Seroepidemiological Survey of Hantavirus Infection in Healthy People in Vallès Occidental,Barcelona

Abstract

Introduction:

Hantaviruses are the etiological agents of hemorrhagic fever with renal syndrome in Europe and Asia, and hantavirus pulmonary syndrome in America. Approximately 150,000 cases are reported annually worldwide. In Spain, some hantavirus infection cases have been described. Besides, rodents that have been described as hantavirus reservoirs are present. The aim of the present study was to determinate the seroprevalence of hantavirus in humans in the northeast of Spain.

Materials and Methods:

During a 5-month period, 217 serum samples were collected. The study population was stratified by age, gender, and residential area. Age, gender, residential area, contact with pets, contact with wild animals, contact with farm animals, and occupation were surveyed. Immunoglobulin G antibodies to Hantaan virus, Seoul virus, or Puumala virus were examined by immunofluorescence assay. Titles ≥1/32 against any of the hantavirus were considered positive.

Results:

Four (1.8%) positive samples were detected. Age ranged from 14 to 67 years. Two subjects were male. Three samples reacted to both Puumala virus and Hantaan virus. The other one reacted against all three hantavirus surveyed. Titles ranged from 32 to 1024. The highest titles were found against Seoul virus.

Conclusions:

Our data show serological evidence about hantavirus infection among population of Catalonia, northeast of Spain. Seroprevalence rate was (around 2%) similar to other regions of Spain.

Introduction

H antaviruses belong to the Bunyaviridae family and cause a great variability in terms of clinical manifestations and severity. Rodents are their main reservoir. Each hantavirus type is carried by a specific rodent species. Humans generally become infected through inhalation of aerosols contaminated with saliva, urine, or feces of infected animals (Rodríguez and Vaqué 1994), although rodent's bite or direct contact of skin lesion with feces could also be transmission routes (Gegúndez and Lledó 2005). Hantavirus are the etiological agents of hemorrhagic fever with renal syndrome (HFRS) in Europe and Asia (Heyman et al. 2008), and hantavirus pulmonary syndrome in America (McCaughey and Hart 2000). Approximately 150,000 cases are reported annually worldwide (Vapalahti et al. 2003, Gegúndez and Lledó 2005, Jonsson et al. 2010).

Until now, >30 serotypes have been identified (Gegúndez and Lledó 2005), half of which are human pathogens. Clinical manifestations vary in severity depending on the causative virus.

Most cases of Hantaan virus (HTNV) have been reported from Asia and cause the most severe form of HFRS, whose mortality is around 10%.

Seoul virus (SEOV) has been reported to cause around 25% HFRS cases in Asia. HFRS caused by SEOV is less severe than that caused by HTNV, presenting 1% of mortality (Vapalahti et al. 2003, Gegúndez and Lledó 2005). Its main reservoir is Rattus sp., which is present worldwide. In fact, it has been detected in rats from Asia, America, and Europe (McCaughey and Hart 2000, Heyman et al. 2004, 2009, Lokugamage et al. 2004). Thus, SEOV could be a cause of human disease worldwide (Clement et al. 1994, 1997, McCaughey and Hart 2000, Lledó et al. 2004, Chow et al. 2005).

Puumala virus (PUUV) is the most prevalent hantavirus in Europe (Vapalahti et al. 2003, Gegúndez and Lledó 2005), and it is found almost everywhere in Europe (Zöller et al. 1995, McCaughey and Hart 2000, Markotić et al. 2002, Lledó et al. 2003, Heyman et al. 2008, Pettersson et al. 2008). PUUV causes the mildest form of HFRS called nephropatia epidemica. Often, this form is subclinical or misdiagnosed. In fact, about 30% PUUV infections present clinical symptoms and only 20% are hospitalized. Fatal cases are rare (0%–0.4%) (Vapalahti et al. 2003). This fact could explain the dissociation between the human seroprevalence rate in Europe, around 2%, and the number of known cases (Rodríguez and Vaqué 1994). Recently, human cases of nephropatia epidemica due to PUUV infection in Europe have increased (Olsson et al. 2003, Clement et al. 2010) and outbreaks have been described (Schilling et al. 2007, Pettersson et al. 2008).

In Spain, some hantavirus infection cases have been described (Segura et al. 2002, Lledó et al. 2003, 2007). Moreover, rodents that have been described as hantavirus reservoirs are present in Spain. However, a few seroepidemiological studies have been done, both in general and in risk population (Lledó et al. 2002, 2003, 2004, Gegúndez et al. 1996) or patients (Lledó et al. 2003, 2007).

The aim of this study was to extend the knowledge of the geographical distribution of human hantavirus infection in Spain. Thus, seroprevalence of hantavirus in a representative sample of the general population from Northeastern Spain will be determined.

Materials and Methods

Geographical area

The study was undertaken in Vallés Occidental. This is a predominantly urban area near the coast in Barcelona, Northern Spain. A total of eleven municipalities participated in the study. Population of these municipalities is around 407,736 inhabitants.

Samples

During a 5-month period, from September to January, 217 serum samples from patients who had been visited at Sabadell Hospital were collected. The samples include 161 adults undergoing minor surgery and 56 children treated for noninfectious diseases in the Pediatrics Emergency Service. Written consent was obtained from all adult participants and from parents or legal guardians of minors.

Taking into account the actual population of Vallés Occidental, the study population was stratified by age (0–14, 15–29, 30–44, 45–64, and ≥65 years), gender, and residential area (rural, semirural, and urban). Municipalities with <5000 inhabitants were included in rural area group, municipalities with 5000–50,000 inhabitants were considered semirural areas, and municipalities with >50,000 inhabitants were regarded as urban areas.

Each patient filled out a questionnaire in which the following variables were registered: age, gender, residential area, contact with pets, contact with wild animals, contact with farm animals, and occupation. Those inhabitants unable to answer the epidemiological survey were excluded.

One milliliter of blood sample was aseptically collected and introduced in a serum-separating tube and stored at −80°C for serologic analyses. A serological survey was carried out according to the ethical guidelines of the Ethics Committee approval from Sabadell Hospital.

Of the 217 subjects, 118 were male and 99 female. The mean age was 34.36 years (0–91 years). Subjects were reported by 11 municipalities, and 144 (66.3%), 59 (27.2%), and 14 (6.5%) subjects live in urban, semirural, and rural areas, respectively; 35 (16.1%) reported contact with pets, and 4 (1.8%) with stray animals, 7 (3.2%) with farm animals and 2 (0.9%) with wild animals. In the group of 161 adults (≥18 years), there were 11 (6.8%) students, 27 (16.8%) retired, 37 (23%) housewives, 53 (32.9%) workers, and 26 (16.1%) unemployed. In seven (4.3%) adults the occupation was unknown.

Serological technique

Serum sample were evaluated by indirect immunofluorescence assay (IFA). Slides coated with Vero E6 cells (ATCC CRL 1586) infected with HTNV (strain 76-118) SEOV (strain 80/39) and PUUV (strain Cg 18-20) mixed with noninfected Vero E6 cells (20:20:20:40) were used to detect human immunoglobulin G (IgG) antibodies against hantavirus. Sera were screened at 1/16 initial dilution. The fluorescein-labeled conjugate used was a rabbit anti-human IgG serum (Sigma, St. Louis, MO) diluted 1/128 in phosphate-buffered saline containing Evan's blue. Spots of uninfected Vero E6 cells were used as negative controls.

When a typical granular pattern of fluorescence in the cytoplasm of the infected cells was observed, sera were titled. Twofold dilutions of serum specimens in phosphate-buffered saline were applied separately on the HTNV, SEOV, or PUUV slides. Binding sera were detected by rabbit anti-human IgG serum (Sigma). The highest dilution, at which distinct and specific fluorescence was seen, was scored as the end-point title for the serum sample. Titles >1/32 against any of the hantavirus were considered positive.

Results

Of 217 samples, IgG antibody titles against hantavirus were detected in 4 (1.8%). In fact, all four sera reacted with PUUV and HTNV: three samples showed titles of 1/32 against PUUV and HTNV, and the other one showed titles of 1/256, 1/512, and 1/1024 against PUUV, HTNV, and SEOV, respectively. Considering these results, 1.8% samples reacted against PUUV and HTNV and 0.5% against SEOV. Serological and demographic data are shown in Table 1.

Table 1.

Serological and Demographic Information from Seropositive Subjects

	Seropositive subjects
Serum sample	8	100	142	158
Serological data
Puumala	1/32	1/256	1/32	1/32
Seoul	<1/32	1/1024	<1/32	<1/32
Hantaan	1/32	1/512	1/32	1/32
Demographic data
Age (years)	14	67	23	18
Gender	Woman	Man	Woman	Man
Residence area	Urban	Urban	Suburban	Urban
Contact with animals	No	No	No	No
Occupation	Student	Retired	Butcher	Unknown

Two seropositive subjects were male and two were female. Age ranged from 14 to 67 years and mean age was 30.5 ± 24.61 years. Three of them live in an urban area and the other one in a semirural area. None of them reported contact with pets, or contact with wild animals, or contact with farm animals.

Discussion

The diseases caused by hantaviruses have been described in various European countries (Gegúndez and Lledó 2005), especially in recent years. Moreover, they have been related to outbreaks (Schilling et al. 2007, Pettersson et al. 2008, Clement et al. 2010).

In Spain, some cases of hantavirus infection have been described (Salavert et al. 1996, Segura et al. 2002, Lledó et al. 2007). The presence of hantavirus infection in Spain has been suggested since the 1990s (Rodríguez and Vaqué 1994, Rodríguez et al. 1995, Gegúndez et al. 1996, Salavert et al. 1996, Saz et al. 1997). However, the number of epidemiological studies of hantavirus in our country is small. For instance, a study in occupational hazard biologists and individuals without apparent risk was carried out. The samples of this study were collected around the country (Lledó et al. 2004). In addition there are some studies in general population, in individuals from different areas (Lledó et al. 2003), or from some specific regions such as Soria (Gegúndez et al. 1996), Catalunya (Rodríguez et al. 1995), Guadalajara (Saz et al. 1997), and Madrid (Lledó et al. 2002).

IFA was chosen to perform the present study in our area. Due to the pattern of cross reactions of the hantavirus, it was possible to obtain to sufficient spectrum to avoid false negatives using PUUV, HTNV, and SEOV antigens (Gegúndez and Lledó 2005).

Consistent with other studies conducted in Spain, our results show a low seroprevalence (1.8%). Thus, studies carried out in the central regions of Spain by IFA (Gegúndez et al. 1996, Saz et al. 1997, Lledó et al. 2002) showed similar seroprevalences (1.35%–2.2%). Another study was performed with >10,000 samples of healthy population from different regions (Lledó et al. 2003). The seroprevalence observed was <0.1% and positive cases were only found in central, north, and northeast Spain. Although part of the samples were collected in our area, there was not any positive case. However, seroprevalences of 2.3% in 657 healthy individuals and of 3.1% in 543 individuals undergoing hemodialysis had been found in an earlier study performed in our region (Rodríguez and Vaqué 1994). Our data support this second study, obtaining a similar percentage of seropositive in healthy population and confirming the presence of hantavirus in our area.

Some countries from center of Europe presented seroprevalences similar to those found in our study: Belgium (1.35%) (Clement and van der Groen 1987), The Netherlands (0.9%) (Groen et al. 1995), Italy (2.3%) (Nuti et al. 1992), and Germany (1.7%) (Zöller et al. 1995). However, others such as Sweden (8%–8.9%) (Ahlm et al. 1997), Finland (5%) (Brummer-Korvenkontio et al. 1999), and Greece (4%–14%) (Papadimitriou and Antoniadis 1994) showed higher seroprevalence.

Our results show three patients with a positive result to PUUV and HTNV with low titles. PUUV virus causes thousands of cases of HFRS each year in Europe (Gegúndez and Lledó 2005). Many European studies have described PUUV infection (Groen et al. 1995, Brummer-Korvenkontio et al. 1999, McCaughey and Hart 2000, Markotić et al. 2002, Olsson et al. 2003, Schilling et al. 2007, Pettersson et al. 2008, Clement et al. 2010). However, this serotype is less frequent in studies conducted in our country. In fact, 2% of the population of Soria presented antibodies against PUUV, and only 0.05% of samples showed reaction only with PUUV antigen (Gegúndez et al. 1996). In our study, there were low titles against PUUV in three patients, as well as titles were twofold lower than titles against SEOV in the other patient. Other studies in our country could corroborate these data. For example, six different patterns were found in a study conducted in Madrid (Lledó et al. 2002). In this study 0.23% of samples presented antibodies against PUUV, 0.23% against SEOV, and 0.18% against HTNV (titles ranged from 1/32 to 1/512). The most common serologic pattern (33.3%) was that in which the reaction was observed with all three serotypes. As it has been pointed by Lledó et al. (2003), Clethrionomys glareolus is the reservoir of PUUV and it is only found in a few northern areas of our country. Therefore, it could be possible that PUUV may not be present in Spain. Considering low titles found, results could be cross-reactions or, if a new rodent vector was found, there could be some other serotype antigenically similar. It would be interesting to conduct more research studies to clarify this point.

On the other hand, one of our patients (0.5%) presented a much higher reactivity against SEOV than that observed against PUUV and HTNV. Taking into account these high titles, this case was reviewed. The patient had presented a compatible clinical. Thus, considering the high titles against SEOV and without the presence of other causes that could justify the disease, this result point to an acute infection whose etiological agent was a hantavirus antigenically similar to SEOV (Segura et al. 2002).

The SEOV has a worldwide distribution and it has been found in rats of the species Rattus rattus and Rattus norvegicus from around the world (Clement et al. 1994, 1997, McCaughey and Hart 2000, Heyman et al. 2004, 2009, Lokugamage et al. 2004). For this reason, it could be cause of human diseases worldwide (McCaughey and Hart 2000, Lledó et al. 2004, Chow et al. 2005). However, few cases of infections caused by this virus have been reported and confirmed outside Asia, where it could be responsible for >25% of human cases described (Lokugamage et al. 2004, Chow et al. 2005). The presence of SEOV in our country was also suggested in 2004 (Lledó et al. 2004). In that study, which analyzed an occupational risk population, only 1 of the 22 subjects (4.54%) showed a positive serology against SEOV. This subject reported contact with rats.

It has been reported that hantavirus IgG antibodies remain detectable for decades after infection (Brummer-Korvenkontio et al. 1999). For this reason, many studies have observed a higher prevalence in older individuals (Lledó et al. 2002). However, predominance toward younger individuals is observed in the study in Soria (Gegúndez et al. 1996). Some studies did not present differences between sexes (Nuti et al. 1992, Gegúndez et al. 1996, Lledó et al. 2002, 2004). In our study three of four individuals are young and a difference between sexes has not been found. However, the very limited number of seropositives is a limitation to obtain conclusions.

No significant differences were found concerning habitat. In the study performed in Madrid (Lledó et al. 2002), significantly higher prevalence was observed in the rural area. Although hantavirus infection is generally associated with rural areas, Gegúndez and Lledó (2005) suggested that some forms of SEOV virus could occur in the urban environment. It is interesting that none of the seropositive cases of our study lived in rural areas. However, due to the low number of positives as well as urban predominance, our data are not conclusive in this way.

In conclusion, in spite of the low prevalence in our population, this study confirms the evidence of hantavirus infection in our area. Physicians and public health personnel should be aware of the possibility of this infection.

Footnotes

Disclosure Statement

Authors do not have any conflict of interest.

References

Ahlm

, Juto

, Stegmayr

, Settergren

et al. Prevalence of serum antibodies to hantaviruses in northern Sweden as measured by recombinant nucleocapsid proteins. Scand J Infect Dis, 1997; 29:349–354.

Brummer-Korvenkontio

, Vapalahti

, Henttonen

, Koskela

et al. Nephropathia epidemica in Finland 1989–1996: an epidemiological study of 6701 cases. Scand J Infect Dis, 1999; 31:427–435.

Chow

, Shu

, Huang

, Wang

et al. A retrospective study of hantavirus infection in Kinmen, Taiwan. J Microbiol Immunol Infect, 2005; 38:343–349.

Clement

, Heyman

, Mc Kenna

, Colson

, Avsic-Zupanc

. The hantaviruses of Europe: from the beside to the bench. Emerg Infect Dis, 1997; 3:205–211.

Clement

, Maes

, van Ypersele de Strihou

, van der Groen

et al. Beechnuts and outbreaks of nephropathia epidémica (NE): of mast, mice and men. Nephrol Dial Transplant, 2010; 25:1740–1746.

Clement

, Mc Kenna

, Avsic-Zupanc

, Skinner

. Rat-transmitted hantavirus diseases in Sarajevo. Lancet, 1994; 344:131.

Clement

, van der Groen

. Acute hantavirus nephropathy in Belgium: preliminary results of a sero-epidemiological study. Adv Exp Med Biol, 1987; 212:251–263.

Gegúndez

, Lledó

. Infection due to Hantavirus and other rodent-borne viruses. Enferm Infecc Microbiol Clin, 2005; 23:492–500.

Gegúndez

, Saz

, Alves

, Merino

et al.

[Hantavirus infection in Spain: seroepidemiologic study in the province of Soria]

Med Clin (Barc), 1996; 106:131–133.

10.

Groen

, Gerding

, Jordans

, Clement

et al. Hantavirus infections in The Netherlands: epidemiology and disease. Epidemiol Infect, 1995; 114:373–383.

11.

Heyman

, Baert

, Plyusnina

, Cochez

et al. Serological and genetic evidence for the presence of Seoul hantavirus in Rattus norvegicus in Flanders, Belgium. Scand J Infect Dis, 2009; 41:51–56.

12.

Heyman

, Plyusnina

, Berny

, Cochez

et al. Seoul hantavirus in Europe: first demonstration of the virus genome in wild Rattus norvegicus captured in France. Eur J Clin Microbiol Infect Dis, 2004; 23:711–717.

13.

Heyman

, Vaheri

. ENIVD Members. Situation of hantavirus infections and haemorrhagic fever with renal syndrome in European countries as of December 2006. Euro Surveill, 2008; 13pii:18925.

14.

Jonsson

, Moraes

, Vapalahti

. A global perspective on hantavirus ecology, epidemiology, and disease. Clin Microbiol Rev, 2010; 23:412–441.

15.

Lledó

, Gegúndez

, Martínez

, Sanfeliu

. [Epidemic nephropathy in Spain: first imported case] Med Clin (Barc), 2003; 121:677.

16.

Lledó

, Gegúndez

, Saz

, Alves

et al. Serological study of hantavirus in man in the Autonomous Community of Madrid, Spain. J Med Microbiol, 2002; 51:861–865.

17.

Lledó

, Gegúndez

, Saz

, Beltrán

. Seroprevalence of antibodies to Seoul virus in occupational risk groups. Enferm Infecc Microbiol Clin, 2004; 22:217–219.

18.

Lledó

, Gegúndez

, Ledesma

, Domingo

et al.

Prevalence of anti-hantavirus antibodies in patients with hypertransaminemia in Madrid (Spain)

Am J Trop Med Hyg, 2007; 77:371–375.

19.

Lledó

, Klingström

, Gegúndez

, Plyusnina

et al. Hantavirus infections in Spain: analysis of sera from the general population and from patients with pneumonia, renal disease and hepatitis. J Clin Virol, 2003; 27:296–307.

20.

Lokugamage

, Kariwa

, Lokugamage

, Iwasa

et al. Epizootiological and epidemiological study of hantavirus infection in Japan. Microbiol Immunol, 2004; 48:843–851.

21.

Markotić

, Nichol

, Kuzman

, Sanchez

et al. Characteristics of Puumala and Dobrava infections in Croatia. J Med Virol, 2002; 66:542–551.

22.

McCaughey

, Hart

. Hantaviruses. J Med Microbiol, 2000; 49:587–599.

23.

Nuti

, Amaddeo

, Autorino

, Crovatto

et al. Seroprevalence of antibodies to hantaviruses and leptospires in selected Italian population groups. Eur J Epidemiol, 1992; 8:98–102.

24.

Olsson

, Dalerum

, Hörnfeldt

, Elgh

et al. Human hantavirus infections, Sweden. Emerg Infect Dis, 2003; 9:1395–1401.

25.

Papadimitriou

, Antoniadis

. Hantavirus nephropathy in Greece. Lancet, 1994; 343:1038.

26.

Pettersson

, Boman

, Juto

, Evander

et al. Outbreak of Puumala virus infection, Sweden. Emerg Infect Dis, 2008; 14:808–810.

27.

Rodríguez

, Vaqué

. [Hantavirus disease: an emerging infection] Enferm Infecc Microbiol Clin, 1994; 12:477–479.

28.

Rodríguez

, Vaqué

, Gurri

. La fiebre hemorrágica con síndrome renal debida a Hantavirus. Med Clin (Barc), 1995; 105:62–67.

29.

Salavert

, López-Hontangas

, Ponz

, Gobernado

. [Hantavirus infection and the pulmonary syndrome: another new entity to look for in our environment?] Enferm Infecc Microbiol Clin, 1996; 14:395–396.

30.

Saz

, Gegúndez

, Beltrán , Hantavirus

. En: Consejería de Sanidad y Bienestar Social. Junta de Castilla y León . Zamora: España, 1997; 7–10.

31.

Schilling

, Emmerich

, Klempa

, Auste

et al. Hantavirus disease outbreak in Germany: limitations of routine serological diagnostics and clustering of virus sequences of human and rodent origin. J Clin Microbiol, 2007; 45:3008–3014.

32.

Segura

, López

, Sanfeliu

, Ponz

et al.

A new case of hantavirus disease in Spain?

Rev Clin Esp, 2002; 202:181.

33.

Vapalahti

, Mustonen

, Lundkvist

, Henttonen

et al. Hantavirus infections in Europe. Lancet Infect Dis, 2003; 3:653–661.

34.

Zöller

, Faulde

, Meisel

, Ruh

et al. Seroprevalence of hantavirus antibodies in Germany as determined by a new recombinant enzyme immunoassay. Eur J Clin Microbiol Infect Dis, 1995; 14:305–313.