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.     

Performance of Polymerase Chain Reaction Analysis of the Amniotic Fluid of Pregnant Women for Diagnosis of Congenital Toxoplasmosis: A Systematic Review and Meta-Analysis

IntroductionCongenital infection caused by Toxoplasma gondii can cause serious damage that can be diagnosed in utero or at birth, although most infants are asymptomatic at birth. Prenatal diagnosis of congenital toxoplasmosis considerably improves the prognosis and outcome for infected infants. For this reason, an assay for the quick, sensitive, and safe diagnosis of fetal toxoplasmosis is desirable.GoalTo systematically review the performance of polymerase chain reaction (PCR) analysis of the amniotic fluid of pregnant women with recent serological toxoplasmosis diagnoses for the diagnosis of fetal toxoplasmosis.MethodA systematic literature review was conducted via a search of electronic databases; the literature included primary studies of the diagnostic accuracy of PCR analysis of amniotic fluid from pregnant women who seroconverted during pregnancy. The PCR test was compared to a gold standard for diagnosis.ResultsA total of 1.269 summaries were obtained from the electronic database and reviewed, and 20 studies, comprising 4.171 samples, met the established inclusion criteria and were included in the review. The following results were obtained: studies about PCR assays for fetal toxoplasmosis are generally susceptible to bias; reports of the tests’ use lack critical information; the protocols varied among studies; the heterogeneity among studies was concentrated in the tests’ sensitivity; there was evidence that the sensitivity of the tests increases with time, as represented by the trimester; and there was more heterogeneity among studies in which there was more time between maternal diagnosis and fetal testing. The sensitivity of the method, if performed up to five weeks after maternal diagnosis, was 87% and specificity was 99%.ConclusionThe global sensitivity heterogeneity of the PCR test in this review was 66.5% (I²). The tests show low evidence of heterogeneity with a sensitivity of 87% and specificity of 99% when performed up to five weeks after maternal diagnosis. The test has a known performance and could be recommended for use up to five weeks after maternal diagnosis, when there is suspicion of fetal toxoplasmosis.     

Diagnosis of congenital toxoplasmosis: pre- and post-natal evaluation in Sicilian (Italy) epidemiological area. Preliminary data

To evaluate the usefulness of conventional serological methods with western blot assay (WB) in congenital toxoplasmosis diagnosis, we prospectively enrolled in a clinical and serological follow-up all pregnant women with Toxoplasma gondii infection and their offspring, referred to us from October 2004. Western blot and standard serological test were performed on sera collected from mother during pregnancy and from mother and child at birth, at postpartum month 1-3-6-9 and 12. At this point in time, 22 pregnant women and 14 infants have completed the follow-up. 4 newborns were infected and 2 had specific toxoplasmosis anomalies at the birth. In mothers without seroconversion, the WB performed during pregnancy demonstrates the highest accordance with postnatal follow-up whereas in 1 case the negative result of PCR analysis was not confirmed by postnatal observation. The detection of anti-T gondii IgG against 8 kDa accessory antigenic band and against the accessory band included between 35 and 40 kDa band in immunoblot assay was useful for diagnosis of acute phase but did not improve the evaluation of comparative postnatal profile. Althougth few infants have concluded the postnatal follow-up, the preliminary results showed a greater value of using a IgM and IgA WB test than other standard method for the early diagnosis of toxoplasmosis at birth also in child born to treated mothers. The comparative anti-T gondii IgG immunoblot profile of mother and child permitted us to reduce the time of ruling out infection in newborns born to mothers with probable or possible infection and/or when prenatal diagnosis is negative or not performed.     

Western blotting for the diagnosis of congenital toxoplasmosis

Toxoplasmosis is a common congenital infection. It does not usually produce recognizable signs of infection at birth so most infected newborns are not detected by routine clinical examination and remain untreated. Infected children without clinical symptoms should nonetheless be identified and treated as early as possible. Serological diagnosis of congenital toxoplasmosis is quite difficult. The aim of this study was to evaluate the utility of Western blot for the diagnosis of congenital toxoplasmosis. We compared the immunological profiles of mothers and children to differentiate between passively transmitted maternal antibodies and antibodies synthesized by the infants in the first three months of life. The method enabled us to diagnose congenital toxoplasmosis in cases in which the infection had not been detected by classical serology techniques.     

Serological studies in Nectomys squamipes demonstrate the low sensitivity of coprological exams for the diagnosis of schistosomiasis

The existence of wild rodents naturally infected by Schistosoma mansoni is a drawback for schistosomiasis control programs. As a consequence, it is necessary to have a precise diagnosis of S. mansoni infection in wild rodents (water rats; Nectomys squamipes), the species seemingly involved in the transmission of schistosomiasis at Sumindouro, Rio de Janeiro, Brazil. A total of 78 specimens of N. squamipes was captured in an endemic area at Vale do Pamparr?o and Porteira Verde, Sumidouro, Brazil; 5 more were born in captivity and experimentally infected. The sensitivity and specificity of the coprological method of Kato-Katz and serological methods, i.e., enzyme-linked immunosorbent assay (ELISA) and western blot (WB), were compared. The rodents were subsequently killed and necropsied to confirm infection. The prevalences observed using ELISA (48%) and WB (41%) were equivalent to those found at necropsy (41%). The ELISA showed a sensitivity of 97% and a specificity of 87%, whereas the WB showed a sensitivity of 87% and a specificity of 89%. The Kato-Katz method exhibited 50% sensitivity and 100% specificity. The differences found among the ELISA, WB, and necropsy, when compared with Kato-Katz, may be related to the low sensitivity of the coprological method. Serological methods should be used for more reliable epidemiological information.     

Western blotting using Strongyloides ratti antigen for the detection of IgG antibodies as confirmatory test in human strongyloidiasis

The present study was conducted to evaluate the frequency of antigenic components recognized by serum IgG antibodies in Western blotting (WB) using a Strongyloides ratti larval extract for the diagnosis of human strongyloidiasis. In addition, the WB results were compared to the enzyme-linked immunosorbent assay (ELISA) and the indirect immunofluorescence antibody test (IFAT) results. Serum samples of 180 individuals were analyzed (80 with strongyloidiasis, 60 with other intestinal parasitoses, and 40 healthy individuals). S. ratti was obtained from fecal culture of experimentally infected Rattus rattus. For IFAT, S. ratti larvae were used as antigen and S. ratti larval antigenic extracts were employed in WB and ELISA. Eleven S. ratti antigenic components were predominantly recognized by IgG antibodies in sera of patients with strongyloidiasis. There was a positive concordance for the three tests in 87.5% of the cases of strongyloidiasis. The negative concordance in the three tests was 94% and 97.5%, in patients with other intestinal parasitoses and healthy individuals, respectively. In cases of positive ELISA and negative IFAT results, diagnosis could be confirmed by WB. ELISA, IFAT, and WB using S. ratti antigens showed a high rate of sensitivity and specificity. In conclusion, WB using S. ratti larval extract was able to recognize 11 immunodominant antigenic components, showing to be a useful tool to define the diagnosis in cases of equivocal serology.     

Toxoplasma gondii: Congenital transmission in a hamster model

The objective of the research was to test the hamster for a model of transmission of congenital toxoplasmosis. A non-invasive method for the diagnosis of pregnancy in hamsters was designed, with a specificity and a sensitivity of 70.2 and 94.7%, respectively (n = 168). Of 32 females with a chronic toxoplasma infection, 3 transmitted Toxoplasma congenitally during their first pregnancy, but not during the subsequent pregnancy. Congenital transmission rates of infections initiated during pregnancy with 2 stages of 2 strains of Toxoplasma were in the range of 33 to 100% of the 76 females inoculated. Only 1 of 17 females transmitted the parasite exclusively via milk. It was concluded that the hamster is a promising species for a model of transmission of congenital toxoplasmosis.     

New prospects in immunology of toxoplasmosis: skin-tests for control of immunity and immuno-assays and agglutination tests for the detection of specific IgM antibodies

An excretory-secretory (ES) antigen was extracted from supernatants of cell cultures infected with Toxoplasma gondii, purified and controlled according to current standards. In 638 volunteers, the correlation with fluorescent antibody was 94.2% and no false positive skin tests were noted. The skin test did not transform an originally negative serological test into a positive one. For the prevention of congenital toxoplasmosis, this sensitive, specific and inexpensive skin test can be widely used for the detection of immunity to Toxoplasma in women before their first pregnancy. During pregnancy, the detection of specific IgM is very important for the diagnosis of a recently acquired toxoplasmosis and allows for an immediate treatment. For this detection and for the diagnosis of congenital toxoplasmosis, five different serological tests were compared: Indirect Fluorescent Antibody-test (IFA), ELISA test, ELISA test After Capture of IgM (ACCAs), Reverse Enzyme Immuno Assay R-EIA), Double-Sandwich Enzyme Linked ImmunoSorbent Assay (DS-ELISA) and ImmunoSorbent AGglutination Assay (ISAGA). For 37 sera of recently acquired toxoplasmosis, IgM were detected in 98.7% with ISAGA, in 89.5% with DS-ELISA and ELISA in 83% with R-EIA and in 59% with IFA test. The best specificity is obtained with ISAGA, DS-ELISA and R-EIA, from controls with non immune patients (99 cases), patients with chronic toxoplasmosis (77 sera), rheumatoid factors (35 sera) or anti-nuclear antibodies (7 sera). In 21 sera from infants with congenital toxoplasmosis, ISAGA was positive in 13 cases (62%), IFA in 5 cases (24%), ELISA and R-EIA in 2 cases (9.5%) and DS-ELISA in 9 cases (43%).(ABSTRACT TRUNCATED AT 250 WORDS)     

Wilm's tumor. Diagnostic capacities of magnetic resonance imaging. MRI-pathomorphological comparison

The accuracy of magnetic resonance imaging (MRI) in the diagnosis of Wilms' tumor (WT) and in the evaluation of preoperative chemotherapy (PCH) efficiency was investigated and compared with histopathological data of 56 children and infants with proven retroperitoneum neoplasma (WT--49, neuroblastoma--6, congenital mesoblastic nephroma--1). The author described the WT MRI-semiotics in general and in particular for its changes during the preoperative chemotherapy. The formula for calculation of tumor reduction index is suggested. The MRI sensitivity (100%), specificity (77.8%) and accuracy (91.1%) are detected. The high positive correlation level between the MRI and pathologic findings, concerning WT dimensions, pseudocapsule presence and safety, tumor structure secondary alterations and tumor spreading was found. At the same time, the specific MRI criteria for the different histological types of WT were not found. MRI is confirmed to be an accurate tool for diagnostic monitoring of patients with WT and other retroperitoneum neoplasms.     

Refinement of the mouse model of congenital toxoplasmosis

The goals of the present investigation, focusing on the BALB/c mouse model of congenital toxoplasmosis, were: (1) to find a method to determine pregnancy in the mouse. The method has 100% sensitivity and 72% specificity; (2) to test congenital transmission during the chronic stage of toxoplasmosis. This occurred in 2 of 10 mice tested; (3) to investigate the relationship between the infective dose and the rate of congenital transmission. This was not demonstrated for doses of 10(2) to 10(3) bradyzoites and oocysts of Prugniaud, M3 and M7741 strains, with transmission rates of 3 of 8 to 6 of 10 mice inoculated; (4) to determine homologous and heterologous protection. Homologous protection was demonstrated with Prugniaud cysts, and heterologous protection was found between ME-49 and M3 cysts. This finding is consistent with the uniform natural protection against congenital toxoplasmosis seen in immune women and ewes.     

Severe obstetric brachial plexus palsies can be identified at one month of age

Objective

To establish whether severe obstetric brachial plexus palsy (OBPP) can be identified reliably at or before three months of age.

Methods

Severe OBPP was defined as neurotmesis or avulsion of spinal nerves C5 and C6 irrespective of additional C7-T1 lesions, assessed during surgery and confirmed by histopathological examination. We first prospectively studied a derivation group of 48 infants with OBPP with a minimal follow-up of two years. Ten dichotomous items concerning active clinical joint movement and needle electromyography of the deltoid, biceps and triceps muscles were gathered at one week, one month and three months of age. Predictors for a severe lesion were identified using a two-step forward logistic regression analysis. The results were validated in two independent cohorts of OBPP infants of 60 and 13 infants.

Results

Prediction of severe OBPP at one month of age was better than at one week and at three months. The presence of elbow extension, elbow flexion and of motor unit potentials in the biceps muscle correctly predicted whether lesions were mild or severe in 93.6% of infants in the derivation group (sensitivity 1.0, specificity 0.88), in 88.3% in the first validation group (sensitivity 0.97, specificity 0.76) and in 84.6% in the second group (sensitivity of 1.0, specificity 0.66).

Interpretation

Infants with OBPP with severe lesions can be identified at one month of age by testing elbow extension, elbow flexion and recording motor unit potentials (MUPs) in the biceps muscle. The decision rule implies that children without active elbow extension at one month should be referred to a specialized center, while children with active elbow extension as well as active flexion should not. When there is active elbow extension, but no active elbow flexion an EMG is needed; absence of MUPs in the biceps muscle is an indication for referral.     

Clinical course of congenital toxoplasmosis in children]

Hundred eleven children with the congenital toxoplasmosis were treated at the Department of Infectious and Parasitic Diseases in Childhood in 1979-1988. Multi-symptomatic toxoplasmosis has been diagnosed in 35 cases, ocular form in 65, oligosymptomatic in 6, and asymptomatic in 5 cases. Clinical symptoms suggesting congenital toxoplasmosis was seen in the majority of children (63 cases) in the first year of life and the disease was diagnosed in 50% of cases (33 children) at this age. Congenital toxoplasmosis in the group of 78 children has been diagnosed later. The majority of cases was ocular form. Diagnosis of the oligo- and asymptomatic congenital toxoplasmosis is possible in the first year of life, only. A titre of antibodies is exclusively an indicator of the immunologic response, not a severity of infection and does not contribute to the prognosis. Antitoxoplasma drugs were administered to 102 children including 33 under the first year of life. Pyrimethamine, sulphonamides, and spiramycin were used in the treatment. Dosage, duration of therapy, and way of administration have been established individually in dependence of patients age and clinical form of the congenital toxoplasmosis. Two out of 35 children with multi-symptomatic congenital toxoplasmosis died whereas 13 demonstrate psychomotor retardation of significant degree despite the fact that 11 of them were treated in the first year of life.     

Bilateral sweat tests with two different methods as a part of cystic fibrosis newborn screening (CF NBS) protocol and additional quality control

Infants with positive CF newborn screening (NBS) results are called to a CF Centre for verification. Those, in whom the sweat test is elevated, undergo further medical procedures. The aim of our study was to evaluate the applicability of Nanoduct - a new system measuring sweat conductivity and giving immediate results in a CF NBS protocol. Measurements with Nanoduct were compared with the classic pilocarpine method. During 3 years 487 infants from CF NBS had both sweat tests performed on the same day, at the same CF centre. CF infants had a mean conductivity of 99.8 ± 1 8.8 mmol/L and a mean chloride concentration of 74.0 ± 18.4 mmol/L. Non-CF infants values were 29.8 ± 7.7 mmol/L and 19.2 ± 6.6 mmol/L respectively. A good correlation between both tests was found (95% confidence level (CI); r=0.87). The optimal cut off, based on follow up experience of screened children, for conductivity tests was 50 mmol/L and for chloride concentration was 34 mmol/L (no lost CF, 11 false positive) with 100% sensitivity and 97.5 % specificity. In conclusion Nanoduct is a very useful and reliable tool in CF NBS protocol, allowing more time efficient organization of the diagnostic and training procedures. Simultaneous bilateral sweat testing with two different methods (concentration and conductivity) provides an extra quality control system.     

First Colombian multicentric newborn screening for congenital toxoplasmosis

Aims

To determine the incidence of congenital toxoplasmosis in Colombian newborns from 19 hospital or maternal child health services from seven different cities of five natural geographic regions (Caribbean, Central, Andean, Amazonia and Eastern).

Materials and Methods

We collected 15,333 samples from umbilical cord blood between the period of March 2009 to May 2010 in 19 different hospitals and maternal-child health services from seven different cities. We applied an IgM ELISA assay (Vircell, Spain) to determine the frequency of IgM anti Toxoplasma. The results in blood cord samples were confirmed either by western blot and repeated ELISA IgM assay. In a sub-sample of 1,613 children that were negative by the anti-Toxoplasma IgM assay, the frequency of specific anti-Toxoplasma IgA by the ISAGA assay was determined. All children with positive samples by IgM, IgA, clinical diagnosis or treatment during pregnancy were recalled for confirmatory tests after day 10 of life.

Results

61 positive samples for specific IgM (0.39%) and 9 positives for IgA (0.5%) were found. 143 questionnaires were positive for a clinical diagnosis or treatment for toxoplasmosis during pregnancy. 109 out of the 218 children that had some of the criteria for postnatal confirmatory tests were followed. Congenital toxoplasmosis infection was confirmed in 15 children: 7 were symptomatic, and three of them died before the first month of life (20% of lethality). A significant correlation was found between a high incidence of markers for congenital toxoplasmosis and higher mean annual rainfall for the city.

Conclusions

Incidence for congenital toxoplasmosis is significantly different between hospitals or maternal child health services from different cities in Colombia. Mean annual rainfall was correlated with incidence of congenital toxoplasmosis.     

Toxoplasma gondii antibody titers in sera of children admitted to the Seoul National University Children's Hospital

A total of 542 children under 10 years of age, admitted to the Seoul National University Children's Hospital, was examined for antibody titers of Toxoplasma gondii using indirect latex agglutination (ILA) test. Among them, 7.7% showed positive titers higher than 1:32, without significant difference between males (7.3%) and females (8.5%). The seropositive rate increased with age although the statistical significance was negligible (0.05 < P < 0.1). By residential areas, the prevalence appeared higher among children from southern provinces (Kyongsang-do and Cholla do) than those from other areas, but the statistical significance was also very low (0.05 < P < 0.1). When the seropositive cases were analyzed by coincidental diseases, the prevalence was significantly higher in patients with congenital diseases than in patients with non-congenital diseases (P < 0.05). The results showed that the seropositive rate of toxoplasmosis in children examined was not high compared with other endemic countries. Some correlations are suggested between toxoplasmosis and congenital anomalies in Korea.     

Toxoplasma gondii: an improved rat model of congenital infection

The objective of this study was to refine the rat model of congenital toxoplasmosis. In Fischer rats we found that visualization of spermatozoa in vaginal exudates and the detection of at least 6 g body weight increase between days 9 and 12 of pregnancy, allowed the diagnosis and timing of pregnancy with 60% specificity and 84% sensitivity. A dose of 10⁴Toxoplasma gondii bradyzoites or 10²T. gondii oocysts of the Prugniaud strain resulted in more than 50% of congenital infection of the rat litters. Transmission of T. gondii via lactation was not detected in rats inoculated with either bradyzoites or oocysts. Bioassays of 51 neonates born from mothers inoculated with bradyzoites (in tissue cysts) and 29 neonates from mothers inoculated with oocysts demonstrated that both liver and lungs can be used for the diagnosis of congenital transmission in this model.     

Repeated isolation of toxoplasma from the cerebrospinal fluid and from the blood,and the antibody response in four cases of congenital toxoplasmosis

Summary In 64 sera from persons with a history of possible toxoplasmosis out of approximately 600 sera, a dye test titer ranging from 1/100 to 1/8192, and a complement fixation titer from 1/2 to 1/256 were found. In four infants with congenital toxoplasmosis Toxoplasma was isolated from the ventricle fluid and from the blood on several occasions during the first two months of life. The antibody response in the children and in their mothers was studied. The neutralizing antibody reached its peak in the first month of life, that of the complement fixing antibody was reached in the second month. In spite of the high titer of neutralizing antibody, and treatment with sulphamethylpyrimidine, virulent Toxoplasma were present in the blood and the ventricle fluid. Two of the patients died, and in the brains extensive granulomatous lesions with necrosis and abundant masses of Toxoplasma were found. Two of the mothers had prepared a hare during pregnancy, but there is no proof, that this potential animal reservoir of toxoplasma has been the source of infection.     

Transplacental measles immunity in infants in the 1st year of life]

A total of 187 parturients (66 with a history of measles and 121 immunized with live measles vaccine, or LMV, in childhood) and their 187 newborn infants, as well as 195 children aged up to 1 year, were examined. Antimeasles antibodies in blood sera were detected in the hemagglutination inhibition test. In all mothers with a history of measles and in their newborn infants antimeasles antibodies in different titers were detected. In mothers, formerly immunized with LMV, antimeasles antibodies were absent in 5.8% and in their newborn infants, in 6.6% of the examinees. Among children aged up to 1 year, born of formerly immunized mothers, more rapid disappearance of passive antimeasles immunity was observed. In cases of contact with measles, the serological examinations of all children born of mothers immunized with LMV should be carried out in order to protect seronegative children by passive or active immunization.     

Age-dependent impairment of functional helper T cell responses to immunodominant epitopes of Toxoplasma gondii antigens in congenitally infected individuals

Human infection with Toxoplasma gondii is generally asymptomatic in immunocompetent adults while it causes significant morbidity in congenitally infected children. Cell mediated immunity plays the main role in host resistance to T. gondii infection and a Th1 cytokine profile is necessary for protection and control of infection. The present work focused on comparing the helper T cell response to the GRA1 antigen of the parasite between children with congenital toxoplasmosis and healthy adults with acquired infection. We demonstrated that in young children with congenital infection the specific T cell response to parasite antigens is impaired and that such hypo-responsiveness is restored during childhood. Also, we provided clear evidence that in individuals with congenital toxoplasmosis the acquisition of functional helper T cell responses is disease-unrelated and indistinguishable in terms of strength, epitope specificity, and cytokine profile from the corresponding responses in immunocompetent adults with asymptomatic acquired T. gondii infection.     

Fine needle aspiration of cystic liver lesions. Cytologic examination and carcinoembryonic antigen assay of cyst contents

Since fine needle aspiration (FNA) cytology has been rarely successful in preoperatively separating neoplastic from nonneoplastic (inflammatory and congenital) liver cysts, the adjunctive use of measurements of cyst fluid carcinoembryonic antigen (CEA) levels for enhancing the accuracy of the cytologic diagnosis was examined. FNA was performed on 17 consecutive cystic lesions from 15 patients. Cytologic examination performed on Papanicolaou-stained smears or Cytospins of the sediment gave a sensitivity of 66% and a specificity of 100%. CEA measurements on the supernatant by enzyme immunoassay showed negative levels (less than 5 ng/mL) in fluids from benign (nonmucinous) cysts and abscesses and elevated levels (greater than 600 ng/mL) in fluids from biliary cystadenoma/cystadenocarcinoma and pseudocystic metastatic carcinoma. CEA assay gave a sensitivity of 100% and a specificity of 94%, thus enhancing the sensitivity of FNA for the detection of malignancy in cystic liver lesions.