Background
Methods
Patients
Stools sample collection
Microbial agents and target sequences
Agent | Forward primer | Reverse primer | Probe | Target gene | Ref |
---|---|---|---|---|---|
Norovirus GG2 | TGGAYTTTTAYGTGCCCAG | CGACGCCATCTTCATTCAC | AGCCAGATTGCGATCGCCC | Polymerase-capsid junction | [16] |
Rotavirusa
| AACCATCTACACATGACCCTCTATGA | GGTCACATAACGCCCCTATAGC | CAATAGTTAAAAGCTAACACTGTCAAA | Non-strutural protein 3 | [17] |
AACCATCTTCACGTAACCCTCTATGA | |||||
Adenovirus | GCCACGGTGGGGTTTCTAAACTT | GCCCCAGTGGTCTTACATGCACATC | TGCACCAGACCCGGGCTCAGGTACTCCGA | Hexon | [15] |
Campylobacter jejuni
| ATGCAAACCATAATTGGGTTTCAAC | CGAGTATCAGCAACTTCTTCTACAGCT | TTGCCACCAAAACCAAAACT | Fibronectin-binding protein (cadF) | [18] |
ETEC-estA
| AAGCATGAATAGTAGCAATTACTGCT | TTAATAGCACCCGGTACAAGCA | AACAACACAATTCAC | Heat-stable enterotoxin | [19] |
ETEC-eltB
| TCCGGCAGAGGATGGTTACA | CCAGGGTTCTTCTCTCCAAGC | AGCAGGTTTCCCACCGGATCACC | Heat-labile enterotoxin | [20] |
E. coli eae
| ACATGACCGATGACAAGGCA | CGCGACTGAAGCTGGCTAC | TCGCCGCCTGTTGTGCCG | Enteroaggregative factor | [21] |
E. coli bfpA
| GGTCTGTCTTTGATTGAATCTGCA | GCAGACTGGTAGTAAAACATCACACC | GCGCTTGCTGCCACCGTTACCG | Bundle-forming pilus | [22] |
Shigella spp
| ACCGGCGCTCTGCTCTC | GCAATGTCCTCCAGAATTTCG | CTGGGCAGGGAAATGTTCCGCC | Invasion plasmid antigen H | [23] |
Cryptosporidium parvum/hominis
| CAAATTGATACCGTTTGTCCTTCTG | TGGTGCCATACATTGTTGTCCT | TGTCCTCCTGGATTCA | Oocyst wall protein (OWP) | [24] |
Sample preparation and nucleic acid extraction
Real-time PCR
Analysis of human gene content in faeces and rectal swabs
Ethical committee approval
Statistics
Results
Detections rates in faeces and rectal swabs
Diarrhoea (n = 207) | No diarrhoea (n = 119) | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Faeces | Rectal swab | P value | Faeces | Rectal swab | P value | |||||
ETEC-eltB
| 38% | (78) | 29% | (60) | 0.06 | 47% | (56) | 42% | (50) | >0.30 |
ETEC-estA
| 16% | (34) | 15% | (31) | >0.30 | 10% | (12) | 13% | (16) | >0.30 |
E. coli bfpA
| 21% | (44) | 17% | (35) | 0.26 | 7.6% | (9) | 9.2% | (11) | >0.30 |
E. coli eae
| 22% | (45) | 22% | (45) | >0.30 | 23% | (27) | 25% | (30) | >0.30 |
Shigella ipaH
| 22% | (46) | 19% | (39) | >0.30 | 17% | (20) | 22% | (26) | >0.30 |
Campylobacter
| 20% | (42) | 20% | (42) | >0.30 | 22% | (26) | 20% | (24) | >0.30 |
Cryptosporidium
| 9.7% | (20) | 9.2% | (19) | >0.30 | 5.0% | (6) | 6.7% | (8) | >0.30 |
Norovirus GG2 | 8.7% | (18) | 8.2% | (17) | >0.30 | 4.2% | (5) | 3.4% | (4) | >0.30 |
Rotavirus | 23% | (48) | 23% | (48) | >0.30 | 0% | (0) | 0.9% | (1) | >0.30 |
Adenovirus | 13% | (26) | 16% | (33) | >0.30 | 50% | (59) | 55% | (65) | >0.30 |
Reactive in faeces or rectal swab | Reactive in faeces | Faeces sensitivitya
| Reactive in rectal swab | Rectal swab sensitivityb
| Reactive in faeces andrectal swab | Discordant samplesc
| Kappa value | |
---|---|---|---|---|---|---|---|---|
ETEC-eltB
| 147 | 134 | 91% | 110 | 75% | 97 | 50 | 0.674 |
ETEC-estA
| 56 | 46 | 82% | 47 | 84% | 37 | 19 | 0.762 |
E. coli bfpA
| 61 | 53 | 87% | 46 | 75% | 38 | 23 | 0.726 |
E. coli eae
| 91 | 72 | 79% | 75 | 82% | 56 | 35 | 0.692 |
Shigella ipaH
| 77 | 66 | 86% | 65 | 84% | 54 | 23 | 0.780 |
Campylobacter
| 76 | 68 | 89% | 65 | 86% | 57 | 19 | 0.821 |
Cryptosporidium
| 31 | 25 | 81% | 28 | 90% | 22 | 9 | 0.815 |
Rotavirus | 60 | 48 | 80% | 49 | 82% | 37 | 23 | 0.721 |
Norovirus GG2 | 27 | 23 | 85% | 21 | 78% | 17 | 10 | 0.756 |
Adenovirus | 122 | 85 | 73% | 95 | 84% | 65 | 53 | 0.452 |
Ct values in faeces and rectal swabs
Median Ct valuea
| dCtb, mean (SE) | P valuec
| ||
---|---|---|---|---|
Rectal swab | Faeces | |||
ETEC-eltB
| 32.0/37.5 | 28.5/37.4 | 2.28 (0.48) | <0.0001 |
ETEC-estA
| 27.7/36.2 | 25.8/40.1 | 2.99 (0.48) | <0.0001 |
E. coli bfpA
| 27.6/34.2 | 26.9/37.5 | 2.10 (0.49) | <0.0001 |
E. coli eae
| 34.8/38.1 | 33.5/38.5 | 0.85 (0.47) | 0.0743 |
Shigella ipaH
| 29.4/37.8 | 28.1/36.7 | 0.91 (0.58) | 0.1245 |
Campylobacter
| 32.6/35.8 | 33.2/37.4 | -0.002 (0.59) | 1.00 |
Cryptosporidium
| 36.2/39.9 | 34.1/40.9 | 2.23 (0.76) | 0.008 |
Rotavirus | 22.5/41.2 | 20.9/36.2 | 2.25 (0.61) | 0.0008 |
Norovirus GG2 | 30.3/38.8 | 29.3/33.8 | 1.45 (0.78) | 0.08 |
Adenovirus | 34.0/39.7 | 35.9/39.9 | -0.48 (0.39) | 0.21 |
Diarrhoea (n = 207) | No diarrhoea (n = 119) | |||||
---|---|---|---|---|---|---|
Mean Ct differencea
| n | P value | Mean Ct differencea
| n | P value | |
ETEC-eltB
| 3.61 | 47 | <0.0001 | 1.03 | 50 | 0.09 |
ETEC-est A | 3.11 | 24 | <0.0001 | 2.76 | 13 | 0.02 |
E. coli bfpA
| 2.27 | 28 | 0.0002 | 1.63 | 10 | 0.20 |
E. coli eae
| 2.40 | 30 | 0.0001 | -0.93 | 26 | 0.1662 |
Shigella
| 2.41 | 34 | <0.0004 | -1.64 | 20 | 0.1008 |
Campylobacter
| -0.61 | 30 | 0.50 | -0.69 | 27 | 0.36 |
Cryptosporidium
| 3.09 | 16 | 0.002 | -0.06 | 6 | 0.96 |
Rotavirus | 2.25 | 37 | 0.0008 | 0 | ||
Norovirus GG2 | 2.21 | 12 | 0.04 | -0.39 | 5 | 0.72 |
Adenovirus | 0.43 | 12 | 0.71 | -0.69 | 53 | 0.087 |