Group A Streptococcus (GAS) is a common cause of acute pharyngitis, responsible for up to 30% and 10% of cases in children and adults respectively.
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Group A Streptococcus (GAS) is a common cause of acute pharyngitis, responsible for up to 30% and 10% of cases in children and adults respectively.1 Group A Streptococcus pharyngitis is mostly self-limiting, with suppurative complications including peritonsillar abscess, mastoiditis and otitis media occurring in a small minority.2 However, most important in the Aotearoa New Zealand setting are the post-infectious autoimmune sequalae of rheumatic fever (RF) and post-streptococcal glomerulonephritis (PSGN).2 Aotearoa New Zealand has a high and inequitably distributed rate of RF, with a pre-COVID (and the associated non-pharmaceutical interventions) incidence of 28.6/100,000 and 83.2/100,000 for Māori and Pacific children (5–14 years) respectively, compared to <1/100,000 in children of European and other ethnicities.3
The Aotearoa New Zealand sore throat guidelines recommend a throat swab for bacterial culture and the prescription of empiric antimicrobials for patients at high risk of RF.4 Laboratory testing is utilised to make a diagnosis, as clinical presentation does not reliably distinguish GAS from other non-RF associated bacterial or viral causes of pharyngitis.5 The extended turn-around time of current culture-based testing favours empiric as opposed to diagnostics directed therapy. The use of nucleic acid amplification tests (NAATs) for GAS has, however, been expanding in recent years, including in Aotearoa New Zealand, and these methods have the advantages of a decreased turn-around time, improved sensitivity and potential labour savings.1,6–8 The use of a molecular GAS assay, with an associated decrease in turn-around time, could also facilitate less unnecessary antimicrobial use (through either less empiric therapy or the ability to stop antimicrobials if GAS PCR is negative) while allowing prompt treatment when required.1,2 The cost of commercial NAATs for GAS are currently prohibitive for implementation of large-scale testing in the community setting. However, the use of a laboratory-developed extraction free PCR assay that can be performed on routinely utilised gel amies throat swabs can help overcome this cost barrier.
Medlab Central is a diagnostic laboratory located in Palmerston North, Aotearoa New Zealand, and tests hospital and community samples from the MidCentral, Tairāwhiti and Whanganui health districts. The annual number of throat swabs processed by the laboratory is approximately 23,000. This study describes the validation of an in-house GAS PCR as compared to routine bacterial culture.
This was a prospective study comparing the performance of an in-house real-time GAS PCR versus bacterial culture using throat swabs collected as part of routine clinical care. The study used a predefined sample size of 1,094 consecutive throat swabs received by the laboratory from 4 September 2023. The sample size was calculated on a 95% confidence level, 2% margin of error and the local 2022 prevalence of GAS in throat swabs of 13.1%. Demographic data (age, sex, ethnicity and health district) for each sample/patient were extracted from clinical records. Throat swabs (Copan 108C rayon swabs, gel amies without charcoal) were collected and underwent routine culture on sheep blood agar with 3% NaCl. Plates were incubated for 48 hours with reading at 24 and 48 hours. After a standard 1-week clinical sample retention period, the swabs were tested on the GAS PCR. The template for PCR was created by swirling the throat swab in 500µL of phosphate-buffered saline (PBS) for 5–10 seconds.
The in-house GAS real-time PCR assay was a multiplex assay with a GAS target and an RNaseP internal control. The GAS primers targeted the SpeB gene with primer and probe sequences and cycling conditions as described by Dunne et al.9 The probe fluorophore was changed from Cy3 to FAM. The internal control utilised RNaseP primer and probe sequences as per the Centers for Disease Control and Prevention (CDC) SARS-CoV-2 multiplex assay with Cal Fluor orange 610 as the flurophore.10 The PCR was run in 10µL reactions with each reaction made to contain: 100nM forward and reverse SpeB primers, 150nM SpeB probe, 200nM forward and reverse RNaseP primers, 300nM RNaseP probe and 1X PerfeCTa ToughMix (Quantabio). Each reaction used 2µL of the PBS template described above. PCR was performed on MIC induction cyclers (BMS) using the following conditions: 95°C for 3 minutes, then 40 cycles of 95°C for 20 seconds followed by 60°C for 20 seconds. To determine the assay limit of detection, DNA extracted from pure GAS culture containing 100,000 copies of GAS was used (with DNA concentration determined by nanodrop). Four tenfold serial dilutions were made, and PCR was performed in duplicate at 100,000, 10,000, 1,000 copies, four times at 100 copies, and eight times at 10 copies. The specificity of the assay was tested using extracted DNA from culture isolates of the following organisms; Group C/G Streptococcus (n=25), Group B Streptococcus (n=10), viridans Streptococci (n=10), Escherichia coli (n=10), Staphylococcus aureus (n=10), Enterococcus species (n=11), Pseudomonas aeruginosa (n=10), Candida albicans (n=10), Neisseria gonorrhoeae (n=1) and Haemophilus influenzae (n=10).
Samples were considered PCR-positive for GAS if there was amplification of the SpeB GAS target regardless of the RNaseP internal control result. Samples were considered negative for GAS if there was no amplification of the SpeB GAS target and a Ct value of <37.00 for the internal control. Samples negative for GAS with an internal control Ct of ≥37.00 were subject to repeat PCR. If repeat PCR gave the same result, they were subject to additional PCR using a spike control containing human DNA to assess for inhibition. 20µL of the spike control was added to the PBS solution for each sample and the PCR repeated in parallel with a PBS blank containing 20µL spike in 500µL PBS. Samples with RNaseP (internal control) Ct >2 cycles above the PBS blank were considered inhibitory and the GAS result invalid. Samples with discordant results between culture and the in-house GAS PCR were tested using the Xpert Group A strep PCR assay (Cepheid).
The percent agreement between culture and the in-house GAS PCR was described. Post-introduction into practice, the turn-around time from receipt in laboratory to reporting of the result was also compared between the culture-based period of testing in January 2023 to PCR-based period of testing in January 2024. Study approval was obtained from the New Zealand Health and Disability Commission Ethics Committee (HDEC approval: 2023 FULL 18386).
The assay could reliably detect 100 copies of GAS and showed good linearity over the range of 102 to 105 copies. No non-specific amplification was observed with any of the non-GAS organisms tested. Of the 1,094 samples tested, 1,083 produced a valid initial PCR result. Eleven SpeB and RNaseP negative samples required additional testing with the spike control, with one sample subsequently excluded due to PCR inhibition. This resulted in 1,093 samples included in the final analysis. The basic demographic information for the study population is shown in Table 1.
View Table 1–2.
Group A Streptococcus was detected by culture and GAS PCR in 262 (24.0%) and 319 (29.2%) of 1,093 throat swabs respectively (Table 2). GAS Ct values had a median of 25.4 (IQR: 23.5–28.2). The RNaseP internal control had a median Ct of 32.7 (IQR: 31.3–34.2). The overall, positive and negative agreement of the in-house GAS PCR with culture was 94.2%, 98.9% and 92.8% respectively. There were 63 discordant results with three culture-positive/PCR-negative samples and 60 culture-negative/PCR-positive samples (Table 2). One (33.3%) of three culture-positive/PCR-negative samples and 56 (93.3%) of 60 of the culture-negative/PCR-positive samples were GAS-positive on the GeneXpert Group A strep assay.
The median laboratory turn-around time between culture-based testing in January 2023 (1,151 swabs) and PCR-based testing in January 2024 (1,191 swabs) reduced from 44 to 16 hours. In January 2023 95% of throat swab results were reported within 65 hours, while in January 2024 95% of results were reported within 24 hours.
The in-house GAS PCR under evaluation had an absolute and relative increase in GAS detection of 5.2% and 21.8% respectively compared to culture. This is consistent with prior descriptions of GAS NAATs.6,8 As the PCR was performed post–swab inoculation onto culture media and with a 7-day delay in testing, the described increase in detection likely represents a lower bound estimate. There were three culture-positive/PCR-negative samples that are presumed to represent false negative PCR results and 60 culture-negative/PCR-positive specimens, 56 of which were also GAS-positive on the GeneXpert Group A strep assay. These culture-negative/PCR-positive results are presumed to represent true positives detected due to the increased analytical sensitivity of PCR. The increased sensitivity can in part be attributed to PCR being relatively unaffected by delays between sample collection and processing, while the sensitivity of culture is likely to be more negatively affected by such delays. The four specimens with a positive in-house PCR/negative Xpert PCR result may represent stochastic detection in specimens with low levels of GAS DNA or false positivity.
In the 11 specimens with no RNaseP detection, the use of an external spike control excluded inhibition in all but one sample. This suggests that the lack of RNaseP detection largely reflects sampling quality or potentially the age of samples at testing. Sampling can be impacted by factors such as patient age, recent eating and drinking and excessive pus, and self-sampling may also contribute.11 Since the methods introduction into routine diagnostic testing, RNaseP has been not detected in only 0.2% of samples, suggesting sample age was a significant contributor in the initial evaluation.
The in-house PCR method presented here has a reagent cost of NZ$1.20 per sample, making it comparable to culture (NZ$1.00 per sample) from a reagent cost perspective. Locally there was no change in staffing (and associated cost), as testing of throat swabs moved from the Microbiology to Molecular departments, where the work was absorbed within existing staffing and hours with minimal additional training required. The method has potential labour savings, with an estimated hands-on time of less than 5 minutes per sample (including preparation of PBS tubes, sample swirling, bulk preparation of PCR mastermix and sample transfer to PCR tubes) compared to culture, which requires multiple plate reads and additional identification steps for positives. These labour savings could be increased with the use of swabs in liquid transport media, and through the use of automation.
A significant advantage of the PCR method is the impact on turn-around time. The MIC cyclers are capable of running 46 samples and two controls per batch with a running time of 1 hour. Testing is performed daily (except Sunday), with there generally being a morning, midday and afternoon run. The in-house PCR reduced median laboratory turn-around time by 63.6% from 44 to 16 hours, which may decrease time to antimicrobial treatment initiation or discontinuation in certain populations. This TAT does not, however, consider the time between sample collection and arrival in the laboratory. For samples from MidCentral, the delay between collection and receipt can be a few hours, while for the most distant samples from Tairāwhiti this delay can be up to 36 hours.
Although full case information (e.g., socio-economic status or occupation) was not available, it is notable that 45% of samples came from European patients and 25% from persons aged over 30 years, which suggests over-testing (and likely over-treatment) in individuals at low risk for complications of GAS pharyngitis. Testing and treatment should largely be targeted to those populations at high risk of post-infectious complications, such as RF, as per the Aotearoa New Zealand sore throat guidelines.4
This work describes the validation of an in-house extraction free real-time polymerase chain reaction (PCR) for the detection of Group A Streptococcus (GAS) in throat swabs collected in gel amies.
Throat swabs received by the laboratory were prospectively tested by routine bacterial culture and an in-house PCR assay targeting the GAS SpeB gene with a multiplexed RNaseP internal control. Samples with discrepant culture/PCR results had additional testing using the commercial Xpert Group A Strep PCR assay (Cepheid). Post–introduction of the in-house GAS PCR the comparative laboratory turn-around time between PCR and historic culture results was determined.
Of the 1,093 throat swabs included in the final analysis, GAS was detected by culture and GAS PCR in 262 (24.0%) and 319 (29.2%) respectively. The overall, positive and negative agreement of the GAS PCR with culture was 94.2%, 98.9% and 92.8% respectively. Of the 63 discordant samples, one (33.3%) of three culture positive/in-house PCR negative samples and 56 (93.3%) of 60 culture negative/in-house PCR positive samples were GAS positive on the Xpert Group A Strep assay. Median turn-around time from laboratory receipt to result decreased from 44 to 16 hours with the introduction of the GAS PCR into routine practice. Forty-five percent of samples came from European patients and 25% from persons aged over 30 years, suggesting over-testing in persons at low risk of GAS pharyngitis complications.
The in-house GAS PCR provided greater and faster detection of GAS from throat swabs compared to culture. However, throat swabbing for GAS needs to be better targeted to those populations at high risk of post-GAS pharyngitis complications.
Rebecca Lucas: HOD Molecular Department, Medlab Central, Palmerston North.
Emma Tapp: Medical Laboratory Scientist, Molecular Department, Medlab Central, Palmerston North.
Rumbi Chimwayange: Medical Laboratory Scientist, Molecular Department, Medlab Central, Palmerston North.
Luiza Hermoso: Medical Laboratory Scientist, Molecular Department, Medlab Central, Palmerston North.
Matthew R Blakiston: Clinical Microbiologist, Microbiology/Molecular Departments, Medlab Central, Palmerston North.
Thank you to Tina Lusher and Natalya Clark for their assistance with the project.
Rebecca Lucas: Molecular Department, Medlab Central, Palmerston North Hospital, Ruahine Street, Palmerston North 4414. Ph: 06 952 3173.
Nil.
Study funded in entirety by Medlab Central.
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