SANDFLY – PAPPATACI FEVER IN BOSNIA AND HERZEGOVINA : THE NEW-OLD DISEASE

Sandfl y fever viruses (SFV) are endemic in the Mediterranean, Middle East, northern African and western Asian countries. Toscana virus (TOSV), serotype of Sandfl y fever Naples virus, is among of the three most prevalent viruses associated with meningitis during the warm seasons in northern Mediterranean countries. Th e historical data of the sandfl y fever (Pappataci fever) indicates its origin in Bosnia and Herzegovina at the end of th century. Th ere is a long period of time for which there are no data on research related to the SFV in Bosnia and Herzegovina. Th e purpose of the study was to investigate the presence of sandfl y fever in Bosnia and Herzegovina in recent years. Th e  of serum samples were obtained from February  until September  from a group of patients with febrile illness of unknown etiology. Th e sera were tested on the presence of IgG and IgM antibodies against TOSV by specifi c serology testrecomLine Bunyavirus IgG/IgM immuno-line assay. Th e recent TOSV-infection was confi rmed in the patients in each year during the study: , (/) in ; , (/) in  and , (/) in . Th e presence of specifi c antibodies to TOSV in the sera of the patients in recent years indicates re-emerging character of the disease in this region. It would be necessary to make biological, epidemiological and clinical research on the TOSV and related phleboviruses to elucidate the problem of SFV in Bosnia and Her-


Introduction
Sandfly fever viruses are classified among family Bunyaviridae, genus Phlebovirus.They are endemic in the Mediterranean, Middle East, northern African and western Asian countries, where they represent a significant health problem, especially for non-native and non-immune visitors ().These viruses are responsible for a human disease known as sandfly fever, phlebotomus fever or Pappataci fever.Although the genus Phlebovirus consists currently of  distinct virus serotypes, eight serotypes, namely Alenquer, Candiru, Chagres, sandfly fever Naples, Punta Toro, Rift Valley fever, SFSV and sandfl y fever Toscana virus (TOSV), have been linked to disease in humans ().
TOSV is endemic in central Italy and described as the most frequent cause of aseptic meningitis in children in that region (, ).As a consequence of the life cycle of Phlebotomus, TOSV is more frequent during summer, with a peak in August.The most common presentation is an acute febrile illness or meningitis, and more rarely a meningoencephalitis (-).Most of the studies on TOSV have been done in central Italy, but the occurrence of TOSV in other countries such as France, Spain, Slovenia, Greece, Cyprus, Turkey and Egypt, has also been reported recently ().

History of sandfl y fever
Th e fi rst who described the disease was Austro-Hungarian military doctor of medicine from Trebinje (Herzegovina), Czech Alois Pick.In , he published an article entitled "Pathology and the therapy of the domestic endemic disease" in "Wiener medizinischen Wochenschrift" ().He rejected the use of the name of the disease "gastroenteritis endemica" which was in use by many physicians, because of its medical nomenclature, and introduced a new term of the disease "the dog disease".Th e new term was commonly used by people and soldiers.In , Pick was intensively involved in study of "the dog disease" (), when the disease occurred around Trebinje and Stolac (Herzegovina).He knew that similar disease occurred in Mostar and Ljubuški (Herzegovina), and in Korčula and Dubrovnik (Croatia), as well.Foreigners were more exposed to the disease than native population.Also, other scientists were study about "the dog disease".Without knowledge of Pick, Gabel investigated the disease in Mostar, Trebinje and Stolac (Herzegovina), and thought on "acclimatization disease", that had never been noted in Spring, Autumn and Winter time ().In , Taussig performed broad clinical and epidemiological research and found out the existance of "Pappataci" mosqitos in some military barracks in Herzegovina ().These "Pappataci" mosqitos hit only during the night."Th e dog disease" was appeared just in places where "Pappataci" mosqitos occurred.In , Giovanni Battista Grassi (-) studied about "phlebotomus papatasii" in Rome and described this type of mosqito ().In summer , leadership of Committee constituted of physicians Franz, Taussig and Doerr took the mission to explore the infective agent of the disease ().Doerr found out that a virus is a causative agent and the infected pappataci mosqito is vector of the disease.Th at was assumed by Taussig.Commettee made the issue to term the disease as "Pappataci fever", respectively, in honor of Grassi "Phlebotomus fever" ().However, at the beginning of the history of development of the disease is the name of Alois Pick, who gave the origin of the disease in Bosnia and Herzegovina.During the World War II, the disease affected large numbers of soldiers serving in the Mediterranean operations, especially USA Army personnel.Th e disease was first recognized among American soldiers when the morbidity rate of febrile cases increased during the period they spent in North Africa (end of April ).Study of the infected military personnel led to the clinical description, the discovery of the vector of the disease (Phlebotomus papatasii) and the isolation of the responsible viruses, namely sandfl y fever Sicilian virus (SFSV) and sandfly fever Naples virus (SFNV) ().After World War II, the disease occurred in Serbia and the first epidemic of pappataci fever in this Republic was described by Karakašević in  ().Th e epidemic aff ected East Serbia, then Belgrade, where there were several thousand sick persons.In the same year, the fi rst cases occurred in Banat, and in  a new epidemic struck the southern part of that region, where over  of the inhabitants were taken ill ().Th e epidemics remained without etiological confi rmation, and the presumptive diagnosis was based on the clinical picture, epidemiological data and entomological fi ndings.
Th ere is a long period of time for which there are no data on research related to the sandfl y fever (phlebotomus fever or Pappataci fever) in Bosnia and Herzegovina.According to the historical data of the phlebotomus fever in Bosnia and Herzegovina and emergence of Toscana Virus in Europe, fi nding disease in this region is to expect.Aim of this study was to investigate the presence of sandfly fever in Bosnia and Herzegovina (B&H) in a group of patients with febrile illness of unknown etiology, from  till .

Material and Methods
Th e study was retrospective.Th e  serum samples were collected from February  until September  from a group of patients with febrile illness of unknown etiology.All the patients were hospitalized in University of Sarajevo Clinics Centre, Bosnia and Herzegovina.Samples sera were tested on the presence of IgG and IgM antibody directed against TOSV by the recomLine Bunyavirus IgG/IgM immuno-line assay (Mikrogen GmbH, Germany) according to the manufacturer's instructions The test is based on specific recombinant antigens for the determination of IgG-and IgM-antibodies against Hantavirus (serotypes Hantaan, Puumala, Seoul and Dobrava) as well as Sandfly Fever Virus (serotype Toscana) in human serum or plasma.According to the manufacturer's instructions a positive IgM-fi nding without a positive IgG-fi nding is evaluated as borderline.A serum with a borderline or even negative IgM result and positive IgG result could indicate a recent infection.In acute cases of the disease, IgG and IgM antibodies should be detected, confi rming the diagnosis.

Results
Anti-TOSV IgG and IgM were analyzed in  human serum samples.Recent infection was found in seven patients (, ).Th e fi ndings are presented in Table ,

Discussion
Before World War II, the papatasi fever in the former Yugoslavia was known mainly in Herzegovina, Dalmatia, Montenegro and especially in Macedonia, where its prevalence coincided with that of Phlebotomus papatasi.Th e fi rst cases of pappataci fever in Bosnia and Herzegovina were detected at the end of th century (, ).Most of the clinical and epidemiologic studies have been conducted in Bosnia and Herzegovina between  and  ().After that period, interest for the pappataci fever has disappeared.Th ere is no published data about the TOSV infection after  in the B&H, the year when the virus was originally isolated in central Italy ().Our study presents the evidence of occurrence of the TOSV infection among the selected group of patients with febrile illness of unknown etiology.We have found IgM positive results in three cases (two in , and one in ).However, no positive findings in IgG were detected for these patients.Th ese results where evaluated as borderline and it was recommended to retest the new serum samples.We did not have the results of the retested samples so we could not confi rm or reject the diagnosis of TOSV-infection.In all other cases, IgG positive fi ndings represented the recent infection.
In , our laboratory experiences showed a large number of bacteriological sterile liquor samples (/; ,) of patients with CNS disease (unpublished data).This could imply that virus was ethological agent of the infection in those patients.
The recent infection was found in , to , of patients during the period of three years ( -).Th e percentage of the positive sera in that period is less then forty years ago.In - Tesh et al. () made the fi rst extensive serological investigations in this fi eld, reported their fi ndings, indicating the high prevalence of the antibodies to Naples and Sicilian sandfly fever viruses in population of Dalmatia, North Croatia and Kosovo.On the island of Brač, , of the population had antibodies for the Naples virus and , for the Sicilian virus; in Kosovo-, had Naples antibodies and , had Sicilian.In this research on the prevalence of the arbovirus infection, Terzin et al. () discussed the data on the detection of residual antibodies for the Naples virus in the inhabitants of Bosnia and Herzegovina in .In subsequent work (), it has been seen that the prevalence of the Naples virus infection reached ,.Th e signifi cant importance was the fi nding of antibodies in , of children aged - years.Th e similar results were obtained by Salja and associates in Kosovo, in  ().Th ese results indicated the continual circulation of Naples virus in nature.Th e results of faunistic and ecological investigations of the sandfl y vectors associated with this virus have been published (,-).Th e oldest data on the study of sandfl ies in former Yugoslavia, exactly in Bosnia and Herzegovina, are in the works of Strobl (,), where the detection of the Phlebotomus papatasi was fi rst mentioned.Th e virus of Naples pappataci fever was the fi rst causative agent of the disease isolated in former Yugoslavia ().
Finding the TOSV positive results as member of the genus Phlebovirus in clinical samples after more than forty years (), means that the virus has still circulating in this region, but has not been the point of interest of the local explorers.It is very important to direct the attention of clinicians to sandfl y fever (pappataci fever) because the disease was unrecognized in B&H in the last time.
It would be necessary to make the research on the TOSV and related phleboviruses in its natural reservoir and in men, as well as to compare the results with data available in literature.

Conclusion
Th is study confi rms that TOSV as a serotype of Sandfl y fever Naples virus circulates in Bosnia and Herzegovina for centuries.Th e presence of such phlebovirus in recent years indicates re-emerging character of the TOSV infection in this region.
Despite increasing evidence of its major role in medicine as an emerging cause of CNS infections, TOSV remains an unstudied pathogen.Th e recent data on TOSV circulation in Mediterranean countries and so in Bosnia and Herzegovina raises concern about potential implications for blood donations, because of the asymptomatic course of illness in majority of cases.
Figure  and .Positive IgM-finding without a positive IgGfinding was found in three samples of sera.They are evaluated as borderline (Figure ).There was not a case with IgM and IgG antibodies against TOSV present at the same time.The recent infection was conformed in the patients in each year during the study: , (/) in ; , (/) in  and , (/) in  (Figure ).

TABLE 1 .
Prevalence of anti-TOSV antibodies detected by recomLine Bunyavirus IgG/IgM immuno-line assay (Mikrogen GmbH, Germany) in group of patients with febrile illness of unknown etiology in BiH during the years 2006-2008.