Imported arboviral infections in Italy, July 2014-October 2015: a National Reference Laboratory report.

BACKGROUND: Imported cases of infections due to Dengue (DENV) and Chikungunya (CHIKV) viruses and, more recently, Zika virus (ZIKV) are commonly reported among travelers returning from endemic regions. In areas where potentially competent vectors are present, the risk of autochthonous transmission of these vector-borne pathogens is relatively high. Laboratory surveillance is crucial to rapidly detect imported cases in order to reduce the risk of transmission. This study describes the laboratory activity performed by the National Reference Laboratory for Arboviruses (NRLA) at the Italian National Institute of Health in the period from July 2014 to October 2015. METHODS: Samples from 180 patients visited/hospitalized with a suspected DENV/CHIKV/ZIKV infection were sent to the NRLA from several Italian Hospitals and from Regional Reference Laboratories for Arboviruses, in agreement with the National Plan on human surveillance of vector-borne diseases. Both serological (ELISA IgM test and Plaque Reduction Neutralization Test-PRNT) and molecular assays (Real Time PCR tests, RT-PCR plus nested PCR and sequencing of positive samples) were performed. RESULTS: DENV infection was the most frequently diagnosed (80 confirmed/probable cases), and all four genotypes were detected. However, an increase in imported CHIKV cases (41 confirmed/probable cases) was observed, along with the detection of the first ZIKV cases (4 confirmed cases), as a consequence of the recent spread of both CHIKV and ZIKV in the Americas. CONCLUSIONS: Main diagnostic issues highlighted in our study are sensitivity limitations of molecular tests, and the importance of PRNT to confirm serological results for differential diagnosis of Arboviruses. The continuous evaluation of diagnostic strategy, and the implementation of laboratories networks involved in surveillance activities is essential to ensure correct diagnosis, and to improve the preparedness for a rapid and proper identification of viral threats.

Lack of usutu virus RNA in cerebrospinal fluid of patients with encephalitis of unknown etiology, Tuscany, Italy.

Usutu virus (USUV) is an African mosquito-borne flavivirus associated with human neurological disorders in Europe. Recently, USUV introduction in Europe has been traced back to Eurasian blackbirds deaths in the Tuscany region of Italy in 1996. Ninety-six cerebrospinal fluid (CSF) samples from patients with encephalitis of unknown etiology diagnosed in 2010-2013 were screened to determine whether USUV circulates in humans in Tuscany. Using real-time polymerase chain reaction, no positive patient was found. USUV does not seem to cause neuroinvasive disorders in humans in Tuscany.

Short-term kinetics of torque teno virus viraemia after induction immunosuppression confirm T lymphocytes as the main replication-competent cells.

Torque teno virus (TTV) is increasingly considered a universal marker of global immune function. The virus is supposed to replicate in lymphocytes, but poor information is available about fluctuations of viraemia after administration of anti-lymphocyte agents. We studied TTV kinetics in a cohort of 70 kidney±pancreas recipients receiving one of two different anti-T-cell induction immunosuppressants. During the first 30 days after anti-T-cell antibody administration, we report kinetics of TTV viraemia compatible with replication in T lymphocytes, and highly dependent on the potency of the anti-T-cell drug administered.

Intracellular detection of interleukin 17 and other cytokines in human bronchoalveolar lavage fluid: a first assessment.

Increasing evidence links pulmonary pathology to cytokines determining an inflammatory environment in the lung. Detection of cells secreting specific cytokines in BALF could be helpful as a diagnostic tool but which cytokines to choose among their great variety may be the first question to solve. The aim of this study was to investigate the Th1, Th2 and Th17 cytokine profile in whole cells within the human bronchoalveolar lavage fluid (BALF) by flow cytometry, with a focus on interleukin (IL)-17-producing cells, in order to assess which cytokines might lend themselves as markers of disease in future studies. BALF and paired peripheral blood samples were collected from 52 patients admitted to hospital for pulmonary pathologies. Cells obtained from BALF and peripheral blood were incubated in vitro in the absence or presence of appropriate stimuli and analyzed for intracellular content of IL-4, -10, -12, -17, interferon (IFN)γ and tumor necrosis factor (TNF)α in association to expression of either HLA-DR or CD4. IL-17-secreting cells were further characterized. Production of IL-17 by unstimulated BALF cells could be detected in 2 of the 32 patients that could be examined; upon PMA/IM stimulation in vitro, IL-17 was produced by varying percentages of lymphocytes, mostly memory CD4(+) cells, in all BALF samples. IL-4 could be detected in a relatively high proportion of unstimulated HLA-DR(+/-), SSC(hi) cells, most probably granulocytes; IL-10 could be found mostly in macrophages in a number of the BALF samples analyzed. Finally, IFNγ and TNFα were only produced by lymphocytes after in vitro stimulation. This study shows that T cells producing IL-17 can be found in the lung of respiratory patients in the absence of ex vivo stimulation, making IL-17 a good candidate marker of specific pathologies of the lung. Upon stimulation, IL-17 production was accounted for by CD4(+) CD45RO(+) cells. Other cytokines are also discussed. An interesting cytokine secretion profile found in BALF from a patient with rheumatoid lung disease is also reported.

Torquetenovirus infection and ciliary dysmotility in children with recurrent pneumonia.

BACKGROUND: The aim of the study was to assess Torquetenovirus (TTV) loads within respiratory ciliated cells and to verify the existence of a correlation between TTV loads and functional or structural ciliary abnormalities, in a group of children with recurrent or persistent pneumonia. METHODS: Nasal brushing samples of 55 children (28 male) were evaluated for ciliary motion and ultrastructural assessment, as well as for detection and quantification of TTV. Moreover, presence and load of TTV within ciliated cells, obtained from 5 patients by laser capture microdissection, were determined. RESULTS: The nasal samples of 47 (85%) children with persistent or recurrent pneumonia resulted positive for TTV (loads = 2.1-7.3 log10 copies/microg total DNA). TTV were demonstrated also within microdissected ciliated cells. No significant difference between primary (11 subjects) and secondary ciliary dyskinesia (44 subjects) for TTV prevalence and mean loads were found. A significant correlation was observed between nasal TTV loads and ciliary beat frequency score (r = 0.305; P < 0.05), but not between TTV loads and presence of abnormal motion patterns, in patients with secondary ciliary abnormalities. As expected no correlations were found between nasal TTV loads and ciliary motion analysis in primary ciliary dyskinesia. CONCLUSIONS: The presence of TTV in nasal samples demonstrates TTV ability to infect respiratory ciliated cells and suggests that these cells are potentially able to support virus replication. Moreover, TTV may behave in respiratory cells in a similar way to other viruses, that is, they disrupt the mucociliary escalator.