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.

Respiratory syncytial virus groups A and B in Porto Alegre, Brazil, from 1990 to 1995 and 1998

We analyzed the respiratory syncytial virus (RSV) groups and their epidemiological pattern that were detected over the course of seven years in southern Brazil. The two RSV groups co-circulated each year, but frequencies of groups A and B varied both between and within yearly outbreaks. In 1991, group A predominated over group B (p=0.0016). RSV outbreaks analyzed showed a temperature-dependent pattern and no association with rainfall, similarly to other countries from southern South America. Knowledge of the variants is important in terms of both diagnosis and definition of a vaccine composition.

Respiratory syncytial virus infections during an epidemic period in Salvador, Brazil: viral antigenic group analysis and description of clinical and epidemiological aspects

Acute respiratory infections (ARI) caused by respiratory syncytial virus (RSV) were studied in 482 children from Salvador, BA, Brazil, over a period of 12 months. The epidemic period of RSV infections in Salvador occurred from February (summer) to August (winter), with peaks in May, June, and July. The grouping characteristics of 84 RSV present in nasopharyngeal secretions of children seen at a reference university hospital were analyzed. RSV represented 17.4% of all cases and 54.5% of the positive samples. Sixty-four RSV strains were assigned to group A and 14 to group B. Both groups circulated in the five months of the epidemic period studied. Infections by both groups of RSV were more frequent in children up to one year of age. The incidence of RSV ARI was slightly more frequent in males, although group B had more infected females.

Respiratory viruses in the pediatric intensive care unit: prevalence and clinical aspects

A survey was conducted in two pediatric intensive care units in hospitals in Porto Alegre, Brazil, in order to monitor the main respiratory viruses present in bronchiolitis and/or pneumonia and their involvement in the severity of viral respiratory infections. Viral respiratory infection prevalence was 38.7%. In bronchiolitis, respiratory syncytial virus (RSV) was detected in 36% of the cases. In pneumonia, the prevalence rates were similar for adenovirus (10.3%) and RSV (7.7%). There was a difference among the viruses detected in terms of frequency of clinical findings indicating greater severity. Frequency of crackles in patients with RSV (47.3%) showed a borderline significance (p = 0.055, Fisher's exact test) as compared to those with adenovirus (87.5%). The overall case fatality rate in this study was 2.7%, and adenovirus showed a significantly higher case fatality rate (25%) than RSV (2.8%) (p = 0.005). Injected antibiotics were used in 49% of the children with RSV and 60% of those with adenovirus. Adenovirus was not detected in any of the 33 children submitted to oxygen therapy.

Molecular epidemiology of human respiratory syncytial virus in Uruguay: 1985-2001 - A review

The variability of the G glycoprotein from human respiratory syncytial viruses (HRSV) (groups A and B) isolated during 17 consecutive epidemics in Montevideo, Uruguay have been analyzed. Several annual epidemics were studied, where strains from groups A and B circulated together throughout the epidemics with predominance of one of them. Usually, group A predominates, but in some epidemics group B is more frequently detected. To analyse the antigenic diversity of the strains, extracts of cells infected with different viruses of group A were tested with a panel of anti-G monoclonal antibodies (MAbs). The genetic variability of both groups was analyzed by sequencing the C-terminal third of the G protein gene. The sequences obtained together with previously published sequences were used to perform phylogenetic analyses. The data from Uruguayan isolates, together with those from the rest of the world provide information regarding worldwide strain circulation. Phylogenetic analyses of HRSV from groups A and B show a model of evolution analogous to the one proposed for influenza B viruses providing information that would be beneficial for future immunization programs and to design safe vaccines.

Therapeutic failure of praziquantel in the treatment of Schistosoma haematobium infection in Brazilians returning from Africa

Several cases of therapeutic failure of praziquantel used for the treatment of urinary schistosomiasis have been reported. Alternative drugs, like niridazol and metrifonate, have shown a lower therapeutic effect and more side effects than praziquantel. Twenty-six Brazilian military men (median age of 29 years) with a positive urine parasitological exam who were part of a United Nation peace mission in Mozambique in 1994 were treated with 40 mg/kg body weight praziquantel, single dose. They swimmed in Licungo river (Mocuba city, Mozambique) during the weekends. After this, they presented haematuria, dysuria, polakiuria, and lumbar pain. Control cystoscopy examinations carried out between 6 and 24 months after each treatment (including two additional treatments at a minimum interval of 6 months) revealed the presence of viable eggs. Granulomas in the vesical submucosa were observed in 46.2% (12/26) of the individuals. A vesical biopsy confirmed the presence of granulomas in all of these patients and the presence of viable eggs in 34.3% (9/26) of individuals who no longer excreted eggs in urine. The eggs filled with miracidia showed characteristics of viability. Histopathological examination using different strains demonstrated therapeutic failure and the need for repeated treatment. In this study, we demonstrated a low efficacy of praziquantel in the treatment of schistosomiasis haematobia, and the necessity of the urinary bladder biopsy as criterion of cure.

Failure of neutrophil migration toward infectious focus in severe sepsis: a critical event for the outcome of this syndrome

Sepsis is a systemic inflammatory response commonly caused by bacterial infection. We demonstrated that the outcome of sepsis induced by cecal ligation and puncture (CLP) correlates with the severity of the neutrophil migration failure towards infectious focus. Failure appears to be due to a decrease in the rolling and adhesion of neutrophil to endothelium cells. It seems that neutrophil migration impairment is mediated by the circulating inflammatory cytokines, such as TNF-alpha and IL-8, which induce the nitric oxide (NO) production systemically. It is supported by the fact that intravenous administration of these cytokines reduces the neutrophil migration induced by different inflammatory stimuli, and in severe sepsis the circulating concentrations of the cytokines and chemokines are significantly increased. Moreover, the neutrophil migration failure and the reduction in the rolling/adhesion were not observed in iNOS-/- mice and, aminoguanidine prevented this event. We also demonstrated that the failure of neutrophil migration is a Toll-4 receptor (TLR4) dependent mechanism, since it was not observed in TLR4 deficient mice. Furthermore, it was also observed that circulating neutrophils obtained from septic patients present failure of neutrophil chemotaxis toward fMLP, IL-8, and LTB4 and an increased in sera concentrations of NO3 and cytokines. In conclusion, we demonstrated that, in sepsis, failure of neutrophil migration is critical for the outcome and that NO is involved in the process.

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.

Respiratory viruses in children younger than five years old with acute respiratory disease from 2001 to 2004 in Uberlândia, MG, Brazil

The main viruses involved in acute respiratory diseases among children are: respiratory syncytial virus (RSV), influenzavirus (FLU), parainfluenzavirus (PIV), adenovirus (AdV), human rhinovirus (HRV), and the human metapneumovirus (hMPV). The purpose of the present study was to identify respiratory viruses that affected children younger than five years old in Uberlândia, Midwestern Brazil. Nasopharyngeal aspirates from 379 children attended at Hospital de Clínicas (HC/UFU), from 2001 to 2004, with acute respiratory disease, were collected and tested by immunofluorescence assay (IFA) to detect RSV, FLU A and B, PIV 1, 2, and 3 and AdV, and RT-PCR to detect HRV. RSV was detected in 26.4% (100/379) of samples, FLU A and B in 9.5% (36/379), PIV 1, 2 and 3 in 6.3% (24/379) and AdV in 3.7% (14/379). HRV were detected in 29.6% (112/379) of the negative and indeterminate samples tested by IFI. RSV, particularly among children less than six months of life, and HRV cases showed highest incidence. Negative samples by both IFA and RT-PCR might reflect the presence of other pathogens, such as hMPV, coronavirus, and bacteria. Laboratorial diagnosis constituted an essential instrument to determine the incidence of the most common viruses in respiratory infections among children in this region.

Isolation of respiratory syncytial virus from nasopharyngeal aspirates stored at 20ºC from one to fifteen months after collection

Cell culture isolation is used for recovering respiratory syncytial virus (RSV) from respiratory specimens. As RSV is a thermolabile virus, specimens destined for inoculation into cell culture require special transport, handling, and storage. The isolation rate of RSV from nasopharyngeal aspirates (NPA) stored at 20ºC for one to 15 months after collection was investigated. A total of 126 samples considered positive for RSV by indirect fluorescence-antibody were tested by virus isolation in HEp-2 cell culture. RSV was isolated from 47/126 specimens (37.3%). These results show that RSV may be recovered from NPA stored at 20ºC by cell culture.

Evaluation of respiratory model employing conventional NIH mice to access the immunity induced by cellular and acellular pertussis vaccines

The increasing number of pertussis cases reported on the last twenty years and the existence of new acellular vaccines reinforce the need of research for experimental models to assure the quality of available pertussis vaccines. In this study, allotments of whole-cell and acellular pertussis vaccines were tested through the Intranasal Challenge Model (INM) using conventional NIH mice. The results have been compared to those achieved by the "Gold standard" Intracerebral Challenge Model (ICM). In contrast to ICM, INM results did not show intralaboratorial variations. Statistical analysis by Anova and Ancova tests revealed that the INM presented reproducibility and allowed identification and separation of different products, including three-component and four-component accellular pertussis vaccines. INM revealed differences between pertussis vaccines. INM provides lower distress to the mice allowing the reduction of mice number including the possibility of using conventional mice (less expensive) under non-aseptic environment. Thus, INM may be used as an alternative method of verifying the consistence of allotment production, including acellular pertussis vaccines.

Antiretroviral resistance in individuals presenting therapeutic failure and subtypes of the human immunodeficiency virus type 1 in the Northeast Region of Brazil

This study aimed to analyze human immunodeficiency virus (HIV) mutation profiles related to antiretroviral resistance following therapeutic failure, and the distribution of hiv subtypes in the Northeast Region of Brazil. A total of 576 blood samples from AIDS patients presenting therapeutic failure between 2002 and 2004 were analyzed. The genotyping kit viroSeq® was used to perform viral amplification in order to identify mutations related to hiv pol gene resistance. An index of 91.1% of the patients presented mutations for nucleoside reverse transcriptase inhibitors (nrti), 58.7% for non-nucleoside reverse transcriptase inhibitors (nnrti), and 94.8% for protease inhibitors (pi). The most prevalent mutations were 184V and 215E for nrti, 103N and 190A for nnrti. Most mutations associated with PIs were secondary, but significant frequencies were observed in codons 90 (25.2%), 82 (21.1%), and 30 (16.2%). The resistance index to one class of antiretrovirals was 14%, to two classes of antiretrovirals 61%, and to three classes 18.9%. Subtype B was the most prevalent (82.4%) followed by subtype F (11.8%). The prevalence of mutations related to nrti and nnrti was the same in the two subtypes, but codon analysis related to PI showed a higher frequency of mutations in codon 63 in subtype B and in codon 36 in subtype F. The present study showed that there was a high frequency of primary mutations, which offered resistance to nrti and nnrti. Monitoring patients with treatment failure is an important tool for aiding physicians in rescue therapy.

Antigenic and genomic characterization of adenovirus associated to respiratory infections in children living in Northeast Brazil

From January to December 1998, nasopharyngeal aspirates were obtained from 482 children with acute respiratory infections attended in emergence department and wards of a teaching hospital in the city of Salvador, Brazil. The samples were tested for the presence of adenovirus by isolation in tissue culture and indirect immunofluorescence assay. Eleven adenoviruses were detected by both methods in the same clinical samples. Infections by adenovirus were observed during seven months of the year without association with rainy season. Genome analysis was performed on these 11 isolates. Species C was represented by serotypes 1, 2 and 5. Within species B, only serotype 7 (Ad7) was detected. Two genomic variants of Ad1, two variants of Ad2, one of Ad5, and one of Ad7 (7h) were identified. This is the first study of molecular epidemiology of adenovirus associated to acute respiratory infections in children living in Northeast Brazil, and contributes to a better understanding of adenovirus infections in the country.

Adenoviruses C in non-hospitalized Mexican children older than five years of age with acute respiratory infection

Adenoviruses (AdV) are commonly involved in acute respiratory infections (ARI), which cause high morbidity and mortality in children. AdV are grouped in six species (A-F), which are associated with a wide range of diseases. The aim of this study was to identify the AdV species infecting non-hospitalized Mexican children with ARI symptoms, attending to the same school. For that, a PCR/RFLP assay was designed for a region of the hexon gene, which was chosen, based on the bioinformatical analysis of AdV genomes obtained from GenBank. A total of 100 children's nasopharyngeal samples were collected from January to June, 2005, and used for viral isolation in A549 cells and PCR/RFLP analysis. Only 15 samples produced cytopathic effect, and in all of them AdV C was identified. AdV C was also identified in eight additional nasopharyngeal samples which were negative for viral isolation. In summary, this outpatient population showed a rate of AdV infection of 23%, and only AdV C was detected.

Use of in-house PCR for identification of Mycobacterium tuberculosis in BACTEC broth cultures of respiratory specimens

We evaluated the ability of a PCR assay to identify Mycobacterium tuberculosis complex (MTBC) from positive BACTEC® 12B broth cultures. A total of 107 sputum samples were processed and inoculated into Ogawa slants and BACTEC® 12B vials. At a growth index (GI) > 30, 1.0 ml of the 12B broth was removed, stored, and assayed with PCR. Molecular results were compared to those obtained by phenotypic identification methods, including the BACTEC® NAP method. The average times required to perform PCR and NAP were compared. Of the 107 broth cultures evaluated, 90 were NAP positive, while 91 were PCR positive for MTBC. Of particular interest were three contaminated BACTEC® 12B broth cultures yielding microorganisms other than acid-fast bacilli growth with a MTBC that were successfully identified by PCR, resulting in a mean time of 14 days to identify MTBC before NAP identification. These results suggest that PCR could be used as an alternative to the NAP test for the rapid identification of MTBC in BACTEC® 12B cultures, particularly in those that contained both MTBC and nontuberculous mycobacteria.