Toxoplasma gondii SAG2, SAG3 and GRA6 alleles and single nucleotide polymorphism in congenital infections with known parasite load and clinical outcome

ABSTRACT Amniotic fluid DNA samples were genotyped by multilocus-nested-PCR-RFLP, but only three of 11 markers amplified 113 of 122 (92.6%) samples, resulting in 12 untyped and 101 partial non-archetypal genotypes. The 101 typed samples were subdivided into four groups: G1 with 73 samples (5'and 3' SAG2 allele I + SAG3 allele III + GRA6 allele III), 53 had parasite load ≤ 102 parasites/mL (43 asymptomatic, 10 mild infections), 17 had load > 102 and ≤ 103 (one mild, 13 moderate and three severe), and three had load > 103 parasites/mL (three severe); G2 with 22 samples (5'and 3' SAG2 allele I + SAG3 allele III), all parasite load levels ≤ 102 parasites/mL (18 asymptomatic and four mild); G3 with five samples (5' and 3' SAG2 allele I + SAG3 allele II), parasite load ≤ 102 parasites/mL (three asymptomatic and two mild); G4 with one sample (5' and 3' SAG2 allele II + SAG3 allele II + GRA6 allele I), a parasite load < 102 parasites/mL in an asymptomatic infant. After DNA sequencing, restriction sites confirmed SAG2, SAG3 and GRA6 alleles in 98.7%, 100% and 100% of the cases, respectively, while single nucleotide polymorphisms confirmed 90% of 5'-SAG2 allele I; 98.7% of 3'-SAG2 allele I; 98% of SAG-3 allele III, but only 40% of GRA6 allele III results. For the moment, partial non-archetypal genotypes of parasites did not show any relationship with either parasite load in amniotic fluid samples or clinical outcome of infants at the age of 12 months.

Coxsackievirus A6 strains causing an outbreak of hand-foot-and-mouth disease in Northeastern Brazil in 2018

ABSTRACT Hand-foot-and-mouth disease (HFMD) is a highly contagious viral disease commonly associated to Enteroviruses (EV). During 2018, Brazil faced massive HFMD outbreaks spread across the country. This study aimed to characterize the EV responsible for the HFMD outbreak that occurred in Paraiba State, Brazilian Northeastern region, in 2018, followed by a phylogenetic analysis to detail information on its genetic diversity. A total of 49 serum samples (one from each patient) collected from children ≤ 15 years old, clinically diagnosed with HFMD were tested for EV using conventional RT-PCR and RT-qPCR. EV infection was confirmed in 71.4% (35/49) of samples. The mean and median ages were 1.83 years and one year old, respectively. Twenty-two EV-positive samples were successfully sequenced and classified as EV-A species; 13 samples were also identified with the CV-A6 genotype. The phylogenetic analysis (VP1 region) of three samples revealed that the detected CV-A6 strains belonged to sub-lineage D3. The CV-A6 strains detected here clustered with strains from South America, Europe and West Asia strains that were also involved in HFMD cases during the 2017-2018 seasons, in addition to the previously detected Brazilian CV-A6 strains from 2012 to 2017, suggesting a global co-circulation of a set of different CV-A6 strains introduced in the country at different times. The growing circulation of the emerging CV-A6 associated with HFMD, together with the detection of more severe cases worldwide, suggests the need for a more intense surveillance system of HFMD in Brazil. In addition, this investigation was performed exclusively on serum samples, and the analysis of whole blood samples should be considered and could have shown advantages when employed in the diagnosis of enteroviral HFMD outbreaks.

Technical performance of a lateral flow immunoassay for detection of anti-SARS-CoV-2 IgG in the outpatient follow-up of non-severe cases and at different times after vaccination: comparison with enzyme and chemiluminescent immunoassays

ABSTRACT This study assessed the technical performance of a rapid lateral flow immunochromatographic assay (LFIA) for the detection of anti-SARS-CoV-2 IgG and compared LFIA results with chemiluminescent immunoassay (CLIA) results and an in-house enzyme immunoassay (EIA). To this end, a total of 216 whole blood or serum samples from three groups were analyzed: the first group was composed of 68 true negative cases corresponding to blood bank donors, healthy young volunteers, and eight pediatric patients diagnosed with other coronavirus infections. The serum samples from these participants were obtained and stored in a pre-COVID-19 period, thus they were not expected to have COVID-19. In the second group of true positive cases, we chose to replace natural cases of COVID-19 by 96 participants who were expected to have produced anti-SARS-CoV-2 IgG antibodies 30-60 days after the vaccine booster dose. The serum samples were collected on the same day that LFIA were tested either by EIA or CLIA. The third study group was composed of 52 participants (12 adults and 40 children) who did or did not have anti-SARS-CoV-2 IgG antibodies due to specific clinical scenarios. The 12 adults had been vaccinated more than seven months before LFIA testing, and the 40 children had non-severe COVID-19 diagnosed using RT-PCR during the acute phase of infection. They were referred for outpatient follow-up and during this period the serum samples were collected and tested by CLIA and LFIA. All tests were performed by the same healthcare operator and there was no variation of LFIA results when tests were performed on finger prick whole blood or serum samples, so that results were grouped for analysis. LFIA's sensitivity in detecting anti-SARS-CoV-2 IgG antibodies was 90%, specificity 97.6%, efficiency 93%, PPV 98.3%, NPV 86.6%, and likelihood ratio for a positive or a negative result were 37.5 and 0.01 respectively. There was a good agreement (Kappa index of 0.677) between LFIA results and serological (EIA or CLIA) results. In conclusion, LFIA analyzed in this study showed a good technical performance and agreement with reference serological assays (EIA or CLIA), therefore it can be recommended for use in the outpatient follow-up of non-severe cases of COVID-19 and to assess anti-SARS-CoV-2 IgG antibody production induced by vaccination and the antibodies decrease over time. However, LFIAs should be confirmed by using reference serological assays whenever possible.

Differences in children and adolescents with SARS-CoV-2 infection: a cohort study in a Brazilian tertiary referral hospital

OBJECTIVES: To compare demographic/clinical/laboratory/treatments and outcomes among children and adolescents with laboratory-confirmed coronavirus disease 2019 (COVID-19). METHODS: This was a cross-sectional study that included patients diagnosed with pediatric COVID-19 (aged <18 years) between April 11, 2020 and April 22, 2021. During this period, 102/5,951 (1.7%) of all admissions occurred in neonates, children, and adolescents. Furthermore, 3,962 severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) detection samples were processed in patients aged <18 years, and laboratory-confirmed COVID-19 occurred in 155 (4%) inpatients and outpatients. Six/155 pediatric patients were excluded from the study. Therefore, the final group included 149 children and adolescents (n=97 inpatients and 52 outpatients) with positive SARS-CoV-2 results. RESULTS: The frequencies of sore throat, anosmia, dysgeusia, headache, myalgia, nausea, lymphopenia, pre-existing chronic conditions, immunosuppressive conditions, and autoimmune diseases were significantly reduced in children and adolescents (p<0.05). Likewise, the frequencies of enoxaparin use (p=0.037), current immunosuppressant use (p=0.008), vasoactive agents (p=0.045), arterial hypotension (p<0.001), and shock (p=0.024) were significantly lower in children than in adolescents. Logistic regression analysis showed that adolescents with laboratory-confirmed COVID-19 had increased odds ratios (ORs) for sore throat (OR 13.054; 95% confidence interval [CI] 2.750-61.977; p=0.001), nausea (OR 8.875; 95% CI 1.660-47.446; p=0.011), and lymphopenia (OR 3.575; 95% CI 1.355-9.430; p=0.010), but also had less hospitalizations (OR 0.355; 95% CI 0.138-0.916; p=0.032). The additional logistic regression analysis on patients with preexisting chronic conditions (n=108) showed that death as an outcome was significantly associated with pediatric severe acute respiratory syndrome (SARS) (OR 22.300; 95% CI 2.341-212.421; p=0.007) and multisystem inflammatory syndrome in children (MIS-C) (OR 11.261; 95% CI 1.189-106. 581; p=0.035). CONCLUSIONS: Half of the laboratory-confirmed COVID-19 cases occurred in adolescents. Individuals belonging to this age group had an acute systemic involvement of SARS-CoV-2 infection. Pediatric SARS and MIS-C were the most important factors associated with the mortality rate in pediatric chronic conditions with COVID-19.

Severe clinical spectrum with high mortality in pediatric patients with COVID-19 and multisystem inflammatory syndrome

OBJECTIVES: To assess the outcomes of pediatric patients with laboratory-confirmed coronavirus disease (COVID-19) with or without multisystem inflammatory syndrome in children (MIS-C). METHODS: This cross-sectional study included 471 samples collected from 371 patients (age<18 years) suspected of having severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The study group comprised 66/371 (18%) laboratory-confirmed pediatric COVID-19 patients: 61 (92.5%) patients tested positive on real-time reverse transcription-polymerase chain reaction tests for SARS-CoV-2, and 5 (7.5%) patients tested positive on serological tests. MIS-C was diagnosed according to the criteria of the Center for Disease Control. RESULTS: MIS-C was diagnosed in 6/66 (9%) patients. The frequencies of diarrhea, vomiting, and/or abdominal pain (67% vs. 22%, p=0.034); pediatric SARS (67% vs. 13%, p=0.008); hypoxemia (83% vs. 23%, p=0.006); and arterial hypotension (50% vs. 3%, p=0.004) were significantly higher in patients with MIS-C than in those without MIS-C. The frequencies of C-reactive protein levels >50 mg/L (83% vs. 25%, p=0.008) and D-dimer levels >1000 ng/mL (100% vs. 40%, p=0.007) and the median D-dimer, troponin T, and ferritin levels (p<0.05) were significantly higher in patients with MIS-C. The frequencies of pediatric intensive care unit admission (100% vs. 60%, p=0.003), mechanical ventilation (83% vs. 7%, p<0.001), vasoactive agent use (83% vs. 3%, p<0.001), shock (83% vs. 5%, p<0.001), cardiac abnormalities (100% vs. 2%, p<0.001), and death (67% vs. 3%, p<0.001) were also significantly higher in patients with MIS-C. Similarly, the frequencies of oxygen therapy (100% vs. 33%, p=0.003), intravenous immunoglobulin therapy (67% vs. 2%, p<0.001), aspirin therapy (50% vs. 0%, p<0.001), and current acute renal replacement therapy (50% vs. 2%, p=0.002) were also significantly higher in patients with MIS-C. Logistic regression analysis showed that the presence of MIS-C was significantly associated with gastrointestinal manifestations [odds ratio (OR)=10.98; 95%CI (95% confidence interval)=1.20-100.86; p=0.034] and hypoxemia [OR=16.85; 95%CI=1.34-211.80; p=0.029]. Further univariate analysis showed a positive association between MIS-C and death [OR=58.00; 95%CI=6.39-526.79; p<0.0001]. CONCLUSIONS: Pediatric patients with laboratory-confirmed COVID-19 with MIS-C had a severe clinical spectrum with a high mortality rate. Our study emphasizes the importance of investigating MIS-C in pediatric patients with COVID-19 presenting with gastrointestinal involvement and hypoxemia.

Accuracy of the QuantiFERON-TB Gold in Tube for diagnosing tuberculosis in a young pediatric population previously vaccinated with Bacille Calmette-Guerin / Precision del quantiferon-tb gold in tube en el diagnostico de tuberculosis en una poblacion pediatrica joven previamente vacunada con Bacille Calmette-Guerin / Acurácia do QuantiFERON-TB Gold in Tube no diagnóstico de tuberculose em uma população pediátrica jovem previamente vacinada com Bacille Calmette-Guérin

Objective: To evaluate the accuracy of an interferongamma release assay (QuantiFERON-TB Gold in Tube) for diagnosing Mycobacterium tuberculosis infection in a young pediatric population. Methods: 195 children previously vaccinated with BCG were evaluated, being 184 healthy individuals with no clinical or epidemiological evidence of mycobacterial infection, and 11 with Mycobacterium tuberculosis infection, according to clinical, radiological, and laboratory parameters. A blood sample was obtained from each child and processed according to the manufacturer's instructions. The assay performance was evaluated by a Receiver Operating Characteristic (ROC) curve. Results: In the group of 184 non-infected children, 130 (70.6%) were under the age of four years (mean age of 35 months). In this group, 177 children (96.2%) had negative test results, six (3.2%) had indeterminate results, and one (0.5%) had a positive result. In the group of 11 infected children, the mean age was 58.5 months, and two of them (18%) had negative results. The ROC curve had an area under the curve of 0.88 (95%CI 0.82-0.92; p<0.001), disclosing a predictive positive value of 81.8% for the test (95%CI 46.3-97.4). The assay sensitivity was 81.8% (95%CI 48.2-97.2) and the specificity was 98.8% (95%CI 96-99.8). Conclusions: In the present study, the QuantiFERON-TB Gold in Tube performance for diagnosing M. tuberculosis infection was appropriate in a young pediatric population. .

Objetivo: Evaluar la precisión de una prueba de liberación de interferón gama (QuantiFERON-TB Gold in Tube) para el diagnóstico de la infección por el Mycobacterium tuberculosis en una población pediátrica. Métodos: Se evaluaron 195 niños previamente vacunados con BCG, siendo 184 niños sanos sin evidencia clínica o epidemiológica de infección por el M. tuberculosis, y 11 niños con infección, definida conforme a criterios clínicos, radiológicos y laboratoriales. Se obtuvo una muestra de sangre de cada niño, que fue procesada conforme a las instrucciones del fabricante. El desempeño del ensayo fue evaluado mediante una curva de características operacionales (curva ROC). Resultados: En el grupo de 184 niños no infectados, 130 (70,6%) tenían menos que cuatro años (promedio de 35 meses). En este grupo control, 177 niños (96,2%) tuvieron un resultado negativo de la prueba, mientras que 6 niños (3,2%) presentaron resultado indeterminado, y un niño (0,5%) tuvo un resultado positivo. En el grupo de 11 niños con infección, el promedio de edad era de 58,5 meses, y 2 niños (18%) presentaron resultado negativo. La curva ROC determinó un área bajo la curva de 0,876 (95%IC 0,82-0,92; p<0,001), evidenciando un valor predictivo positivo del 81,8% para la prueba (95%IC 46,3-97,4). La sensibilidad de la prueba fue de 81,8% (95%IC, 48,2-97,2) y la especificidad de 98,8% (95%IC, 96-99,8). Conclusión: En el presente estudio, el desempeño del QuantiFERON-TB Gold in Tube para el diagnóstico de la infección por el M. tuberculosis fue adecuado cuando utilizado en una población pediátrica joven. .

Objetivo: Avaliar a acurácia de um teste de liberação de interferon-gama (QuantiFERON-TB Gold in Tube) para diagnosticar a infecção pelo Mycobacterium tuberculosis em uma população pediátrica. Métodos : Avaliaram-se 195 crianças previamente vacinadas com BCG, sendo 184 sadias, sem evidência clínica ou epidemiológica de infecção pelo M. tuberculosis, e 11 com infecção, definida de acordo com critérios clínicos, radiológicos e laboratoriais. Obteve-se uma amostra de sangue de cada criança, processada conforme as instruções do fabricante. Avaliou-se o desempenho do ensaio por meio de uma curva de características operacionais (curva Receiver Operating Characteristic - ROC). Resultados : No grupo de 184 crianças não infectadas, 130 (70,6%) eram menores de quatro anos (média de 35 meses). Nesse grupo, 177 crianças (96,2%) tiveram resultado negativo do teste, seis (3,2%) apresentaram resultado indeterminado e uma (0,5%) teve resultado positivo. No grupo de 11 crianças com infecção, a idade média era de 58,5 meses e duas (18%) apresentaram resultado negativo. A curva ROC determinou uma área sob a curva de 0,88 (IC95% 0,82-0,92; p<0,001), evidenciando um valor preditivo positivo de 81,8% para o teste (IC95% 46,3-97,4). A sensibilidade do teste foi de 81,8% (IC95% 48,2-97,2) e a especificidade, de 98,8% (IC95% 96-99,8). Conclusões: No presente estudo, o desempenho do QuantiFERON-TB Gold in Tube para o diagnóstico da infecção pelo M. tuberculosis foi adequado quando utilizado em uma população pediátrica jovem. .

USEFULNESS OF kDNA PCR IN THE DIAGNOSIS OF VISCERAL LEISHMANIASIS REACTIVATION IN CO-INFECTED PATIENTS / Utilidade da kDNA PCR no diagnostico de reativacao de leishmaniose visceral em pacientes co-infectados sintomaticos

SUMMARY It is important to develop new methods for diagnosing relapses in the co-infection of visceral leishmaniasis (VL) and HIV to enable earlier detection using less invasive methods. We report a case of a co-infected patient who had relapses after VL treatment, where the qualitative kDNA PCR showed a good performance. The kDNA PCR seems to be a useful tool for diagnosing VL and may be a good marker for predicting VL relapses after treatment of co-infected patients with clinical symptoms of the disease. .

RESUMO É importante a pesquisa de novos métodos laboratoriais para o diagnóstico de recidivas em casos de co-infecção leishmaniose visceral (LV) e vírus da imunodeficiência humana (HIV), que permitam o diagnóstico precoce das recidivas, utilizando métodos menos invasivos. Descrevemos aqui, o caso de paciente co-infectada que apresentou recidivas após o tratamento da LV e onde a PCR qualitativa demonstrou bom desempenho. A kDNA PCR parece ser ferramenta útil para o diagnóstico de recidivas de LV após o tratamento em pacientes co-infectados com sintomas clínicos da doença. .

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%). .

Interleukin 10 (IL10) and transforming growth factor 1 (TGF1) gene polymorphisms in persistent IgE-mediated cow's milk allergy

OBJECTIVES: The aim of this cross-sectional study was to evaluate whether interleukin 10 (IL10) and transforming growth factor β1 (TGFβ1) gene polymorphisms were associated with persistent IgE-mediated cow's milk allergy in 50 Brazilian children. The diagnostic criteria were anaphylaxis triggered by cow's milk or a positive double-blind, placebo-controlled food challenge. Tolerance was defined as the absence of a clinical response to a double-blind, placebo-controlled food challenge or cow's milk exposure. METHOD: The genomic DNA of the 50 patients and 224 healthy controls (HCs) was used to investigate five IL10 gene polymorphisms (-3575A/T, -2849A/G, -2763A/C, -1082G/A, -592C/A) and one TGFβ1 polymorphism (-509C/T). RESULTS: Among the five IL10 polymorphisms analyzed, homozygosis for the G allele at the -1082 position was significantly higher in the patients compared with the healthy controls (p = 0.027) and in the persistent cow's milk allergy group compared with the healthy controls (p = 0.001). CONCLUSIONS: Homozygosis for the G allele at the IL10 -1082G/A polymorphism is associated with the persistent form of cow's milk allergy. .

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.

Factors associated with hyperglycemia and low insulin levels in children undergoing cardiac surgery with cardiopulmonary bypass who received a single high dose of methylprednisolone

OBJECTIVES: Administering steroids before cardiopulmonary bypass in pediatric heart surgery modulates systemic inflammatory response syndrome and improves postoperative recovery. However, the use of steroids aggravates hyperglycemia, which is associated with a poor prognosis. Adult patients with systemic inflammatory response syndrome usually evolve with hyperglycemia and high insulin levels, whereas >90% of pediatric patients exhibit hyperglycemia and low insulin levels. This study aims to determine: A) the metabolic and inflammatory factors that are associated with hyperglycemia and low insulin levels in children who underwent cardiac surgery with cardiopulmonary bypass and who received a single high dose of methylprednisolone and B) the best predictors of insulin variation using a mathematical model. METHODS: This preliminary study recruited 20 children who underwent heart surgery with cardiopulmonary bypass and received methylprednisolone (30 mg/kg) immediately after anesthesia. Among the 20 patients initially recruited, one was excluded because of the absence of hyperglycemia and lower insulin levels after surgery. However, these abnormalities were confirmed in the remaining 19 children. The C-peptide, CRP, IL-6, and adrenomedullin levels were measured before surgery, immediately after cardiopulmonary bypass, and on the first, second, and third days after cardiac surgery. RESULTS: IL-6, CRP, and adrenomedullin increments were observed, whereas the C-peptide levels remained within reference intervals. CONCLUSION: The multiple regression model demonstrated that in addition to age and glycemia (two well-known factors that are directly involved in glucose metabolism), adrenomedullin and IL-6 levels were independent factors associated with lower insulin concentrations. These four parameters were responsible for 64.7% of the observed insulin variances. In addition, the fact that C-peptide levels did not fall together with insulin could have grounded the medical decision not to administer insulin to patients.

Longitudinal study of Cystatin C in healthy term newborns

OBJECTIVE: The purpose of this study was to determine the levels of Cystatin C in healthy term newborns in the first month of life. INTRODUCTION: Cystatin C may be a suitable marker for determining the glomerular filtration rate because it is not affected by maternal renal function. METHODS: Cohort study. Inclusion: term newborns with appropriate weight; mother without renal failure or drugs that could affect fetal glomerular filtration rate. Exclusion: malformations; hypertension or any condition that could affect glomerular filtration rate. Cystatin C (mg/L)and creatinine (rng/dl) were determined in the mother (Mo) and in the newborn at birth (Day-0), 3rd (Day-3), 7th(Day-7) and 28t>h(Day-28) days. Statistics: one way ANOVA and Pearson's correlation tests. Sample size of 20 subjects for a = 5 percent and a power test = 80 percent (p<0.05). RESULTS: Data from 21 newborns were obtained (mean + standard deviation): MoCystatin C=1.00 ± 0.20; Day-0 Cystatin C 1.70 ± 0.26; Day-3 Cystatin C = 1.51±0.20; Day-7 Cystatin C = 1.54 ± 0.10; Day-28 Cystatin C= 1.51±0.10. MoCystatin C was smaller than Day-0 Cystatin C (p<0.001), while MoCreatinine was not different from Day-0 Creatinine. Cystatin C only decreased from Day-0 to Day-3 (p = 0.004) but newborns Creatinine decreased along the time. Correlations were obtained between MoCystatin C and MoCreatinine (p = 0.012), as well as Day-3 (p = 0.047) and Day-28 (p = 0.022) Cystatin C and Creatinine values. CONCLUSION: Neonatal Cystatin C values were not affected by MoCystatin C and became stable from the 3rd day of life.

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.

Application of the pediatric risk of mortality (PRISM) score and determination of mortality risk factors in a tertiary pediatric intensive care unit

INTRODUCTION: To establish disease severity at admission can be performed by way of the mortality prognostic. Nowadays the prognostic scores make part of quality control and research. The Pediatric Risk of Mortality is one of the scores used in the pediatric intensive care units. OBJECTIVES: The purpose of this study is the utilization of the pediatric risk of mortality to determine mortality risk factors in a tertiary pediatric intensive care units. METHODS: Retrospective cohort study, in a period of one year, at a general tertiary pediatric intensive care unit. The pediatric risk of mortality scores corresponding to the first 24 hours of hospitalization were recorded; additional data were collected to characterize the study population. RESULTS: 359 patients were included; the variables that were found to be risk factors for death were multiple organ dysfunction syndrome, mechanical ventilation, use of vasoactive drugs, hospital-acquired infection, parenteral nutrition and duration of hospitalization (p < 0,0001). Fifty-four patients (15 percent) died; median pediatric risk of mortality score was significantly lower in patients who survived (p=0,0001). The ROC curve yielded a value of 0.76 (CI 95 percent 0,69-0,83) and the calibration was shown to be adequate. DISCUSSION: It is imperative for pediatric intensive care units to implement strict quality controls to identify groups at risk of death and to ensure the adequacy of treatment. Although some authors have shown that the PRISM score overestimates mortality and that it is not appropriate in specific pediatric populations, in this study pediatric risk of mortality showed satisfactory discriminatory performance in differentiating between survivors and non-survivors. CONCLUSIONS: The pediatric risk of mortality score showed adequate discriminatory capacity and thus constitutes a useful tool for the assessment of prognosis for pediatric patients admitted to a tertiary pediatric intensive care units.

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.

Mediadores pró-inflamatórios e antinflamatórios na sepse neonatal: associação entre homeostase e evolução clínica / Pro-inflammatory and anti-inflammatory mediators in neonatal sepsis: association between homeostasis and clinical outcome

OBJETIVO: avaliar a utilidade de citocinas pró-inflamatórias (TNF-±, IL-1² e IL-6) e de citocinas anti-inflamatórias (IL-10 e IL-1Ra) no diagnóstico da sepse neonatal, e verificar se a homeostase entre estes mediadores poderia ser determinante para a evolução clínica da doença. MÉTODO: coorte prospectiva compreendendo 31 recém-nascidos (RN) com diagnóstico de sepse neonatal, classificados em dois grupos: sepse e sepse grave, com evolução complicada (choque, falência múltipla de órgãos, óbito). Os níveis séricos de TNF-±; IL-1²; IL-6; IL-10 e IL-1Ra foram mensurados nos dias 0 (diagnóstico), 3 e 7 (evolutivos)...

Objectives: to evaluate the utility of pro-inflammatory cytokines (TNF-a, IL1-b, and IL-6) and anti-inflammatory cytokines (IL-10 and IL-1Ra) for the diagnosis of neonatal sepsis, and toverify if the homeostasis of these mediators might determine the clinical outcome. Method:prospective cohort study including 31 newborns with neonatal sepsis whose diagnosis wasmade on the basis of clinical signs and positive blood culture, or high C-reactive protein. Newbornswere classified in two groups: sepsis and favorable outcome, and severe sepsis with unfavorableoutcome (septic shock and/or DIVC and/or FMOS and/or death). On days 0 (diagnosis), 3 and 7after diagnosis, serum levels of TNF-a, IL-1b, IL-6, IL-10, and IL-1Ra were measured...

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.

Diagnosis of neonatal group B Streptococcus sepsis by nested-PCR of residual urine samples

Group B streptococcus (GBS) remains the most common cause of early-onset sepsis in newborns. Laboratory gold-standard, broth culture methods are highly specific, but lack sensitivity. The aim of this study was to validate a nested-PCR and to determine whether residue volumes of urine samples obtained by non invasive, non sterile methods could be used to confirm neonatal GBS sepsis. The nested-PCR was performed with primers of the major GBS surface antigen. Unavailability of biological samples to perform life supporting exams, as well as others to elucidate the etiology of infections is a frequent problem concerning newborn patients. Nevertheless, we decided to include cases according to strict criteria: newborns had to present with signs and symptoms compatible with GBS infection; at least one of the following biological samples had to be sent for culture: blood, urine, or cerebrospinal fluid; availability of residue volumes of the samples sent for cultures, or of others collected on the day of hospitalization, prior to antibiotic therapy prescription, to be analyzed by PCR; favorable outcome after GBS empiric treatment. In only one newborn GBS infection was confirmed by cultures, while infection was only presumptive in the other three patients (they fulfilled inclusion criteria but were GBS-culture negative). From a total of 12 biological samples (5 blood, 3 CSF and 4 urine specimen), eight were tested by culture methods (2/8 were positive), and 8 were tested by PCR (7/8 were positive), and only 4 samples were simultaneously tested by both methods (1 positive by culture and 3 by PCR). In conclusion, although based on a restricted number of neonates and samples, our results suggest that the proposed nested-PCR might be used to diagnose GBS sepsis as it has successfully amplified the three types of biological samples analyzed (blood, urine and cerebrospinal fluid), and was more sensitive than culture methods as PCR in urine confirmed diagnosis...

O estreptococo do grupo B (GBS) constitui a causa mais freqüente de sepse neonatal precoce. O teste de referência continua sendo o isolamento em cultura, apesar de apresentar problemas de sensibilidade. O objetivo do presente estudo foi validar uma técnica de dupla amplificação e determinar a possibilidade do uso de amostras residuais de urina colhidas por método não invasivo, não estéril, para a confirmação da sepse por GBS em recém-nascidos. As amostras foram amplificadas com primers do principal gene de superfície do GBS. A insuficiência de volume de material biológico para a realização de exames para suporte de vida, além de outros necessários à identificação do agente etiológico de infecções é muito freqüente em recém-nascidos. Mesmo assim, decidimos definir critérios bastante rigorosos para a inclusão de pacientes na casuística: os recém-nascidos deveriam apresentar sinais e sintomas compatíveis com infecção pelo GBS; deveriam ter tido ao menos uma amostra enviada para cultura, podendo ser sangue, urina ou líquor; disponibilidade de volumes residuais dessas amostras, ou de outras colhidas no dia da hospitalização, antes da introdução da antibioticoterapia, de forma a possibilitar a análise por PCR, e evolução favorável com a antibioticoterapia empírica. Em apenas um dos quatro recém-nascidos a infecção foi confirmada por cultura, enquanto nos outros três casos a infecção foi considerada presuntiva (pacientes preencheram os critérios de inclusão, mas o GBS não foi isolado). De um total de 12 amostras dos quatro pacientes (5 de sangue, 3 de líquor e 4 de urina), 8 foram testadas por cultura (2 foram positivas), 8 foram testadas por PCR (7 foram positivas), e apenas 4 pelos dois métodos simultaneamente (1 positiva por cultura e 3 por PCR). Concluímos que apesar do número restrito de pacientes e de amostras testadas, os resultados apresentados sugerem que a amplificação proposta poderia ser usada para o diagnóstico...