ABSTRACT
In the context of poliomyelitis eradication, a reinforced supplementary laboratory surveillance of enteroviruses was implemented in Greece. Between 2008 and 2014, the Hellenic Polioviruses/Enteroviruses Reference Laboratory performed detailed supplementary surveillance of circulating enteroviruses among healthy individuals in high-risk population groups, among immigrants from countries in which poliovirus is endemic, and in environmental samples. In total, 722 stool samples and 179 sewage water samples were included in the study. No wild-type polioviruses were isolated during these 7 years of surveillance, although two imported vaccine polioviruses were detected. Enterovirus presence was recorded in 25.3 and 25.1% of stool and sewage water samples, respectively. Nonpolio enteroviruses isolated from stool samples belonged to species A, B, or C; coxsackievirus A24 was the most frequently identified serotype. Only enteroviruses of species B were identified in sewage water samples, including four serotypes of echoviruses and four serotypes of coxsackie B viruses. Phylogenetic analysis revealed close genetic relationships among virus isolates from sewage water samples and stool samples, which in most cases fell into the same cluster. To the best of our knowledge, this is the first study to compare enterovirus serotypes circulating in fecal specimens of healthy individuals and environmental samples, emphasizing the burden of enterovirus circulation in asymptomatic individuals at high risk. Given that Greece continues to receive a large number of short-term arrivals, students, migrants, and refugees from countries in which poliovirus is endemic, it is important to guarantee high-quality surveillance in order to maintain its polio-free status until global eradication is achieved.
IMPORTANCE This article summarizes the results of supplementary poliovirus surveillance in Greece and the subsequent characterization of enteroviral circulation in human feces and the environment. The examination of stool samples from healthy refugees and other individuals in “high-risk” groups for poliovirus enables the identification of enterovirus cases and forms the basis for further investigation of the community-level risk of viral transmission. In addition, the examination of composite human fecal samples through environmental surveillance links poliovirus and nonpoliovirus isolates from unknown individuals to populations served by the sewage or wastewater system. Supplementary surveillance is necessary to comply with the prerequisites imposed by the World Health Organization for monitoring the emergence of vaccine-derived polioviruses, reemergence of wild polioviruses, or disappearance of all vaccine-related strains in order for countries such as Greece to maintain their polio-free status and contribute to global poliovirus eradication.
KEYWORDS: environmental sewage, high-risk populations, laboratory surveillance, nonpolio enteroviruses, phylogenetic analysis, polioviruses
INTRODUCTION
Enteroviruses (EVs) are common human pathogens belonging to the genus Enterovirus and the family Picornaviridae. EV serotypes have been classified as echoviruses, coxsackievirus (CV) group A or B, polioviruses (PV), or new enterovirus types. So far, human EVs comprise more than 100 types which are classified, based on phylogenetic analysis, into four species, EV-A to EV-D (1, 2). EVs are transmitted through the fecal-oral route and infect millions of people worldwide each year, particularly children. Although EV infection is usually asymptomatic, sometimes it is associated with diverse clinical syndromes ranging from minor febrile illness to severe, potentially fatal diseases, such as aseptic meningitis, encephalitis, paralysis, myocarditis, and neonatal enteroviral sepsis (2–4). In some cases, individual EV serotypes can cause particular symptoms; CV type A24 (CVA24) has been associated with acute hemorrhagic conjunctivitis (5, 6), while enterovirus 71 (EV71) has been associated with major outbreaks of hand, foot, and mouth disease (HFMD) and concurrent fatal encephalitis among very young children (7, 8).
PV infection is known to be associated with acute paralytic poliomyelitis, a clinical manifestation which is very rare or absent (silent virus circulation) in highly immune populations. Since the launch of the Global Polio Eradication Initiative (GPEI) in 1988, more than 99% of its goal to eradicate and contain all wild polioviruses (WPVs), vaccine-derived polioviruses (VDPVs), and Sabin-like (SL) polioviruses has been achieved. The main strategy recommended by the World Health Organization (WHO) for PV surveillance is the investigation of acute flaccid paralysis (AFP) cases in children, which is a sensitive marker for poliomyelitis (9). Additional WHO prerequisites for strengthening PV surveillance include environmental surveillance as a useful tool for monitoring of PV activity and population-based alerts. This is based on the observation that infected people, including those who are asymptomatic, shed large amounts of virus into the wastewater system, making this type of detection effective (10, 11). That was the case in Israel in 2013 where silent introduction and circulation of WPV type 1 (WPV1) occurred in a highly immune population in which an inactivated polio vaccine (IPV) had been used exclusively since 2005, and AFP surveillance alone had not detected it because viral shedding was not accompanied by any paralytic symptoms (12, 13). As the GPEI moves toward achieving the goal of PV eradication, environmental surveillance might also play a significant role in providing evidence for certification of polio-free status (11). For countries that receive large numbers of asylum seekers, the WHO further supports the screening of stool samples from refugees and healthy individuals in groups at high risk for PV (i.e., those with low vaccination coverage and/or living in inadequate sanitation facilities) in order to complement disease-based AFP surveillance.
Greece, along with the WHO European region, was declared polio free in 2002. The last case of poliomyelitis due to indigenous WPV occurred in Greece in 1982 (14). Since then, an outbreak of paralytic disease caused by WPV1 occurred in the neighboring country of Albania in 1996, which resulted in five imported cases of poliomyelitis in nonvaccinated Gypsy children (15). Even if poliomyelitis is not a problem in polio-free countries, high-quality surveillance is important in countries such as Greece, which receive a large number of short-term arrivals, students, migrants, and refugees from countries in which poliovirus is endemic and countries that still use oral poliomyelitis vaccine (OPV), until global eradication is achieved. In this article, we present the results of a supplementary investigation of enterovirus serotypes circulating in fecal specimens of healthy individuals at high risk and environmental samples during the years 2008 to 2014. Furthermore, molecular characterization of the nonpolio enterovirus isolates was carried out to identify specific serotypes, their diversity and circulation patterns.
RESULTS
Enterovirus surveillance.
Out of 415 stool samples from healthy Roma children, 105 (25.3%) were positive for EV by real-time reverse-transcription PCR (rRT-PCR) and/or culture (Table 1). Of these positive samples, 53 were positive by both rRT-PCR and culture, while the remaining 52 were positive by only rRT-PCR. Typing based on seroneutralization was successful in 26 out of 53 culture isolates (49%), as serotyping of several EVs was not supported by the provided antisera. On the other hand, molecular typing based on the VP1 region was successful in 86 out of 105 (81.9%) EV-positive stool samples with high viral load (threshold cycle [CT] ≤ 32 in the 5′ untranslated region [UTR] rRT-PCR), while the remaining 19 remained untypeable. In all cases, serotyping results by seroneutralization were identical with those obtained by VP1 molecular typing.
TABLE 1.
Enteroviral presence in stool and sewage water samples in different regions
| District | Region | No. of stool samples (% positive) | No. of sewage water samples (% positive) |
|---|---|---|---|
| Eastern Macedonia and Thrace | Evros | 94 (16) | 24 (20.8) |
| Rodopi | 39 (20.5) | ||
| Xanthi | 35 (40) | 3 (66.7) | |
| Kavala | 28 (3.6) | ||
| Drama | 11 (9.1) | ||
| Central Macedonia | Thessaloniki | 6 (50) | |
| Chalkidiki | 9 (33.3) | ||
| Pieria | 7 (57.1) | ||
| Imathia | 69 (24.6) | ||
| Western Macedonia | Grevena | 1 (0) | |
| Thessaly | Larisa | 50 (28) | 20 (20) |
| Trikala | 14 (35.7) | 4 (0) | |
| Magnisia | 15 (6.7) | 1 (100) | |
| Karditsa | 4 (25) | ||
| Epirus | Ioannina | 5 (0) | |
| Ionian islands | Corfu | 17 (82.4) | |
| Central Greece | Fokida | 6 (0) | |
| Western Greece | Ileia | 10 (20) | |
| Peloponnese | Lakonia | 17 (41.2) | |
| Messinia | 31 (32.3) | ||
| Argolida | 34 (8.8) | ||
| Korinthia | 8 (75) | ||
| Attica | Attiki | 10 (60) | 4 (0) |
| Crete | Irakleio | 10 (0) | |
| South Aegean Islands | Dodecanese (Rodos) | 8 (37.5) | |
| Sum | 415 (25.3) | 179 (25.1) |
Sequence analysis revealed a variety of enteroviral strains in healthy Roma children that belong to species EV-A, EV-B, or EV-C, all of which circulated throughout Greece and accounted for 10.5% (9 of 86), 48.8% (42 of 86), and 40.7% (35 of 86) of the strains, respectively. No EV-D viruses were identified. EV-B was the major species found during most years of the study period. EV-B and EV-C were identified during almost the whole period of surveillance (2008 to 2013 and 2008 to 2014, respectively), while EV-A viruses were identified during the period 2010 to 2012. Sequences obtained were assigned to four serotypes within the EV-A species, 14 serotypes within the EV-B species, and eight serotypes within the EV-C species (Table 2). All samples were negative for PVs, either wild or vaccine derived. CVA24 was the most frequently identified serotype (13/86; 15.1%), followed by echovirus type 25 (Echo25) (7/86; 8.1%), and CVA1 or CVB4 (6/86; 7%). Almost all geographical regions presented a mixture of EVs (Fig. 1). The prefecture of Corfu recorded the highest rates of enteroviral presence (82.4%), followed by those of Attiki (60%), Lakonia (41.2%), and Xanthi (40%) (Table 1).
TABLE 2.
Temporal distribution of EV serotypes identified in stool and sewage water samples
| EV species | Serotype | 2008 (stool) | 2010 (stool) | 2011 (stool) | 2012 |
2013 |
2014 |
|||
|---|---|---|---|---|---|---|---|---|---|---|
| Stool | Sewage | Stool | Sewage | Stool | Sewage | |||||
| A | CVA2 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| CVA4 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
| CVA5 | 0 | 0 | 0 | 4 | 0 | 0 | 0 | 0 | 0 | |
| CVA16 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | |
| B | CVA9 | 0 | 0 | 0 | 5 | 0 | 0 | 0 | 0 | 0 |
| CVB1 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CVB2 | 0 | 0 | 0 | 0 | 0 | 1 | 4 | 0 | 0 | |
| CVB3 | 0 | 0 | 0 | 0 | 1 | 1 | 0 | 0 | 4 | |
| CVB4 | 0 | 4 | 0 | 2 | 2 | 0 | 0 | 0 | 2 | |
| CVB5 | 0 | 0 | 0 | 0 | 1 | 0 | 2 | 0 | 1 | |
| Echo1 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 0 | 0 | |
| Echo6 | 0 | 1 | 2 | 0 | 4 | 0 | 0 | 0 | 0 | |
| Echo7 | 0 | 0 | 4 | 0 | 5 | 0 | 0 | 0 | 0 | |
| Echo11 | 1 | 2 | 0 | 2 | 7 | 0 | 7 | 0 | 1 | |
| Echo13 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | |
| Echo14 | 1 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Echo17 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| Echo25 | 0 | 2 | 1 | 0 | 0 | 4 | 0 | 0 | 0 | |
| Echo30 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | |
| EV82 | 0 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | |
| C | CVA1 | 0 | 0 | 0 | 0 | 0 | 6 | 0 | 0 | 0 |
| CVA13 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 2 | 0 | |
| CVA19 | 0 | 0 | 0 | 0 | 0 | 1 | 0 | 0 | 0 | |
| CVA21 | 0 | 3 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
| CVA22 | 0 | 0 | 0 | 0 | 0 | 2 | 0 | 2 | 0 | |
| CVA24 | 3 | 2 | 1 | 2 | 0 | 4 | 0 | 1 | 0 | |
| EV96 | 0 | 0 | 1 | 0 | 0 | 0 | 0 | 0 | 0 | |
| EV99 | 0 | 2 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | |
FIG 1.
Spatial distribution of EV serotypes identified in stool and sewage water samples. (Base map from the Union of Greek Regions [EN.P.E.].)
Phylogenetic analysis indicated that the majority of the Greek enteroviral strains identified in this study clustered most closely to strains circulated during the past 6 years in other European countries, such as France, Germany, Italy, Finland, and Netherlands (Fig. 2A through C). Interestingly, the Echo30 strain isolated from a Roma child in 2012 in the prefecture of Evros was genetically related to other Echo30 viruses identified during a meningitis outbreak in children in the same prefecture during 2012 (16).
FIG 2.
Evolutionary relationships of Greek enteroviruses species A (A), B (B), and C (C) with other European and reference strains, based on partial VP1 gene. The evolutionary history was inferred using the neighbor-joining method. The percentages of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) are shown next to the branches. Only values above 70% are shown. Greek strains are indicated with a filled circle, reference strains are in bold type, and other GenBank strains are in italic type. Strains derived from sewage water samples are indicated by [S].
Among the immigrants, only 6 out of 307 (2%) were found to be positive for EVs, four of them by rRT-PCR and cell culture and the remaining two by only rRT-PCR. Typing by sequence analysis and/or seroneutralization was successful for four of these positive cases, revealing the existence of CVA and CVB (CVA13 and CVB3) and two PVs (types 1 and 3). These two PV strains were detected in June 2014 in two children hosted in a reception site in northern Greece (prefecture of Evros). These two children had been vaccinated with OPV and belonged to a Roma population coming from Iraq. Other family members, as well as sewage water samples collected from the same reception site, were negative for PVs. rRT-PCR intratypic differentiation revealed that these isolates were SL strains; that was further confirmed by sequence analysis of the VP1 gene, as these strains shared >99% similarity with the PV vaccine strains (17). These results were also confirmed by the regional poliomyelitis reference laboratory in Rome, Italy.
Environmental surveillance.
Forty-five out of 179 sewage water samples (25.1%) were positive for EVs by rRT-PCR and/or culture (Table 1). Enteroviral typing was successful in 43 (95.6%) of these positive samples via sequence analysis and 35 (77.8%) via culture and subsequent seroneutralization, revealing the circulation of EVs within the EV-B species, including four serotypes of Echo (57.8%) and four serotypes of CVB viruses (37.8%) (Table 2). Rates of viral isolation and successful typing were higher in sewage water samples than in stool samples, reflecting the higher EV load obtained through sample concentration. Echo11 was the most frequently identified serotype (33.3%), however rarely seen in stool samples (4.8%), followed by Echo7 and CVB3 (11.1%). The prefecture of Magnisia recorded the highest rate of enteroviral positivity (100%), followed by those of Korinthia (75%), Xanthi (66.7%), and Pieria (57.1%) (Table 1).
Phylogenetic analysis revealed close genetic relationships among virus isolates from sewage water and stool samples, which in most cases fell into the same cluster (Fig. 2C). Echo11 and CVB4 strains circulated in the same region and during the same period in environmental and clinical samples. Both serotypes were detected during 2012 in the northern part of Greece (Fig. 1 and Table 2). Sequence analysis revealed that Echo11 viruses share a nucleotide identity of 93.4 to 99.7% and a deduced amino acid identity of 97.4 to 100%, while CVB4 viruses share a nucleotide identity of 88.6 to 99.7% and a deduced amino acid identity of 97.4 to 100%. On the other hand, Echo6 and Echo7 were initially identified in stool samples during 2010 to 2011 from northern and central Greece, respectively. Environmental surveillance in 2012 revealed circulation of these viruses in sewage water samples from different regions of central and northern Greece, respectively. Genomic sequencing of the partial VP1 gene showed 94 to 99.4% nucleotide and 88.8 to 100% amino acid homology among the Echo6 strains and 94 to 100% nucleotide and 94.8 to 100% amino acid homology among the Echo7 strains.
DISCUSSION
To the best of our knowledge, our report is the first to compare EV serotypes circulating in fecal specimens of healthy individuals with those circulating in environmental samples, emphasizing the burden of enterovirus circulation in asymptomatic individuals at high risk. Between 2008 and 2014, the Hellenic Polioviruses/Enteroviruses Reference Laboratory (HPRL) performed detailed supplementary surveillance of the circulating EVs throughout Greece. In addition to AFP surveillance, the establishment of supplementary surveillance for PV in stool and sewage water samples provides an additional means of monitoring the polio-free status of Greece. No WPVs were isolated during this period, although two imported SL polioviruses were detected in humans.
Supplementary EV surveillance targets populations at high risk for either transmitting poliovirus (e.g., areas where refugees settle or are in reception/detention centers) or acquiring polio (e.g., areas with a suboptimally immunized population). In that respect, the results of our study reveal the circulation of a plethora of nonpolio EVs (25.3%) among healthy children living under poor sanitation conditions throughout Greece. Cristanziano et al. reported the circulation of EVs belonging to species EV-A, EV-B, or EV-C in 22.8% of healthy individuals aged 0 to 53 years between June 2013 and December 2014 (18), which is similar to our results. A lower EV presence (10.6%) was recorded in a study conducted among healthy children ≤5 years old in China during 2010 to 2011, where EV-B species were detected more frequently, with an overall prevalence of 47.1%, which are findings similar to ours (48.8%) (19). On the other hand, cocirculation of various enteroviral serotypes in the same country and, in some cases, in the same region has also been found in other surveillance studies from different European countries, such as France and Germany (20, 21).
The incidence of EV among immigrant populations was much lower (2%) than that recorded for Roma children. With regard to the supplementary EV detection in fecal samples, only a few studies on immigrants have been published (22–24). In one of them, Bottcher et al. (24) revealed a frequency of EV (14.8%) among 629 Syrian refugees/asylum seekers between November 2013 and April 2014 that was higher than that found in our study. This difference may be related to the fact that the Bottcher et al. study was conducted mainly among children less than 18 years old (70.7% less than 3 years old). In our study, only 25.1% of the specimens were collected from children less than 18 years old, and the positive EV detection rate in the target group was slightly higher (3.9%) than that of all samples combined (2%). In another study, Tafuri et al. (22) examined 152 fecal samples from immigrants (30% of whom came from countries in which poliovirus was endemic) with a mean age of 22 years. Their results indicated that all fecal samples were negative for EVs.
The supplementary EV surveillance system also involves environmental surveillance, which serves as an early warning system for PV circulation in areas where high-risk groups reside. Environmental surveillance in Greece revealed echoviruses to be the most frequently isolated, followed by CVBs. CVBs have generally been reported to be the most frequently isolated EVs from environmental samples worldwide (25–27). It is noteworthy that of the 26 EV serotypes isolated from stool samples, only six were detected in sewage water samples, all belonging to species B. On the other hand, six out of eight serotypes detected in sewage water samples were also observed in stool isolates (Table 2).
The combination of methods used in our study might have underestimated the true diversity of enteroviruses observed in sewage water samples, as it relies on cell culture for the identification and typing of EVs. Some of them, particularly CVA from EV-A species, do not grow well in cell culture and are usually underdetected by traditional cell culture–based surveillance systems. Similar to us, Richter et al. identified several serotypes of EV-A and EV-B species in clinical samples, but only some strains of EV-B species were detected in environmental samples (26). Likewise, Khetsuriani et al. reported the circulation of serotypes of EV-A and EV-B species in human specimens only, while only serotypes of EV-B species were recovered from environmental specimens (28). Interestingly, Sedmak et al. reported that even when a system of seven cell lines was used for EV surveillance in environmental specimens, only one out of 399 EV serotypes was characterized as species A, in contrast to clinical specimens where a higher number of serotypes of EV-A and EV-B species was identified (29). Contrary to our study, in which fecal specimens were collected from healthy individuals, clinical samples analyzed in the above-referenced studies were obtained from patients with enterovirus-compatible disease. A wider introduction of molecular methods of enterovirus typing could provide more accurate information on circulating enteroviruses, as Harvala et al. indicated (30). Implementation of molecular methods for EV screening could be beneficial in terms of increased sensitivity of detection and speed with which results can be reported, as well as reduced workload and total cost due to the exclusion of EV-negative samples from further analysis. In any case, we should bear in mind that a different magnitude of EV shedding from humans or a different viability of specific serotypes in environmental samples cannot be excluded.
EV detection in raw sewage is a powerful tool for environmental monitoring, since EVs are excreted in high numbers by infected, symptomatic, or asymptomatic people, and they are resistant to a variety of environmental conditions (11). Intensified environmental (sewage) surveillance in Israel succeeded in identifying the introduction and sustained silent spread of WPV1, in 2013 which had circulated among a highly immune population (12, 13). Importations of WPV to Switzerland from Chad in 2007 and to Egypt from Pakistan in 2013 were also identified through environmental surveillance (31, 32). In December 2012, Egypt reported the detection of WPV1 in two sewage samples collected in Cairo; Pakistan was identified as the source of the virus (33).
Echo11 and CVB4 were the only EV serotypes detected in the same region and period in the analyzed sewage water and stool samples. A previous study conducted in 2013 and 2014 by our laboratory revealed the circulation of a similar combination of viruses in the population of Greece. However, the majority of viruses at that time belonged to serotypes Echo30 (33.3%) and CVA6 (18.1%), which were almost absent from the Roma and immigrant populations but also from environmental samples (unpublished data). In a healthy Roma child from the prefecture of Evros in 2012, supplementary EV surveillance revealed only an Echo30 strain which was genetically similar to other Echo30 virus strains identified at the same time and in the same region of an Echo30 outbreak that resulted in a high meningitis attack rate in children (16). In the rest of the country, Echo30 virus strains that circulated in 2013 through 2014 formed four clusters which were different from the one mentioned above, suggesting a separate introduction and evolution of the virus (data not published). Overall, genetic sequencing of the Greek enteroviral strains revealed that the majority of them might have shared a common evolutionary history with other strains isolated in Europe (Fig. 2B).
In our study, PCR proved to be a powerful tool for detecting and subtyping EVs. Although cell culture is considered the gold standard method for the identification of PVs, it enabled the detection of only 58.3% of positive samples. Reliable rRT-PCR results were obtained even in the absence of infectious virus and in samples with a low viral load. In addition, seroneutralization supported only specific EV subtypes and thus failed to successfully type 29.7% of EVs which circulated throughout Greece. In contrast, RT-PCR based on the VP1 region supported the characterization of 86.5% of the EVs identified in our study.
Given that Greece is a key entry point of migrants into Europe, there is a constant risk to other European Union countries for the importation of WPV from areas in which polio is endemic and for the reintroduction of SL or circulating VDPVs from countries where OPV vaccination is still implemented. An active supplementary EV surveillance system, in addition to an AFP investigation for wild and SL polioviruses and the vaccination of migrants, represent key risk assessment strategies. Even if global polio eradication is achieved, EV surveillance should continue indefinitely in Greece to improve the epidemiological understanding of other important enteroviruses, such as EV71.
MATERIALS AND METHODS
Ethics statement.
This study was approved by the institutional ethics committee of the Hellenic Centre for Disease Control & Prevention (HCDCP). Patient records were coded and deidentified prior to analysis. No identifying details are included in this article.
Enterovirus surveillance in stool specimens.
Supplementary EV surveillance was initially implemented in Greece in 2008 as a pilot study until its establishment at the end of 2010 to complement AFP surveillance. Between 2008 and mid-2014, 415 stool samples were collected from healthy children ≤15 years old belonging to high-risk population groups with low vaccination coverage and poor sanitation and living conditions in representative geographical areas of Greece. The study was performed in collaboration with the Hellenic Centre for Disease Control & Prevention (HCDCP) and local support centers for Roma and underserved populations that provide medical and social services to local (stable and mobile) Roma populations and other vulnerable population groups. The samples were collected from eight regions throughout Greece (Table 3) and sent to the national poliovirus/enterovirus reference laboratory (HPRL) for further processing.
TABLE 3.
Spatiotemporal and numerical distribution of stool and sewage water samples included in the study
| Region | Prefecture | 2008 (stool) | 2010 (stool) | 2011 (stool) | 2012 |
2013 |
2014 |
|||
|---|---|---|---|---|---|---|---|---|---|---|
| Stool | Sewage | Stool | Sewage | Stool | Sewage | |||||
| Eastern Macedonia and Thrace | Evros | 30 | 0 | 37 | 16 | 19 | 11 | 0 | 141a | 5 |
| Rodopi | 0 | 18 | 2 | 0 | 0 | 19 | 0 | 0 | 0 | |
| Xanthi | 0 | 0 | 0 | 26 | 3 | 0 | 0 | 9 | 0 | |
| Kavala | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 28 | 0 | |
| Drama | 0 | 0 | 0 | 0 | 0 | 7 | 0 | 4 | 0 | |
| Central Macedonia | Thessaloniki | 0 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 0 |
| Chalkidiki | 0 | 0 | 0 | 0 | 9 | 0 | 0 | 0 | 0 | |
| Pieria | 0 | 0 | 0 | 0 | 6 | 0 | 0 | 0 | 1 | |
| Imathia | 0 | 20 | 19 | 20 | 0 | 10 | 0 | 0 | 0 | |
| Western Macedonia | Grevena | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 |
| Thessaly | Larisa | 0 | 0 | 20 | 0 | 12 | 13 | 0 | 17 | 8 |
| Trikala | 0 | 14 | 0 | 0 | 3 | 0 | 0 | 0 | 1 | |
| Magnisia | 15 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 1 | |
| Karditsa | 0 | 0 | 0 | 0 | 3 | 0 | 0 | 0 | 1 | |
| Epirus | Ioannina | 0 | 0 | 0 | 0 | 5 | 0 | 0 | 0 | 0 |
| Ionian islands | Corfu | 0 | 10 | 7 | 0 | 0 | 0 | 0 | 0 | 0 |
| Central Greece | Fokida | 6 | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| Western Greece | Ileia | 0 | 0 | 0 | 0 | 0 | 10 | 0 | 0 | 0 |
| Peloponnese | Lakonia | 0 | 0 | 0 | 0 | 0 | 17 | 0 | 19a | 0 |
| Messinia | 0 | 0 | 0 | 0 | 0 | 0 | 23 | 0 | 8 | |
| Argolida | 0 | 0 | 0 | 0 | 22 | 0 | 0 | 0 | 12 | |
| Korinthia | 0 | 0 | 0 | 0 | 0 | 0 | 8 | 0 | 0 | |
| Attica | Attiki | 0 | 0 | 10 | 0 | 4 | 0 | 0 | 0 | 0 |
| Crete | Irakleio | 0 | 0 | 0 | 0 | 10 | 0 | 0 | 0 | 0 |
| North Aegean Islands | Lesvos | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 54a | 0 |
| Samos | 0 | 0 | 0 | 0 | 0 | 0 | 0 | 93a | 0 | |
| South Aegean Islands | Dodecanese (Rodos) | 0 | 0 | 0 | 0 | 8 | 0 | 0 | 0 | 0 |
Includes samples obtained from immigrant populations.
Supplementary EV surveillance was reinforced in 2014 by the additional study of 307 fecal samples collected from immigrant populations who resided in four different campus areas of Eastern Macedonia and Thrace, the North Aegean Islands, and Peloponnese (Table 3). Participants provided samples voluntarily. The age of participants ranged from 0 to 53 years (mean age, 23 years), and 73.9% of them came from countries where PV was still endemic (Afghanistan, Nigeria, and Pakistan). The remaining 26.1% arrived from Iraq, Somalia, or Syria. In almost all cases, vaccination history of the participants was unknown.
Environmental surveillance in sewage water samples.
Environmental surveillance was initiated in 2012. Collection of environmental samples was accomplished following the WHO guidelines for environmental poliovirus surveillance (11). In total, 179 sewage water samples were collected from areas where high-risk populations resided and from immigrant concentration camps from nine regions throughout Greece by 17 regional public health laboratories (RPHLs) and sent to the HPRL for further processing (Table 3).
Treatment of clinical and environmental samples.
All samples were kept at −20°C until they were processed. Two grams of each fecal sample was homogenized in a plastic tube containing 90% Dulbecco's modified Eagle medium (DMEM), 10% chloroform, and sterile glass beads. After homogenization and centrifugation at 2.000 rpm for 20 min, viral RNA was extracted from 140 μl of the supernatant using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany) according to manufacturer instructions. Sewage water samples were concentrated by the two-phase polyethylene glycol (PEG)-dextran separation method and decontaminated by chloroform extraction, as recommended by the WHO (11). A cell culture step followed, as described in “Cell Culture and Virus Isolation,” and 140 μl of the culture supernatant was subjected to viral RNA extraction as previously mentioned for fecal samples.
Enterovirus molecular detection.
Stool specimens and cell culture supernatants from sewage water samples were analyzed for EV presence by an in-house real-time reverse-transcription PCR (rRT-PCR) targeting the 5′ UTR. The enterovirus group-specific (panEV) primers and probe were used as described by Kilpatric et al. (34), with minor protocol modifications concerning reagent concentrations. Optimization was performed on a poliovirus type 1 genome with a detection limit of 32 copies/reaction. The assay was accredited according to ISO 15189 by the Hellenic Accreditation System for implementation in clinical samples. For the specific detection of PVs, an in-house pan-poliovirus rRT-PCR targeting the VP1 region based on primers and probe sequences proposed by the WHO (35) was performed. Additional rRT-PCR experiments were applied for intratypic differentiation between WPV and SL strains and for VDPV identification (35).
To detect the presence of inhibitors in the RNA extracts and to indicate template loss during processing, the potato Solanum tuberosum phyB gene was copurified and coamplified with each sample as previously described (36). To avoid contamination in PCR experiments, all necessary controls and precautions were taken, including use of a three-room separation system.
Cell culture and virus isolation.
Fecal samples that tested positive by rRT-PCR were inoculated into rhabdomyosarcoma (RD) cell cultures (from a human cell line that supports the replication of most prototype strains of EVs). RD cells were kindly provided by the regional reference WHO laboratory (Rome, Italy) and treated according to standard WHO methodology (37). Two other cell lines were also used for sample inoculations, L20B, a genetically engineered mouse cell line expressing the human poliovirus receptor, and Hep-2, human epithelial type 2 cells. Cultures were observed daily, and cytopathic effects (CPEs) were recorded.
Sewage water samples were initially inoculated in the three cell lines after their concentration. Supernatants from the first passage of positive and negative cell cultures were subjected to rRT-PCR, while a second passage was performed to record CPEs. Culture supernatants from all fecal and sewage water samples were collected and stored at −20°C.
Enterovirus typing.
EV cell culture isolates were typed by seroneutralization utilizing pooled PV and CVB antisera, as well as pools of A-G antisera for echovirus typing (RIVM/National Institute of Public Health and the Environment, Bilthoven, Netherlands) according to WHO methodology (37).
Genotypic identification of enterovirus strains was performed by seminested RT-PCR amplification of the VP1 region. Extracted RNA was used as a template for cDNA synthesis, and seminested RT-PCR was performed as described by Nix et al. (38). PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) and the MinElute gel extraction kit (Qiagen) and were sequenced bidirectionally using BigDye Terminator cycling conditions and the GenomeLab DTCS quick start sequencing kit (Beckman Coulter, Inc., USA) on a CEQ 8000 genetic analyzer (Beckman Coulter).
Phylogenetic analysis.
Sequences obtained were identified in terms of closest homology sequence using BLAST (see http://blast.ncbi.nlm.nih.gov/Blast.cgi). Multiple sequence alignments with the respective reference strains from GenBank database sequences were made by BioEdit Sequence Alignment Editor (Ibis Biosciences, USA). Molecular Evolutionary Genetics Analysis (MEGA) software, version 5 (see http://www.megasoftware.net/), was used for the phylogenetic analysis. Phylogenetic trees were constructed by the neighbor-joining method (bootstrap resampling with 1,000 replicates).
Accession number(s).
Distinct partial VP1 sequences from isolates in this study were submitted to GenBank, EMBL, and DNA Databank of Japan nucleotide sequence databases under the accession numbers KX187341 to KX187425 (as well as APU50822 to APU50906).
ACKNOWLEDGMENTS
This study was partially supported by the Hellenic Centre for Disease Control & Prevention (HCDCP), Athens.
The active involvement of the personnel of the Department for the Epidemiological Surveillance and Intervention, Panagiotis Katsaounos, Pandelis Mavraganis, and Anastasia Andreopoulou, is gratefully acknowledged. We thank the staff of the water authorities from (i) the Central Public Health Laboratory of Athens, (ii) the Peripheral Public Health Laboratories of Eastern Macedonia and Thrace, Central Macedonia, Epirus, Thessaly, Western Greece, South Aegean (Rhodos), and Crete, (iii) the General Directorate of Public Health and Social Care, Region of Epirus and Peloponnese, (iv) the Directorate of Public Health, Prefecture of Corinthos and Messinia, and (v) the D.E.Y.A of Nafplio, Argos and Mycenae, and Epidaurus for their involvement in the environmental surveillance. We also thank the staffs of (i) the Support Centre for Roma and vulnerable population groups (municipality of Trikala, prefecture of Trikala; municipality of Pyrgos, prefecture of Elia), (ii) the medical health center for Roma population (municipality of Alexandreia, prefecture of Imatheia; municipality of Drama, prefecture of Drama; municipality of Farsala, prefecture of Larisa; municipality of Orestiada, prefecture of Evros; municipality of Drosero, prefecture of Xanthi; municipality of Nestos, prefecture of Kavala; municipality of Sapes, prefecture of Rodopi), (iii) the Medico-social Centre (municipality of Komotini, prefecture of Komotini; municipality of Kipaki, prefecture of Trikala; municipality of Delta, prefecture of Thessaloniki; municipality of Aigeiros, prefecture of Rodopi; municipality of Corfu, prefecture of Corfu; municipality of Larisa, prefecture of Larisa; municipality of Saint-Paraskevi Volos, prefecture of Magnisia; municipality of Amfissa, prefecture of Phocis), and (iv) the Health Centre (municipality of Markopoulo, prefecture of Attica) for their participation in the stool surveillance.
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