RS virus diagnosis: comparison of isolation, immunofluorescence and enzyme immunoassay

Two techniques for rapid diagnosis, immunofluorescence (IFAT) and enzyme immunoassay (EIA), have been compared with virus isolaion in tissue culture for the detection of respiratory syncytial virus (RSV) in specimens of nasopharyngeal secretions. The specimens were obtained from children under five years of age suffering from acute respiratory iliness, during a period of six months from January to June 1982. Of 471 specimens examined 54 (11.5%) were positive by virus isolation and 180 (38.2%) were positive by immunofluorescence. The bacterial contamination of inoculated tissue cultures unfortunately prevented the isolation of virus from many samples. Specimens from 216 children were tested to compare enzyme immunoassay and immunofluorescence. Of these 60 (27%) were positive by EIA and 121 (56%) were positive by IFAT. Our results suggest that the EIA technique although highly specific is rather insensitive. This may be because by the time these tests were done the originl nasopharyngeal secretions were considerably diluted and contained more mucus fragments than the call suspension used for IFAT. Of the three techniques, IFAT gives the best results although EIA may be useful where IFAT is not possible.

Immunohistochemical detection of virus through its nuclear cytopathic effect in idiopathic interstitial pneumonia other than acute exacerbation

Idiopathic interstitial pneumonias include complex diseases that have a strong interaction between genetic makeup and environmental factors. However, in many cases, no infectious agent can be demonstrated, and these clinical diseases rapidly progress to death. Theoretically, idiopathic interstitial pneumonias could be caused by the Epstein-Barr virus, cytomegalovirus, adenovirus, hepatitis C virus, respiratory syncytial virus, and herpesvirus, which may be present in such small amounts or such configuration that routine histopathological analysis or viral culture techniques cannot detect them. To test the hypothesis that immunohistochemistry provides more accurate results than the mere histological demonstration of viral inclusions, this method was applied to 37 open lung biopsies obtained from patients with idiopathic interstitial pneumonias. As a result, immunohistochemistry detected measles virus and cytomegalovirus in diffuse alveolar damage-related histological patterns of acute exacerbation of idiopathic pulmonary fibrosis and nonspecific interstitial pneumonia in 38 and 10% of the cases, respectively. Alveolar epithelium infection by cytomegalovirus was observed in 25% of organizing pneumonia patterns. These findings were coincident with nuclear cytopathic effects but without demonstration of cytomegalovirus inclusions. These data indicate that diffuse alveolar damage-related cytomegalovirus or measles virus infections enhance lung injury, and a direct involvement of these viruses in diffuse alveolar damage-related histological patterns is likely. Immunohistochemistry was more sensitive than the histological demonstration of cytomegalovirus or measles virus inclusions. We concluded that all patients with diffuse alveolar damage-related histological patterns should be investigated for cytomegalovirus and measles virus using sensitive immunohistochemistry in conjunction with routine procedures.

L’étude de l’impact des protéines non structurales NS1 et NS2 du virus respiratoire syncitial sur la réponse immunitaire innée

Le virus respiratoire syncytial (RSV) est un virus à ARN de polarité négative. Les études démontrent que toute la population sera infectée par ce virus au moins deux fois avant l’âge de 3 ans. Le RSV peut provoquer plusieurs pathologies respiratoires telles que la bronchiolite aiguë et la pneumonie. Les infections sévères corrèlent avec le développement de l’asthme. Lors d’une infection virale, les particules du RSV sont détectées par le senseur RIG-I qui induit l’activation des facteurs de transcription NF-κB et IRF-3. Respectivement, les facteurs de transcription activeront les réponses inflammatoire et antivirale. Au coeur des pathologies induites par le RSV se trouve une réponse immunitaire mal adaptée. Plus précisément, par l’entremise de NF-κB, le RSV provoque une production exagérée de cytokines et chimiokines qui induisent une réponse inflammatoire démesurée provoquant du dommage tissulaire. Paradoxalement, le RSV est capable d’échapper à la réponse antivirale. Ces deux phénomènes sont contrôlés par l’entremise des protéines non structurales NS1 et NS2. Le mécanisme délimitant le mode d’action de NS1 et NS2 sur la réponse antivirale reste à être déterminé. Avec pour objectif d’élucider comment NS1 et NS2 inhibent la réponse antivirale, nous avons investigué le mécanisme de reconnaissance de l’hôte vis-à-vis de RSV. Nous démontrerons, pour la première fois, que le senseur cytosolique MDA5 est impliqué dans la réponse antivirale contre le RSV. Nous présenterons des résultats préliminaires qui suggèrent que le rôle de MDA5 est non redondant à RIG-I. À l’aide d’ARN interférant dirigé contre RIG-I et de transfection de MDA5, nous démontrerons que MDA5 ne contribue pas à la phosphorylation d’IRF-3, mais plutôt qu’elle régit la stabilité du facteur de transcription. Nous démontrerons aussi que, contrairement à l’hypothèse actuelle sur le fonctionnement de NS1 et NS2, l’inhibition de ces derniers ne provoque pas une augmentation de la cytokine antivirale IFN−β. Cependant, l’expression ectopique de NS1 et NS2 réduit l’activité du promoteur de l’IFN-β et de la protéine cytoplasmic antivirale ISG56 lorsqu’elle est mesurée par essai luciférase.

Modular alpha-helical mimetics with antiviral activity against respiratory syncitial virus

A 13-residue peptide sequence from a respiratory syncitial virus fusion protein was constrained in an alpha-helical conformation by fusing two back-to-back cyclic alpha-turn mimetics. The resulting peptide, Ac-(3 -> 7; 8 -> 12)-bicyclo-FP[KDEFD][KSIRD]V-NH2, was highly alpha-helical in water by CD and NMR spectroscopy, correctly positioning crucial binding residues (F488, I491, V493) on one face of the helix and side chain-side chain linkers on a noninteracting face of the helix. This compound displayed potent activity in both a recombinant fusion assay and an RSV antiviral assay (IC50 = 36 nM) and demonstrates for the first time that back-to-back modular alpha-helix mimetics can produce functional antagonists of important protein-protein interactions.

Infección por virus sincitial respiratorio y su relación con valores de IGG en niños críticos

Antecedente: La infección por el virus sincitial respiratorio (VSR) representa una elevada morbimortalidad, y en algunos casos necesidad de manejo en unidades de cuidado intensivo pediátrico (UCIP). La respuesta inmunológica influye de manera directa en la expresión de la severidad y pronóstico de los pacientes con infección respiratoria. Metodología: Estudio de una cohorte retrospectiva de pacientes con infección respiratoria grave secundaria a VSR, sin historia de inmunodeficiencia, atendidos en la UCIP del Hospital Universitario Clínica San Rafael. Se realizó análisis descriptivoglobaly de acuerdo a la categorización de las prueba de IgG. Resultados: De 188 pacientes que ingresaron a la UCIP, 13% presentaron infección por VSR (24), con una edad promedio de 7,3 (DE=3,6) meses. Pertenecían al sexo masculino79,83%. Se encontró que 12,5% tenían un valor de IgGbajo para su edad, 58,33% tenían valores en límite inferior y el 29,17% dentro de rangos normales para su edad. En los pacientes con IgG baja, fue mayor la presentación de choque séptico que no responde a líquidos (100 vs 92 vs 86%), la mediana de días de ventilación mecánica fue mayor (8 vs 6 vs 5 respectivamente), así como la mortalidad (67 vs 7,1 vs 0%). Conclusión: Nuestra serie encontró que aquellos pacientes con niveles bajos o valores en el límite inferior de IgG sérica tuvieron mayor compromiso sistémico, mayor duración de ventilación mecánica y mayor mortalidad. Se necesitan estudios prospectivos que relaciones niveles bajos de IgG con severidad y pronostico en estos pacientes con infección grave por VSR.

Respiratory Viruses Are Continuously Detected In Children With Chronic Tonsillitis Throughout The Year.

To evaluate the oscillations on the viral detection in adenotonsillar tissues from patients with chronic adenotonsillar diseases as an indicia of the presence of persistent viral infections or acute subclinical infections. Cross-sectional prospective study. Tertiary hospital. The fluctuations of respiratory virus detection were compared to the major climatic variables during a two-year period using adenoids and palatine tonsils from 172 children with adenotonsillar hypertrophy and clinical evidence of obstructive sleep apnoea syndrome or recurrent adenotonsillitis, without symptoms of acute respiratory infection (ARI), by TaqMan real-time PCR. The rate of detection of at least one respiratory virus in adenotonsillar tissue was 87%. The most frequently detected viruses were human adenovirus in 52.8%, human enterovirus in 47.2%, human rhinovirus in 33.8%, human bocavirus in 31.1%, human metapneumovirus in 18.3% and human respiratory syncytial virus in 17.2%. Although increased detection of human enterovirus occurred in summer/autumn months, and there were summer nadirs of human respiratory syncytial virus in both years of the study, there was no obvious viral seasonality in contrast to reports with ARI patients in many regions of the world. Respiratory viruses are continuously highly detected during whole year, and without any clinical symptomatology, indicating that viral genome of some virus can persist in lymphoepithelial tissues of the upper respiratory tract.

Vírus respiratório sincicial humano em crianças hospitalizadas por infecções agudas das vias aéreas inferiores

OBJETIVO: Avaliar a prevalência e a sazonalidade do vírus respiratório sincicial humano (VRSH) em crianças de 0 a 6 anos hospitalizadas por infecção aguda das vias aéreas inferiores (IVAI) em São José do Rio Preto (SP) e a associação entre faixa etária, diagnóstico e VRSH. MÉTODOS: Entre maio de 2004 e setembro de 2005, foram estudados 290 episódios consecutivos de IVAI adquiridos na comunidade em crianças de 0 a 6 anos internadas no Hospital de Base de São José do Rio Preto. Para identificação do VRSH, foram coletadas amostras de secreção de nasofaringe e realizou-se análise molecular por meio da técnica de RT-PCR. RESULTADOS: A prevalência de VRSH foi de 29,3% nos episódios de IVAI hospitalizados. A IVAI foi frequente em lactentes (mediana de idade = 13,5 meses). O VRSH foi mais frequente nos casos de bronquiolite (64%) e no primeiro ano de vida (35%). Os episódios de infecção por VRSH ocorreram entre o outono e a primavera, com frequência maior em 2004 do que em 2005. Os critérios clínicos e radiológicos não foram suficientes para o diagnóstico de infecção pelo VRSH. Em 78,8% dos episódios de VRSH, houve tratamento com antibiótico. CONCLUSÕES: A prevalência do VRSH em crianças de 0 a 6 anos hospitalizadas por IVAI foi elevada, com predomínio nas mais jovens ou com bronquiolite. A circulação do vírus variou nos dois anos estudados. Os resultados sugerem necessidade de diagnóstico laboratorial do VRSR na prática clínica.

Association of haplotypes in the IL8 gene with susceptibility to chronic periodontitis in a Brazilian population

Background: Interleukin 8 (IL-8) is a chemokine related to the initiation and amplification of acute and chronic inflammatory processes. Polymorphisms in the IL8 gene have been associated with inflammatory diseases. We investigated whether the - 845(T/C) and - 738(T/A) single nucleotide polymorphisms (SNPs) in the IL8 gene, as well as the haplotypes they form together with the previously investigated -353(A/T), are associated with susceptibility to chronic periodontitis. Methods: DNA was extracted from buccal epithelial cells of 400 Brazilian individuals (control n =182, periodontitis n=218). SNPs were genotyped by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Disease associations were analyzed by the chi(2) test, Exact Fisher test and Clump program. Haplotypes were reconstructed using the expectation-maximization algorithm and differences in haplotype distribution between the groups were analyzed to estimate genetic susceptibility for chronic periodontitis development. Results: When analyzed individually, no SNPs showed different distributions between the control and chronic periodontitis groups. Although, nonsmokers carrying the TTA/CAT (OR = 2.35, 95% CI = 1.03-5.36) and TAT/CTA (OR= 6.05, 95% CI = 1.32-27.7) haplotypes were genetically susceptible to chronic periodontitis. The ITT/TAA haplotype was associated with protection against the development of periodontitis (for nonsmokers OR= 0.22, 95% CI = 0.10-0.46). Conclusion: Although none of the investigated SNPs in the IL8 gene was individually associated with periodontitis, some haplotypes showed significant association with susceptibility to, or protection against, chronic periodontitis in a Brazilian population. (C) 2010 Elsevier B.V. All rights reserved.

Human bocavirus respiratory infections in children

Human bocavirus (HBoV) was recently identified in respiratory samples from patients with acute respiratory infections and has been reported in different regions of the world. To the best of our knowledge, HBoV has never been reported in respiratory infections in Brazil. Nasopharyngeal aspirates were collected from patients aged <5 years hospitalized in 2005 with respiratory infections in Ribeirao Preto, southeast Brazil, and tested by polymerase chain reaction (PCR) for HBoV. HBoV-positive samples were further tested by PCR for human respiratory syncytial virus, human metapneumovirus, human coronaviruses 229E and OC43, human influenza viruses A and B, human parainfluenza viruses 1, 2 and 3, human rhinovirus and human adenovirus. HBoV was detected in 26/248 (10.5%) children of which 21 (81%) also tested positive for other respiratory viruses. Despite the high rates of co-infections, no significant differences were found between HBoV-positive patients with and without co-infections with regard to symptoms.

Frequency of human metapneumovirus infection in hematopoietic SCT recipients during 3 consecutive years

Respiratory viruses can cause significant morbidity in immunocompromised hosts. Human metapneumovirus (hMPV) has been increasingly associated with lower respiratory tract infection in hematopoietic SCT (HSCT) recipients, with mortality rates up to 50%. No data on the occurrence of hMPV infection in HSCT recipients have been reported in the southern hemisphere. We conducted a retrospective study including 228 nasal wash samples from 153 HSCT recipients with respiratory symptoms during 2001, 2002 and 2003. hMPV was detected by real-time PCR with primers complementary to the nucleocapsid region of hMPV genome. Eleven of the 153 patients (7.2%) acquired hMPV infection during the study period (6.4% in 2001, 4.7% in 2002 and 11.1% in 2003). Among the 11 HSCT recipients with hMPV infection, 1 died 8 days after the diagnosis, but the role of hMPV in the patient`s death could not be established. In 2001 and 2003, hMPV group A prevailed over group B. In 2002, both groups were detected equally. hMPV infections were diagnosed in late winter and spring. The frequency of hMPV infection in HSCT recipients living in Brazil was similar to those observed in the northern hemisphere. Sensitive techniques to detect hMPV should be included in the diagnostic assessment of HSCT recipients with respiratory symptoms.

Human cytomegalovirus reinfection is associated with intrauterine transmission in a highly cytomegalovirus-immune maternal population

OBJECTIVE: To determine contribution of reinfection with new strains of cytomegalovirus in cytomegalovirus seromimmune women to incidence of congenital cytomegalovirus infection. STUDY DESIGN: In 7848 women studied prospectively for congenital cytomegalovirus infection from a population with near universal cytomegalovirus seroimmunity, sera from 40 mothers of congenitally infected infants and 109 mothers of uninfected newborns were analyzed for strain-specific anticytomegalovirus antibodies. RESULTS: All women were cytomegalovirus seroimmune at first prenatal visit. Reactivity for 2 cytomegalovirus strains was found in 14 of 40 study mothers and in 17 of 109 control mothers at first prenatal visit (P=.009). Seven of 40 (17.5%) study women and 5 of 109 (4.6%) controls (P=.002) acquired antibodies reactive with new cytomegalovirus strains during pregnancy. Evidence of infection with more than 1 strain of cytomegalovirus before or during current pregnancy occurred in 21 of 40 study mothers and 22 of 109 controls (P<.0001). CONCLUSION: Maternal reinfection by new strains of cytomegalovirus is a major source of congenital infection in this population.

The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions

Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.

Avaliação da atividade anti-hRSV da quercetina e seus derivados acetilados

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Longitudinal study of acute respiratory diseases in Rio de Janeiro: occurrence of respiratory viruses during four consecutive years

The occurrence of different viruses in nasopharyngeal secretions from children less than 5 years old with acute respiratory infections (ARI) was investigated over a period of 4 years (1982-1985) in Rio de Janeiro. Of the viruses known to be associated with ARI, all but influenza C and parainfluenza types 1, 2 and 4 were found. Viruses were found more frequently in children attending emergency or pediatric wards than in outpatients. This was clearly related to the high incidence of respiratory syncytial virus (RSV) in the more severe cases of ARI. RSV positive specimens appeared mainly during the fall, over four consecutive years, showing a clear seasonal ocurrence of this virus. Emergency wards provide the best source of data for RSV surveillance, showing sharp increase in the number of positive cases coinciding with increased incidence of ARI cases. Adenovirus were the second most frequent viruses isolated and among these serotypes 1,2 and 7 were predominant. Influenza virus and parainfluenza virus type 3 were next in frequency. Influenza A virus were isolated with equal frequency in outpatient departments, emergency and pediatric wards. Influenza B was more frequent among outpatients. Parainfluenza type 3 caused outbreaks in the shanty town population annually during the late winter or spring and were isolated mainly from outpatients. Herpesvirus, enterovi-rus and rhinovirus were found less frequently. Other viruses than RSV and parainfluenza type 3 did not show a clear seasonal incidence.

Etiology and severity of community acquired pneumonia in children from Uruguay: a 4-year study

The 4-year study (1987-1990) covered the major clinical-epidemiological characteristics of pneumonia in children as diagnosed at the emergency service of the Children's Hospital, as well as etiologies, and factors involved in the most severe cases. Etiology was determined in 47.7% of the 541 pneumonia cases, involving 283 pathogens of which 38.6% were viruses and 12.6% bacteria. Viral and mixed etiologies were more frequent in children under 12 months of age. Bacteria predominated in ages between 6 and 23 months. Among the viruses, respiratory syncytial virus predominated (66%). The bacterial pneumonias accounted for 12.2% of the recognized etiologies. The most important bacterial agents were S. pneumoniae (64%) and H. influenzae (19%). H. influenzae and mixed infections had a relevant participation during the 1988 season, pointing to annual variations in the relative participation of pathogens and its possible implication in severity of diseases. Correlation of severity and increased percentage of etiological diagnosis was assessed: patients with respiratory rates over 70 rpm, or pleural effusion and/or extensive pulmonary parenchyma compromise yielded higher positive laboratory results. Various individual and family risk factors were recognized when comparing pneumonia children with healthy controls.