Inflammatory Cytokine Expression Is Associated with Chikungunya Virus Resolution and Symptom Severity

The Chikungunya virus infection zones have now quickly spread from Africa to parts of Asia, North America and Europe. Originally thought to trigger a disease of only mild symptoms, recently Chikungunya virus caused large-scale fatalities and widespread economic loss that was linked to recent virus genetic mutation and evolution. Due to the paucity of information on Chikungunya immunological progression, we investigated the serum levels of 13 cytokines/chemokines during the acute phase of Chikungunya disease and 6- and 12-month post-infection follow-up from patients of the Italian outbreak. We found that CXCL9/MIG, CCL2/MCP-1, IL-6 and CXCL10/IP-10 were significantly raised in the acute phase compared to follow-up samples. Furthermore, IL-1β, TNF-α, Il-12, IL-10, IFN-γ and IL-5 had low initial acute phase levels that significantly increased at later time points. Analysis of symptom severity showed association with CXCL9/MIG, CXCL10/IP-10 and IgG levels. These data give insight into Chikungunya disease establishment and subsequent convalescence, which is imperative to the treatment and containment of this quickly evolving and frequently re-emerging disease

West Nile virus in Italy: a further threat to blood safety, a further challenge to the blood system

This report describes the epidemiology of WNV in an heavily populated area where this infection appeared recently in horses and humans. In particular, the implementation of a strategy for assuring the safety of blood donation is described. An epidemiological surveillance system directed at the rapid detection of WNV has been established since the last decade in Italy, due to the high diffusion of mosquitoes vectors. This system is based on the monitoring of sentinel animals located in different areas of the country. The data obtained from this activity indicated a possible circulation of WNV in the flat area of the lower Po river valley during the summer of 2008. In September 2008 the first cases of WNV related diseases in horses from this area were confirmed by laboratory investigations at the National Veterinary Reference Centre for exotic disease. Based on these data a surveillance for the human meningo-encephalitis was established. On September 20th 2008, the Laboratory of the Regional Reference Centre for Microbiological Emergency (CRREM), in Bologna, reported the first case of neuro-invasive WNV disease in a female patient living in a small village located in the middle of the area where the horses infection have been reported. This patient suffered from sudden onset of meningo-encephalitis and a specific IgM and IgG response to WNV was identified. Up to mid November 2008, two additional cases of WNV neuro-invasive disease have been identified by CRREM. All these cases have been further confirmed by the National Reference Laboratory for Arboviruses in Rome. As of November 14th 2008, the total number of horses that showed the presence of a specific antibody response to WNV was 354, with 21 animals also positive for viral RNA by RT-PCR. These epidemiological data prompted for the immediate necessity of a system to assess the safety of blood donations harvested in the area of WNV diffusion, that accounts for a total at risk population of about 1.8 millions subjects. Based on guidelines from the National Blood Centre, a nucleic acid amplification test (NAAT) based and CE marked method, (PROCLEIX West Nile Virus, Chiron) was chosen a screening technique by the CRREM in Bologna. Due to the high number of samples, including plasma specimens from donors patients for solid organs and tissues (> 200) that need to be tested per day, this methods has been performed by using the PROCLEIX TIGRIS automated system. Since the beginning of the automated screening testing (October 10th, 2008) more than 6000 plasma samples from blood and organs/tissues donors have been evaluated, with a mean daily number of samples tested of 204. Up to November 15th no positive samples among the blood or organ donors were detected. The use of the automated \u201cwalk-away\u201d instrumentation allowed the release of results within 6 hours from the arrival of the samples and required only one unit of personnel to perform the test. In addition, the results have been reported in a time schedule that fit the requirements for solid organ donation in six cases. The screening activity for the safety assessment of blood and organ donations is presently scheduled until November 30th, due to the seasonal decline of the vectors activity. A plan of activities for the next season is currently under development. The screening season will be from May 1st to November 30th and the activation would be dependent on the results of a seroprevalence studies that has been undertaken by CRREM on a 11.000 blood donors population from the WNV affected area. The identification of the exact sero-prevalence ratio and the geographical distribution of the seropositive population would be extremely useful in order to define the WNV NAAT screening activity for the next vector season

Acute phase CHIKV disease was associated with high levels of IL-6, CXCL9, CCL2, CXCL10.

<p>Cytokine Bead Array analysis of CHIKV patient serum samples showed high levels of IL-6, CXCL9, CCL2 and CXCL-10 are associated with acute disease phase and decreased with patient convalescence. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute levels. All samples were also analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value less than 0.05 for acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).</p

The stages of CHIKV Acute phase were marked by changes in CXCL10 and IL-10.

<p>Acute CHIKV patients were categorized into Viral stage (V), Antibody Initiation stage (AI) or Seroconversion stage (SC) according to the presence of CHIKV, IgM and IgG antibodies. Cytokine Bead Array analysis of the serum samples showed a significant decrease in CXCL10 and IL-10 from the Viral stage to the Seroconversion stage of the Acute phase. A Mann-Whitney U test was used to determine significance among the phases. The star symbol indicates a p-value less than 0.05 compared to the Viral phase.</p

CHIKV disease severity is associated with high CXCL10, CXCL9 and IgG levels at the 6-month time point.

<p>CHIKV patients were determined to be nonsymptomatic (N), to have mild symptoms (M) or to have severe symptoms (S). The cytokine and IgG levels were then grouped by symptom level and a Mann-Whitney U test was used to determine significance among the severity groups. CXCL10, CXCL9 and IgG were found to be significantly increased in the patients with mild and severe symptoms at 6 months following initial infection. The cross symbol indicates a p-value less than 0.05.</p

IL-2, IL-8 and IL-4 were not associated with acute or convalescence phase of CHIKV disease.

<p>Cytokine Bead Array analysis of CHIKV patient serum samples showed that IL-2, IL-8 and IL-4 were not associated with acute phase or convalescence of CHIKV disease in patients. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. Samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).</p

CHIKV patient convalescence was associated with increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10.

<p>Cytokine Bead Array analysis of CHIKV patient serum samples showed that following the acute phase of CHIKV disease patients had increasing levels of TNF-α, IL-5, IL-1β, IL-12, IFN-γ and IL-10. Six-month and one-year cytokine levels were analysed for statistical significance using the Wilcoxon test for Significance by comparing with acute phase values. As well, samples were analyzed for significance against healthy controls by the Mann-Whitney U test. The cross symbol indicates a p-value (Wilcoxon test) less than 0.05 for 6- and 12-month groups compared to acute values and star symbol indicates a p-value (Mann-Whitney U test) less than 0.05 acute, 6- and 12-month groups compared to control values. The dotted line indicates the median of healthy control cytokine levels. Acute (A), 6-month follow-up (6), and 12-month follow-up (12).</p

The stages of CHIKV 6- and 12-month follow-up phases are marked by differentials in CXCL10, CXCL9, IL-6 and CXCL9, IL-10 respectively.

<p>IgG levels in 6- and 12-month patient serum samples were determined by ELISA. The samples were then grouped by IgG level; a low IgG level group (L) and a high IgG level group (H). Cytokine Bead Array analysis of the serum samples showed a significant difference in CXCL9, CXCL10 and IL-6 between patients with high and low IgG levels at the 6-month time point. Twelve-month follow-up CHIKV patient samples showed a significant difference in CXCL9 and IL-10 between low (1) and high (2) IgG groups. A Mann-Whitney U test was used to determine significance among the IgG groups. The cross symbol indicates a p-value less than 0.05.</p