Background
Methods
Subjects of this study
Specimen and data collection
Laboratory test for enteric pathogens
Bacterial detection
Enteric pathogens | Target gene | Primer (5′–3′) | Amplicon sizes (bp) | Remarks | Source |
---|---|---|---|---|---|
EPEC | eae | CCACGGTTTATCAAACTGATAACG | 105 | Each stool specimen was inoculated to MAC media to culture DEC at 37 °C for 18 h, And then ten putative DEC colonies were selected to mix with 150 μL water to extract DNA at 100 °C for 10 min, and then the 20 μL volume of qPCR system is composed of 10 μL master mix (Takara Bio Inc, Shiga, Japan), 1 μL forward primer (10 μmol), 1 μL reverse primer (10 μmol), 1 μL DNA template and 7 μL H2O. The cycling conditions for each subtype DEC was 95 °C for 5 min, 40 cycles of 95 °C for 5 s, 60 °C for 30 s. The fluorescence recorded was at the annealing stage | |
EHEC | stx1 | ACTTCTCGACTGCAAAGACGTATG | 132 | ||
ACAAATTATCCCCTGAGCCACTATC | |||||
stx2 | CCACATCGGTGTCTGTTATTAACC | 93 | |||
GGTCAAAACGCGCCTGATAG | |||||
ETEC | elt | TTCCCACCGGATCACCAA | 62 | ||
CAACCTTGTGGTGCATGATGA | |||||
estA | CCTTTCGCTCAGGATGCTAAAC | 128 | |||
CAGTAATTGCTACTATTCATGCTTTCAG | |||||
estB | CTTTCCCCTCTTTTAGTCAGTCAACT | 137 | |||
GCAGTAAAATGTGTTGTTCATATTTTCTG | |||||
EAEC | aggR | CAGCGATACATTAAGACGCCTAAAG | 116 | ||
CGTCAGCATCAGCTACAATTATTCC | |||||
EIEC | ipaH | ACCATGCTCGCAGAGAAACT | 175 | ||
TCAGTACAGCATGCCATGGT | |||||
RVA | VP6 | GACGGVGCRACTACATGGT | 382 | RVA, NoV GI, NoV GII, SaV and As were RNA viruses, complementary DNA (cDNA) was synthesized using a random primer (Takara Bio Inc, Shiga, Japan) at 55 °C for 1.5 h, followed by 100 °C for 10 min, and holding at 4 °C. The reaction condition of RVA was 94 °C for 5 min, followed by 40 cycles at 94 °C for 1 min, 42 °C for 1 min, 72 °C for 1 min, and with final extension at 72 °C for 10 min. Multiplex RT-PCR was used to detect the presence of NoV GI, NoV GII, and SaV, the thermal profile consisted of 94 °C for 5 min, 40 cycles of 94 °C for 70 s, 49 °C for 70 s, and 72 °C for 1 min, followed by 72 °C for 10 min. The thermal profile of As was 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min | [22] |
GTCCAATTCATNCCTGGTGG | |||||
NoV GI NoV GII SaV | Polymerase | TGACGATTTCATCATCACCATA | 331/319 | [23] | |
TGACGATTTCATCATCCCCGTA | |||||
GATTACTCCAGGTGGGACTCCAC | |||||
GATTACTCCAGGTGGGACTCAAC | |||||
GATTACTCCAGGTGGGATTCAAC | |||||
GATTACTCCAGGTGGGATTCCAC | |||||
As | Capsid | CAACTCAGGAAACAGGGTGT | 449 | [24] | |
TCAGATGCATTGTCATTGGT | |||||
Ad | Hexon | TTCCCCATGGCICAYAACAC | 482 | The thermal profile was 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min | [25] |
CCCTGGTAKCCRATRTTGTA | |||||
Blastocistis hominis
| SSU-rRNA | CGAATGGCTCATTATATCAGTT | 260 | The thermal profile was 94 °C for 5 min, 40 cycles of 94 °C for 30 s, 53 °C for 30 s, and 72 °C for 1 min, followed by 72 °C for 10 min | [26] |
TCTTCGTTACCCGTTACTGC | |||||
Cryptosporidium spp. | 18S-rRNA | TTCTAGAGCTAATACATGCG | The primary cycle consisted of 94 °C for 5 min, 35 cycles of 94 °C for 50 s, 55 °C for 1 min and 72 °C for 90 s, followed by 72 °C for 10 min, the annealing step for a second reaction was 58 °C | [27] | |
CCCATTTCCTTCGAAACAGGA | |||||
GGAAGGGTTGTATTTATTAGATAAAG | 840 | ||||
CTCATAAGGTGCTGAAGGAGTA | |||||
Giardia lamblia
| Tim | AAATIATGCCTGCTCGTCG | The thermal profile of first round was 94 °C for 1 min, 53 °C for 1 min, and 72 °C for 1 min, followed by 72 °C for 10 min. A second reaction was carried out similarly | [28] | |
CAAACCTTITCCGCAAACC | |||||
CCCTTCATCGGIGGTAACTT | 530 | ||||
GTGGCCACCACICCCGTGCC |
Virus detection
Enteric protozoan detection
Data analysis
Results
Basic information and clinical symptoms
Characteristic | Diarrhea | Control |
---|---|---|
n (%) | n (%) | |
n | 271 | 149 |
Age | ||
5–15 years | 21 (7.7) | 9 (6.0) |
15–50 years | 175 (64.6) | 94 (63.1) |
≥50 years | 75 (27.7) | 46 (30.9) |
Sex | ||
Male | 133 (49.1) | 73 (49.0) |
Female | 138 (50.9) | 76 (51.0) |
Residence | ||
Urban | 184 (67.9) | 99 (66.4) |
Rural–urban fringe zone | 68 (25.1) | 35 (23.5) |
Rural | 19 (7.0) | 15 (10.1) |
Seasons | ||
Spring (Feb–Apr) | 87 (32.1) | 32 (21.5) |
Summer (May–Jul) | 65 (24.0) | 42 (28.2) |
Autumn (Aug–Oct) | 59 (25.5) | 45 (30.2) |
Winter (Nov–Jan) | 50 (18.5) | 30 (37.5) |
Symptom | ||
Fever (>37.3 °C) | 23 (8.5) | – |
Abdominal pain | 73 (26.9) | – |
Nausea | 91 (33.6) | – |
Vomiting | 58 (21.4) | – |
Dehydration | 3 (1.1) | – |
Tenesmus | 5 (1.8) | – |
Diarrhea | – | |
Watery stool | 70 (25.8) | – |
Mucus stool | 173 (63.8) | – |
Other stool | 28 (10.3) | – |
The prevalence of enteropathogen in subjects with diarrhea or not
Enteropathogen | Diarrhea cases n = 271 n (%) | Healthy controls n = 149 n (%) | Univariate analysis | Multivariate analyses | ||
---|---|---|---|---|---|---|
p value | OR (95% CI) |
p value | OR (95% CI) | |||
At least one enteropathogen | 79 (29.2) | 18 (12.1) |
p < 0.0001 | 3.00 (1.71–5.23) | – | – |
At least one enteric bacterial pathogens | 37 (13.7) | 5 (3.4) |
p = 0.001 | 4.55 (1.75–11.85) | – | – |
DEC | 34 (12.5) | 5 (3.4) |
p = 0.002 | 4.13 (1.58–10.80) | – | – |
EAEC | 20 (7.4) | 2 (1.3) |
p = 0.008 | 5.86 (1.35–25.41) |
p = 0.198 | 5.95 (1.33–26.63) |
EPEC | 15 (5.5) | 3 (2.0) |
p = 0.088 | 2.85 (0.81–10.01) |
p = 0.107 | 2.86 (0.80–10.27) |
ETEC | 1 (0.4) | 0 (0.0) | – | – | – | – |
EIEC | 0 (0.0) | 0 (0.0) | – | – | – | – |
EHEC | 0 (0.0) | 0 (0.0) | – | – | – | – |
NTS | 2 (0.7) | 0 (0.0) |
p = 0.541 | – | – | – |
Plesiomonas spp. | 1 (0.4) | 0 (0.0) | – | – | – | – |
Vibrio parahaemolyticus
| 0 (0.0) | 0 (0.0) | – | – | – | – |
Vibrio cholera
| 0 (0.0) | 0 (0.0) | – | – | – | – |
Aeromonas spp. | 0 (0.0) | 0 (0.0) | – | – | – | – |
Shigella spp. | 0 (0.0) | 0 (0.0) | – | – | – | – |
At least one enteric virus pathogens | 39 (14.4) | 6 (4.0) |
p = 0.001 | 4.00 (1.66–9.70) | – | – |
NoV GII | 27 (10.0) | 3 (2.0) |
p = 0.002 | 5.38 (1.60–18.06) |
p = 0.0794 | 3.86 (0.85–17.48) |
RVA | 20 (7.4) | 2 (1.3) |
p = 0.008 | 5.86 (1.35–25.41) |
p = 0.0166 | 4.50 (1.31–15.43) |
NoV GI | 1 (0.4) | 0 (0.0) | – | – | – | – |
SaV | 1 (0.4) | 0 (0.0) | – | – | – | – |
As | 0 (0.0) | 1 (0.7) |
p = 0.355 | – | – | – |
Ad | 0 (0.0) | 0 (0.0) | – | – | ||
At least one enteric parasite pathogens | 13 (4.8) | 9 (6.0) |
p = 0.584 | 0.78 (0.33–1.88) | ||
B. hominis
| 13 (4.8) | 9 (6.0) |
p = 0.584 | 0.78 (0.33–1.88) |
p = 0.412 | 0.68 (0.27–1.71) |
Cryptosporidium spp. | 1 (0.4) | 0 (0.0) | – | – | – | – |
Giardia lamblia
| 0 (0.0) | 0 (0.0) | – | – | – | – |
Temporal distribution of enteric pathogen in diarrhea cases
Enteropathogen | Spring (Feb–Apr) n = 87 n (%) | Summer (May–Jul) n = 65 n (%) | Autumn (Aug–Oct) n = 69 n (%) | Winter (Nov–Jan) n = 50 n (%) | χ2
|
p value |
---|---|---|---|---|---|---|
EAEC | 1 (1.2) | 9 (13.8) | 8 (11.6) | 2 (4.0) | – |
p = 0.0045 |
EPEC | 7 (8.0) | 7 (10.8) | 1 (1.4) | 0 (0.0) | – |
p = 0.0156 |
RVA | 4 (4.6) | 0 (0.0) | 8 (11.6) | 8 (16.0) | – |
p = 0.0015 |
NoV | 5 (5.7) | 7 (10.8) | 10 (14.5) | 5 (10.0) | 3.359 |
p = 0.341 |
B. hominis
| 0 (0.0) | 7 (10.8) | 6 (8.7) | 0 (0.0) | – |
p < 0.0001 |
Prevalence of enteric pathogens in diarrhea cases in different age group
Enteropathogen | Total n = 271 n (%) | 5–15 years n = 21 n (%) | 15–50 years n = 175 n (%) | ≥50 years n = 75 n (%) | χ2
|
p value |
---|---|---|---|---|---|---|
At least one enteropathogens | 79 (29.2) | 9 (42.9) | 52 (29.7) | 18 (24.0) | 2.90 |
p = 0.234 |
At least one bacterium | 37 (13.7) | 5 (23.8) | 27 (15.4) | 5 (6.7) | 5.41 |
p = 0.0668 |
At least one virus | 39 (14.4) | 4 (19.0) | 23 (13.1) | 12 (16.0) | 0.748 |
p = 0.688 |
At least one parasite | 13 (4.8) | 1 (4.8) | 10 (5.7) | 2 (2.7) | – |
p = 0.654 |
EAEC | 20 (7.4) | 1 (4.8) | 16 (9.1) | 3 (4.0) | 2.56 |
p = 0.323 |
EPEC | 15 (5.5) | 4 (19.0) | 9 (5.0) | 2 (2.7) | – |
p = 0.031 |
NoV | 27 (10.0) | 4 (19.0) | 15 (8.6) | 8 (10.7) | 2.35 |
p = 0.309 |
RVA | 20 (7.4) | 2 (9.5) | 13 (7.4) | 5 (6.7) | 0.198 |
p = 0.906 |
B. hominis
| 13 (4.8) | 1 (4.8) | 10 (5.7) | 2 (2.7) | – |
p = 0.654 |
Co-infection of enteric pathogen in diarrhea cases and healthy cases
Co-infections of enteric pathogens | Diarrhea cases n = 271 n (%) | Healthy controls n = 149 n (%) |
p value | OR (95% CI) |
---|---|---|---|---|
Any two any enteric pathogens | 20 (7.4) | 2 (1.3) |
p = 0.0079 | 5.86 (1.35–25.41) |
Virus–virus | 9 (3.3) | 0 (0.0) |
p = 0.0298 | – |
RVA–NoV GII | 8 (3.0) | 0 (0.0) |
p = 0.0549 | – |
Bacteria–virus | 5 (1.8) | 0 (0.0) |
p = 0.166 | – |
DEC–NoV GII | 4 (1.5) | 0 (0.0) |
p = 0.302 | – |
DEC–RVA | 3 (1.1) | 0 (0.0) |
p = 0.556 | – |
Bacteria–protozoan | 3 (1.1) | 2 (1.3) |
p = 0.999 | 0.83 (0.14–5.00) |
DEC–B. hominis
| 3 (1.1) | 2 (1.3) |
p = 0.999 | 0.83 (0.14–5.00) |
Any three enteric pathogens | 1 (0.4) | 0 (0.0) |
p = 0.999 | – |
DEC–RVA–Cryptosporidium spp. | 1 (0.4) | 0 (0.0) |
p = 0.999 | – |
Total | 21 (7.7) | 2 (1.3) |
p = 0.0059 | 6.17 (1.43–26.71) |