Autochthonous Human and Canine Strongyloides stercoralis Infection in Europe: Report of a Human Case in An Italian Teen and Systematic Review of the Literature

Autochthonous human and canine strongyloidiasis is reported in Europe but is unclear whether the transmission of infection still occurs. We report a previously unpublished human case in an Italian teen and perform a systematic review of literature on autochthonous human and canine strongyloidiasis in Europe to investigate the current dynamic of transmission. Overall, 109 papers published after 1987 were included and one previously unpublished Italian case was added. Eighty case reports were retrieved and 42 of them (52.5%) had severe strongyloidiasis. Most cases were diagnosed in Spain, Italy and France. The median age was 58, the most represented age group was 61–70 years, 11 patients were under 30, and 7 of them were diagnosed after 2000. Epidemiological studies on human strongyloidiasis showed prevalence ranging from 0.56% to 28%. Overall, agriculture work, mine work and walking barefoot were the most commonly reported risk factors for infection. Canine strongyloidiasis was reported mainly in Italy (68 cases), but a few cases occurred also in Iceland, Finland, England, Germany, France, Switzerland, Russia, Slovakia, Romania and Greece. Autochthonous strongyloidiasis is still reported in Europe and sporadic transmission still occurs. Health care professionals should be aware of this issue to identify infected subjects and avoid adverse outcomes, especially in immunosuppressed patients. Further investigations are needed to clarify the zoonotic transmission of this nematode.


Introduction
Strongyloides stercoralis is a parasitic nematode able to infect humans, non-human primates, dogs and wild canids. Parasite transmission occurs through direct skin (or mucosae in the case of shore. Since the age of 14 the complete blood count showed mild anaemia, without eosinophilia, which was treated with oral iron therapy. In 2014, at the age of 15, hypereosinophilia was detected for the first time, with an eosinophil count ranging between 1000 and 1600 cell/mm 3 in the following years and with no other symptoms associated. In our clinic she underwent further investigations. The examinations showed a normal immunophenotype, negative autoimmunity tests, negative serology for Trichinella spp. with Western Blot technique (performed at the Istituto Superiore di Sanità, Rome, Italy). Serology for Echinococcus spp. (indirect haemoagglutination Cellognost Echinococcosis IHA, Siemens Healthcare Diagnostics, Marburg, Germany) and Schistosoma spp. (Schisto II Western Blot IgG, LD-BIO Diagnostics, Lyon France) were both negative. Surprisingly, a borderline result for Strongyloides IgG ELISA (Bordier Affinity Products SA, Crissier, Switzerland) was obtained (index 1, reference values ≤0.9 negative, 0.9-1.1 borderline, ≥1.1 positive) and the diagnosis of strongyloidiasis was confirmed through detection of the rhabdoid larvae with a prominent genital primordium and adult male in agar plate stool culture ( Figure 1). Serology for S. stercoralis with SeroELISA Strongyloides IgG (IVD Research Carlsbad, Canada) was negative (index 0.41, reference value > 1 positive) as well as with the in house Indirect Immune Fluorescent Assay Test previously described by Boscolo et al. (title 0, reference value > 20) [23]. HIV 1-2 Ab/Ag and HTLV 1-2 Ab were negative. PCR for S. stercoralis on stool was performed in three different laboratories in Florence (amplification performed with RT-PCR homemade method), in Rome (PCR-based methods targeting the cox1 gene [24] and Negrar (qPCR performed as described previously by Verweij et al. [25]) and the results were negative. The girl was treated with ivermectin 200 mcg/Kg/day for two days plus another two days of therapy two weeks apart. Blood samples performed after one, six and 12 months showed normal eosinophil count and negative serology for S. stercoralis (with the Bordier kit) and agar plate stool culture.   Out of the 109 papers included, 67 were single human strongyloidiasis case reports or case series reporting detailed data on each case, eight were papers reporting human aggregated data, 17 were human epidemiological studies, seven were canine case reports or case series and 11 were canine epidemiological studies. One paper was considered both among canine and human epidemiological studies [16].
Considering all the kinds of studies included (case reports and case series, paper reporting aggregated data, epidemiological studies), human strongyloidiasis was reported in 19 countries: Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, England, France, Germany, Greece, Italy, Macedonia, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Spain and Turkey. The countries where at least 20 human cases were reported were: Spain (565 cases), Italy (264 cases), France (33 cases), Slovakia (25 cases) and Romania (23 cases). Among these countries, the most represented areas were the eastern coast of Spain, especially Valencia province, the north of Italy (Piedmont, Lombardy, Veneto and Friuli-Venezia-Giulia), and the south-west and the north-east of France. Canine strongyloidiasis cases were reported in 12 countries, most of them in Italy (68 cases) and fewer in Belgium, England, Finland, France, Iceland, Germany, Switzerland, Russia, Slovakia, Romania and Greece. The European distribution of human and canine cases, according to the information available from the papers, is illustrated in Figure 3. Out of the 109 papers included, 67 were single human strongyloidiasis case reports or case series reporting detailed data on each case, eight were papers reporting human aggregated data, 17 were human epidemiological studies, seven were canine case reports or case series and 11 were canine epidemiological studies. One paper was considered both among canine and human epidemiological studies [16].
Considering all the kinds of studies included (case reports and case series, paper reporting aggregated data, epidemiological studies), human strongyloidiasis was reported in 19 countries: Austria, Belgium, Bosnia and Herzegovina, Bulgaria, Croatia, England, France, Germany, Greece, Italy, Macedonia, Netherlands, Norway, Poland, Portugal, Romania, Slovakia, Spain and Turkey. The countries where at least 20 human cases were reported were: Spain (565 cases), Italy (264 cases), France (33 cases), Slovakia (25 cases) and Romania (23 cases). Among these countries, the most represented areas were the eastern coast of Spain, especially Valencia province, the north of Italy (Piedmont, Lombardy, Veneto and Friuli-Venezia-Giulia), and the south-west and the north-east of France. Canine strongyloidiasis cases were reported in 12 countries, most of them in Italy (68 cases) and fewer in Belgium, England, Finland, France, Iceland, Germany, Switzerland, Russia, Slovakia, Romania and Greece. The European distribution of human and canine cases, according to the information available from the papers, is illustrated in Figure 3.
Pathogens 2020, 9, x FOR PEER REVIEW 5 of 28 Figure 3. Regional distribution of human (red) and canine (green) autochthonous strongyloidiasis cases in Europe reported in the literature from 1988 to 2018. If multiple countries of exposure were reported, the case was not inserted in the map. One canine case was reported from an unspecified area of Russia and it was arbitrarily highlighted the geographical corresponding to the Russia capital city Moscow [24]. Two countries (Belgium, 2 cases; and Bulgaria, 1 case) were fully coloured since the regions of exposure of respective cases were not specified.

Human Case Reports and Case Series
Sixty-seven papers reporting case reports and case series, plus the previously unpublished case reported above, were identified. Overall, 80 autochthonous human strongyloidiasis cases were retrieved in this category. Demographic and clinical details are showed in Tables 1 and 2. n (%) Figure 3. Regional distribution of human (red) and canine (green) autochthonous strongyloidiasis cases in Europe reported in the literature from 1988 to 2018. If multiple countries of exposure were reported, the case was not inserted in the map. One canine case was reported from an unspecified area of Russia and it was arbitrarily highlighted the geographical corresponding to the Russia capital city Moscow [24]. Two countries (Belgium, 2 cases; and Bulgaria, 1 case) were fully coloured since the regions of exposure of respective cases were not specified.
The most frequent risk factor for complicated strongyloidiasis was use of steroid therapy followed by chemotherapy, other immunosuppressive therapy, chronic obstructive pulmonary disease (COPD), oncohematological malignancy or solid tumor, solid organ transplant recipient and HIV infection. Concerning patients with HIV infection and severe strongyloidiasis, six had CD4+ lymphocytes < 60mm 3 and one had 420 total lymphocytes. Possible factors linked to risk of exposure to strongyloidiasis were reported in 29 subjects: 15 patients were agriculture workers, four were miners, six subjects walked barefoot and two patients had mental disorder; no one was reported to be a dog owner. Interestingly, as it has recently been a matter of debate, two cases of strongyloidiasis in MSM were reported and one of them was supposed to be infected through homosexual intercourse.
The diagnosis of strongyloidiasis after solid organ transplantation (kidney, pancreas, heart and liver) was done in 12 subjects in Spain (seven cases), Norway (two cases), in Portugal (one case), in Turkey (one case) and in Netherland (one case). Seven transplant recipients most likely acquired the infection through the graft since their donors were originally born in endemic countries and retrospective testing of pre-donation donors serum resulted positive for S. stercoralis. In the remaining cases it was not possible to ascertain whether the infection was acquired before the transplant or through the graft.
The most frequently used diagnostic technique was fresh stool examination. Out of 62 patients (77.5%) tested with direct faecal smear examination and/or concentration methods, 47 (75.8%) had a positive result. Details on diagnostic test performed, when available, are listed in Table 2. Male subjects were 60 (75%) and the median age was 58 years. The most represented age group was between 61 and 70 years (21 patients, 26.2%), 11 patients were under 30 (13.7%), and 7 of them were diagnosed after 2000. Most of the subjects were exposed to infection in Spain (21 cases), followed by Italy (18 cases), France (11 cases) and Turkey (8 cases). Out of the seven patients aged less than 30 years and diagnosed after 2000, two were exposed to infection in Spain, two in Turkey, and one each in France, Italy and Romania. The majority of subjects were symptomatic (72, 90%), as shown in Table 1. Hyperinfection syndrome was recognized in 42 patients (47%), eight of them had a disseminated strongyloidiasis, and 15 had a fatal outcome.

Human Aggregated Cases
The most frequent risk factor for complicated strongyloidiasis was use of steroid therapy followed by chemotherapy, other immunosuppressive therapy, chronic obstructive pulmonary disease (COPD), oncohematological malignancy or solid tumor, solid organ transplant recipient and HIV infection. Concerning patients with HIV infection and severe strongyloidiasis, six had CD4+ lymphocytes < 60mm 3 and one had 420 total lymphocytes. Possible factors linked to risk of exposure to strongyloidiasis were reported in 29 subjects: 15 patients were agriculture workers, four were miners, six subjects walked barefoot and two patients had mental disorder; no one was reported to be a dog owner. Interestingly, as it has recently been a matter of debate, two cases of strongyloidiasis in MSM were reported and one of them was supposed to be infected through homosexual intercourse.
The diagnosis of strongyloidiasis after solid organ transplantation (kidney, pancreas, heart and liver) was done in 12 subjects in Spain (seven cases), Norway (two cases), in Portugal (one case), in Turkey (one case) and in Netherland (one case). Seven transplant recipients most likely acquired the infection through the graft since their donors were originally born in endemic countries and retrospective testing of pre-donation donors serum resulted positive for S. stercoralis. In the remaining cases it was not possible to ascertain whether the infection was acquired before the transplant or through the graft.
The most frequently used diagnostic technique was fresh stool examination. Out of 62 patients (77.5%) tested with direct faecal smear examination and/or concentration methods, 47 (75.8%) had a positive result. Details on diagnostic test performed, when available, are listed in Table 2.

Human Aggregated Cases
Data on aggregated human strongyloidiasis cases were obtained from eight papers (Table 3), published between 1992 and 2018. Seven Spanish papers described 61 cases, most of which occurred in the Valencian area, where many of the male subjects had been agriculture workers. Rivasi F. reported 15 cases diagnosed with gastro-duodenal biopsy among a group of immunocompromised subjects living in the Po valley (northern Italy), a region considered endemic for strongyloidiasis. Other two Italian cases have been reported in Florence betwen 2012 and 2014 and the area of exposure was Tuscany and Umbria (central Italy), respectively.

Human Prevalence and Epidemiology Studies
Prevalence data were reported from epidemiological studies conducted at the hospital or community level ( Table 4). The prevalence demonstrated a large variability, both within different countries and within different areas of the same country. The highest prevalence has been reported by an Italian paper where 37 out of 132 subjects over 60 (28%) living in the north of Italy (Lombardy and Veneto regions) with eosinophilia had also positive serology (IFAT technique). This was the pilot study of a more extended work by Buonfrate et al. which revealed a prevalence of 8% among subjects over 60 with eosinophilia (cases) and 1% among those without eosinophilia (controls) born in Lombardy, Veneto and Friuli-Venezia-Giulia regions. A prevalence of 7.78% is reported among 120 hospitalized children in Dabrowa Bialostocka District (Poland) with symptoms characteristic to parasite infections.

Discussion
The European countries where most of autochthonous strongyloidiasis cases have been reported are Spain, Italy and France. In Spain, as confirmed by a review by Barroso et al., most cases have been diagnosed in the Valencia region among elderly patients that worked as farmer or in other jobs typically done barefoot [127]. The review showed that the peak of diagnosis occurred from 2001 to 2010, but even if drastically reduced new diagnoses are still reported. In Italy most cases have been reported in the last 20 years, due to prevalence studies conducted among the elderly population living in agricultural areas in the north of the country, where the transmission was frequent in the past. Regarding France, data from a retrospective study by Magnaval et al. suggest that infestation still exists in the Midi-Pyrenees region (southwestern France) [93], while sporadic cases have been reported in almost all other regions from the north to the south. Interestingly, besides the Mediterranean area, a few cases have been described also in colder countries of Eastern Europe, such as Romania, Bulgaria and Poland. Norway is the country at the highest latitude where strongyloidiasis has been described, although the transmission of the infection seemed to be related to kidney transplantation from a donor that was born in an endemic area. The epidemiological findings vary widely among European countries, where the different climate areas and humidity degrees can be more or less favourable for larvae survival in the soil.
Most of cases were diagnosed with direct stool examination and about half of them were severe cases. Since direct stool examination has a low sensitivity and severe strongyloidiasis develops in a minority of patients, it is probable that there are fewer reported cases compared to the real number of infected people. On the other hand, high seroprevalence reported by some studies, such as the one carried out among Slovakian children with no positive direct stool examinations, should be interpreted with caution since serological false positive results cannot be ruled out [16]. Concerning the transmission of infection through transplantation, we found twelve cases of strongyloidiasis diagnosed after solid organ transplantation in subjects without history of travel outside Europe. All of them were diagnosed in the last decade. In seven cases, the infection was most likely transmitted through the graft since the donors were born in tropical or subtropical endemic countries and resulted positive to S. stercoralis serology. In the remaining five cases the infection could have been acquired by the recipient before transplantation or through the graft.
Canine strongyloidiasis is not frequently reported in Europe. Italy is the country that has recorded the highest number of cases. Epidemiological studies have been conducted in different regions among kennel, wild and domestic dogs, finding prevalence ranging within 3% and 0.2%, with a single study conducted in an Italian shelter reporting a prevalence of 56% [112]. The higher prevalence in the latter study could be due to the diagnostic test performed, since overall prevalence was detected with fecal examination and/or rt-PCR plus serology (ELISA and IFAT). Sporadic canine infections have been described in England, Germany, Greece, Iceland, Romania, Russia, Slovakia and Switzerland.
Concerning human strongyloidiasis case reports, the maximum peak of diagnosis occurred within 60 and 70 years of age from the 1980s to the present. Given the typical chronicity of the infestation, most of these elderly subjects may have been infected in the post-war period when the scarce hygienic conditions could have facilitated the transmission. Although the improvement of health and working conditions probably led to a reduction in transmission, a stronger clinician's awareness of the disease can explain the stable trend of cases reported. Moreover, some sporadic infections among young patients have been reported, even in the last decade. Several hypotheses can explain these recent cases including soil transmitted cases sustained by persistent free-living cycle in the environment; canine or, less likely, human reservoir; or direct interhuman transmission (for example through sexual intercourse).
The role of strongyloidiasis as a zoonotic disease has been largely debated [128] under the historical evidence that S. stercoralis can adapt from human to dog hosts, suggesting that the reverse could also be possible [4,129]. Recent phylogenetic studies [18,19] indicated that S. stercoralis consists of two genetically distinct lineages, one isolated from both dogs and humans, the other found exclusively in dogs. Moreover, the study conducted by Jaleta et al. on humans and dogs living in the same household in rural Cambodia demonstrated that dogs carry two genetically different populations of S. stercoralis one of which is shared with humans [130]. Thus, this study is the first to provide convincing evidence of natural zoonotic transmission of S. stercoralis between dogs and humans. According to this evidence, some authors suggested concomitant treatment of dogs alongside mass drug administration programs to control strongyloidiasis in humans [19,131,132]; however, this approach could be more useful in highly endemic countries rather than in Europe.
Another issue of concern is direct interhuman transmission. Infestation with S. stercoralis through homosexual intercourse has been described since 1981 [133], and it has been recognized by the scientific community that sexual contact, especially oral-anal contact, can facilitate the transmission of intestinal parasites [134].
When the disease is not recognized, the characteristic mechanism of autoinfection can lead to chronic infestation persisting for decades, posing infected subjects to ongoing risk of severe manifestations in cases of immunosuppression. According to our findings, strongyloidiasis must be taken in account in elderly European subjects living in endemic areas, with or without clinical symptoms or eosinophilia and whether they are immunosuppressed or a candidate for immunosuppression [135]. The risk of development of a severe form of strongyloidiasis has been associated with many immunosuppressive conditions such as steroid therapy, other immunosuppressants, solid tumour or oncohematologic malignancy, transplantation, and chemotherapy [136]. The COHEMI guidelines by Requena-Méndez et al. provide indications for the screening and management of Strongyloides in subjects living in non-endemic countries. Concerning immunosuppressed patients, screening for strongyloidiasis or pre-emptive therapy where a strong clinical suspicious exists is strongly recommended in every subject at risk, including elderly European patients that have lived in endemic areas [137]. In 2013, the American Society of Transplantation provided guidelines recommending the evaluation for Strongyloides infection in transplant candidates and donors with epidemiologic risk factors or unexplained eosinophilia during pretransplant evaluation [138]. In our opinion, deeper efforts are needed to improve European screening programs among transplant centers, with a focus on donors' origins and risk factors for infection. According to this review, cases might occur, although seldom, in young patients. Hence, strongyloidiasis cannot be excluded in young autochthonous patients especially in cases of unexplained eosinophilia or in cases with risk factors such as working as a farmer or miner, walking barefoot, having at risk sexual intercourse, or mental disorders.
A limitation of our review is the exclusion of grey literature and papers written in languages other than Italian, English, French, Spanish or German, probably underrepresenting the total number of papers. Another limitation is that several cases have probably been misdiagnosed and are not published in the literature. Moreover, a publication bias can be present, due to the higher interest of journals in publishing papers reporting cases of severe strongyloidiasis rather than an asymptomatic infection.

Conclusions
Traditionally, strongyloidiasis is considered a disease related to tropical and subtropical areas and is often underdiagnosed and underestimated. In Europe transmission of the disease still occurs in humans and dogs, although rarely. Clinicians need to be aware of the epidemiological features of autochthonous infection in order to promptly recognize it and avoid severe complications in immunosuppressed patients. Further studies must be performed to better understand the possible zoonotic transmission of this infection.