Introduction
Materials and methods
Quantitative genomic DNAs and stool specimens
Vibrant gastrointestinal pathogens panel (GPP)
Bacteria | Clostridium difficile toxin A/B |
Campylobacter spp. (jejuni, upsaliensis) | |
Plesiomonas shigelloides
| |
Yersinia enterocolitica
| |
Salmonella spp. | |
Vibrio parahaemolyticus
| |
Vibrio cholerae
| |
Vibrio vulnificus
| |
Enteroaggregative E. coli (EAEC) | |
Enteropathogenic E. coli (EPEC) | |
Enterotoxigenic E. coli (ETEC) lt/st | |
Shiga-like toxin producing E. coli (STEC) stx1/stx2 | |
E. coli O157:H7 | |
Shigella/Enteroinvasive E. coli (EIEC) | |
Helicobacter pylori
| |
Listeria spp. | |
Virus | Norovirus GI/GII |
Rotavirus A | |
Adenovirus F 40/41 | |
Astrovirus | |
Sapovirus (I, II, IV, V) | |
Parasite |
Giardia lamblia
|
Cryptosporidium spp. (parvum, hominis) | |
Entamoeba histolytica
| |
Cyclospora cayetanensis
| |
Fungi | Candida spp. |
Microsporidium spp. |
Pathogen-specific primer design
Nucleic acid extraction
Multiplex PCR amplification
GPP array hybridization
GPP array on-chip extension and labelling
GPP array target detection
Results
Precision study
Organism Source/isolate ID | Target concentration | Agreement |
---|---|---|
E. coli O157:H7 | 6.0 × 104 cells/mL | 100% (20/20) |
ATCC 43895 | ||
Enteroaggregative E. coli (EAEC) | 4.8 × 106 cells/mL | 100% (20/20) |
ATCC 23501 | ||
Enteropathogenic E. coli (EPEC) | 3.6 × 105 cells/mL | 100% (20/20) |
ATCC 43887 | ||
Enterotoxigenic E. coli (ETEC) lt/st | 3.6 × 107 cells/mL | 100% (20/20) |
ATCC 35401 | ||
Shiga-like toxin producing E. coli (STEC) stx1/stx2 | 2.8 × 105 cells/mL | 100% (20/20) |
ATCC BAA-2196, ATCC 43895 | ||
Plesiomonas shigelloides
| 6.0 × 105 cells/mL | 100% (20/20) |
ATCC 14029 | ||
Vibrio parahaemolyticus
| 6.0 × 105 cells/mL | 100% (20/20) |
ATCC 17802 | ||
Vibrio vulnificus
| 2.4 × 108 cells/mL | 100% (20/20) |
ATCC 27562 | ||
Helicobacter pylori
| 3.6 × 108 cells/mL | 100% (20/20) |
ATCC 700392 | ||
Listeria spp. | 6.0 × 106 cells/mL | 100% (20/20) |
ATCC 19111 | ||
Vibrio cholerae
| 6.0 × 105 cells/mL | 100% (20/20) |
ATCC 14035 | ||
C. difficile Toxin A/B | 3.6 × 105 cells/mL | 100% (20/20) |
ATCC 9689, Clinical Specimen | ||
Salmonella spp. | 4.8 × 106 cells/mL | 100% (20/20) |
ATCC 700623 | ||
Shigella/Enteroinvasive E. coli (EIEC) | 2.4 × 105 cells/mL | 100% (20/20) |
ATCC 29930 | ||
Yersinia enterocolitica
| 4.8 × 108 cells/mL | 100% (20/20) |
ATCC 9610 | ||
Campylobacter jejuni
| 4.8 × 106 cells/mL | 100% (20/20) |
ATCC BAA-1234 | ||
Campylobacter upsaliensis
| 4.8 × 103 cells/mL | 100% (20/20) |
ATCC 43954 | ||
Giardia lamblia
| 3.6 × 104 cells/mL | 100% (20/20) |
ATCC 30957 | ||
Cryptosporodium spp. | 1.5 × 104 oocycts/mL | 100% (20/20) |
Waterborne P102C | ||
Entamoeba histolytica
| 2.4 × 103 cells/mL | 100% (20/20) |
ATCC 30459 | ||
Cyclospora cayetanensis
| 2.4 × 105 RNA copies/mL | 100% (20/20) |
Zeptometrix control | ||
Norovirus GI/GII | 1.1 × 105 RNA copies/mL | 100% (20/20) |
Zeptometrix control, Clinical Specimen | ||
Rotavirus A | 1.0 TCID50/mL | 100% (20/20) |
ATCC VR-2104 | ||
Adenovirus F 40/41 | 1.0 TCID50/mL | 100% (20/20) |
ATTC VR-930/931 | ||
Astrovirus | 1.2 × 103 RNA copies/mL | 100% (20/20) |
ATCC VR-3238SD | ||
Sapovirus (I, II, IV, V) | 2.1 × 105 RNA copies/mL | 100% (20/20) |
Zeptometrix control | ||
Candida spp. | 2.4 × 103 cells/mL | 100% (20/20) |
ATCC 10231 | ||
Microsporidium spp. | 2.2 × 105 DNA copies/mL | 100% (20/20) |
Clinical Specimen |
Limit of detection analysis
Organism | Source/isolate ID | LoD concentration | Agreement | |
---|---|---|---|---|
Bacteria | E. coli O157:H7 | ATCC 43895 | 1.0 × 102 cells/mL | 100% (5/5) |
Enteroaggregative E. coli (EAEC) | ATCC 23501 | 1.0 × 102 cells/mL | 100% (5/5) | |
Enteropathogenic E. coli (EPEC) | ATCC 43887, Clinical Specimen | 1.5 × 103 cells/mL | 100% (5/5) | |
Enterotoxigenic E. coli (ETEC) lt/st | ATCC 35401 | 2.0 × 103 cells/mL | 100% (5/5) | |
Shiga-like toxin producing E. coli (STEC) stx1/stx2 | ATCC BAA-2196, ATCC 43895, Clinical Specimen | 1.0 × 102 cells/mL | 100% (5/5) | |
Plesiomonas shigelloides
| ATCC 14029 | 3.0 × 102 cells/mL | 100% (5/5) | |
Vibrio parahaemolyticus
| ATCC 17802 | 3.0 × 103 cells/mL | 100% (5/5) | |
Vibrio vulnificus
| ATCC 27562 | 1.0 × 104 cells/mL | 100% (5/5) | |
Helicobacter pylori
| ATCC 700392 | 1.5 × 104 cells/mL | 100% (5/5) | |
Listeria spp. | ATCC 19111 | 3.0 × 103 cells/mL | 100% (5/5) | |
Vibrio Cholerae
| ATCC 14035 | 2.0 × 102 cells/mL | 100% (5/5) | |
C. difficile Toxin A/B | ATCC 9689 | 1.0 × 103 cells/mL | 100% (5/5) | |
Salmonella spp. | ATCC 700623 | 2.0 × 103 cells/mL | 100% (5/5) | |
Shigella/Enteroinvasive E. coli (EIEC) | ATCC 29930 | 1.0 × 102 cells/mL | 100% (5/5) | |
Yersinia enterocolitica
| ATCC 9610 | 2.0 × 104 cells/mL | 100% (5/5) | |
Campylobacter jejuni
| ATCC BAA-1234 | 3.0 × 102 cells/mL | 100% (5/5) | |
Campylobacter upsaliensis
| ATCC 43954 | 1.0 × 102 cells/mL | 100% (5/5) | |
Parasite |
Giardia lamblia
| ATCC 30957 | 4.0 × 102 cells/mL | 100% (5/5) |
Cryptosporodium spp. | Waterborne P102C | 1.0 × 102 oocycts/mL | 100% (5/5) | |
Entamoeba histolytica
| ATCC 30459 | 1.0 × 102 cells/mL | 100% (5/5) | |
Cyclospora cayetanensis
| Zeptometrix control | 2.0 × 103 DNA copies/mL | 100% (5/5) | |
Virus | Norovirus GI/GII | Zeptometrix control | 1.0 × 103 RNA copies/mL | 100% (5/5) |
Rotavirus A | ATCC VR-2104 | 3.1 × 102 RNA copies/mL | 100% (5/5) | |
Adenovirus F 40/41 | ATTC VR-930/931 | 1.0 × 102 RNA copies/mL | 100% (5/5) | |
Astrovirus | Zeptometrix control | 1.2 × 103 RNA copies/mL | 100% (5/5) | |
Sapovirus (I, II, IV, V) | Zeptometrix control | 2.1 × 102 RNA copies/mL | 100% (5/5) | |
Fungi | Candida spp. | ATCC 10231 | 1.0 × 102 cells/mL | 100% (5/5) |
Microsporidium spp. | Clinical specimen | 2.0 × 102 DNA copies/mL | 100% (5/5) |
Performance evaluation of validated positive specimens
Organism | Comparator (culture or RT-PCR) | Vibrant GPP array | Agreement (95% CI) | ||
---|---|---|---|---|---|
Culture isolates | Clinical samples | Culture isolates | Clinical samples | ||
Bacteria | |||||
E. coli O157:H7 | 2 | 7 | 2 | 7 | 100 (66.4–100) |
Enteroaggregative E. coli (EAEC) | 2 | 3 | 2 | 3 | 100 (47.8–100) |
Enteropathogenic E. coli (EPEC) | 1 | 4 | 1 | 4 | 100 (47.8–100) |
Enterotoxigenic E. coli (ETEC) lt/st | 2 | 6 | 2 | 6 | 100 (63.1–100) |
Shiga-like toxin producing E. coli (STEC) stx1/stx2 | 2 | 9 | 2 | 9 | 100 (71.5–100) |
Plesiomonas shigelloides | 1 | 8 | 1 | 8 | 100 (66.4–100) |
Vibrio parahaemolyticus | 1 | 7 | 1 | 7 | 100 (63.1–100) |
Vibrio vulnificus | 1 | 6 | 1 | 6 | 100 (59.0–100) |
Helicobacter pylori | 2 | 3 | 2 | 3 | 100 (47.8–100) |
Listeria spp. | 1 | 6 | 1 | 6 | 100 (59.0–100) |
Vibrio cholerae | 1 | 3 | 1 | 3 | 100 (39.8–100) |
C. difficile toxin A/B | 1 | 9 | 1 | 9 | 100 (69.2–100) |
Salmonella spp. | 3 | 8 | 3 | 8 | 100 (71.5–100) |
Shigella/Enteroinvasive E. coli (EIEC) | 3 | 5 | 3 | 5 | 100 (63.1–100) |
Yersinia enterocolitica | 1 | 5 | 1 | 5 | 100 (54.1–100) |
Campylobacter jejuni | 2 | 3 | 2 | 3 | 100 (47.8–100) |
Campylobacter upsaliensis | 1 | 3 | 1 | 3 | 100 (39.8–100) |
Parasite | |||||
Giardia lamblia | 1 | 3 | 1 | 3 | 100% (39.8–100) |
Cryptosporodium spp. | 2 | 8 | 2 | 8 | 100% (69.2–100) |
Entamoeba histolytica | 7 | 2 | 7 | 2 | 100% (66.4–100) |
Cyclospora cayetanensis | 2 | 4 | 2 | 4 | 100% (54.1–100) |
Virus | |||||
Norovirus GI | 2 | 9 | 2 | 9 | 100% (71.5–100) |
Norovirus GII | 2 | 3 | 2 | 3 | 100% (47.8–100) |
Rotavirus A | 1 | 7 | 1 | 7 | 100% (63.1–100) |
Adenovirus F 40/41 | 1 | 10 | 1 | 10 | 100% (71.5–100) |
Astrovirus | 1 | 9 | 1 | 9 | 100% (69.2–100) |
Sapovirus (I, II, IV, V) | 1 | 2 | 1 | 2 | 100% (29.2–100) |
Fungi | |||||
Candida spp. | 1 | 6 | 1 | 6 | 100% (59.0–100) |
Microsporidium spp. | 1 | 1 | 1 | 1 | 100% (15.8–100) |
Accuracy study
Vibrant GPP panel | No. of positive samples | No. of negative samples | Sensitivity % (95% CI) | Specificity % (95% CI) | PPV % (95% CI) | NPV % (95% CI) |
---|---|---|---|---|---|---|
E. coli O157:H7 | 25 | 1028 | 96.2 (80.4–99.9) | 99.9 (99.5–100) | 96.2 (80.4–99.9) | 99.9 (99.5–100) |
Enteroaggregative E. coli (EAEC) | 8 | 1046 | 100 (63.1–100) | 99.9 (99.5–100) | 88.9 (63.1–100) | 100 (99.5–100) |
Enteropathogenic E. coli (EPEC) | 17 | 1038 | 100 (80.5–100) | 100 (99.6–100) | 100 (80.5–100) | 100 (99.6–100) |
Enterotoxigenic E. coli (ETEC) lt/st | 6 | 1047 | 100 (54.1–100) | 99.8 (99.3–100) | 75.0 (54.1–100) | 100 (99.3–100) |
Shiga-like toxin producing E. coli (STEC) stx1/stx2 | 12 | 1042 | 92.3 (64.0–99.8) | 100 (99.6–100) | 100 (64.0–99.8) | 99.9 (99.6–100) |
Plesiomonas shigelloides
| 4 | 1050 | 80 (28.4–99.5) | 100 (99.6–100) | 100 (28.4–99.5) | 99 (99.6–100) |
Helicobacter pylori
| 4 | 1051 | 100 (39.8–100) | 100 (99.6–100) | 100 (39.8–100) | 100 (99.6–100) |
Listeria spp. | 7 | 1048 | 100 (59.0–100) | 100 (99.6–100) | 100 (59.0–100) | 100 (99.6–100) |
Vibrio cholerae
| 5 | 1048 | 100 (47.8–100) | 99.8 (99.3–100) | 71.4 (47.8–100) | 100 (99.3–100) |
C. difficile Toxin A/B | 10 | 1043 | 91.7 (61.5–99.8) | 99.9 (99.5–100) | 91.7 (61.5–99.8) | 99.9 (99.5–100) |
Salmonella spp. | 13 | 1042 | 100 (75.3–100) | 100 (99.6–100) | 100 (75.3–100) | 100 (99.6–100) |
Shigella/Enteroinvasive E. coli (EIEC) | 5 | 1049 | 83.3 (35.9–99.6) | 100 (99.6–100) | 100 (35.9–99.6) | 99.9 (99.6–100) |
Yersinia enterocolitica
| 12 | 1042 | 92.3 (64.0–99.8) | 100 (99.6–100) | 100 (64.0–99.8) | 99.9 (99.6–100) |
Campylobacter jejuni
| 7 | 1047 | 87.5 (47.4–99.7) | 100 (99.6–100) | 100 (47.4–99.7) | 99.9 (99.6–100) |
Campylobacter upsaliensis
| 3 | 1051 | 100 (30.0–100) | 99.9 (99.5–100) | 75.0 (30.0–100) | 100 (99.5–100) |
Giardia lamblia
| 8 | 1047 | 100 (63.1–100) | 100 (99.6–100) | 100 (63.1–100) | 100 (99.6–100) |
Entamoeba histolytica
| 6 | 1049 | 100 (54.1–100) | 100 (99.6–100) | 100 (54.1–100) | 100 (99.6–100) |
Cyclospora cayetanensis
| 3 | 1050 | 100 (30.0–100) | 99.8 (99.3–100) | 60.0 (30.0–100) | 100 (99.3–100) |
Norovirus GI/GII | 12 | 1042 | 92.3 (64.0–99.8) | 100 (99.6–100) | 100 (64.0–99.8) | 99.9 (99.6–100) |
Rotavirus A | 4 | 1051 | 100 (39.8–100) | 100 (99.6–100) | 100 (39.8–100) | 100 (99.6–100) |
Adenovirus F 40/41 | 7 | 1048 | 100 (59.0–100) | 100 (99.6–100) | 100 (59.0–100) | 100 (99.6–100) |
Astrovirus | 4 | 1052 | 100 (39.8–100) | 100 (99.6–100) | 100 (39.8–100) | 100 (99.6–100) |
Sapovirus (I, II, IV, V) | 3 | 1050 | 100 (47.8–100) | 100 (99.6–100) | 100 (47.8–100) | 100% (99.6–100) |
Candida spp. | 26 | 1027 | 96.3 (81.0–99.9) | 99.8 (99.3–100) | 92.9 (81.0–99.9) | 99.9 (99.3–100) |
Vibrant GPP panel | No: positive samples tested | No: negative samples tested | Sensitivity % (95% CI) | Specificity % (95% CI) | PPV % (95% CI) | NPV % (95% CI) |
---|---|---|---|---|---|---|
Vibrio parahaemolyticus
| 3 | 1055 | 100 (29.2–100) | 100 (99.6–100) | 100 (29.2–100) | 100 (99.6–100) |
Vibrio vulnificus
| 3 | 1055 | 100 (29.2–100) | 100 (99.6–100) | 100 (29.2–100) | 100 (99.6–100) |
Cryptosporodium spp. | 1 | 1055 | 100 (2.5–100) | 100 (99.6–100) | 100 (2.5–100) | 100 (99.6–100) |
Microsporidium spp. | 2 | 1055 | 100 (15.8–100) | 100 (99.6–100) | 100 (15.8–100) | 100 (99.6–100) |
Detection of multiple pathogens
Multiple detection | Number of specimens |
---|---|
E. coli O157:H7 + Norovirus (GI/GII) | 10 |
Campylobacter jejuni + E. coli O157:H7 + STEC stx1/stx2 | 1 |
C. difficile toxin A/B + STEC stx1/stx2 | 3 |
Adenovirus 40/41 + EPEC | 5 |
Candida spp. + EPEC | 3 |
EAEC + EPEC | 6 |
E. coli O157:H7 + STEC stx1/stx2 | 7 |
Stability study
Assay | Number of total samples | Number of correct samples | Percent of correct calls |
---|---|---|---|
Before shipping | |||
Culture and RT-PCR | 28 | 28 | 100 |
After shipping | |||
Culture and RT-PCR | 28 | 28 | 100 |