Evaluation of eleven immunochromatographic assays for SARS-CoV-2 detection: investigating the dengue cross-reaction

ABSTRACT COVID-19 disease is spread worldwide and diagnostic techniques have been studied in order to contain the pandemic. Immunochromatographic (IC) assays are feasible and a low-cost alternative especially in low and middle-income countries, which lack structure to perform certain diagnostic techniques. Here we evaluate the sensitivity and specificity of eleven different IC tests in 145 serum samples from confirmed cases of COVID-19 using RT-PCR and 100 negative serum samples from blood donors collected in February 2019. We also evaluated the cross-reactivity with dengue using 20 serum samples from patients with confirmed diagnosis for dengue collected in early 2019 through four different tests. We found high sensitivity (92%), specificity (100%) and an almost perfect agreement (Kappa 0.92) of IC assay, especially when we evaluated IgG and IgM combined after 10 days from the onset of symptoms with RT-PCR. However, we detected cross-reactivity between dengue and COVID-19 mainly with IgM antibodies (5 to 20% of cross-reaction) and demonstrated the need for better studies about diagnostic techniques for these diseases.

Burkholderia cepacia, cystic fibrosis and outcomes following lung transplantation: experiences from a single center in Brazil

OBJECTIVES: To evaluate the impact of Burkholderia cepacia complex colonization in cystic fibrosis patients undergoing lung transplantation. METHODS: We prospectively analyzed clinical data and respiratory tract samples (sputum and bronchoalveolar lavage) collected from suppurative lung disease patients between January 2008 and November 2013. We also subtyped different Burkholderia cepacia complex genotypes via DNA sequencing using primers against the recA gene in samples collected between January 2012 and November 2013. RESULTS: From 2008 to 2013, 34 lung transplants were performed on cystic fibrosis patients at our center. Burkholderia cepacia complex was detected in 13 of the 34 (38.2%) patients. Seven of the 13 (53%) strains were subjected to genotype analysis, from which three strains of B. metallica and four strains of B. cenocepacia were identified. The mortality rate was 1/13 (7.6%), and this death was not related to B. cepacia infection. CONCLUSION: The results of our study suggest that colonization by B. cepacia complex and even B. cenocepacia in patients with cystic fibrosis should not be considered an absolute contraindication to lung transplantation in Brazilian centers.

Detection of Bartonella henselae DNA in clinical samples including peripheral blood of immune competent and immune compromised patients by three nested amplifications / Detecção de DNA de Bartonella henselae em amostras clínicas, incluindo sangue periférico, de pacientes imunocompetentes e imunodeprimidos por meio de três amplificações duplas

Bacteria of the genus Bartonella are emerging pathogens detected in lymph node biopsies and aspirates probably caused by increased concentration of bacteria. Twenty-three samples of 18 patients with clinical, laboratory and/or epidemiological data suggesting bartonellosis were subjected to three nested amplifications targeting a fragment of the 60-kDa heat shock protein (HSP), the internal transcribed spacer 16S-23S rRNA (ITS) and the cell division (FtsZ) of Bartonella henselae, in order to improve detection in clinical samples. In the first amplification 01, 04 and 05 samples, were positive by HSP (4.3%), FtsZ (17.4%) and ITS (21.7%), respectively. After the second round six positive samples were identified by nested-HSP (26%), eight by nested-ITS (34.8%) and 18 by nested-FtsZ (78.2%), corresponding to 10 peripheral blood samples, five lymph node biopsies, two skin biopsies and one lymph node aspirate. The nested-FtsZ was more sensitive than nested-HSP and nested-ITS (p < 0.0001), enabling the detection of Bartonella henselae DNA in 15 of 18 patients (83.3%). In this study, three nested-PCR that should be specific for Bartonella henselae amplification were developed, but only the nested-FtsZ did not amplify DNA from Bartonella quintana. We conclude that nested amplifications increased detection of B. henselae DNA, and that the nested-FtsZ was the most sensitive and the only specific to B. henselae in different biological samples. As all samples detected by nested-HSP and nested-ITS, were also by nested-FtsZ, we infer that in our series infections were caused by Bartonella henselae. The high number of positive blood samples draws attention to the use of this biological material in the investigation of bartonellosis, regardless of the immune status of patients. This fact is important in the case of critically ill patients and young children to avoid more invasive procedures such as lymph nodes biopsies and aspirates.

Bactérias do gênero Bartonella constituem patógenos emergentes detectados em biópsias de linfonodos e secreções de gânglios provavelmente devido a maior concentração de bactérias. Vinte e três amostras de 18 pacientes com dados clínicos, laboratoriais e/ou epidemiológicos sugestivos de bartonelose foram submetidas a três amplificações duplas para a detecção de fragmento da proteína de choque térmico de 60-kDa (HSP), do espaçador interno 16S-23S rRNA (ITS) e da proteína de divisão celular (FtsZ) de Bartonella henselae, para melhorar a detecção em amostras clínicas. Na primeira amplificação, uma, quatro e cinco amostras, respectivamente, foram positivas pelo HSP (4,3%), FtsZ (17,4%) e pelo ITS (21,7%). Com a segunda amplificação foram identificadas seis amostras positivas pelo nested-HSP (26%), oito pelo nested-ITS (34,8%) e 18 pelo nested- FtsZ (78,2%), correspondentes a 10 amostras de sangue periférico, cinco biópsias de linfonodos, duas biópsias de pele e um aspirado de gânglio. A nested-FtsZ foi mais sensível que a nested-HSP e a nested-ITS (p < 0,0001), possibilitando a detecção de DNA de Bartonella henselae em 15 de 18 pacientes (83,3%). No presente estudo, três nested-PCR, consideradas específicas para a amplificação da Bartonella henselae, foram desenvolvidas, porém somente a nested-FtsZ não amplificou o DNA de Bartonella quintana. Concluímos que amplificações duplas aumentaram a detecção de DNA de B. henselae, e que a nested-FtsZ foi a mais sensível e a única específica para B. henselae em diferentes amostras biológicas. Como todas as amostras detectadas pelo HSP-nested e nested-ITS foram também pela nested-FtsZ, inferimos que, em nossa casuística, as infecções foram causadas por Bartonella henselae. A elevada positividade de amostras de sangue chamou a atenção para a utilização deste material biológico na investigação de bartoneloses, independentemente do estado imune dos pacientes. Este fato é importante no caso de pacientes criticamente enfermos e crianças pequenas para evitar procedimentos mais invasivos, como biópsias e punções de gânglios.

Frequência de polimorfismo de nucleotídeo único de alguns genes da resposta imune em amostra populacional da cidade de São Paulo, Brasil / Frequency of single nucleotide polymorphisms of some immune response genes in a population sample from São Paulo, Brazil

Objective: To present the frequency of single nucleotide polymorphisms of a few immune response genes in a population sample from São Paulo City (SP), Brazil. Methods: Data on allele frequencies of known polymorphisms of innate and acquired immunity genes were presented, the majority with proven impact on gene function. Data were gathered from a sample of healthy individuals, non-HLA identical siblings of bone marrow transplant recipients from the Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, obtained between 1998 and 2005. The number of samples varied for each single nucleotide polymorphism analyzed by polymerase chain reaction followed by restriction enzyme cleavage. Results: Allele and genotype distribution of 41 different gene polymorphisms, mostly cytokines, but also including other immune response genes, were presented. Conclusion: We believe that the data presented here can be of great value for case-control studies, to define which polymorphisms are present in biologically relevant frequencies and to assess targets for therapeutic intervention in polygenic diseases with a component of immune and inflammatory responses.

Objetivo: Apresentar a frequência de polimorfismo de nucleotídeo único de alguns genes da resposta imune em amostra populacional da cidade de São Paulo (SP). Métodos: Foram apresentadas as frequências de alelos de conhecidos polimorfismos de genes de imunidade inata e adquirida, a maioria com impacto funcional comprovado. Os dados foram coletados a partir de amostras de indivíduos saudáveis, irmãos não-HLA idênticos, de receptores de transplante de medula óssea do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo, obtidos entre 1998 e 2005. O número de amostras variou para cada polimorfismo de nucleotídeo único analisado por reação em cadeia pela polimerase seguida de clivagem com enzimas de restrição. Resultados: Apresentou-se a distribuição de alelos e genótipos de 41 polimorfismos genéticos, a maioria de genes para citocinas, mas também incluindo outros genes de resposta imune. Conclusão: Acreditamos que os dados apresentados aqui possam ser de grande valor para definir quais os polimorfismos presentes em frequências relevantes, para estudos caso-controle e para avaliar alvos de intervenção terapêutica nas doenças poligênicas com componente de resposta imune ou inflamatória.

Uso de corticosteróides no recém-nascido com choque séptico: estado da arte / The use of corticosteroids in the newborn with sepsis shock: state of art

Objetivo: Verificar se a utilização de corticosteróide no recém-nascido com choque séptico e insuficiência adrenal tem influência na sobrevida dos pacientes. Fontes Pesquisadas: MEDLINE e LILACS no período de 1996 a 2006. Os artigos foram selecionados desde que fornecessem informações sobre a utilização de corticosteróides em recém-nascidos com choque séptico. Síntese dos Dados: A revisão foi dividida em tópicos que abordaram o recém-nascido com choque séptico, a utilização de corticosteróides neste tipo de choque e na insuficiência adrenal associada a este quadro e a relação do tratamento com a evolução dos pacientes. Conclusões: A insuficiência adrenal também ocorre no recém-nascido com choque séptico e os corticosteróides devem ser considerados, mesmo nos pré-termos, não respondentes ao tratamento convencional como expansão de volume e terapia inotrópica.

Objective: To review the use of corticosteroids in the treatment of newborns with septic shock and adrenal failure and the role in survival of the patients. Data Sources: Scientific articles were searched in the data base MEDLINE and LILLACS between 1996 and 2006. The articles were selected whether they provided information about the association of the use of corticosteroids in newborn infants with septic shock. Data Synthesis: This review is structured in topics, in which the septic shock is defined, as well the use of corticosteroids in the adrenal failure and their role in the survival of this patients. Conclusions: There are a very few studies including the use of corticosteroids in critically ill term or premature newborn infants. However, the reposition of adrenal cortex hormones must be considered in the treatment of septic shock with adrenal failure when these patients did not have a good response to the conventional treatment how volume expansion and inotropic therapy.

Polimorfismo de interleucina 10 e persistência da alergia ao leite de vaca / Interleukin 10 polymorphism and cow's milk allergy persistence

Justificativa: Alergia à leite de vaca (ALV) afeta 2,5% das crianças menores de 3 anos, sendo que a maioria dos pacientes desenvolvem tolerância até 3 anos de idade. No entanto, na ALV IgE-mediada cerca de 35% desses pacientes persistem sintomáticos. O objetivo deste estudo foi determinar se polimorfismos no gene que codifica a IL-lO estariam associados à ALV persistente mediada por IgE em crianças brasileiras com cinco anos. Métodos: Neste estudo, 50 pacientes com ALV com idade de 5 anos foram avaliados, sendo 36 persistentes e 14 tolerantes. Um grupo controle composto por 224 indivíduos saudáveis foi incluído no estudo. Os critérios de diagnóstico foram: anafilaxia desencadeada pelo leite de vaca (LV) ou reação clínica imediata para o Teste Duplo Cego Placebo Controlado (DCPC). A tolerância foi definida como a ausência de resposta clínica ao DBPC ou durante a exposição acidental ao LV. Os dados utilizados na análise dos resultados clínicos e laboratoriais foram aqueles na época do diagnóstico. Todos os pacientes e os controles foram avaliados pelo PCR-RFLP para os seguintes polimorfismos de IL-lO: -3575A/T, -2849A/G, -763A/C, 592C/A e pelo PCR-SSP para o polimorfismo IL-lO -1082G/A. Resultados: Houve diferença estatisticamente significante apenas para o polimorfismo IL-l0 -1082G/A, sendo a homozigose para o alelo A estatisticamente significante comparando-se pacientes do grupo ALV total versus grupo controles (p = 0,027) e a homozigose para o alelo G entre grupo persistente versus grupo controle (p = 0,001). Conclusão: Nos pacientes avaliados, o polimorfismo de IL-lO 1082G/A foi associado ao fenótipo da ALV persistente.

Rationale: Cow's milk allergy (CMA) affects 2.5% of children under 3 years and the majority of patients develop tolerance at age 3. However, in IgE-mediate CMA about 35% of them persist symptomatic. The aim of this study is to determine if interleukin 10 (IL-l0) gene polymorphisms are associated to persistent IgE-mediated CMA in Brazilian children at age five. Methods: In this study, 50 IgE-mediated CMA patients were evaluated at age 5, being 36 persistent and 14 tolerant to cow's milk (CM). A control group with 224 healthy individuals was included. The diagnosis criteria were: anaphylaxis triggered by CM or immediate clinical reaction to double blind placebo control test (DBPCT). The tolerance was defined as the absence of clinical response to the DBPCT or during the accidental exposure to CM. The data used about clinical and laboratorial findings were from the diagnosis work up. All patients and the controls were typed by PCR-RFLP for the following IL-l0 polymorphisms: -3575A/T, -2849A/G, -763A/C, -592C/A and by SSP for -1082G/A. Results: There was differences statistically significant only for IL-lO polymorphisms -1082G/A. Homozygosis to A allele was statistically significant comparing CMA total patients with controls (p = 0.027) and homozygosis to G allele between persistent group versus control group (p = 0.001). Conclusion: In these patients evaluated the IL-lO -1082G/A polymorphism was associated to CMA persistent phenotype.

Epstein-Barr virus nuclear antigen-2 detection and typing in immunocompromised children correlated with lymphoproliferative disorder biopsy findings

Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, plays a significant role as a cofactor in the process of tumorigenesis, and has consistently been associated with a variety of malignancies especially in immunocompromised patients. Forty-four children and adolescents (21 liver transplant patients, 7 heart transplant, 5 AIDS, 3 autoimmune hepatitis, 2 nephritic syndromes, 2 medullar aplasia, 2 primary immunodeficiency disorder patients, 1 thrombocytopenic purpura and 1 systemic lupus erythematosus) presenting with chronic active EBV infection (VCA-IgM persistently positive; VCA-IgG > 20 AU/mL and positive IgG _ EBNA) had peripheral blood samples obtained during clinically characterized EBV reactivation episodes. DNA samples were amplified in order to detect and type EBV on the basis of the EBNA-2 sequence (EBNA2 protein is essential for EBV-driven immortalization of B lymphocytes). Although we have found a predominance of type 1 EBNA-2 virus (33/44; 75 percent), 10 patients (22.73 percent) carried type 2 EBNA-2, and one liver transplant patient (2.27 percent) a mixture of the two types, the higher proportion of type 2 EBV, as well as the finding of one patient bearing the two types is in agreement with other reports held on lymphoproliferative disorder (LPD) patients, which analyzed tumor biopsies. We conclude that EBNA-2 detection and typing can be performed in peripheral blood samples, and the high prevalence of type 2 in our casuistic indicates that this population is actually at risk of developing LPD, and should be monitored.

Estudo do poliformismo genético na hepatite auto-imune na infância: busca de genes e haplótipos de suscetibilidade / Study of genetic polymorphism in children: searching for susceptibility genes and haplotypes

A hepatite auto-imune (HAI) é uma doença inflamatória crônica do fígado, de etiologia desconhecida, que acomete preferencialmente mulheres, com destruição progressiva do parênquima hepático e que, sem tratamento imunossupressor, evolui freqüentemente para cirrose. É uma doença rara na infância, com menos de 10% dos pacientes com doença hepática crônica, porém de alta mortalidade. Caracteriza-se pela presença de hipergamaglobulinemia, auto-anticorpos não órgãos-específicos e infiltrado inflamatório portal linfoplasmocitário. Cerca da metade dos pacientes atendidos no Instituto da Criança, apresenta também níveis elevados de IgE, sem causas aparentes como parasitoses ou atopia. A suscetibilidade genética à doença está principalmente associada a genes que codificam as moléculas de histocompatibilidade (HLA). A presença do HLA-DR é importante, mas não suficiente para o desenvolvimento dessa doença rara, fazendo inclusive supor um forte componente externo/ambiental no desencadeamento da doença. Genes recém descritos, localizados na região de classe III do Complexo Principal de Histocompatibilidade (CPH) e ligados ao controle da resposta imune, em especial, alguns próximos à junção com a região de classe I, têm sido investigados como "loci" secundários para o desenvolvimento de doenças auto-imunes. A forte associação da HAI com genes na região do MHC, bastante comuns na população, aliada a incidência muito baixa da doença, leva à questão da presença de genes adicionais de suscetibilidade, que, junto com o HLA-DR, seriam responsáveis pela suscetibilidade genética observada. Dessa forma, o HLADRB1* 13, além de um fator por si, também pode ser um marcador da região cromossômica, ou seja, de um haplótipo específico e que, portanto, carrega mais de um gene de suscetibilidade. Estudamos polimorfismos, tipo SNP, de xx genes próximos ao HLA-DRB1, como TNFA, LTA, NFKBIL1 e BAT1, buscando haplótipos de susceptibilidade à doença, em pacientes HAI-1 (n=105) e...

Autoimmune hepatitis (AIH) is an inflammatory chronic liver disease of unknown etiology found predominantly in females, leading when untreated, to cirrhosis. It is a rare disease in the childhood, corresponding to about 10% of patients with chronic hepatitis, but exhibits high mortality. It is characterized by hipergammaglobulinemia, organ nonspecific circulating autoantibodies, and an inflammatory liver-infiltrating lymphocytes and plasma cells. Almost half of patients investigated at Instituto da Criança had increased plasma IgE levels, without any apparent cause such as parasite infestation or atopy. Genetic predisposition to AIH has been mainly linked to genes coding for HLA class II molecules. HLA-DR is important but not sufficient to explain this rare disease, suggesting there is an external/environmental component triggering the disease. Recently, several genes in the class III region of MHC, linked to immune responses, especially near the junction with the MHC class I region have been investigated as secondary loci for autoimmune disease susceptibility. The strong association of AIH with genes in the MHC region, common in the population, but a disease with the very low incidence, suggests additional genes linked with HLA-DR could add to the disease susceptibility. So, HLA-DR*13 besides being a factor by itself, could also be a chromosomal region marker, taking part of a specific haplotype carrying more than one susceptibility gene. We studied single nucleotide polymorphisms (SNP) in genes near HLA-DRB1, like TNFA, LTA, NFKBIL1 and BAT1 searching for disease susceptibility haplotypes, in HAI-1 patients (n=105) compared to healthy controls (n=227). The ancestral haplotype 8.1, which includes HLA-DRB1*03 and the rare TNFA allele at position -308 was increased (p=0.0005). However, HLA-DRB1*13, though present in the majority of the patients, did not show any specific haplotype associated to it. xxii We also analyzed cytokine genes involved in IgE...