Novice

ILLUMIGENE STREP A

Background

illumigene® Group A Streptococcus (not FDA cleared) is a rapid and simple molecular assay (Meridian Bioscience Inc., Cincinnati, OH) that uses loop-mediated isothermal amplification (LAMP) technology to detect Streptococcus pyogenes. It is designed to target a conserved exotoxin gene of the Streptococcus pyogenes genome. The reaction is performed on the Meridian illumipro-10™ incubator/reader under isothermal conditions. Following DNA amplification, magnesium-pyrophosphate is produced as a by-product resulting in turbidity which is measured by the instrument.

Method

345 total specimens were tested by illumigene® and culture. Culture and illumigene® were positive on 30 samples, negative on 287 samples and there were 26 samples positive by illumigene® that were culture negative. Culture did detect 2 Group A Strep that was not detected by illumigene®. Results with a reference PCR assay confirmed Group A Strep in 23/26 discrepant samples. The overall resolved sensitivity and specificity performance was 96.4% and 99.0% respectively. A LoD of approximately 490 cfu/swab was determined for S. pyogenes. No inhibition or cross-reactivity observed with the bacteria and human genomic DNA.

Results

345 total specimens were tested by illumigene® and culture. Culture and illumigene® were positive on 30 samples, negative on 287 samples and there were 26 samples positive by illumigene® that were culture negative. Culture did detect 2 Group A Strep that was not detected by illumigene®. Results with a reference PCR assay confirmed Group A Strep in 23/26 discrepant samples. The overall resolved sensitivity and specificity performance was 96.4% and 99.0% respectively. A LoD of approximately 490 cfu/swab was determined for S. pyogenes. No inhibition or cross-reactivity observed with the bacteria and human genomic DNA.

Conclusions

The illumigene® Group A Streptococcus assay is a rapid and sensitive method for the direct detection of Streptococcus pyogenes from throat swab samples.
• The sensitivity and specificity of illumigene® Group A Strep compared to culture was 93.8% and 91.7%, respectively
• The resolved sensitivity and specificity of illumigene® Group A Strep compared to culture/PCR was 96.4% and 99.0%, respectively
• illumigene® Group A Strep detected 23 more positive samples than did culture
• illumigene® Group A Strep can report results in 1 hour compared to standard 48 hour culture

Primer Design

The illumigene® Group A Strep assay composed of 6 LAMP (Loop mediated isothermal amplification) primers that specifically amplify a 206 base pair (bp) sequence of the Streptococcus pyogenes genome. The target DNA sequence resides in the 416-621 base pair region of the gene Streptococcus pyogenes pyrogenic exotoxin B (speB). The product of the speB gene is pyrogenic exotoxin B whose function determined as cysteine protease precursor. The sequence region encompassing the target region of the illumigene® Group A Strep was screened against the National Center for Biotechnology Information (NCBI) DNA sequence database that includes Nucleotide collection (nr/nt) and Whole-Genome- Shotgun (wgs) reads. The target sequence for BLAST alignments was derived from the following NCBI database entry: GenBank ID: L26149.1. The target sequence was found in 58 S. pyogenes strains comprising 14 complete genome and 44 speB gene targets of Streptococcus pyogenes strains.
illumigene® Group A Strep Assay Work Flow: The illumigene® Group A Strep assay involves a simplified workflow for ease of use. Clinical throat swabs are placed in the sample preparation apparatus which contains the assay control. Sample is collected and heat treated at 95 C for 10 minutes. 50 uL of the heated sample are added directly to the test and control reaction beads. The device is placed into an illumipro-10 and results are automatically reported in 40 minutes. The work flow is outlined in Figure 1.

Assays

Duel throat swab samples were collected and submitted to the laboratory in a non-nutritive transport medium. A swab was tested directly in the illumigene® assay with a simple workflow without any sample enrichment. 48 hour cultures were performed on the other swab following the laboratory standard protocol. When a single throat swab was submitted to the laboratory, the swab was used first to support the culture process and then the same swab was later tested by the illumigene® Group A Strep assay. Discrepant results were assessed using real-time PCR assay (ASR) at a reference laboratory.

Limit of Detection

Blood-Agar grown and quantified stocks of two S. pyogenes strains were used to determine the Limit of
detection (LoD). ATCC Stains 19615 and 12344 were diluted in PBSA containing tRNA and formalin treated cells harboring spa