Use of polymerase chain reaction (PCR) in the diagnosis of congenital toxoplasmosis.

Congenital toxoplasmosis (CT) is a parasitic disease that causes serious fetal and neonatal harm or death. In countries that do not have antenatal screening programs, the initiation of CT treatment relies on a postnatal diagnosis. Until recently, diagnosis was based on clinical signs and immunoglobulin seropositivity, which is fraught with difficulty. In these cases, diagnosis was often delayed or treatment, which carries risk, started empirically. We highlight the use of polymerase chain reaction to diagnose a case of congenital toxoplasmosis, allowing early treatment and justifying the treatment burden.

Genotyping of Toxoplasma gondii strain directly from human CSF samples of congenital toxoplasmosis clinical case.

This report describes a case of congenital toxoplasmosis in a newborn in Southern Italy. A pregnant mother had been admitted at the 20th week of her pregnancy on account of pharyngodynia and laterocervical lymphadenopathy. Although serological testing of the mother's serum documented a seroconversion with positive IgG and IgM anti-Toxoplasma antibodies during II trimester, the woman refused to perform prenatal diagnosis for congenital toxoplasmosis. Fetal ultrasound scan already showed mild asymmetrical triventricular hydrocephaly and cerebral calcifications. After birth, real-time PCR on cerebrospinal fluid and blood samples of the newborn showed a positive result for 529bp-repeat element DNA of T. gondii, In addition brain magnetic resonance imaging and computed tomography showed a characteristic diffuse brain tissue loss associated with hydrocephalus. For the first time molecular characterization of T. gondii isolate was performed directly from the newborn's CSF samples by using nested-PCR-RFLP of sag-2 and pk1 genes. The PCR-RLFP analysis revealed that the isolate belongs to the clonal type II, the predominant lineage causing human toxoplasmosis, as confirmed by DNA sequencing.

Polymerase chain reaction in cerebrospinal fluid for the diagnosis of congenital toxoplasmosis.

BACKGROUND: Congenital toxoplasmosis can result in visual impairment, hearing loss, serious neurologic sequelae and death in the infant. We studied the potential of the polymerase chain reaction (PCR) in cerebrospinal fluid (CSF) for diagnosis of congenital toxoplasmosis. METHODS: For this purpose, we studied both congenitally infected (diagnosed clinically and serologically) and noninfected infants born to untreated mothers. RESULTS: The infants ranged in age from 0 to 180 days. CSF PCR was positive in 27 of the 58 (46.5%) congenitally infected infants and was negative in each of the 103 infants without congenital toxoplasmosis. The frequency of positive CSF PCR varied according to whether infants had major clinical signs of the disease; PCR was positive in 70.9%, 53.3% and 50.9% of those with hydrocephalus, cerebral calcifications and/or eye disease, respectively. Of 6 infants who were negative for both IgM and IgA antibodies, 3 had a positive PCR in their CSF as the confirmatory test for diagnosis of congenital toxoplasmosis. IgM and IgA antibodies and CSF PCR, when combined, yielded a higher sensitivity for diagnosis of congenital toxoplasmosis when compared with the performance of each test alone. CONCLUSIONS: Our findings reveal that in infants with clinical and serologic findings suggestive of congenital toxoplasmosis and born to untreated mothers, CSF PCR has the potential to increase the frequency of cases in which the diagnosis is confirmed.

Isolation of Toxoplasma gondii strains similar to Africa 1 genotype in Turkey.

INTRODUCTION: Toxoplasma gondii is a protozoon parasite that has a worldwide dissemination. It can cause serious clinical problems such as congenital toxoplasmosis, retinochoroiditis, and encephalitis. Currently, T. gondii genotypes are being associated with these clinical presentations which may help clinicians design their treatment strategy. CASE REPORTS: Two T. gondii strains named Ankara and Ege-1 were isolated from newborns with congenital toxoplasmosis in Central and Western Anatolia, respectively. Ankara and Ege-1 strains were isolated from the cerebrospinal fluid of newborns. According to microsatellite analysis, Ankara and Ege-1 strains were sorted as Africa 1 genotype. CONCLUSION: T. gondii strains isolated in Turkey were first time genotyped in this study. Africa 1 genotype has previously been isolated in immunosuppressed patients originating from sub-Saharan Africa. The reason of detecting a strain mainly detected in Africa can be associated with Turkey's specific geographical location. Turkey is like a bridge between Asia, Europe and Africa. Historically, Anatolia was on the Silk Road and other trading routes that ended in Europe. Thus, detecting Africa 1 strain in Anatolia can be anticipated. Consequently, strains detected mainly in Europe and Asia may also be detected in Anatolia and vice versa. Therefore, further studies are required to isolate more strains from Turkey.

Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis.

Toxoplasma gondii has a clonal population genetic structure with three (I, II, and III) lineages that predominate in North America and Europe. Type II strains cause most cases of symptomatic human infections in France and the United States, although few other regions have been adequately sampled. Here we determined the parasite genotype in amniotic fluid and cerebrospinal fluid samples from congenital toxoplasmosis cases in Poland. Nineteen confirmed congenital cases of toxoplasmosis were analyzed, including both severe and asymptomatic cases. The genotype of parasite strains causing congenital infection was determined by direct PCR amplification and restriction fragment length polymorphism analysis. Nested multiplex PCR analysis was used to type four independent polymorphic markers. The sensitivity of multiplex nested PCR was >/=25 parasites/ml in amniotic fluid and cerebral spinal fluid samples. Parasite DNA was successfully amplified in 9 of 19 samples (eight severely affected and one asymptomatic fetus). Only genotype II parasites were identified as the source of T. gondii infection based on restriction fragment length polymorphism analysis. Strains causing congenital infections were also typed indirectly based on detection of antibodies to strain-specific peptides. Serotyping indicated that 12 of 15 cases tested were caused by type II strains and these positives included both symptomatic and asymptomatic infections. Overall, the combined analysis indicated that 14 of the cases were caused by type II strains. Our results are consistent with the hypothesis that parasite burden is associated with severity of congenital toxoplasmosis and indicate that serological testing provides a promising method for genotypic analysis of toxoplasmosis.

[Analysis of a case of severe congenital toxoplasmosis]. / Análisis de un caso de toxoplasmosis congénita grave.

BACKGROUND: To describe a case of severe congenital toxoplasmosis because of inadequate surveillance of a seronegative pregnant woman and to evaluate the usefulness of different microbiological diagnostic methods after birth. METHODS: We applied serology, DNA amplification by one-tube semi-nested PCR, cell culture and mice inoculation analysis. RESULTS: Anti. T. gondii serology was useful for the diagnosis of congenital toxoplasmosis. PCR analysis of neonate cerebrospinal fluid and peripheral blood were positive, and yielded negative results after a few days of specific treatment. Cellular culture and mice inoculation yielded negative results. CONCLUSIONS: Our results suggest that serology and PCR are useful methods for the diagnosis of toxoplasmosis in newborns. Prenatal toxoplasmosis screening and suitable follow up of the seronegative pregnant women are necessary to prevent cases of severe infection in our area.

Value of cerebrospinal fluid cytochemical examination for the diagnosis of congenital toxoplasmosis at birth in France.

BACKGROUND: Because of routine screening and treatment of pregnant women for Toxoplasma infection in France, most neonates born to mothers who seroconverted during pregnancy are either not infected or asymptomatic. Early diagnosis relies mainly on radiologic, ophthalmologic and biologic tests. Cerebrospinal fluid (CSF) cytochemical evaluation is one of several tests performed in parallel to increase the overall sensitivity of the diagnostic evaluation. Our goal was to assess the value of cytochemical examination and to confirm whether using a portion of available CSF for this analysis is legitimate. METHODS: The individual performance of each of the two cytochemical tests and their combined value when used in parallel were assessed. These findings were then compared with the anti-Toxoplasma IgM and IgA serum titers and the clinical, ophthalmologic and radiologic findings at birth. RESULTS: CSF cytochemical analysis was possible in only 52% of the 233 children in the study. Our results in 112 children indicated poor sensitivity estimates. There was no significant change in the posttest probability of infection compared with the pretest estimation of risk in cases of a negative finding. After a mean follow-up of 80 months there was no evidence that CSF cytochemistry helped predict the risk of sequelae. CONCLUSION: In our setting cytochemical examination did not significantly contribute to the diagnosis of congenital infection at birth. Because of the limited quantity of CSF available, we suggest the use of other methods with higher yield.

Possible reasons for failure of conventional tests for diagnosis of fatal congenital toxoplasmosis: report of a case diagnosed by PCR and immunoblot.

Diagnosis of subclinical congenital toxoplasmosis has to rely on serological methods or isolation of the parasite. We present a case of congenital toxoplasmosis, in which conventional tests failed to establish the diagnosis. It was shown that this infant developed an intrathecal antibody response that was directed only against one of two Toxoplasma gondii strains used for routine diagnosis. In contrast to conventional tests, the diagnosis of cerebral toxoplasmosis could be established by using immunoblot and polymerase chain reaction (PCR). We therefore suggest that in unclarified cases, PCR and immunoblot, using at least two different strains of T. gondii, should be considered as additional tools for diagnosis of an infection with Toxoplasma and that examination of cerebrospinal fluid may be critical.

Detection of IgA specific for toxoplasmosis in serum and cerebrospinal fluid using a non-enzymatic IgA-capture assay.

A non-enzymatic immuno assay was optimized for detection of immunoglobulin A in serum and cerebrospinal fluid specific in acquired and congenital toxoplasmosis. An antihuman IgA monoclonal antibody was coated onto a polystyrene to capture total IgA. Suspensions of Toxoplasma gondii were used as a visible antigen. Eight hundred specimens (sera, cord blood serum, and cerebrospinal fluid) were tested. These were collected from 300 patients with acquired toxoplasmosis and from 28 children with congenital toxoplasmosis. In acquired toxoplasmosis, the assay allowed early detection of specific IgA, with kinetics similar to those of specific IgM expression. In congenital toxoplasmosis, anti-T. gondii IgA could be detected in the neonatal period. This assay was useful for diagnosis, follow-up, and posttherapeutic evaluation of toxoplasmosis. Specific IgA was also detected in the cerebrospinal fluid of infected newborn children. This simple IgA capture assay improves serological diagnosis of acquired and congenital toxoplasmosis when used in combination with analysis of T. gondii specific IgG and IgM.

[Toxoplasmosis. Clinical, radiological and pathological aspects. Report of 44 cases]. / Toxoplasmosis. Aspectos clínicos radiológicos y patológicos. Presentación de 44 casos.

Forty four cases of toxoplasmosis seen in a 20 years span are reviewed. The largest number of cases corresponded to the neonatal group. The most important clinical findings usually involve the central nervous system, reticulum endothelium, skin and eyes. The disease is compared to an equivalent group of congenital lues (Torch complex). Clinical and radiological criteria for the differentiation are established. The most important finding is the presence of intracraneal calcifications. The different patterns and distribution are discussed. The radiologist plays an important role in the diagnosis of this condition, particularly, in the neonatal group of patients where the incidence of intravenous calcification appears to be higher.

[Subdural hygroma in infants with congenital toxoplasmosis (author's transl)]. / Die subduralen Hygrome bei den Säuglingen mit angeborener Toxoplasmose.

Cytological examination of the fluid from subdural hygromas revealed congenital Toxoplasmosis in 14 of 43 infants (32,5%). Conversely, subdural hygroma was a significant clinical symptom in 42 cases of congenital Toxoplasmosis. In 9 cases trophozoites or cystes of Toxoplasma Gondii were found in the subdural fluid, and in 4 of these in the cerebrospinal fluid as well. Since serological investigations in young infants and particularly severe infections often remain inconclusive the cytological examination of cerebrospinal fluid is emphasized importance of.

The fractionation of cerebrospinal fluid proteins by cellulose acetate electrophoresis in children with infectious diseases of the central nervous system (author's transl). / Die Celluloseacetatfolien-Elektrophoreses der Liquorproteine von Kindern mit entzündlichen Erkrankungen des Zentralnervensystems

The increased permeability of the blood-brain barrier during acute inflammation of the central nervous system leads to changes of the cerebrospinal fluid (C.S.F.) protein pattern. Initially, in the cases of bacterial meningitis, cellulos acetate electrophoresis revealed decreased prealbumin, albumin and tau-globulin fraktion whereas alpha- and gamma-globulin fractions were found increased. In later stages of purulent inflammation a hydrocephalus occurred in five children, associated with an increased amount of albumin in the C.S.F. Cases of viral meningoencephalitis had a characteristic decrease of prealbumin and increase of gamma-globulin, the lowered prealbumin values were found more often. In three cases of congenital encephalitis pathological patterns of C.S.F. proteins were still found 1--1 1/2 years postpartum. Children with acute peripheral facial palsy and febrile convulsions had a normal C.S.F. protein profile.

Toxoplasmosis. Historical review, direct diagnostic microscopy, and report of a case.

A case of congenital toxoplasmosis diagnosed by the detection of Toxoplasma gondii organisms in the ventricular fluid of a living patient, confirmed by mouse inoculation, is reported. The subject of toxoplasmosis is reviewed historically, especially in regard to cases diagnosed by direct microscopic examination of cerebrospinal or ventricular fluids. Although several such cases have been reported in the world medical literature, an extensive survey failed to reveal such a case previously reported in the United States.