Introduction

Giardiasis is a disease caused by a microscopic parasite; a protozoan that affects many animal species, including domestic carnivores and humans. The disease is often asymptomatic and can be the cause of chronic enteritis with a syndrome of poor digestion and absorption, which may be accompanied by stunted growth (Wielinga et al. 2015; Squire and Ryan 2017). Giardia intestinalis is the only species within the genus Giardia that infects humans, causing intestinal infections. Children are at most risk of Giardia intestinalis infection, mostly those in developing countries and living in poor community settings (Sirize et al. 2008; Ramírez et al. 2015). Molecular characterization of Giardia intestinalis has classified it into seven assemblages depending on particular genes (Saksirisampant et al. 2012; Skhal et al. 2016). Assemblages A and B infect a broad range of hosts including humans, livestock, cats, dogs, and wild mammals (de Lucio et al. 2015).

In Algeria, intestinal parasites are of public health concern and still a reason for consultation in medical practice (Hamaidi et al. 2010). Most of the epidemiological studies are based on microscopic examination (Hamaidi et al. 2010; Benouis et al. 2013). So far only one study was performed using molecular tools (Lalle et al. 2009). Therefore, this present study aimed to determine the different genotypes of G. intestinalis isolates from a sample of school children in the city of Djelfa, in an effort to attain a better understanding of the genetic diversity and transmission of this disease.

Materials and methods

Population and sampling

The present study was carried out at the Djelfa province of Algeria. It is considered as the seventh-most-populous city in Algeria. Djelfa is the capital of Djelfa Province, approximately 300 km from Algiers, it has a population of about three hundred thousand people in the 2008 census. Djelfa has a chilly semi-dry climate (Fig. 1). Official approval was obtained from the Directorate of Education in the province state of Djelfa for obtaining samples from school children. An information sheet for each patient has been filled out by an adult family member, containing information about age, gender, socioeconomic background, personal hygiene, specifically regarding hand washing and food consumption, types of water supply, sewage disposal system, and if there is close contact with household pets.

Fig. 1
figure 1

Map of Algeria where the location of the study is marked by a star

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Stool collection and microscopic examination

A total of 355 fecal samples were collected from three different schools at the city of Djelfa with parental consent during the first semester of the school year 2018–2019, in a labeled and sterile container. Stool smears were stained by Lugol’s iodine and examined under a light microscope at × 40 and × 100 magnifications. The intensity of infection was estimated by average cyst count per high power field (HPF) of a light microscope. Samples’ score was divided into three categories: 1–5 per field (+), 6–10 (++) and more than 10 cysts per field (+++). All positive samples for Giardia were stored at 4 °C in 2.5% potassium dichromate solution (1:3) for further analysis.

Molecular analysis

Potassium dichromate was washed off by distilled water, and samples were centrifuged at 3800 rpm for 10 min. This procedure was repeated between 8 and 10 times approximately, to ensure the removal of all dichromate. Genomic DNA was extracted from almost 250 mg of each fecal sample using QIAamp DNA stool mini kit (QIAamp® DNA Stool Mini Kit, QIAGEN) as described by Skhal et al. (2016). We applied PCR–RFLP method using beta-giardin gene to differentiate Giardia assemblages. Briefly, a nested PCR method was used to amplify a 514 bp fragment of the gene using the following primer pairs: G7/G759 and GiarF/GiarR respectively (Caccio et al. 2002; Lalle et al. 2005). The PCR reaction (25 µl final volume) contained 12.5 µl One PCR™ master mix 2X (GeneDirex Inc, Taiwan ROC), 1 µl of each primer pairs, 10.5 µl nuclease- free water, and 3 µl of the extracted gDNA. PCR conditions were previously described by Skhal et al. (2016). The amplified products were digested by HaeIII restriction enzyme (Thermo Fisher Scientific, USA) for 1 h at 37 °C followed by 20 min inactivation at 80 °C. Restriction fragments for all samples were electrophoresed in 2% agarose gel stained with ethidium bromide (Sigma-Aldrich, USA) along with a 100 bp DNA ladder (GeneDirex Inc, Taiwan ROC) as a size standard. The final results were visualized under a UV transilluminator and photographed for documentation.

Results

Our 30 positive samples consisted of 70% males and 30% females. The presence of weight loss (23.3%) was the most significant symptom in all symptomatic cases. However, most of our studied cases were asymptomatic (Table 1).

Table 1 Summary of the studied samples from Djelfa city, by age, sex and clinical symptoms
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Cysts and/or trophozoites of Giardia, were observed in all fecal samples after staining by Lugol’s iodine (Fig. 2). Among the 30 children infected with giardiasis, co-infection with other parasites was found in 5 cases (one case Entamoeba coli plus Entamoeba spp; one case: Blastocystis hominis plus Taenia spp; two cases: Blastocystis hominis alone and one case Chilomastix).

Fig. 2
figure 2

Giardia intestinalis in stool sample. a Cysts; b trophozoites (× 100)

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PCR amplification of the 514 bp fragment of the β-giardin gene was successfully obtained from 28 out of the 30 studied cases (Fig. 3a), while only two samples showed no amplification. Furthermore, only 20 samples have shown strong PCR products suitable for RFLP analysis. Our genotyping results showed different restriction patterns (Fig. 3b); 70% of the samples were belonging to assemblage A (14/20) and 30% belonging to assemblage B (6/20). Our total results are summarized in Table 2.

Fig. 3
figure 3

Ethidium Bromide-Stained Agarose Gel 2%. a PCR Products. Lane M, molecular weight marker (100 bp); lanes 1–4, PCR products from different samples. b Enzyme digestion of β-giardin- PCR products. Lane M, molecular weight marker (100 bp); lane 1, uncut PCR product; lanes 2.4.5 represent Assemblage A; lanes 3,6, represent Assemblage B

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Table 2 Microscopic and molecular results
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Our data revealed that the most age group associated with Giardiasis was 6–7 years old (56.7%) followed by 8–9 then 10–11 (30% and 13.3% respectively) (Table 1).

No assemblage B was noted in females of any age groups. We noted the dominance of male sex regarding assemblages distributions (Table 3).

Table 3 Distribution of Giardia assemblage A and B according to gender and age
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Regarding the relation between assemblages and symptoms, our results revealed the presence of assemblages A at 7 symptomatic and 7 asymptomatic cases also. The symptomatic cases were characterized by the frequent presence of weight loss (57.1%), diarrhea (42.9%), anorexia and fever (28.6%) and abdominal cramps (14.3%). On the other hand, assemblages B were detected at 4 asymptomatic and 2 symptomatic cases. The latter shows weight loss, anorexia, abdominal cramps, and fever.

The comparison between assemblages A and B according to close contact with animals showed that there was a difference between the two assemblages. Six cases of assemblage A were associated with domestic animals (cats), poultry and birds; however, there were no cases for assemblage B.

Discussion

This study provides for the first time, information about the distribution of G. intestinalis genotypes in the city of Djelfa. G. intestinalis is the most common intestinal parasite of humans in several countries (Caccio and Ryan 2008; Lane and Lloyd 2002; Abdullah et al. 2016). Recently, this protozoa is included in the ‘Neglected Diseases Initiative’ by the WHO (Savioli et al. 2006).

The routine diagnostic method for giardiasis is the microscopic detection of Giardia cysts and/or trophozoites in stools. Our data revealed that most of microscopic positive samples were identified by nested PCR, only two samples (6.7%) with low numbers of cysts were negative after nested PCR. False-negative results using PCR were mentioned in many previous studies (Amar et al. 2002; Bertrand et al. 2005; Hatam Nahavand et al. 2011). Although there is no clear explanation for such false- negative results, it could be due to low concentration level of DNA, the presence of PCR inhibitors in some of the fecal samples and the existence of a robust wall that inhibits release of DNA from the cysts or degradation of parasite material during storage (Babaei et al. 2008; Roointan et al. 2013).

PCR–RFLP is usually a sensitive and powerful analytical tool, that is capable of discriminating between and within assemblages by targeting some loci such as gdh, tpi, and β-giardin (Amar et al. 2002; Cedillo-Rivera et al. 2003; Read et al. 2004; Itagaki et al. 2005; Skhal et al. 2016). Despite the small number of samples studied, our data indicated that assemblage A (14/20; 70%) was more prevalent than assemblage B (6/20; 30%). This result is in agreement with many previous studies conducted in different countries as in Iran (Roointan et al. 2013; Hooshyar et al. 2017; Kashinahanji et al. 2019); Syria (Skhal et al. 2016); Saudi Arabia (Al-Mohammed 2011); Eygpt (Helmy et al. 2009) and South Asia, including India (Bertrand et al. 2005; Geurden et al. 2009). Also, the predominance of assemblage A in wastewater has been reported in Italy (Yong et al. 2000; Cedillo-Rivera et al. 2003). On the other hand, the results of a study conducted in the south west of Iran showed that the majority of cases belonging to assemblage B (Rafiei et al. 2013). Similar results were obtained in Egypt (Soliman et al. 2011) and in Baghdad (Qader and Bakir 2011), as well as in Algeria, results of the study conducted on 120 stool samples from Sahrawi children indicated the dominance of assemblage B (56.2%) comparing to assemblage A (37.5%) (Lalle et al. 2009). It has been reported that these differences in the prevalence of assemblages may be attributed to the geographical location, or the contamination of drinking water, in addition, the lifestyle of the studied population who may be in close contact with animal wastes, especially in rural areas. (Lalle et al. 2005; Feng and Xiao 2011; Gasparinho et al. 2017; Al-Shehri et al. 2018).

Our data indicated the presence of assemblages A and B in both asymptomatic and symptomatic patients, so it is difficult to link the clinical outcome to a specific assemblage. However, no consensus is currently available on the correlation between the occurrence of symptoms and the genetic variations of G. intestinalis involved in the infection (Kasaei et al. 2018). Nonetheless, the prevalence of assemblage A in symptomatic cases was more than symptomatic cases at assemblage B in our study. This result is in agreement with some previous studies which have correlated assemblage A with the occurrence of symptoms (Sahagun et al. 2008; Robertson et al. 2010; Cardona et al. 2011; Laishram et al. 2012; Anuar et al. 2015; Skhal et al. 2017; Kasaei et al. 2018). Despite the fact that in some other studies, assemblage A has been associated with asymptomatic patients (Sarkari et al. 2012; Nasiri Goorabi et al. 2017). It is worth to mention that, other studies showed a clear association between assemblage B and symptoms like diarrhea, abdominal pain, and flatulence (Lebbad et al. 2011; Jerez-Puebla et al. 2015).

Our data have shown that the most age group associated with Giardiasis was 6–7 years old (56.7%), this could be due to the young age of this group of patients and the lack of awareness of personal hygiene.

Co-infection with other gastrointestinal parasites is commonly noticed. In this study, 16.6% of our studied cases had one or two parasites along with G. intestinalis. The reason for such co-infections is due to drinking or eating contaminated food or water, this case is of great significance for child health especially in countries with limited resources (Acosta et al., 2014). Previous studies revealed that in such countries, infection with Giardia appeared to protect children against diarrhea via unknown mechanisms (Veenemans et al. 2011; Muhsen et al. 2014). This can explain the limited number of diarrhea cases observed in our study sample.

It is well known that G. intestinalis assemblages have a broad spectrum of hosts including domestic animals, and livestock (Sprong et al. 2009; Pestehchian et al. 2012; Skhal et al. 2017). Our data revealed that only assemblage A was associated with close contacts with domestic animals and birds.

In conclusion, determining the genetic base of G. intestinalis is a useful way to understand the infection route, to prevent infection effectively, and to reveal the critical issues in the molecular epidemiology of this parasite in Algeria. Further studies are recommended on a larger number of individuals as well as animals. In addition, more studies are required in other regions of Algeria in order to determine the distribution pattern of this parasite in the country.