PCR em tempo real no diagnóstico da toxoplasmose congênita / Real-time PCR in the diagnosis of congenital toxoplasmosis

O diagnóstico da toxoplasmose congênita apresenta limitações sendo, portanto, necessárias novas opções de exames. A análise do líquido aminiótico pela PCR em tempo real já se mostrou eficaz para confirmação da infecção fetal. No entanto, o seu desempenho em outras amostras biológicas ainda não está claro. O objetivo deste estudo é avaliar a PCR em tempo real no sangue da mãe e do recém-nascido assim como no líquido amniótico e placenta, no diagnóstico da toxoplasmose congênita. Esse é um estudo descritivo de gestantes com toxoplasmose acompanhadas no Rio de Janeiro, Brasil. Foi realizada PCR em tempo real em amostras de sangue materno, líquido amniótico, placenta e sangue dos recém-nascidos e o exame histopatológico das placentas. Também foram coletados dados clínicos e laboratoriais dos recém-nascidos. Foram acompanhadas 116 gestantes e analisadas 298 amostras. Uma (0,9%) gestante apresentou PCR positiva no sangue, três (3,5%) no líquido amniótico, uma (2,3%) na placenta e nenhum recém-nascido apresentou PCR positiva no sangue. O estudo histopatológico foi sugestivo de infecção por toxoplasmose em 24 (49%) placentas. Seis (5,2%) recém-nascidos foram diagnosticados com toxoplasmose congênita e apenas os casos com PCR positiva no líquido amniótico tinham associação do resultado da PCR com o diagnóstico de infecção congênita. Tanto as amostras de sangue materno quanto as de sangue dos recém-nascidos e placenta, não demonstraram ser promissoras no diagnóstico da toxoplasmose congênita. Novos estudos são necessários para avaliar o real papel do diagnóstico molecular em outros materiais biológicos que não o líquido amniótico.

The diagnosis of congenital toxoplasmosis has limitations so new options are needed. Real-time PCR analysis of amniotic fluid has proven effective for confirming fetal infection. However, its performance in other biological samples still needs to be determined. This study aims to evaluate the real-time PCR role in the blood of the mother and newborn as well as in the amniotic fluid and placenta, in congenital toxoplasmosis diagnosis. It is a descriptive study of pregnant women with toxoplasmosis followed in Rio de Janeiro, Brazil. Real-time PCR was performed on maternal blood, amniotic fluid, placenta, and newborn blood samples. In addition, a histopathological examination of the placentas was performed and data from the babies were collected. One hundred and sixteen pregnant women were followed and 298 samples were analyzed. One (0.9%) pregnant woman had positive PCR in the blood, three (3.5%) in the amniotic fluid, one (2.3%) in the placenta, and any newborn had positive PCR in the blood. The histopathological study suggested toxoplasmosis infection in 24 (49%) placentas. Six (5.2%) newborns were diagnosed with congenital toxoplasmosis and only the cases with positive PCR in amniotic fluid associated with the diagnosis of congenital infection. Neither maternal nor newborn blood and placenta samples have not shown promise in diagnosing congenital toxoplasmosis. Further studies are needed to evaluate the fundamental role of molecular diagnostics in others biological materials than amniotic fluid.

Condução da toxoplasmose gestacional / Management of gestational toxoplasmosis

A toxoplasmose é uma doença proveniente do Toxoplasma gondii, um protozoário que tem os felinos como seu hospedeiro definitivo e os mamíferos e aves como seu hospedeiro intermediário. Tem um curso benigno e autolimitado quando acomete um indivíduo imunocompetente, no entanto a infecção durante a gestação acarreta até 50% de chance de toxoplasmose congênita, podendo causar danos severos ao feto. A virulência dos genótipos encontrados nas Américas Central e do Sul é a mais alta, comparada a Europa e América do Norte, tendo a doença um comportamento mais agressivo. Os estudos relatam a diminuição da infecção fetal em até 60% com o uso da espiramicina, usada ainda na profilaxia. Este artigo discute sobre a triagem materna pré-natal e sua necessidade, a profilaxia e o tratamento da infecção fetal ainda intraútero, com o objetivo de diminuir a transmissão vertical e as sequelas neonatais com suas implicações ao longo da vida.(AU)

Toxoplasmosis it is a disease originating from Toxoplasma gondii, a protozoan that has felines at as ultimate host and mammals and birds at as intermediate host. Has a benign and self-limiting course when affects immunocompetent individual, however, infection during pregnancy leads 50% chance of congenital toxoplasmosis and can cause severe damage to the fetus. The virulence of genotypes found in Central and South America is the highest compared to Europe and North America, having the disease a more aggressive behavior. Studies report a reduction in fetal infection 60% with the use spiramycin still used for prophylaxis. This article discusses prenatal maternal screening, prophylaxis and treatment of fetal infection still in utero with the objective of decreasing vertical transmission and neonatal sequelae with their lifelong implications.(AU)

The performance of four molecular methods for the laboratory diagnosis of congenital toxoplasmosis in amniotic fluid samples

Introduction Toxoplasmosis may be life-threatening in fetuses and in immune-deficient patients. Conventional laboratory diagnosis of toxoplasmosis is based on the presence of IgM and IgG anti-Toxoplasma gondii antibodies; however, molecular techniques have emerged as alternative tools due to their increased sensitivity. The aim of this study was to compare the performance of 4 PCR-based methods for the laboratory diagnosis of toxoplasmosis. One hundred pregnant women who seroconverted during pregnancy were included in the study. The definition of cases was based on a 12-month follow-up of the infants. Methods Amniotic fluid samples were submitted to DNA extraction and amplification by the following 4 Toxoplasma techniques performed with parasite B1 gene primers: conventional PCR, nested-PCR, multiplex-nested-PCR, and real-time PCR. Seven parameters were analyzed, sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), positive likelihood ratio (PLR), negative likelihood ratio (NLR) and efficiency (Ef). Results Fifty-nine of the 100 infants had toxoplasmosis; 42 (71.2%) had IgM antibodies at birth but were asymptomatic, and the remaining 17 cases had non-detectable IgM antibodies but high IgG antibody titers that were associated with retinochoroiditis in 8 (13.5%) cases, abnormal cranial ultrasound in 5 (8.5%) cases, and signs/symptoms suggestive of infection in 4 (6.8%) cases. The conventional PCR assay detected 50 cases (9 false-negatives), nested-PCR detected 58 cases (1 false-negative and 4 false-positives), multiplex-nested-PCR detected 57 cases (2 false-negatives), and real-time-PCR detected 58 cases (1 false-negative). Conclusions The real-time PCR assay was the best-performing technique based on the parameters of Se (98.3%), Sp (100%), PPV (100%), NPV (97.6%), PLR (∞), NLR (0.017), and Ef (99%). .

Evaluation of polymerase chain reaction on amniotic fluid for diagnosis of congenital toxoplasmosis

The present study assessed sensitivity and specificity of PCR targeting P30 gene in diagnosis of congenital toxoplasmosis using amniotic fluid samples. A total of 358 pregnant women in their first trimester of pregnancy, most of them were asymptomatic while the others suffered lymphadenopathy, fever and/or malaise. The serum sample from each woman was screened for Toxoplasma-specific ELISA IgM and IgG. Sero-negative females were then screened in 2[nd] and 3[rd] trimesters for sero-conversion. Amniocentesis was performed to seropositive and sero-converted women. Detection of Toxoplasma DNA in AF was done by animal inoculation and PCR targeting P30 gene. 85/358 women were sero-positive for Toxoplasma. Congenital infection was detected in 14/85 fetuses by MI. One mouse had tachyzoite in peritoneal exudate while other 13 showed cysts in histpathological sections of mice. PCR test targeting P30 gene was positive in 13 with additional four fetuses, only PCR gave positive results, and serologic follow-up of suspected fetuses [17] by IgM ELISA confirmed congenital toxoplasmosis. Sixteen cases of congenitally infected newborn were a symptomatic. One was clinically diagnosed [ventricular dilatation] by the ultrasound. The PCR drastically changed the diagnostic repertoire for prenatal diagnosis. The sensitivity and specificity of PCR targeting P30 gene on AF samples were 92.9% and 94.4% respectively while positive predictive value [PPV] was 76.5%, and negative predictive value [NPV] was 98.5%. Its disadvantages were in fact that negative result cannot exclude acute infection, and thus must be confirmed by MI and it is also an expensive technique

Significant performance variation among PCR systems in diagnosing congenital toxoplasmosis in São Paulo, Brazil: analysis of 467 amniotic fluid samples

INTRODUCTION: Performance variation among PCR systems in detecting Toxoplasma gondii has been extensively reported and associated with target genes, primer composition, amplification parameters, treatment during pregnancy, host genetic susceptibility and genotypes of different parasites according to geographical characteristics. PATIENTS: A total of 467 amniotic fluid samples from T. gondii IgM- and IgG-positive Brazilian pregnant women being treated for 1 to 6 weeks at the time of amniocentesis (gestational ages of 14 to 25 weeks). METHODS: One nested-B1-PCR and three one-round amplification systems targeted to rDNA, AF146527 and the B1 gene were employed. RESULTS: Of the 467 samples, 189 (40.47 percent) were positive for one-round amplifications: 120 (63.49 percent) for the B1 gene, 24 (12.69 percent) for AF146527, 45 (23.80 percent) for both AF146527 and the B1 gene, and none for rDNA. Fifty previously negative one-round PCR samples were chosen by computer-assisted randomization analysis and re-tested (nested-B1-PCR), during which nine additional cases were detected (9/50 or 18 percent). DISCUSSION: The B1 gene PCR was far more sensitive than the AF146527 PCR, and the rDNA PCR was the least effective even though the rDNA had the most repetitive sequence. Considering that the four amplification systems were equally affected by treatment, that the amplification conditions were optimized for the target genes and that most of the primers have already been reported, it is plausible that the striking differences found among PCR performances could be associated with genetic diversity in patients and/or with different Toxoplasma gondii genotypes occurring in Brazil. CONCLUSION: The use of PCR for the diagnosis of fetal Toxoplasma infections in Brazil should be targeted to the B1 gene when only one gene can be amplified, preferably by nested amplification with primers B22/B23.

Diagnóstico antenatal de sífilis congénita por PCR en líquido amniótico y sus posibles aplicaciones / Prenatal diagnosis of congenital syphilis by PCR in amniotic liquid and its possible applications

Se presenta un caso clínico de sífilis congénita diagnosticada antenatalmente mediante el uso de la reacción de la polimerasa en cadena (PCR) en líquido amniótico. La PCR permitiría identificar la espiroqueta en diferentes medios, como en sangre, líquido amniótico y líquido céfalo-raquídeo. Deberán desarrollarse nuevos protocolos para poder probar la efectividad de los tratamientos en base a esta técnica diagnóstica.

PCR technique for detecting Toxoplasma gondii in animal amniotic fluid.

The goal of diagnosing congenital toxoplasmosis is early detection of maternofetal transmission, for early treatment to prevent unwanted sequelae. Polymerase chain reaction (PCR) is a method used recently for detecting toxoplasmosis during pregnancy. Amniotic fluid is a the clinical specimen used, since it provides a rapid, simple and safe method to obtain accurate results. The advantages of the PCR technique are high sensitivity, specificity and positive predictive value compared with other laboratory methods. To determine the sensitivity, specificity and lower detection limits in our laboratory, amplification of the B1 gene by nested PCR was performed on Toxoplasma gondii tachyzoites added to animal amniotic fluid samples. From 48 samples, our technique detected T. gondii in 30 out of 41 positive samples, and gave negative results for all the negative samples. The sensitivity for this nested PCR was 73%, the specificity was 100%, and the efficiency of the test was 77.1%. The nested PCR technique is recommended as a diagnostic method for detecting T. gondii in suspected congenital toxoplasmosis animals.

Hallazgo de tripomastigoto en estudio citoquímico de líquido amniótico / Clinical finding of trypomastigote in citochemical study of amniotic fluid

Chagas disease is an important endemic disease in Latinamerica. The transmitions mechanism of Trypanosoma cruzi, etiologic agent of Chagas disease are multiples, this comunication is refering to congenital infection or transplacental infection. The aim of the present report was to describe the finding of trypomastigote in citochemical study of amniotic fluid from pregnant women with 32 weeks of gestation. Clinical findings of trypomastigote in amniotic fluid, shows the importance of microscopics tools in orger to detect and diagnose de Chagas disease in pregnant patients. Diagnosis of Chagas disease by citochemical study is an unreported and unexploited technique, easy to perform in an ordinary laboratory, allowing the possibility to explore events of early detection in the newborn

Diagnostic prenatal de la Toxoplasmose congenitale: Apport du PCR test sur liquide amniotique

The PCR test on amniotic fluid is safe, rapid and highly performant in the prenatal diagnosis of congenital toxoplasmosis. We report six cases of pergravidic toxoplasmosis seroconversion. Prenatal diagnosis was based exclusively on the PCR test, which was positif in only one case. The foetal infection rate was 17%. In the five other cases, the PCR test was negative and five infants were born at term. At a year, the neonatal survey, didn't found any toxoplasmic manifestation

Determinación de glucosa en líquido amniótico como valor predictivo de infección intraamniótica en pacientes con trabajo de parto pretérmino

OBJETIVO: Determinar la utilidad de la concentraciónde glucosa en líquido amniótico en pacientes con trabajo de parto pretermino, como factor predictivo de infección intraamniótica. MATERIAL Y METODOS: Se practicó amniocentesis a 56 gestantes con trabajo de parto pretérmino y membranas íntegras, enviándose muestra para Gram, cultivo y concentración de glucosa. Simultáneamente se obtuvieron muestras de líquido amniótico en 62 pacientes a quienes se les indicó amniocentesis por otras patologías y que sirvieron de grupo de control. Se correlacionaron los resultados con la presencia de parto pretérmino y/o evidencia clínica de corioamnionitis utilizando métodos estadisticos de Chi cuadrado y "t" test. CONCLUSION: La determinación de glucosa en líquido amniótico es una alternativa diagnóstica rápida y certera en la identificación de infección intraamniótica en pacientes con actividad uterina pretérmino(Truncado 2500 caracteres)

Enfermedad de Chagas congenita: evaluación inmuni-parasitología del líquido amniotico / Congenital Chagas disease, inmunoparasitologic study of the amniotic fluid

Se realizo un estudio inmunoparasitológico del Líquido Amniótico (L.A.) en 135 pacientes que llegaron al Hospital Materno Infantil Germán Urquidi, en trabajo de parto, entre Junio y Diciembre de 1993. Se analizarón en sangre de la madre y del cordon umbilical y en el L.A. obtenido por amniocentesis transvaginal, la presencia de parásitos (T. Cruzi) y de anticuerpos: Is G en la madre por la técnicas de HAI y TIF, Is G e Is M en sangre de cordon por la técnica de ELISA y Ac Is y en el L.A. también por la Técnica de ELISA. No fue posible realizar PCR (Polimerasa Chain Reacction) ni identificación de Is M en el L.A. por problemas técnicos que se tratan de superar, actualmente en el LABIMED.