Prevalence of Intestinal Parasitism and Associated Risk Factors in Rural and Urban Areas of Southern Algeria: a Four-year Community Based Study

Background: Intestinal parasitic infections are amongst the most common infections worldwide and have been identied as one of the most signicant causes of morbidity and mortality among disadvantaged populations. This community based study was conducted to assess the magnitude and pattern of intestinal parasitism and to identify the key risk factors associated with intestinal parasitic infections in Laghouat province, south Algeria. Methods: Stool samples were collected from 2277 symptomatic and asymptomatic patients aged 1-89 years old. Structured questionnaire were used to identify environmental, socio demographic and behavioral factors. Stool specimens were collected and examined for intestinal parasitic infection. Results: More than 33% of participants were found to be infected for at least one protozoan and/or helminth parasite. Of these, the most common parasites were Blastocystis spp. (17.79%), followed by Entamoeba histolytica/dispar (8.78%), Giardia intestinalis (4.22%), Entamoeba coli (4.08%) and more rarely Endolimax nana, Cryptosporidium spp, Trichomonas intestinalis, Enterobius vermicularis, Taenia spp., and a single case of Trichuris trichiura. Multivariate analysis revealed signicant associations between prevalence and species richness of combined protozoan parasites and contact with animal, living in rural areas and relying primarily on tap water, particularly in young individuals. Analysis of temporal consistency showed a high rate of infection in rural locations in the spring of 2018 and a rm relationship between school children and tap water was also detected. Conclusions: The high prevalence of intestinal parasitic infections among the Laghouat population, with identication of several risk factors indicates that parasitic infections are important public health problems. Therefore, several strategies are recommended in order to effectively reduce these infections including good animal husbandry practices, health education focused on good personal hygiene practices especially for school children, availability of safe drinking water and adequate sanitation.


Introduction
Intestinal parasitic infections are amongst the most common infections worldwide causing signi cant morbidity and mortality [1]. It is estimated that some 3.5 billion people are affected annually, and that 450 million develop clinical illness as a result of these infections and of those, at least 50% are schoolage children [2]. These infections cause serious public health problems ranging from diarrhea and stunting in children to impaired cognitive development, iron de ciency anemia and other physical and mental health problems [3]. The distribution and prevalence of various species of intestinal parasites differ from country to country and even regionally within countries. However, many studies have revealed high frequencies of parasitic infections in rural areas compared with urban areas [4,5]. This difference may be linked causally to any, or a combination of factors including, lack of sanitation, poverty, close contact with animals, lack of access to safe water, poor socioeconomic conditions and poor hygiene in rural communities [6].
Common intestinal parasites with direct life cycles, are transmitted mostly by the fecal oral route through direct contact with infected persons or animals, or indirectly through ingestion of contaminated water or food. Giardiasis, caused by Giardia intestinalis is a frequent cause of diarrhea, affecting approximately 200 million people worldwide [7], whereas Blastocystis spp. is currently estimated to infect up to 1 billion humans worldwide [8]. Among parasitic diseases, deaths attributable to intestinal amebiasis, caused by the protozoan Entamoeba histolytica, are only exceeded by those from malaria and schistosomiasis [9]. Approximately 40 million people, worldwide suffer from this disease per year and 40,000 die due to the resulting dysentery and liver abscesses [10]. The opportunistic protozoan, Cryptosporidium spp. has also emerged as an important cause of diarrheal illnesses worldwide, particularly in young children and immunocompromised patients, with a prevalence of 4% in developed countries and three to four times more frequent infections in developing countries [11]. These parasites constitute serious health challenges particularly in economically developing countries and are regarded as the most common and important etiological agents for more than 900 waterborne outbreaks of epidemic and endemic human diseases [12,13].
In this context, surveys carried out in Algeria and neighboring countries have highlighted the presence of protozoan cysts and helminths eggs in treated wastewater used for irrigation in Médéa Algeria [14], and even in water stored in private cisterns in rural areas of Tunisia [15]. These transmission stages of parasites have also been recorded in faecal samples of exposed populations that use wastewater in agriculture in rural areas of Morocco [16]. No major waterborne or foodborne outbreaks of giardiasis, amoebiasis or cryptosporidiosis have been reported so far in Algeria, but the presence of these protozoan genera has been recorded in epidemiological studies of human populations [17][18][19][20] and in domestic animals highlighting the high risk of zoonotic transmission [21][22][23].
To the best of our knowledge, no current information is available on the potential risk factors associated with these protozoan infections for communities living in the Algerian steppe area. Laghouat province is characterized by a diversity of life styles and includes urban, rural and Bedouin communities. It is also known as the province with the second most numerous livestock ownership in Algeria, so this makes Laghouat an interesting area for parasitological research although to date there are no published reports on the extent of prevalence and distribution of human intestinal parasites among people living in this region. Thus, the objective of the current study was to determine the prevalence of intestinal parasites and to identify associated risk factors among the Laghouat population.

Materials And Methods
Study area and study subject The study was carried out in the province of Laghouat, the province with the second most numerous livestock and one of the important socio-economical spot in Algeria, located in the center of the country at 400 km to the south of the capital Algiers (33° 48' N, 02° 53' E). The province of Laghouat is affected by three types of climates: semi-arid in the north, arid in the center and Saharan in the extreme south of the province. Daily temperatures average − 5°C in winter months and over 40°C in summer. The average annual rainfall is 151.21 mm per year [24]. Laghouat province is located on the banks of the Mzee valley in the Amour Range of the Saharan Atlas mountains, and constitutes an oasis on the northern edge of the Sahara Desert. This province covers about 25,052 km 2 and had a population estimated at 603.876 inhabitant in 2016, of which 81.5% lived in an urban environment and 4.9% in rural areas of the province [25]. These two populations live under essentially similar conditions with respect to local infrastructure and provision of drinking water, which is made available through boreholes and taps (supplied by wells or public water delivery systems). However, the rural population is mainly engaged in agricultural activities such as livestock husbandry and breeding (sheep, goats, cattle, horses, camels, chicken) [26]. The remaining 13.6% of the population, the Bedouin (Nomads), live in sparsely populated parts of the province in relatively isolated communities. This population lives without infrastructure, with unsafe water supplies and inadequate levels of sanitation [25].
We carried out a community based study (March 2015 to July 2018) involving symptomatic and asymptomatic patients who attended the laboratory of public hospital (LPH) and a private laboratory for medical analysis (Dr. Debagha) for parasitological examination. Patients were from two municipalities in urban areas; Laghouat (n = 916), Ksar El Hiran (n = 321) and from four municipalities in rural areas; Kheng (n = 408), Sidi Makhlouf (n = 216), Tadjmout (n = 226) and Hassi Delaa (n = 190) (Fig. 1). In general, parasite assessments are requested by doctors following the presence of digestive disorders and may also be requested for provision of a medical certi cate of health status required for job applications and for conscripted soldiers joining the army.

Sample collection and stool examination
We assessed intestinal parasitic infections among 2.277 hospitalized subjects and outpatients, aged 1-89 years (27.4 ± 18.66) who had been referred to the two laboratories referred to above for routine stool tests in Laghouat province. The objectives of this study were explained to the participants and a standard questionnaire summarizing personal information was completed by each individual or his/her guardian (i.e. age, sex, place of residence (urban or rural), the nature of the water consumed, the presence of digestive disorders, immune status, and possible contact with animals).
Fresh fecal specimens were collected from each subject in 25 ml clean wide-mouth, covered plastic containers and then immediately examined in normal saline (0.9%) and Lugol's iodine preparations, using direct microscopy as part of a routine parasitological examination. After completing the direct stool examination, the formol ether concentration and Willis otation techniques were performed in order to increase the likelihood of detection of parasites [27]. Additional individual fecal smears were xed with methanol and stained by the modi ed Ziehl-Neelsen for detection of intestinal coccidian parasites. Stool samples suspected of containing Blastocystis were inoculated into 5ml of modi ed Boeck and Drbohlav Locke-egg medium supplemented with 10% horse serum and incubated at 37°C. The cultures were checked for the presence of Blastocystis, by direct microscopy on the third day after inoculation. We performed also the Graham technique (Scotch test) for detection of pinworm infection in children.

Statistical analysis
Data are summarized as prevalence (percentage of infected subjects ± 95% con dence limits [Cl 95 ]) and as mean protozoan species richness (PSR, mean number of protozoan species harbored per subject ± standard error of the mean (S.E.M.). The 95% con dence limits were calculated by bespoke software based on the tables of Rohlf and Sokal (1995) [28]. Analysis of data for each of the protozoan infection measures (prevalence and PSR) was undertaken in two phases. First, for prevalence univariate log-linear models were tted in IBM-SPSS (version 24) with each factor in turn and infection (combined protozoan infections at 2 levels, present or absent), as described elsewhere [29]. For PSR we tted non-parametric tests (Mann-Whitney U test or Kruskal Wallis test). We tted in turn the following seven factors SEX, (at two levels, male or female), AGE (11 levels corresponding to the following age limits, level 1 = 1-4 years; In a second phase we tted multi-factorial models. For analysis of prevalence we used maximum likelihood techniques based on log-linear analysis of contingency tables as described previously [29], beginning with the full factorial model, and then employing the backward selection procedure in SPSS we simpli ed the model until only signi cant terms remained. For each level of analysis in turn, beginning with the most complex model, involving all possible main effects and interactions, those combinations that did not contribute signi cantly to explaining variation in the data were eliminated in a stepwise fashion beginning with the highest level interaction (backward selection procedure). A minimum su cient model was then obtained, for which the likelihood ratio of chi-square was not signi cant, indicating that the model was su cient in explaining the data. The importance of each term in interactions involving infection in the nal model was assessed by the probability that its exclusion would alter the model signi cantly and these values are given in the text, assessed by a likelihood ratio test between nested models with and without each factor of interest.
For analysis of PSR we implemented a multifactorial generalized linear model (GLM), tted with a Poisson log link in SPSS 24, incorporating all the main effects and all relevant 2-way interactions. As above, the model was simpli ed in steps beginning with the full factorial model. Model simpli cation was by backward selection, deleting the least signi cant interaction in turn at each successive cycle, until only signi cant 2-way interactions, and relevant main effects remained. Signi cance was based on the Wald χ 2 output of the minimum su cient model thus generated.
Relationships between prevalence of infection or PSR, and levels within speci c factors that showed a directional trend (meaningful increase across levels e.g. change with host age) were examined by the non-parametric Spearman's test in SPSS 24, and rs is given. P values less than 0.05 were considered to indicate statistical signi cance.

Results
The overall prevalence of parasitic infections in the study population was 33.3% [31.40-35.27], the majority of which were attributable to protozoan species (prevalence = 32.5% [30.62-34.47]), helminth infections in this population being relatively rare (prevalence = 0.79% [0.469-1.249]). The prevalence of each of the ten species of parasitic organisms identi ed is given in Table 1. Table 1. Prevalence of protozoan and helminth species.
The most common species was Blastocystis spp. and the rarest was the nematode T. trichiura, which was recorded in only one subject. Because helminths were so rare in this group of subjects, further analysis is focused on infections by protozoan species, but it is of interest to note that of the 18 subjects infected with helminths, 16 were under 14 years in age, and 13 of these were attributable to E. vermicularis. Only two adult subjects were diagnosed with helminths, both in their 30s and both with infections with Taenia spp.
Combined species richness (both protozoa and helminths) was 0.39 ± 0.013 species/host, and mostly attributable to the protozoan species (0.38 ± 0.013). Single species protozoan infections were the most common (27.8% of total population, and 85.6% among the protozoa infected subjects), and multiple species protozoan infections much rarer (two species = 4.1%, three species = 0.57%, and four species 0.44%, of all subjects).
Intrinsic factor affecting prevalence and species richness of combined protozoan parasites Prevalence of combined protozoan infections varied signi cantly with host age ( Fig. 2A; χ 2 10 = 34.15, P < 0.001), increasing from age class 1 to age class 2 by 11.4% and then falling as host age increased to a low of 23.1% among the oldest age class. Although the fall in prevalence with increasing host age was modest (a reduction of 21.7% from age class 2 to 11), it was nevertheless signi cant (r s = -0.095, n = 2.277, P < 0.001). Prevalence was very similar and not signi cantly different between the sexes (Table 2; χ 2 1 = 0.173, P = 0.68).

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Protozoan species richness (PSR) also varied signi cantly between the age classes ( Fig. 2B; Kruskal-Wallis test, H 10 = 35.42, P < 0.001), and showed declining values with increasing host age (r s = -0.099, n = 2.277, P < 0.001), much as for prevalence. Interestingly, however despite the lower mean values in subjects in age classes 7-11, corresponding to subjects who were older than 29 years of age, eight of the 13 cases of 3-species infections occurred in these age classes. The single case of a 4-species infection was in a 21-year old male. Mean PSR was very similar in both sexes and not signi cantly different (Table 2; z = 0.781, P = 0.44).
Seasonal, annual and location-speci c effects on prevalence and species richness of combined protozoan parasites Table 2 shows the prevalence of combined protozoan infections in each year, season and area in which the subjects lived. Analysis of each factor in turn revealed that prevalence differed signi cantly between subjects living in rural and urban areas (χ 2 1 = 26.67, P < 0.001), with prevalence among subjects from rural sites 10.2% higher than that for subjects living in urban sites. Table 2. Prevalence of combined protozoan infections, by year, season and area.
As Table 2 shows, overall prevalence was similar in each of the years of the study varying only between 28.7% (2015) and 35.7% (2017). Prevalence was even more constant when examined by season, varying only from 30.2-33.9%. Neither of these was signi cant. However, when all three factors were tted in a multifactorial model, the highest order interaction (YEAR x SEASON x AREA x INFECTION) could not be simpli ed (χ 2 9 = 20.673, P = 0.014), indicating signi cant interactions at this level. These are illustrated in Fig. 3, which shows that whereas prevalence in most data subsets centred around 30%, unusually low prevalence was detected in 2018 in spring and summer among people living in urban areas and a very high prevalence was seen also in 2018 in the spring in rural areas.
Much the same outcome was seen when PSR was analysed by non-parametric tests: there was a highly signi cant difference in mean PSR between subjects from urban and rural areas (Mann-Whitney-U test, z = 5.406, P < 0.001), but none between the seasons (Kruskal-Wallis test, H 3 = 2.19, P = 0.535) or between years (Kruskal-Wallis test, H 3 = 5.862, P = 0.5118).
Prevalence and species richness of combined protozoan parasites in relation to sources of drinking water and ownership of animals Subjects who relied primarily on tap water were more likely to experience infections with protozoan organisms than those who used bottled water (χ 2 1 = 4.04, P = 0.045), but the difference in prevalence, while signi cant, was not marked between these two groups (Table 2). However, prevalence was more than twice as high among those who had animals compared to those who did not (Table 2; χ 2 1 = 224.6, P < 0.001).
A similar outcome was found for PSR. The difference between subjects relying on bottled and tap water was signi cant ( Table 2; Mann-Whitney U test, z = 2.818, P = 0.005). Mean PSR was also signi cantly higher among those who had animals compared with those who did not (Table 2; z = 15.52, P < 0.001).
The key factors affecting prevalence of combined protozoan infections As a nal stage to this analysis we tted a statistical model that incorporated all the key signi cant factors from stages described above. This included the effects of AGE, AREA, YEAR, WATER, ANIMALS and INFECTION. The minimum su cient model comprised ve terms that included INFECTION. The rst term was ANIMALS x AREA X INFECTION (χ 2 1 = 4.81, P = 0.028) and this is illustrated in Fig. 4A, where it is clearly apparent that the possession of animals was a key determinant of prevalence. Whether living in an urban or rural area, prevalence was much higher among subjects that had animals. Although in both cases prevalence was higher among the inhabitants of rural areas, the difference between subjects from rural and urban areas was more marked in those without animals. While possession or contact with animals greatly enhanced the likelihood of infection, the difference in prevalence between subjects who worked with animals and those that did not, also varied between years (ANIMALS x YEAR x INFECTION, χ 2 3 = 27.232, P < 0.001). However, as Table 3 shows, prevalence was always higher in each year of the study among those with animals, but the effect was not totally consistent and the difference in prevalence between the two groups varied in magnitude. The difference between areas also varied between years (Table 3; AREA x YEAR x INFECTION, χ 2 3 = 38.53, P < 0.001), and whilst higher among rural inhabitants in three years, especially in 2018, there was little difference between subjects from these two areas in 2015, and therefore the effect of where the subjects lived was not consistent from year to year. Table 3. Temporal variation in prevalence of combined protozoan infections in subjects who owned or looked after animals and those that had no contact with animals, those living in rural and urban regions of the province and those that relied on tap or bottled water.
While overall reliance on tap rather than bottled water resulted in a higher prevalence of protozoan infections, the difference between the two groups was dependent on host age (WATER x AGE x INFECTION, χ 2 10 = 22.83, P = 0.011). As Fig. 4B shows the earlier nding of declining prevalence with age was mostly driven by subjects who relied on tap water and among them there was a signi cant negative correlation between age and prevalence of infection with combined protozoa (r s = -0.176, n = 1.201, P < 0.001). Among those that used bottled water, prevalence was much the same in all age classes and did not correlate signi cantly with host age (r s = -0.029, n = 1.076, P = 0.34). There was also a marked difference between years in the extent of the difference in prevalence between those that used bottled or tap water (Table 3; WATER x YEAR x INFECTION, χ 2  3 = 14.431, P = 0.002). In two years (2015 and 2018) prevalence was higher among those relying on tap water, and in the other two prevalence was higher among bottled water users.
The key factors affecting protozoan species richness As a nal step in exploring the factors that in uenced PSR, we tted a GLM (with Poisson errors, main effects and all 2-way interactions) with all the factors (SEX, AGE, AREA, SEASON, YEAR, WATER and ANIMALS). This con rmed the signi cance of differences in mean PSR between age classes, although with all other factors and their 2-way interactions taken into account signi cance was only marginal (Wald χ 2 10 = 18.11, P = 0.05). The difference between areas in which subjects lived was also signi cant (Wald χ 2 1 = 19.11, P < 0.001), as was the hugely signi cant effect of ownership of animals (Wald χ 2 1 = 119.25, P < 0.001). As in the earlier analysis the difference between the sexes was not signi cant (Wald χ 2 1 = 0.051, P = 0.82), nor was that between seasons (Wald χ 2 3 = 3.34, P = 0.34). However, with all other factors and their 2-way interactions taken into account there was no independent effect of quality of water (Wald χ 2 1 = 1.58, P = 0.21), and the difference between years gained signi cance (Wald χ 2 3 = 11.912, P = 0.008).
This analysis identi ed ve signi cant interactions, which are illustrated in Fig. 5. The most marked interaction was that between AREA and YEAR (Wald χ 2 3 = 26.212, P < 0.001). While mean PSR was generally higher among subjects living in rural compared with urban areas, when the data were broken down by year of the study, it became apparent that this effect was not consistent from year to year (Fig. 5A). In 2015 there was no difference, in 2016 and 2017, there was only a marginal difference while in 2018 there was a huge difference with mean PSR being almost four times higher among subjects in rural areas. Ownership of animals ( Fig. 5C) was another key factor. While mean PSR was considerably higher in all four years of the study among subjects that owned animals, compared to those that did not, the extent of the difference between the two groups varied from year to year (Wald χ 2 3 = 17.960, P < 0.001), but was always in the same direction. Therefore, the effect of ownership of animals showed some temporal consistency and was clearly an important risk factor predisposing people to infection with protozoan parasites. While the earlier univariate analysis indicated that people who relied on tap water had a higher mean PSR than those who used bottled water (see above), when other factors had been taken into account the difference was no longer signi cant, but there was a signi cant interaction between WATER and YEAR (Wald χ 2 3 = 16.498, P = 0.001; Fig. 5B). Subjects relying on tap water had a higher mean PSR in 2015 and there was a marked difference 2018, but not in 2016 and 2017, so the difference between people relying on tap versus bottled water was not consistent from year to year. The two remaining signi cant interactions were weaker (Figs. 5D and 5E). Although there was no overall signi cant difference between the sexes, and this was particularly the case in summer and autumn, in spring mean PSR was higher in female subjects while in winter it was higher in males (Wald χ 2 3 = 11.727, P = 0.008). Finally, the difference in mean PSR between subjects who lived in urban versus rural areas was greater when they relied on bottled water compared with tap water (Wald χ 2 1 = 6.361, P = 0.012).
Prevalence and species richness of combined protozoan parasites in relation to symptoms experienced by the subjects Perhaps as expected, prevalence of combined protozoan infections was considerably higher among subjects who showed some symptoms (79.5% [76.55-82.12]; χ 2 1 = 849.3, P < 0.001) compared to asymptomatic subjects (14.0% [13.16-16.59]). Among the 623 subjects with symptoms, no evidence of protozoan infection was found in 128, but 18 of these subjects harboured helminth infections. Table 4 shows the breakdown of symptoms reported by the subjects and/or con rmed at clinical inspection, and the associated prevalence of combined protozoan infections. In all symptomatic categories prevalence was much higher than in symptomless subjects, although sample size was small in some cases.

Discussion
In this community based study we performed an analysis of intestinal parasitosis among symptomatic and asymptomatic patients who presented at medical health laboratories for parasitological examination, and then evaluated the results according to the demographic, spatio-temporal, behavioral and occupational factors that had been recorded. Risk factors (age, patient's provenance, clinical symptoms, drinking water resource and contact with animals) were considered for the rst time in an Algeria population to identify the key drivers affecting the spread of intestinal parasites in the country.
The mean prevalence of intestinal parasitic infections was high in Laghouat province (33.33%) with one or more intestinal parasites. The overall prevalence found in this study was much lower than had been reported earlier in Blida, north Algeria (60.61%) [18] and was slightly higher compared to those reported in Oran west Algeria (19.96%) [17], in Tunisia (26.6%) [30] in Morocco (14.15%) [31], in Italy (11.1%) [32] and in Spain (13.7%) [33]. However, overall prevalence in our study was comparable to other communitybased studies conducted in Saudi Arabia (32.2%) [34], in Qatar (33.9%) [35] and in Morocco (34.5%) [36]. These differences in overall prevalence among North African countries may be attributable to variation in the sampling techniques used, differences in the quality of drinking water, personal and community hygiene, and variation in the environmental conditions in the different study locations. Furthermore, our results also showed that protozoan infections were more common compared to helminth infections, with the majority attributable to Blastocystis spp., E. histolytica/dispar and G. intestinalis and these were the most common enteric parasites associated with symptoms in the study population. This nding is in agreement with studies conducted in Algeria [17,18], in North Africa [5,30,31] and in Europe [32,33] but differs from studies carried out further south in Africa and elsewhere in the tropics, in which soiltransmitted helminthiasis have been reported to be the dominant parasites [4,37]. The explanation for this contrast in the species of intestinal parasites involved lies in the geoclimatic differences between North Africa and further south, notably the extreme aridity of northern regions (high ambient temperatures, low humidity and infrequent poor rainfall) compared to elsewhere. Other factors, that also contribute, most likely include soil textures and farming ecosystems. Human intestinal helminths, the soiltransmitted species, cannot easily complete their life cycles in the absence of an appropriate soil environment or under the extremely arid environmental conditions in Laghouat province [24].
Nevertheless, the overall pattern of the prevalence of combined protozoan infections in our study was clearly in uenced by the pattern of prevalence of Blastocystis spp., E. histolytica/dispar and G. intestinalis. The high prevalence of Blastocystis spp. in this study con rms the results of a previous survey in the same study area in which a prevalence of 32.1% was recorded [19]. Today, Blastocystis spp. is considered an under-reported parasite, but has a worldwide distribution. The highest prevalence recorded to date is 100%, which was recorded in Senegalese children [38]. However, the infection rates of E. histolytica/dispar were signi cantly higher than observed in Blida and Oran, Algeria (1.67%-4.83%) respectively [17,18], while the incidence of G. intestinalis is considerably lower than that in other provinces in Algeria (15.32%-41,67%) respectively [17,18] and in North African countries with similar standards of living, e.g. Tunisia (17%) [30] and Morocco (22.71%) [31]. The higher prevalence of G. intestinalis in this provinces could be due to higher rainfall in this areas compared to Laghouat province. Although at the whole population level there was relative stability in the prevalence of combined protozoan infections and PSR from year to year, and virtually no differences between the values of both parameters for the four seasons, more in depth analysis revealed that there were some marked consistent trends and some unexpected differences at the data subset level. The strongest and most consistent in uence on both prevalence of infection and PSR was ownership of animals. Subjects who owned or looked after animals had a markedly higher prevalence of infection and harbored more species of protozoan parasites (i.e. had a higher value for PSR). The higher values of these two parameters in those with animals were evident in each year of the study, and whether people lived in urban or rural locations. As emphasized earlier, Laghouat province is known as the province with the second most numerous livestock ownership in Algeria [24] and farms are located always in rural areas with a high diversity of animals species (sheep, goat, bovine, horse, camel and chicken). Additionally, nomadic and pastoral populations live close to their animals and this close contact increases the risk of zoonotic transmission. Livestock, have often been implicated as a major source of environmental contamination and potential reservoirs for zoonotic infection [21,22]. In Algeria G. intestinalis and C. parvum have been reported in domesticated animal species [21][22][23]. These parasites are potentially zoonotic pathogens and close contact with animals is believed to be the major risk factor for human infections.
The next in uence on prevalence and PSR in terms of the magnitude of its effect was the location in which subjects lived. Overall data showed that those living in rural locations had both higher prevalence of protozoan infections and higher PSR, but the magnitude of this effect was not nearly as marked as that of ownership of animals. Similar nding in Morocco showed a high intestinal infection in rural area (53.3%) compared to urban area (29.5%) [36]. This may be explained by the precarious hygienic conditions in rural areas where access to adequate sanitation and the supply of drinking water is di cult, and the most important factor is close contact with livestock. Indeed in our study the relatively small difference in prevalence between people living in rural and urban areas contrasts with the much larger discrepancy in prevalence between those who owned animals and those that did not. Regarding the seasonality there were signi cant inconsistencies with the inhabitants of rural locations showing an expectedly high prevalence in the spring of 2018 compared to springs in particular, but also seasons in other years. Those living in urban locations showed particularly low prevalence in both spring and summer of 2018. There were also inconsistencies in the magnitude of the difference in PSR between rural and urban inhabitants between the years of the study. In 2015 the values for PSR were almost identical for these two areas, in 2016 and 2017, PSR was marginally higher among rural inhabitants, but the biggest difference occurred in 2018, when there was a huge difference between these two data subsets. The overall difference in PSR between the inhabitants of rural and urban areas was therefore driven largely by the hugely skewed results of 2018, and was not consistent across the years of the study. These within-year uctuations in prevalence are most likely explained by seasonal changes in eating habits in particular years, with an increase in the consumption of water and raw foods during the hot seasons and on variation in climatic conditions which favour the embryonation and development of the infective stages in ova and oocysts during their external phase. Another pertinent factor centres around agricultural practices in the spring in the Algerian steppe. This is the best time for pasture improvement and in this period livestock owners and breeders are in close contact with their livestock, because this is the season when goat kids and lambs are born and when sheep are sheared. Transhumance of animals in this period is very frequent in search of fresh pastures on which to graze and this close contact between livestock and pastoralists increases the risk of zoonotic transmission in spring and in rural area, in contrast to urban area where pets are not moved seasonally to different locations and are frequently monitored medically.
There was no signi cant overall variation in the prevalence of combined protozoan infections between years in the present study. Our results therefore contrast with other studies which have reported signi cant temporal changes in the prevalence of protozoan infections. In Morocco, for example prevalence was found to have fallen from 10 [40], to 4.89% during 2012-2014 [41]. Such changes in prevalence of protozoan infections are most likely attributable to improvements in health and social conditions of the relevant populations. However, the more stable overall annual prevalence in this study suggests that the subjects in the current work did not experience any major changes/improvements in their health and social conditions. Nevertheless, annual prevalence data may hide underlying uctuations in subsets of the population in the study and in the current work these were revealed by multifactorial statistical models.
As expected our data showed variation in both prevalence of protozoan infections and PSR with age. The prevalence of combined protozoan infection was observed to be signi cantly higher in children in the age range 6-10 years, which is in line with studies performed elsewhere [5,30,37]. Nevertheless, multivariate analysis showed that there was a strong association between the quality of water consumed and age with prevalence of combined protozoan infections declining with increasing host age among those who relied on tap water. Such high rates in school children could be explained by the fact that children in this age are more susceptible to intestinal infectious diseases than adults because of their poor hygiene habits, poor health education, consumption of school tap water, immature immunity of young children and close personal contact in crowded classrooms [37]. The majority of houses and schools in Laghouat province are supplied by public water delivery systems from boreholes and then stored in water tanks and provided via taps. In Algeria, this tap water is treated by advanced phyisco-chemical techniques to make it suitable for human consumption, with xed limits for the chemical, physical, organoleptic, radionuclide and bacteriological parameters. However, to the best of our knowledge, it is not subjected to parasitological inspection and relevant treatment, as has been reported in some Arab countries [15,42].
According to the World Health Organization statistical series for Algeria, 16% of the population does not have access to improved sources of drinking water and 5% of the population does not have access to improved sanitation [43]. It is interesting that the mean PSR was higher in subjects living in rural areas whether they relied on bottled or tap water, compared to those living in urban areas, among whom reliance on bottled water did confer some modest advantage in terms of lower PSR (Fig. 5E, This result is consistent with our nding that ownership of animals in Laghouat province is a far more important risk factor predisposing people to infection with protozoan parasites.
In our study, we noticed that the sex of subjects did not in uence the prevalence of the intestinal parasites, this may be due to the fact that people are exposed to the same source of infection regardless of their sex and this nding is in agreement with some other studies [17,31], although it contrasts with a previous study reported in Morocco [36] in which a signi cant association between sex and infection was found. However, when examined at a subset level, our data did in fact provide some support for variation in prevalence between the sexes. Thus in spring the mean PSR was higher in female subjects, while in winter it was higher in males, and this difference between the sexes may have arisen because in winter, men tend to eat fast food, while in spring women are more frequently in contact with raw vegetables of the season than men, when preparing food.
Our application of multivariate analyses in the present study established that contact with animals was the most important risk factor and thus predictor of intestinal protozoan parasitic infections in Laghouat province. This analysis also revealed that living in rural rather urban areas, reliance on tap water and age, whilst showing less temporal stability, were nevertheless additional secondary risk factors that also need to be taken into consideration. Further molecular investigations of the exact genotypes of each of the intestinal parasites recorded here in humans, and extended additionally to animals and tap water samples, are required to con rm these conjectures.
We have demonstrated in our study that the most important risk factor for infection with intestinal protozoan parasites among the subjects that were referred for tests was ownership of and work with animals. While those living in rural versus urban environments and those relying primarily on tap versus bottled water, also showed higher prevalence of intestinal protozoan infections overall, these effects were not consistent from year to year. Our study emphasises the need to examine closely variation in parameters within subsets of data and not to rely just on global statistics that do not control for other in uences.