RESUMO Objetivo: Validar a quantificação de T-cell receptor excision circles (TRECs) e kappa-deleting recombination circles (KRECs) por reação em cadeia de polimerase (polymerase chain reaction, PCR) em tempo real (qRT-PCR), para triagem neonatal de imunodeficiências primárias que cursam com defeitos nas células T e/ou B no Brasil. Métodos: Amostras de sangue de recém-nascidos (RN) e controles foram coletadas em papel-filtro. O DNA foi extraído e os TRECs e KRECs foram quantificados por reação duplex de qRT-PCR. O valor de corte foi determinado pela análise de Receiver Operating Characteristics Curve, utilizando-se o programa Statistical Package for the Social Sciences (SSPS) (IBM®, Armonk, NY, EUA). Resultados: 6.881 amostras de RN foram analisadas quanto à concentração de TRECs e KRECs. Os valores de TRECs variaram entre 1 e 1.006 TRECs/µL, com média e mediana de 160 e 139 TRECs/µL, respectivamente. Três amostras de pacientes diagnosticados com imunodeficiência grave combinada (severe combined immunodeficiency, SCID) apresentaram valores de TRECs abaixo de 4/µL e um paciente com Síndrome de DiGeorge apresentou TRECs indetectáveis. Os valores de KRECs encontraram-se entre 10 e 1.097 KRECs/µL, com média e mediana de 130 e 108 KRECs/µL, e quatro pacientes com diagnóstico de agamaglobulinemia tiveram resultados abaixo de 4 KRECs/µL. Os valores de corte encontrados foram 15 TRECs/µL e 14 KRECs/µL, e foram estabelecidos de acordo com a análise da Receiver Operating Characteristics Curve, com sensibilidade de 100% para detecção de SCID e agamaglobulinemia, respectivamente. Conclusões: A quantificação de TRECs e KRECs foi capaz de diagnosticar crianças com linfopenias T e/ou B em nosso estudo, validando a técnica e dando o primeiro passo para a implementação da triagem neonatal em grande escala no Brasil.

ABSTRACT Objective: To validate the quantification of T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) by real-time polymerase chain reaction (qRT-PCR) for newborn screening of primary immunodeficiencies with defects in T and/or B cells in Brazil. Methods: Blood samples from newborns and controls were collected on filter paper. DNA was extracted and TRECs, and KRECs were quantified by a duplex real-time PCR. The cutoff values were determined by receiver operating characteristic curve analysis using SPSS software (IBM®, Armonk, NY, USA). Results: Around 6,881 samples from newborns were collected and TRECs and KRECs were quantified. The TRECs values ranged between 1 and 1,006 TRECs/µL, with mean and median of 160 and 139 TRECs/µL, respectively. Three samples from patients with severe combined immunodeficiency (SCID) showed TRECs below 4/µL and a patient with DiGeorge syndrome showed undetectable TRECs. KRECs values ranged from 10 to 1,097 KRECs/µL, with mean and median of 130 and 108 KRECs/µL. Four patients with agammaglobulinemia had results below 4 KRECs/µL. The cutoff values were 15 TRECs/µL and 14 KRECs/µL and were established according to the receiver operating characteristic curve analysis, with 100% sensitivity for SCID and agammaglobulinemia detection, respectively. Conclusions: Quantification of TRECs and KRECs was able to diagnose children with T- and/or B-cell lymphopenia in our study, which validated the technique in Brazil and enabled us to implement the newborn screening program for SCID and agammaglobulinemia.

A tetravalent rhesus-human reassortant rotavirus (RRV-TV) vaccine (4 x 10(4) plaque-forming units/dose) was evaluated for safety, immunogenicity and efficacy in a prospective, randomized, double-blind, placebo-controlled trial involving 540 Brazilian infants. Doses of vaccine or placebo were given at ages, 1, 3 and 5 months. No significant differences were noted in the occurrence of diarrhoea or vomiting in vaccine and placebo recipients following each dose. Low-grade fever occurred on days 35 in 23% of vaccinees after the first dose, but not after the second or third doses of vaccine. An IgA antibody response to rhesus rotavirus (RRV) occurred in 58% of vaccinees and 33% of placebo recipients. Neutralizing antibody responses to individual serotypes did not exceed 20% when measured by fluorescent focus reduction, but exceeded 40% when assayed by plaque reduction neutralization. There were 91 cases of rotavirus diarrhoea among the 3-dose (vaccine or placebo) recipients during two years of follow-up, 36 of them among children given the vaccine. Overall vaccine efficacy was 8% (P = 0.005) against any diarrhoea and 35% (P = 0.03) against any rotavirus diarrhoea. Protection during the first year of follow-up, when G serotype 1 rotavirus predominated, was 57% (P = 0.008), but fell to 12% in the second year. Similar results were obtained when analysis was restricted to episodes in which rotavirus was the only identified pathogen. There was a tendency for enhanced protection by vaccine against illness associated with an average of 6 or more stools per day. These results are sufficiently encouraging to warrant further studies of this vaccine in developing countries using a higher dosage in an attempt to improve its immunogenicity and efficacy.

Se evaluó la inocuidad, inmunogenicidad y eficacia de una vacuna tetravalente obtenida por recombinación genética de rotavirus aislados de monos rhesus y seres humanos (RRV-TV) (4 x 10(4) unidades formadoras de placas por dosis) en un ensayo prospectivo, aleatorio, a doble ciego y controlado con placebo que se efectuó con 540 lactantes brasileños. Se administraron dosis de vacuna o de placebo a la edad de 1, 3 y 5 meses. No se observaron diferencias significativas en la frecuencia de diarrea o vómito en los bebés de ninguno de los dos grupos después de administrar la dosis correspondiente. De 2 a 3% de los vacunados tuvieron fiebre baja los días tercero a quinto después de recibir la primera dosis, pero no después de las dosis segunda o tercera. Se observó una respuesta de anticuerpos del tipo IgA al rotavirus aislado de monos rhesus (RRV) en 58% de los vacunados y en 33% de quienes recibieron placebo. La respuesta de anticuerpos neutralizantes a cada serotipo no pasó de 20% cuando se determinó con la prueba de reducción de focos de fluorescencia, pero fue superior a 40% al medirse con la prueba de neutralización a base de reducción de placas. Se presentaron 91 casos de diarrea causada por rotavirus entre los niños que recibieron las tres dosis (de vacuna o de placebo) durante un seguimiento de 2 años, 36 de ellos en los niños vacunados. La eficacia general de la vacuna fue de 8% (P = 0,005) contra toda clase de diarrea y de 35% (P = 0,03) contra la diarrea causada por rotavirus. La protección durante el primer año de seguimiento, cuando predominó el rotavirus G del serotipo 1, fue de 57% (P = 0,008), pero se redujo a 12% en el segundo año. Se obtuvieron resultados similares al restringir el análisis a episodios en que el rotavirus fue el único agente patógeno identificado. Se observó en la vacuna una mayor tendencia a proteger contra casos de enfermedad con un promedio de seis o más deposiciones diarias. Estos resultados son lo suficientemente alentadores para justificar que en países en desarrollo se hagan otros estudios de esta vacuna con una dosis mayor para tratar de mejorar su inmunogenicidad y eficacia.