Wioletta Wujcicka

Age-associated prevalence of Toxoplasma gondii in 8281 pregnant women in Poland between 2004 and 2012

This study aimed to describe Toxoplasma gondii prevalence in Polish pregnant women and the incidence rates of congenital infections in their neonates observed between 2004 and 2012. Serological tests for T. gondii-specific IgG and IgM antibodies were performed on serum samples of 8281 pregnant women treated at the Polish Mothers Memorial Hospital Research Institute in Lodz. The yearly seroconversion rate for T. gondii IgG antibodies was estimated using a mathematical model to determine the dependency between age and prevalence. Mean prevalence of IgG antibodies between 2004 and 2012 in pregnant women was 40·6% [95% confidence interval (CI) 39·6-41·7] and increased with age with a yearly seroconversion rate of 0·8% (95% CI 0·6-1·0, P<0·001). Assuming a T. gondii materno-fetal transmission rate of 30% gave an estimate of 1·80/1000 neonates as congenitally infected. The increased mean age (28·7 vs 26·7 years, P<0·001) of pregnant women was probably the most important factor in abolishing the effect of falling prevalence rates.

Do the placental barrier, parasite genotype and Toll-like receptor polymorphisms contribute to the course of primary infection with various Toxoplasma gondii genotypes in pregnant women?

Toxoplasma gondii has a highly clonal genetic structure classified into three major genetic types, I, II, and III, plus additional recombinant and atypical strains. In humans, type I and atypical strains usually associate with severe toxoplasmosis. Type II strains, predominantly identified in European countries and the United States, correlate with a differential course of toxoplasmosis. During pregnancy, the important protective role of the placenta against maternal–fetal T. gondii transmission has been reported. T. gondii preferentially colonizes extravillous trophoblasts as compared to syncytiotrophoblasts. The latter compartment was suggested to act as the real barrier to the fetal dissemination of T. gondii. Alterations in immune response to particular T. gondii strains were observed. Higher transcription levels of IP-10, IL-1β, IL-6, IL-10, IL-12 cytokines, and NF-κB translocation to the nucleus were more often documented for type II strains than type I strains. Since the induction of IL-12 during type II infection was Myd88-dependent, the involvement of Toll-like receptors (TLRs) in the immunity against these strains was suggested. Differential expression of TLRs depends on placental cell types and gestational age. The expression of TLR2 and TLR4 in the first trimester of pregnancy was reported only for villous cytotrophoblasts and extravillous trophoblasts, but not for syncytiotrophoblasts. The involvement of single-nucleotide polymorphisms (SNPs) in the TLR genes in infectious pathogenicity, including toxoplasmic retinochoroiditis, points at a possible involvement of TLR alterations in immunity against T. gondii. We conclude that studies on TLR contributions in the maternal–fetal transmission of particular parasite strains and congenital toxoplasmosis are warranted.

TLR9 2848 GA Heterozygotic Status Possibly Predisposes Fetuses and Newborns to Congenital Infection with Human Cytomegalovirus

Background Some single nucleotide polymorphisms (SNP), located in Toll-like receptor (TLR) genes, were reported to be associated with human cytomegalovirus (HCMV) infections. The study was aimed to assess the correlation of SNPs at TLR4 and TLR9 genes with the occurrence of congenital cytomegaly, based on available samples. Methods Reported case-control study included both HCMV infected and non-infected fetuses and newborns. The specimens were classified to the molecular analyses, based on serological features of the recent infection and HCMV DNAemia in body fluids. TLR SNPs were studied, using multiplex nested PCR-RFLP assay, and determined genotypes were confirmed by sequencing. Hardy-Weinberg equilibrium was assessed for the identified genotypes. The linkage disequilibrium was also estimated for TLR4 SNPs. A relationship between the status of TLR genotypes and congenital cytomegaly development was estimated, using a logistic regression model. Results Hardy Weinberg equilibrium was observed for almost all SNPs, both infected and non-infected patients, with exception of TLR4 896 A>G polymorphism in the control group (P≤0.050). TLR4 896 A>G and 1196 C>T SNPs were found in linkage disequilibrium in both study groups (P≤0.050). The CC genotype at TLR4 1196 SNP and the GA variant at TLR9 2848 G>A SNP were significantly associated with HCMV infection (P≤0.050). The risk of congenital cytomegaly was higher in heterozygotes at TLR9 SNP than in the carriers of other genotypic variants at the reported locus (OR 4.81; P≤0.050). The GC haplotype at TLR4 SNPs and GCA variants at TLR4 and TLR9 SNPs were significantly associated with HCMV infection (P≤0.0001). The ACA variants were more frequent among fetuses and neonates with symptomatic, rather than asymptomatic cytomegaly (P≤0.0001). Conclusions TLR4 and TLR9 polymorphisms may contribute to the development of congenital infection with HCMV in fetuses and neonates. The TLR9 2848 GA heterozygotic status possibly predisposes to HCMV infection, increasing the risk of congenital cytomegaly development.

Possible role of TLR4 and TLR9 SNPs in protection against congenital toxoplasmosis

The purpose of this investigation was the determination of the distribution of genotypes at single nucleotide polymorphisms (SNPs) of the toll-like receptor 4 (TLR4) and the toll-like receptor 9 (TLR9) in fetuses and newborns congenitally infected with Toxoplasma gondii and the identification of genetic changes predisposing to infection development. The study involved 20 fetuses and newborns with congenital toxoplasmosis and 50 uninfected controls. The levels of IgG and IgM antibodies against T. gondii, as well as IgG avidity, were estimated by enzyme-linked fluorescent assay (ELFA) tests. T. gondii DNA loads in amniotic fluids were assayed by the real-time (RT) quantitative polymerase chain reaction (Q PCR) technique for parasitic B1 gene. TLR4 and TLR9 SNPs were identified using a self-designed multiplex nested PCR-restriction fragment length polymorphism (RFLP) assay. Randomly selected genotypes at SNPs were confirmed by sequencing. All the genotypes were tested for Hardy–Weinberg equilibrium and TLR4 genotypes were analyzed for linkage disequilibrium. A correlation was studied between the genotypes or haplotypes and the development of congenital toxoplasmosis using a logistic regression model. Single SNP analysis showed no statistically significant differences in the distribution of distinct genotypes at the analyzed TLR4 and TLR9 SNPs between T. gondii-infected fetuses and newborns and the controls. Taking into account the prevalence of alleles residing within polymorphic sites, similar prevalence rates were observed in both of the studied groups. The multiple SNP analysis indicated GTG variants at the TLR4 and TLR9 SNPs to be significantly less frequent in offspring with congenital toxoplasmosis than in uninfected offspring (p ≤ 0.0001). TLR4 and TLR9 SNPs seem to be involved in protection against congenital toxoplasmosis.

Mixed infections with distinct cytomegalovirus glycoprotein B genotypes in Polish pregnant women, fetuses, and newborns

The purpose of this investigation was to describe a distribution of cytomegalovirus (CMV) single and multiple genotypes among infected pregnant women, their fetuses, and newborns coming from Central Poland, as well as congenital cytomegaly outcome. The study involved 278 CMV-seropositive pregnant women, of whom 192 were tested for viral DNAemia. Human cytomegalovirus (HCMV) genotyping was performed for 18 of 34 pregnant women carrying the viral DNA and for 12 of their 15 offspring with confirmed HCMV infections. Anti-HCMV antibodies levels were assessed by chemiluminescence immunoassay (CLIA) and enzyme-linked fluorescence assay (ELFA) tests. Viral DNA loads and genotypes were determined by real-time polymerase chain reaction (PCR) assays for the UL55 gene. In the pregnant women, we identified HCMV gB1, gB2, gB3, and gB4 genotypes. Single gB2, gB3, or gB4 genotypes were observed in 14 (77.8 %) women, while multiple gB1–gB2 or gB2–gB3 genotypes were observed in four (22.2 %). Maternal HCMV genotypes determined the genotypes identified in their fetuses and newborns (p ≤ 0.050). Half of them were infected with single HCMV gB1, gB2, or gB3 genotypes and the other half with multiple gB1–gB2 or gB2–gB3 genotypes. Single and multiple genotypes were observed in both asymptomatic and symptomatic congenital cytomegaly, although no gB3 genotype was identified among asymptomatic cases. In Central Poland, infections with single and multiple HCMV strains occur in pregnant women, as well as in their fetuses and neonates, with both asymptomatic and symptomatic infections. HCMV infections identified in mothers seem to be associated with the viral genotypes in their children.

Alterations in TLRs as new molecular markers of congenital infections with Human cytomegalovirus

Toll-like receptors (TLRs) play a crucial role in non-specific immunity against various infections. The most common intrauterine infection, caused by Human cytomegalovirus (HCMV), results in perinatal morbidity and mortality of primary infected fetuses. The induction of immune response by TLRs was observed in HCMV infections in murine models and cell lines cultured in vitro. Studies reported an immunological response in pregnant women with primary HCMV infection and TLR2 activity in collecting of HCMV particles in placental syncytiotrophoblasts (STs) in vivo and cultured ST, and in stimulation of tumor necrosis factor (TNF)-α expression and damage of villous trophoblast. Expression levels of TLRs are associated with cell type, stage of pregnancy and response to microorganisms. We show the effect of HCMV infection on the development of pregnancy as well as the effect of TLR single-nucleotide polymorphisms on the occurrence and course of infectious diseases, immune response and diseases of pregnancy. We report the impact of TLRs on the function of miRNAs and the altered expression levels of these molecules, as observed in HCMV infections. We suggest that the methylation status of TLR gene promoter regions as epigenetic modifications may be significant in the immune response to HCMV infections. We conclude that it is important to study in detail the molecular mechanisms of TLR function in the immune response to HCMV infections in pregnancy.

Toll-like receptors genes polymorphisms and the occurrence of HCMV infection among pregnant women

BackgroundHuman cytomegalovirus (HCMV) is the most common cause of intrauterine infections worldwide. The toll-like receptors (TLRs) have been reported as important factors in immune response against HCMV. Particularly, TLR2, TLR4 and TLR9 have been shown to be involved in antiviral immunity. Evaluation of the role of single nucleotide polymorphisms (SNPs), located within TLR2, TLR4 and TLR9 genes, in the development of human cytomegalovirus (HCMV) infection in pregnant women and their fetuses and neonates, was performed.MethodsThe study was performed for 131 pregnant women, including 66 patients infected with HCMV during pregnancy, and 65 age-matched control pregnant individuals. The patients were selected to the study, based on serological status of anti-HCMV IgG and IgM antibodies and on the presence of viral DNA in their body fluids. Genotypes in TLR2 2258 A > G, TLR4 896 G > A and 1196 C > T and TLR9 2848 G > A SNPs were determined by self-designed nested PCR-RFLP assays. Randomly selected PCR products, representative for distinct genotypes in TLR SNPs, were confirmed by sequencing. A relationship between the genotypes, alleles, haplotypes and multiple variants in the studied polymorphisms, and the occurrence of HCMV infection in pregnant women and their offsprings, was determined, using a logistic regression model.ResultsGenotypes in all the analyzed polymorphisms preserved the Hardy-Weinberg equilibrium in pregnant women, both infected and uninfected with HCMV (P > 0.050). GG homozygotic and GA heterozygotic status in TLR9 2848 G > A SNP decreased significantly the occurrence of HCMV infection (OR 0.44 95% CI 0.21–0.94 in the dominant model, P ≤ 0.050). The G allele in TLR9 SNP was significantly more frequent among the uninfected pregnant women than among the infected ones (χ2 = 4.14, P ≤ 0.050). Considering other polymorphisms, similar frequencies of distinct genotypes, haplotypes and multiple-SNP variants were observed between the studied groups of patients.ConclusionsTLR9 2848 G > A SNP may be associated with HCMV infection in pregnant women.

TLR2 2258 G>A single nucleotide polymorphism and the risk of congenital infection with human cytomegalovirus

BackgroundHuman cytomegalovirus (HCMV) is responsible for the most common intrauterine infections, which may be acquired congenitally from infected pregnant woman to fetus. The research was aimed to estimate the role of three single nucleotide polymorphisms (SNPs) located in TLR2 gene, and the common contribution of TLR2, and previously studied TLR4 and TLR9 SNPs, to the occurrence of congenital HCMV infection in fetuses and newborns.MethodsThe study was performed in 20 Polish fetuses and newborns, congenitally infected with HCMV, and in 31 uninfected controls, as well as with participation of pregnant women, the mothers of 16 infected and 14 uninfected offsprings. Genotypes in TLR2 SNPs were determined, using self-designed nested PCR-RFLP assays, and confirmed by sequencing. The genotypes were tested for Hardy-Weinberg (H-W) equilibrium, and for their relationship with the development of congenital cytomegaly, using a logistic regression model. The common influence of TLR2, TLR4 and TLR9 SNPs on the occurrence of congenital disease was estimated by multiple-SNP analysis.ResultsDistribution of the genotypes and alleles in TLR2 1350 T>C and 2029 C>T SNPs was similar between the studied groups of fetuses and neonates. In case of 2258 G>A polymorphism, the GA heterozygotic status was significantly more frequent in the infected cases than among the uninfected individuals (25.0% vs. 3.2%, respectively), and increased the risk of HCMV infection (OR 10.00, 95% CI 1.07–93.44; P ≤ 0.050). Similarly, the A allele within 2258 G>A polymorphism was significantly more frequent among the infected offsprings than in the uninfected ones (12.5% vs. 1.6%; P ≤ 0.050). Complex AA variants for both TLR2 2258 and TLR9 2848 G>A polymorphisms, were estimated to be at increased risk of congenital HCMV infection (OR 11.58, 95% CI 1.19–112.59; P ≤ 0.050). Additionally, significant relationships were observed between the occurrence of complex AA or GA variants for both TLR2 and TLR9 SNPs and the increased viral loads, determined in fetal amniotic fluids and in maternal blood or urine specimens (P ≤ 0.050).ConclusionsAmong various TLR2, TLR4 and TLR9 polymorphisms, TLR2 2258 G>A SNP seems to be an important factor associated with increased risk of congenital HCMV infection in Polish fetuses and neonates.

Genetic modifications of cytokine genes and Toxoplasma gondii infections in pregnant women

PURPOSE Toxoplasma gondii causes one of the most common intrauterine infections worldwide, thus being a severe threat during pregnancy. IL1, IL6, IL10, IL12, and TNF-α cytokines were reported to be involved in immune responses to infections with T. gondii. The research was aimed to reveal relationships between genetic changes within the polymorphisms of these cytokine genes and the incidence of T. gondii infection among pregnant women, as well as congenital transmission of the parasite to the foetuses of their infected mothers. METHODS The primary study was performed in 148 Polish pregnant women, including 74 T. gondii-infected patients and 74 age-matched uninfected individuals; and further analysis - among the additional 142 pregnant women. Genotypes within IL1A -889 C>T, IL1B +3954 C>T, IL6 -174 G>C, IL10 -1082 G>A, IL12B -1188 A>C and TNFA -308 G>A single nucleotide polymorphisms (SNPs) were determined, using self-designed nested PCR-RFLP assays. Randomly selected PCR products, representing distinct genotypes in the analyzed polymorphisms, were confirmed by sequencing, using the Sanger method. A statistical analysis was carried out of relationships between genetic alterations within studied SNPs and the occurrence of T. gondii infection, using the following tools: cross-tabulation, Pearsons Chi-square test and the logistic regression model to estimate genetic models of inheritance. A power analysis of statistically significant outcomes was performed by Cramérs V test. RESULTS A multiple-SNP analysis showed TC haplotype for IL1A and IL1B SNPs to be significantly associated with a decreased risk of the parasitic infection (OR 0.41, P≤0.050). The association remained important after power analysis (Cramérs V = 0.39, χ2 = 7.73, P≤0.050), and the additional analysis with larger groups of patients (OR 0.47, P≤0.050). Moreover, the CCCAGA complex variants were for all the studied polymorphisms at an increased risk of T. gondii infection (OR 8.14, P≤0.050), although this strong relationship was not significant in the further analysis (Cramérs V = 0.76, χ2 = 26.81, P = 0.310). Regarding the susceptibility to congenital transmission of T. gondii from mothers to their foetuses among the infected pregnant women, the presence of GA heterozygotic status within IL10 polymorphism significantly increased the risk of parasitic transmission (OR 5.73 in the codominant model and OR 5.18 in the overdominant model; P≤0.050). The correlation stayed important in the power analysis (Cramérs V = 0.29, χ2 = 6.03, P≤0.050), although it was non-significant in larger groups of patients. Important relationships specific for the first study cohort remained non-significant in the second group of studied pregnant women. CONCLUSIONS Within the analyzed cohort of Polish pregnant women, the genetic modifications from SNPs of genes, encoding both the proinflammatory IL1α, IL1β, IL6, IL12 and TNF-α, and anti-inflammatory IL10 cytokines, may have been associated with susceptibility to T. gondii infection. It is the first study on the contribution of cytokine genes polymorphisms to the occurrence of T. gondii infection during pregnancy. Further studies for other populations of pregnant women would be justified to reveal a detailed role of the analyzed polymorphisms for the occurrence of T. gondii infections during pregnancy.

Genetic alterations within TLR genes in development of Toxoplasma gondii infection among Polish pregnant women

PURPOSE The research was conducted to evaluate the role of genotypes, haplotypes and multiple-SNP variants in the range of TLR2, TLR4 and TLR9 single nucleotide polymorphisms (SNPs) in the development of Toxoplasma gondii infection among Polish pregnant women. MATERIAL AND METHODS The study was performed for 116 Polish pregnant women, including 51 patients infected with T. gondii, and 65 age-matched control pregnant individuals. Genotypes in TLR2 2258 G>A, TLR4 896 A>G, TLR4 1196 C>T and TLR9 2848 G>A SNPs were estimated by self-designed, nested PCR-RFLP assays. Randomly selected PCR products, representative for distinct genotypes in the studied polymorphisms, were confirmed by sequencing. All the genotypes were calculated for Hardy-Weinberg (H-W) equilibrium and TLR4 variants were tested for linkage disequilibrium. Relationships were assessed between alleles, genotypes, haplotypes or multiple-SNP variants in TLR polymorphisms and the occurrence of T. gondii infection in pregnant women, using a logistic regression model. RESULTS All the analyzed genotypes preserved the H-W equilibrium among the studied groups of patients (P>0.050). Similar distribution of distinct alleles and individual genotypes in TLR SNPs, as well as of haplotypes in TLR4 polymorphisms, were observed in T. gondii infected and control uninfected pregnant women. However, the GACG multiple-SNP variant, within the range of all the four studied polymorphisms, was correlated with a decreased risk of the parasitic infection (OR 0.52, 95% CI 0.28-0.97; P≤0.050). CONCLUSIONS The polymorphisms, located within TLR2, TLR4 and TLR9 genes, may be involved together in occurrence of T. gondii infection among Polish pregnant women.