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Soil contamination by Toxocara canis and human seroprevalence in the Attica region, Greece.

Introduction

Toxocariasis is a human infection caused by larvae (immature worms) of either Toxocara canis or Toxocara cati. Humans normally become infected by ingestion of embryonated eggs (each containing a fully developed larva, L2) from contaminated sources (soil, fresh or unwashed vegetables, or improperly cooked paratenic hosts). (1) Three syndromes are commonly recognized: visceral larva migrants, ocular larva migrants and covert toxocariasis. (2) Covert toxocariasis is the most common form of the disease and is frequently asymptomatic or can cause mild symptoms such as headache, cough, fever, and wheezing. Consequently, many Toxocara infections remain underdiagnosed and underappreciated.

According to several studies worldwide, T. canis human infection rates are linked mostly with soil contamination with infectious stages of the parasite. (3) Due to the growth in the number of animals kept as domestic pets, public areas of urban recreation, such as parks, playgrounds and squares have become serious risk factors for the transmission of such zoonotic parasites to humans. (4,5)

In Greece, apart from the increased number of dog owners, a significant raise in the number of stray dogs that freely circulate in public places has become an important issue in recent years, constituting a significant threat to the spread of parasitic infections to humans, mainly in urban areas. However, only limited number of Greek studies report data on either human or animal Toxocara spp. infection, mostly referring to rural Greek areas. (6) Furthermore, no studies exist reporting data with reference to soil contamination by Toxocara spp. in any part of Greece.

The aim of the present study was to investigate the presence of T. canis ova in soils of public areas of a broader region of Athens, the capital and most populated city of Greece, as well as to estimate the seroprevalence of human toxocariasis in healthy residents.

Methods

Collection sites and sampling design

From March 2014 to April 2014, soil samples were collected from 33 most popular public places of six large regions throughout the total area of the Attica region: (A) Central Athens area, (B) West Attica area (C) North suburbs area (D) Southern suburbs area (E) Piraeus area (F) Elefsina area (Figure 1). Those areas differ in terms of: 1) social status of the inhabitants and 2) population density, block of apartments or house availability, free spaces and parks, number of persons who visit open spaces or parks, and presence of stray or domestic dogs. They also differ in terms of personal hygiene habits (hand washing), profession (e.g., jobs related to soil such as gardeners, land workers, etc.) or hobbies related to plants, flowers or herb collection.

The number of soil samples taken from each place was calculated on the basis of the selected areas' dimensions (Table 1). Sample takers carried out a rough calculation of the place dimensions by measuring its diagonal with steps (one step = approximately one meter). Places where the ground was covered by gravel, cement or asphalt-based materials were excluded from the sampling procedure.

With a small garden shovel marked down with color lines to a depth of three cm, sample-takers dug square-shaped holes three cm deep, then collected the soil sample and placed it into plastic bags. (7,8) Where sample-takers noted that the soil was less dense, they dug below 3 cm to get a sample. They used air-tight plastic bags to store the samples. The soil volume kept in each bag was about 70 [cm.sup.3] and weighed slightly over 50 grams. Both the name of the place and the sample-taking date were marked on each bag, which was then stored in a dry and cool place at about 25[degrees]C until soil examination (which was planned to be carried out soon after collection).

Soil processing for isolation and identification of Toxocara ova

The ova detection in soil samples was based on the technique proposed by Kazacos. (9) In this method a saturated solution of NaNO3 was used as flotation medium. About 30 g of carefully mixed soil aliquot was placed into a 50 mL conical centrifuge tube and 30 mL of 0.5 percent Tween 40 solution was added. The whole mixture was shaken thoroughly for 4 min and sieved through a nylon sieve of 1 [mm.sup.2] pore size. The filtrate was later centrifuged at 1500 rpm (327xg) for 3 min and the supernatant was discarded. Afterwards the sediment was washed twice with distilled water. After centrifugation, the supernatant was discarded and the sediment was suspended in 30 mL of saturated NaNO3 solution (sp.gr-1.35) and centrifuged for 3 min at 1500 rpm (327xg).

All samples were examined microscopically using x10 and x40 magnification lenses for parasitic ova. Two laboratory clinicians examined in parallel and independently the samples. When recognition of T. canis or T. cati eggs was impossible they were classified to Toxocara genus.

The criteria used for recognition of the ova were the following: 1) oval or round shape; 2) cortex (thickness); 3) color (light-colored shell compared with the egg interior); 4) existence of pronymph; 5) size between 65-90 pm (measured by the microscope graded scale). A sample with at least a single egg detected was considered positive. Discrimination between T. canis and T. cati ova by microscopic examination was based on the ova sizes (85 by 75 pm and 75 by 65 pm, respectively) verified by an ocular micrometer, the shape, color and the wall thickness. T. canis eggs are thick-walled and have oval or spherical shapes with pits on the surface bigger than those observed on the eggs of T. cati.

Residents' healthcare records

Blood samples were collected from residents routinely visiting the examined areas for at least a decade, selected while they were undergoing specific activities such as playing soccer, collection of plants, herbs, etc. from the sampled places. The selection of residents and the collection of blood samples were carried out in parallel with soil investigation for Toxocara ova. All residents that were present at the time of soil sampling and were appropriate for the study, were asked to participate. Twenty-three of them refused to participate. Sera were collected from 200 adults (104 males, 96 females, from 16 to 56 years) and 50 children <14 years (32 males, 18 females) present at all examined areas throughout Attica during the soil sampling. Each participant signed an informed consent form and filled an anonymous questionnaire including the following data: A) date of birth, sex, age, place of residence, profession, and education level; B) evidence of fever, cough, wheezing, hepatomegaly, and splenomegaly; C) personal hygiene habits that may raise the likeliness of infection by Toxocara parasite such as: 1) not washing hands before meals; 2) indirect geophagia e.g., when cleaning herbs, vegetables, etc.; 3) presence of dogs in the house or in the backyard/ front yard. Blood samples for serology were collected after appointment and the serum was stored at -20[degrees]C until the day of the test.

Serological testing procedure

Serum samples were treated following the instructions of RIDASCREEN[R] Toxocara IgG test (r-Biopharm, Darmstadt, Germany), which is an enzyme immunoassay for the qualitative determination of IgG antibodies against T. canis in human serum. The test is performed through the binding of serum antibodies to purified antigens coated to a micro-well plate. The result is obtained by dividing the absorption value of the sample by the cut-off value. According to the procedure, ratios are accepted as negative (<0.9), equivocal (0.9-1.1) and positive (>1.1). The specificity of the test is considered 98.4% and the sensitivity 100%.

Statistical analysis

Categorical variables were compared between groups using the Chi-square test. Continuous parametric variables were compared between groups using the T-test. A p value of <0.05 was considered statistically significant. Statistical analysis was performed with SPSS 17.0 (SPSS Inc., Chicago, IL, USA).

Results

Soil contamination

Based on the dimensions of the 33 public areas, a total of 1,510 soil samples were collected. According to our results, all 6 regional areas of Attica were contaminated with T. canis ova. More specifically, T. canis eggs were isolated from 31 out of the 33 examined public areas (94%). Of the total 1,510 examined samples, T. canis ova were recovered from 258 samples, suggesting an overall T. canis ova contamination percentage of 17.2%. Sixteen samples contained Toxocara eggs that none of the laboratory clinicians could confidently classify in species, therefore they were excluded from the positive T. canis samples.

Statistically significant differences in soil contamination rates were established between the six regional areas of Attica. The highest contamination rate was observed in West Attica area (22.8%) and the lowest in Piraeus area (10%) (Table 2). In particular, a difference in contamination rates was established among Piraeus area and North Athens area (p=0.055), West Attica and Piraeus area (p=0.004), and among West Attica area and Elefsina area (p=0.005). No differences in contamination rates were established among West Attica, Central Athens and North Athens areas. Similarly, no differences in contamination rates were established among South Athens, Piraeus and Elefsina areas.

No correlation could be made between dogs' population visiting the examined areas and the Toxocara ova soil contamination since the presence of dogs at the sampling sites was infrequent during the study conduct.

Toxocara seroprevalence

The epidemiological characteristics of the participants are presented in Table 3. The average age was 19.9 [+ or -] 5.8 years in the T. canis IgG seropositive group and 47.8 [+ or -] 5.4 years in the T. canis IgG seronegative group (t-value=22.549, df=248, p<0.001).

Positive T. canis antibody titers were found in 40/250 (16%) of serum samples in total. The proportion of seropositive samples was 48% (24 out of 50) in the group of children and 8% (16 out of 200) among adults (p<0.001). Differences in rates of T. canis antibody elevated IgG titers were observed between the various areas of Attica, although not in a statistically significant level. The highest T. canis seropositivity rate was observed in West Attica area (25%) and the lowest in Piraeus area (9.4%) (Table 2). Within the same geographical areas, no statistically significant differences were established between percentages of T. canis seropositivity and soil contamination rates (Table 2).

Discussion

Soil transmitted and zoonotic helminths, such as Toxocara spp., are still part of the most important health issues in the world, mainly in developing countries. (10) In several cities, there are countless dogs walking freely in public places and it is very common that, stimulated by their owners, they defecate in these places contaminating the soil with several types of parasites with zoonotic importance. A consequence of this phenomenon is the widespread contamination of the public areas with the eggs of T. canis originating from defecation of dogs. Several studies regarding soil sampling from different parts of the world have a wide variation in contamination rates by Toxocara ova in public areas. (11) One of the main reasons T. canis prevalence reports vary significantly across the various parts of the world is the striking difference between urban, semi-urban and rural areas. Moreover, the lack of standardization of methods by means of the number and volume of samples, depth of sampling, season of examination, method of egg recovery, preservation of samples and laboratory skills, (12) hamper the comparison of findings of different studies, (13) and the evaluation of their impact to public health.

In Greece, the environmental contamination by Toxocara spp. is practically unknown. This is the first Greek study providing data regarding soil contamination by T. canis. The analysis of our sampled areas confirmed a considerably high T. canis contamination level, revealing T. canis positive soil samples in almost all the examined public regions in Attica except for two (94%). However, the percentage of positive soil samples was 17.21%, lower than the rates reported by most other European countries. More specifically, Toxocara spp. prevalence in sandpit samples from Toulouse, France was estimated at 38%, 50% in the Marche region of Italy, (14) 5-45% in Prague, Czech Republic, (15) and research also suggests that the prevalence of the disease in North America is significant. (16) Our results are similar to those from other European cities and countries such as Lisbon, Portugal, where the rate of T. canis contaminated fecal samples collected from public sandpits and public parks was 16%, (17) Poland where reports demonstrate soil contamination percentages of 16%, (18) and Madrid, Spain reporting Toxocara spp. as the most frequent parasite species found in the examined soil samples (16.4%). (19)

The microenvironmental conditions were relatively similar throughout the various areas examined, whereas the type of Attica soils is predominantly clay. Moreover, all soil samples were collected during a short time-period (two-month) in order to avoid considerable weather changes.

The method implemented was that of flotation in a saturated and hypertonic solution of NaN[O.sub.3] (sodium nitrate) with specific gravity 1.35. This technique is considered the most suitable for the detection of ova of various roundworms. Furthermore, the use of saturated NaN[O.sub.3] is now efficient for detecting Toxocara ova in the soil with a success rate of 65% to 82.5%. (12) We have chosen to apply this method, even though a number of previous studies have used molecular techniques to diagnose Toxocara infections, mainly to analyze genetic variations. However, the gold standard to diagnose infections caused by protozoa or helminths remains the microscopic or macroscopic detection of the parasite, particularly in epidemiological studies, similar to ours.

Statistically significant differences in soil contamination rates were established between the six areas of Attica. These areas differ in terms of population density and socioeconomic status of the inhabitants. The areas of higher socioeconomic status presented lower percentages of soil contamination in a statistically significant level, compared to the areas of lower socioeconomic status. Previous reports have also shown that toxocariasis mainly affects socioeconomically disadvantaged populations. (20)

Finally, our serological findings indicate that human T. canis infection actually occurs in the Attica Region. A proportion of 16% of examined sera samples had elevated T. canis IgG antibodies, suggesting a contact with the larva migrans of the nematode. We also demonstrated a major Toxocara IgG seroprevalence in children. Indeed, children are widely considered to be particularly vulnerable for acquiring infection, since they are likely to be more exposed than adults in contaminated environments (21) by playing and putting their fingers in their mouths, either deliberately or by accident. Prevalence of toxocariasis in children ranges from very low to over 50% (22-24) in studies from different parts of the world and is generally considered to be underestimated.

An interesting finding is that similar percentages were established among T. canis seropositivity and soil contamination within the same geographical areas. High human seroprevalence has been indicated in areas with documented soil contamination, while the risk for transmission may be increased in proportion analogous to the degree of environmental contamination.

Conclusion

The level of environmental T. canis contamination found in the Attica region calls for a greater awareness towards this public issue and the implementation of specific preventive measures, such as maintaining dogs free of worms by regular administration of anthelmintics, removal of dog feces from the environment before the eggs are able to embryonate and good personal hygiene. Also, a program to control the stray dog population should be implemented and may include the improvement of health and welfare of the stray dog population, the reduction of stray dogs' numbers to an acceptable level and the minimization of zoonotic diseases risk.

doi: 10.18683/germs.2018.1143

Received: 24 July 2018; revised: 02 September 2018; accepted: 02 September 2018.

Authors' contributions statement: RHR performed the laboratory experiments, collected the data and performed the statistical analysis. NAL collected the data, interpreted the findings and drafted the manuscript. AAE designed the study, supervised the laboratory experiments and drafted the manuscript. All authors read and approved the final version of the manuscript.

Conflicts of interest: All authors--none to disclose.

Funding: None to declare.

References

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(6.) Papavasilopoulos V, Bonatsos G, Elefsiniotis I, Birbas C, Panagopoulos P, Trakakis E. Seroepidemiological investigation of Toxocara canis in a female Greek pregnant population in the area of Athens. Clin Exp Obstet Gynecol 2016;43:384-7.

(7.) Mizgajska H. Eggs of Toxocara spp. in the environment and their public health implications. J Helminthol 2001;75:147-51.

(8.) Mizgajska H, Uga S. Exposure and environmental contamination. In: Holland CV, Smith SV, Eds. Toxocara the enigmatic parasite. Oxfordshide-Cambridge: GABI Publishing; 2006; p.210-33.

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(12.) Rosa Xavier I, Ramos BC, Santarem VA. Recovery threshold of Toxocara canis eggs from soil. Vet Parasitol 2010;167:77-80. [Crossref]

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(14.) Habluetzel A, Traldi G, Ruggieri S, et al. An estimation of Toxocara canis prevalence in dogs, environmental egg contamination and risk of human infection in the Marche region of Italy. Vet Parasitol 2003;113:243-52. [Crossref]

(15.) Dubna S, Langrova I, Jankovska I, et al. Contamination of soil with Toxocara eggs in urban (Prague) and rural areas in the Czech Republic. Vet Parasitol 2007;144:816. [Crossref]

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(17.) Otero D, Alho AM, Nijsse R, Roelfsema J, Overgaauw P, Madeira de Carvalho L. Environmental contamination with Toxocara spp. eggs in public parks and playground sandpits of Greater Lisbon, Portugal. J Infect Public Health 2018;11:94-8. [Crossref]

(18.) Mizgajska-Wiktor H, Jarosz W, Fogt-Wyrwas R, Drzewiecka A. Distribution and dynamics of soil contamination with Toxocara canis and Toxocara cati eggs in Poland and prevention measures proposed after 20 years of study. Vet Parasitol 2017;234:1-9. [Crossref]

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(21.) Paul M, King L, Carlin EP. Zoonoses of people and their pets: a US perspective on significant pet-associated parasitic diseases. Trends Parasitol 2010;26:153-4. [Crossref]

(22.) Fan CK, Liao CW, Cheng YC. Factors affecting disease manifestation of toxocarosis in humans: genetics and environment. Vet Parasitol 2013;193:342-52. [Crossref]

(23.) Nunez CR, Martinez GDM, Arteaga SY, et al. Prevalence and risk factors associated with Toxocara canis infection in children. Sci World J 2013;2013:572089. [Crossref]

(24.) Manini MP, Marchioro AA, Colli CM, Nishi L, Falavigna-Guilherme AL. Association between contamination of public squares and seropositivity for Toxocara spp. in children. Vet Parasitol 2012;188:48-52. [Crossref]

Vasilios Papavasilopoulos [1], Vassiliki Pitiriga [2], Konstantinos Birbas [3], John Elefsiniotis [4], Gerasimos Bonatsos [5], Athanasios Tsakris [6], *

[1] MD, PhD, National School of Public Health, 196 Leoforos Alexandras Avenue, 11521 Athens, Greece; [2] Assist Prof, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece; [3] Assoc Prof, Faculty of Nursing, School of Public Health, National and Kapodistrian University of Athens, 123 Papadiamantopoulou Street, 11526 Athens, Greece; [4] Assoc Prof, Faculty of Nursing, School of Public Health, National and Kapodistrian University of Athens, 123 Papadiamantopoulou Street, 11526 Athens, Greece; [5] Prof, Faculty of Nursing, School of Public Health, National and Kapodistrian University of Athens, 123 Papadiamantopoulou Street, 11526 Athens, Greece; [6] Prof, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece.

* Corresponding author: Athanasios Tsakris, Prof, MD, PhD, FRCPath, Department of Microbiology, Medical School, National and Kapodistrian University of Athens, 75 Mikras Asias Street, 11527 Athens, Greece. atsakris@med.uoa.gr

Caption: Figure 1. Demonstration of the examined areas throughout Attica region
Table 1. The estimated number of the soil
samples taken, according to the size of the
examined areas

Size of area   Place size   No. of samples
([m.sup.2])

50-150           Small            4
200-100          Medium           8
250-350          Large            12

Table 2. Distribution of T. canis contaminated soil
samples and residents' T. canis IgG seropositivity
among the tested geographical Attica regions

Region             T. canis (+)      T. canis IgG
                   soil samples     seropositivity

Central Athens    16.3% (44/270)      16% (8/50)
West Attica       22.8% (89/390)      25% (20/80)
North Athens      17.4% (89/510)      10% (5/50)
South Athens      12.2% (11/90)       10% (2/20)
Piraeus Area       10% (15/150)       9.4% (3/32)
Elefsina Area      12% (12/100)      11.1% (2/18)
Total            17.2% (260/1510)    16% (40/250)

Region            Chi-square     P-value
                 value (df= 1)

Central Athens       0.003        0.998
West Attica          0.177        0.674
North Athens         1.824        0.176
South Athens         0.078        0.780
Piraeus Area         0.012        0.922
Elefsina Area        0.012        0.922
Total                0.120        0.728

Table 3. Epidemiological characteristics among
T. canis IgG seropositive and seronegative
residents

Residents'          T. canisIgG    T. canis IgG
characteristics     seropositive   seronegative
                       (n=40)        (n=210)

Sex (male/female)      23/17          113/97
Fever                    1              2
Cough                    2              4
Wheezing                 3              4
Hepatomegaly             0              1
Splenomegaly             0              2
Washing hands            14             65
  before meals
Indirect                 7              43
  geophagia
Dog ownership            23             78

Residents'           Chi-square    P-value
characteristics     value (df=1)

Sex (male/female)      0.184        0.667
Fever                  0.693        0.405
Cough                  1.374        0.241
Wheezing               3.865        0.049
Hepatomegaly           1.734        0.187
Splenomegaly           0.679        0.410
Washing hands          0.255        0.613
  before meals
Indirect               0.186        0.666
  geophagia
Dog ownership          5.783        0.016
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Title Annotation:Original article
Author:Papavasilopoulos, Vasilios; Pitiriga, Vassiliki; Birbas, Konstantinos; Elefsiniotis, John; Bonatsos,
Publication:GERMS
Article Type:Report
Geographic Code:4EUGR
Date:Sep 1, 2018
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