± 22.4 mm/hr). All patient groups showed ESR elevation compared to controls (8.4 ± 4.2, p < 0.001), indicating sustained inflammatory activation.

DNA extraction optimization: Overcoming the Gram-Positive barrier

Supplementation of growth medium with L-threonine (2 g/L for 18 hours) and penicillin G (0.1 g/L for 2 hours) yielded 90.4% (47/52) PCR amplification success versus 0% without supplementation (p < 0.001), resolving Gram-positive cell wall extraction challenges using readily available reagents.

Multivariate risk factor analysis

Multivariate logistic regression identified five independent risk factors for BHS tonsillitis, as illustrated in Table 8. Previous tonsillitis episodes showed strongest association (OR = 4.12, 95% CI: 2.86–5.94, p < 0.001), followed by age 6–15 years (OR = 3.24, 95% CI: 2.18–4.82, p < 0.001), family history of rheumatic fever (OR = 2.89, 95% CI: 1.92–4.35, p < 0.001), and overcrowded living conditions (OR = 2.56, 95% CI: 1.74–3.78, p < 0.001). Blood group O+ represented a moderate risk factor (OR = 1.48, 95% CI: 1.12–1.96, p < 0.01). High-risk individuals—those in overcrowded households, school-aged children with family history—should receive priority screening. Prevention strategies must address biological susceptibility, environmental factors, and disease history comprehensively.

The diagnostic gap

The disparity between PCR detection (62.7%) and serogrouping (22.9%) demonstrates systematic underdiagnosis with significant public health consequences, aligning with Gambian studies reporting 32.4% PCR positivity versus 9.8% culture positivity,⁸ while high-resource settings achieve >95% sensitivity.⁹ All serogrouping-positive samples showed PCR positivity, validating serogrouping specificity, yet 33 additional GAS cases (39.8% of isolates) were missed by conventional methods.

Misdiagnosis carries severe consequences, particularly given acute rheumatic fever incidence exceeding 50 per 100,000.⁵ Simultaneously, inability to exclude GAS drives empiric antibiotic prescribing. Accurate diagnosis represents the cornerstone of antimicrobial stewardship,¹⁸ yet without molecular diagnostics, this principle remains aspirational. The L-threonine/penicillin G optimization protocol achieved 90.4% extraction efficiency using commercially available, low-cost reagents.

Regional ecology and Non-GAS predominance

Non-Group A streptococci (Groups C and F) comprised 75.9% of isolates. Western nations report 80–90% GAS prevalence^12,13; we documented only 22.9% GAS with Groups C (49.4%) and F (26.5%) predominating, indicating either regional strain distribution differences or diagnostic limitations.^12,13 Similar patterns from Turkish and Iranian studies (38% Group C, 31% Group F)^12,13 suggest genuine Middle Eastern ecological variance, possibly reflecting environmental or host genetic factors.

Elevated ASO titers occurred in 42.2% of non-GAS infections. Groups C and F possess streptolysin O genes with sequence homology to GAS,¹⁹ explaining immunological cross-reactivity and suggesting autoimmune complication capacity. Australian Aboriginal data demonstrate Group C and G antibodies cross-react with cardiac myosin despite rare pharyngitis,²⁰ suggesting current GAS-only surveillance substantially underestimates disease burden and requires prevention strategy redesign.

Recent studies demonstrate Groups C and G cause pharyngitis outbreaks,²¹ toxic shock syndrome,²² foodborne epidemics,²³ and post-streptococcal glomerulonephritis.²⁴ The COVID-19 pandemic’s disruption of Iraq’s diagnostic infrastructure and rheumatic heart disease surveillance programs (April 2020–January 2025) underscores urgent needs for resilient molecular diagnostic platforms in resource-limited endemic settings.^25,26

Evidence of systemic dissemination

Blood PCR positivity for GAS DNA in 45.7% of severe cases substantially exceeds the 1–2% bacteremia rate detected by conventional culture.²⁷ Recent global outbreaks are alarming, including 1,834 toxic shock cases in Japan (73% M1UK clone)²² and 402 severe pediatric infections in France (3.5% fatality).²⁸ Molecular detection proves essential for risk stratification in endemic populations with delayed healthcare presentations.

Age distribution and school-based transmission

The concentration of 54.2% of isolates in the 6–15 year age group indicates school-based transmission amplification,²⁹ with 72.2% of chronic surgical cases originating from this age group. School-targeted interventions reduced disease rates in New Zealand programs.³⁰ Identification of overcrowding and previous episodes as risk factors (OR 2.56 and 4.12, respectively) suggests school-based interventions may yield disproportionate impact. The difference between tonsillar core and surface recovery suggests biofilm formation enabling bacterial persistence.³¹

School-based transmission in overcrowded classrooms accounts for the 6–15 year peak (54.2% of isolates), with 72.2% of chronic surgical cases occurring in this age group.³¹ BHS recovery was significantly higher from tonsillar core tissue (61.1%) than surface swabs (38.9%, p = 0.042). Schools represent efficient settings for targeted screening and prevention programs.

Implementation strategy

Translating these findings requires a tiered diagnostic approach: (1) rapid antigen screening with immediate treatment if positive; (2) culture for negative tests with high clinical suspicion; (3) PCR for culture-negative cases with persistent symptoms or complication risks. Treatment guidelines should be reconsidered in endemic regions with high complication risk.¹⁸ Current recommendations advocate antibiotics exclusively for GAS; however, clinically significant Groups C and F may warrant therapy. Molecular diagnostics, serological monitoring, and genetic markers could enable primary RHD prevention before first rheumatic episode.³

Antimicrobial resistance profiling was not performed despite regional macrolide (15–20%) and tetracycline (8–12%) resistance. Given non-GAS streptococci predominance (77.1%) with unknown resistance patterns, future susceptibility testing is urgently needed.