Secondary Autochthonous Outbreak of Chikungunya, Southern Italy, 2017.

In 2017, a chikungunya outbreak in central Italy later evolved into a secondary cluster in southern Italy, providing evidence of disease emergence in new areas. Officials have taken action to raise awareness among clinicians and the general population, increase timely case detection, reduce mosquito breeding sites, and promote mosquito bite prevention.

Epidemiological and clinical suspicion of congenital Zika virus infection: Serological findings in mothers and children from Brazil.

The emergence of Zika virus in the Americas has caused an increase of babies born with microcephaly or other neurological malformations. The differential diagnosis of Zika infection, particularly serological diagnosis, is an important but complex issue. In this study, we describe clinical manifestations of 94 suspected cases of congenital Zika from Bahia state, Brazil, and the results of serological tests performed on children and/or their mothers at an average of 71 days after birth. Anti-Zika immunoglobulin M (IgM) antibodies were detected in 44.4% and in 7.1% of samples from mothers and children, respectively. Nearly all the IgM, and 92% of immunoglobulin G positive results were confirmed by neutralization test. Zika specific neutralizing antibodies were detected in as much as 90.4% of the cases. Moreover, dengue specific neutralizing antibodies were detected in 79.0% of Zika seropositive mothers. In conclusion, Zika IgM negative results should be considered with caution, due to a possible rapid loss of sensitivity after birth, while the NS1-based Zika IgM enzyme-linked immunosorbent assay test we have used has demonstrated to be highly specific. In a high percentage of cases, Zika specific neutralizing antibodies were detected, which are indicative of a past Zika infection, probably occurred during pregnancy in this population.

The Italian 2017 Outbreak Chikungunya Virus Belongs to an Emerging Aedes albopictus-Adapted Virus Cluster Introduced From the Indian Subcontinent.

BACKGROUND: Chikungunya virus is an emerging mosquito-borne pathogen with a wide global distribution. With the severe morbidity that it causes, chikungunya virus is a major public health problem in the affected areas and poses a considerable risk for unaffected areas hosting competent vector populations. In the summer of 2017, Italy experienced a chikungunya virus outbreak that spread in the Lazio region and caused a secondary outbreak in the Calabrian village of Guardavalle, with a final case number of 436. The causative strain was recognized as an Indian Ocean lineage (IOL) virus. METHODS: To understand the underlying genetic and molecular features of the outbreak virus, viruses from mosquito pools and clinical samples were isolated in cell culture and subjected to whole-genome sequencing and genetic analyses. RESULTS: All 8 characterized genomes shared a high sequence identity. A distinct substitution pattern in the Italian 2017 viruses (including mutations in E1, E2, and nsP4) was partly shared with the Pakistani 2016 outbreak viruses. Evolutionary analyses indicate that these 2 recent outbreaks and several geographically widely distributed, travel-associated viruses form a cluster of rapidly emerging Indian-origin IOL viruses. CONCLUSIONS: Our analyses show that the 2017 Italian outbreak virus belongs to a cluster of novel IOL chikungunya viruses originating in India. Their emergence calls for enhanced monitoring and strengthened preparedness measures, including vector control programs and raised awareness among general practitioners in countries potentially at risk.

Seroprevalence of West Nile and dengue virus in the human population of the Bolivian Chaco.

To determine the seroprevalence of antibodies against dengue virus (DENV) and West Nile virus (WNV) in the human population of the Bolivian Chaco, we tested 256 inhabitants of two rural communities. The seroprevalence, confirmed by plaque reduction neutralization test, was 7.8% and 2.7% for DENV and WNV, respectively.

Vector competence of Aedes albopictus for the Indian Ocean lineage (IOL) chikungunya viruses of the 2007 and 2017 outbreaks in Italy: a comparison between strains with and without the E1:A226V mutation.

We compared the vector competence of an Italian population of Aedes albopictus for two strains of chikungunya virus (CHIKV), with and without E1:A226V mutation, responsible for outbreaks in 2007 in the Emilia Romagna region and 2017 in the Lazio and Calabria regions, respectively. Ae. albopictus showed similar vector competence for both viral strains indicating that E1:A226V mutation is not exclusively responsible for ability of CHIKV to replicate well in this mosquito species.

Prevalence of Usutu and West Nile virus antibodies in human sera, Modena, Italy, 2012.

A collection of 3069 human sera collected in the area of the municipality of Modena, Emilia Romagna, Italy, was retrospectively investigated for specific antibodies against Usutu (USUV) and West Nile viruses (WNV). All the samples resulting positive using a preliminary screening test were analyzed with the plaque reduction neutralization test. Overall, 24 sera were confirmed as positive for USUV (0.78%) and 13 for WNV (0.42%). The results suggest that in 2012, USUV was circulating more than WNV in North-eastern Italy.

Seroprevalence survey of arboviruses in workers from Tuscany, Italy.

BACKGROUND: Arthropod-borne viruses (Arbovirus) play an important role among emerging and re-emerging infectious diseases and in the spreading of infections in new geographic areas. Although some arboviral infections may be asymptomatic or mild flu-like illnesses, many occur as severe forms of meningitis and meningoencephalitis. OBJECTIVES: To assess whether arboviral infections may be associated with occupational risk, in a population of agricultural and forestry workers potentially at high risk for arthropods bite and sting. METHODS: A seroprevalence survey for arboviruses belonging to the genera Flaviviruses (West Nile, Tick-borne encephalitis and Usutu viruses) and Phlebovirus (Toscana virus) was carried out in Grosseto province (Tuscany, Italy). One hundred and one serum samples of occupationally exposed workers and 100 serum samples of not exposed workers were analyzed using commercial and home-made serological assays. Serological data were obtained in 2012 and analyzed according to demographic characteristics, recollection of insect-bites, and time spent in outdoor activities. RESULTS: A total seropositivity of 10% (21/201) was observed for Toscana virus. No difference in seroprevalence for Toscana virus was observed among the exposed (10/101) versus the not exposed (11/100) workers. No seropositivity for West Nile, Usutu and Tick-borne encephalitis viruses was detected. CONCLUSIONS: Although circulation of Toscana virus is recognized in the study area, our results did not reveal a higher risk for workers exposed to arthropods bite and sting. Health surveillance programs remain useful to monitor the potential emergence of arboviruses.

Vector competence of Italian Aedes albopictus populations for the chikungunya virus (E1-226V).

BACKGROUND: Chikungunya virus (CHIKV) is an emerging arbovirus, belonging to the Togaviridae family, Alphavirus genus, transmitted by Aedes spp. mosquitoes. Since 2007, two different CHIKV strains (E1-226A and E1-226V) have been responsible for outbreaks in European countries, including Italy, sustained by Ae. albopictus mosquitoes. FINDINGS: In this study, we assessed the susceptibility to the CHIKV E1-226V, strain responsible for the Italian 2007 outbreak, of eight Ae. albopictus populations collected in Northern, Central, Southern, and Island Italy, by experimental infections. Vector competence was evaluated by estimating infection, dissemination, and transmission rates (IR, DR, TR), through detection of the virus in the bodies, legs plus wings, and saliva, respectively. Additionally, vertical transmission was evaluated by the detection of the virus in the offspring. The results of our study demonstrated that the Italian populations of Ae. albopictus tested were susceptible to CHIKV infection, and can disseminate the virus outside the midgut barrier with high values of IR and DR. Viral infectious RNA was detected in the saliva of three populations from Central, Southern, and Island Italy, also tested for TR and population transmission rate (PTR) values. No progeny of the first and second gonotrophic cycle were positive for CHIKV. CONCLUSIONS: This study strongly confirms the role of Ae. albopictus as a potential CHIKV vector in Italy. This may represent a threat, especially considering both the high density of this species, which is widespread throughout the country, and the increasing number of cases of imported arboviruses.

Detection of a chikungunya outbreak in Central Italy, August to September 2017.

An autochthonous chikungunya outbreak is ongoing near Anzio, a coastal town in the province of Rome. The virus isolated from one patient and mosquitoes lacks the A226V mutation and belongs to an East Central South African strain. As of 20 September, 86 cases are laboratory-confirmed. The outbreak proximity to the capital, its late summer occurrence, and diagnostic delays, are favouring transmission. Vector control, enhanced surveillance and restricted blood donations are being implemented in affected areas.

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.

Climate change, vector-borne diseases and working population.

INTRODUCTION: Risks associated with climate change are increasing worldwide and the global effects include altered weather and precipitation patterns, rising temperatures and others; human health can be affected directly and indirectly. This paper is an overview of literature regarding climate changes, their interaction with vector-borne diseases and impact on working population. MATERIALS AND METHODS: Articles regarding climate changes as drivers of vector-borne diseases and evidences of occupational cases have been picked up by public databank. Technical documents were also included in the study. RESULTS: Evidences regarding the impact of climate changes on vector-borne diseases in Europe, provided by the analysis of the literature, are presented. DISCUSSION: Climate-sensitive vector-borne diseases are likely to be emerging due to climate modifications, with impacts on public and occupational health. However, other environmental and anthropogenic drivers such as increasing travelling and trade, deforestation and reforestation, altered land use and urbanization can influence their spread. Further studies are necessary to better understand the phenomenon and implementation of adaptation strategies to protect human health should be accelerated and strengthened.

Experimental investigation of the susceptibility of Italian Culex pipiens mosquitoes to Zika virus infection.

We investigated the susceptibility of an Italian population of Culex pipiens mosquitoes to Zika virus (ZIKV) infection, tested in parallel with Aedes aegypti, as a positive control. We analysed mosquitoes at 0, 3, 7, 10, 14, 20 and 24 days after an infectious blood meal. Viral RNA was detected in the body of Cx. pipiens up to three days post-infection, but not at later time points. Our results indicate that Cx. pipiens is not susceptible to ZIKV infection.

Experimental studies of susceptibility of Italian Aedes albopictus to Zika virus.

We report a study on vector competence of an Italian population of Aedes albopictus for Zika virus (ZIKV). Ae. albopictus was susceptible to ZIKV infection (infection rate: 10%), and the virus could disseminate and was secreted in the mosquito's saliva (dissemination rate: 29%; transmission rate: 29%) after an extrinsic incubation period of 11 days. The observed vector competence was lower than that of an Ae. aegypti colony tested in parallel.

Phleboviruses detection in Phlebotomus perniciosus from a human leishmaniasis focus in South-West Madrid region, Spain.

BACKGROUND: Phlebotomus-borne (PhB-) viruses are distributed in large areas of the Old World and are widespread throughout the Mediterranean basin, where recent investigations have indicated that virus diversity is higher than initially suspected. Some of these viruses are causes of meningitis, encephalitis and febrile illnesses. In order to monitor the viral presence and the infection rate of PhB-viruses in a recently identified and well characterized human zoonotic leishmaniasis focus in southwestern Madrid, Spain, a sand fly collection was carried out. METHODS: Sand fly insects were collected in four stations using CDC light traps during 2012-2013 summer seasons. Screening for Phlebovirus presence both via isolation on Vero cells and via polymerase chain reaction (PCR), using degenerated primers targeting a portion of the L segment, was performed. The serological identity and phylogenetic relationships on the three genomic segments of the viral isolates were carried out. RESULTS: Six viral isolates belonging to different serological complexes of the genus Phlebovirus were obtained from fifty pools on a total of 963 P. perniciosus (202 females). Phylogenetic analysis and serological assays allowed the identification of two isolates of Toscana virus (TOSV) B genotype, three isolates strongly related to Italian Arbia virus (ARBV), and one isolate of a novel putative Phlebovirus related to the recently characterized Arrabida virus in South Portugal, tentatively named Arrabida-like virus. Positive male sand fly pools suggested that transovarial or venereal transmission could occur under natural conditions. CONCLUSIONS: Our findings highlighted the presence of different Phlebovirus species in the South-West area of the Madrid Autonomous Community where an outbreak of cutaneous and visceral human leishmaniasis has been recently described. The evidence of viral species never identified before in Spain, as ARBV and Arrabida-like virus, and TOSV B genotype focus stability was demonstrated. Environmental aspects such as climate change, growing urbanization, socio-economic development could have contributed to the genesis of this wide ecological niche of PhB-viruses and Leishmania spp. The potential role of vertebrates as reservoir for the phleboviruses identified and the possibility of Phleboviruses-Leishmania co-infection in the same sand fly should be assessed. Furthermore the PhB-viruses impact on human health should be implemented.

An autochthonous case of Zika due to possible sexual transmission, Florence, Italy, 2014.

We report a case of Zika virus infection imported in Florence, Italy ex-Thailand, leading to a secondary autochthonous case, probably through sexual transmission. The two cases occurred in May 2014 but were retrospectively diagnosed in 2016 on the basis of serological tests (plaque reduction neutralisation) performed on stored serum samples. Our report provides further evidence that sexual transmission of Zika virus is possible.

Experimental evaluation of sand fly collection and storage methods for the isolation and molecular detection of Phlebotomus-borne viruses.

BACKGROUND: Several viruses have been recently isolated from Mediterranean phlebotomine sand flies; some are known to cause human disease while some are new to science. To monitor the Phlebotomus-borne viruses spreading, field studies are in progress using different sand fly collection and storage methods. Two main sampling techniques consist of CDC light traps, an attraction method allowing collection of live insects in which the virus is presumed to be fairly preserved, and sticky traps, an interception method suitable to collect dead specimens in high numbers, with a risk for virus viability or integrity. Sand flies storage requires a "deep cold chain" or specimen preservation in ethanol. In the present study the influence of sand fly collection and storage methods on viral isolation and RNA detection performances was evaluated experimentally. METHODS: Specimens of laboratory-reared Phlebotomus perniciosus were artificially fed with blood containing Toscana virus (family Bunyaviridae, genus Phlebovirus). Various collection and storage conditions of blood-fed females were evaluated to mimic field procedures using single and pool samples. Isolation on VERO cell cultures, quantitative Real time-Retro-transcriptase (RT)-PCR and Nested-RT-PCR were performed according to techniques commonly used in surveillance studies. RESULTS: Live engorged sand flies stored immediately at -80 °C were the most suitable sample for phlebovirus identification by both virus isolation and RNA detection. The viral isolation rate remained very high (26/28) for single dead engorged females frozen after 1 day, while it was moderate (10/30) for specimens collected by sticky traps maintained up to 3 days at room temperature and then stored frozen without ethanol. Opposed to viral isolation, molecular RNA detection kept very high on dead sand flies collected by sticky traps when left at room temperature up to 6 days post blood meal and then stored frozen in presence (88/95) or absence (87/88) of ethanol. Data were confirmed using sand fly pools. CONCLUSIONS: While the collection and storage methods investigated had not much impact on the ability to detect viral RNA by molecular methods, they affected the capacity to recover viable viruses. Consequently, sand fly collection and handling procedures should be established in advance depending on the goal of the surveillance studies.

Experimental studies on comparison of the vector competence of four Italian Culex pipiens populations for West Nile virus.

BACKGROUND: West Nile virus (WNV) is a vector-borne disease responsible for causing epidemics in many areas of the world. The virus is maintained in nature by an enzootic bird-mosquito-bird cycle and occasionally transmitted to other hosts, such as equines and humans. Culex species, in particular the ubiquitous species Culex pipiens is thought to play a major vector role both in enzootic and epizootic maintenance and transmission of WNV. Introduced in Europe in recent years, since 2008 WNV has been stably circulating mainly in the Northeastern regions of Italy, although sporadic equine and/or human cases, as well as WNV infected Cx. pipiens pools, have been recorded in other Italian areas. The scope of our study was to evaluate the potential competence of some Italian populations of Cx. pipiens to transmit WNV and to assess their ability for vertical transmission of the virus. For this purpose four Italian populations, from different areas, were experimentally infected. METHODS: After the infectious blood meal, fed females were monitored for 32 days to determine the length of viral extrinsic incubation period. WNV titre of infected mosquitoes was evaluated both by quantitative Real Time PCR and viral titration by Plaque Forming Units/ml (PFU/mL) in VERO cells. Potential Infection, Dissemination, Transmission rates (IR, DR, TR) were assessed by detection of the virus in body, legs plus wings and saliva of the fed females, respectively. RESULTS: All tested populations were susceptible to the WNV infection. The viral presence in legs and wings demonstrated the ability of WNV to disseminate in the mosquitoes. Viral RNA was detected in the saliva of tested populations. No significant differences in TR values were observed among the four studied populations. The offspring of the Cx. pipiens infected females were WNV negative. CONCLUSIONS: Our study addressed an important issue in the knowledge on the complex WNV-vector relationships in Italy, indicating that all Italian Cx. pipiens populations tested exhibited vector competence for WNV. Further studies should be performed in order to better clarify the role of other factors (vector density, climatic conditions, reservoir presence etc.) in order to predict where and when WNV outbreaks could occur.