Evaluation of Diagnostic Performance of BD Max EBP Assay in Patients with Diarrheal Illness.

Background: Detection of the etiological agents in patients with acute diarrhea is challenging due to a wide variety of pathogens. Detection and identication of the pathogens of acute diarrhea are important for both individual patient care and public health. The aim of this study is to evaluate the diagnostic performance of BD Max Enteric Bacterial Pathogens (EBP) PCR assay in patients with diarrheal illness. Methods: Between 1 January 2014 and 31 May 2015, one thousand two hundred twenty four stool samples from pediatric or adult patients with diarrhea submitted for routine analysis of bacterial stool pathogens were included in the study. We compared the BD Max EBP PCR assay to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA (Enzym Immun Assay) for Shiga toxins 1 and 2. Discordant results were adjudicated by either antigen detection methods or Film array GI (Gastro Intestinal) Panel. Coinfections were excluded. The chi-square test was used for comparisons of PPA and NPA. Results: The PPA (positive percent agreement) values for the BD Max EBP assay was 100% and NPA (negative percent agreement) was between 98.0%-99.7%, when compared with culture and EIA. After discrepant analysis, the PPA values for the BD Max EBP assay was 100% and NPA was between 99.5%-100%. Conclusion: The BD Max EBP assay showed a high correlation rate with conventional and molecular methods for the detection of stool pathogens.


Introduction
Infectious diarrheal diseases cause substantial morbidity and mortality worldwide. A wide variety of pathogens lead to infectious diarrhea, which makes the diagnosis of bacterial pathogens particularly challenging given the large amounts of background normal gastrointestinal ora [1,2]. Viral agents such as the noroviruses are responsible for most of the acute infectious diarrhea, while bacteria are responsible for most cases with more aggresive and in amatory diarrhea. [3] Salmonella, Campylobacter, Shigella, and Shiga toxin-producing Escherichia coli (STEC) are the most common diarrheagenic bacteria and routine stool culture is designed to detect these pathogens in most laboratories [4].
Detection and identi cation of the pathogens of acute diarrhea are important for both individual patient care and public health investigation. Furthermore, some infectious diarrheal pathogens can lead to longterm complications such as Hemolytic uremic syndrome and Guillain-Barre syndrome. [5].
Conventional stool culture is the gold standard for the diagnosis of bacterial gastroenteritis [6]. On the other hand stool cultures are either insensitive or labor intensive with long turnaround time. For the diagnosis of bacterial diarrhea, a wide variety of culture protocols involving multiple selective media and reagents are available in the microbiological laboratory [1,7].However, the use of antibiotics affects the culture result and frequently causes low yield for identi cation of enteropathogens [7].Molecular methods can increase sensitivity and speci city compared to stool culture [8].

Materials And Methods
The aim of this study is to evaluate the diagnostic performance of BD Max Enteric Bacterial Pathogens (EBP), assay which is a Polymerase Chain Reaction (PCR) test, in patients with diarrheal illness in Turkey.
Between 1 January 2014 and 31 May 2015 in Akdeniz University in Antalya, one thousand two hundred twenty four stool samples from pediatric or adult patients with diarrhea submitted for routine analysis of bacterial stool pathogens were included in the study. Duplicate specimens from the same patient were not enrolled. We compared the BD Max EBP assay to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an Enzym Immun Assay (EIA) for Shiga toxins 1 and 2. Discordant results were adjudicated by either antigen detection methods or Film array GI (Gastro Intestinal) Panel.
Culture and Enzyme Immunoassay (EIA): Fresh stool specimens were inoculated onto Mac Conkey agar, Xylose Lysine Deoxycholate (XLD) Agar for Salmonella and Shigella, and incubated at 37°C for 24 hours in an aerobic incubator. Lactose, xylose nonfermenting colonies with or without black centers on these media were screened phenotypically on triple sugar iron agar, motility medium, urea agar, Simmon's citrate agar and lysine iron agar. Suspected colonies were tested with Wellcolex™ Color Salmonella Rapid Latex Agglutination Test Kit and Wellcolex™ Color Shigella (ThermoFisher, UK). BD Max EBP automated PCR: The BD Max EBP is a multiplex nucleic acid ampli cation assay which detects DNA from Campylobacter spp. (jejuni and coli), Salmonella spp., Shigella spp./Enteroinvasive E. coli (EIEC), Shiga toxin1(stx1)/Shiga toxin2(stx2) genes in stool specimens with the BD Max system less than three hours. (BD Diagnostics, Baltimore, MD, USA) (Harrington). The BD MAX™ System is a fullyautomated, closed system which allows for simultaneous processing of up to 24 individual tests. Fresh stool samples were tested daily with the BD Max EBP assay, according to the manufacturer's instructions.
We accepted conventional culture as the reference method for the detection of Shigella spp., Campylobacter spp and Salmonella spp. and EIA as the reference method for the detection of Shiga toxins for the calculation of Negative percent agreement (NPA) and Positive percent agreement (PPA) of BD Max EBP assay.
In addition, BD Max EBP assay positive and conventional method negative results were adjudicated by either antigen detection methods or Film array GI Panel.
Stool samples with discordant results between Campylobacter culture and the BD Max EBP assay were tested by using an enzyme immunoassay (RIDASCREEN®, Campylobacter, r-biopharm, Germany) according to the manufacturer's instructions. Samples that gave different results between the BD MAX EBP assay and Campylobacter EIA were subject to FilmArray Gastrointestinal (GI) Panel (BioFire-BioMeriux, France).
Samples with discordant results between Salmonella and Shigella culture or Shiga toxin EIA and the BD Max EBP assay were tested by FilmArray GI Panel following the manufacturer's instructions.

Statistics
PPA and NPA and their 95% con dence intervals were calculated, as reported previously [9].

Results
One thousand two hundred twenty four stool samples were included in the study, 46 of which were excluded due to inhibition by BD Max EBP assay.
Culture and Shiga toxin EIA results: 14 (1.19%) specimens were positive for Campylobacter spp, 22 (1.87%) were positive for Salmonella spp. and two (0.17%) were positive for Shigella/EIEC by culture. 21 (1.78%) were positive for Shiga toxins by EIA. These were also positive with BD Max EBP assay. Coinfection was not detected by culture. The positivity rate of investigated pathogens was 5.01% (59/1178) by culture and EIA.
Of the 1178 samples, 30 had Salmonella spp., 6 had Shigella / EIEC, 37 had Campylobacter spp., and 38 had Shiga-like toxin genes (stx1 and / or stx2) by BD Max EBP assay. In addition, BD Max EBP assay identi ed coinfections in two samples (in one sample Salmonella + Shiga-like toxin genes and in another Campylobacter + Shiga-like toxin genes).
When coinfections were excluded, the NPA of the BD Max EBP assay was 99.3% for Salmonella spp., 99.7 % for Shigella / EIEC, and 98.0% for Campylobacter spp. when compared with culture. NPA was 98.5% for Shiga toxins using EIA as a reference method. PPA was 100% for all targets (Table 1).  When the samples with coinfection were examined, there was no growth in culture and EIA tests results were negative. When these samples were studied with FilmArray GI Panel, Salmonella and Shiga-like toxin genes were found to be negative in one sample and only Campylobacter gene was positive in another sample with Campylobacter + Shiga-like toxin genes. Harrington et al conducted a multicenter evaluation of the BD Max EBP assay in comparison to culture for the detection of Salmonella spp., Shigella spp., Campylobacter jejuni, and Campylobacter coli and an EIA for Shiga toxins 1 and 2 with stool culture for fresh and preserved stool specimen. Following discrepant analysis, PPA and NPA values were 97.3% and 99.8% for Salmonella spp., 99.2% and 100% for Shigella spp., 97.5% and 99.0% for C.jejuni and C. coli, and 100% and 99.7% for Shiga toxins, respectively. They concluded that, the BD Max EBP assay with superior sensitivity compared to conventional methods and excellent speci city, may improve the detection of bacterial stool pathogens and time to reporting of results [9].

Discussion
In a prospective study including 971 stool samples, the PPA of the BD MAX EBP assay and stool culture or In our study, BD Max EBP assay showed excellent performance for the detection of Salmonella spp., Shigella spp., Campylobacter spp. and Shiga toxins. The NPA of BD MAX EBP in our study was similar to previous reports. Since we did not have a BD MAX EBP negative but the reference test positive sample, PPA of BD MAX EBP in our study was slightly higher than previous studies. The reasons for this difference may be due to interlaboratory technical variance, specimen transport and processing practices such as unemployment of enrichment broth.
This is a single center, laboratory-based, prospective study with a high number of samples. The clinical features of patients were not included. The study was done only in fresh stool samples but not Cary Blairpreserved specimens.

Conclusion
We concluded that, the BD Max EBP showed a high correlation rate with conventional and molecular methods for the detection of stool pathogens. In addition, our detection rates increased with BD Max EBP which has high PPV and NPV. On the other hand, BD Max EBP assay detect DNA and not necessarily viable organisms which may lead to increased appreciation of asymptomatic infections and prolonged shedding.
For this reason the results should be interpreted with consideration of clinical information.