Seroprevalence of Toscana Virus Infection in Tunisia

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International Journal of Infectious Diseases 17 (2013) e1172–e1175

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International Journal of Infectious Diseases

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Seroprevalence of Toscana virus infection in Tunisia

a b c b a,

Ons Fezaa , Olfa Bahri , Nissaf Ben Alaya Bouaﬁf , Henda Triki , Ali Bouattour *

Laboratoire d’Epide´miologie et Microbiologie Ve´te´rinaire, Service d’Entomologie Me´dicale, Institut Pasteur de Tunis, Universite´ Tunis El Manar, BP74, 1002

Tunis-Be´lve´de`re, Tunisia

Laboratoire de Recherche ‘‘Diversite´ Ge´ne´tique des Ente´rovirus et Virus des He´patites’’ Institut Pasteur de Tunis, Faculte´ de Me´decine de Tunis, Universite´

Tunis El Manar, Tunis, Tunisia

Observatory of New and Emerging Diseases, Ministry of Health, Tunis, Tunisia

A R T I C L E I N F O S U M M A R Y

Article history: Objectives: The main objective of this study was to assess the prevalence of IgG antibodies against

Received 10 June 2013

Toscana virus (TOSV) by an ELISA test and to determine the extent of its circulation in Tunisia.

Received in revised form 2 August 2013

Methods: An indirect ELISA test was performed to detect anti-TOSV IgG. The results were compared to

Accepted 13 August 2013

those of an indirect ﬂuorescent antibody (IFA) test.

Corresponding Editor: Eskild Petersen,

Results: The survey tested 494 healthy people from various regions of Tunisia by ELISA for anti-TOSV

Aarhus, Denmark

IgG; 47 people (9.5%) were found to be positive. Seroprevalence varied by bioclimatic region and gender.

Two hundred and twelve samples, randomly chosen from the same selected population and tested with

Keywords: ELISA, were retested using an IFA for IgG antibodies. An 85% concordance between the IFA and ELISA was

Toscana virus

obtained (kappa = 0.650).

Phlebotomus

Conclusions: These serological data conﬁrm the circulation of TOSV in different bioclimatic zones in

ELISA

Tunisia where the vector sand ﬂies are found. The detection of IgG against TOSV suggests that the

IFA

diagnosis of TOSV infection is often neglected, as this virus often causes asymptomatic infections, with

Tunisia

only a few patients developing severe illnesses involving neurological manifestations.

1. Introduction Mediterranean region, however, the epidemiology of TOSV

infection is still largely unknown. A study conducted in Morocco

Toscana virus (TOSV) is an arbovirus transmitted to humans by showed the presence of TOSV in the sand ﬂy vectors, but included

the bites of Phlebotomine sand ﬂies, mainly Phlebotomus no data on the epidemiology of the virus in humans. We have

1,2

perniciosus and Phlebotomus perﬁliewi. TOSV belongs to the recently shown that TOSV is present in Tunisia and is responsible

8,9

Phlebovirus genus of the Bunyaviridae family, and can cause brain for approximately 10% of cases of neurological disease. In

injuries. Many reported cases have shown that TOSV has a tropism addition, sand ﬂies taken from pools in northern Tunisia have been

for the central nervous system (CNS) and represents a major cause shown to carry strains of TOSV. The TOSV infection can be

1,3

of meningitis and meningo-encephalitis. In Italy, for example, diagnosed using direct methods, such as virus isolation, vital

the virus appears to be one of the three major viral pathogens antigen detection by immunoﬂuorescence, and RT PCR, or by

involved in aseptic meningitis, whose clinical manifestations indirect methods, detecting the presence of speciﬁc IgM and IgG

4 11

resemble those of other viral agents. The cases of meningitis or antibodies using immunoenzymatic techniques.

meningo-encephalitis caused by TOSV are regularly observed The main objective of this study was to assess the prevalence of

during the summer season, with a peak in August when sand ﬂy IgG antibodies against TOSV in Tunisia by ELISA test and to

activity is high. The incidence of neurological damage varies from determine the extent of its circulation in the country. As an

year to year and is directly related to differences in the annual alternative to the ELISA test, the indirect ﬂuorescent antibody test

density of the vector, which is greatly inﬂuenced by changes in (IFA) was used, and results were compared to those of the ELISA

climatic conditions. technique.

TOSV has been reported in several European countries.

5,6

Seroprevalence varies from country to country. In the southern

2. Materials and methods

2.1. Population tested

* Corresponding author. Tel.: +216 71 893 340; fax: +216 71 791 833.

The seroprevalence study was conducted using sera collected

E-mail addresses: [email protected], [email protected]

(A. Bouattour). from 494 anonymous, healthy individuals aged 5 to 85 years. Data

O. Fezaa et al. / International Journal of Infectious Diseases 17 (2013) e1172–e1175 e1173

Table 1

Distribution of the positive cases to TOSV according to geographical origin, age group, and sex

a b

Bioclimatic zones Governorates 95% CI Localities Age, No. positive/No. tested Sex

No. positive/No. tested (%) No. positive/No. tested (%)

5–19 20–39 40–59 60 M F

Humid Jendouba 15/274 (5.5%) 3.1–8.87% Jbel el Jouza 0/33 0 7 15 11 22 11

El Faija 5/89 (6%) 0 25 38 5/26 64 25

Tbe´nia 0/32 0 1 23 8 23 9

Ain Draham 9/63 (14%) 0 5 7/42 2/16 48 15

Tabarka 1/57 (2%) 1 16 1/25 15 40 17

Semi-arid Mahdia 26/112 (23%) 15.76–32.14% - 2/35 8/32 12/30 4/15 51 61

Arid Tataouine 6/108 (5.6%) 2.07–11.70% - 4/58 1/32 1/16 0/2 54 54

Total 47/494 (9.5%) 7.07–12.45% 6/94 10/118 21/189 10/93 302 192

CI, conﬁdence interval; M, male; F, female.

a 3

p < 10 .

p = 0.002.

related to age, sex, previous medical history, and geographical where the individuals had attended appointments for various

origin for each person were recorded, and subjects were classiﬁed examinations with non-infectious pathological indications.

into four age groups (Table 1). Sera were taken from volunteer The population studied came from three governorates

blood donors from whom informed consent was obtained. Sera (Figure 1) – Jendouba, Mahdia, and Tataouine – in different

from persons younger than 20 years and older than 60 years came bioclimatic zones. In Jendouba Governorate, located in a humid

from the serum banks of regional hospitals of each governorate, bioclimatic zone that gets more than 800 mm of annual rainfall,

274 serum samples were taken from people living in ﬁve randomly

selected localities: Jbel el Jouza, Tbe´nia, Ain Draham, El Faija, and

Tabarka. In Mahdia Governorate, located in central Tunisia in a

lower semi-arid bioclimatic zone with annual rainfall between

200 and 300 mm, serum samples were taken from 112 individuals.

In Tataouine Governorate, located in the south in an arid

bioclimatic area with annual rainfall between 88 and 157 mm,

108 samples were collected for antibody testing.

2.2. ELISA detection of IgG antibodies against TOSV

An indirect ELISA test was performed to detect anti-TOSV IgG

antibodies, as described above. Wells of polystyrene plates were

coated with a predetermined optimum quantity of positive and

negative antigens (ISS Phl.3), in phosphate-buffered saline (PBS)

(pH 7.2) and incubated overnight at 4 8C. Human sera were diluted

at 1:100 in PBS–TM (PBS, 0.05% Tween 20, 3% milk). Two negative

and two positive control sera were included in each assay. Goat

anti-human IgG (Fc-speciﬁc) peroxidase conjugated antibody

(Sigma) was diluted at 1:1000 and added to each well. The color

reaction was developed by adding a substrate solution containing

0 0

3,3 ,5,5 -tetramethylbenzidine (TMB) (Sigma). At each step, the

reaction mixture was incubated for 75 min at 37 8C and washed

extensively with PBS–Tween. H2SO4 was added at a ﬁnal

concentration of 1 N to stop the reaction. All serum samples were

tested in duplicate with viral and negative control antigens. The

IgG ELISA cut-off value was determined by the mean plus three

standard deviations of the optical densities (OD) read, calculated as

the average OD with antigen minus the average OD with negative

control antigen. The borderline between positive and negative

samples was calculated as the mean of the test results of 30 known

negative sera plus three standard deviations. The OD of each

sample was read at two wavelengths – 450 and 620 nm.

2.3. Comparison of ELISA and IFA techniques

Of the 494 samples tested by ELISA, only 212 were randomly

chosen and re-tested by IFA because of a limited number of slides

and an insufﬁcient quantity of serum left in some samples.

The IFA was performed in accordance with the procedure

described by Valassina et al. (1996). Brieﬂy, Vero cells infected

with a TOSV isolate (kindly provided by Dr M. Bouloy of the Institut

Pasteur, Paris) were harvested and collected on spot slides (10

Figure 1. Map of Tunisia: location of the regions studied. cells per spot); uninfected cells were collected in the same manner

e1174 O. Fezaa et al. / International Journal of Infectious Diseases 17 (2013) e1172–e1175

and used as negative control. The slides were air-dried and ﬁxed in kappa equal to 0.650 (standard error of kappa = 0.080; 95% CI

cold acetone for 10 min. When not immediately used, the slides 0.493–0.808) was obtained. The strength of the agreement is

were stored at 30 8C. The sample sera were diluted at 1:100 and considered to be good (Table 2).

screened for speciﬁc IgG antibodies along with negative and

positive control sera included in each assay. Specimens were 4. Discussion

incubated for 30 min at 37 8C, then washed three times with PBS

for 10 min at room temperature. A goat anti-human IgG (whole Several studies have demonstrated TOSV infection to be

molecule)FITC antibody conjugate (Sigma) was subsequently endemic in Mediterranean countries. TOSV is considered to be

added at a ﬁnal concentration of 1:1000, with Evans blue as a the most frequent viral agent implicated in neuromeningeal

counter-stain. The slides were incubated for 30 min at 37 8C and human infections in endemic countries.

washed with PBS for 10 min. Two operators read each slide twice To investigate the prevalence of IgG antibodies against TOSV in

and positive results were characterized by small patches of Tunisia, serum samples were collected from three different

intranuclear and intracytoplasmic ﬂuorescence. bioclimatic zones. The ELISA test showed that 9.5% (47/494) of

healthy people have antibodies against TOSV; this rate is roughly

2.4. Statistical analysis similar to that reported in blood donors from the south of France

(12%), but lower than that observed in hyper-endemic areas such

To compare the seroprevalence obtained in each region, as Italy. Indeed, earlier serological surveys have shown that TOSV is

statistical differences among groups were determined by Pearson’s endemic in the Mediterranean Basin with varying seroprevalences:

Chi-square test. Epi Info version 6.04 was used. A probability (p) 80% in Italy, 35–60% in Greece, 25% in Spain, and 20% in

3,5,15

value of less than 0.05 was considered as statistically signiﬁcant. In Cyprus. By contrast, the seroprevalence found in this

addition, the kappa coefﬁcient measuring the agreement between investigation is higher than that observed in Portugal: 3.1% in

two rates was used to evaluate the agreement between the ELISA the group showed no neurological signs and 4.2% of patients had

and the IFA test. If the raters are in complete agreement then kappa neurological signs. In the absence of data concerning the TOSV

= 1. If there is no agreement among the raters other than what reservoir host, serological variations are strictly correlated with

would be expected by chance (as deﬁned by Pr(e)), kappa = 0. favorable habitats for the sand ﬂy vectors, with a seroprevalence

distribution based on socioeconomic status. Indeed, in Tuscany,

3. Results TOSV seroprevalence was found to be higher in forestry workers

(77.2%) than in the urban population (22.7%).

3.1. Serological survey of TOSV In this study, we noted regional variations in seroprevalence:

5.5% and 5.6% in humid and arid zones, respectively, and 23% in the

A total of 494 subjects (302 male, 192 female) with an average semi-arid zone. The signiﬁcantly higher rate observed in Mahdia

age of 45 years were tested. Of the 494 samples, 47 (9.5%, 95% Governorate can be explained by the exposure of people in this

conﬁdence interval (CI) 7.07–12.45%) tested positive for anti-TOSV region to diverse and abundant sand ﬂy populations, predomi-

IgG by ELISA. The obtained seroprevalence varied between nantly P. perniciosus and P. perﬁliewi species, considered to be the

governorates and sex, and no signiﬁcant difference was seen main vectors of TOSV; these species are more active in warm,

between age groups (p = 0.441). humid environments. During the hot season, populations living in

Among the population tested, 14.6% of women (28/192, 95% CI this area are active outdoors and therefore at greater risk of being

9.91–20.38%) and 6.3% of men (19/302, 95% CI 3.38–9.65%) tested bitten by sand ﬂies.

positive for TOSV infection; this difference is signiﬁcant (p = 0.003). Seventeen species of sand ﬂy circulate in the different

Regional differences were found to be statistically signiﬁcant bioclimatic zones of Tunisia, where their distribution differs from

3 18,19

(p < 10 , Table 1). Of the 274 serum samples tested from one zone to another. In Jendouba, a humid zone, P. perniciosus

Jendouba, 15 were positive for IgG anti-TOSV corresponding to a and P. perﬁliewi are the predominant species, but the seropreva-

seroprevalence of 5.5% (95% CI 3.1–8.87%); this varied signiﬁcantly lence of TOSV was only 5.5%. This might be explained by a low

by locality (Ain Draham, Jbel El Jouza, El Faija, Tbe´nia, Tabarka), human population density and the lower sand ﬂy densities

with a signiﬁcantly higher value in Ain Draham (n = 9/63; 95% CI previously reported in this zone. In Tataouine, Phlebotomus

6.75–25.39%; p = 0.002). The seroprevalence rate in Jendouba was papatasi and Sergentomyia minuta are the dominant species,

similar to that of Tataouine, where of 108 samples, six were although P. perniciosus exists but at a lower rate. The low sand

positive for anti-TOSV IgG (5.6%, 95% CI 2.07–11.70%). By contrast, ﬂy population density of this arid zone could explain the low

in Mahdia, the prevalence was 23% (n = 26/112; 95% CI 15.76– seroprevalence of TOSV. Indeed in the Mediterranean Basin, P.

32.14%). perniciosus is predominant in the sub-humid and semi-arid zones.22

23,24

3.2. Comparison between ELISA and IFA TOSV is mainly transmitted by P. perniciosus, the dominant

species in semi-arid and arid bioclimatic zones, which is also

Among the 212 serum samples tested with the two tests, 18 involved in transmitting Leishmania infantum to dogs and humans

tested positive with both techniques. Of the remaining 194 in Tunisia. The incidence of this protozoan is also higher in the

25,26

samples, 12 were positive by IFA only and four by ELISA only. A semi-arid zone.

Our study showed no signiﬁcant difference among age groups.

There was, however, an age-dependent increase in infection in

Table 2 Jendouba and Mahdia, while in Tataouine, infection was more

Comparison of the results of the two serological techniques frequent among children. A study conducted in Granada, Spain

ELISA showed an anti-TOSV IgG rate of 4% in persons 15 years to 60.4%

in those aged >65 years, however this difference was not

IFA Number positive Number negative 24

signiﬁcant. In Tuscany the seroprevalence rate was found to

Number positive 18 12 be 19.8% in adults and 5.8% in children. A lower TOSV

Number negative 4 178

seroprevalence in children could be related to lower exposure to

Total 22 190 27

the vector.

O. Fezaa et al. / International Journal of Infectious Diseases 17 (2013) e1172–e1175 e1175

2. Peyreﬁtte CN, Devetakov I, Pastorino B, Villeneuve L, Bessaud M, Stolidi P, et al.

The serological results showed that the infection seems to occur

Toscana virus and acute meningitis, France. Emerg Infect Dis J 2005;11:778–80.

much less frequently in men than in women (p = 0.003). Women

3. Charrel RN, Gallian P, Navarro-Marı´ JM, Nicoletti L, Papa A, Sa´nchez-Seco MP,

living in rural areas are usually most involved in cleaning and et al. Emergence of Toscana virus in Europe. Emerg Infect Dis 2005;11:1657–63.

4. Braito A, Corbisiero R, Corradini S, Marchi B, Sancasciani N, Fiorentini C, et al.

feeding animals and therefore near the habitats of sand ﬂies;

Evidence of Toscana virus infections without central nervous system involve-

consequently they are much more exposed to Phlebotomus bites.

ment: a serological study. Eur J Epidemiol 1997;13:761–4.

The prevalence of detection of IgG antibodies against TOSV 5. Valassina M, Valentini M, Pugliese A, Valensin PE, Cusi MG. Serological survey of

Toscana virus infections in a high-risk population in Italy. Clin Diagn Lab suggests that the diagnosis of TOSV infection is oftenneglected, since

Immunol 2003;10:483–4.

this virus usually causes asymptomatic infections, with only a few

6. Cusi MG, Savellini GG, Zanelli G. Toscana virus epidemiology: from Italy to

patients developing severe illnesses that include neurological beyond. Open Virol J 2010;4:22–8.

manifestations (meningitis and encephalitis). Indeed, our recent 7. Es-Sette N, Nourlil J, Hamdi S, Mellouki F, Lemrani M. First detection of Toscana

virus RNA from sand ﬂies in the genus Phlebotomus (Diptera: Phlebotomidae)

study showed that 10% of patients with neurological diseases (31

8 naturally infected in Morocco. J Med Entomol 2012;49:1507–9.

cases) tested positive for TOSV IgM.

8. Bahri O, Fazaa O, Ben Alaya-Bouaﬁf N, Bouloy M, Triki H, Bouattour A. Roˆle du

We used an ELISA, considered to be the most sensitive virus Toscana dans les infections neurome´ninge´es en Tunisie. Pathol Biol (Paris)

28 2010;59:125–7.

technique, to detect IgG antibodies against TOSV, and then carried

9. Sghaier W, Bahri O, Kedous E, Fazaa O, Rezig D, Touzi H, et al. Retrospective

out an IFA test to compare the results. These two techniques are

study of viral causes of central nervous system infections in Tunisia (2003–

sensitive for detecting anti-TOSV antibodies as compared to other 2009). Med Sante Trop 2012;22:373–8.

10. Bichaud L, Dachraoui K, Piorkowski G, Chelbi I, Moureau G, Cherni S, et al.

serological tools. They are known to be easy to use and rapid in

29 Toscana virus isolated from sandﬂies, Tunisia. Emerg Infect Dis 2013;19:322–4.

reporting results, bearing in mind that positivity for IgG antibodies

11. Verani P, Nicoletti L. Phlebovirus infections. In: Porteﬁeld JS, editor. Kass

does not distinguish between current and past viral infections. handbook of infectious diseases: exotic viral infection. London: Chapman and

Hall; 1995. p. 295–318.

We estimated a concordance greater than 85% between the

12. Institut National de Recherches Forestie`res. Carte bioclimatique de la Tunisie

results of the IFA and ELISA (kappa = 0.650). However, a

selon la classiﬁcation d’Emberger. Etages et variantes. INRF; 1976. Available at:

discrepancy between the two techniques was observed and a http://eusoils.jrc.ec.europa.eu/esdb_archive/eudasm/africa/images/maps/

download/afr_tnbicl.jpg (accessed April 2013).

positive result obtained for 12 of 212 sera by IFA. This could result

13. Valassina M, Cusi MG, Valensin PE. Rapid identiﬁcation of Toscana virus by

from a false secondary reaction previously reported for IFA,

nested PCR during an outbreak in the Siena area of Italy. J Clin Microbiol

especially inherent to reading microscope slides. This suggests the 1996;34:2500–2.

utility of routine double-reading by two technicians. 14. Zehender G, Bernini F, Delogu M, Cusi MG, Rezza G, Galli M, et al. Bayesian

skyline plot inference of the Toscana virus epidemic: a decline in the effective

These serological data conﬁrm the circulation of TOSV in different

number of infections over the last 30 years. Infect Genet Evol 2009;9:562–6.

bioclimatic zones in Tunisia where the vector sand ﬂies are found.

15. De Lamballerie X, Tolou H, Durand JP, Charrel RN. Prevalence of Toscana virus

Physicians should consider this neglected disease in patients with antibodies in volunteer blood donors and patients with central nervous system

infections in southeastern France. Vector Borne Zoonotic Dis 2007;7:275–8.

unexplained meningitis and aseptic cerebrospinal ﬂuid. ELISA

16. Amaro F, Luz T, Parreira P, Marchi A, Ciufolini MG, Alves MJ. Serological

diagnostic assays are recommended to carry out the epidemiological

evidence of Toscana virus infection in Portuguese patients. Epidemiol Infect

study. In addition, the development of an RT-PCR is necessary for 2012;140:1147–50.

17. Boudabous R, Amor S, Khayech F, Marzouk M, Bdira S, Mezhoud H, et al. The

research on TOSV and other Naples virus complexes (Naples and

phlebotomine fauna (Diptera: Psychodidae) of the eastern coast of Tunisia. J Med

Sicily virus) to identify the viral strains circulating in patients and

Entomol 2009;46:1–8.

vectors. An investigation of sand ﬂy vectors is key to understanding 18. Chelbi I, Zhioua E. Conﬁrmation of the presence of Sergentomyia (Sintonius)

clydei (Sinton, 1928) in Tunisia. B Soc Pathol Exot 2012;105:396–8.

the epidemiology of the Phlebovirus-related diseases. Investigations

19. Ghrab J, Rhim A, Bach-Hamba D, Chahed MK, Aoun K, Nouira S, et al. Phlebo-

of the bio-ecology and the behavior of each vector species and the

tominae (Diptera: Psychodidae) of human leishmaniosis sites in Tunisia. Parasite

identiﬁcation of the reservoir hosts of such viruses, would help us 2006;13:23–33.

20. Croset H, Rioux JA, Maistre ME, Bayar N. Les phle´botomes de Tunisie (Diptera,

understand and monitor these phleboviruses and develop appro-

Phlebotomidae), mise au point syste´matique, chorologique et e´thologique. Ann

priate control programs.

Parasitol Hum Comp 1978;53:711–49.

21. Tabbabi A, Ghrab J, Aoun K, Ready PD, Bouratbine A. Habitats of the sandﬂy

Acknowledgements vectors of Leishmania tropica and L. major in a mixed focus of cutaneous

leishmaniasis in southeast Tunisia. Acta Trop 2011;119:131–7.

22. Rioux JA, Rispail P, Lanotte G, Lepart J. Relations phle´botomes—bioclimats en

We are grateful to Dr Michele Bouloy for providing us with the e´cologie des leishmanioses. Corollaires e´pide´miologiques. L’exemple du Maroc.

Bull Soc Botanique Fr 1984;131:549–57.

IFI slides and the Toscana antigen. We are also grateful to all the

23. Verani P, Nicoletti L, Ciufolini MG. Antigenic and biological characterization of

hospital medical staff of Jendouba, Mahdia, and Tataouine for their

Toscana virus, a new Phlebotomus fever group virus isolated in Italy. Acta Virol

help. We also thank Dr Sparagano O.A.E., Dr A. Ghram and Dr 1984;28:39–47.

24. Sanbonmatsu-Ga´mez S, Pe´rez-Ruiz M, Collao X, Sa´nchez-Seco MP, Morillas-

Deborah Glassman for constructive comments and English

Ma´rquez F, de la Rosa-Fraile M, et al. Toscana virus in Spain. Emerg Infect Dis

corrections on early drafts of the manuscript. This work was

2005;11:1701–7.

conducted with ﬁnancial support from Inter-Pasteurien Concerted 25. Dedet J, Ben Osman F, Chadli A, Croset H, Rioux JA. La leishmaniose canine en

Actions (ACIP-08). Tunisie. Ann Parasitol Hum Comp 1973;48:653–60.

26. Diouani MF, Ben Alaya Bouaﬁf N, Bettaib J, Louzir H, Jedidi S, Ftaiti A, et al. Dogs

Ethical approval: The Bioethics Committee of Institut Pasteur de

L. infantum infection from an endemic region of the north of Tunisia: a

Tunis approved this study.

prospective study. Arch Inst Pasteur Tunis 2008;85:55–61.

Conﬂict of interest: The authors declare that they have no 27. Terrosi C, Olivieri R, Bianco C, Cellesi C, Cusi MG. Age-dependent seroprevalence

of Toscana virus in central Italy and correlation with the clinical proﬁle. Clin

competing interests.

Vaccine Immunol 2009;16:1251–2.

28. Ergu¨ nay K, Litzba N, Lo MM, Aydog˘an S, Saygan MB, Us D, et al. Performance of

References

various commercial assays for the detection of Toscana virus antibodies. Vector

Borne Zoonotic Dis 2011;11:781–7.

1. Echevarria JM, de Ory F, Eulalia Guisasola M, Sanchez-Seco MP, Tenorio A, 29. Ciufolini MG, Fiorentini C, Di Bonito P, Mochi S, Colomba G. Detection of

Lozano A, et al. Acute meningitis due to Toscana virus infection among patients Toscana virus-speciﬁc immunoglobulins G and M by an enzyme-linked immu-

from both the Spanish Mediterranean region and the region of Madrid. J Clin nosorbent assay based on recombinant viral nucleoprotein. J Clin Microbiol

Virol 2003;26:79–84. 1999;37:2010–2.