[Show abstract][Hide abstract]ABSTRACT: Only a small percentage of individuals living in endemic areas develop severe malaria suggesting that host genetic factors may play a key role. This study has determined the frequency of single nucleotide polymorphisms (SNPs) in some pro and anti-inflammatory cytokine gene sequences: IL6 (-174; rs1800795), IL12p40 (+1188; rs3212227), IL4 (+33; rs2070874), IL10 (-3575; rs1800890) and TGFb1 (+869; rs1800470), by means of PCR-RFLP. Blood samples were collected from 104 symptomatic and 37 asymptomatic subjects. Laboratory diagnosis was assessed by the thick blood smear test and nested-PCR. No association was found between IL6 (-174), IL12p40 (+1188), IL4 (+33), IL10 (- 3575), TGFb1 (+869) SNPs and malaria symptoms. However, regarding the IL10 -3575 T/A SNP, there were significantly more AA and AT subjects, carrying the polymorphic allele A, in the symptomatic group (c2 = 4.54, p = 0.01, OR = 0.40 [95% CI - 0.17- 0.94]). When the analysis was performed by allele, the frequency of the polymorphic allele A was also significantly higher in the symptomatic group (c2 = 4.50, p = 0.01, OR = 0.45 [95% CI - 0.21-0.95]). In conclusion, this study has suggested the possibility that the IL10 - 3575 T/A SNP might be associated with the presence and maintenance of malaria symptoms in individuals living in endemic areas. Taking into account that this polymorphism is related to decreased IL10 production, a possible role of this SNP in the pathophysiology of malaria is also suggested, but replication studies with a higher number of patients and evaluation of IL10 levels are needed for confirmation.
Preview · Article · Apr 2016 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract]ABSTRACT: Host and parasite genotypes are among the factors associated with congenital toxoplasmosis pathogenesis. As HLA class II molecules play a key role in the immune system regulation, the aim of this study was to investigate whether HLA-DQA1/B1 alleles are associated with susceptibility or protection to congenital toxoplasmosis. One hundred and twenty-two fetuses with and 103 without toxoplasmosis were studied. The two study groups were comparable according to a number of socio-demographic and genetic variables. HLA alleles were typed by PCR-SSP. In the HLA-DQA1 region, the allele frequencies showed that *01:03 and *03:02 alleles could confer susceptibility (OR= 3.06, p = 0.0002 and OR= 9.60, p= 0.0001, respectively) as they were more frequent among infected fetuses. Regarding the HLA-DQB1 region, the *05:04 allele could confer susceptibility (OR = 6.95, p < 0.0001). Of the 122 infected fetuses, 10 presented susceptibility haplotypes contrasting with only one in the non-infected group. This difference was not statistically significant after correction for multiple comparison (OR = 9.37, p=0.011). In the casuistic, there were two severely damaged fetuses with high parasite loads determined in amniotic fluid samples and HLA-DQA1 susceptibility alleles. In the present study, a discriminatory potential of HLA-DQA1/B1 alleles to identify susceptibility to congenital toxoplasmosis and the most severe cases has been shown.
[Show abstract][Hide abstract]ABSTRACT: To monitor the bacterial load in newborns with proven infections on the day of admission, 48 h and 7 days after treatment.
Real-time PCR (qPCR) targeting the 16S rDNA.
The study recruited 17 newborns and the bacterial load was in general low (<50 CFU/mL). In three of four deaths, the bacterial load values increased, and in 11 of the 13 survivors the values decreased until the third evaluation.
Considering the extreme sensitivity and high negative predictive value of qPCR, this test could help to monitor the treatment of neonatal sepsis and to assist in medical decision to discontinue antibiotics.
Preview · Article · Sep 2015 · The journal of maternal-fetal & neonatal medicine: the official journal of the European Association of Perinatal Medicine, the Federation of Asia and Oceania Perinatal Societies, the International Society of Perinatal Obstetricians
[Show abstract][Hide abstract]ABSTRACT: Background
Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients.
DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures.
The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients.
Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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).
In the present study, the QuantiFERON-TB Gold in Tube performance for diagnosing M. tuberculosis infection was appropriate in a young pediatric population.
[Show abstract][Hide abstract]ABSTRACT: SUMMARYIt 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.
Full-text · Article · Dec 2013 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract]ABSTRACT: 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.
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).
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).
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 (âˆž), NLR (0.017), and Ef (99%).
Full-text · Article · Oct 2013 · Revista da Sociedade Brasileira de Medicina Tropical
[Show abstract][Hide abstract]ABSTRACT: 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.
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).
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).
Homozygosis for the G allele at the IL10 -1082G/A polymorphism is associated with the persistent form of cow's milk allergy.
[Show abstract][Hide abstract]ABSTRACT: BACKGROUND: For many years fluconazole has been commonly used to treat Candida infections. However, the indiscriminate use of this antimycotic therapy has favored the emergence of resistant isolates. Mutations in the ERG11 gene have been described as one of the primary mechanisms of resistance in Candida species. In this study we investigated missense mutations in ERG11 genes of C. albicans, C. glabrata and C. tropicalis isolates previously evaluated by susceptibility testing to fluconazole. METHODS: Screening for these mutations was performed on 19 Candida clinical isolates (eight C. albicans, five C. glabrata and six C. tropicalis) resistant and susceptible to fluconazole. The ERG11 gene was amplified by PCR with specific primers for each Candida species and analyzed by automated sequencing. RESULTS: We identified 14 different missense mutations, five of which had not been described previously. Among them, a new mutation L321F was identified in a fluconazole resistant C. albicans isolate and it was analyzed by a theoretical three-dimensional structure of the ERG11p. CONCLUSION: The L321F mutation in C. albicans ERG11 gene may be associated with fluconazole resistance.
[Show abstract][Hide abstract]ABSTRACT: 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.
Full-text · Article · Feb 2013 · Revista do Instituto de Medicina Tropical de São Paulo
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
IL-6, CRP, and adrenomedullin increments were observed, whereas the C-peptide levels remained within reference intervals.
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.
Full-text · Article · Jan 2013 · Clinics (São Paulo, Brazil)
[Show abstract][Hide abstract]ABSTRACT: Nutrition therapy (NT) is essential for the care of critically ill children. Inadequate feeding leads to malnutrition and may increase the patient's risk of morbidity and mortality. The aim of this study was to describe the NT used in a tertiary pediatric intensive care unit (PICU).
The authors evaluated NT administered to 90 consecutive patients who were hospitalized for 7 days in the PICU of Instituto da Criança, Hospital das Clínicas, Universidade de São Paulo, Brazil. NT was established according to the protocol provided by the institution's NT team. NT provided a balance of fluids and nutrients and was monitored with a weekly anthropometric nutrition assessment and an evaluation of complications.
NT was initiated, on average, within 72 hours of hospitalization. Most children (80%) received enteral nutrition (EN) therapy; of these, 35% were fed orally and the rest via nasogastric or postpyloric tube. There were gastrointestinal complications in patients (5%) who needed a postpyloric tube. Parenteral nutrition (PN) was used in only 10% of the cases, and the remaining 10% received mixed NT (EN + PN). The average calorie and protein intake was 82 kcal/kg and 2.7 g/kg per day. Arm circumference and triceps skinfold thickness decreased.
The use of EN was prevalent in the tertiary PICU, and few clinical complications occurred. There was no statistically significant change in most anthropometric indicators evaluated during hospitalization, which suggests that NT probably helped patients maintain their nutrition status.
Full-text · Article · Jul 2011 · Journal of Parenteral and Enteral Nutrition
[Show abstract][Hide abstract]ABSTRACT: The purpose of this study was to determine the levels of Cystatin C in healthy term newborns in the first month of life.
Cystatin C may be a suitable marker for determining the glomerular filtration rate because it is not affected by maternal renal function.
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% and a power test = 80% (p<0.05).
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.
Neonatal Cystatin C values were not affected by MoCystatin C and became stable from the 3rd day of life.
Full-text · Article · Feb 2011 · Clinics (São Paulo, Brazil)
[Show abstract][Hide abstract]ABSTRACT: The use of improved microbiological procedures associated with molecular techniques has increased the identification of Candida bloodstream infections, even if the isolation of more than one species by culture methods remains uncommon. We report the
cases of two children presenting with severe gastrointestinal disorders and other risk factors that contribute to Candida infections. In the first patient, C. albicans DNA was initially detected by a nested-amplification and C. tropicalis was found later during hospitalization, while blood cultures were persistently negative. In the second child, there was amplification
of C. albicans and C. glabrata DNA in the same samples, but blood cultures yielded only C. albicans. Both patients received antifungal therapy but had unfavorable outcomes. These two cases illustrate that PCR was more successful
than culture methods in detecting Candida in the bloodstream of high risk children, and was also able to detect the presence of more than one species in the same patient
that might impact therapy when the fungi are resistant to azole compounds.
Preview · Article · Dec 2010 · Medical mycology: official publication of the International Society for Human and Animal Mycology
[Show abstract][Hide abstract]ABSTRACT: 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.
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.
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.
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%) 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% 0,69-0,83) and the calibration was shown to be adequate.
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.
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.
Full-text · Article · Oct 2010 · Clinics (São Paulo, Brazil)
[Show abstract][Hide abstract]ABSTRACT: There is scarce information on the potential benefits of immunosuppression in children with myocarditis and viral genomes in myocardium. We investigated the occurrence of myocarditis in children with a preliminary diagnosis of dilated cardiomyopathy, the frequency of cardiotropic viruses in the myocardium, and the response to immunosuppression.
Thirty patients (nine months to 12 years) with left ventricular ejection fraction of 22.8 ± 4.1% were subjected to right cardiac catheterization and endomyocardial biopsy. Specimens were analyzed for the presence of inflammatory elements (Dallas criteria) and viral genome (polymerase chain reaction). Patients with active myocarditis received immunosuppressants (azatioprine and prednisone) and were re-catheterized nine months later. A historical control group of nine patients with myocarditis who did not receive immunosuppressants was included.
Active myocarditis was diagnosed in ten patients (five with viral genomes detected). Immunosuppression resulted in a significant increase in left ventricular ejection fraction from 25.2 ± 2.8% to 45.7 ± 8.6% (versus 20.0 ± 4.0% to 22.0 ± 9.0% in historical controls, p<0.01) and cardiac index from 3.28 ± 0.51 L/min/m(2) to 4.40 ± 0.49 L/min/m(2) (versus 3.50 ± 0.40 L/min/m(2) to 3.70 ± 0.50 L/min/m(2) in controls, p<0.01), regardless of the presence of viral genomes (p=0.98 and p=0.22, respectively for the two variables). No relevant clinical events were observed. Non-inflammatory cardiomyopathy was diagnosed in 20 patients (seven with viral genomes). While on conventional therapy, there were four deaths and three assignments to transplantation, and no improvement of left ventricular ejection fraction in the remaining ones (22.5 ± 3.6% to 27.5 ± 10.6%).
Children with chronic myocarditis seem to benefit from immunosuppressive therapy, regardless of the presence of viral genome in the myocardium.
No preview · Article · Nov 2009 · International journal of cardiology