Introduction
Materials and methods
Serological tests
DNA isolation
Detection and quantification of T. gondii DNA
Determination of SNPs located in TLR4 and TLR9 genes
Gene | SNPa name | Primer sequences (5′-3′) | Annealing temperature (°C) | Amplicon length (bps)b
| Restriction enzyme | Profile (bps) | |
---|---|---|---|---|---|---|---|
TLR4
| 896 A>G (1063 A>G, rs4986790) | External | For: AAAACTTGTATTCAAGGTCTGGC | 52 | 355 | NcoI | AA: 188 AG: 188, 168, 20 GG: 168, 20 |
Rev: TGTTGGAAGTGAAAGTAAGCCT | |||||||
Internal | For: AGCATACTTAGACTACTACCTCCATG | 61 | 188 | ||||
Rev: AGAAGATTTGAGTTTCAATGTGGG | |||||||
1196 C>T (1363 C>T, rs4986791) | External | For: AGTTGATCTACCAAGCCTTGAGT | 52 | 510 | HinfI | CC: 407 CT: 407, 378, 29 TT: 378, 29 | |
Rev: GGAAACGTATCCAATGAAAAGA | |||||||
Internal | For: GGTTGCTGTTCTCAAAGTGATTTTGGGAGAA | 59 | 407 | ||||
Rev: ACCTGAAGACTGGAGAGTGAGTTAAATGCT | |||||||
TLR9
| 1635 G>A (2848G>A, rs352140) | External | For: GTCAATGGCTCCCAGTTCC | 52 | 292 | BstUI | GG: 135, 42 GA: 177, 135, 42 AA: 177 |
Rev: CATTGCCGCTGAAGTCCA | |||||||
Internal | For: AAGCTGGACCTCTACCACGA | 59 | 177 | ||||
Rev: TTGGCTGTGGATGTTGTT |
Statistical analysis
Results
T. gondii DNA loads in fetal and neonatal body fluids
Hardy–Weinberg equilibrium, linkage disequilibrium
Prevalence rates of the genotypes at the TLR4 896 A>G, 1196 C>T, and TLR9 1635 G>A SNPs
Gene polymorphism | Genetic model | Genotype | Genotype prevalence rates; n (%)a
| ORb (95 % CI)c
|
p-Valued
| |
---|---|---|---|---|---|---|
Infected cases | Uninfected controls | |||||
TLR4 896 A>G | Codominant | AA | 18 (94.7 %) | 47 (94 %) | 1.00 | 0.420 |
AG | 1 (5.3 %) | 1 (2 %) | 2.61 (0.15–44.01) | |||
GG | 0 (0 %) | 2 (4 %) | 0.00 (0.00–NA) | |||
Dominant | AA | 18 (94.7 %) | 47 (94 %) | 1.00 | 0.910 | |
AG-GG | 1 (5.3 %) | 3 (6 %) | 0.87 (0.08–8.92) | |||
Recessive | AA-AG | 19 (100 %) | 48 (96 %) | 1.00 | 0.250 | |
GG | 0 (0 %) | 2 (4 %) | 0.00 (0.00–NA) | |||
Overdominant | AA-GG | 18 (94.7 %) | 49 (98 %) | 1.00 | 0.490 | |
AG | 1 (5.3 %) | 1 (2 %) | 2.72 (0.16–45.85) | |||
Log-additive | – | – | – | 0.66 (0.12–3.75) | 0.620 | |
TLR4 1196 C>T | Codominant | CC | 17 (94.4 %) | 36 (87.8 %) | 1.00 | 0.450 |
CT | 1 (5.6 %) | 3 (7.3 %) | 0.71 (0.07–7.3) | |||
TT | 0 (0 %) | 2 (4.9 %) | 0.00 (0.00–NA) | |||
Dominant | CC | 17 (94.4 %) | 36 (87.8 %) | 1.00 | 0.410 | |
CT-TT | 1 (5.6 %) | 5 (12.2 %) | 0.42 (0.05–3.91) | |||
Recessive | CC-CT | 18 (100 %) | 39 (95.1 %) | 1.00 | 0.220 | |
TT | 0 (0 %) | 2 (4.9 %) | 0.00 (0.00–NA) | |||
Overdominant | CC-TT | 17 (94.4 %) | 38 (92.7 %) | 1.00 | 0.800 | |
CT | 1 (5.6 %) | 3 (7.3 %) | 0.75 (0.07–7.69) | |||
Log-additive | – | – | – | 0.43 (0.07–2.78) | 0.300 | |
TLR9 1635 G>A | Codominant | AA | 5 (25 %) | 15 (34.1 %) | 1.00 | 0.760 |
GA | 11 (55 %) | 21 (47.7 %) | 1.57 (0.45–5.47) | |||
GG | 4 (20 %) | 8 (18.2 %) | 1.50 (0.31–7.21) | |||
Dominant | AA | 5 (25 %) | 15 (34.1 %) | 1.00 | 0.460 | |
GA-GG | 15 (75 %) | 29 (65.9 %) | 1.55 (0.47–5.09) | |||
Recessive | AA-GA | 16 (80 %) | 36 (81.8 %) | 1.00 | 0.860 | |
GG | 4 (20 %) | 8 (18.2 %) | 1.12 (0.3–4.28) | |||
Overdominant | AA-GG | 9 (45 %) | 23 (52.3 %) | 1.00 | 0.590 | |
GA | 11 (55 %) | 21 (47.7 %) | 1.34 (0.46–3.87) | |||
Log-additive | – | – | – | 1.25 (0.59–2.68) | 0.560 |
The prevalence rates of the alleles located at the TLR4 and TLR9 SNPs
Gene polymorphism | No.a of carriers with TLR alleles (%) |
p-Valueb
| ||
---|---|---|---|---|
Congenital toxoplasmosis | Uninfected control | |||
TLR4 896 A>G | ||||
Alleles | A | 37 (97.4) | 95 (95.0) | 0.542 |
G | 1 (2.6) | 5 (5.0) | ||
TLR4 1196 C>T | ||||
Alleles | C | 35 (97.2) | 75 (91.5) | 0.252 |
T | 1 (2.8) | 7 (8.5) | ||
TLR9 1635 G>A | ||||
Alleles | G | 19 (47.5) | 51 (58.0) | 0.271 |
A | 21 (52.5) | 37 (42.0) |