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BACKGROUND Besides seasonal influenza viruses (IV), several other pathogens-including respiratory syncytial virus (RSV)-are involved in clinically undistinguished influenza-like illnesses (ILIs). This study aimed at investigating the contribution of RSV in ILI cases in Lombardy (Northern Italy) during four consecutive winter seasons. MATERIALS AND METHODS In the framework of influenza surveillance, respiratory samples from ILI outpatients were collected from 2014-2015 to 2017-2018 season. IV-negative swabs were included in the study and analyzed to detect and molecularly characterize RSV-A and RSV-B. RESULTS A total of 12.9% (135/1047) of samples were positive to RSV that was mostly detected among children ≤5 years (51/183, 27.8%) and those aged 6 to 15 years (30/158, 18.9%), whereas elderly >65 years accounted for 12% of RSV cases (15/125). The median start of RSV epidemic was in the end of November, with a peak in mid-February and a width of nearly 4 months, almost overlapping seasonal influenza epidemic. RSV-A and RSV-B co-circulated in all considered seasons, with RSV-B predominating on RSV-A (63.6% vs 36.4%; P

An Italian 13-year-old boy immunosuppressed due to kidney transplant presented in November 2018 with acute flaccid paralysis with anterior horn cell involvement resembling the clinical, radiological, and laboratory features of poliomyelitis. Enterovirus was molecularly identified in cerebral spinal fluid and stool samples and the sequence analysis of the VP1 gene of enterovirus genome revealed the presence of Echovirus 30 both in CSF and in stool samples. Echovirus 30 is an emerging neurotropic virus able to cause outbreaks of aseptic meningitis and meningoencephalitis all over the world, but acute flaccid paralysis is not a classical manifestation. A 6-month follow-up revealed a poor outcome with severe motor deficits and only slight improvement in disability. Clinicians must be aware of the possible role of Echovirus 30 in acute flaccid paralysis and active surveillance should consider the possible influence of immunosuppression on the symptoms caused by the widening spectrum of enterovirus infections.

Enteroviruses (EVs) and Hepatitis A Viruses (HAVs) are human pathogens with a wide spectrum of clinical manifestations. The monitoring of sewage samples enables to monitor the EVs and HAVs in circulation among the general population and recognize possible outbreaks. This study focused on the molecular characterization and phylogenetic analysis of the EVs and HAVs identified in 33 sewage samples collected every 15 days at the influent of a wastewater treatment plant located in Northern Italy from March to October 2016. According to the results of the molecular characterization, the most frequently identified viruses were Echovirus 6 (E-6), E-11 and HAV-IA. The phylogenetic analyses indicated the rapid genetic evolution of E-6 and E-1; noteworthy, most E-11 strains clustered with a strain isolated from a clinical sample collected in the same geographical area over the same period by our laboratory. Most of the HAV strains detected clustered with epidemic HAV-IA strains identified during the European hepatitis A outbreak that occurred in 2016-2017 affecting men who have sex with men (MSM). The detection of environmental HAV strains before and at the beginning of its spread amongst humans demonstrated that this outbreak could have been predicted by monitoring sewage samples. Moreover, conducting a genetic comparison between the HAV and EV strains identified in sewage and clinical samples may improve knowledge of viral epidemiology. EV and HAV molecular environmental surveillance may prove useful for identifying viral circulation and for issuing early warning alerts on possible outbreaks among the human population.

Background. Enteroviruses (EVs) can cause infections and outbreaks of mild to severe diseases, such as central nervous system (CNS) and systemic infections. The contribution of EVs to acute CNS/systemic infections requiring hospitalization was assessed by analysing data extracted from virology laboratory database. Methods. Real-life data obtained from two molecular virology laboratories located in Northern Italy were retrieved from databases and analysed retrospectively. The queries used to extract the data were (i) requests for EV-RNA detection in clear cerebrospinal fluid (CSF) specimens collected from hospitalized patients with suspected acute CNS (including aseptic meningitis, encephalitis, and acute flaccid myelitis/paralysis) or systemic infections (sepsis-like illness or fever (≥ 38°C) of unknown origin), (ii) CSF samples collected from January 1st, 2015, to December 31st, 2017. Results. 582 requests of EV-RNA detection in CSF samples collected from as many patients of any age were recorded. EV-RNA was detected in 4.5% of the CSF samples; 92.3% of EV-positive cases were patients<15 years, 58.3% of whom were < 3 months. EVs circulated all-year-round, and the highest EV-positive rates were observed from May to August. The risk of EV infection and the relative illness ratio value among children<1−year−old were significantly higher than those observed for older patients. Conclusions. EV surveillance should be carried out for all pediatric patients<15 years and especially children less than 1 year of age with clinically suspected CNS infection/systemic infections. The implementation of a laboratory-based surveillance established for analysing the virological data provided by laboratories that routinely perform EV molecular testing may enable us to determine the impact of EVs that can cause infections requiring hospitalization.

BackgroundCongenital Cytomegalovirus (cCMV) is a serious global public health issue that can cause irreversible fetal and neonatal congenital defects in symptomatic or asymptomatic newborns at birth. In absence of universal cCMV screening, the retrospective diagnosis of cCMV infection in children is only possible by examining Dried Blood Spot (DBS) samples routinely collected at birth and stored for different time spans depending on the newborn screening regulations in force in different countries. In this article, we summarize the arguments in favor of long-term DBS sample storage for detecting cCMV infection.Main textCMV infection is the most common cause of congenital infection resulting in severe defects and anomalies that can be apparent at birth or develop in early childhood. Sensorineural hearing loss is the most frequent consequence of cCMV infection and may have a late onset and progress in the first years of life. The virological diagnosis of cCMV is essential for clinical research and public health practices. In fact, in order to assess the natural history of CMV infection and distinguish between congenital or acquired infection, children should be diagnosed early by analyzing biological samples collected in the first weeks of life (3 weeks by using viral culture and 2 weeks by molecular assays), which, unfortunately, are not always available for asymptomatic or mildly symptomatic children. It now seems possible to overcome this problem since the CMV-DNA present in the blood of congenitally infected newborns can be easily retrieved from the DBS samples on the Guthrie cards routinely collected and stored within 3 days from birth in the neonatal screening program for genetic and congenital diseases. Early collection and long-term storage are inexpensive methods for long-term bio-banking and are the key points of DBS testing for the detection of cCMV.ConclusionDBS sampling is a reliable and inexpensive method for long-term bio-banking, which enables to diagnose known infectious diseases - including cCMV - as well as diseases not jet recognized, therefore their storage sites and long-term storage conditions and durations should be the subject of political decision-making.

A previously healthy 6-year-old boy was admitted to hospital with hypotonia and hyposthenia of lower limbs. Electromyography and slow motor nerve conduction velocity test identified a lower limb acute motor axonal neuropathy. Brain and spinal cord magnetic resonance imaging demonstrated multifocal cortical gray matter lesions in both cerebral hemispheres consistent with gray matter acute disseminated encephalitis otherwise with viral/Mycoplasma pneumoniae encephalitis, and signs of involvement of anterior nerve roots of the cauda equina consistent with Guillain-Barré syndrome. The patient resulted negative to routinely bacterial and viral investigations but positive to human parechovirus that sequence analyses confirmed as type 6. Intravenous immunoglobulins and methylprednisolone treatment were administered but did not relieve the symptoms of Guillain-Barré syndrome. The disease improved gradually over the next 3-month follow-up with a complete remission of both central and peripheral nervous system symptoms.

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.

Aim: This paper discusses the unexpected findings from the RotaNet-Italy hospital-based surveillance study carried out in northern Italy. Materials & methods: From September 2015 to August 2016, 51 rotavirus-A (RVA) positive fecal samples were collected from children aged less than 15 years, who were hospitalized for acute gastroenteritis in Lombardy, northern Italy. Results: Molecular characterization revealed the predominance of the uncommon G12P[8] RVA strain, which was detected in 49% of cases. Phylogenetic analysis showed that these G12 strains clustered into lineage 3. Conclusion: To our knowledge, this is the first study on the G12P[8] genotype’s introduction in northern Italy. Our findings emphasize the importance of the surveillance of RVA gastroenteritis with the aim of obtaining new insight into the unusual newly emerging RVA strains.

Background/aim: Autism spectrum disorders (ASD) are neurodevelopmental disorders without a definitive etiology in most cases. Environmental factors, such as viral infections, have been linked with anomalies in brain growth, neuronal development, and functional connectivity. Congenital cytomegalovirus (CMV) infection has been associated with the onset of ASD in several case reports. The aim of this study was to evaluate the prevalence of congenital CMV infection in children with ASD and in healthy controls. Patients and methods: The CMV genome was tested by polymerase chain reaction (PCR) on dried blood spots collected at birth from 82 children (38 with ASD and 44 controls). Results: The prevalence of congenital CMV infection was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212). Conclusion: The infection rate was about 10-fold higher in patients with ASD than in the general Italian population at birth. For this reason, detection of CMV-DNA on dried blood spots could be considered in the work-up that is usually performed at ASD diagnosis to rule-out a secondary form. Given the potential prevention and treatment of CMV infection, this study could have intriguing consequences, at least for a group of patients with ASD. Keywords: Cytomegalovirus; congenital infection; nervous system.

Besides the influenza virus (IV), several other viruses are responsible for influenza-like illness (ILI). Although human parechoviruses (HPeVs) and enteroviruses (EVs) may impact on ILI, limited data on their epidemiological characteristics are available. During seven consecutive winter seasons (from 2010–2011 to 2016–2017), within the framework of an influenza surveillance system (InfluNet), 593 respiratory swabs were collected from children ≤5 years of age with ILIs. Molecular detection showed that 58.3 % of swabs were positive for at least one of the viruses under study: 46 % for IV, 13 % for EV and 5.4 % for HPeV. A single virus was identified in 51.3 % of samples while more than one virus was detected in 7 % of the samples. The risk of contracting IV was higher than the risk associated with EV, which in turn was higher than the risk of contracting HPeV. The risk of developing an IV infection was twofold greater in children >3 years than in those ≤3 years, who had higher risk of EV/HPeV infection. The frequency of EV/HPeV-positive swabs increased significantly during the 2016–2017 winter season compared to the previous six seasons. Sixteen EV genotypes were identified belonging to species A and B. HPeV-1 was the most frequently detected genotype, followed by -6 and -3. In this study, IV was mainly responsible for ILI, however EV and HPeV were also involved and particularly affected children ≤3 years of age. Influenza surveillance samples could provide us with valuable insight into the epidemiological features of viruses involved in ILI.

Aim: Several authors have hypothesized an association between congenital viral infections and the onset of autism spectrum disorders (ASD). We aimed to assess the prevalence of congenital varicella zoster virus (VZV) infection in patients with ASD. Patients & methods: Congenital infection by VZV was evaluated in a cohort of 38 children with ASD and in 44 healthy controls. PCR for VZV-DNA performed on dried blood spots collected at birth. Results & conclusion: No VZV infection was detected in both groups. With the limitation of the small sample size of this study, the results are not in favor of a role of VZV in the etiology of ASD.

Background/aim Autism spectrum disorders (ASD) are neurodevelopmental disorders without a definitive etiology in most cases. Environmental factors, such as viral infections, have been linked with anomalies in brain growth, neuronal development, and functional connectivity. Congenital cytomegalovirus (CMV) infection has been associated with the onset of ASD in several case reports. The aim of this study was to evaluate the prevalence of congenital CMV infection in children with ASD and in healthy controls. Patients and methods The CMV genome was tested by polymerase chain reaction (PCR) on dried blood spots collected at birth from 82 children (38 with ASD and 44 controls). Results The prevalence of congenital CMV infection was 5.3% (2/38) in cases and 0% (0/44) in controls (p=0.212). Conclusion The infection rate was about 10-fold higher in patients with ASD than in the general Italian population at birth. For this reason, detection of CMV-DNA on dried blood spots could be considered in the work-up that is usually performed at ASD diagnosis to rule-out a secondary form. Given the potential prevention and treatment of CMV infection, this study could have intriguing consequences, at least for a group of patients with ASD.

Background Congenital Cytomegalovirus (cCMV) is the most common cause of non-genetic hearing loss in childhood. A newborn hearing screening program (NHSP) is currently running in Italy, but no universal cCMV nor statewide hearing-targeted CMV screening programs have been implemented yet. This observational monocentric study was aimed at estimating the rate of cCMV infections identified by CMV-DNA analysis on Dried Blood Spots (DBS) samples in deaf children identified via NHSP in Northern Italy in the period spanning from 2014 to 2018. Methods Children with a confirmed diagnosis of deafness and investigated for CMV-DNA by nucleic acid extraction and in-house polymerase-chain reaction (PCR) on stored newborns screening cards (DBS-test) were included in this study. Deafness was defined by a hearing threshold ≥20 decibel (dB HL) by Auditory Brainstem Responses (ABR); all investigated DBS samples were collected within 3 days of life. Results Overall, 82 children were included (median age: 3.4 months; lower-upper quartiles: 2–5.3 months; males: 60.9%). Most of them (70.7%) presented bilateral hearing loss with a symmetrical pattern in 79.3% of the cases. ABR thresholds were ≥ 70 dB HL (severe/profound deafness) in 46.5% of children. Among all tested children, 6.1% resulted positive for cCMV. The rate of severe/profound deafness was statistically higher in children with cCMV infection. Conclusions The addition of DBS-test to the NHSP allowed the identification, in their first months of life, of a cCMV infection in 6.1% of children who had failed NHS. The introduction of a targeted CMV screening strategy could help clinicians in the differential diagnosis and in the babies’ management. DBS samples can be considered a “universal newborns biobank”: their storage site and duration should be the subject of political decision-making.

Between September and October 2018, an enterovirus D68 (EV-D68) outbreak occurred in patients hospitalised with severe acute respiratory infection in northern Italy; 21 laboratory-confirmed cases were reported. Phylogenetic analysis revealed that 16/20 of the EV-D68 sequences belonged to a divergent group within the sub-clade D1. Since its upsurge, EV-D68 has undergone rapid evolution with the emergence of new viral variants, emphasising the need for molecular surveillance that include outpatients with respiratory illness.

JC virus (JCV) is a widespread member of the Polyomaviridae family. Following primary infection, which occurs asymptomatically during childhood, JCV establishes latency in the host. JCV seroprevalence can reach 80 % in healthy adults, but the age of viral exposure has not been yet characterized. This study was conducted to define JCV seroprevalence in Italian infants and to estimate the date of primary infection. A JCV viral protein 1 (VP1)-GST fusion protein was used in conjunction with a homemade indirect enzyme-linked immunosorbent assay (ELISA) to test for the presence of IgG antibodies to JCV in 981 serum samples collected from 644 Italian infants of different ages (1 day to 3 years old) and in 102 breast milk samples. IgM antibody presence was also evaluated in longitudinally collected samples from 17 selected children. JCV antibody prevalence and normalized optical density (nOD) were calculated. For the longitudinal analysis, generalized estimating equation techniques and spline functions were used to estimate the possible non-linear effects of time on antibody production kinetics. JCV IgG was detected in 71.8 % of the sera. Prevalence increased over time from 46.1 % (1 month old) to 80.7 % (12 months old), 85.9 % (24 months old), and 85.5 % (36 months old). As determined by nOD, the longitudinal analysis of serum IgG amounts in children of this study (ages 1 day to 3 years old) illustrated IgG kinetic changes with statistically significant trends (p = 0.001). One-month-old children were largely negative for JCV IgM (82.4 %), and 58.8 % of children produced JCV IgM within the second and sixth months of life. JCV IgG was detected in 27.3 % of breast milk samples. JCV primary infection likely occurs before 6 months of age, and a sizeable percentage of Italian infants will become JCV seropositive within 2 years of age. This study can be used to determine the optimal age for potential future JCV vaccination in infants.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis in young (5 years of age) children, causing up to 450.000 deaths worldwide, mostly in developing countries. VP7 (G-type) and VP4 (P-type) genotypes are the basis for the binary RVA classification. Although at least 27 G-types and 37 P-types of rotavirus are presently known, most RVA infections in humans worldwide are associated with the five major G/P combinations G1P[8], G2P[4], G3P[8], G4P[8] and G9P[8]. During RVA gastroenteritis surveillance in Italy, a total of 1112 samples collected from children hospitalized with acute gastroenteritis in 2013 were RVA positive and were genotyped following standardized protocols from the EuroRotaNet. Most strains analyzed belonged to the five major human genotypes. Among these common strains, 22 G4P[8] RVA strains from different Italian regions were subjected to nucleotide sequencing of their VP4, VP6, VP7 and NSP4 genes to investigate their evolution. The phylogenetic analysis showed that the Italian strains belonged to lineage G4-I for VP7 and to lineage P[8]-III for VP4, in line with the modern G4P[8] RVA strains detected in children worldwide. The phylogenetic trees revealed high degrees of nucleotide identity between the RVA strains involved in this study and G4P[8] strains detected previously in Europe, Asia and Africa, but also demonstrated at least three separate evolution clusters within the same lineage. Based on the amino acid sequences deduced for their hypervariable regions, both the VP7 and VP8* proteins of the Italian G4P[8] RVA strains presented amino acid substitutions near known neutralizing epitopes.

Background: Polyomavirus (PyV) infection is common, ranging from 60% to 100% depending on the virus. The urinary excretion rates of JC virus (JCV) and BK virus (BKV) have been extensively studied, but less is known about the more recently discovered PyVs. Objectives: To investigate the urinary excretion of Merkel cell PyV (MCPyV), which is associated with Merkel cell carcinoma (MCC), in kidney transplant recipients and healthy subjects, as well as those of lymphotropic polyomavirus (LPV), which was isolated from the B-cells of African green monkeys but has also been found in human blood, JCV and BKV. Study design: Urine samples were collected from 62 adult (ATP) and 46 pediatric (PTP) kidney transplant recipients and from 67 adult (AHC) and 40 pediatric (PHC) healthy controls. DNA was isolated and analyzed using real-time PCR (Q-PCR) to determine the viral loads of MCPyV, LPV, JCV and BKV. Results: MCPyV DNA was more frequently detected (p

Background Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young (aged

Although in the last years poliovirus (PV) transmission has been reported at the lowest levels ever recorded, the spread of virus from endemic countries endures; the high levels of immigration flows across the Mediterranean Sea jeopardize Italy for PV reintroduction. The World Health Organization (WHO) strategic plan for global poliomyelitis (polio) eradication indicates the nationwide surveillance of Acute Flaccid Paralysis (AFP) as the gold standard for detecting cases of polio. In addition, the Environmental Surveillance (ES), seeking the presence of PV and Non-Polio Enterovirus (NPEV) in sewage, is recognized as a powerful tool to confirm PV circulation in absence of AFP cases, especially in polio-free countries. Here we report the results of AFP surveillance (AFPS) and ES in Lombardy (Northern Italy) from 2012 to 2015. Forty-eight AFP cases were identified during the study period. No AFP case was caused by PV infection. NPEVs were identified in 6.3% (3/48) of AFP cases. The annual AFP incidence rate was 0.87/100'000 children15 y in 2012, 1.42/100'000 in 2013, 1.02/100'000 in 2014, and 0.47/100'000 in 2015; according to WHO indicators, the sensitivity of AFPS was adequate in 2013 and 2014. Completeness of case investigation raised progressively during the study period to achieve the WHO standards in 2014 (92.3%) and 2015 (100%). Completeness of follow-up increased from 72.7% in 2012 to 100% in 2014. In the framework of the ES conducted in Milan, 268 wastewater samples were collected from 2012 to 2015 and no PVs were isolated. In contrast, NPEVs were detected in 65.3% (175/268) of samples. All NPEVs characterized belonged to enterovirus species B: echovirus type 11, 6 and 3 were the most frequently detected viruses, representing 29.1% (41/141), 20.6% (29/141) and 9.2% (13/141) of genotyped NPEVs, respectively. Keeping strong and encouraging both AFPS and ES is crucial to ensure that PV will not return unnoticed in Italy - as well as in other polio-free countries - and, as a final point, to achieve the global polio eradication goal.

Aims Human Enteroviruses (HEVs) infections have a significant impact on public health, being implicated in outbreaks of meningitis, encephalitis, hand-foot-mouth disease and other acute and chronic manifestation. In the strategic plan for poliomyelitis eradication, the environmental surveillance of poliovirus (PV) has been identified by the World Health Organization (WHO) as an activity that can complement the surveillance of polio. Having wastewater samples available for PV surveillance allows us to study nonpolio enteroviruses (NPEVs) circulating in the study population, which are widely spread. Methods and Results This study was carried out according to the WHO guidelines for environmental surveillance of PV and analysed the circulation of PV and NPEVs through the isolation of viruses in cell cultures in Milan area; from 2006 to 2010, 321 wastewater samples were collected, regularly over time, at the inlet of three diverse waste water treatment plants (WWTPs). Culturable HEVs were isolated in 80% of sewage samples: all isolates belonged to the HEV-B group and those circulating more intensely were CVB5 and Echo 6, while CVB4 was the predominant serotype found in 2010. In this study, two type 2 PVs were isolated, both characterized as Sabin like. Conclusion Environmental monitoring of HEVs in Milan has proved to be an interesting tool to investigate the circulation and distribution of viruses. Significance and Impact of the Study The detection of PV and other NPEV could be predictive of possible re-emergence of these viruses with an impact on public health. NPEV monitoring could also be a powerful public health tool to investigate the possible role of NPEV in different clinical manifestations.

Group A rotaviruses (RVA) are the leading cause of acute gastroenteritis (AGE) in young children worldwide. The RVA outer capsid layer is composed of the VP7 and VP4 proteins. The VP7 (G-type) and VP4 (P-type) genotypes are the basis for the binary RVA nomenclature. At least 27 G-types and 37 P-types of RVA are currently known, but most of human infections are related to the five major genotypes G1P[8], G2P[4], G3P[8], G4P[8], and G9P[8]. Every year G1P[8] strains cause approximately 50% of all symptomatic RVA infections reported in children in Italy. Fifteen G1P[8] RVA strains identified in different areas of Italy between 2010 and 2014 were selected. Strains were subjected to nucleotide sequencing of the VP7, VP4, VP6 and NSP4 genes to investigate their genetic variability with respect to geographic area and date of detection. Phylogenetic analyses showed that the 15 G1P[8] RVA strains belonged to two different lineages for both the VP7 and NSP4 genes, and showed some intra-lineage diversity in VP4 and VP6 genes. Similarities between strains correlated by either area or date of detection were also evaluated. The results obtained by phylogenetic analyses were confirmed analyzing the deduced amino acid sequences of the VP7, VP4, VP6 and NSP4 proteins of the G1P[8] RVA strains, detecting several substitutions in all proteins. The genetic variability observed between common G1P[8] RVAs highlights the constant evolution of the RVA genome through random point mutations (genetic drift) and intra-genotype reassortment (genetic shift). The evolution and diversity of the G1 RVA strains observed in this study can be related to the naturally acquired herd immunity, which represents the main mechanism of selective pressure in Italy, where mass anti-rotavirus vaccination was missing during the years of the study.

We report 2 neonates with human parechoviruses type 3 encephalitis. Both newborns presented with fever, irritability and seizures. Cerebrospinal fluid analyses were normal, but magnetic resonance imaging revealed white matter damage, suggesting human parechoviruse infection. Human parechoviruses type 3-RNA was detected in cerebrospinal fluid samples and in blood, stool, urine and respiratory samples, indicating the dissemination of the virus.

Nowadays, several screening strategies are available to prevent cervical cancer, but inadequate resources, sociocultural barriers, and sampling issues impede their success in low-income countries. To overcome these issues, this study aimed to evaluate the performance of human papillomavirus (HPV) testing from dried urine spots (DUS). Eighty-eight urine samples (including 56 HPV DNA positive specimens) were spotted on filter paper, dried, and stored in paper-bags. HPV DNA was detected from the DUS after 1 week and 4 weeks of storage using a polymerase chain reaction (PCR) assay. The sensitivity, specificity, and concordance of the DUS-based HPV test were evaluated by comparing the results with those of HPV testing on fresh urine samples as the gold standard. The sensitivity of the test was 98.21% (95% CI: 90.56–99.68) for DUS stored for 1 week and 96.42% (95% CI: 87.88–99.01) for DUS stored for 4 weeks. The specificity was 100% (95% CI: 89.28–100) at both time points. The concordance between DUS and fresh urine HPV testing was “almost perfect” using theκstatistic. These preliminary data suggest that a DUS-based assay could bypass sociocultural barriers and sampling issues and therefore could be a suitable, effective tool for epidemiological surveillance and screening programs, especially in low-income countries.

Cytomegalovirus (CMV) infection is the most frequent congenital infection in humans. Its prevalence and the frequency of disabling sequelae must be assessed in different populations to permit the formulation or assessment of preventive measures.To check the prevalence of congenital infection and seroprevalence in Italy; to verify the rate of sensorineural hearing loss (SNHL) in infected infants; and to assess the proportion of children with SNHL attributable to congenital CMV infection.Diagnosis of congenital CMV infection was sought in 9032 children born between March 2002 and February 2003 by testing for viral DNA [CMV dried blood spot (DBS) test] in each newborn's Guthrie card and confirmation by isolation of CMV from urine collected in the first 3 weeks of life; CMV IgG testing in 1200 women of childbearing age; clinical and audiologic tests in the first 24 months for infected children; CMV DBS tests on the Guthrie cards collected from screening centers for 77 children (3 months-5 years) presenting SNHL of 40 dB or more.CMV infection was diagnosed in 14 asymptomatic and 2 symptomatic newborns (0.18%). CMV seroprevalence was 80%. In 2 infected infants, transient, unilateral SNHL was found. Nineteen of the 71 children with SNHL70 dB were congenitally infected.The prevalence of congenital CMV infection is low in Italy. Population characteristics limiting the circulation of CMV strains in adult women might explain this. The fact that CMV contributes to significant SNHL highlights the need for preventive measures.

Measles and rubella are infectious diseases and humans are the only reservoir of these infections. Effective vaccines are available with the potential for measles (MV) and rubella (RuV) virus eradication. According to the World Health Organisation guidelines, a national plan was approved in Italy in 2013 to achieve the MV/RuV elimination by 2015, and active MV/RuV integrated surveillance initiated. Towards this purpose, a regional laboratory centre was set up on 1 September 2013 in Lombardy, Northern Italy. This paper aimed at: (1) evaluating measles-mumps-rubella (MMR) vaccine coverage and MV/RuV notified cases retrospectively; and (2) presenting the results of MV/RuV integrated surveillance (laboratory confirmed and viral genetic profiles). The 95% target for MMR vaccine coverage was achieved in 2001, and coverage increased until 2007 (96.6%), but then a decreasing trend was observed. Since 2000 to 2014, 3026 rubella cases were notified, with nearly 58% of them in the 2002 epidemic. From 2009, less than 45 RuV cases per year were reported. From 2000 to 2014, 5024 measles cases were notified. Since 2008, three large outbreaks (in 2008, 2011, and 2013) were observed. From data obtained during our surveillance activity, there were no rubella cases, and 57.5% (46/80) collected samples were MV-positive by real-time RT-PCR. A fragment of the MV N gene was sequenced from 37 MV-positive samples; D8, D9, and B3 genotypes were detected. Data obtained retrospectively and from active surveillance underline the necessity to achieve and maintain high vaccination coverage and to improve surveillance and the effectiveness of healthcare actions.

As the regional influenza reference centre operating within the Italian network InfluNet, here we report data on virological and epidemiological surveillance of influenza, as well as on the vaccination coverage rates achieved in Lombardy (Northern Italy) over 10 consecutive winter seasons (2004–2014). Over the past 10 years, influenza vaccine coverage declined both in the general population (from 15.7% in 2004–2005 to 11.7% in 2013–2014) and in the vaccine-target population of individuals ≥65-y-of-age (from 65.3% in 2004–2005 to 48.6% in 2013–2014) and is far below the minimum planned threshold level (75%). The highest influenza-like illness (ILI) rates were recorded during the 2004–2005 and 2009–2010 epidemics (peak incidence: 12.04‰ and 13.28‰, respectively). Both seasons were characterised by the introduction of novel viral strains: A/Fujian/411/2002(H3N2) (a drifted hemagglutinin variant) and A/California/7/2009(H1N1) pandemic virus (a swine origin quadruple reassortant), respectively. Because the antigenic match between vaccine and circulating strains was good in both of these seasons, a relevant proportion of cases may have been prevented by vaccination. A different situation was observed during the 2011–2012 season, when ILI morbidity rates in individuals ≥65-y-of-age were 1.5–6-fold higher than those registered during the other epidemics under review. The higher morbidity resulted from the circulation during the 2011–2012 season of an A/Victoria/361/2011(H3N2)-like variant that presented a reduced genetic match with the A(H3N2) strain included in the 2011–2012 vaccine composition. The continuous surveillance of the characteristics of circulating viruses is an essential tool for monitoring their matching with seasonal vaccine strains. Strategies to increase coverage rates are warranted.

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Diagnosis of this infection is important because 5-17% of asymptomatic infected babies will develop late sequelae and should be followed closely. Most of these children will remain undetected, since screening of all newborns by viral culture is too expensive. The aim of this study was to demonstrate that pool testing could be used to detect HCMV congenital infection in newborns. For this purpose, a nested-PCR technique was tested in urine pools. In phase 1, urine specimens were tested alone by nested-PCR and compared with viral culture, followed by cross experiments to test the reliability of detecting one positive specimen in a 20 samples in a urine pool. In phase 2, this pool method was applied to all urine specimens from children received in the virology laboratory of the Centro Hospitalar Cova da Beira for diagnosis of HCMV infection, between January 2002 and March 2003. In phase 1, 74 urine specimens were tested simultaneously by shell-vial culture and nested-PCR; 17 were positive and the remaining 57 negative by both methods. The negative specimens were divided into three pools and each pool was tested alone and crossed with each of the positive specimens by nested-PCR. Although the three pools were negative when tested alone, all 51 crossed results were positive. In phase 2, 15 out of the 180 urine samples tested positive by shell-vial culture and were detected by this pool method. These results suggest that urine pools can be used to detect HCMV positive urines in children, with similar sensitivity and specificity when compared with the standard method, but with a substantial labour reduction. This significant reduction in labour and consequently in cost per test, opens the possibility of applying PCR to urine pools for screening the HCMV congenital infection in newborns.

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

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.

Background: The role of the virulence of the infecting cytomegalovirus (CMV) strain in the transmission of the virus from mother to fetus and the outcome of the fetal infection has not received much attention yet. Molecular analysis of the gene coding for the surface glycoprotein B (gB) has been used to investigate the relationship between genotype and virulence in groups of immunosuppressed patients. Objectives: (1) to assess the prevalence of different gB genotypes in babies with congenital CMV infection; (2) to investigate the possible relationship between genotype and severity of congenital CMV disease; (3) to evaluate the possibility of using dried blood on Guthrie cards (DBS) for genotyping. Study design: CMV DNA was extracted from DBS and from urine/saliva samples collected in the first two weeks of life of 98 congenitally infected babies, half of which were symptomatic at birth. Genotyping was performed through RFLP analysis of the region corresponding to the cleavage site of the gB protein. Results: The most prevalent genotype was gB1 (42%) followed by gB3 (26%), gB2 (19%) and gB4 (13%). Rates of disease and CNS damages were higher among children infected by gB1 (35%, 17%) and gB3 (31%, 28%) than in those infected by gB2 and gB4 (20%, 17% and 13%, 15%, respectively). These differences however did not reach the statistical significance. The parallel typing of DBS and urine/saliva strains gave a full concordance of results. Conclusions: All four major CMV gB genotypes (gB1–4) can cause a congenital infection but none seems to be associated to the development and the severity of disease. The possibility of using the neonatal DBS for genotyping opens a way to the examination of large numbers of cases of congenital CMV infection.

Background: A simple and reliable diagnosis of congenital cytomegalovirus infection is necessary both for clinical and epidemiological purposes. This could be accomplished through the demonstration of cytomegalovirus (CMV) DNA in blood spots (DBS) on Guthrie cards. Objectives: (1) To assess the sensitivity and specificity of the method (DBS test) in diagnosing congenital CMV infection compared with viral isolation and (2) to evaluate the applications of the test to the late diagnosis of congenital CMV. Study design: The method was tested on the cards of (1) 509 babies examined through viral isolation within their third week of life (72 positive cases) and (2) 191 children studied after 3 weeks of life (25 days to 5 years). Blood was eluted from Guthrie cards and heat extracted. The products of a nested polymerase chain reaction (PCR) amplifying one region in the CMV glycoprotein B (gB) gene were detected by agarose gel electrophoresis. Results: DBS test was positive in all 72 congenitally infected babies and in four of the 437 negative at cytomegalovirus isolation (sensitivity 100%, specificity 99%). Infection in 16 of the 92 infants with a late viral isolation was demonstrated to be congenital by the test, which also detected congenital infection in 18 of 83 children in whom viral culture was not performed (13 with and five without symptoms). Fifty-six additional control cases tested negative. Conclusions: DBS test is a reliable assay for diagnosing congenital cytomegalovirus infection and could be used as an alternative to viral culture. It is able to reveal whether ascertained CMV infection is congenital or postnatal at an age when viral isolation is not able to do so. It can assess the role of risky procedures such as transfusion and it can ascertain the etiology of morbid conditions diagnosed late or of controversial origin.

Knowledge of the prevalence of congenital cytomegalovirus infection is necessary to evaluate the need for prevention. We performed a multicentre one-year study involving 11 neonatology divisions to ascertain the prevalence in Lombardy. Cytomegalovirus was isolated by culturing saliva samples from all babies born (n = 1268) of two 15-day sample periods and from 185 neonates with suspected congenital CMV based on clinical and laboratory findings and the history. The overall prevalence of congenital infection was 0.47% (6/1268) in the sample period group and 5% (9/185) in the second group. Clinical monitoring revealed sequelae in two of three children with symptomatic infection and no asymptomatic child at age two years. In a subgroup of 205 babies including 14 of the infected infants we also evaluated a test to detect cytomegalovirus DNA in the Guthrie cards obtained in neonatal screening for genetic and metabolic disorders. The test's sensitivity was 100% and specificity 98.5%, encouraging its use for early identification of infected neonates and for large epidemiological studies.

We report the case of a woman with natalizumab-treated Multiple Sclerosis (MS) that developed progressive multifocal leukoencephalopathy (PML) with atypical MRI features at early onset. This case shows that PML can have variable radiological patterns in natalizumab-treated MS patients thus expanding the possible MRI patterns at onset in these patients.

Background: The reference method of cytomegalovirus (CMV) isolation from urine or saliva is not a feasible routine technique for all newborns, and laboratory diagnosis of this infection would be useful both for epidemiological purposes and to enable prompt institution of adequate measures to identify and correct late sequelae. Extraction and amplification of viral DNA from dried blood spots (DBS) collected from babies in the first days of life during routine screening for genetic and metabolic disorders has been proposed for the early diagnosis of viral congenital infections. Objectives: To test the method for CMV DNA extraction from DBS and to evaluate the results obtained in newborns with and without a diagnosis of congenital infection based on viral isolation from urine and or saliva at birth. Study design: DBS from Guthrie cards collected in babies who underwent virological tests for CMV infection were tested for CMV DNA by observers blinded to the virological results. DNA was extracted from DBS both in water and in cell culture medium according to Shibata et al. with minor modifications. The products of nested polymerase chain reactions (PCR) amplifying two regions in the IE1 and gp58 genes were detected by agarose gel electrophoresis. Strict control measures were adopted to avoid carryovers and contaminations. Results: DBS from the eight symptomatic and 11 asymptomatic congenitally infected babies were positive when extraction was performed in medium, whereas extraction in water failed to identify two of the asymptomatic cases. The results obtained with the two extraction methods agreed in the remaining cases; the 71 CMV negative control babies were negative and two out of 21 cases of supposed postnatal infection were diagnosed as congenital on the basis of a positive DBS. All positive cases were identified by gp58 PCR but only slightly over half of them by IE1 PCR. Extraction in medium was more efficient than in water. Conclusions: The method of CMV DNA extraction in medium followed by amplification of the gp58 region showed 100% sensitivity and specificity compared with isolation in cell culture. Therefore, we propose this procedure to diagnose congenital CMV infection at birth and also later.

Viral DNA detection in dried blood spotted on filter paper, dried blood spots (DBS), is valuable in the diagnosis of viral infections, with at the moment congenital cytomegalovirus (CMV) being the most common application. CMV detection in clinical samples taken within the first 2-3 weeks after birth differentiates congenital CMV infection from the in general harmless postnatal acquired cytomegalovirus infection. DBS render the possibility to diagnose congenital CMV infection retrospectively, e.g., when late-onset hearing loss, the most frequently encountered symptom of congenital CMV infection, becomes manifest. Additionally, CMV DNA detection in DBS can be of usage in recently advocated newborn screening on congenital CMV infection. The procedure of CMV DNA detection in DBS consists of two separate steps: (1) DNA extraction from the DBS, followed by (2) CMV DNA amplification. Here, we describe two efficient methods for the extraction of DNA from DBS. Sensitivity, specificity, and applicability of the methods for high-throughput usage are discussed.

Background Many congenitally cytomegalovirus-infected (cCMV) neonates are at risk for severe consequences, even if they are asymptomatic at birth. The assessment of the viral load in neonatal blood could help in identifying the babies at risk of sequelae. Methods In the present study, we elaborated the results obtained on blood samples collected in the first two weeks of life from 22 symptomatic and 48 asymptomatic newborns with cCMV diagnosed through urine testing. We evaluated the performances of two quantitative methods (pp65 antigenemia test and plasma Real-time PCR) and the semi-quantitative results of dried blood sample (DBS) test in the aim of identifying a valid method for measuring viral load. Results Plasma qPCR and DBS tests were positive in 100% of cases, antigenemia in 81%. Only the latter test gave quantitatively different results in symptomatic versus asymptomatic children. qPCR values of 103 copies/ml were found in 52% of newborn. "Strong" DBS test positivity cases had higher median values of both pp65 positive PBL and DNA copies/ml than cases with a "weak" positivity. Conclusions As expected antigenemia test was less sensitive than molecular tests and DBS test performed better on samples with higher rates of pp65 positive PBL and higher numbers of DNA copies/ml. The prognostic significance of the results of these tests will be evaluated on completion of the ongoing collection of follow-up data of these children.

Cytomegalovirus (CMV) infection is the most frequent congenital infection in humans and can cause permanent damage--particularly neurological--in about 20% of those infected, with or without symptoms at birth. Laboratory diagnosis is essential on account of the relatively non-specific clinical manifestations in symptomatic newborns but also because of the high frequency of asymptomatic cases that are nevertheless at risk of lesions later in life. However, these tests need samples taken within 3 weeks of birth to distinguish congenital infection from the more common, but clinically benign, perinatal infection. Tests for viral DNA have proved a valid means of diagnosing congenital CMV infection in neonatal blood dried on paper (DBS) widely used in screening for metabolic and genetic diseases, as an alternative to the conventional urine culture method. The DBS test is simpler, faster and less costly than viral isolation; in addition the samples can be safely stored for long periods, so diagnosis can be made even after several years. The sensitivity and specificity of the DBS test, compared to the reference method, have been reported to range between 71 and 100% and 99 and 100%, respectively, depending on the different studies and diagnostic criteria applied. The most interesting applications reported so far involve retrospective determination of the impact of congenital CMV in sensorineural deafness, abnormalities of cortical development, neonatal cholestasis and surveys of the prevalence of this infection in various populations. The test might be useful in the future for neonatal screening with a view to treating neonates and so avoiding the damage this disease can cause.

Background Congenital cytomegalovirus infection causes 20–30% of congenital sensorineural hearing loss (SNHL) cases. Early identification of CMV attributable cases and their successful treatment are often hampered by the late appearance of the damage in a high proportion of children both symptomatic and asymptomatic at birth. Objective To discuss the feasibility of a screening program aimed at finding congenitally infected babies followed by their audiological monitoring. Study design Opinion—review article. Results and conclusions Frequency and severity of hearing loss due to congenital CMV suggest it maybe worthwhile setting up neonatal screening campaigns. Structures where SNHL can be diagnosed and treated exist already in many countries as part of universal neonatal audiological screening schemes. A test based on viral DNA extraction from neonatal dried blood spots on Guthrie cards and its amplification by means of a nested PCR (DBS test) seems to offer the best characteristics for use in screening. Clearly it will be necessary to calculate whether the costs of screening, diagnosis and follow-up, and the financial and emotional burden on the families of infected children, are up to the potential gain.

Background: Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases. Objective: To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection. Study design: We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA. Results: The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml. Conclusions: The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.

Background. Diagnostic problems in identifying congenital infection cases in infancy have thus far impaired the assessment of the role of congenital cytomegalovirus (CMV) infection in the etiology of sensorineural hearing loss (SNHL). Objective. To estimate the impact of congenital infection in children with SNHL by detection of CMV DNA in stored samples of neonatal dried blood (dried blood spots test). Methods. The Guthrie cards of 130 children with hearing loss >40 dB hearing loss were retrieved from the regional screening center. CMV DNA was extracted by thermal shock and amplified by PCR. Results. The percentage of SNHL cases attributable to congenital CMV infection was 10% (9 of 87) in infants whose SNHL had been diagnosed in their first 2 months of life and 34.2% (13 of 38) in children with deafness of unidentified cause that was diagnosed in early childhood. In the latter group 42.7% (12 of 28) of the children with a hearing loss of >70 dB were CMV-positive. Conclusions. The results suggest that congenital CMV infection has a more relevant role in the etiology of SNHL than previously reported. The data obtained in both groups suggest that 20 to 30% of all deafness cases are caused by CMV. The percent of congenital CMV cases alone appears to account for all the cases previously attributed to all congenital infections. More than 40% of deafness cases with an unknown cause, needing rehabilitation, are caused by congenital CMV.

Recurrent herpes simplex labialis represents a disease still difficult to treat, despite the availability of many established antiviral drugs used in clinical research since 30 years ago. Although differences between the human disease and that obtained in experimental animal suggest caution in predicting an effective clinical response from the experimental results, some of the animal models seem to be useful in optimising the topical formulation of single antiviral drugs. In the present work the dorsal cutaneous guinea pig model was used to compare 5 different topical antiviral formulations with clinical promise (active molecule: 5 % w/w micronized aciclovir, CAS 59277-89-3), using both roll-on and lipstick application systems. The aim being to evaluate which vehicle (water, oil, low melting and high melting fatty base) and application system (roll-on, lipstick) enhances the skin penetration and the antiviral activity of the drug, after an experimental intradermal infection with Herpes simplex virus type 1 (HSV-1). As reference, a commercial formulation (5 % aciclovir ointment) was used. The cumulative results of this study showed that the formulation A, containing 5 % aciclovir in an aqueous base in a roll-on application system, has the better antiviral efficacy in reducing the severity of cutaneous lesions and the viral titer; among the lipsticks preparations, the formulation D, containing 5 % aciclovir in a low melting fatty base, demonstrates a very strong antiviral activity, though slightly less than formulation A. This experimental work confirms the validity of the dorsal cutaneous guinea pig model as a rapid and efficient method to compare the antiviral efficacy of new formulations, with clinical promise, to optimise the topical formulation of the active antiviral drugs.

Background. Cytomegalovirus (CMV) is the most frequent agent of viral infection in the fetus ; it causes varying damage, particularly neurologic, which becomes evident at birth or in infancy in about 20% of infected individuals. Postnatal acquisition is usually asymptomatic and without sequelae. Laboratory diagnosis of congenital and postnatal infection is based on the demonstration of virus in urine. Objectives. To investigate the systemic spread of CMV in neonates with congenital or postnatal infection and to evaluate its significance in diagnosis and in monitoring anti-CMV treatments. Design. Quantitative determinations of infective CMV (viremia) and viral antigen pp65 (antigenemia) were performed on peripheral blood leukocytes (PBL) from the buffy coat of heparinized blood from children with a diagnosis of congenital (n = 19) or postnatal (n = 19) infection based on viral isolation from urine. Results. Antigen pp65 in PBL was detected particularly in children with symptomatic infection, both congenital (100%) and postnatal (79% ; P > 0.05), and significantly less frequently (50% ; P < 0.001) in those with asymptomatic infection. Viremia was observed less often but always in association with antigenemia. Both tests became negative within 6 months. Neither viral titer nor persistent positivity was related to clinical manifestations. In the nine infants given anti-CMV therapy (ganciclovir and/or hyperimmune gamma-globulins) an early suspension of treatment resulted in the appearance of antigenemia and/or viremia. Conclusions. Cytomegalovirus was detected in PBL mainly in the most severely affected children. Monitoring antigenemia and viremia in CMV-infected infants is recommended to demonstrate persistent systemic infection and to evaluate virologic results of treatment.

Immunity to tetanus toxoid and polioviruses was studied in 34 (27 allografted, 7 autografted) children who underwent bone marrow transplantation (BMT). At a median time of 3 years after BMT, only one recipient was seronegative for tetanus toxoid. On the contrary 73% of children were seronegative for at least one of the three poliovirus types and 30% for all virus types. Undetectable antibody titers were more frequently found against type 3 than the other two types. We recommend that reimmunizations of children after BMT be based on serologic tests for antibody titers.