Identification of toxocara canis antigens by Western blot in experimentally infected rabbits

Toxocariasis is a frequent helminthiasis that can cause visceral and ocular damage in humans specially in children. The identification of specific antigens of Toxocara canis is important in order to develop better diagnostic techniques. Ten rabbits were infected orally with a dose of 5000 Toxocara canis embryonated eggs. Rabbits were bled periodically and an ELISA assay was performed to determine levels of specific Toxocara IgG antibodies. ELISA detected antibodies at day 15 after infection. Western blot (WB) assay was performed using excretory/secretory antigens (E/S) of T. canis second stage larvae. Different antigen concentrations were evaluated: 150, 200, 250 and 300 µg/mL. The concentration of 250 µg/mL was retained for analysis. Rabbit sera were diluted 1:100. Secondary antibody was used at a dilution of 1:1000. Results of WB indicated that in the first month after infection specific antibodies against the 200 KDa, 116 KDa, 92 KDa and 35 KDa antigens were detected; antibodies against the 92 KDa, 80 KDa, 66 KDa, 45 KDa, 31 KDa and 28 KDa antigens appeared later. All positive sera in the ELISA test were also positive in WB. Two antigen bands, 92 KDa and 35 KDa, were identified since the beginning and throughout the course of infection. These antigens merit further evaluation as candidates for use in diagnosis.

EVALUATION OF A RAPID SCREENING ASSAY FOR BACTERIAL IDENTIFICATION (DOT-ELISA) IN FECAL SAMPLES FROM CHILDREN

With the objective of standardizing a Dot Enzyme-Linked Immunosorbent Assay (Dot-ELISA) to detect antigens of fecal bacterial enteropathogens, 250 children, aged under 36 months and of both sexes, were studied; of which 162 had acute gastroenteritis. The efficacy of a rapid screening assay for bacterial enteropathogens (enteropathogenic Escherichia coli "EPEC", enteroinvasive Escherichia coli "EIEC", Salmonella spp. and Shigella spp.) was evaluated. The fecal samples were also submitted to a traditional method of stool culture for comparison. The concordance index between the two techniques, calculated using the Kappa (k) index for the above mentioned bacterial strains was 0.8859, 0.9055, 0.7932 and 0.7829 respectively. These values express an almost perfect degree of concordance for the first two and substantial concordance for the latter two, thus enabling this technique to be applied in the early diagnosis of diarrhea in infants. With a view to increasing the sensitivity and specificity of this immunological test, a study was made of the antigenic preparations obtained from two types of treatment: 1) deproteinization by heating; 2) precipitation and concentration of the lipopolysaccharide antigen (LPS) using an ethanol-acetone solution, which was then heated in the presence of sodium EDTA

Genotypic identification of Cryptosporidium spp. isolated from hiv-infected patients and immunocompetent children of São Paulo, Brazil

Cryptosporidium isolates identified in fourteen stool samples, collected from five HIV-infected patients and nine immunocompetent children, living in the Sate of São Paulo, Brazil, were submitted to a molecular analysis using a nested PCR followed of restriction fragment length polymorphism (RFLP), for genetic characterization. The analysis was based on digestion with RsaI restriction enzyme of a DNA fragment amplified from the Cryptosporidium oocyst wall protein (COWP) gene. Based on this analysis, four samples were identified as Cryptosporidium parvum, eight as Cryptosporidium hominis and two presented a profile that correspondedto Cryptosporidium meleagridis when compared to the standards used in the analysis. The use of molecular methods can be helpful to identify source of infections and risk factors related to Cryptosporidium infection in our communities.

Current status of herpesvirus identification in the oral cavity of HIV-infected children

INTRODUCTION: Some viruses of the Herpesviridae family are frequently the etiologic agents of oral lesions associated with HIV. The aim of this study was to identify the presence of herpes simplex virus types 1 and 2 (HSV-1, HSV-2), Varicella Zoster virus (VZV), Epstein-Barr virus (EBV), human cytomegalovirus (HCMV), human herpesvirus type 6, type 7 and type 8 (HHV-6, HHV-7 and HHV-8) in the oral cavity of HIV-infected children/adolescents and verify the association between viral subtypes and clinical factors. METHODS: The cells of oral mucosa were collected from 50 HIV infected children/adolescents, 3-13 years old (mean age 8.66). The majority (66%) of selected were girls, and they were all outpatients at the pediatric AIDS clinic of a public hospital in Rio de Janeiro. Nested-PCR was used to identify the viral types. RESULTS: Absence of immunosuppression was observed in 66% of the children. Highly active antiretroviral therapy (HAART) was used by 72.1% of selected and moderate viral load was observed in 56% of the children/adolescents. Viral types were found in 86% of the children and the subtypes were: HSV-1 (4%), HSV-2 (2%), VZV (4%), EBV (0%), HCMV (24%), HHV6 (18%), HHV-7 (68%), HHV8 (0%). CONCLUSIONS: The use of HAART has helped to reduce oral lesions, especially with herpes virus infections. The health professionals who work with these patients should be aware of such lesions because of their predictive value and the herpes virus can be found circulating in the oral cavity without causing lesions.

Isolation and identification of Adenovirus in hospitalized children, under five years, with acute respiratory disease, in Havana, Cuba

Nine Adenovirus (Ad) strains isolated in Cuba, from 128 nasopharingeal swab specimens of children below five years old, with acute respiratory diseases, during 1996 and 1997, were studied by restriction enzyme analysis of genomic DNA with two endonucleases BamH I and Sma I. All different fragment patterns were compared with the respective prototypes. The identified adenoviruses were Ad 1 (n=4), Ad 2 (n=1) and Ad 6 (n=4). Males were more frequently infected than females. The analysis of the occurrence of these Adenovirus strains of subgenus C revealed that Ad 1 and Ad 6 were the predominant serotypes in 1996 and in 1997, respectively.

Assessment of a strategy for the control of respiratory diseases in children

A programme for the control of respiratory diseases in children was conceived for the State of S. Paulo, Brazil, in 1986. Its progress thereafter and the epidemiology of the diseases concerned are examined. Apart from an inquiry into the 64 existing State local health authorities, a sample of 18,255 cases of children assisted by the programme at different levels, including both in-patient and outpatient care, is analysed. Each case record included information about identification (child, doctor and health facility), reasons for calling, diagnoses made and outcome of treatment. Further data were also sought from hospitals and from State mortality records. The programme was found to be poorly implemented in the State but, where implemented, it showed itself capable of resolving problems (only 0.5% of the cases could not be handled) as also of changing ongoing trends (more than 50% reduction in hospital admission rates). Individual assessment of each item of the programme indicated its bottlenecks. Regarding the epidemiology of respiratory diseases, it is observed that the major burden to health services comes from children aged less than five, and that the most important diseases are wheezing illnesses and pneumonia. Morevoer, they were found to be significantly associated (p = 0.000) so that a child in the community presenting wheezing diseases is 5 times more likely to develop pneumonia than a child with any other respiratory diagnosis. Similarly, among the under five deaths it was found that the risk for pneumonia is 3 times greater for children who died presenting wheezing diseases than it is for children with any other sort of diagnosis. In conclusion, the programme is deemed to be efficient and effective but its efficacy is marred by administrative flaws. The successful control of respiratory problems in childhood is related to a proper appreciation of the importance of wheezing diseases.

Isolation and serological identification of enteropathogenic Escherichia coli in pasteurized milk in Brazil

OBJECTIVE: To evaluate the microbiological quality of pasteurized milk commercialized in Rio de Janeiro, Brazil, and determine serologically enteropathogenic Escherichia coli (EPEC) strains in E. coli isolates obtained from milk samples. METHODS: Ninety samples of pasteurized milk -- types B and C -- of three different commercial brands, purchased in supermarkets and bakeries in Rio de Janeiro, were examined. The amount of total and fecal coliform bacteria was estimated using the Most Probable Number technique. Mesophilic, psychrotrophic, and thermoduric microorganism counts were determined by the Standard Plate Count technique. Isolation and identification of E. coli were carried out using conventional physiological tests. Commercial antisera were used for serological characterization of EPEC. RESULTS: The three milk brands analyzed revealed bacterial counts above the regulated values of the Brazilian government. It was found that among 208 strains of E. coli isolated, 46 (22.1%) were serologically classified as EPEC. The most common EPEC serogroup was O55 (15.2%). CONCLUSIONS: Though recent studies on virulence factors indicate that not all strains serologically classified as EPEC are able to attaching/effacing lesion, it is believed that the isolation of EPEC serogroups from pasteurized milk represent a potential risk for children, as well as an indicative of the presence of other enteropathogens.

Identification of respiratory virus in infants with congenital heart disease by comparison of different methods

Respiratory virus infections are the main cause of infant hospitalization and are potentially severe in children with congenital heart disease (CHD). Rapid and sensitive diagnosis is very important to early introduction of antiviral treatment and implementation of precautions to control transmission, reducing the risk of nosocomial infections. In the present study we compare different techniques in the diagnosis of respiratory viruses in CHD infants. Thirty-nine samples of nasopharyngeal aspirate were obtained from CHD infants with symptoms of respiratory infection. The Multiplex PCR (Seeplex® RV 12 ACE Detection) driven to the detection of 12 respiratory viruses was compared with the direct immunofluorescence assay (DFA) and PCR, both targeting seven respiratory viruses. The positivity found by DFA, Multiplex and PCR was 33.3%, 51.3% and 48.7%, respectively. Kappa index comparing DFA and Multiplex, DFA and PCR and PCR and Multiplex PCR was 0.542, 0.483 and 0.539, respectively. The concordance between techniques was considered moderate. Both Multiplex PCR (p = 0.001) and PCR (p = 0.002) detected significantly more respiratory virus than DFA. As the performance of the tests may vary, the combination of two or more techniques may increase diagnostic sensitivity favoring the diagnosis of co-infections, early introduction of antiviral therapy and implementation of appropriate measures.

FUNGEMIA CAUSED BY Candida SPECIES IN A CHILDREN'S PUBLIC HOSPITAL IN THE CITY OF SÃO PAULO, BRAZIL: STUDY IN THE PERIOD 2007-2010

Candidemia remains a major cause of morbidity and mortality in the health care environment. The epidemiology of Candida infection is changing, mainly in relation to the number of episodes caused by species C. non-albicans. The overall objective of this study was to evaluate the frequency of yeasts of the genus Candida, in a four-year period, isolated from blood of pediatric patients hospitalized in a public hospital of the city of São Paulo, Brazil. In this period, yeasts from blood of 104 patients were isolated and, the identified species of Candida by phenotypic and genotypic methods were: C. albicans (39/104), C. tropicalis (25/104), C. parapsilosis (23/104), Pichia anomala (6/104), C. guilliermondii (5/104), C. krusei (3/104), C. glabrata (2/104) and C. pararugosa (1/104). During the period of the study, a higher frequency of isolates of C. non-albicans (63.55%) (p = 0.0286) was verified. In this study we verified the increase of the non-albicans species throughout the years (mainly in 2009 and 2010). Thus, considering the peculiarities presented by Candida species, a correct identification of species is recommended to lead to a faster diagnosis and an efficient treatment.

Prevalence of intestinal parasites in preschool children in the region of Uberlândia, State of Minas Gerais, Brazil

INTRODUCTION: Children are an important high-risk group for helminth and protozoa infections. Daycare centers are environments where children have proven to be more susceptible to acquiring intestinal parasites. Thus, the purpose of this study was to verify the prevalence of intestinal parasites in children who attended the two daycare centers maintained by the local government of Uberlândia, State of Minas Gerais, Brazil. METHODS: Fecal samples were collected from 133 children (73 children at the Public Preschool for Early Childhood Education, PPECE A, and 60 at the PPECE B) following identification according to sex and age and agreement to participate by parents or guardians who signed the free, informed consent form. The samples were examined by the Lutz method. RESULTS: Coproparasitological tests performed on 133 children showed that 29.3% of them were parasitized for enteroparasites or commensals, 6.7% of the children presented polyparasitism. Among the protozoa, Giardia lamblia were the most prevalent and Hymenolepis nana were the most frequent among the helminths. CONCLUSIONS: Thus, analysis of the results showed that intestinal parasites still represent a public health problem, especially among children and in areas where the socioeconomic and educational conditions are less favorable.

Premature thelarche: identification of clinical and laboratory data for the diagnosis of precocious puberty

PURPOSE: Two groups of girls with premature breast development were studied retrospectively. We tried to identify clinical, radiological, and hormonal parameters that could distinguish between a benign, nonprogressive premature thelarche and a true precocious puberty. METHODS: The clinical outcome of 88 girls with breast enlargement before 6.1 years of age was analyzed. Taking into account the progression of their sexual maturation, we allocated the children into 2 groups: "Isolated Premature Thelarche" (n = 63) and "Precocious Puberty" (n = 25) groups. Chronological and bone ages, height and growth velocity centiles, computerized tomography of hypothalamus-pituitary area, pelvic ultrasonography, gonadotropin response to luteinizing hormone-releasing hormone stimulation as well as basal levels of luteinizing hormone, follicle-stimulating hormone, estradiol, and prolactin were studied in both groups. Statistical analysis were performed using the Student t test to compare the sample means. Fisher's exact test and chi² test were used to analyze the nonparametric variables. RESULTS: Isolated premature thelarche most frequently affected girls younger than 2 years who presented exaggerated follicle-stimulating hormone response to luteinizing hormone-releasing hormone stimulation test. The precocious puberty group had higher initial stature, accelerated growth rate and bone age, increased uterine and ovarian volumes, high spontaneous luteinizing hormone levels by immunofluorimetric assay, as well as a high luteinizing hormone response and peak luteinizing hormone/follicle-stimulating hormone ratio after luteinizing hormone-releasing hormone stimulation. CONCLUSION: At initial presentation, girls who undergo true precocious puberty present advanced bone age, increased uterine and ovarian volumes in addition to breast enlargement, as well as an luteinizing hormone-predominant response after a luteinizing hormone-releasing hormone stimulation test.

Hypophosphatemia in critically ill children

The purpose of this paper is to review clinical studies on hypophosphatemia in pediatric intensive care unit patients with a view to verifying prevalence and risk factors associated with this disorder. We searched the computerized bibliographic databases Medline, Embase, Cochrane Library, and LILACS to identify eligible studies. Search terms included critically ill, pediatric intensive care, trauma, sepsis, infectious diseases, malnutrition, inflammatory response, surgery, starvation, respiratory failure, diuretic, steroid, antiacid therapy, mechanical ventilation. The search period covered those clinical trials published from January 1990 to January 2004. Studies concerning endocrinological disorders, genetic syndromes, rickets, renal diseases, anorexia nervosa, alcohol abuse, and prematurity were not included in this review. Out of 27 studies retrieved, only 8 involved pediatric patients, and most of these were case reports. One clinical trial and one retrospective study were identified. The prevalence of hypophosphatemia exceeded 50%. The commonly associated factors in most patients with hypophosphatemia were refeeding syndrome, malnutrition, sepsis, trauma, and diuretic and steroid therapy. Given the high prevalence, clinical manifestations, and multiple risk factors, the early identification of this disorder in critically ill children is crucial for adequate replacement therapy and also to avoid complications.

Virulence markers and antimicrobial susceptibility of bacteria of the Bacteroides fragilis group isolated from stool of children with diarrhea in São Paulo, Brazil

Bacteroides fragilis has been isolated from several human and non-human monomicrobial and mixed infections. In this study, some virulence markers and the antimicrobial susceptibility of bacteria of the B. fragilis group isolated from children's stools were evaluated. All the 64 isolates showed the following characteristics: capsulated, beta-hemolytic, hydrophilic, and serum-resistant. Only, 24 (37.5%) strains were resistant at 60ºC, for 30 min, and among them, 12 (18.75%) were resistant at 60ºC, for 60 min. Also, none strain was resistant at 100ºC. Four strains were able to hemagglutinate erythrocytes and D-mannose, D-galactose, D-arabinose, and D-xylose inhibited hemagglutination in 2 B. fragilis strains (p76a, p76b). The hemagglutination in the strain B. uniformis p3-2 was inhibited by D-xylose and D-galactose. The bft gene detection and the enterotoxin production were observed only in 13 EF-enterotoxigenic species. Fragilysin activity was confirmed on HT-29 cells. The antimicrobial determination confirmed that both imipenem and metronidazole were efficient against B. fragilis species; all the strains were resistant to lead and nickel. Plasmids of 2.9, 4.4, 4.8, and 8.9 kb were observed in 6 tested strains. These results show the values of the species identification from clinical infections, as well as of the periodic evaluation of the resistance patterns of the B. fragilis group at Brazilian medical institutions.

Frequency of viruses associated with acute respiratory infections in children younger than five years of age at a locality of Mexico City

A locality in the district of Tlalpan, Mexico City, was selected in order to identify the viral agents in children younger than 5 years of age with acute respiratory infection (ARI). A total of 300 children were randomly selected and were included in this study for a period of 13 months. During this period nasopharyngeal exudates were collected for the isolation of viral agents. Monoclonal fluorescent antibodies were used for viral identification after cell culture. Viral infection was detected in 65% of the specimens. The respiratory syncytial virus (RSV) was the most common virus agent detected. Children required an average of two consultations during the study period. Two high incidence peaks were observed, one during the summer and the other during winter; the most frequent viruses during these seasons were influenza A and RSV, respectively. The largest number of viruses was isolated in the group of children between 1 and 2 years of age and in the group between 4 and 5 years of age. This study demonstrated the presence of ARI and of different viruses in a period of 13 months, as well as the most frequent viruses in children younger than 5 years of age from a community of Mexico City.

Detection of diarrheagenic Escherichia coli from children with and without diarrhea in Salvador, Bahia, Brazil

We identified different diarrheagenic (DEC) Escherichia coli pathotypes isolated from 1,207 children with and without acute endemic diarrhea in Salvador, Bahia, Brazil collected as part of a case-control study. Since the identification of DEC cannot be based on only biochemical and culture criteria, we used a multiplex polymerase chain reaction developed by combining five specific primer pairs for Enteropathogenic Escherichia coli (EPEC), Shiga toxin-producing E. coli/ Enterohaemorrhagic E. coli (STEC/EHEC), Enterotoxigenic E. coli (ETEC) and Enteroaggregative E. coli (EAEC) to detect these pathotypes simultaneously in a single-step reaction. In order to distinguish typical and atypical EPEC strains, these were tested for the presence of EAF plasmid. The prevalence of diarrheagenic E. coli in this sample of a global case-control study was 25.4% (259 patients) and 18.7% (35 patients) in the diarrhea group (1,020 patients) and the control group (187 patients), respectively. The most frequently isolated pathotype was EAEC (10.7%), followed by atypical EPEC (9.4%), ETEC (3.7%), and STEC (0.6%). Typical EPEC was detected only in one sample. The prevalence of the pathotypes studied in children with diarrhea was not significantly different from that in children without diarrhea.