Gabriele Buttinelli

Waterborne Outbreak of Norwalk-Like Virus Gastroenteritis at a Tourist Resort, Italy

In July 2000, an outbreak of gastroenteritis occurred at a tourist resort in the Gulf of Taranto in southern Italy. Illness in 344 people, 69 of whom were staff members, met the case definition. Norwalk-like virus (NLV) was found in 22 of 28 stool specimens tested. The source of illness was likely contaminated drinking water, as environmental inspection identified a breakdown in the resort water system and tap water samples were contaminated with fecal bacteria. Attack rates were increased (51.4%) in staff members involved in water sports. Relative risks were significant only for exposure to beach showers and consuming drinks with ice. Although Italy has no surveillance system for nonbacterial gastroenteritis, no outbreak caused by NLV has been described previously in the country.

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.

Importation and circulation of poliovirus in Bulgaria in 2001

OBJECTIVE To characterize the circumstances in which poliomyelitis occurred among three children in Bulgaria during 2001 and to describe the public health response. METHODS Bulgarian authorities investigated the three cases of polio and their contacts, conducted faecal and serological screening of children from high-risk groups, implemented enhanced surveillance for acute flaccid paralysis, and conducted supplemental immunization activities. FINDINGS The three cases of polio studied had not been vaccinated and lived in socioeconomically deprived areas of two cities. Four Roma children from the Bourgas district had antibody titres to serotype 1 poliovirus only, and wild type 1 virus was isolated from the faeces of two asymptomatic Roma children in the Bourgas and Sofia districts. Poliovirus isolates were related genetically and represented a single evolutionary lineage; genomic sequences were less than 90% identical to poliovirus strains isolated previously in Europe, but 98.3% similar to a strain isolated in India in 2000. No cases or wild virus isolates were found after supplemental immunization activities were launched in May 2001. CONCLUSIONS In Bulgaria, an imported poliovirus was able to circulate for two to five months among minority populations. Surveillance data strongly suggest that wild poliovirus circulation ceased shortly after supplemental immunization activities with oral poliovirus vaccine were conducted.

Antigenic sites of coxsackie A9 virus inducing neutralizing monoclonal antibodies protective in mice

A panel of murine IgG monoclonal antibodies (MAbs) was produced against coxsackievirus A9 (CAV9). Fifty-nine MAbs reactive in ELISA with purified CAV9 were identified. Eighteen of them could efficiently inhibit infection by CAV9 but not coxsackieviruses B. Neutralization-resistant CAV9 variants to four different MAbs were isolated and tested for resistance to neutralization by other MAbs of the panel. Three groups of reactivity including 10, 7, and 1 MAbs were thus identified. Sequencing of neutralization-escape virus mutants showed that neutralization by one MAb group was affected by change of VP3 amino acids 62 or 69. For the second group of reactivity, mutations included amino acids 154 or 165 of VP2. The only MAb of the third group selected for a change at residue 70 of VP2. Protection studies in a newborn mouse model of myositis suggested that either epitopes in VP2 or in VP3 mediate protection from CAV9 infection in vivo.

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.

Three cases of paralytic poliomyelitis associated with type 3 vaccine poliovirus strains in Bulgaria

Oral poliovirus vaccine (OPV) can cause, in extremely rare cases vaccine‐associated paralytic poliomyelitis in recipients, or contacts of vaccinees. Three cases of vaccine‐associated paralytic poliomyelitis (two contacts and one recipient) occurred in the Bourgas region of Bulgaria in the spring of 2006. The first two cases, notified as acute flaccid paralysis, were 55 days old unvaccinated twin brothers, having been in contact with vaccinees. The third case concerned a 4‐month‐old infant who had received the first OPV dose 37 days prior to the onset of illness. Complete clinical, epidemiological, virological, serological and molecular investigations of the children with paralysis and their contacts were undertaken. In all the three cases type 3 polioviruses were isolated from fecal samples and characterized as Sabin‐like poliovirus strains. Type 3 polioviruses isolated from the twin brothers demonstrated by sequence analysis U‐to‐C back mutation at nt 472 of the 5′ UTR, known to correlate with neurovirulence, and mutation in the VP1 region. Type 3 poliovirus isolated from the third child demonstrated in the 3D sequenced region a recombination with Sabin type 1 poliovirus. In the latter region, three silent mutations and one, resulting in amino acid substitution, were also observed. The clinical, epidemiological and virological data and the neurological sequelae observed 60 days following the onset of paralysis, confirmed the diagnosis of vaccine‐associated paralytic poliomyelitis in all the three patients. J. Med. Virol. 81:1661–1667, 2009.

Severe pertussis infection in infants less than 6 months of age: Clinical manifestations and molecular characterization

ABSTRACT We conducted a study to determine the main traits of pertussis among unimmunized infants less than 6 months of age. From August 2012 to March 2015, 141 nasopharyngeal aspirates (NPAs) were collected from infants with respiratory symptoms attending 2 major hospitals in Rome. Clinical data were recorded and analyzed. Lab-confirmation was performed by culture and realtime PCR. B. pertussis virulence-associated genes (ptxP, ptxA and prn), together with multilocus variable-number tandem repeat analysis (MLVA), were also investigated by the sequence-based analysis on the DNAs extracted from positive samples. Antibiotic susceptibility with Etest was defined on 18 viable B. pertussis isolates. Samples from 73 infants resulted positives for B. pertussis. The median age of the patients was 45 d (range 7–165); 21 infants were treated with macrolides before hospital admission. Cough was reported for a median of 10 d before admission and 18 d after hospital discharge among infected infants, 84% of whom showed paroxysmal cough. No resistance to macrolides was detected. Molecular analysis identified MT27 as the predominant MLVA profile, combined with ptxP3-ptxA1-prn2 associated virulence genes. Although our data may not be generalized to the whole country, they provide evidence of disease severity among infants not vaccinated against pertussis. Moreover, genetically related B. pertussis strains, comprising allelic variants of virulence associated genes, were identified.

Pertussis in infants less than 6 months of age and household contacts, Italy April 2014

We report pertussis cases in 4 infants less than 6 months admitted with symptoms compatible with pertussis to the intensive care unit of the Università Cattolica del Sacro Cuore in Rome, April 2014. Realtime PCR confirmed pertussis diagnosis for the 4 infants, 2 of them were cousins, and for the household contacts of 1 of them. Analysis of pertussis toxin, its promoter and pertactin was also performed. First of all, this report emphasizes the need to investigate household contact of infants with pertussis; secondly, to evaluate the selective vaccination of household members of newborns as an effective program to reduce pertussis in infants.

Protection against Pertussis in Humans Correlates to Elevated Serum Antibodies and Memory B Cells

Pertussis is a respiratory infection caused by Bordetella pertussis that may be particularly severe and even lethal in the first months of life when infants are still too young to be vaccinated. Adults and adolescents experience mild symptoms and are the source of infection for neonates. Adoptive maternal immunity does not prevent pertussis in the neonate. We compared the specific immune response of mothers of neonates diagnosed with pertussis and mothers of control children. We show that women have pre-existing pertussis-specific antibodies and memory B cells and react against the infection with a recall response increasing the levels specific serum IgG, milk IgA, and the frequency of memory B cells of all isotypes. Thus, the maternal immune system is activated in response to pertussis and effectively prevents the disease indicating that the low levels of pre-formed serum antibodies are insufficient for protection. For this reason, memory B cells play a major role in the adult defense. The results of this study suggest that new strategies for vaccine design should aim at increasing long-lived plasma cells and their antibodies.

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.