Claudio Giuseppe Molteni

Toll-like receptor 3 gene polymorphisms and severity of pandemic A/H1N1/2009 influenza in otherwise healthy children

BackgroundToll-like receptors (TLRs) form an essential part of the innate immune system, which plays a fundamental role in rapidly and effectively controlling infections and initiating adaptive immunity. There are no published data concerning the importance of polymorphisms of TLRs in conditioning susceptibility to influenza or the severity of the disease. The aim of this study was to evaluate whether selected polymorphisms of TLR2, TLR3 and TLR4 influence the incidence and clinical picture of pandemic A/H1N1/2009 influenza.ResultsThe study involved 272 healthy children attending our Emergency Room for influenza-like illness (ILI), including 51 (18.8%) with pandemic A/H1N1/2009 influenza as revealed by real-time polymerase chain reaction, and 164 healthy controls examined after minor surgery. Genomic DNA was extracted from whole blood samples and five single-nucleotide polymorphisms (SNPs) were studied: TLR2 rs5743708, TLR3 rs5743313, TLR3 rs5743315, TLR4 rs4986790 and TLR4 rs4986791. The TLR3 rs5743313/CT polymorphism was found in all of the children with pneumonia and influenza infection, but in a significantly smaller number of those with A/H1N1/2009 influenza without pneumonia (<0.0001). TLR2, TLR3 rs5743315/AC and TLR4 polymorphisms were equally distributed in all of the groups regardless of the presence of the pandemic A/H1N1/2009 virus and clinical diagnosis. Viral load was comparable in all of the study groups.ConclusionsThere is a close relationship between the presence of TLR3 rs5743313/CT and an increased risk of pneumonia in children infected by the pandemic A/H1N1/2009 influenza virus.

Collection by trained pediatricians or parents of mid-turbinate nasal flocked swabs for the detection of influenza viruses in childhood.

This study evaluated the efficiency of pediatric mid-turbinate nasal flocked swabs used by parents in 203 children aged 6 months to 5 years with signs and symptoms of respiratory disease. Two nasal samples were collected from each child in a randomised sequence: one by a trained pediatrician and one by a parent. The real-time polymerase chain reaction influenza virus detection rates were similar in the samples collected using the two methods (Cohens kappa = 0.86), as were the cycle threshold values. In comparison with the pediatrician-collected samples, the sensitivity and specificity of the parental collections were respectively 89.3% (95% confidence interval [CI]: 77.8-100%) and 97.7% (95% CI: 95.5-100%), and the positive and negative predictive values were respectively 86.2% (95% CI: 73.7-95.1%) and 98.2% (95% CI: 96.4-100%). The children were significantly more satisfied with the parental collections (median values ± standard deviation, 1.59 ± 0.55 vs 3.51 ± 0.36; p < 0.0001). These findings show that mid-turbinate nasal flocked swabs specifically designed for infants and children can be used by parents without reducing the influenza virus detection rate. Moreover, the direct involvement of parents significantly increases patient acceptance, thus simplifying collection and suggesting that this novel swab design should be considered for epidemiological surveys and vaccine efficacy studies.

Role of polymorphisms of toll-like receptor (TLR) 4, TLR9, toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A genes in malaria susceptibility and severity in Burundian children

BackgroundMalaria caused by Plasmodium falciparum is one of the leading causes of human morbidity and mortality from infectious diseases, predominantly in tropical and sub-tropical countries. As genetic variations in the toll-like receptors (TLRs)-signalling pathway have been associated with either susceptibility or resistance to several infectious and inflammatory diseases, the supposition is that single nucleotide polymorphisms (SNPs) of TLR2, TLR4, TLR9, Toll-interleukin 1 receptor domain containing adaptor protein (TIRAP) and FCGR2A could modulate malaria susceptibility and severity.MethodsThis study was planned to make a further contribution to solving the problem of the real role of the most common polymorphisms of TLR4, TLR9, TIRAP and FCGR2A genes in modulating the risk of malaria and disease severity in children from Burundi, Central Africa. All the paediatric patients aged six months to 10 years admitted to the hospital of Kiremba, Burundi, between February 2011 and September 2011, for fever and suspicion of acute malaria were screened for malaria parasitaemia by light microscopy of thick and thin blood smears. In children with malaria and in uninfected controls enrolled during the study period in the same hospital, blood samples were obtained on filter paper and TLR4 Asp299Gly rs4986790, TLR9 G1174A rs352139, T-1486 C rs187084 TLR9 T-1237 C rs5743836, TIRAP Ser180Leu rs8177374 and the FCGR2A His131Arg rs1801274 polymorphisms were studied using an ABI PRISM 7900 HT Fast Real-time instrument.ResultsA total of 602 patients and 337 controls were enrolled. Among the malaria cases, 553 (91.9 %) were considered as suffering from uncomplicated and 49 (8.1 %) from severe malaria. TLR9 T1237C rs5743836CC was associated with an increased risk of developing malaria (p = 0.03), although it was found with the same frequency in uncomplicated and severe malaria cases. No other differences were found in all alleles studied and in genotype frequencies between malaria cases and uninfected controls as well as between uncomplicated and severe malaria cases.ConclusionsTLR9 T1237C seems to condition susceptibility to malaria in Burundian children but not its severity, whereas none of the assessed SNPs of TLR4, TIRAP and FCGR2A seem to influence susceptibility to malaria and disease severity in this population.

Oseltamivir-induced resistant pandemic A/H1N1 influenza virus in a child with cystic fibrosis and Pseudomonas aeruginosa infection

BACKGROUND Oseltamivir is considered the drug of choice for patients with pandemic influenza for whom drug treatment is recommended because adamantanes seem to be ineffective against pandemic A/H1N1 influenza virus and zanamivir is contraindicated in people with underlying respiratory conditions and difficult to administer in younger children. OBJECTIVES To increase knowledge on oseltamivir resistance emergence in pandemic A/H1N1 influenza. STUDY DESIGN Description of the case of an 8-year-old boy with cystic fibrosis and Pseudomonas aeruginosa infection in whom an oseltamivir-resistant pandemic A/H1N1 influenza virus was demonstrated. RESULTS On the basis of clinical and virological failure (nasopharyngeal secretions remained positive for pandemic A/H1N1 influenza virus and appearance of 275Y mutation in 100% virus population) on fifth day of treatment, oseltamivir was replaced by zanamivir inhalation (5mg to be inhaled twice a day). This change was associated with a rapid improvement in the patients general condition, respiratory findings and laboratory data (including disappearance of pandemic A/H1N1 influenza virus) in the absence of any adverse event. CONCLUSIONS The emergence of oseltamivir-resistant strains is related to the administration of the drug, supporting the restriction of oseltamivir use to carefully defined high-risk groups. Infection due to pandemic virus with the H275Y mutation can be associated with a severe clinical course, supporting the systematic monitoring of antiviral susceptibility in pandemic influenza-positive high-risk patients whose influenza is not resolved by oseltamivir treatment. Zanamivir inhalation can be successfully used in patients with cystic fibrosis without causing adverse respiratory events, highlighting that the risks and benefits of this drug must be considered on a patient by patient basis.

Clinical importance and impact on the households of oseltamivir-resistant seasonal A/H1N1 influenza virus in healthy children in Italy

A resistance of A/H1N1 influenza viruses to oseltamivir has recently emerged in a number of countries. However, the clinical and socioeconomic importance of this resistance has not been precisely defined. As children have the highest incidence of influenza infection and are at high risk of severe disease, the aim of this study was to evaluate the clinical importance and the impact on the households of oseltamivir-resistant seasonal A/H1N1 influenza virus in an otherwise healthy pediatric population. A total of 4,726 healthy children younger than 15 years with influenza-like illness were tested for influenza viruses by real-time polymerase chain reaction in the winters of 2007-2008 and 2008-2009 in Italy. The influenza A virus-positive samples underwent neuraminidase gene analysis using pyrosequencing to identify mutations H275Y and N294 S in A/H1N1, and E119V, R292K, and N294 S in A/H3N2. Among the A/H1N1 subtypes, the H275Y mutation was found in 2/126 samples taken in 2007-2008 (1.6%) and in all 17 samples (100%; p < 0.0001) taken in 2008-2009. No other mutation was identified in any of the A/H1N1 or A/H3N2 influenza viruses. No significant differences were found in terms of clinical importance or impact on the households between the children with oseltamivir-resistant seasonal A/H1N1 influenza virus and those with the wild-type. The spread of H275Y-mutated A/H1N1 seasonal influenza virus is a common phenomenon and the clinical importance and impact on the households of the mutated virus is similar to that of the wild-type in an otherwise healthy pediatric population.

Paramyxovirus Infection Regulates T Cell Responses by BDCA-1+ and BDCA-3+ Myeloid Dendritic Cells

Respiratory syncytial virus (RSV) and human Metapneumovirus (hMPV), viruses belonging to the family Paramyxoviridae, are the most important causes of lower respiratory tract infection in young children. Infections with RSV and hMPV are clinically indistinguishable, and both RSV and hMPV infection have been associated with aberrant adaptive immune responses. Myeloid Dendritic cells (mDCs) play a pivotal role in shaping adaptive immune responses during infection; however, few studies have examined how interactions of RSV and hMPV with individual mDC subsets (BDCA-1+ and BDCA-3+ mDCs) affect the outcome of anti-viral responses. To determine whether RSV and hMPV induce virus-specific responses from each subset, we examined co-stimulatory molecules and cytokines expressed by BDCA-1+ and BDCA-3+ mDCs isolated from peripheral blood after infection with hMPV and RSV, and examined their ability to stimulate T cell proliferation and differentiation. Our data show that RSV and hMPV induce virus-specific and subset-specific patterns of co-stimulatory molecule and cytokine expression. RSV, but not hMPV, impaired the capacity of infected mDCs to stimulate T cell proliferation. Whereas hMPV-infected BDCA-1+ and BDCA-3+ mDCs induced expansion of Th17 cells, in response to RSV, BDCA-1+ mDCs induced expansion of Th1 cells and BDCA-3+ mDCs induced expansion of Th2 cells and Tregs. These results demonstrate a virus-specific and subset-specific effect of RSV and hMPV infection on mDC function, suggesting that these viruses may induce different adaptive immune responses.

Reactive oxygen and nitrogen species during viral infections

Abstract Oxygen and nitrogen radicals are frequently produced during viral infections. These radicals are not only a physiological mechanism for pathogen clearance but also result in many pathological consequences. Low concentrations of radicals can promote viral replication; however, high concentrations of radicals can also inhibit viral replication and are detrimental to the cell due to their mitogenic activity. We reviewed the detailed mechanisms behind oxygen and nitrogen radical production and focused on how viruses induce radical production. In addition, we examined the effects of oxygen and nitrogen radicals on both the virus and host. We also reviewed enzymatic and chemical detoxification mechanisms and recent advances in therapeutic antioxidant applications. Many molecules that modulate the redox balance have yielded promising results in cell and animal models of infection. This encourages their use in clinical practice either alone or with existing therapies. However, since the redox balance also plays an important role in host defence against pathogens, carefully designed clinical trials are needed to assess the therapeutic benefits and secondary effects of these molecules and whether these effects differ between different types of viral infections.