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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 56  |  Issue : 3  |  Page : 244-251

Human visceral leishmaniasis in northern Greece: Seroepidemiology and risk factors in endemic region


1 Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Komotini, Greece
2 Laboratory of Hygiene and Environmental Protection, Medical School of Alexandroupolis, Democritus University of Thrace, Komotini, Greece
3 Department of Microbiology, Medical School of Aristotle University of Thessaloniki, Thessaloniki, Greece

Date of Submission08-Apr-2018
Date of Acceptance28-Aug-2018
Date of Web Publication09-Jul-2020

Correspondence Address:
Ms Dionysia Theocharidou
Department of Internal Medicine, University Hospital of Alexandroupolis, Democritus University of Thrace, Komotini
Greece
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/0972-9062.289399

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  Abstract 


Background & objectives: Visceral leishmaniasis is endemic in Greece, with sporadic cases reported annually both in the mainland and in coastal areas. Seroepidemiological studies across Greece report seropositivity rates from 0.5 to 15%, in different parts of the country. The aim of the present study was to evaluate the Leishmania seropositivity rate of the general population of Drama prefecture, a rich in water supply region, in northern Greece.
Methods: Serum samples collected from 347 healthy individuals were tested for IgG Leishmania infantum antibodies. Furthermore, 132 domestic dogs, clinically suspected to suffer from canine leishmaniasis, from all across the prefecture, were also evaluated.
Results: Among 347 healthy individuals tested, 24 (6.9%) were positive for IgG L. infantum antibodies. Age, gender, occupational and leisure time activities didn’t show significant relation to IgG seropositivity, whereas low altitude of place of residency and residency at places with surface water were significantly related to IgG seropositivity. All seropositive individuals follow a geographic pattern, gathering themselves in Drama basin (rich in surface and underground water bodies), whereas canine leishmaniasis cases show a wide distribution across the prefecture.
Interpretation & conclusion: Evaluation of both human seroprevalence and high incidence of canine leishmaniasis, as well as favorable landscape and climatic conditions of the study area, indicates that high level of clinical awareness need to be employed by physicians, as human and canine visceral leishmaniasis constitutes a serious public health concern.

Keywords: Asymptomatic leishmaniasis; Greece; Leishmania; seroepidemiology


How to cite this article:
Theocharidou D, Maltezos E, Constantinidis TC, Papa A. Human visceral leishmaniasis in northern Greece: Seroepidemiology and risk factors in endemic region. J Vector Borne Dis 2019;56:244-51

How to cite this URL:
Theocharidou D, Maltezos E, Constantinidis TC, Papa A. Human visceral leishmaniasis in northern Greece: Seroepidemiology and risk factors in endemic region. J Vector Borne Dis [serial online] 2019 [cited 2020 Oct 25];56:244-51. Available from: https://www.jvbd.org/text.asp?2019/56/3/244/289399




  Introduction Top


Leishmaniasis is caused by protozoans of the genus Leishmania and acquired by the bite of a Phlebotomus or Lutzomyia sandfly. The disease is usually divided into three clinically distinct main syndromes, visceral leishmaniasis (VL), cutaneous leishmaniasis (CL) and mucosal leishmaniasis (ML)[1].

Studies conducted on humans and animal models support that most cases of Leishmania spp. inoculation are easily controlled by the host immune response, without presenting clinical manifestations[2]. Concerning visceral leishmaniasis, an increasing interest is attributed to asymptomatic infections, which provide the population with Leishmania infantum asymptomatic carriers. Various seropositivity rates, reaching 47%, have been reported among asymptomatic individuals in France, Greece, Italy, Spain and Croatia[3],[4],[5].

Four Leishmania species are found in the Mediterranean basin: L. infantum, the most frequent species, responsible mainly for visceral leishmaniasis, L. major and L. tropica, which cause cutaneous leishmaniasis occurring in north Africa and the Middle East, and the newly introduced, in the Mediterranean, L. donovani, observed in Cyprus, which has been linked to both visceral and cutaneous forms of the disease[6]. Furthermore, L. infantum is the most frequent species observed in Greece causing human VL and canine leishmaniasis. Dogs are the major reservoir of the disease, with incidence rates[5],[7],[8] ranging from 2.05 to 50.2%.

Visceral leishmaniasis is endemic in Greece with L. infantum being the predominant species transmitted by Phlebotomus neglectus, P. tobbi, and P. perfiliewi[9]. However, sporadic cutaneous leishmaniasis is also present in certain coastal areas—such as the Ionian Islands and Crete, transmitted by P. sergenti. According to reported and laboratory-confirmed cases from1998 to 2011, the incidence of VL is 0.36 per 100,000 population[10]. However, asymptomatic infections may be much more frequent, playing a significant role in the disease transmission. Studies conducted in certain regions of Greece, evaluating anti-L. infantum antibodies, have shown different rates[5],[6],[11],[12] among asymptomatic individuals rating from 0.5 to 15%.

Drama, a prefecture lying in northern Greece, covering 3468 km2 of non-coastal area, inhabited by 98,287 people (based on 2011 demographic census), is crossed by one of the main Greek rivers (the Nestos) and is rich in underground water supplies. Furthermore, the region is surrounded by three mountains, forming the Drama basin. About 16% of the working population lives on agricultural and livestock farming occupations. VL is endemic in Drama, with 3–4 reported cases, annually. On the other hand, there haven’t been reported cases of cutaneous leishmaniasis in this region[10].

Symptomatic VL represents only the tip of the iceberg, given the fact that the vast majority of infected individuals remain asymptomatic or progress to a subclinical infection. Seroprevalence of L. infantum antibodies is thought to be indicative of VL transmission intensity, especially in southern Europe[3]. In endemic areas, asymptomatic leishmaniasis could be estimated by the use of direct and indirect laboratory methods[3]. Indirect methods, as enzyme-linked immunosorbent assay (ELISA), immunofluorescent antibody test (IFAT) and direct agglutination test (DAT), are capable of diagnosing both long-term ongoing infections and cases of recent contact to the parasite, which may result to parasite clearance and infection resolution. However, based on the fact that L. infantum infection after its resolution is accompanied by short-term immunity, IgG seropositivity at a certain area and period can be indicative of human transmission rates. Furthermore, persisting IgG seropositivity, over a certain period, for inhabitants living in endemic areas, is representative of ongoing asymptomatic infection, rather than repetitive cycles of infection—parasite clearance—reinfection[13]. On the other hand, direct methods, such as PCR, are capable of identifying ongoing both symptomatic and asymptomatic infections, although asymptomatic infections may appear through transit parasitaemia.

The obj ective of this study was to determine the seroprevalence of VL in Drama prefecture (north Greece) using a commercial ELISA kit with high specificity and sensitivity for L. infantum antibodies and explore the associated risk factors related toVL. Furthermore, canine leishmaniasis data are evaluated to provide information concerning vector abundance and human behavioral habits, which can lead to L. infantum infection.


  Material & Methods Top


The population studied consisted of 347 healthy residents of Drama Province, northern Greece. The whole region was divided into further sectors based on both geographical and residential habits—urban and rural populations.

All the participants were asked to fill in a questionnaire concerning basic demographic data—gender, age, residency, and further epidemiological data—occupation, spare-time activities, traveling history (time and location).

During a period of 5 years (2011–2016), 366 serum samples were collected from 347 healthy immunocompetent individuals (62% women), with a median age of 54 yr (age range 11–94 yr). They showed no clinical signs of visceral or cutaneous leishmaniasis, or any other febrile infection. Blood was drawn, centrifuged and serum samples were stored at –20 °C. All the sera were tested for L. infantum IgG antibodies, using a commercially available ELISA kit (Cypress Ab panel, Belgium), with 100% sensitivity and 96% specificity, according to the manufacturer’s instructions. As clearly declared by the manufacturer, a sample was considered positive, when the ratio between the sample and cut-off value (sample/cut-off) was greater than 1.1, and a grey zone, when the ratio of sample/cut-off was 0.9–1.1.

Furthermore, in collaboration with a local veterinary office, 158 blood samples were collected from 132 domestic dogs, with suspected canine visceral leishmaniasis, and tested using the commercially available immunochromatography kit (Agrolabo IC), identifying L. infantum antibodies, according to the manufacturer’s instructions. The dogs were domestic animals living with their owners in Drama helping in rural activities: shepherding, hunting, guarding, etc.

Questionnaire data were gathered and inserted into a Microsoft Excel sheet. The SPSS 14.0 for Windows package was used for statistical analysis. To evaluate possible risk factors related to VL, the laboratory results were compared to all categorical variables on a univariate analysis using the Pearson’s chi-squared test. The non-parametric Mann-Whitney U-test was used to describe non-parametric variables, such as altitude.

Google Earth data were used for map drawing, and maps were also provided by the Drama city hall electronic archive. Furthermore, geospatial characteristics of the region, such as altitude and existence of surface water— both artificial and natural, such as springs or watering channels, were also evaluated.

Ethical statement

The study was approved by the Democritus University of Thrace Ethics Committee. All the participants were informed about the purpose of the study and their written participation consent was obtained accordingly. Each participant had full access to the study findings at any given time and was provided with the right to withdraw his/her participation at any time he/she wished. Concerning children, their parents provided their written consent, in order to enable them to participate in the study.


  Results Top


Among the 347 individuals tested, 24 (6.9%) were positive for IgG L. infantum antibodies. The basic demographic characteristics of IgG seropositive individuals are shown in [Table 1]. [Figure 1] represents the geographical distribution of IgG seropositive individuals.
Table 1: Epidemiological data of the population studied in correlation to IgG L. infantum seropositivity

Click here to view
Figure 1: Leishmaniasis seroprevalence map indicating locations of seropositive cases. The red dots represent IgG L. infantum seropositive individuals, yellow dots represent previously confirmed VL cases, and blue dots represent places where canine leishmaniasis cases (dog) were diagnosed.

Click here to view


Seropositivity among males did not differ significantly statistically to seropositivity among females (8 and 6%, respectively, p = 0.415). The age distribution of seropositive persons showed a low rate (3%) in the 20–40 yr age group, whereas higher rates were observed among the <20, 60–80 and >80 yr age groups (10, 12 and 11%, respectively, p = 0.400).

Among the 24 IgG positive individuals, 9 resided at urban places, 12 were rural citizens and 3 did not declare a place of residency. There was no significant difference in seropositivity rates between the rural (7%) and the urban (6%) population (p = 0.696). Furthermore, the IgG positive individuals show a wide geographical distribution throughout the prefecture (see [Figure 1]). The study population consisted of 147 inhabitants of the Drama city, while 116 were suburban residents (Doxato 18, Kalampaki 24, Rodolivos 19,Nevrokopi 6, Paranesti 5) and 12 individuals did not provide their residency. Concerning seropositivity, 9 out of 24 individuals lived in Drama city (6%) and 13 in the suburbs (Kalampaki 8 and Rodolivos 6%). However, the residency was not statistically significant in relation to seropositivity rates (p = 0.653). Among the people studied from Nevrokopi, Paranesti and Doxato, none had IgG L. infantum antibodies [Figure 1].

Occupational data were available for 309, out of 347 individuals. Among those, the majority (n = 187) worked indoors (office workers), 31 were farmers, 7 were cattle breeders and 76 worked outdoors, mainly as lumberjacks. Overall, seropositivity observed among indoor workers was not significantly lower to the seropositivity rates among outdoor workers (6 and 7%, respectively, p = 0.961). Further, data concerning leisure time activities were available for 250 individuals. Out of 152 participants, who enjoyed outdoor sports and activities, such as camping, trekking, gardening and hiking, 12 (8%) had anti-IgG L. infantum antibodies, but this was not significantly different from the 7% observed among the individuals, whose leisure-time activities took place indoors (p = 0.827). Taking a closer look at human habits— both occupational and spare time activities— it is concluded that 7% (15 out of 210) of the individuals that employed both occupational and spare time rural activities were IgG seropositive, whereas the seropositivity rate was 6% (4 out of 66) among the individuals with indoor activities (p = 1.000).

Concerning travel history, data were available for 241 participants and 34% reported a previous journey to foreign countries. The most frequently visited countries were Turkey, Bulgaria, Great Britain, and Germany. Further, 23% of the participants visited leishmaniasis endemic countries. The IgG seropositivity of travelers in endemic countries was 7%, reaching no significant difference, when compared to the 6% observed among people who didn’t travel at all, or traveled to non-endemic countries (p = 0.769).

The prevalence of IgG L. infantum antibodies among citizens of areas, where surface water existed, was 12%, whereas, only 4% of the inhabitants of places where no surface water existed, were found to be IgG seropositive (p = 0.013). The median altitude of residency areas, where the seropositive individuals were diagnosed was 118 m, statistically significantly lower to the residency areas, where no seropositive individuals were found at 160 m (p = 0.001).

Out of132 dogs suspected to suffer from canine leishmaniasis, 86 (65%) were found to have anti-IgG L. infantum antibodies. The location where seropositive dogs were diagnosed is shown in [Figure 1].


  Discussion Top


In Greece, visceral (caused by L. infantum) and cutaneous (caused by L. tropica) leishmaniasis cases are annually reported. Leishmania infantum is the predominant species observed in the Mediterranean European countries, presenting recently a remarkable northward shift towards non-endemic regions, such as northern Italy. Population genetic studies in the Mediterranean and Balkan region, demonstrate a clear molecular differentiation between southwestern and southeastern strains, but Greek L. infantum strains show a quite stable pattern, without presenting significant genetic diversity. Besides, both disease forms are abundant in neighboring Turkey, following exactly the same pattern whereas cutaneous leish- maniasis is predominantly observed in eastern countries, such as Syria, Iran and Iraq[14],[15]. The disease constitutes a great public health issue in Iran caused by L. tropica and L. major. Molecular studies conducted in highly endemic regions showed different heterogeneity patterns for L. tropica strains and high genetic variation for L. major strains obtained from skin lesions isolated from cutaneous leishmaniasis patients[16],[17].

Regarding leishmaniasis in Greece, there has been a significant increase in canine and human VL cases[7]. From 1981–1997, a total of 688 VL cases were reported in Greece (incidence rate 0.36/100,000 population), with higher incidence rates in the Ionian Islands and central Greece. The majority of cases reported were males and the age group 0–4 yr old had significantly higher disease incidence than the other age groups[10].

The Drama prefecture is located in northern Greece, at the Greek-Bulgarian borders. Its climate is characterized as semi-continental, with cold winters and warm summers, with frequent rainfalls during February, May to June and October (mean annual rainfall: 532 mm). Its mean altitude is 115 m above the mean sea level, with the highest elevation levels noticed at the Nevrokopi area, 658 m. The region is rich in both underground and surface water. On its southern and western borders, with Kavala and Serres, respectively, lakes and swamps used to exist, which favored mosquito populations, resulting to the 1922–23 malaria epidemic in the region. Immediately after the first antimalarial combat in Greece, swamp drying, which ended in 1937, and DDT spraying after World War II, reduced both mosquito and sandfly populations, ending up to malaria elimination, which was accompanied by a drastic leishmaniasis reduction. The lake and swamp drainage was accompanied by the construction of an artificial network of drainage channels, which is used for farmland irrigation of the whole plain up to the present day.

Visceral leishmaniasis is endemic in the Drama region with 3–4 cases reported annually, mainly among inhabitants of the Drama Municipality and neighboring villages. The attached map demonstrates that out of the 13 cases of human VL diagnosed in the Drama Municipality during 1985–96, the majority were citizens of Drama town (n = 3) and its nearby northern suburbs Xiropotamos (n = 7) and Monastiraki (n = 1). Two cases were also reported in villages lying at Drama and Serres borders.

According to the present study, seropositivity among healthy individuals of the Drama prefecture was 6.9%. Published data from southern Europe reports variable rates, such as from 4.9 to 52.8% among certain population groups in Spain[3],[18],[19],[20], 7.4 to 27.5% in Italy[3],[13], 30 to 58% in France[21], 11.4% in Croatia[22]. In Greece, there have also been attempts to evaluate the seropositivity of certain geographic areas. In a study that took place in a zoonosis high-risk Cretan village in Rethimno, in two different periods (1985–1987 and 1998), no seropositive individual was recognized in either period tested[23]. A survey conducted in northern Greece (Macedonia) in 1992 reported L. infantum seroprevalence of 9.2% among asymptomatic individuals[24]. On the contrary, much lower rates (0.5%) among healthy individuals were reported between 1994–2001 from Epirus (northwestern Greece)[5]. A study conducted in 2003 in Lasithi, Crete, among a population of healthy blood donors, revealed that 15% had anti-IgG L. infantum antibodies[12]. Similarly, in a study conducted among healthy individuals from 2001 to 2006 in northern Greece (including Drama prefecture), seropositivity of 2.8% was reported for IgG L. infantum antibodies[11]. Relating to Drama prefecture, 2 out of 62 sera tested, proved to be positive, reaching an IgG positivity rate of 3.2%. Our study revealed that 6.9% of the healthy individuals tested in Drama prefecture were seropositive.

Furthermore, factors related to vector ecology and human habits were evaluated in order to point out whether any of them play a significant role to acquire VL. Rural settings are thought to be favorable for parasite transmission, due to high populations of sandfly vectors. Earlier published studies, conducted in endemic areas support the fact that inhabitants of rural areas are at higher risk[13],[19]. We hereby report that 7% of the rural inhabitants tested were IgG positive, contrary to 6% of the urban population. As a result, our study population does not seem to differ significantly concerning the place of residency and IgG positivity.

Taking a closer look at the map where the IgG seropositive individuals are presented, we could clearly point out that all the cases were gathered in the Drama basin, with no seropositive individuals presented at the mountainous areas of Nevrokopi or Paranesti. On the other hand, a lot of seropositive individuals, 10 out of 24, were residents of Drama city—a town rich in underground water supplies, with two large springs surrounded by recreational parks, where outdoor concerts and plays take place during the summer evenings. Further, given the previous history of the disease endemicity in the city of Drama and neighboring villages, we could assume that these are high- risk places. Although seropositivity among habitants of low-risk regions reaches 6% and the seropositivity among high-risk region habitants is 7%, this difference is not statistically significant (p = 0.630), whereas lower altitude (115 m), a factor associated to vector abundance (two generations of sandfly populations during summer in Drama basin), is significantly related to IgG seropositivity. This finding is tightly connected to the geographical distribution of seropositive individuals and is in total compliance with earlier published studies conducted nationwide[7].

In the present study, men did not seem to have significantly higher rates of IgG L. infantum antibodies than women (8 vs 6%,p = 0.415). This finding comes in compliance with other studies published both in Greece[5],[11] and in Europe[8],[22]. On the other hand, there have been previous studies that reported IgG seropositivity to be significantly higher among men than women[13],[19].

Another assumption that connects both vector abundance and individual habits is occupational activities[13]. According to the questionnaire, the population was divided into two categories: the people whose job required long-term outdoor activities (farming, cattle breeders, lumberjacks, housewives who reported gardening and professional gardeners, etc), and the people who work indoors (office workers). It was found that 7% of the outdoor working population had IgG (+) L. infantum antibodies, whereas 6% of the indoor working individuals were seropositive, indicating that occupational habits in the Drama region are not connected to higher seropositivity. The same pattern was observed when we tried to evaluate whether recreational outdoor activities such as trekking, camping, hiking, and gardening could constitute a risk factor concerning L. infantum seropositivity: 12 out of 152 people (8%), who experienced rural outdoor sports for leisure time activities turned to be sero-positive, whereas, the same figure was 7% among people who were interested in indoor sports. Further, given the fact that both occupational and recreational activities may oblige a person to be exposed to rural settings, a combination of these two was also evaluated. Among 210 individuals who spent a certain amount of time outdoors for occupational and/or recreational reasons, 15 (7%) were IgG L. infantum positive. Although the same percentage is lower (6%) among people whose jobs and sports are conducted indoors, this difference is not statistically significant (p = 1.000). Traveling has previously been linked to acquiring “imported” infections. More precisely, northern European countries have reported “imported” visceral leishmaniasis cases from travelers to endemic countries[25], however, in our study there has been no significant difference between the seropositivity of travelers to endemic countries, to people who did not travel at all, or visited only non-endemic countries.

Interestedly, a 24-yr-old woman participant, who suffered from VL during childhood, was IgG seronegative. It has to be pointed out that cell-mediated immunity, which is tested through the Montenegro test, provides life-long immunity to the pathogen, whereas humoral immunity reaches a high level after infection and declines significantly after the disease resolution. However, among previously published studies, the time required for both cell-mediated immunity (Montenegro Skin Test) to develop and for humoral immunity (anti- Leishmania antibodies) to suppress, remains unclear. Recently published data on post-treatment individuals, support this previously mentioned pattern. A study conducted recently in Brazil, which tested 42 post-treatment VL patients concluded that cell-mediated immunity is persistent even 10 yr after successful treatment, whereas in the second year after cure most individuals lose humoral immunity, represented by anti- Leishmania antibody titre[26]. Similarly, a pediatric population post-treatment follow up study in Turkey, showed that anti- L. infantum antibody titres decline abruptly after successful treatment and disease remission, whereas, an increase in antibody titres was accompanied by disease relapses[27]. On the other hand, surveys conducted in high endemicity settings, where VL is caused by L. donovani, support that a significant percentage of the patients preserve high levels of antibodies many years after disease remission, probably due to the continual exposure to the parasite or incomplete treatment[28],[29].

According to the present study, 65% of the dogs suspected to have canine leishmaniasis were positive. Canine visceral leishmaniasis is endemic in Greece and reported seroprevalence among clinically healthy dogs ranged from 2.05 to 30.12% in different areas[8]. Canine leishmaniasis is widely observed in the Drama region, with cases diagnosed even in mountainous regions, such as Nevrokopi, Bathytopos, etc. Although, most cases of IgG seropositive humans were residents of places where canine leishmaniasis was also diagnosed, the widespread of canine cases interferes with concluding to a grounded connection. As a result, there was no significant difference in canine seropositivity among the places where seropositive humans resided, and the places where no seropositive individuals were found (p = 0.383). The widespread distribution of canine leishmaniasis reported (throughout the municipality), dictates that the parasite, as well as the vector, is prevalent in every part of the region.


  Conclusion Top


Seroprevalence of VL in Drama prefecture using a commercial ELISA kit was relatively high (6.9%), indicating that the risk of VL continues in this region. Seropositivity was significantly higher among habitants of low altitude, rural areas, where surface water exists, which comes in total compliance with the vector’s ecology in the region. Seropositivity was observed both in rural and urban settings, showing no correlation to age, gender, occupational and leisure-time activities, and travel history. Evaluation of human seroprevalence and high incidence of canine leishmaniasis indicates that high levels of clinical awareness need to be employed by physicians, even when a patient presents mild or non-specific symptoms. The increasing number of stray dogs in the region underlines the need of employing preventive surveillance measures, both by the authorities (seasonal spraying) and the residents of the high-risk areas, including the use of insect repellents, and clothing that covers all parts of the body, especially during the evenings from May to October.

Conflict of interest

The authors declare no conflict of interests.


  Acknowledgements Top


This study was supported by the Democritus University of Greece (Research Account of the Second Department of Internal Medicine – Medical School). The authors sincerely appreciate the help of medical staff involved in selection of human study population and veterinary doctors for collecting canine blood samples and providing patients’ records.



 
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