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

Abstract

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.

Keywords: :

• Acute Flaccid Paralysis
• Polio Eradication Initiative
• Poliovirus
• Enterovirus
• Surveillance

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Corrigendum

Introduction

Although the Americas, the Western Pacific, Europe and Southeast Asia have been declared polio-free by the World Health Organisation (WHO), endemic polio persists in Pakistan, Afghanistan, and Nigeria. Until poliovirus (PV) transmission is halted in these nations, all countries are at risk of polio importation. Strengthening immunization protocols and implementing acute flaccid paralysis (AFP) surveillances are essential for polio eradication.

The Global Polio Eradication Initiative (GPEI) included four principal strategies: (1) Immunize infants at high rates with four doses of oral polio vaccine (OPV) in the first year of life in developing and endemic countries, and immunize routinely with OPV and/or inactivated polio vaccine (IPV) elsewhere; (2) Organise “National Immunization Days” to provide supplementary doses of OPV to all children less than five years of age; (3)Actively survey for wild PV through reporting and laboratory testing of all AFP cases among children <15 y of age. 4) Target “mop-up” campaigns once wild PV transmission is limited to specific focal areas.¹

All pathogen eradication initiatives rely on highly sensitive surveillance protocols, and this is particularly necessary for polio eradication. In fact, 99% of PV infections are mild, show few symptoms, and often go unrecognized,² in contrast to smallpox in which each infected person develops clinical symptoms. Since most PV infected people are unaware that they carry the virus, PV spreads widely before cases arise. For this reason, in 1996, WHO extended polio surveillance to all AFP cases, including Guillain-Barré Syndrome (GBS), traumatic neuritis, myelitis/encephalitis, other Enterovirus (EV) infections, and several paralytic syndromes.²

The surveillance system is considered to be sensitive if at least one case of AFP per 100 000 children <15 y of age is detected annually. It is also important to collect stool samples for each case soon after symptoms appear to determine whether PV is involved in the clinical manifestations.

In Italy, the last case of indigenous polio caused by wild PV was reported in 1982, and Italy, which is part of the WHO European Region, was declared polio-free in 2002.³ However, because Italy and other countries remain at risk for PV importation, it is crucial that the sensitivities of AFP surveillance protocol meet the performance indicators set by the WHO, to detect any polio case early and before outbreaks can develop. Moreover, the spread of PV can be prevented if more than 90% of people in a community are vaccinated.

Since 2002, the polio vaccination schedule in Italy has consisted of 4 IPV doses per person.

This article describes the results of the AFP surveillance conducted from 1997 to 2011 in the Lombardy region (Northern Italy) in the context of the national AFP surveillance program, and discusses the critical parameters demanded to achieve the performance indicators required by the WHO.

Results

The number of children aged under 15 y in Lombardy grew from 1 100 000 persons in 1997 to 1 300 000 persons in 2011. The number of AFP cases in Lombardy was predicted to range from 11 to 13 per year.

In Lombardy, from 1997 to 2011, 131 AFP cases were reported with an average of 8.7 cases per year and a mean incidence rate of 0.7/100 000 children <15 y of age (range: 0.3/100 000–1.1/100 000) (Table 1). The median age of AFP cases was 5 y and 75/131 (57.2%) cases were in children ≤5 y old, and no gender difference was observed. The monthly distribution of AFP cases was typical with peaks in November, in January, and in March (Fig. 1).

Table 1. Acute flaccid paralysis incidence per 100 000 children aged <15 y in Lombardy, 1997–2011

Years	1997	2000	2001	2002	2003	2004	2005	2006	2007	2008	2009	2010	2011	Tot.
Incidence index	0.7	0.7	1.1	1.1	1.1	0.5	0.7	0.8	0.9	0.7	0.3	0.9	0.3	0.7

Figure 1. Monthly distribution of AFP cases in Lombardy, 1997–2011.

Paediatric wards in the vicinity of Milan reported the majority of the AFP cases. The most common admission diagnoses were GBS (40%) and encephalomyelitis/myelitis (13%). Two cases were admitted as suspected Vaccine-Associated Paralytic Paralysis (VAPP) because symptoms began 28 d after OPV administration (Table 2). Conclusive diagnoses were available for 110 (84%) of the AFP patients, and follow-ups within 90 d of the onset of paralysis were obtained for 84 (64%) of the AFP cases. The common final diagnoses were GBS (51/110, 46%) and encephalomyelitis/myelitis (16/110, 14.5%). Residual paralysis after 90 d was present in just the two patients admitted as suspected VAPP (Table 2).⁴

Table 2. Diagnoses of acute flaccid paralysis cases at hospital admission and at follow-up in Lombardy, 1997–2011

	Admission diagnoses		Follow-up diagnoses
	n.	%	n.	%
Guillain-Barrè Syndrome	52	39.7%	51	46.4%
Encephalomyelitis/myelitis	17	13.0%	16	14.5%
Other diagnoses	31	23.7%	22	20.0%
No definite diagnoses	29	22.1%	19	17.3%
VAPP*	2	1.5%	2	1.8%
Total	131	100%	110	100%

* Vaccine-Associated Paralytic Paralyses (VAPP) cases at hospital admission (paralysis onset within 28 d from Sabin OPV administration).

At least one stool sample was collected from 115 (88%) of the AFP cases and a second stool sample was taken after 24–48 h for 97 (74%) of the AFP cases.

During this study period, no wild or neurovirulent circulating Vaccine-Derived PV (cVDPV) were isolated from the stool samples of the AFP patients examined. Between 1997–2001, when OPV was still used in Italy, Sabin-like type 3 PV was detected in two VAPP patients (both occurred in 1997; a 3-y-old male and a 3-y-old female). Sequence analyses of the 5′NCR of the type 3 PV strains isolated from the VAPP patients showed a retro-mutation at nucleotide 472 (U to C), which is associated with a neurovirulent phenotype.⁵

In 2000, Sabin-like type 1 PV was detected in a 13-y-old male, and in 2001, Sabin-like type 1 and Sabin-like type 2 PVs were detected in a 1-y-old male. Neither child had residual paralysis at follow-up and the conclusive diagnoses were GBS and AFP, respectively. Moreover, the sequence analyses of these PV strains showed no retro-mutation.⁵

Since the introduction of IPV vaccination in Italy in 2002, no wild and Sabin-like PV were isolated from AFP cases in Lombardy. At least one serological sample was collected from 80% (104/131) of the AFP cases and protective antibody levels against all PV serotypes were assessed for ~96% (100/104) of the cases.

Conclusion

The annual number of wild PV cases has decreased by >99% worldwide since 1988, but three countries, Afghanistan, Nigeria and Pakistan, still have endemic PV and uninterrupted wild PV transmission. In 2013, wild PVs were exported from three countries in three major epidemiological zones: in central Asia (from Pakistan to Afghanistan), in Central Africa (from Cameroon to Equatorial Guinea), and in the Middle East (from Syria to Iraq), and these cases represented ~60% of the worldwide polio cases.⁶ Based on these data, on 5 May 2014, the Director-General of the WHO declared the international spread of wild PV a public health emergency of international concern, and the Emergency Committee provided additional advise to reduce the risk of further international spread and to achieve global polio eradication.⁶

Because of its geographical position and high immigration rate, Italy is at risk for the importation of wild PV from areas with endemic polio and for the reintroduction of Sabin-like PV or cVDPVs from countries where OPV vaccination still occurs.⁷ In addition, Italy is considered ‘a door to Europe’ and this may facilitate the reintroduction of wild PV to other European Union (EU) countries. Therefore, high sensitivity polio surveillances are crucial for the early detection of polio reintroductions worldwide.^7,8

During the AFP surveillance in Lombardy, the AFP median incidence rate was lower than expected (0.7/100 000 vs.1/100 000), but was similar to rates in other Italian regions.^5,9-11 However, during the study period, variations in the rates of AFP were reported, and performance variability was probably due to the long absence of polio cases in Italy and the minimal priority given to AFP, compared with more frequent or emerging diseases, by physicians.⁴ Moreover, the medical sentinels often did not record AFP because a differential diagnosis, such as GBS or encephalomyelitis/myelitis, is often made directly on the basis of symptoms. But, high surveillance performances were recorded for 2001–2003, the years surrounding the polio-free declaration.

When AFP surveillances do not meet WHO performance indicators and especially in countries where IPV is used, WHO has proposed surveillances of sewage samples collected at the inlets of water treatment plants.¹² An environmental monitoring of raw sewage is a powerful tool to detect EVs because of the high faecal excretion of viruses from infected, symptomatic or asymptomatic people, and because EVs are resistant to a variety of environmental conditions.¹²

Since 2006, environmental surveillances for PV and other EVs have been conducted in Lombardy in accordance with WHO protocols,¹² and no wild PV or cVDPV has been detected, supporting the epidemiological data from environmental surveillances in other Italian regions.^13,14

In contrast, Sabin-like type 2 PV was isolated in Lombardy and other Italian regions in 2007 and in 2008. Because the full IPV Salk schedule was introduced in Italy in 2002, the isolation of Sabin-like PVs were probably due to the presence of individuals who came from countries that still use OPV.^13,14 The identification of Sabin-like PV in Italy underscores the risk of virus reintroduction due to globalization and highlights the need to perform high-quality PV surveillances.

Serological surveillances of PV and epidemiological studies of EVs elimination from healthy children have been performed in Lombardy^15-18 to endorse polio surveillance as recommended by the WHO.

In our surveillance, as in other studies,^19,20 no difference was observed in AFP rates between males and females, and more than half of the AFP cases were in children ≤ 5 y of age. In our study, the number of AFP cases peaked in November, in January and in March, but D’Errico et al. have found that AFP cases peaked between May and October in Marche region (central Italy).¹⁸

GBS is the most common manifestation recognized of AFP onset, especially when transmission of wild PV has been interrupted.²¹ In this study, half of all of the AFP patients were admitted with a GBS diagnosis that was ultimately confirmed. As expected, no wild PV, and Sabin-like PV was identified during the time period, 1997–2001, when OPV was still provided, thus our data supports the national AFP surveillance data.⁵ In 1997, two AFP cases were confirmed as VAPP,⁴ and there were 15 cases of VAPP in Italy from 1990 to 2001 making the incidence of VAPP approximately 1 case per 2.5 million OPV doses administered.²²

The spread of PV can only be prevented if >90% of the people in a community are vaccinated. As expected, the Italian national PV immunization program has provided sero-protection against PV in the children analysed (~96% of AFP cases), as well as in the entire Italian population <15 y old.^16,18 In Italy, ~96.5% of children <2 y old are protected with three doses of IPV,²² nevertheless, some parents delay and refuse vaccination. If PV is introduced into Italy, individuals without protective antibodies against PV will be at risk of infection because IPV stimulates strong humoral immunity but weak mucosal immunity (IgA). As a result, when a person who was immunized with IPV is infected with wild PV, the wild virus will replicate inside the intestines and will be shed in the faeces, thus circulation of PV will continue. It would be interesting to evaluate the titres of PV neutralizing antibodies in adults to confirm the eventual reduction in immunity seen in other studies^16,24 and to investigate immunological memory responses to PV challenges.

The limitations identified in this study were that only 88% of AFP cases were investigated virologically and conclusive diagnoses occurred for only 84% of the AFP cases, thus definite diagnoses could not be made for some of the cases.

Improvements in our surveillance system are ongoing and AFP surveillance will be maintained until global poliovirus eradication will be declared.

Material and Methods

AFP surveillance system in Italy

An AFP case is defined as a child <15 y of age showing clinical AFP of one or more limbs with deceased or absent deep tendon reflexes or with bulbar paralysis. In Italy, AFP surveillance tightly follow WHO guidelines,¹ and AFP notifications became mandatory in 1995 (circular Letter n. 400.2/28/911 of 7th February 1995). In January 1996, a program of active AFP surveillance targeting children aged <15 y was set up as a pilot study limited to four representative regions of Italy. The surveillance was extended to the entire nation in 1997 and was conducted by the National Institute of Health (ISS) and the Ministry of Health (MOH) through reference laboratories, primarily the University laboratories of hygiene, located in each of the 20 Italian regions. The Regional Reference Centre (RRC) coordinated directly with local, collaborating hospitals.⁴ The RRC was required to report every case of AFP in individuals aged <15 y to ISS and MOH using preliminary questionnaires with clinical and epidemiological information. In agreement with WHO guidelines, clinical samples for virological investigations were collected for each case and consisted of 1) two stool specimens taken 24–48 h apart, and 2) acute (within 14 d since the onset of paralysis) and convalescent paired sera for serological investigation.¹ After case notification, a follow-up questionnaire with more detailed information was required to be sent to ISS and MOH to determine whether residual paralysis or death occurred 60–90 d after diagnosis, as well as to clarify the conclusive diagnoses. Virological investigations are usually performed by ISS, but six of the 20 Italian RRCs also have the skills and the laboratory facilities to organize networks and to perform preliminary virological and clinical PV investigations.⁴

AFP surveillance system in Lombardy

The Laboratory of Diagnostic Virology, Department of Biomedical Science for Health, University of Milan, was one of the 6 sub-national reference laboratories (SNRL) that performed preliminary virological investigations on clinical samples collected from AFP cases in Lombardy, Northern Italy. Every year, the laboratory is evaluated by the National Polio Reference Laboratory (NPRL) for proficiency in isolation and typing of EVs. The proficiency test involves testing panels of coded stool samples prepared ad hoc with EVs mixtures.⁴ Methods recommended by the WHO were strictly followed.¹

The SNRL enrolled and coordinated with approximately 45 sentinel hospitals (with paediatric, neurological, intensive therapy, and infectious disease wards) that reported all AFP cases admitted into their units by sending monthly “zero AFP cases; reports to the SNRL. If an AFP case was identified, stool and serum samples were collected at the hospital and transported to the SNRL in refrigerated containers. According to the WHO standard protocol, virological investigations were started within 48 hours of specimen collection and consist to detection of viral growth after two blind passages in a rhabdomyosarcoma (RD) cell line, which is permissive to EVs, and in a L20B mouse cell line, which expresses the human cellular receptor for PV and is sensitive to PV and a few other EVs. A negative result refers to the absence of viral growth. Nested polymerase chain reaction (PCR) with primers to the 5′NCR (nucleotides 179-575)¹³ was performed on stool samples and supernatants from RD and L20B cells or confirmation of PV and EVs infections.

If PV was detected, it was serotyped using monospecific anti-PV pooled sera RIVM/WHO kit (National Institute of Public Health and Environment, RIVM, Bilthoven, Holland) in accordance with the WHO protocol. Intratypic differentiation between Sabin-like or non-Sabin-like PV was performed by either an enzyme-linked immunoassay (ELISA, RIVM) or by PCR amplification with specific primers.¹ After amplification, the 5′NCR, the VP1 coding, the VP1/A junction, and the 3D regions were sequenced.¹

PV neutralizing antibody titres against serotypes 1, 2, and 3 were determined by standard neutralization tests (NT) using Sabin vaccine strains as challenge viruses in accordance with WHO recommendations.¹ Acute and convalescent (14 d after the onset of symptoms) phase sera from AFP cases were tested to verify the polio immunization statuses of patients.

This study was performed in compliance with the Helsinki Declaration, 1975.

Abbrevations	=	AFP, Acute Flaccid Paralysis
cVDPV	=	circulating Vaccine-Derived Poliovirus
EU	=	European Union
EV	=	Enterovirus
GBS	=	Guillain-Barré Syndrome
GPEI	=	Global Polio Eradication Initiative
IPV	=	Inactivated Polio Vaccine
ISS	=	National Institute of Health
MOH	=	Ministry of Health
NPRL	=	National Polio Reference Laboratory
NT	=	Neutralization Test
OPV	=	Oral Polio Vaccine
PCR	=	Polymerase Chain Reaction
Polio	=	Poliomyelitis
PV	=	Poliovirus
RD	=	Rhabdomyosarcoma
RRC	=	Regional Reference Centre
SNRL	=	Sub-National Reference Laboratory
VAPP	=	Vaccine-Associated Paralytic Paralysis
WHO	=	World Health Organization

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

The authors thank medical and paramedical staff involved in the acute flaccid paralysis surveillance network, all children in study and their parents.