Diagnosis of congenital toxoplasmosis: prenatal and neonatal evaluation of methods used in Toulouse University Hospital and incidence of congenital toxoplasmosis

The aim of this study was to determine the incidence of congenital toxoplasmosis (CT) and to assess the performances of prenatal and neonatal diagnoses. From 1994-2005, in Toulouse University Hospital, France, amniocentesis was performed on 352 pregnant women who were infected during pregnancy. All women were treated with spiramycin and pyrimethamine-sulfadoxine when prenatal diagnosis was positive. Among the 275 foetuses with follow-up, 66 (24%) were infected. The transmission rates of Toxoplasma gondii were 7%, 24% and 59% in the first, second and third trimesters, respectively. The sensitivity and specificity of PCR on amniotic fluid (AF) were 91% and 99.5%, respectively. One case was diagnosed by mouse inoculation with AF and six cases were diagnosed by neonatal or postnatal screening. The sensitivity and specificity of PCR on placentas were 52% and 99%, respectively. The sensitivity of tests for the detection of specific IgA and IgM in cord blood was 53% and 64%, respectively, and specificity values were 91% and 92%. In conclusion, PCR performed on AF had the highest levels of sensitivity and specificity for the diagnosis of CT. This permits an early diagnosis of most cases and should be recommended.

Congenital toxoplasmosis: evaluation of serological methods for the detection of anti-Toxoplasma gondii IgM and IgA antibodies

A study was carried out to evaluate the presence of serological markers for the immunodiagnosis of the vertical transmission of toxoplasmosis. We tested the sensitivity, specificity and predictive values (positive and negative) of different serological methods for the early diagnosis of congenital toxoplasmosis. In a prospective longitudinal study, 50 infants with suspected congenital toxoplasmosis were followed up in the ambulatory care centre of Congenital Infections at University Hospital in Goiânia, Goiás, Brazil, from 1 January 2004-30 September 2005. Microparticle Enzyme Immunoassay (MEIA), Enzyme-Linked Fluorescent Assay (ELFA) and Immune-Fluorescent Antibody Technique (IFAT) were used to detect specific IgM anti-Toxoplasma gondii antibodies and a capture ELISA was used to detect specific IgA antibodies. The results showed that 28/50 infants were infected. During the neonatal period, IgM was detected in 39.3% (11/28) of those infected infants and IgA was detected in 21.4% (6/28). The sensitivity, specificity and predictive values (positive and negative) of each assay were, respectively: MEIA and ELFA: 60.9%, 100%, 100%, 55.0%; IFAT: 59.6%, 91.7%, 93.3%, 53.7%; IgA capture ELISA: 57.1%, 100%, 100%, 51.2%. The presence of specific IgM and IgA antibodies during the neonatal period was not frequent, although it was correlated with the most severe cases of congenital transmission. The results indicate that the absence of congenital disease markers (IgM and IgA) in newborns, even after confirming the absence with several techniques, does not constitute an exclusion criterion for toxoplasmosis.

The IFN-³+874T/A gene polymorphism is associated with retinochoroiditis toxoplasmosis susceptibility

Toxoplasmosis is a worldwide zoonosis that generally produces an asymptomatic infection. In some cases, however, toxoplasmosis infection can lead to ocular damage. The immune system has a crucial role in both the course of the infection and in the evolution of toxoplasmosis disease. In particular, IFN-³ plays an important role in resistance to toxoplasmosis. Polymorphisms in genes encoding cytokines have been shown to have an association with susceptibility to parasitic diseases. The aim of this work was to analyse the occurrence of polymorphisms in the gene encoding IFN-³ (+874T/A) among Toxoplasma gondii seropositive individuals, including those with ocular lesions caused by the parasite, from a rural population of Santa Rita de Cássia, Barra Mansa, state of Rio de Janeiro, Brazil. Further, we verified which of these polymorphisms could be related to susceptibility to the development of ocular toxoplasmosis. This study included 34 individuals with ocular toxoplasmosis (ocular group) and 134 without ocular lesions (control group). The differences between A and T allele distributions were not statistically significant between the two groups. However, we observed that a higher frequency of individuals from the ocular group possessed the A/A genotype, when compared with the control group, suggesting that homozygocity for the A allele could enhance susceptibility to ocular toxoplasmosis in T. gondii infection.

Anti-Toxoplasma gondii secretory IgA in tears of patients with ocular toxoplasmosis: immunodiagnostic validation by ELISA

Toxoplasma gondii causes posterior uveitis and the specific diagnosis is based on clinical criteria. The presence of anti-T. gondii secretory IgA (sIgA) antibodies in patients' tears has been reported and an association was found between ocular toxoplasmosis and the anti-T. gondii sIgA isotype in Brazilian patients. The purpose of this study was to provide an objective validation of the published ELISA test for determining the presence of anti-T. gondii sIgA in the tears of individuals with ocular toxoplasmosis. Tears from 156 patients with active posterior uveitis were analysed; 82 of them presented characteristics of ocular toxoplasmosis (standard lesion) and 74 patients presented uveitis due to other aetiologies. Cases of active posterior uveitis were considered standard when a new inflammatory focus satellite to old retinochoroidal scars was observed. The determination of anti-T. gondii sIgA was made using an ELISA test with crude tachyzoite antigenic extracts. Tears were collected without previous stimulation. Detection of sIgA showed 65.9% sensitivity (95% CI = 54.5-74.4), 71.6% specificity (95% CI = 59.8-81.2), a positive predictive value of 72% (95% CI = 60.3-81.5) and a negative predictive value of 65.4% (95% CI = 54.0-75.4). sIgA reactivity was higher in the tears of patients with active posterior uveitis due to T. gondii (p < 0.05). The test is useful for differentiating active posterior uveitis due to toxoplasmosis from uveitis caused by other diseases.

Candidate gene analysis of ocular toxoplasmosis in Brazil: evidence for a role for toll-like receptor 9 (TLR9)

Toxoplasma gondii infection is an important mediator of ocular disease in Brazil more frequently than reported from elsewhere. Infection and pathology are characterized by a strong proinflammatory response which in mice is triggered by interaction of the parasite with the toll-like receptor (TLR)/MyD88 pathway. A powerful way to identify the role of TLRs in humans is to determine whether polymorphisms at these loci influence susceptibility to T. gondii-mediated pathologies. Here we report on a small family-based study (60 families; 68 affected offspring) undertaken in Brazil which was powered for large effect sizes using single nucleotide polymorphisms with minor alleles frequencies > 0.3. Of markers in TLR2, TLR5 and TLR9 that met these criteria, we found an association Family Based Association Tests [(FBAT) Z score = 4.232; p = 1.5 x 10-5; p corrected = 1.2 x 10-4] between the C allele (frequency = 0.424; odds ratio = 7; 95% confidence interval 1.6-30.8) of rs352140 at TLR9 and toxoplasmic retinochoroiditis in Brazil. This supports the hypothesis that direct interaction between T. gondii and TLR9 may trigger proinflammatory responses that lead to severe pathologies such as the ocular disease that is associated with this infection in Brazil.

IgG and IgM western blot assay for diagnosis of congenital toxoplasmosis

The aim of this study was to evaluate the utility of western blot (WB) analysis as a diagnostic tool for congenital toxoplasmosis in 215 newborn infants. The children were submitted to clinical examinations to assess macular, neurological and hearing signals. The WB results obtained were compared to the persistence of IgG antibodies at the end of 12 months, which is regarded as the "gold standard" diagnosis of congenital toxoplasmosis. Association between the WB results and the clinical signs presented by the infants was also assessed. Of the 215 children, 177 had a confirmed congenital toxoplasmosis diagnosis and 38 were uninfected. IgG-WB showed a sensitivity of 73.5% and a specificity of 97.4%. IgM-WB showed a sensitivity of 54.8% and a specificity of 94.7%. The IgG-WB and IgM-WB combination increased the sensitivity to 86.5%. The IgM-WB-positive children had a 1.4-fold greater risk of presenting active macular lesions than did those that were IgM-WB-negative. This study showed that the WB assay is a useful tool to confirm a diagnosis of congenital toxoplasmosis and that the IgM-WB-positive results can indicate active macular lesions in newborn infants.

Ocular toxoplasmosis: evaluation of lacrimal - specific secretory IgA levels in both patients with active and inactive phases of the disease

Ocular toxoplasmosis can result in recurrent uveitis. Studies have shown that a correlation between active ocular toxoplasmosis and the presence of anti-Toxoplasma gondii secretory IgA (SIgA) in tears. This study compares anti-T. gondii SIgA levels in patients' tears during the acute and inactive phases of toxoplasmic uveitis. Twenty-nine positive tear specific SIgA for T. gondii patients with acute toxoplasmic uveitis were selected and were followed-up for at least two years, when the anti-T. gondii SIgA tears levels were determined. Specific SIgA for T. gondii was negative in 22 patients (75.86%) and positive in seven patients (24.13%) of whom six (85.7%) were followed over three years. Average SIgA levels during the acute phase are 1.54 and decrease significantly to 0.72 (p = 0.0001) during the inactive phase of disease. Because anti-T. gondii SIgA in the tear is negative in 75.86% of patients after the acute phase of infection, T. gondii SIgA levels may be used as a complementary diagnostic marker for active ocular toxoplasmosis.

Depletion of CD25+ cells during acute toxoplasmosis does not significantly increase mortality in Swiss OF1 mice

The interleukin (IL)-2R alpha chain (CD25) is expressed on regulatory T cells (Treg), which constitute more than 85% of the CD25+ T cell population in a naïve mouse. CD25 is also expressed on effector T cells in mice suffering from an acute infection by the obligate intracellular protozoan parasite, Toxoplasma gondii. Lethal toxoplasmosis is accompanied by a significant loss of Treg in mice naturally susceptible to toxoplasmosis. The present study was done to explore the role of Treg cells using an anti-CD25 antibody-mediated depletion in mice naturally resistant to toxoplasmosis. Although a significant decrease in the percentage of Treg cells was observed following anti-CD25 monoclonal antibody injections, the depletion of CD25+ cells during acute toxoplasmosis did not significantly increase the mortality of Swiss OF1 mice and no significant difference was observed in the brain parasitic load between the mice in the depleted-infected and isotype-infected groups. We found no significant difference between the titres of total IgG in the sera of the mice from the two groups in the chronic phase. However, CD25+ cells depletion was followed by significantly higher levels of IL-12 in the serum of depleted mice than in that of mice injected with the isotype control antibody.

Early diagnosis of congenital toxoplasmosis in newborn infants using IgG subclasses against two Toxoplasma gondii recombinant proteins

The aim of this work was to evaluate the utility of ELISA-based testing of total IgG (IgGt) antibodies and its subclasses (IgG1, IgG2, IgG3 and IgG4) against soluble (STAg) and recombinant (rSAG1 and rMIC3) antigens of Toxoplasma gondii for diagnosing congenital toxoplasmosis. Sera from 217 newborns initially testing positive for specific IgM in filter paper dried blood spots were tested for specific IgM and IgG by ELFA-VIDAS®. Congenital toxoplasmosis was confirmed in 175 and ruled out in 42 infants. The validity of the ELISA tests was determined using the persistence of IgG antibodies (ELFA-VIDAS® kit) at the end of 12 months, which is considered the reference test for the diagnosis of congenital toxoplasmosis. The frequency of positivity with IgGt against STAg, rSAG1 and rMIC3 was found in 97.2%, 96.3% and 80.2%, respectively, of the newborns with confirmed congenital toxoplasmosis. IgG1 reacted with all three antigens, while IgG3 and IgG4 reacted preferentially with rMIC3. Higher mean values of reactivity (sample optical density/cut-off) were found for all subclasses when using rMIC3. All of the antigens showed high sensitivity and low specificity in detecting anti-T. gondii IgGt and IgG1 and low sensitivity and high specificity in detecting IgG3 and IgG4. In conclusion, the combined detection of IgG antibody subclasses against recombinant toxoplasmic antigens may be useful for the early diagnosis of congenital toxoplasmosis.

Time-based distribution of Staphylococcus saprophyticus pulsed field gel-electrophoresis clusters in community-acquired urinary tract infections

The epidemiology of urinary tract infections (UTI) by Staphylococcus saprophyticus has not been fully characterised and strain typing methods have not been validated for this agent. To evaluate whether epidemiological relationships exist between clusters of pulsed field gel-electrophoresis (PFGE) genotypes of S. saprophyticus from community-acquired UTI, a cross-sectional surveillance study was conducted in the city of Rio de Janeiro, Brazil. In total, 32 (16%) female patients attending two walk-in clinics were culture-positive for S. saprophyticus. Five PFGE clusters were defined and evaluated against epidemiological data. The PFGE clusters were grouped in time, suggesting the existence of community point sources of S. saprophyticus. From these point sources, S. saprophyticus strains may spread among individuals.

Toxoplasmosis seroprevalence in urban rodents: a survey in Niamey, Niger

A serological survey of Toxoplasma gondii was conducted on 766 domestic and peridomestic rodents from 46 trapping sites throughout the city of Niamey, Niger. A low seroprevalence was found over the whole town with only 1.96% of the rodents found seropositive. However, differences between species were important, ranging from less than 2% in truly commensal Mastomys natalensis, Rattus rattus and Mus musculus, while garden-associated Arvicanthis niloticus displayed 9.1% of seropositive individuals. This is in line with previous studies on tropical rodents - that we reviewed here - which altogether show that Toxoplasma seroprevalence in rodent is highly variable, depending on many factors such as locality and/or species. Moreover, although we were not able to decipher statistically between habitat or species effect, such a contrast between Nile grass rats and the other rodent species points towards a potentially important role of environmental toxoplasmic infection. This would deserve to be further scrutinised since intra-city irrigated cultures are extending in Niamey, thus potentially increasing Toxoplasma circulation in this yet semi-arid region. As far as we are aware of, our study is one of the rare surveys of its kind performed in Sub-Saharan Africa and the first one ever conducted in the Sahel.

Single nucleotide polymorphisms in the interferon gamma gene are associated with distinct types of retinochoroidal scar lesions presumably caused by Toxoplasma gondii infection

The association of single nucleotide polymorphisms (SNPs) in the interferon (IFN)-γ gene ( IFNG ) with different types of retinal scar lesions presumably caused by toxoplasmosis were investigated in a cross-sectional population-based genetic study. Ten SNPs were investigated and after Bonferroni correction, only the associations between SNPs rs2069718 and rs3181035 with retinal/retinochoroidal scar lesions type A (most severe scar lesions) and C (least severe scar lesions), respectively, remained significant. The associations of two different IFNG SNPs with two different types of retinal lesions attributable to toxoplasmosis support the hypothesis that different inflammatory mechanisms underlie the development of these lesions. The in vitro analysis of IFN-γ secretion by peripheral blood mononuclear cells stimulated with Toxoplasma gondii antigens was also investigated. The association between SNP rs2069718 and type A scar lesions revealed that differential IFN-γ levels are correlated with distinct genotypes. However, no correlation was observed with IFN-γ secretion levels and the SNP rs3181035 , which was significantly associated with type C scar lesions. Our findings strongly suggest that immunogenetic studies of individuals with congenital or postnatally acquired infection are needed to better understand the role of IFN-γ and its polymorphisms in the pathogenesis of ocular toxoplasmosis.

Prevalence of human papillomavirus infection, distribution of viral types and risk factors in cervical samples from human immunodeficiency virus-positive women attending three human immunodeficiency virus-acquired immune deficiency syndrome reference centres in northeastern Brazil

Human immunodeficiency virus (HIV)-positive patients have a greater prevalence of coinfection with human papillomavirus (HPV) is of high oncogenic risk. Indeed, the presence of the virus favours intraepithelial squamous cell lesion progression and may induce cancer. The aim of this study was to evaluate the prevalence of HPV infection, distribution of HPV types and risk factors among HIV-positive patients. Cervical samples from 450 HIV-positive patients were analysed with regard to oncotic cytology, colposcopy and HPV presence and type by means of polymerase chain reaction and sequencing. The results were analysed by comparing demographic data and data relating to HPV and HIV infection. The prevalence of HPV was 47.5%. Among the HPV-positive samples, 59% included viral types of high oncogenic risk. Multivariate analysis showed an association between HPV infection and the presence of cytological alterations (p = 0.003), age greater than or equal to 35 years (p = 0.002), number of partners greater than three (p = 0.002), CD4+ lymphocyte count < 200/mm3 (p = 0.041) and alcohol abuse (p = 0.004). Although high-risk HPV was present in the majority of the lesions studied, the low frequency of HPV 16 (3.3%), low occurrence of cervical lesions and preserved immunological state in most of the HIV-positive patients were factors that may explain the low occurrence of precancerous cervical lesions in this population.