Concetta Amato

Rapid methods for identification of poliovirus isolates and determination of polio neutralizing antibody titers in human sera

A new rapid method for identification and determination of the titer of polioviruses and other enteroviruses in cell monolayers grown in microtiter plates is described. The method is based on immunoperoxidase staining of infected cells with commercial monoclonal antibodies (MAb) and biotin-labeled secondary antibody. The presence of poliovirus or other enteroviruses was established as the appearance of at least one focus of cells with stained cytoplasm 6 h post-infection. Viral titers determined by this method were expressed as focus forming unit (FFU) per ml which was found to correspond approximately to 10 TCID(50)/ml. The suitability of this technique to determine poliovirus antibody titers in human sera was also tested comparing the immunocytochemical neutralization assay with a conventional neutralization in microtiter plates. The test was standardized using reference human sera in order to produce antibody titers expressed in international units (IU). In addition to high reproducibility, the new neutralization test appears to be sensitive, specific and rapid, and might thus represent a useful tool for the diagnosis of polio and other enterovirus infections.

Response to poliovirus immunization and type of feeding in babies of atopic families

The aim of the present study was to investigate the antibody response to oral poliovirus immunization, and to evaluate the infection morbidity in a group of infants with a positive family history of atopy who were soy‐protein‐formula fed during the first 6 months of life. We have selected and followed from birth to 4 yr, 107 babies, 18 of whom were exclusively soy‐protein‐formula fed during the first 6 months of life. All infants were seen at our clinic at the ages of 1, 3, 6, 9, and 12 months, and once‐a‐year afterwards. They regularly received the oral vaccine against polioviruses at the ages of 3, 5 and 12 months. At age 6, 12 and 24 months, poliovirus type 1, 2 and 3 antibodies were detected. There was no significant difference of seroconversion in the babies, breast, soy or breast and soy fed. Furthermore, no increase in infection morbidity was observed in the soy‐protein‐formula fed infants. Our data shows that soy‐protein‐formula feeding in the first 6 months of life does not induce any abnormal antibody response to the oral poliovirus vaccination, as well as no increase in infection morbidity.

Surveillance of acute flaccid paralysis in Italy: 1996-1997

The last case of poliomyelitis due to transmission of indigenous wild poliovirus occurred in Italy in 1982. To achieve the certification of the eradication of poliomyelitis in Italy, an active surveillance of acute flaccid paralysis (AFP) in the population aged less than 15 years was set up following the World Health Organization (WHO) guidelines. The survey started in 1996 with a pilot study involving 4 out of 21 regions, and was gradually extended to a national level in 1997. The two-year survey identified five patients with diagnosis of vaccine associated paralytic poliomyelitis (VAPP). Polioviruses type 2 and 3 Sabin-like were isolated and characterized in three of them. In the remaining two cases, samples were collected late after the onset of symptoms, and poliovirus could not be isolated. No wild polioviruses were detected during the survey. The rate of non-polio AFP found in Italy in 1997 was 0.61 cases per 100,000, which is lower than the level of 1.0 case per 100,000 considered as acceptable by the WHO. This was mainly due to the delay in organizing the hospital network and starting the active search of AFP cases in the largest and most densely populated regions. Therefore, the overall rate of AFP found in Italy underestimates the global effectiveness of the program, which however will be better evaluated in the next few years. This study is the first systematic attempt to determine the rate of AFP in Italy.

Paralytic poliomyelitis in Italy (1981–85)

Fifteen cases of presumptive poliomyelitis occurring in Italy between 1981–85 were studied in order to differentiate between paralysis caused by poliovirus and that of different etiology. Out of seven confirmed cases three were ≪ temporally associated with vaccination ≫.Three aspects are discussed: i) the need for a careful differential diagnosis of paralytic cases; ii) the overconcern about the problem of vaccine-associated cases: iii) the risk connected with re-importation of wild poliovirus strains.

Sporadic isolation of sabin-like polioviruses and high-level detection of non-polio enteroviruses during sewage surveillance in seven Italian cities, after several years of inactivated poliovirus vaccination.

ABSTRACT Sewage surveillance in seven Italian cities between 2005 and 2008, after the introduction of inactivated poliovirus vaccination (IPV) in 2002, showed rare polioviruses, none that were wild-type or circulating vaccine-derived poliovirus (cVDPV), and many other enteroviruses among 1,392 samples analyzed. Two of five polioviruses (PV) detected were Sabin-like PV2 and three PV3, based on enzyme-linked immunosorbent assay (ELISA) and PCR results. Neurovirulence-related mutations were found in the 5′ noncoding region (5′NCR) of all strains and, for a PV2, also in VP1 region 143 (Ile > Thr). Intertypic recombination in the 3D region was detected in a second PV2 (Sabin 2/Sabin 1) and a PV3 (Sabin 3/Sabin 2). The low mutation rate in VP1 for all PVs suggests limited interhuman virus passages, consistent with efficient polio immunization in Italy. Nonetheless, these findings highlight the risk of wild or Sabin poliovirus reintroduction from abroad. Non-polio enteroviruses (NPEVs) were detected, 448 of which were coxsackievirus B (CVB) and 294 of which were echoviruses (Echo). Fifty-six NPEVs failing serological typing were characterized by sequencing the VP1 region (nucleotides [nt] 2628 to 2976). A total of 448 CVB and 294 Echo strains were identified; among those strains, CVB2, CVB5, and Echo 11 predominated. Environmental CVB5 and CVB2 strains from this study showed high sequence identity with GenBank global strains. The high similarity between environmental NPEVs and clinical strains from the same areas of Italy and the same periods indicates that environmental strains reflect the viruses circulating in the population and highlights the potential risk of inefficient wastewater treatments. This study confirmed that sewage surveillance can be more sensitive than acute flaccid paralysis (AFP) surveillance in monitoring silent poliovirus circulation in the population as well as the suitability of molecular approaches to enterovirus typing.

Serum antibodies against coxsackie B1–6 viruses in type 1 diabetics

SummaryTwenty-two newly diagnosed insulin-dependent diabetics and 46 control subjects have been examined for Coxsackie B1–6 serum antibodies. Evidence for CoxB4 neutralizing antibodies at titers ≥ 16 and two seroconversions for this single virus type were shown in the diabetic group (31.8% of patients with titers ≥ 16 in comparison with 10% of controls; p=0.08). Reduced titers against CoxB2 and CoxB5 in diabetics were also observed. In agreement with the majority of publications, these data suggest the hypothesis that CoxB4 infections are frequently associated with type 1 diabetes, and the positive relationship found only for this type of virus may be related to greater tropism for the pancreas. The specificity of this viral infection, as shown by our serologic results, could suggest a possible causative role in the development of type 1 diabetes only in a limited number of cases. The authors propose new studies with monoclonal and IgM specific antibodies, obtained from new isolates, to detect more accurately the qualiquantitative differences between type 1 diabetics and controls.

Surveillance of acute flaccid paralysis (AFP) in Lombardy, Northern Italy, from 1997 to 2011 in the context of the national AFP surveillance system

An Acute Flaccid Paralysis (AFP) surveillance system was set up in Lombardy (Northern Italy) in 1997 in the framework of the national AFP surveillance system, as part of the polio eradication initiative by the World Health Organization (WHO). This surveillance system can now be used to detect Poliovirus (PV) reintroductions from endemic countries. This study aimed at describing the results of the AFP surveillance in Lombardy, from 1997 to 2011. Overall, 131 AFP cases in Lombardy were reported with a mean annual incidence rate of 0.7/100 000 children <15 years of age (range: 0.3/100 000–1.1/100 000). The sensitivity of the surveillance system was optimal from 2001–2003. The monthly distribution of AFP cases was typical with peaks in November, in January, and in March. The major clinical diagnoses associated with AFP were Guillain-Barré Syndrome (GBS, 40%) and encephalomyelitis/myelitis (13%). According to the virological results, no poliomyelitis cases were caused by wild PV infections, but two Vaccine-Associated Paralytic Paralysis (VAPP) cases were reported in 1997 when the Sabin oral polio vaccine (OPV) was still being administered in Italy. Since a surveillance system is deemed sensitive if at least one case of AFP per 100,000 children <15 years of age is detected each year, our surveillance system needs some improvement and must be maintained until global poliovirus eradication will be declared.

Large-Scale Survey of Human Enteroviruses in Wastewater Treatment Plants of a Metropolitan Area of Southern Italy

Human enteroviruses (HEVs) occur in high concentrations in wastewater and can contaminate receiving environmental waters, constituting a major cause of acute waterborne disease worldwide. In this study, we investigated the relative abundance, occurrence, and seasonal distribution of polio and other enteroviruses at three wastewater treatment plants (WWTPs) in Naples, Southern Italy, from January 2010 to December 2014. Influent and effluent samples from the three WWTPs were collected monthly. One hundred and sixty-one of the 731 wastewater samples collected (22.0%) before and after water treatment were CPE positive on RD cells; while no samples were positive on L20B cells from any WWTPs. Among the 140 non-polio enterovirus isolated from inlet sewage, 69.3% were Coxsackieviruses type B and 30.7% were Echoviruses. Among these, CVB3 and CVB5 were most prevalent, followed by CVB4 and Echo6. The twenty-one samples tested after treatment contained 6 CVB4, 5 CVB3, 3 Echo11, and 2 Echo6; while other serotypes were isolated less frequently. Data on viral detection in treated effluents of WWTPs confirmed the potential environmental contamination by HEVs and could be useful to establish standards for policies on wastewater management.

Acute flaccid paralysis surveillance in bosnia and herzegovina: Recent isolation of two sabin like type 2 poliovirus

The WHO Regional Commission for the Certification of Poliomyelitis Eradication has recently indicated Bosnia and Herzegovina (B&H) as a high risk country for transmission, following importation, of wild poliovirus (WPV) or circulating vaccine‐derived poliovirus (cVDPV). We analyzed data on Acute Flaccid Paralysis (AFP) surveillance between 2007 to 2016, and the trend of polio immunization coverage in B&H. The majority of AFP cases was recorded in 2016 suggesting an enhancement of the AFP surveillance activities. However, the decline in the immunization coverage, around 74%, and the isolation of two Sabin‐like poliovirus type 2 strains, one of them close to a VDPV, require a particular attention in the area. Although B&H has successfully maintained its polio‐free status since 2002 several challenges need to be addressed.

Poliovirus and Other Enteroviruses from Environmental Surveillance in Italy, 2009–2015

Within the initiatives for poliomyelitis eradication by WHO, Italy activated an environmental surveillance (ES) in 2005. ES complements clinical Acute Flaccid Paralysis (AFP) surveillance for possible polio cases, detects poliovirus circulation in environmental sewage, and is used to monitor transmission in communities. In addition to polioviruses, the analyses comprised: (i) the monitoring of the presence of non-polio enteroviruses in sewage samples and (ii) the temporal and geographical distribution of the detected viruses. From 2009 to 2015, 2880 sewage samples were collected from eight cities participating in the surveillance. Overall, 1479 samples resulted positive for enteroviruses. No wild-type polioviruses were found, although four Sabin-like polioviruses were detected. The low degree of mutation found in the genomes of these four isolates suggests that these viruses have had a limited circulation in the population. All non-polio enteroviruses belonged to species B and the most frequent serotype was CV-B5, followed by CV-B4, E-11, E-6, E-7, CV-B3, and CV-B2. Variations in the frequency of different serotypes were also observed in different seasons and/or Italian areas. Environmental surveillance in Italy, as part of the ‘WHO global polio eradication program’, is a powerful tool to augment the polio surveillance and to investigate the silent circulation or the re-emergence of enteroviruses in the population.