Rapid Multi-target Detection of Gastrointestinal Pathogens ... · The FilmArray ® GI Detection...
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Presented at the Society of Armed Forces Medical Laboratory Scientists, April 2012 CONTACT INFORMATION Mike Vaughn Idaho Technology, Inc. [email protected] See all of ITI’s scientific posters by scanning the QR code to access the Scientific Poster Page M. Vaughn 1 , L. Banks 1 , J. Gardner 1 , B. Harrel 1 , R. Wallace 1 , R. Crisp 1 , A. Pavia 2 , T. Barney 3 , G. Alger 3,2 , J. Daly 3,2 ; M. Rogatcheva 1 1 Idaho Technology Inc, Salt Lake City, UT, 2 Univ. of Utah, Salt Lake City, UT, 3 Primary Children’s Med. Ctr., Salt Lake City, UT Rapid Multi-target Detection of Gastrointestinal Pathogens in Patients Presenting with Diarrhea INTRODUCTION An estimated 211–375 million episodes of diarrheal illness occur each year in the United States, resulting in 73 million physician consultations, 1.8 million hospitalizations, and 3100 deaths [1-3]. Estimates for the cost of food-borne infections range from $6 to $23 billion in the United States alone [4-6]. The overwhelming challenge in diagnosing infectious diarrhea is the large number of pathogens that are known to cause diarrhea, including viruses, bacteria and protozoa. Present diagnostic practice is inefficient as it requires multiple tests which are labor-intensive, expensive, and slow. Additionally, many GI pathogens have limited test availability and/or low sensitivity. The goal of the FilmArray GI panel is to provide fast and effective pathogen detection from stool of individuals with diarrhea. The system (Figure 1) is intended for use mainly in clinical laboratories by individuals trained in laboratory practices and on the use of the FilmArray system. The coverage of pathogens by the FilmArray GI panel make its targeted population very broad, including not only US residents, but also travelers and US military troops around the world. ACKNOWLEDGEMENTS Development of FilmArray GI Detection System is supported by NIH Grant #5R01AI089489. This poster contains information regarding assays that have not been cleared by the FDA for in vitro diagnostic use. CONCLUSION The FilmArray GI Detection System may provide rapid, accurate, and comprehensive detection of GI pathogens and improve treatment of patients presenting with diarrhea. ABSTRACT The FilmArray ® GI Detection System was developed for rapid simultaneous identification of a broad range of gastrointestinal (GI) pathogens including Food- and Water-borne Category B pathogens. Targeted organisms are viruses, protozoa, and bacteria including 5 diarrheagenic E. coli pathotypes. The performance of the FilmArray GI System was compared to conventional diagnostic methods using 118 residual pediatric stool specimens. FilmArray GI System testing was 97% (451/464) concordant with conventional methods and yielded a 26% positivity rate in previously undiagnosed specimens. Multi-target detection revealed multiple infections in about 30% of specimens with up to 5 pathogens in a single sample. The FilmArray GI Detection System detected 43 additional pathogens in specimens where corresponding testing was not requested or not available. The FilmArray GI System may provide rapid and accurate detection of GI pathogens and improve treatment of patients presenting with diarrhea. RESULTS Clinical testing results of 620 sequential patient stool samples submitted to the Primary Children’s Medical Center laboratory for standard-of-care testing were evaluated; it was found that 13.7% of patients were diagnosed with a causative pathogen leaving 86.3% undiagnosed (Figure 2). A subset of 118 residual samples out of the 620 submitted samples were tested using the FilmArray GI Detection System. This subset consisted of 75 negative specimens and 43 specimens in which a causative pathogen had been identified. FilmArray GI pouch testing among these 118 samples resulted in 95 positively detected organisms compared to 45 detected by standard clinical methods as ordered by a physician (Figure 3). The increased rate of positivity can be attributed to two factors: an improved detection sensitivity of the FilmArray GI Detection System (Figure 4), and simultaneous testing for multiple pathogens for which testing was not requested by a physician (Figure 5) or was not available (Figure 6). Overall concordance of the FilmArray System and conventional methods was 97%. Detection of multiple pathogens was found in approximately 30% of the clinical samples when tested using the FilmArray GI Detection System (Figure 7). When using the FilmArray GI pouch, 20 of the 75 previously undiagnosed specimens were found positive with at least one pathogen leading to a potential 26% increase in pathogen detection. Currently Targeted Organisms Bacteria • Diarrheagenic E. coli • ETEC • EPEC • STEC/EHEC -O157:H7 • EIEC • EAEC • Aeromonas spp. • Salmonella spp. • Vibrio spp. -V. cholorae • Shigella spp. -S. dysenteriae • Campylobacter group • Yersinia enterocolitica • Clostridium difficile -C.difficile Nap1* • Plesiomonas shigelloides* Viruses • Norovirus (GI, GII, and GIV) • Adenovirus F (40/41) • Rotavirus (A, B, and C) • Human Astrovirus* • Sapovirus* Protozoa • Cryptosporidium group • Giardia lamblia • Entamoeba histolytica • Cyclospora cayetanensis* *Assays added to panel subsequent to presented data set. Figure 3: FilmArray GI pouch total results Organism Laboratory Detection FilmArray GI Pouch Detection STEC/EHEC 5 8 0157:H7 3 5 EAEC N/T 3 EPEC N/T 8 ETEC N/T 2 C. difficile 18 27 Aeromonas 1 3 Campylobacter 1 3 Salmonella 7 9 Shigella/EIEC 3 7 S. dysenteriae N/T 0 Yersinia 0 0 Vibrio ssp 0 0 V. cholera N/T 0 Cryptosporidium 3 4 G. Iamblia 3 6 Adenovirus 40/41 3 2 Rotavirus A 1 3 Rota B/C N/T 0 NoVGI N/T 2 NoVGII/NovGIV N/T 8 Total 45 95 Figure 5: FilmArray GI pouch detections for tests that were available, but not ordered Organism # of specimens tested by FA GI only FilmArray GI pouch detected Adenovirus F40/41 97 2 C. difficile 69 8 STEC/EHEC 64 0 G. Iamblia 88 2 Cryptosporidium 91 1 Rotavirus A 91 1 Aeromonas 62 0 C. jejuni 62 2 Salmonella 62 2 Shigella/EIEC 62 3 Yersinia 95 0 Total: 21 Figure 6: FilmArray GI pouch detections of less commonly-evaluated pathogens Organism Laboratory detection methods FilmArray GI pouch result Prevalence EAEC N/A 3 2.54% EPEC N/A 8 6.78% ETEC N/A 2 1.69% NoVGI N/A 2 1.69% NoVGII/NovGIV N/A 8 6.78% S. dysenteriae N/A 0 V. cholera N/A 0 Rota B/C N/A 0 Total 23 Figure 7a: FilmArray GI pouch detects multiple GI infections Specimens with positive detections positive for five pathogens positive for three pathogens positive for two pathogens positive for one pathogen 70 60 50 40 30 20 10 0 Lab detection FilmArray GI Pouch Figure 1 A. Fitment with freeze-dried reagents B. Plungers- deliver reagents to blisters C. Sample lysis and bead collection D. Wash station E. Magnetic bead collection blister F. Elution Station G. Multiplex Outer PCR blister H. Dilution blister I. Inner Nested PCR array ITI has developed a lab-in-a-pouch system called “FilmArray”. It is a medium-scale fluid manipu- lation system performed in a self-contained, disposable, thin-film plastic pouch. The FilmArray platform processes a single sample, from nucleic acid purification to result, in a fully automated fashion. These system characteristics are ideal for the multiplex testing of pathogens in standard diagnostic sample matrices. The FilmArray Test System A FilmArray test is initiated by injecting re- hydration solution and a patient sample into the FilmArray pouch and placing it in the FilmArray instrument. The user enters the sample and pouch type (using a barcode reader) into the software and initiates a run. Results are provided in ~ 1 hour. The FilmArray pouch has a fitment (see label A) containing all needed freeze-dried reagents. Figure 2: Results of testing by conventional methods (620 patients) Adenovirus F40/41, 3 C. difficille, 52 G. Iamblia, 3 Cryptosporidium, 3 Entamoeba, 0 Rotavirus A, 5 STEC/EHEC, 5 Aermonas, 1 Campylobacter, 1 Salmonella, 13 Shigella, 4 Yersinia, 0 Figure 4: FilmArray GI pouch performance compared to conventional methods Organism Number of specimens tested FA GI positive/Lab methods positive Positive by FA GI only Adenovirus F40/41 21 0/3* 0 C. difficile 58 18/18 1 EHEC 54 5/5 3 G. Iamblia 30 3/3 1 Cryptosporidium 27 2/3 0 Rotavirus A 27 1/1 1 Aeromonas 56 1/1 2 Campylobacter 56 1/1 0 Salmonella 56 7/7 0 Shigella/EIEC 56 3/3 1 Yersinia 23 0/0 0 * Discrepancies resolved by sequence analysis Figure 7b REFERENCES 1. Herikstadt H, Vergia D, Hadler J, et al. Population-based estimate of the burden of diarrheal illnesses: FoodNet 1996-1997. 1st International Conference on Emerging Infectious Diseases (Atlanta), March 1998. 2. LeClere FB, Moss AJ, Everhart JE, Roth HP. Prevalence of major digestive disorders and bowel symptoms, 1989. Adv Data 1992; 212:1-15. 3. Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999; 5:607-25. 4. National Institute of Allergy and Infectious Diseases (NIAID). Food-borne disease fact sheet. 1999. Available at: http://www.niaid.nih.gov/factsheet/foodbornedis.htm. 5. Buzby JC, Roberts T, Jordan Lin C-T, MacDonald JM. Bacterial food-borne disease: medical costs and productivity losses. 1996. 6. Garthright WE, Archer DL, Kvenberg JE. Estimates of incidence and costs of intestinal infectious diseases in the United States. Public Health Reports 1988; 103:107-15. The film portion of the pouch has stations for: 1. Cell lysis (Blister C) 2. Magnetic-bead based nucleic acid purification (D & E) 3. First-stage multiplex PCR (F & G) 4. Array of 102, second-stage nested PCRs (I) PCR primers are dried into the wells of the array and each primer set amplifies a unique product of the first-stage multiplex PCR. The second stage PCR product is detected in real-time using a fluorescent-double-stranded DNA binding dye, LCGreen ® . C D E F G I
Transcript of Rapid Multi-target Detection of Gastrointestinal Pathogens ... · The FilmArray ® GI Detection...
Presented at the Society of Armed Forces Medical Laboratory Scientists, April 2012
CONTACT INFORMATIONMike VaughnIdaho Technology, [email protected]
See all of ITI’s scientific posters by scanning the QR code to access the Scientific Poster Page
M. Vaughn1, L. Banks1, J. Gardner1, B. Harrel1, R. Wallace1, R. Crisp1, A. Pavia2, T. Barney3, G. Alger3,2, J. Daly3,2; M. Rogatcheva1
1 Idaho Technology Inc, Salt Lake City, UT, 2 Univ. of Utah, Salt Lake City, UT, 3 Primary Children’s Med. Ctr., Salt Lake City, UT
Rapid Multi-target Detection of Gastrointestinal Pathogens in Patients Presenting with Diarrhea
INTRODUCTIONAn estimated 211–375 million episodes of diarrheal illness occur each year in the United States, resulting in 73 million physician consultations, 1.8 million hospitalizations, and 3100 deaths [1-3]. Estimates for the cost of food-borne infections range from $6 to $23 billion in the United States alone [4-6]. The overwhelming challenge in diagnosing infectious diarrhea is the large number of pathogens that are known to cause diarrhea, including viruses, bacteria and protozoa. Present diagnostic practice is inefficient as it requires multiple tests which are labor-intensive, expensive, and slow. Additionally, many GI pathogens have limited test availability and/or low sensitivity.
The goal of the FilmArray GI panel is to provide fast and effective pathogen detection from stool of individuals with diarrhea. The system (Figure 1) is intended for use mainly in clinical laboratories by individuals trained in laboratory practices and on the use of the FilmArray system. The coverage of pathogens by the FilmArray GI panel make its targeted population very broad, including not only US residents, but also travelers and US military troops around the world.
ACKNOWLEDGEMENTSDevelopment of FilmArray GI Detection System is supported by NIH Grant #5R01AI089489.
This poster contains information regarding assays that have not been cleared by the FDA for in vitro diagnostic use.
CONCLUSIONThe FilmArray GI Detection System may provide rapid, accurate, and comprehensive detection of GI pathogens and improve treatment of patients presenting with diarrhea.
ABSTRACTThe FilmArray® GI Detection System was developed for rapid simultaneous identification of a broad range of gastrointestinal (GI) pathogens including Food- and Water-borne Category B pathogens. Targeted organisms are viruses, protozoa, and bacteria including 5 diarrheagenic E. coli pathotypes. The performance of the FilmArray GI System was compared to conventional diagnostic methods using 118 residual pediatric stool specimens. FilmArray GI System testing was 97% (451/464) concordant with conventional methods and yielded a 26% positivity rate in previously undiagnosed specimens. Multi-target detection revealed multiple infections in about 30% of specimens with up to 5 pathogens in a single sample. The FilmArray GI Detection System detected 43 additional pathogens in specimens where corresponding testing was not requested or not available. The FilmArray GI System may provide rapid and accurate detection of GI pathogens and improve treatment of patients presenting with diarrhea.
RESULTSClinical testing results of 620 sequential patient stool samples submitted to the Primary Children’s Medical Center laboratory for standard-of-care testing were evaluated; it was found that 13.7% of patients were diagnosed with a causative pathogen leaving 86.3% undiagnosed (Figure 2). A subset of 118 residual samples out of the 620 submitted samples were tested using the FilmArray GI Detection System. This subset consisted of 75 negative specimens and 43 specimens in which a causative pathogen had been identified. FilmArray GI pouch testing among these 118 samples resulted in 95 positively detected organisms compared to 45 detected by standard clinical methods as ordered by a physician (Figure 3). The increased rate of positivity can be attributed to two factors: an improved detection sensitivity of the FilmArray GI Detection System (Figure 4), and simultaneous testing for multiple pathogens for which testing was not requested by a physician (Figure 5) or was not available (Figure 6). Overall concordance of the FilmArray System and conventional methods was 97%. Detection of multiple pathogens was found in approximately 30% of the clinical samples when tested using the FilmArray GI Detection System (Figure 7). When using the FilmArray GI pouch, 20 of the 75 previously undiagnosed specimens were found positive with at least one pathogen leading to a potential 26% increase in pathogen detection.
Currently Targeted Organisms
Bacteria• Diarrheagenic E. coli • ETEC • EPEC • STEC/EHEC -O157:H7 • EIEC • EAEC• Aeromonas spp.• Salmonella spp.• Vibrio spp. -V. cholorae• Shigella spp. -S. dysenteriae• Campylobacter group• Yersinia enterocolitica• Clostridiumdifficile -C.difficileNap1*• Plesiomonasshigelloides*
Viruses• Norovirus (GI, GII, and GIV)• Adenovirus F (40/41)• Rotavirus (A, B, and C)• Human Astrovirus*• Sapovirus*
Protozoa• Cryptosporidium group• Giardia lamblia• Entamoeba histolytica• Cyclosporacayetanensis*
*Assays added to panel subsequent to presented data set.
Figure 3: FilmArray GI pouch total results
Organism Laboratory Detection FilmArray GI Pouch Detection
STEC/EHEC 5 80157:H7 3 5EAEC N/T 3EPEC N/T 8ETEC N/T 2
C.difficile 18 27Aeromonas 1 3
Campylobacter 1 3Salmonella 7 9
Shigella/EIEC 3 7S. dysenteriae N/T 0
Yersinia 0 0
Vibrio ssp 0 0V. cholera N/T 0
Cryptosporidium 3 4G. Iamblia 3 6
Adenovirus 40/41 3 2
Rotavirus A 1 3
Rota B/C N/T 0
NoVGI N/T 2
NoVGII/NovGIV N/T 8
Total 45 95
Figure 5: FilmArray GI pouch detections for tests that were available, but not ordered
Organism # of specimens tested by FA GI only FilmArray GI pouch detected
Adenovirus F40/41 97 2C.difficile 69 8
STEC/EHEC 64 0G. Iamblia 88 2
Cryptosporidium 91 1Rotavirus A 91 1Aeromonas 62 0
C. jejuni 62 2Salmonella 62 2
Shigella/EIEC 62 3Yersinia 95 0Total: 21
Figure 6: FilmArray GI pouch detections of less commonly-evaluated pathogens
Organism Laboratory detection methods
FilmArray GI pouch result Prevalence
EAEC N/A 3 2.54%EPEC N/A 8 6.78%ETEC N/A 2 1.69%
NoVGI N/A 2 1.69%NoVGII/NovGIV N/A 8 6.78%
S. dysenteriae N/A 0
V. cholera N/A 0Rota B/C N/A 0
Total 23
Figure 7a: FilmArray GI pouch detects multiple GI infections
Specimens with positive detections
positive for five pathogens
positive for three pathogens
positive for two pathogens
positive for one pathogen
70
60
50
40
30
20
10
0Lab detection FilmArray
GI Pouch
Figure 1
A. Fitment with freeze-dried reagentsB. Plungers- deliver reagents to blistersC. Sample lysis and bead collectionD. Wash stationE. Magnetic bead collection blisterF. Elution StationG. Multiplex Outer PCR blisterH. Dilution blisterI. Inner Nested PCR array
ITI has developed a lab-in-a-pouch system called “FilmArray”. It is a medium-scale fluid manipu-lation system performed in a self-contained, disposable, thin-film plastic pouch. The FilmArray platform processes a single sample, from nucleic acid purification to result, in a fully automated fashion. These system characteristics are ideal for the multiplex testing of pathogens in standard diagnostic sample matrices.
The FilmArray Test SystemA FilmArray test is initiated by injecting re-hydration solution and a patient sample into the FilmArray pouch and placing it in the FilmArray instrument. The user enters the sample and pouch type (using a barcode reader) into the software and initiates a run. Results are provided in ~ 1 hour.
The FilmArray pouch has a fitment (see label A) containing all needed freeze-dried reagents.
Figure 2: Results of testing by conventional methods (620 patients)
AdenovirusF40/41, 3
C.difficille,52 G.Iamblia,3 Cryptosporidium,3Entamoeba,0
Rotavirus A, 5STEC/EHEC, 5
Aermonas, 1
Campylobacter, 1Salmonella, 13
Shigella,4Yersinia,0
Figure 4: FilmArray GI pouch performance compared to conventional methods
Organism Number of specimens tested
FA GI positive/Lab methods positive
Positive by FA GI only
Adenovirus F40/41 21 0/3* 0C.difficile 58 18/18 1
EHEC 54 5/5 3G. Iamblia 30 3/3 1
Cryptosporidium 27 2/3 0Rotavirus A 27 1/1 1Aeromonas 56 1/1 2
Campylobacter 56 1/1 0
Salmonella 56 7/7 0Shigella/EIEC 56 3/3 1
Yersinia 23 0/0 0
* Discrepancies resolved by sequence analysis
Figure 7b
REFERENCES1. Herikstadt H, Vergia D, Hadler J, et al. Population-based estimate of the burden of diarrheal illnesses: FoodNet 1996-1997. 1st International Conference on Emerging Infectious Diseases (Atlanta), March 1998.
2. LeClere FB, Moss AJ, Everhart JE, Roth HP. Prevalence of major digestive disorders and bowel symptoms, 1989. Adv Data 1992; 212:1-15.
3. Mead PS, Slutsker L, Dietz V, et al. Food-related illness and death in the United States. Emerg Infect Dis 1999; 5:607-25.
4. National Institute of Allergy and Infectious Diseases (NIAID). Food-borne disease fact sheet. 1999. Available at: http://www.niaid.nih.gov/factsheet/foodbornedis.htm.
5. Buzby JC, Roberts T, Jordan Lin C-T, MacDonald JM. Bacterial food-borne disease: medical costs and productivity losses. 1996.
6. Garthright WE, Archer DL, Kvenberg JE. Estimates of incidence and costs of intestinal infectious diseases in the United States. Public Health Reports 1988; 103:107-15.
The film portion of the pouch has stations for:
1. Cell lysis (Blister C)2. Magnetic-bead based nucleic acid purification (D & E)3. First-stage multiplex PCR (F & G) 4. Array of 102, second-stage nested PCRs (I)
PCR primers are dried into the wells of the array and each primer set amplifies a unique product of the first-stage multiplex PCR. The second stage PCR product is detected in real-time using a fluorescent-double-stranded DNA binding dye, LCGreen®.
C D
E
F
G
I
