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Introduction: Solid organ transplantation (SOT) is an effective treatment modality for children and young adults with end-stage organ failure with 5-year overall survival ranging from 25 to 95% depending on the transplanted organ. Patients subsequently require lifelong immunossupression to maintain graft health and limit the risk of graft rejection. Commonly used immunosuppressants to inhibit organ recipient T-lymphocyte function include calcineurin inhibitors (CNI), mTOR inhibitors, thiopurines, and antimetabolites. Most cases of monomorphic Post-Transplantation Lymphoproliferative Disorders (m-PTLD) are related to the Epstein Barr virus (EBV) and occur in patients who were EBV seronegative before the transplant. Objective: Our aim is to assess the overall survival in patients diagnosed with monomorphic Post-Transplantation Lymphoproliferative Disorders after SOT. Methods: We present retrospective data on 46 pediatric patients diagnosed with monomorphic Post-Transplantation Lymphoproliferative Disorders with Non-Hodgkin Lymphoma (NHL) after SOT in our Institution between 2001 and 2024. Results: Twenty four of the 46 were male, and 22 were female, and mean age at the time of diagnosis of PTLD was 8 years (age range, 11 months to 17 years). PTLD was diagnosed at a median time of 36 months after the organ transplant, with a significantly shorter interval in liver (20/46), 31 months, versus heart (15/46) or kidney (11/46) graft recipients (48- and 42-months years, respectively p < 0,05). Most common histopathology subtypes were Burkitt in 20/46 (44%), Diffuse Large B-cell lymphoma (DLBCL) in 19/46 (41%) and other NHL in 7/46 (15%). Most common sites were abdominal (17/46), followed by cervical and axillary lymph nodes (12/46). Murphy staging system revealed: Stage I (5/46 - 11%), Stage II (5/46 - 11%) and Stage III (33/46 -72%). Stage IV diseases with bone marrow (1/46 – 2%) and/or Central nervous system Involvement (CNS) (2/46 - 4%). The treatment carried out was reduction of immunosuppression and rituximab in 18 patients, with chemotherapy, rituximab and reduction of immunosuppression in 28 patients. In one relapse there was a need for bone marrow transplantation. No differences in outcome were observed between Early (22/46) and late-onset (24/46) PTLD and EBV-positive (14/46) or EBV-negative (25/46) PTLD, respectively. The 5-year overall and event-free survival were 91% and 83%, respectively. Conclusion: The outcome of m-PTLD has clearly improved as a result of the introduction of more uniform treatment protocols and improved supportive care, instaging and response monitoring. The majority of had a good response with chemotherapy and/or rituximab.

Introduction: Central nervous system (CNS) involvement is a poor prognostic factor of non-Hodgkin's lymphoma (NHL) in children. Therefore, early diagnosis and treatment of CNS disease is important to improve long-term disease-free survival rates in children. With the continuous progress of science and technology, the methods and technologies used for the diagnosis of CNS disease are constantly improving, from imaging and cerebrospinal fluid (CSF) morphology studies to cerebrospinal fluid CSF flow pattern and genetics studies, and so on. With the continuous progress of diagnosis technology, the clinical staging of patients, which has a vital role in follow-up treatments, has also become more accurate. Most patients with early CNS invasion have no obvious clinical manifestations. Therefore, to improve the diagnostic rate of CNS invasion, flow cytometry (FC) and conventional cytomorphology (CC) of CSF were performed in newly diagnosed NHL patients for the first time of intrathecal chemotherapy. We report the examination findings and flow cytometry (FC) and conventional cytomorphology (CC) of two patients diagnosed with a Burkitt lymphoma and treatment response. Case Report: : Case 1: Adolescent, male, 16 years old, with stage IV Burkitt lymphoma. In first evaluation cerebrospinal fluid, absence of atypical cells in cytological examination, immunophenotyping, presence of 38 cells/mL with immunophenotype: CD10+, CD19+, CD20+, CD38+, CD45+, Kappa+, CD34-, Lambda-. In the second CSF, absence of atypical cells and in the immunophenotyping, absence of monoclonal B lymphoid cells. Currently, the patient is 7 months out of treatment in remission. Case 2: Adolescent, male, 17 years old, with stage IV Burkitt lymphoma; in the first evaluation of the CSF with absence of atypical cells on cytology, on immunophenotyping, presence of 40% of anomalous B cells with immunophenotype: CD10+, CD19+, strong CD20, CD34 weak, CD38 strong. In a second CSF evaluation, the patient presented absence of atypical cells on cytological examination and absence of monoclonal B lymphoid cells. Currently, the patient is undergoing treatment with no evidence of CSF involvement. Conclusion: Central invasion is more common to be found in pediatric NHL patients with bulky disease and/or BM involvement and/or involvement of more than 4 organs. It was also common in the EBV with Burkitt Lymphoma. CSF detection by FC has important clinical significance in the diagnosis of CNS invasion in children with NHL, as it can significantly improve the detection rate of CNS involvement and evaluation during the treatment.

Introduction: Lymphomas are cancers that arise from the white blood cells and have been traditionally divided into two large subtypes: Hodgkin and non-Hodgkin lymphoma. B-cell lymphoma is the most common subtype of non-Hodgkin lymphoma; almost 75% of patients with lymphoma have this variant. Lymphomas can potentially arise from any lymphoid tissue located in the body; however, secondary involvement in the adrenal, pancreas and thyroid is very uncommon. We report the history, examination findings, staging images results of a 17 -year-old adolescent man diagnosed with a Burkitt lymphoma. Case report: Adolescent, male, 17 years old, with the appearance of a subclavicular nodule on the right since November 2023, extending to the axillary region; without presenting fever, weight loss, night sweats, abdominal pain. Admitted to the service in June 2024 with a previous lymph node biopsy result with a reactional appearance. Evolving from April 2024 with a significant increase in the mass, associated with paresthesia of the hand and pain when moving the right upper limb. Chest tomography showed a solid mass in the right axillary region (16.4 × 12.7 × 15.5 cm); with involvement of the axillary and subclavian vessels on the right, maintaining contact with the right lateral and anterior chest wall up to the skin; nodular lesions in the bilateral thyroid parenchyma; lesions compatible with lymphoproliferative disease. Abdominal tomography showed a solid mass in the body of the pancreas (5.8 × 5.2 cm), a solid mass in the topography of the left adrenal gland (10.4 × 6.2 cm), solid nodular lesions in the cortex of the bilateral kidneys (up to 3.2cm on the left); all lesions compatible with metastases. Performed on the lymph node conglomerate that diagnosed Burkitt's lymphoma, the patient concluded the stage without bone marrow involvement but with involvement in the cerebrospinal fluid, defining it as stage IV. Chemotherapy treatment was started with good clinical response and images. Conclusion: Based on this case and a review of the literature, secondary involvement of the adrenal, pancreas and thyroid does not affect the patient's prognosis like involvement of the cerebrospinal fluid or bone marrow. However, this case characterizes an unusual presentation of Burkitt's lymphoma with involvement of three endocrinological organs simultaneously.

A Leucemia Linfoide Aguda (LLA) é a neoplasia mais frequente na faixa etária pediátrica. Fibrose medular é uma alteração estromal que pode ocorrer em medula óssea de forma primária, como na mielofibrose ou associada a outras neoplasias hematológicas. É uma alteração rara nos pacientes com LLA, sendo descrito em torno de 30% das leucemias. Objetivo: Descrever o caso de um paciente que se apresentou com fibrose medular severa ao diagnóstico de LLA, dificultando diagnóstico inicial. Metodologia: descrição de dados clínicos e laboratoriais baseada em revisão de prontuário. Resultados: Paciente masculino, 6 anos de idade, proveniente de Manaus - Amazonas, com antecedente de mal formação em polegares; negou história de neoplasias na família. Deu entrada neste serviço com relato de que há 8 meses da consulta inicial evoluiu com icterícia e palidez na origem, sendo internado para investigação e identificado bicitopenia em hemograma. Tentado mielograma, sem sucesso, e realizado biópsia de medula óssea (BMO) complementar que evidenciou neste momento medula óssea aplasica, com 20% de celularidade e população imatura, fibrose medular grau 3 com hipótese inicial de síndrome mielodisplásica/falência medular congênita; na ocasião recebeu transfusão de hemoderivados e realizado Deb teste, que foi negativo. No período paciente apresentou diversas internações por infecções graves. Devido manutenção dos achados realizada nova avaliação medular que mostrou 100% de celularidade com células de aspecto imaturo, imunohistoquimica compatível com infiltração medular por linfoma/leucemia linfoblástica B. Encaminhado para seguimento neste serviço. Ao exame físico: dismorfismos faciais, emagrecido, com hepatomegalia. Na admissão hemograma com Hb 4,9 g/dL/Ht 13,6%/ VCM 80fL / HCM 29pg / RDW 13,2%, Leucócitos 960/mm3 e Plaquetas 63k/mm3. Avaliação medular hipocelular por diluição com comprometimento de análise e cerca de 5% de blastos; BMO favoreceu diagnóstico de linfoma/leucemia linfoblástica de imunofenótipo B em meio a fibrose reticulínica grau 3. Complementação diagnóstica: cariótipo inconclusivo, painel de translocações negativo e exoma em andamento. Paciente classificado como LLA B, SNC 1, risco intermediário por uso prévio de corticoide, atualmente em quimioterapia na fase de consolidação, respondendo bem ao tratamento. Discussão: A medula óssea é composta por células hematopoiéticas dispostas entre uma matriz extracelular e células estromais, dentre elas os fibroblastos. A fibrose medular é caracterizada por uma desordem arquitetural da medula óssea, gerando prejuízo para a homeostase do ambiente medular. A fibrogênese se dá através da sinalização de fatores de crescimento e citocinas que excitam vias formadoras de fibrose das células tronco mesenquimais na própria medula, culminando com o acúmulo de reticulina, associado ou não ao colágeno. A fibrose medular secundária surge de uma alteração que pode ou não ser clonal. A LLA é uma importante causa de fibrose medular difusa e esta geralmente se resolve com o próprio tratamento da leucemia. Conclusão: A LLA pode se apresentar com fibrose medular e a dificuldade de obtenção de material adequado pode retardar o diagnóstico. É relatado, inclusive, o ressurgimento da fibrose previamente ao aparecimento de blastos na recaída, reforçando a necessidade de um acompanhamento mais minuncioso nestes pacientes.

O gene GATA2 está altamente expresso em células hematopoiéticas imaturas, permitindo proliferação e manutenção de células tronco-hematopoiéticas. A mutação do gene GATA2 (haploinsuficiência) está associada a fenótipos síndromicos variados, e envolve clínica de imunodeficiência e predisposição a neoplasias mieloides. Objetivo: Descrever o caso de um lactente com mutação de GATA2 que se apresentou com mieloproliferação. Metodologia: Descrição de dados clínicos e laboratoriais baseada em revisão de prontuário. Resultados: Paciente de 6 meses de idade, procedente de Palmas-TO, previamente hígido, desenvolvimento adequado. Na história familiar pai com quadro de leucopenia e plaquetopenia, sem investigação prévia. Sem histórico familiar de neoplasia. Há 1 mês da admissão apresentou celulite em pé direito secundária à estrófulo, com necessidade de drenagem de abscesso e antibiótico endovenoso; em exames de controle foi evidenciado anemia (9,5g/dL), leucocitose (95k/mm3) e plaquetose (1.5k/mm3), sustentadas após melhora do quadro infeccioso. Devido alteração laboratorial optado por avaliação medular que evidenciou medula normocelular, com discreta disgranulopoiese, aumento de blastos; imunofenotipagem com presença de 6,5% de celulas imaturas mieloides. Sugerido diagnóstico de leucemia mielomonocítica juvenil (LMMJ), sendo então encaminhado ao Hospital de Câncer Infantojuvenil de Barretos para seguimento. No hemograma inicial: Hb 9,3g/dL / Ht 27,4% / VCM 81fL / HCM 27,4pg / RDW 15,7%, Leucócitos 74.460/mm3 (N 48.399 / E 1489 / B 745 / L 18.615 / M 5.212) Plaquetas 1.435k/mm3. Exame físico sem alterações; ausência de visceromegalias. Ampliado pesquisa: Cariótipo 46, XY, PCR para pesquisa de mutações associadas à LMMJ negativo e em NGS detectado mutação em GATA2 exon 5 VAF 49,5%. À princípio tentado citoredução com hidroxiureia. Discutido caso com equipe do Hospital St. Jude e devido poucos relatos de proliferação celular associado ao gene GATA2 orientado seguir observação, suspensão da hidroxiuréia, visto mieloproliferação provavelmente transitória. Realizada pesquisa de mutação de GATA2 no pai, positivo. Paciente segue assintomático, em acompanhamento ambulatorial, com reavaliação medular sem evidência de SMD, com plaquetometria e leucometria oscilantes. Discussão: Cerca de 80% dos pacientes com haploinsuficiência de GATA2 irão desenvolver neoplasias mieloides; no contexto da SMD pediátrica é o gene mais prevalente e os pacientes se manifestam com citopenia e infecções de repetição. O tratamento na fase de SMD instalada é o transplante de células tronco-hematopoiéticas (TCTH); pacientes assintomáticos podem ser seguidos com reavaliações medulares e de imunidade e neste contexto TCTH preemptivo controverso. A mieloproliferação descrita em raros pacientes com esta mutação é atribuída a uma resposta medular reativa, com relatos de resolução espontânea. O paciente relatado não apresentava, além da monocitose, outros critérios diagnósticos de LMMJ e foi exluída essa hipótese diagnóstica. Conclusão: O reconhecimento neste paciente de histórico de infecções e alteração hematológica associado à história familiar de citopenia foram importantes fatores considerados para investigação de mutações de predisposição germinativa a neoplasias mieloides.

Introduction: Hodgkin lymphoma (HL), unlike non-Hodgkin lymphoma, rarely involves the central nervous system (CNS). The incidence has been reported as 0.2% to 0.5% of all HL cases, but a recent review of 14 868 patients identified only 2 cases of HL involving the CNS. Consequently, there is no consensus to guide therapeutic decision-making. Involvement of the brain or spinal cord by HL can be primary or, more frequently, secondary to the compromise of adjacent lymph nodes. Lesions are more frequently intraspinal than intracranial. We describe a patient with systemic HL who presented with involvement of the CNS at diagnosis. Case Report: A six year -old female with a history of progressive cervical lymphadenopathy, presented in October 2023 with isolated cervical lymphadenopathy on the right, in addition to fever, pruritus in the upper limbs and night sweats. Initial pediatric evaluation did not conclude that the cause was infectious. In December 2023 cervical lymphadenomeglia increased in size (2x2cm) but in January 2024 with a progressive increase of 3x2 cm in addition to fever, night sweats and worsening itching, a biopsy was performed which defined as Hodgkin's Lymphoma subtype nodular sclerosis. Admitted to our service in March 2024, in addition to enlarged lymph nodes, fever, night sweats and pain in the lower limbs. After 48 hours of admission to this hospital to start the staging, she developed loss of strength in the left lower limbs. She mainly started Dexamethasone 10mg/m2/day and underwent an MRI. Magnetic core scan of the total spine and brain that showed: Solid neoplastic lesion inside the vertebral and sacral canal, from L4 to S3, involving the roots of the cauda equina, infiltrating the neuroforamens L5-S1, S1-S2 and S2-S3 on the left and compressing the corresponding roots. Solid paravertebral neoplastic lesion on the left, infiltrating the spinal canal at the level of T1-T2 and T6-T7 on the left. Diffuse heterogeneous change in the bone marrow signal of the vertebral bodies, which may represent neoplastic involvement. Solid neoplastic lesions with a dural base, one in the left temporal lobe, measuring 4.3 x 3.8 x 1.7 cm and another in the right frontal lobe, measuring 3.9 x 2.3 x 1.7 cm (CC x TT x AP), showing intense homogeneous post-contrast enhancement and diffusion restriction, the latter associated with vasogenic edema in the frontal white matter on the right. These findings may be related to the spread of the underlying neoplastic lesion. PET-CT with 18F-FDG showed - Metabolism in supra and infradiaphragmatic lymphadenopathy, spleen and bones compatible with active lymphoproliferative disease. Twenty-four hours after starting Dexamethasone, the patient experienced improvement in neurological symptoms, then underwent the 1st cycle of chemotherapy with improvement in neurological and systemic symptoms. She showed an excellent response after two cycles and is currently undergoing chemotherapy with no evidence of disease progression. Conclusion: Finally, unusual and aggressive extranodal presentations of HL should alert to the possibility of HIV infection. Whole brain irradiation and systemic chemotherapy remain the treatment of choice for CNS involvement in patients with HL.

A Leucemia Mieloide Aguda (LMA) corresponde a até 50% das mortes relacionadas à leucemia. Embora nas últimas décadas tenha sido observada significativa melhora no prognóstico com avanços no diagnóstico e terapias propostas, em países de renda média a baixa como Brasil a sobrevida de pacientes pediátricos com LMA ainda é de 30-40%. Objetivos: Descrever a sobrevida global e livre de eventos dos pacientes pediátricos diagnosticados com LMA no Hospital Infantojuvenil de Barretos, no período de janeiro de 2002 a junho de 2022. Materiais e métodos: Foi realizado um estudo de coorte retrospectivo. A análise de sobrevida foi estratificada em dois períodos distintos: Entre janeiro/2002 a maio/2013 (tratamento realizado na unidade de adultos do Hospital de Câncer de Barretos); e junho/2013 a junho/2022 (tratamento e suporte realizados na unidade infantil do Hospital). As sobrevidas global (SG) e livre de eventos (SLE) foram avaliadas considerando todos os pacientes LMA, LMA exceto leucemia promielocítica aguda (LPA) e, também, considerando apenas as LPA. Resultados: Foram incluídos 83 pacientes, sendo 25 no primeiro período e 58 no segundo período. Analisando o período de 20 anos, incluindo todos os pacientes pediátricos com LMA, encontrou-se SG de 37,7% e SLE de 35,8%. Excluindo LPA, a SG foi de 31,9% e a SLE foi de 31,2%. Já dentre os pacientes com LPA, a SG foi 64,3%. Ao avaliar a sobrevida nos dois períodos separadamente, a sobrevida global foi de 28% no primeiro período e 41,4% no segundo período. O óbito durante fase de indução chegou a 52,6% no primeiro período, e quase 50% dos pacientes tinham hiperleucocitose; a maioria dos pacientes desse período analisado foram tratados segundo protocolo BFM; 26,5% dos pacientes tiveram recaída de doença. O protocolo brasileiro GELMAI foi implantado recentemente e dos 10 pacientes incluídos não houve nenhum óbito na fase de indução. Quando a sobrevida foi estratificada segundo grupo de risco genético, pacientes de baixo risco tiveram uma sobrevida global de 50%, superior quando comparado aos risco intermediário e alto, com 30 e 28,6% de sobrevida, respectivamente. Discussão: Em nosso serviço, foram observadas taxas elevadas de óbito indutório nos pacientes com LMA. O diagnóstico tardio de muitos pacientes devido a dificuldade em serem referenciados a um centro oncológico, assim como quimioterapia intensiva indutória de protocolos internacionais e cuidado suportivo deficiente podem ser considerados para esse achado. Certamente, o cuidado às crianças realizado por pediatras – oncologistas, intensivistas, equipe multidisciplinar – em hospital pediátrico influenciou na melhora sobrevida global e redução de óbito indutório no serviço a partir de 2013. Conclusão: Pode-se inferir, com os dados obtidos, a importância de um serviço de tratamento especializado em conduzir crianças graves, com suporte necessário e de fácil acesso à disposição, além de profissionais treinados ao atendimento à população pediátrica. A morbimortalidade relacionada ao tratamento, entretanto, permanece um desafio nos países em desenvolvimento, inclusive neste Hospital, com a proposição de tratamentos de intensidade reduzida na indução se monstrando uma realidade fáctivel em países de baixa e média renda como o Brasil.

Tropical Bovine Theileriosis is an important tick-borne disease. This study aims to assess the occurrence of Theileria annulata infection in two indigenous Portuguese cattle breeds. A total of 843 blood samples collected from animals of Alentejana (n = 420) and Mertolenga (n = 423) breeds were analyzed. The detection of Theileria annulata was determined by amplification of a fragment of the merozoite-pyroplasm surface antigen gene with 319 base pairs (bp). The prevalence found (10.8%) is lower than that reported in previous studies (21.3%). A statistically significant difference was found for positivity between breeds ( p < 0.05). There is also a higher probability of older animals being positive compared to younger ones ( p < 0.05). The region where Mertolenga animals are located is shown to have a significant impact on positivity ( p < 0.05). Thus, the development of sustainable T. annulata control strategies and their implementation, adapted to the epidemiological conditions of higher risk, will be extremely important.

Genomic studies may generate massive amounts of data, bringing interpretation challenges. Efforts for the differentiation of benign and pathogenic variants gain importance. In this article, we used segregation analysis and other molecular data to reclassify to benign or likely benign several rare clinically curated variants of autosomal dominant inheritance from a cohort of 500 Brazilian patients with rare diseases. This study included only symptomatic patients who had undergone molecular investigation with exome sequencing for suspected diseases of genetic etiology. Variants clinically suspected as the causative etiology and harbored by genes associated with highly-penetrant conditions of autosomal dominant inheritance underwent Sanger confirmation in the proband and inheritance pattern determination because a "de novo" event was expected. Among all 327 variants studied, 321 variants were inherited from asymptomatic parents. Considering segregation analysis, we have reclassified 51 rare variants as benign and 211 as likely benign. In our study, the inheritance of a highly penetrant variant expected to be de novo for pathogenicity assumption was considered as a non-segregation and, therefore, a key step for benign or likely benign classification. Studies like ours may help to identify rare benign variants and improve the correct interpretation of genetic findings., (© 2022. The Author(s).)

Background: Several metabolic disorders follow an autosomal recessive inheritance pattern. Epidemiological information on these disorders is usually limited in developing countries. Our objective is to assess carrier frequencies of rare autosomal recessive metabolic diseases in a cohort of Brazilian patients that underwent molecular investigation with exome sequencing and estimate the overall frequency of these diseases using the Hardy-Weinberg equation., Methods and Results: We reviewed the molecular findings of 320 symptomatic patients who had carrier status for recessive diseases actively searched. A total of 205 rare variants were reported in 138 different genes associated with metabolic diseases from 156 patients, which represents that almost half (48.8%) of the patients were carriers of at least one heterozygous pathogenic/likely pathogenic (P/LP) variant for rare metabolic disorders. Most of these variants are harbored by genes associated with multisystemic involvement. We estimated the overall frequency for rare recessive metabolic diseases to be 10.96/10,000 people, while the frequency of metabolic diseases potentially identified by newborn screening was estimated to be 2.93/10,000., Conclusions: This study shows the potential research utility of exome sequencing to determine carrier status for rare metabolic diseases, which may be a possible strategy to evaluate the clinical and social burden of these conditions at the population level and guide the optimization of health policies and newborn screening programs., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Quercetin is a flavonoid found in a great variety of foods such as vegetables and fruits. This compound has been shown to inhibit the proliferation of various types of cancer cells, as well as the growth of tumors in animal models. In the present study, we analyze morphological and mechanical changes produced by quercetin in T24 bladder cancer cells. Decreased cell viability and cell number were observed following quercetin treatment at 40 μM and 60 μM, respectively, as observed by the MTT assay and trypan blue exclusion test, supporting the hypothesis of quercetin anticancer effect. These assays also allowed us to determine the 40, 60, and 80 μM quercetin concentrations for the following analyses, Lactate Dehydrogenase assay (LDH); Nuclear Morphometric Analysis (NMA); and atomic force microscopy (AFM). The LDH assay showed no cytotoxic effect of quercetin on T24 cancer cells. The AFM showed morphological changes following quercetin treatment, namely decreased cell body, cytoplasmic retraction, and membrane condensation. Following quercetin treatment, the NMA evidenced an increased percentage of nuclei characteristic to the apoptotic and senescence processes. Cells also presented biophysical alterations consistent with cell death by apoptosis, as increased roughness and aggregation of membrane proteins, in a dose-dependent manner. Cellular elasticity, obtained through force curves, showed increased stiffness after quercetin treatment. Data presented herein demonstrate, for the first time, in a quantitative and qualitative form, the morphological and mechanical alterations induced by quercetin on bladder cancer cells., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Next-generation sequencing (NGS) has altered clinical genetic testing by widening the access to molecular diagnosis of genetically determined rare diseases. However, physicians may face difficulties selecting the best diagnostic approach. Our goal is to estimate the rate of possible molecular diagnoses missed by different targeted gene panels using data from a cohort of patients with rare genetic diseases diagnosed with exome sequencing (ES). For this purpose, we simulated a comparison between different targeted gene panels and ES: the list of genes harboring clinically relevant variants from 158 patients was used to estimate the theoretical rate of diagnoses missed by NGS panels from 53 different NGS panels from eight different laboratories. Panels presented a mean rate of missed diagnoses of 64% (range 14%-100%) compared to ES, representing an average predicted sensitivity of 36%. Metabolic abnormalities represented the group with highest mean of missed diagnoses (86%), while seizure represented the group with lowest mean (46%). Focused gene panels are restricted in covering select sets of genes implicated in specific diseases and they may miss molecular diagnoses of rare diseases compared to ES. However, their role in genetic diagnosis remains important especially for well-known genetic diseases with established genetic locus heterogeneity.

Several Mendelian disorders follow an autosomal recessive inheritance pattern. Epidemiological information on many inherited disorders may be useful to guide health policies for rare diseases, but it is often inadequate, particularly in developing countries. We aimed to calculate the carrier frequencies of rare autosomal recessive Mendelian diseases in a cohort of Brazilian patients using whole exome sequencing (WES). We reviewed the molecular findings of WES from 320 symptomatic patients who had carrier status for recessive diseases. Using the Hardy-Weinberg equation, we estimated recessive disease frequencies (q 2 ) considering the respective carrier frequencies (2pq) observed in our study. We calculated the sensitivity of carrier screening tests based on lists of genes from five different clinical laboratories that offer them in Brazil. A total of 425 occurrences of 351 rare variants were reported in 278 different genes from 230 patients (71.9%). Almost half (48.8%) were carriers of at least one heterozygous pathogenic/likely pathogenic variant for rare metabolic disorders, while 25.9% of epilepsy, 18.1% of intellectual disabilities, 15.6% of skeletal disorders, 10.9% immune disorders, and 9.1% of hearing loss. We estimated that an average of 67% of the variants would not have been detected by carrier screening panels. The combined frequencies of autosomal recessive diseases were estimated to be 26.39/10,000 (or ~0.26%). This study shows the potential research utility of WES to determine carrier status, which may be a possible strategy to evaluate the clinical and social burden of recessive diseases at the population level and guide the optimization of carrier screening panels., (© 2021 Wiley Periodicals LLC.)

Rare diseases comprise a diverse group of conditions, most of which involve genetic causes. We describe the variable spectrum of findings and clinical impacts of exome sequencing (ES) in a cohort of 500 patients with rare diseases. In total, 164 primary findings were reported in 158 patients, representing an overall diagnostic yield of 31.6%. Most of the findings (61.6%) corresponded to autosomal dominant conditions, followed by autosomal recessive (25.6%) and X-linked (12.8%) conditions. These patients harbored 195 variants, among which 43.6% are novel in the literature. The rate of molecular diagnosis was considerably higher for prenatal samples (67%; 4/6), younger children (44%; 24/55), consanguinity (50%; 3/6), gastrointestinal/liver disease (44%; 16/36) and syndromic/malformative conditions (41%; 72/175). For 15.6% of the cohort patients, we observed a direct potential for the redirection of care with targeted therapy, tumor screening, medication adjustment and monitoring for disease-specific complications. Secondary findings were reported in 37 patients (7.4%). Based on cost-effectiveness studies in the literature, we speculate that the reports of secondary findings may influence an increase of 123.2 years in the life expectancy for our cohort, or 0.246 years/cohort patient. ES is a powerful method to identify the molecular bases of monogenic disorders and redirect clinical care., (© 2020 Wiley Periodicals LLC.)

Introduction: The use of the Self-Image Form (SIF) expands the identification of active leprosy cases to neighbors of index cases., Methods: The SIF was used to screen two groups: case (neighbors of index cases of leprosy) and control (individuals residing next to houses without leprosy) group. A specialist investigated suspected leprosy cases for disease confirmation., Results: New cases of leprosy were diagnosed in the case group (n = 7, 8.6%), but not the control group., Conclusions: The new surveillance strategy is inexpensive, efficient, and feasible within a primary health strategy. Future studies can help improve the use of the SIF.

Objective: To detect markers for minimal residual disease monitoring based on conventional polymerase chain reaction for immunoglobulin, T-cell receptor rearrangements and the Sil-Tal1 deletion in patients with acute lymphocytic leukemia., Methods: Fifty-nine children with acute lymphocytic leukemia from three institutions in Minas Gerais, Brazil, were prospectively studied. Clonal rearrangements were detected by polymerase chain reaction followed by homo/heteroduplex clonality analysis in DNA samples from diagnostic bone marrow. Follow-up samples were collected on Days 14 and 28-35 of the induction phase. The Kaplan-Meier and multivariate Cox methods were used for survival analysis., Results: Immunoglobulin/T-cell receptor rearrangements were not detected in 5/55 children screened (9.0%). For precursor-B acute lymphocytic leukemia, the most frequent rearrangement was IgH (72.7%), then TCRG (61.4%), and TCRD and IgK (47.7%); for T-acute lymphocytic leukemia, TCRG (80.0%), and TCRD and Sil-Tal deletion (20.0%) were the most common. Minimal residual disease was detected in 35% of the cases on Day 14 and in 22.5% on Day 28-35. Minimal residual disease on Day 28-35, T-acute lymphocytic leukemia, and leukocyte count above 50 x 10(9)/L at diagnosis were bad prognostic factors for leukemia-free survival in univariate analysis. Relapse risk for minimal residual disease positive relative to minimal residual disease negative children was 8.5 times higher (95% confidence interval: 1.02-70.7)., Conclusion: Immunoglobulin/T-cell receptor rearrangement frequencies were similar to those reported before. Minimal residual disease is an independent prognostic factor for leukemia-free survival, even when based on a non-quantitative technique, but longer follow-ups are needed.

Stroke is a known cause of cognitive impairment but the relationship between asymptomatic carotid artery stenosis and cognitive function is not clear. The main risk factors for vascular disease are also related to carotid stenosis and cognitive impairment. The association of high-grade stenosis of the internal carotid artery with cognitive impairment is related to silent embolization and hypoperfusion, but it may also be present without evidence of infarction on magnetic resonance imaging. Carotid stenosis treatment may lead to a decline in cognitive function due to complications related to the procedures (endarterectomy or stenting). On the other hand, reperfusion may improve cognitive impairment. The best treatment choice is unclear, considering possible deterioration of cognitive function related to carotid artery stenosis. There is insufficient evidence to consider cognitive impairment an important factor in determining the therapy for carotid stenosis., Competing Interests: Disclosure: The authors report no conflicts of interest.

Background: Around 15% of patients die or become dependent after cerebral vein and dural sinus thrombosis (CVT)., Method: We used the International Study on Cerebral Vein and Dural Sinus Thrombosis (ISCVT) sample (624 patients, with a median follow-up time of 478 days) to develop a Cox proportional hazards regression model to predict outcome, dichotomised by a modified Rankin Scale score >2. From the model hazard ratios, a risk score was derived and a cut-off point selected. The model and the score were tested in 2 validation samples: (1) the prospective Cerebral Venous Thrombosis Portuguese Collaborative Study Group (VENOPORT) sample with 91 patients; (2) a sample of 169 consecutive CVT patients admitted to 5 ISCVT centres after the end of the ISCVT recruitment period. Sensitivity, specificity, c statistics and overall efficiency to predict outcome at 6 months were calculated., Results: The model (hazard ratios: malignancy 4.53; coma 4.19; thrombosis of the deep venous system 3.03; mental status disturbance 2.18; male gender 1.60; intracranial haemorrhage 1.42) had overall efficiencies of 85.1, 84.4 and 90.0%, in the derivation sample and validation samples 1 and 2, respectively. Using the risk score (range from 0 to 9) with a cut-off of >or=3 points, overall efficiency was 85.4, 84.4 and 90.1% in the derivation sample and validation samples 1 and 2, respectively. Sensitivity and specificity in the combined samples were 96.1 and 13.6%, respectively., Conclusions: The CVT risk score has a good estimated overall rate of correct classifications in both validation samples, but its specificity is low. It can be used to avoid unnecessary or dangerous interventions in low-risk patients, and may help to identify high-risk CVT patients., ((c) 2009 S. Karger AG, Basel.)

The authors prospectively studied transcranial Doppler changes in patients with refractory congestive heart failure before and after cardiac transplantation. They evaluated 22 patients preoperatively and 14 patients after transplantation. Mean postoperative flow velocity increased by 53.3% (p < 0.0001). Preoperative waveform changes became normal after transplantation.

The objective of the present investigation was to study the expression of c-erbB-2 and MIB-1 and try to associate them with morphological features of the cell such as nuclear pleomorphism, mitotic count and histological grade in a series of 70 canine mammary gland tumors, 22 of them benign and 48 malignant. Tumors were collected at the Veterinary Hospital of UFMG (Brazil) and the Veterinary Faculty of Porto University (Portugal). c-erbB-2 expression was determined according to the guidelines provided by the manufacturer of the HercepTest system and nuclear pleomorphism, mitotic count and histological grade according the Elston and Ellis grading system. The HercepTest is the FDA-approved in vitro diagnostic test marketed by Dako. It is a semi-quantitative immunohistochemical assay used to determine overexpression of HER2 protein (human epidermal growth factor receptor) in breast cancer tissue. MIB-1 expression was also evaluated in 28 malignant tumors. Seventeen (35.4%) of the malignant tumors were positive for c-erbB-2 expression, which was positively associated with nuclear pleomorphism (P < 0.0001), histological grade (P = 0.0017) and mitotic count (P < 0.05). Nuclear pleomorphism also showed a positive association with MIB-1 index (P < 0.0001). These results suggest that some of the biological and morphological characteristics of the tumor are associated in canine mammary gland tumors, as also reported for human breast cancer. It was also possible to show that the immunoexpression of c-erbB-2 can be a factor in mammary carcinogenesis. This fact opens the possibility of using anti-c-erbB-2 antibodies in the treatment of canine mammary tumors.

We tested four isolates of Trypanosoma cruzi to assess parasite virulence and ability to cause myocarditis, cardiac sympathetic denervation, and histopathologic alterations in organs of the digestive system. The susceptibility of rats depends on the population of T. cruzi, with the ABC strain and the CL-Brener clone killing all animals, regardless of the parasitemic pattern. All tested T. cruzi populations caused acute myocarditis, but failed to induce histologic alterations in the intestine. The Cl-Brener and ABC isolates caused esophageal myositis. The myocarditis caused by the ABC, CL-Brener, and Y isolates was severe, but resolution started at the end of the acute phase. In contrast, the Col 1.7 G2 clone induced mild and sustained myocarditis. Our results also showed that T. cruzi populations able to induce severe acute myocarditis caused marked sympathetic denervation, but recovery of normal cardiac histology and innervation occurred. The sustained myocarditis induced by Col 1.7 G2 clone failed to cause sustained denervation.

We report on a longitudinal study concerning the incidence of malaria in a riverine population (Portuchuelo) settled on the riverbanks of Rio Madeira, in the State of Rondonia, Brazil. We found the incidence of malaria to be seasonal, prevailing in the dry months of June and July. The Annual Parasite Index (API) was 292/1000 inhabitants, almost three times that of the state of Rondonia for the same period. In contrast with other studied Rondonian populations, malaria in Portuchuelo was more prevalent in youngsters < 16 years old, particularly in the 0-1 year age group. Adults were relatively spared, particularly those over 50 years. Besides being indicative of indoor transmission, these facts may suggest the existence of a certain degree of acquired resistance to infection and/or of lessened symptoms in older people. Riverine populations are spread over the entire Amazon region where most of its members were born. Due to the permanent presence of malaria among riverine populations, we are proposing that they may act as perennial reserves of malaria and, therefore, as sources of infection for migrants or eventual settlers at their vicinity. To date, the opposite view has been generally held. Anopheles darlingi, the main vector species in the area, is essentially sylvatic, which contributes to make the control of malaria highly problematic. The only hopes for control rest on permanent surveillance and the prompt treatment of patients, which are also problematic considering the vastness of the Amazon region and the remoteness of some of its riverine settlements.

Quinine was the first antimalarial drug to be employed and also the first resistance was noticed to. After 1960 quinine urged to be reintroduced in routine therapy alone or in combination. Aiming at evaluating the effectiveness of different schedules we studied 484 patients seen at the Malaria Laboratory. We used quinine alone in 126 patients, quinine plus sulfadoxine and pyrimethamine in 119 patients and quinine plus tetracycline in 239 patients. The results shown that 81% of all patients were treated with success and only 0.6% were R2. and there is no R3. We emphasize a high resistance rate to quinine either alone (23.1%) or associated to sulfadoxine and pyrimethamine (37.8%). A higher resistance rate seen with the combination might be linked to the smaller dose of quinine used in that instance. It is worth noting the high cure rate with the quinine-tetracycline association.

A longitudinal study on the naturally acquired humoral immune responses against the merozoite surface protein 1 of Plasmodium vivax (PvMSP-1) was performed in malaria patients from the Brazilian Amazon region of Rondonia. We have previously cloned and expressed a recombinant protein, ICB2-5, that encodes 508 amino acids from the N-terminal portion of the PvMSP-1 protein. This affinity-purified polypeptide was tested by an enzyme-linked immunosorbent assay in a one-year longitudinal study using sera from 34 patients who had at least one malaria infection during the study period. The results demonstrated that more than 90% of the sera from patients having experienced more than three previous malaria infections contained antibodies to ICB2-5 at the time of a new clinical episode. Unexpectedly, more than half of these multiple-infected patients had an antibody response to ICB2-5 in which the predominant isotype was IgM. In contrast, more than 83% of the sera from these same patients contained predominantly IgG antibodies against total blood-stage antigen preparations. To determine if these results were due to the lack of boosting against this portion of the PvMSP-1 molecule, the presence of IgG antibodies to ICB2-5 in the sera from 11 patients who had consecutive malarial episodes during the study year was investigated. Five of these eleven patients failed to produce IgG antibodies to ICB2-5 even after 1-3 infections. Thus, these results suggest that no boosting against this region of the PvMSP-1 molecule was achieved by natural infections among these patients.

The cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax of individuals from malaria-endemic areas of Brazil was studied. We examined the in vitro proliferative response of the peripheral blood mononuclear cells (PBMC) of 22 individuals when stimulated with a CS recombinant protein (rPvCS-2) and two other synthetic peptides based on the sequence of the P. vivax CS protein. Seven of the individuals from malaria-endemic area displayed an antigen-specific in vitro proliferative response to the recombinant protein PvCS-2 and one out of 6, proliferative response to the peptide 308-320. In contrast, none of the individuals displayed a proliferative response when stimulated with the D/A peptide which represent some of the repeated units present in this CS protein. Our study, therefore, provides evidence for the presence, within the major surface antigen of P. vivax sporozoites, of epitopes capable to induce proliferation of human PBMC.

The murine cellular immune response to the circumsporozoite (CS) protein of Plasmodium vivax was characterized using five synthetic peptides, some of which we identified as corresponding to T cell epitopes. The peptides P308-320, P344-355 and P353-364 were immunogenic, inducing a genetically restricted proliferative response, due to the activation of CD4+ T cells. The peptide P308-320 was recognized only by the lymphocytes of B10 (H-2b) mice. The other two peptides were recognized by primed lymphocytes of H-2a and H-2k mice. Of interest was the finding that one of these peptides, P353-364, induced a proliferative response of a large percentage of immune outbred Swiss mice. Our data provide evidence that, at least in mice, there is recognition of multiple T cell epitopes within the major surface antigen of P. vivax sporozoites.

Very few well-established information is available about the frequency and timeliness of relapses in cases of Plasmodium vivax malaria acquired in Brazil. So, we analysed a series of correctly treated patients observed out of endemic areas. The rate of relapses seen in São Paulo, which may represent that of the parasitosis in the whole country, was high, ranging from 7.5% to 24.5%, and early in most cases, i.e. appearing by three months, what anticipates a high endemicity.

We examined the extent of variation of the 3' region of the circumsporozoite gene among Plasmodium falciparum isolates through amplification of a selected DNA fragment followed by DNA sequencing. A total of 32 isolates were analyzed, of which 24 were from Amazon endemic areas in Brazil and 8 from widely separated geographical regions in the world. Among Brazilian isolates only 2 variants were detected: 19 displayed the same sequence of strain 7G8 whereas the 4 remaining isolates differed from the 7G8 strain at five nucleotide positions which also led to amino acid changes. Variation was restricted to one of the T-helper epitopes while the sequence identified as a cytotoxic T cell epitope was conserved in all Brazilian isolates. P. falciparum samples from other geographical regions in the world showed sequences distinct from those of Brazilian isolates. However, some constancy could be observed within that variation. For instance, the most frequent nucleotide substitutions, from A and C at nucleotide positions 1015 and 1024, were the same in all isolates.