Comparative study of GenePOC GBS LB Assay and GeneXpert GBS LB Assay for the detection of Group B Streptococcus in prenatal screening samples

Group B Streptococcal (GBS) infections in the United States are a leading cause of meningitis and sepsis in newborns. The CDC, therefore recommends GBS screening for all pregnant women at 35–37 weeks of gestation and administration of intrapartum prophylaxis (in those that tested positive) as an effective means of controlling disease transmission. Several FDA approved molecular diagnostic tests are available for rapid and accurate detection of GBS in antepartum women. In this study, we report a clinical comparison of the Xpert GBS LB assay and a novel FDA-cleared test, GenePOC GBS LB assay. A total of 250 vaginal-rectal swabs from women undergoing prenatal screening were submitted to the University of Wisconsin’s clinical microbiology laboratory for GBS testing. We found 96.8% of samples were concordant between the two tests, while 3.2% were discordant with higher sensitivity observed for the GenePOC GBS LB assay, and higher specificity for the GeneXpert GBS LB assay.


Introduction 23
Streptococcus agalactiae, often referred to as Group B Streptococcus (GBS), is a gram-positive 24 bacterium found in the rectum and vagina of approximately 25% of pregnant women (1). While 25 GBS is an asymptomatic colonizer of most healthy adults, it can cause severe infections in 26 neonates, including sepsis, pneumonia, and meningitis (2, 3). GBS early onset disease (EOD) 27 are infections that occur in the first week of life and can be extremely dangerous to the newborn; 28 EOD occurred in 3 per 1000 live births and was associated with a high mortality rate prior to the 29 1990s (4). Due to the high neonatal mortality rate caused by GBS infections, the Center for 30 Disease Control (CDC) implemented a universal guideline in 1996 with recommended screening 31 of all pregnant women at 35-37 weeks of gestation and administration of intrapartum 32 prophylaxis (IAP) in pregnant women that tested positive (1,5). 33 Despite these guidelines, infection with S. agalactiae remains a leading cause of morbidity in 34 neonates born in the United States and therefore the implementation of more rapid and sensitive 35 screening techniques for GBS detection may further reduce transmission of GBS infection 36 intrapartum (1,6,7). The current gold standard for GBS detection is enrichment of the primary 37 specimen, a vaginal-rectal swab, followed by subculture onto a blood agar plate with phenotypic 38 characterization (1). This gold standard method has an extensive turnaround time and GBS 39 colonization sensitivity is only 54-87% (8, 9). Furthermore, bacterial culture requires an 40 experienced technician to further identify and test characteristics of GBS such agglutination and 41 beta hemolysis (1). 42 Although culture remains the gold standard in GBS diagnostics, the 2010 CDC revision for GBS 43 testing allows for nucleic acid amplification tests (NAAT) such as polymerase chain reaction 44 (PCR) as an option for GBS testing (1). A PCR method based on amplification of the CAMP 45 factor encoding gene (cfb), a fragment that is present in nearly all GBS strains, was developed by 46 Ke et al. (3) in 2000. Since then, several rapid and sensitive DNA probes and NAAT assays for 47 GBS have been developed and approved or cleared by the Food and Drug Administration (FDA) 48 (3, 10, 11). In our study, we report a clinical comparison of two FDA cleared NAATs for the 49 detection of GBS in antepartum women: Xpert ® GBS LB (Cepheid Inc., Sunnyvale, CA, USA) 50 and a novel recently FDA-cleared test, GenePOC ™ GBS LB (GenePOC inc, Québec City, 51 Canada). Both systems perform an automated nucleic acid extraction, real-time PCR amplification, and detection of the target nucleic acid sequences after LIM broth enrichment (12, 53 13 The hands-on setup time of each assay was timed and averaged over five individual runs. List 88 price of each test and instrument were provided by vendors. 89

Data analysis 90
Results from both GeneXpert ® and GenePOC ™ systems were compared for accuracy. 91 Concordant results between the assays were recorded, and no additional testing was performed. 92 Discordant results between the initial tests were resolved in two ways: repeating the tests on both 93 instrument platforms and culturing the enriched specimens to check for GBS presence. Results 94 from these additional tests were compared against two definitions of true positivity: 3 of 4 95 molecular tests yielding a positive GBS result or GBS presence in culture. These definitions will 96 be referred to as standard A and B, respectively. 97

98
From the 250 total samples tested, 242 samples were concordant (96.8%), and eight samples 99 were discrepant (3.2%) between the two molecular assays. Among the eight discrepant results, 100 seven specimens tested negative by GeneXpert ® , but positive by GenePOC ™ and one tested 101 positive by GeneXpert ® , but negative by GenePOC ™ with initial testing. All discrepant results 102 were repeated by both molecular assays and set up for culture. Upon repeat testing, one 103 discrepant sample that initially tested positive by GeneXpert ® , tested negative by both standard 104 A and standard B definitions. Upon repeat of the seven discrepant samples that initially tested 105 negative by GeneXpert ® , three samples tested positive and three samples tested negative by 106 standard A; and these six samples tested negative by standard B. Interestingly, one of the 107 samples that had initially tested negative on the GeneXpert ® tested negative upon repeat on 108 GeneXpert ® but tested positive on GenePOC ™ (initial and repeat) and had growth in culture. The 109 strain isolated from that specimen was confirmed to be Streptococcus agalactiae by sequencing 110 the entire 16S rRNA gene and by MALDI-TOF MS analysis. The results are summarized in 111 Table 1A. For all discrepant results, Ct value (listed in Table 1B) were obtained on repeat test 112 from either the instrument (GeneXpert ® ) or by contacting the vendor (GenePOC ™ ).
Sensitivity and specificity of each of the assays were assessed according to the two standards of 114 true positivity and the results are displayed in Table 2. When standard A was applied to define 115 true positives for the detections of GBS, GenePOC ™ demonstrated a sensitivity and specificity of 116 100% and 98%, respectively, while GeneXpert ® demonstrated a sensitivity and specificity of 117 94% and 99%, respectively. When standard B was applied, GenePOC ™ demonstrated a 118 sensitivity and specificity of 100% and 97%, respectively, while GeneXpert ® demonstrated a 119 sensitivity and specificity of 98% and 100%, respectively. 120 Time and cost of each assay were also assessed as displayed in Table 3. The GenePOC ™ GBS 121 LB test resulted in an average of 2.7 minutes to setup with a run time of 70 mins, while the Xpert 122 GBS LB assay resulted in an average of 1.3 minutes to setup with a run time of 59 min per test. 123 The list price of each test was $28 for GenePOC ™ and $30 for GeneXpert ® . The list price of the 124 instrument holding an equal number of testing modules was $35,000 for GenePOC ™ and 125 $110,000 for GeneXpert ® . 126

Discussion 127
The purpose of our study was to compare the newly FDA-cleared GenePOC ™ GBS LB assay to 128 the GeneXpert ® GBS LB assay. Whereas, the small consumable and equipment size of 129 GenePOC ™ are a significant advantage as it translates to minimum space requirements and better 130 waste management, GeneXpert ® allows the flexibility of adding on more modules, has a more 131 extensive test menu, and specimens are tested individually; the GenePOC ™ instrument allows 132 batched testing of 1 to 8 samples per run (12, 13). One shortcoming of the GenePOC ™ 133 instrument during our testing was that we were not notified of any error when loading the pie 134 onto the instrument until run was complete; this caused error/invalid rates of 2.4% for 135 GenePOC ™ , while GeneXpert ® had a 0% error/invalid rate. An additional challenge of 136 GenePOC ™ instrument during this study was that we were unable to readily view Ct values, 137 which could have been useful for troubleshooting and discrepancy analysis; we were notified 138 that this feature will be made available in the future. A major advantage of GenePOC ™ was the 139 higher sensitivity, although there were more false positives based on our two standards of true 140 positivity. It is noteworthy to highlight the single discrepant sample that resulted in GBS-141 negative GeneXpert ® results compared to GBS-positive GenePOC ™ and culture results (Table  142 1A). Both the GeneXpert ® and the GenePOC ™ GBS assays' primers and probes detect a target 143 within or adjacent to the CAMP factor encoding gene (cfb) of S. agalactiae (4,12,13). This cfb gene is noted to be present in almost all group B streptococci, yet recent findings have described 145 cases of cfb-negative isolates that were missed by the GeneXpert ® system (15,16). It is likely 146 that this isolate is either missing or has a mutation in the primer binding region for the primer 147 pair of the GeneXpert ® , and GenePOC ™ GBS assay's primers and probe amplify a cfb site that is 148 different from that of GeneXpert ® . Sequencing of the cfb gene and adjacent sites of this strain 149 may provide further clarification on why this strain was missed by the Xpert assay. The 150 GeneXpert ® false negative GBS strain raises an alarming concern and will need to be explored in 151 future studies. 152 To summarize, we observed higher sensitivity for the GenePOC ™ assay, and higher specificity 153 for the GeneXpert ® assay when applying the two standards of true positivity. Both the setup and 154 run times were slightly longer on the GenePOC ™ assay compared to that of the GeneXpert ® 155 assay. The extra dilution and vortexing step added additional time and material, such as variable 156 volume pipettes to the setup of the GenePOC ™ GBS LB assay, in comparison to the Xpert ® GBS 157 LB assay. The list price per test was less for the GenePOC ™ assay compared to that of 158 GeneXpert ® assay. For the same number of testing modules, the GenePOC ™ instrument costs 159 less than the GeneXpert ® Dx System based on list prices. Overall, both assays perform well and 160 agree in results, however there were a few discrepancies and specific differences listed in our 161 study between the platforms that labs should consider when deciding between the two platforms 162 and GBS assays. 163

Acknowledgment 164
GenePOC provided the revogene instrument and GenePOC GBS LB reagents used in this study. 165 166 167  *Cost is based on the list price provided by the vendors, however each vendor works with individual labs to try to make these kits and instrument affordable within their budgets **The test price is listed as per test; however the tests are purchased as kits