Number of negative swabs and percentage of ILI consultations declared by the sentinel GPs in 2011 and 2012 in Réunion Island.

<p>Specimens from outpatient (n  =  222) that consult for ILI were analyzed using multiplex RT-PCR. Bars represent the number of specimen that tested negative for all pathogens analyzed. The curve showing the weekly proportion of ILI among total visit based on data collected from sentinel network practitioners in 2011–2012 was added.</p

Etiology of Influenza-Like Illnesses from Sentinel Network Practitioners in Réunion Island, 2011-2012

<div><p>In Réunion Island, despite an influenza surveillance established since 1996 by the sentinel general practitioner’s network, little is known about the etiology of Influenza like-illness (ILI) that differs from influenza viruses in a tropical area. We set up a retrospective study using nasal swabs collected by sentinel GPs from ILI patients in 2011 and 2012. A total of 250 swabs were randomly selected and analyzed by multiplex reverse transcriptase polymerase chain reaction (RT-PCR) including research of 18 viruses and 4 bacteria. We detected respiratory viruses in 169/222 (76.1%) samples, mostly rhinovirus (23.4%), influenza A virus (21.2%), influenza B virus (12.6%), coronavirus (4.9%) and Human metapneumovirus (3.6%). Nine swabs (5.3% of positive swabs) revealed co-infections with two viruses identified, among which six concerned co-infections with influenza viruses. We observed important seasonal differences, with circulation of Human Metapneumoviruses, RSV A and B and coronavirus only during summer; whereas parainfluenza viruses were identified only during winter. In conclusion, this study highlights a substantial circulation of multiple respiratory pathogens in Réunion Island throughout the year. It shows that ILI are not only attributable to influenza and underlines the need for biological surveillance. As the use of multiplex RT-PCR showed its efficacy, it is now used routinely in the surveillance of ILI.</p></div

Identified pathogens in nasal swabs analyzed by multiplex PCR, Réunion Island, 2011 and 2012.

<p>Identified pathogens in nasal swabs analyzed by multiplex PCR, Réunion Island, 2011 and 2012.</p

Identified co-infections in nasal swabs analyzed by multiplex PCR, Réunion Island, 2011 and 2012.

<p>Identified co-infections in nasal swabs analyzed by multiplex PCR, Réunion Island, 2011 and 2012.</p

Identified viruses and percentage of ILI consultations declared by the sentinel GPs in 2011 and 2012 in Réunion Island.

<p>Specimens from outpatient (n  =  222) that consult for ILI were analyzed using multiplex RT-PCR. Each panel shows the weekly incidence of one virus. For each virus, bars represent the number of specimen detected. The curve showing the weekly proportion of ILI among total visit based on data collected from sentinel network practitioners in 2011–2012 was added to each panel.</p

Immunomodulatory drug methotrexate used to treat patients with chronic inflammatory rheumatisms post-chikungunya does not impair the synovial antiviral and bone repair responses

<div><p>Chikungunya virus (CHIKV) is a mosquito-transmitted RNA alphavirus causing major outbreaks of infectious chronic inflammatory rheumatisms (CIR). Recently, methotrexate (MTX), a disease modifying anti-rheumatic drug has been used successfully to treat patients suffering from rheumatoid-like arthritis post-CHIK but its immunomodulatory activity in the context of viral persistence has been a matter of concerns. We herein used a model of primary human synovial fibroblasts (HSF) and the synthetic molecule polyriboinosinic:polyribocytidylic acid (PIC) to mimic chronic infectious settings in the joints of CHIKV infected patients. The innate antiviral immune and inflammatory responses were investigated in response to MTX used at the therapeutic concentration of 1 μM. We found that MTX did not affect cellular viability as indicated by the LDH release assay. By quantitative RT-PCR, we observed that HSF responded robustly to PIC by increasing ISG15 and IFNβ mRNA levels. Furthermore, PIC upregulated the mRNA expression of two of the major pattern recognition receptors, RIG-I and MDA5 involved in the innate immune detection of viral RNA. MTX did not impact the antiviral response of PIC on ISG15, IFNβ, RIG-I and MDA5 mRNA expressions. MTX alone or combined with PIC did not affect the expression of proinflammatory CCL2 and CXCL8 chemokines. PIC strongly upregulated the mRNA and protein expression of osteoclastogenic factors (IL-6, GM-CSF but not RANKL). Critically, MTX treatment alone or combined with PIC did not affect the expression of all three tested osteoclastogenic cytokines. We found that MTX alone did not increase the capacity of CHIKV to infect and replicate in HSF. In conclusion, our study argues for a beneficial effect of MTX to treat CIR post-CHIKV given that it does not critically impact the antiviral, the proinflammatory and the bone tissue remodeling responses of synovial cells.</p></div

MTX treatment does not affect CHIKV replication in HSF.

<p>Relative expression of viral NSP1 (A) and E2 (B) mRNA from HSF infected with different MOIs of CHIKV for 24 hours after exposure or not to MTX 1μM treatment (qRT-PCR data). Experiments were done in triplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

MTX treatment does not affect the expression of GM-CSF in HSF stimulated with PIC.

<p>HSF were exposed to PIC 100μg/mL and treated or not with MTX 1μM. <b>A)</b> GM-CSF mRNA levels from HSF stimulated by PIC100μg/mL in the absence and presence of MTX 1μM treatment were assessed by qRT-PCR. <b>B)</b> Levels of GM-CSF in HSF culture supernatants harvested after 6 hours, 24 hours, 48 hours and 72 hours were measured by ELISA assay. All experiments were done in triplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

MTX effects on the expression of proinflammatory chemokines in response to PIC stimulation.

<p><b>A)</b> Relative expression of CCL2 and CXCL8 from HSF stimulated by PIC100μg/mL for 6 hours and 24 hours in the absence and presence of MTX 1μM treatment was assessed by qRT-PCR. <b>B)</b> HSF were exposed to PIC 100μg/mL and treated or not with MTX 1μM. Supernatants were harvested after 6 hours and 24 hours and levels of CCL2 and CXCL8 were quantitated by ELISA assay. All experiments were done in quadruplicates and results are expressed as mean ± standard error. *: p-values ≤ 0.05, **: p-values ≤ 0.01, ***: p-values ≤ 0.001 and ****: p-values ≤ 0.0001.</p

Increased levels of soluble forms of E-selectin and ICAM-1 adhesion molecules during human leptospirosis

<div><p>Leptospirosis is a multisystemic zoonotic disease with infiltration of visceral organs by <i>Leptospira</i>. The capacity of the vascular endothelium to grant immune cell recruitment and activation in target organs during the disease course remains poorly characterized. We ascertained the levels of expression of several soluble cell adhesion molecules (CAM) notably expressed by endothelial cells in human leptospirosis. We prospectively enrolled 20 hospitalized patients and compared them to 10 healthy controls. Disease severity was defined by one or more organ failures, or death. Plasmatic concentrations of soluble CAM were assessed by multiplex bead assay at the time of patient presentation (M0) and 1 month after hospital discharge. The levels of soluble E-selectin (sCD62E) and soluble intercellular adhesion molecule 1 (sICAM-1, sCD53) were significantly increased in patients compared to controls (p<0.0001) and at 1 month (p<0.0001) with median values at 978 ng/ml (interquartile ranges 787–1164; sCD62E) and 1021 ng/ml (690–1428; sCD53). At M0, Soluble P-selectin level (sCD62P) was found to be decreased with levels at 60 ng/ml (0–631) versus 711 ng/ml (343–1113) for healthy controls (p<0.05). Levels of sICAM-3 (sCD50), sVCAM-1 (vascular cell adhesion molecule, sCD106) and sPECAM-1 (platelet endothelial cell adhesion molecule, sCD31) were not different from healthy subjects at M0. This study shows that two adhesion molecules, shed as soluble forms, are elevated during the acute phase of leptospirosis: E-selectin and s-ICAM1. These molecules may interfere with the process of immune cell recruitment to clear <i>Leptospira</i> at tissue levels.</p></div