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Occurrence and assemblage distribution of Giardia Duodenalis in symptomatic and asymptomatic patients in southeastern Iran (2019–2022)

Gut Pathogens volume 16, Article number: 68 (2024) Cite this article

Abstract

Background

The ubiquitous protozoan parasite Giardia duodenalis is a major contributor to the global burden of diarrhoea, particularly in young children living in poor-resource regions. Although rarely mortal, giardiasis is associated with growth retardation and cognitive impairment in early childhood. Here we investigate the epidemiology of human giardiasis in Iranshahr (south-eastern Iran), a region where this information was previously lacking.

Methods

Stool samples were collected from 17,455 outpatients and inpatients attended at three major hospital settings during April 2020 and March 2022. Microscopy was used as a screening method for the presence of Giardia cysts, and the identification of G. duodenalis assemblages was carried out using PCR and Sanger sequencing.

Results

The overall prevalence of giardiasis was 1.87 (326/17,455; 95% CI: 1.7–2.1). Being female was positively associated with higher odds of giardiasis (p = 0.014). Individuals without diarrhoea were less likely to have giardiasis (p = 0.022). Individuals attending the Iran Hospital were more likely to harbour G. duodenalis infections compared to those attending at the Khatam Hospital and the Clinical Reference Laboratory (p = 0.001). Our sequence analyses revealed the presence of assemblages A (56.5%, 13/23), B (39.1%, 9/23), and A + B (4.4%, 1/23). No association was observed between the occurrence of a given assemblage and the occurrence of diarhroea.

Conclusions

Giardia infections were found at relatively low prevalence rates in both symptomatic and asymptomatic individuals seeking medical attention. Being female, having diarrhoea, and being sampled during 2020–21 were predictors of giardiasis. Although limited, our molecular data indicate that some Giardia infections may be zoonotic in nature. These data should be corroborated and expanded in future epidemiological studies targeting simultaneously human, animal, and environmental (water) samples to improve our understanding of the epidemiology of giardiasis in Iran.

Background

Giardia duodenalis is a cosmopolitan enteric protozoan parasite which can cause gastroenteritis in a wide range of mammalian host species globally [1, 2]. In humans, G. duodenalis infections are estimated to cause 1.84 × 108 symptomatic cases annually and 171,100 disability adjusted life years [3]. The prevalence of human giardiasis has been reported to be 2–5% and 20–30% in developed and developing countries, respectively [4]. Young children in low-resource settings with little or no access to safe drinking water and sanitation are at higher risk of infection, particularly those living in tropical and subtropical regions [5, 6]. Early childhood giardiasis has been linked with a wide range of disorders and clinical manifestations including malnutrition, persistent diarrhoea, cognitive deficits, and stunting [7]. Other symptoms reported include nausea, anorexia, bloating, belching, abdominal pain, malaise, anaemia, steatorrhea, and weight loss [8]. Because of its close association with conditions of poverty, giardiasis (together with cryptosporidiosis) joined the ‘Neglected Diseases Initiative’ launched by the World Health Organization in 2004 [9]. Intriguingly, recent prospective, large cohort studies in endemic areas have found Giardia significantly more often in non-diarrhoeal than diarrhoeal infected children [10, 11]. These findings strongly suggest that G. duodenalis infection outcomes are likely the result of a multifactorial process involving nutritional, microbial, metabolic, and pathogen-strain variables [12,13,14].

Transmission of giardiasis occurs via the faecal-oral route, either by direct contact with an infected host or indirectly through ingestion of infective cysts in faecal-contaminated water or food [15]. Giardia duodenalis infections usually occur sporadically, although waterborne [16] and foodborne [17] outbreaks of gastrointestinal giardiasis are well documented globally. Based on the genetic variability of the small subunit ribosomal RNA (ssu rRNA) gene, G. duodenalis comprises eight (A to H) distinct genotypes (the so-called assemblages) with marked differences in host specificity and range [18, 19]. Humans are primarily infected by zoonotic assemblages A and B, although infections by canine- (C and D), ungulate- E, and feline-adapted (F) assemblages are also sporadically documented globally [2]. No human infections by G. duodenalis assemblage H have been reported to date. Assemblages A and B (particularly the latter) exhibit large genetic variations, allowing the categorization into sub-assemblages AI-AIII and BIII-BIV, respectively. AI and AIII are mostly restricted to infect non-human animal species, whereas AII, BIII, and BIV are regarded as zoonotic genetic variants [2, 20, 21]. AII within assemblage A and BIV within assemblage B are the predominant genetic variants circulating in humans [22, 23]. The most common genotyping tools for assessing intra-assemblage genetic variability are based on sequence analyses of the glutamate dehydrogenase (gdh), beta-giardin (bg) and triosephosphate isomerase (tpi) genes [2], although this multilocus sequence typing (MLST) scheme has limitations to resolve the molecular diversity within assemblage B isolates [24]. The G. duodenalis assemblage causing the infection has been investigated as a potential determinant of virulence/pathogenicity in large epidemiological studies, including matched case-control surveys, with uncertain results [25,26,27].

In Iran, the pooled prevalence of G. duodenalis infection at the community level has been estimated at 10.6% (95% CI: 9.6–11.5%) in a recent systematic review [28]. In symptomatic populations, G. duodenalis infections have been documented at variable rates including 2–19% in patients with diarrhoea [29,30,31], 39% in immunocompromised individuals [32, 33], and in 1–7% in HIV-positive patients [34,35,36]. Molecular-based studies aiming at investigating potential associations between the genetic variant of the parasite and diarrhoea generated contradictory data: assemblage A was more prevalent than assemblage B in patients with diarrhoea in some surveys [37], whereas the opposite [38] or inconclusive [39, 40] results were obtained in other studies.

In this study, we investigated G. duodenalis infection rate in symptomatic and asymptomatic patients attended at two large public hospitals in Iranshahr, Sistan and Baluchestan Province in southeastern Iran, a region where information on the epidemiology and public health relevance of the parasite was limited. Additionally, we investigated the molecular diversity of G. duodenalis in a subset of positive samples and assessed associations between genotypes and potential risk factors with the outcome of the infection.

Methods

Study design and population

This study was carried out in Iranshahr County, in the central area of the Sistan and Baluchestan province in southeastern Iran [41]. The latest census in 2016 showed a population of 113,750 people [42]. Iranshahr is one of the most socio-economically deprived areas of Iran. This is an observational cross-sectional study among outpatients and inpatients with and without gastrointestinal manifestations referred to three different health centers (Iran Hospital, Khatam Hospital, and Clinical Reference Laboratory) affiliated to Iranshahr University of Medical Sciences (IRSHUMS) in Iranshahr (Sistan and Baluchestan Province) for coproparasitological examination. The participants’ demographic and clinical data from Khatam and Iran Hospitals were gathered via the hospital information system. However, at the Clinical Reference Laboratory, due to the lack of a health information system, a short questionnaire was designed to collect similar information from participants referred to this health center. Individuals with no age restrictions with or without diarrhoea were included in the study. Patients who took standard intestinal parasite treatment were excluded from it. The study was conducted from April 2020 to March 2022 during the COVID-19 pandemic and included individuals with or without COVID-19. Volunteer asymptomatic individuals (mostly requesting a health card to access health care services) were also included in the study.

Sample size estimation

According to the formula (n = Z2 P (1 – P) / d2) [43], the minimum sample size needed for this study was estimated at ~ 16,000 individuals, considering a 95% confidence level (Z), a precision (d) < 0.0012, and a predicted prevalence (P) of human giardiasis of 0.6% [44].

Stool sample collection

A single stool sample was collected from each participant in a sterile plastic screw-topped specimen container labelled with a unique identifier code. Samples were transported in refrigerated containers to the Department of Parasitology and Mycology at IRSHUMS within 24–48 h of collection. Stool specimens improperly labelled or mixed with urine were discarded. Eventually, 17,455 stool specimens meeting the inclusion criteria were collected during the study period.

Data collection

At the time of specimen collection, the participant’s demographic (e.g., age, gender, health centre of origin) and clinical (e.g., diarrhoea) data were recorded together with the sampling date and the unique identifier code. Giardia infection was considered as the dependent variable, whereas age, gender, health centre of origin, occurrence of clinical manifestations, and sampling year were considered as the independent variables.

Macroscopic and microscopic examination

Stool samples were assessed macroscopically in terms of colour and consistency (1: formed, 2: soft, 3: loose, 4: liquid) at reception. Direct wet mount microscopy was used for the detection of G. duodenalis trophozoites and cysts in fresh stools. Briefly, one drop of saline (0.85% NaCl) was placed on a clean glass slide, mixed with a small amount of faecal material, and covered with a coverslip. Slides were immediately examined under light microscopy at 100× and 400× magnifications using an Eclipse E100 microscope (Nikon, Tokyo, Japan). Identification of cysts and trophozoites of Giardia was made according to standard bench aids [45]. Negative samples were not retested with additional conventional techniques, such as concentration methods or staining. Giardia-positive samples at microscopy examination were shipped to the Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences (Tehran) for downstream molecular testing. Stool samples were stored at 4 °C without preservatives until molecular analysis, for a maximum period of 20 weeks between sample collection and DNA extraction and purification.

DNA extraction and purification

Faecal DNA was isolated from about 250 mg of 28 randomly selected stool samples that were positive by microscopy using the FavorPrep™ Stool DNA Extraction Mini Kit (Favorgen, Ping Tung, Taiwan) according to the manufacturer’s instructions, except that a preliminary freeze/thaw cycle was added to help disrupting the cyst wall [46, 47]. Extracted and purified DNA samples were eluted in 60 µl of AE buffer and kept at − 20 °C until further PCR analyses.

Molecular characterization of G. duodenalis isolates

For assessing the molecular diversity of G. duodenalis isolates, we adopted a sequence-based multilocus genotyping (MLST) scheme targeting the genes encoding for the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) proteins of the parasite. Nested PCR protocols were used to amplify a 432-bp fragment of the gdh gene [48], a 511-bp fragment of the bg gene [49, 50], and a 530-bp fragments of the tpi gene [51]. Detailed information on the PCR cycling conditions and oligonucleotides used in these PCR protocols is presented in Additional file 1: Table S1 and Additional file 2 Table S2, respectively.

All PCR mixes (final volume: 25 µl) consisted of 12 µl of Taq 2× Master Mix RED (Ampliqon, Odense, Denmark), 10 pmol of each forward and reverse primer, and 2–5 µl of template DNA. PCR reactions were run on a Mastercycler Gradient 5331 machine (Eppendorf AG, Hamburg, Germany). Nuclease-free distilled water and laboratory-confirmed positive DNA samples of human origin were used as negative and positive control in all PCR runs, respectively. Separation of PCR products was done by electrophoresis on 1.5% agarose gels (Invitrogen™, Life Technologies GmbH, Leipzig, Germany) stained using SYBR® Safe DNA gel stain (Thermo Fisher Scientific, Waltham, MA, USA).

Sequence and phylogenetic analyses

PCR amplicons of the expected size were purified using the AccuPrep® PCR Purification Kit (Bioneer, Daejeon, South Korea) and send for sequencing in both directions using appropriate internal primer sets (Additional file 2 Table S2) to Bioneer Company (Daejeon, South Korea). DNA sequencing was conducted by capillary electrophoresis on an ABI 3730 automated DNA sequencer (Applied Biosystems, Foster City, CA USA). Obtained sequences were assembled and edited using Bioedit software version 7.2.6.1.

Generated nucleotide consensus sequences were compared with homologous sequences in GenBank using the Basic Local Alignment Search Tool (BLAST) to determine G. duodenalis assemblages and sub-assemblages. To confirm BLAST search results, multiple nucleotide sequence alignments were also carried out using the Clustal W algorithm in MEGA XI software [52].

Nucleotide sequences at the gdh, bg, and tpi markers generated in the present study and appropriate G. duodenalis reference nucleotide sequences obtained from GenBank were included to generate phylogenetic trees. Nucleotide sequences were aligned with the Clustal W algorithm using MEGA XI [52]. Phylogenetic analyses were performed using the Maximum Likelihood (ML) method and pairwise distances were calculated with the Tamura 3-parameter model using MEGA XI [52]. All ambiguous positions were removed for each sequence pair (pairwise deletion option). Bootstrapping with 1,000 replicates was used to determine support for the clades generated. Representative nucleotide sequences obtained in the present study were deposited in GenBank under the accession numbers OQ202298–OQ202307 (gdh marker), OQ202284–OQ202297 (bg marker), and OQ202308–OQ202323 (tpi marker).

Statistical analysis

Statistical analysis was performed with SPSS® Statistics for Windows software, version 25 (IBM Corp., Armonk, N.Y., USA). The participants’ baseline data were summarized as frequencies or percentage into categorical variables. Binomial logistic regression analysed the crude and adjusted associations between G. duodenalis infection and determinants [53]. The association strength between outcome and risk factors was evaluated using the adjusted odds ratio (aOR) and the corresponding 95% confidence interval (95% CI). A probability value < 0.05 was regarded as statistically significant.

Results

Demographic profile

Table 1 summarizes the main demographic and clinical features of the population under study. A total of 17,455 stool samples were collected from diarrhoeal (29.7%, 5,187/17,455) and non-diarrhoeal (70.3%, 12,268/17,455) patients seeking medical attention in three clinical settings affiliated with IRSHUMS: Iran Hospital (n = 5,720), Khatam Hospital (n = 628), and Clinical Reference Laboratory (n = 11,107). The male/female ratio was 3.7. The age of the recruited patients ranged from two days to 100 years (median: 60.0 months; standard deviation: 150.3 months). Young children under seven years of age accounted for 32.4% (5,663/17,455) of the investigated clinical population. Most stool samples (36.9%, 6,446/17,455) were collected during the winter months of 2020–21 and 2021–22.

Table 1 Main demographic and clinical features of the surveyed symptomatic and asymptomatic populations investigated in the present study
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Prevalence of G. duodenalis infection

The overall, microscopy-based prevalence of G. duodenalis infection was 1.87% [326 of 17,455; 95% Confidence Interval (CI): 1.7–2.1]. The higher infection rate was observed in patients from Iran Hospital (3.44%, 197 of 5,720; 95% CI: 3.0–4.0) followed by patients from Khatam Hospital (1.59%, 10 of 628; 95% CI: 0.77–2.91) and Clinical Reference Laboratory (1.07%, 119 of 11,107; 95% CI: 0.89–1.28).

Giardia duodenalis infections were more frequently detected in female (3.0%, 113 of 3,738) than in male (1.6%, 213 of 13,717) patients, regardless of the clinical setting of origin (Table 2), even though females accounted for nearly one out of four patients participating in the survey. Overall, children under the age of seven harboured the highest G. duodenalis infection rates (3.2%, 180 of 5,663). This trend was true for all hospital settings except for Iran Hospital, where the parasite was more common in individuals over seven years of age (5.0%, 26/525) than in children younger than that age (3.3%, 171 of 5,195) (Table 2). Giardia duodenalis was more frequently detected in patients presenting with diarrhoea (2.8%, 143 of 5,187) than in those without it (1.5%, 183 of 12,268), although the opposite trend (2.8% vs. 5.3%) was observed in patients recruited from Iran Hospital (Table 2). The occurrence of the parasite was similar in the two sampling periods considered, being higher in the 2020–2021 campaign (2.1%, 163 of 7,870) than in the 2021–2022 campaign (1.7%, 163 of 9,585). The opposite trend was observed (3.1% vs. 3.7%) for participating patients from Iran Hospital (Table 2).

Table 2 Frequency distribution of Giardia duodenalis infection by hospital setting according to gender, age group, presence/absence of diarrhoea, and sampling period of the cohort of patients investigated in the present study. 95% confidence intervals (CI) are indicated
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Detailed information on the temporal distribution of G. duodenalis cases according to gender and age group or gender and presence/absence of diarrhoea can be found in Additional file 3 Table S3 and Additional file 4 Table S4, respectively. The average positive rate was higher during the spring-summer months (2.1%, 124 of 5,894) than in the autumn-winter months (1.7%, 202 of 11,561). The highest monthly positive rate of infection was recorded in June 2020 (3.7%, 8 of 219), while the lowest positive rate was recorded in November 2021 (0.7%, 6 of 813).

Risk association analysis: univariate analysis

Results of the univariate analysis are summarised in Table 3. Being female increased the odds of G. duodenalis infection (OR: 1.97; 95% CI: 1.55–2.50). Children younger than seven years of age had significantly higher odds of being infected with the parasite (OR: 2.61; 95% CI: 2.09–3.26). Individuals without diarrhoea were less likely to have giardiasis (OR: 0.53; 95% CI: 0.42–0.67). In contrast, the occurrence of the protozoon was similarly distributed through the whole period of the study irrespectively of the sampling year and season. Finally, patients attended at the Iran Hospital were more likely to harbour G. duodenalis infections (OR: 3.21; 95% CI: 2.55–4.05).

Table 3 Univariate analysis of factors associated with Giardia duodenalis infection in the cohort of patients investigated in the present study. Crude odds ratios (OR) and 95% confidence intervals (95% CI) are indicated. Statistically significant values are bolded
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Risk association analysis: multivariate analysis

Results of the multivariate analysis are summarised in Table 4. In the multivariate model, factors that significantly increased the odds of G. duodenalis infection were being female (aOR: 1.36; 95% CI: 1.06–1.74), and being attended at the Iran Hospital (aOR: 3.26; 95% CI: 2.11–5.03). The odds of infection among people without diarrhoea were approximately 33% lower than those with diarrhoea (aOR: 0.67; 95% CI: 0.48–0.94).

Table 4 Multivariate analysis of factors associated with Giardia duodenalis infection in the cohort of patients investigated in the present study. Statistically significant values are bolded
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Molecular characterization of G. duodenalis

We assessed the genetic diversity of 28 randomly selected Giardia-positive samples by conventional microscopy using a MLST scheme based on the amplification of three (gdh, bg, and tpi) genetic markers. Successful PCR amplifications and sequencing data were generated for 35.7% (10/28, gdh), 50.0% (14/28, bg), and 57.1% (16/28, tpi) of the samples investigated at the three loci (Table 5). Overall, 82.1% (23/28) of the Giardia-positive samples were successfully genotyped at one locus at least. MLST data at the three assessed loci was available for six samples (21.4%, 6/28). Subtyping data at a single locus and two loci were available for 42.9% (12/28) and 17.9% (5/28) of samples, respectively. No genotyping data could be obtained for 17.9% (5/28) of the Giardia-positive samples. Assemblage A (56.5%, 13/23) was more prevalent than assemblage B (39.1%, 9/23), while A + B mixed infections were detected in a single isolate (4.4%, 1/23). No host-adapted assemblages of canine (C, D), feline (F), or livestock (E) origin were identified circulating in the surveyed clinical population (Table 5). Overall, 53.9% (7/13) of the patients infected by assemblage A had diarrhoea, while this figure was 44.4% (4/9) for patients infected with assemblage B (OR: 1.46; 95% CI: 0.26–8.05).

Table 5 Multilocus sequence typing results of the 28 Giardia duodenalis-positive samples randomly selected for genotyping purposes in the present survey. Main demographic (gender, age) and clinical (hospital of origin, diarrhoea) data of the patients are indicated. GenBank accession number are provided
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Table 6 shows the frequency and molecular diversity of G. duodenalis at the gdh, bg, and tpi loci. Out of the 10 gdh sequences, five (50.0%) were assigned to the sub-assemblage AII, all of them showing 100% identity with reference sequence L40510. One isolate was identified as sub-assemblage BIII and showed 100% identity with reference sequence AF069059. The remaining four sequences belonged to sub-assemblage BIV and differed by 4–8 single nucleotide polymorphisms (SNPs) from reference sequence L40508. No ambiguous (double peak) positions were observed during chromatogram inspection.

Table 6 Frequency and molecular diversity of G. Duodenalis isolates identified at the Bg, gdh, and Tpi loci in the clinical population under study. GenBank accession numbers are provided
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Out of the 14 bg sequences generated in the present study, one was identified as sub-assemblage AII, being identical to reference sequence AY072723. A total of six sequences were assigned to the sub-assemblage AIII of the parasite. Of them, five showed 100% identity with reference sequence AY072724, while the remaining one differed from it by a single SNP. The seven sequences identified as assemblage B differed by 1–3 SNPs from reference sequence AY072727. As in the case of the gdh locus, all SNPs detected corresponded to clear mutations without evidence of double peaks at chromatogram inspection. All seven assemblage B sequences have a distinctive nucleotide (A) deletion in position 137 of reference sequence AY072727.

Out of the 16 sequences successfully analysed at the tpi locus, 11 were identified as sub-assemblage AII and differed by a single SNP (always a C to A mutation at position 794) of reference sequence U57897. Two isolates were assigned to sub-assemblage BIII. One of them being identical to reference sequence AF069561 and the other one differing form it by a single SNP. Two isolates belonged to sub-assemblage BIV and differed from reference sequence AF069560 by 2–3 SNPs. None of the SNPs detected corresponded to ambiguous double peak) positions. An additional isolate was identified as assemblage B due to the impossibility of clearly assign it to sub-assemblages BIII or BIV.

Phylogenetic analyses conducted at the gdh (Fig. 1), bg (Fig. 2) and tpi (Fig. 3) loci clearly showed that our G. duodenalis sequences formed well-supported clades with appropriate reference sequences retrieved from GenBank.

Fig. 1
figure 1

Phylogenetic relationship among Giardia duodenalis assemblages and sub-assemblages revealed by a maximum likelihood analysis of the partial gdh rDNA gene. Substitution rates were calculated by using the general time reversible model. Numbers on branches are percent bootstrapping values over 70% using 1,000 replicates. The red human silhouettes indicate the nucleotide sequences generated in the present study. The filled red, blue, black, and green circles indicate the nucleotide sequences of sub-assemblages AI, AII, BIII, and BIV, respectively. The world map logos indicate reference sequences. Human, animal, and environmental sequences retrieved from GenBank were included in the analysis for comparative purposes

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Fig. 2
figure 2

Phylogenetic relationship among Giardia duodenalis assemblages and sub-assemblages revealed by a maximum likelihood analysis of the partial bg rDNA gene. Substitution rates were calculated by using the general time reversible model. Numbers on branches are percent bootstrapping values over 70% using 1,000 replicates. The red human silhouettes indicate the nucleotide sequences generated in the present study. The filled red, blue, and yellow circles indicate the nucleotide sequences of sub-assemblages AI, AII, and AIII, respectively. The world map logos indicate reference sequences. Human, animal, and environmental sequences retrieved from GenBank were included in the analysis for comparative purposes

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Fig. 3
figure 3

Phylogenetic relationship among Giardia duodenalis assemblages and sub-assemblages revealed by a maximum likelihood analysis of the partial tpi rDNA gene. Substitution rates were calculated by using the general time reversible model. Numbers on branches are percent bootstrapping values over 70% using 1,000 replicates. The red human silhouettes indicate the nucleotide sequences generated in the present study. The filled red, blue, black, and green circles indicate the nucleotide sequences of sub-assemblages AI, AII, BIII, and BIV, respectively. The world map logos indicate reference sequences. Human, animal, and environmental sequences retrieved from GenBank were included in the analysis for comparative purposes

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Discussion

Despite sustained effort to improve the performance indicators of health assistance and quality of life in Iran, shortcomings in sanitation infrastructures (i.e., public toilets, wastewater treatment facilities) remain common particularly in the southeastern regions of the country. Although overall prevalence rates of parasitosis in Iran have declined significantly over the last few decades, giardiasis by G. duodenalis remains among the most common infections diagnosed [54]. The overall prevalence of giardiasis in Iran was estimated at 14.7% in the last systematic review available [55]. However, this figure should be interpreted with caution, as most of the studies considered in the associated meta-analysis were based on relatively small sample sizes and focused on symptomatic populations, where the prevalence differs between asymptomatic and symptomatic patients. To overcome this limitation, we present here giardiasis prevalence data among outpatients and inpatients with and without gastrointestinal manifestations referred to medical centres in Iranshahr (Sistan and Baluchestan Province). No previous studies have evaluated the presence of human giardiasis in this Iranian region.

We found a microscopy-based Giardia infection rate of 1.87%. This figure is very likely an underestimation of the true prevalence value for several reasons including (i) the limited diagnostic sensitivity of microscopy due to discontinuous cyst shedding in the stools of infected individuals and the low parasite burdens typically found in asymptomatic carriers, (ii) motile trophozoites may go undetected if wet mounts are not examined quickly after preparation [56], and (iii) misdiagnosis due to the limited skill of laboratory personnel and microscopists [57]. The prevalence of infection in this study was very close to the results of some other large-scale studies conducted on symptomatic patients visiting medical centers in Ahvaz (southwestern Iran) (1.77%, 887/50,000) [58], Karaj (north-central Iran) (3.84%, 534/13,915) [59], Shiraz (southwestern Iran) (0.53%, 73/13,686) [44], Rasht (north-central Iran) (2.47%, 206/8,356) [60], and Yazd (0.93%, 376/40,351) [61]. In addition, our result was in contrast with those obtained from previous small-scale studies conducted in Mazandaran (north-central Iran) (10.18%, 87/855) [62], Khuzestan (southwestern Iran) (10.9%, 163/1,494) [63], Shiraz (southwestern Iran) (10.7%, 107/1,000) [54], Kerman (southeastern Iran) (10.6%, 92/861) [64], Yasuj (southwestern Iran) (17.46%, 179/1,025) [65], Kamyaran (northwestern Iran) (11.0%, 22/245) [66], and Shushtar County (southwestern Iran) (7.74%, 90/1,163) [67]. The discrepancies in Giardia infection rates reported in the above-mentioned studies are likely associated with differences in (i) techniques used for detection purposes, (ii) sources of infection (i.e., drinking water quality, contact with domestic animals or individuals with diarhroea), (iii) personal hygiene practices (i.e., hand washing after defecation), (iv) access to sanitation infrastructures (i.e., toilets), and (v) the socio-economic status of the investigated communities. In the only previously report conducted in southeast Iran, a G. duodenalis infection rate of 10.12% (158/1,562) was observed in patients with gastrointestinal complaints in Zahedan, the northern part of Sistan and Baluchestan Province [68]. This figure is five-fold higher than that (1.87%) found in the present study. The difference may be attributed to the fact that, despite Zahedan City being larger and having more urban facilities than Iranshahr, it is less ethnically homogeneous due to the presence of many unauthorized foreign nationals with poor hygiene lifestyles.

Individuals who attended Iran Hospital had a higher rate of infection compared to those at the Khatam Hospital or the Clinical Reference Laboratory. This result can be explained considering that 90.82% (5,195/5,720) of the patients attended at the Iran Hospital were children ≤ 7 years of age, the age group more vulnerable to giardiasis. This result was confirmed by univariate statistical analyses (OR: 2.61; 95% CI: 2.09–3.26) and is in line with previous studies published in Iran [64, 65, 69, 70] and other Asian countries including Malaysia [71] and Pakistan [72] showing that young age is a predisposing factor to giardiasis. Increased exposure to sources of infection and poor personal hygiene have been suggested as the main factors explaining higher prevalence of Giardia infections in young children [71, 73,74,75].

Univariate statistical analyses also demonstrated a significant association between G. duodenalis infection and diarrhoea (OR: 0.53; 95% CI: 0.42–0.67). Similar findings have been reported in other surveys conducted in Cuba [74], Iran [29], Malaysia [71], and South Africa [76]. Remarkably, some studies have shown an inverse correlation between Giardia infection in children and diarrhoea [77,78,79]. This may be the result of an acquired protective immunity in response to regular infection and re-infection events in endemic areas or of a protective effect of Giardia colonization against other diarrhoea-causing intestinal pathogens [80]. Indeed, findings from prospective longitudinal cohort studies conducted in sub-Saharan African countries concluded that Giardia was not an independent risk factor for diarrhoea in children [10, 11, 81].

Being female was a significant risk for Giardia infection both in the present univariate (OR: 1.97; 95% CI: 1.55–2.50) and multivariate (aOR: 1.36; 95% CI: 1.06–1.74) analyses. This finding is in line with earlier studies conducted in Iran [82], Ethiopia [83], and South Africa [76], which reported a higher prevalence of Giardia infection in females compared to males. In contrast, males were more likely to have giardiasis in previous studies conducted in Iran [37, 65, 84, 85], Malaysia [75], and India [86]. However, Giardia distribution was gender-independent in most studies conducted in Iran [29, 59, 60, 62, 63, 68,69,70, 87] and other Asian countries including Malaysia [71], Pakistan [72], and Tajikistan [88]. This variation of exposure among the different sex groups may be attributed to differences in exposure levels across various communities and study areas.

A seasonal pattern of Giardia infections was identified, with a peak occurring from July to September. Similar trends have also been reported in other studies [60, 89, 90], coinciding with increased outdoor activities, particularly at the beginning of the school year.

The molecular diversity of G. duodenalis was investigated in 28 microscopy-positive samples at three markers (gdh, bg, and tpi) commonly used for genotyping and sub-genotyping purposes. Assemblage A was found to be more prevalent than assemblage B (56.5% vs. 39.1%) in the subset of samples investigated, with mixed infections A + B also detected at lower rates (4.4%). The predominance of assemblage A over assemblage B is the norm in most molecular studies conducted previously in Iran in patients with intestinal disorders (68.1% vs. 31.9%) [91], patients with and without gastrointestinal symptoms (92.6% vs. 7.4%) [60], food handlers (65.0% vs. 35.0%) [92], individuals with HIV or cancer (81.8% vs. 18.2%) [93], and samples submitted for laboratory testing (59.7% vs. 37.3%) [37], among others. In our recent study on 200 domestic animals in Iranshahr [10], we found that 19.3% (17/88) of cattle and 6.7% (2/30) of camels tested positive by qPCR, with one cattle isolate successfully genotyped as G. duodenalis assemblage B (accession no. PQ139658), displaying 99.46% sequence identity with a human isolate (LC184469) from Tehran (unpublished data). This suggests that an unknown proportion of human giardiasis cases detected in the present survey may be zoonotic in nature. Of note, assemblages A (37.5%) and E (58.3%) have been identified in slaughterhouse wastewaters in Tehran [94], strongly suggesting that untreated waters can be an important source of environmental contamination with Giardia cysts. Regarding pathogenicity, we did not find a clear association between the presence of a given Giardia assemblage and the occurrence of clinical manifestations. Information from large epidemiological surveys investigating children with and without diarrhoea and matched by age and sex revealed unclear results. Whereas diarrhoea was not associated to a given G. duodenalis assemblage in Mozambique [26], assemblage A was more prevalently found in children with diarrhoea in Bangladesh [25].

The main strengths of this study include (i) the reporting of epidemiological data on human giardiasis from a geographical region in Iran where this information was previously lacking, (ii) the large sample size used and the coverage of three main medical centres in the Southeast of Iran, and (iii) the assessment of the impact of the COVID-19 restrictions on the epidemiology of G. duodenalis. The study has also limitations that might have compromised the accuracy of some of the results obtained. First, the limited diagnostic sensitivity of microscopy (together with the analysis of a single stool sample per patient recruited) suggest that the prevalence data presented here is an underestimation of the true one. Second, negative samples examined with direct smear were not subjected to further testing using conventional techniques (e.g., concentration and staining methods). Third, the associated metadata available was restricted to basic variables, so we were unable to assess the impact of important factors (i.e., drinking water source, sanitary and hygiene habits, latrine availability) that could be predictors of Giardia infection. Fourth, long-term storage of faecal samples (up to 12 weeks) at 4 °C might have affected (i) the morphology of Giardia cysts, impacting the diagnostic performance of conventional microscopy, and (ii) the quality and quantity of parasitic DNA, compromising the performance of the genotyping PCR tools. Lastly, due to financial constraints, molecular analyses were only feasible for a limited number of samples, so the observed frequency and diversity of assemblages/sub-assemblages might not be representative of the one present at the whole population level.

Conclusion

This is the first study (i) investigating the epidemiology of giardiasis in south-eastern Iran, and (ii) systematically assessing risk factors potentially associated with higher odds of Giardia infection in Iran. Giardia infections were found at relatively low prevalence rates in both symptomatic and asymptomatic individuals seeking medical attention in Iranshahr. Being female and having diarrhoea were predictors of giardiasis. Individuals attended the Iran Children’s Hospital were more likely to have giardiasis. Infections were caused by both assemblages A and B, with a higher frequency of A than B. Although limited, our molecular data indicate that an unknown proportion of Giardia infections may be zoonotic. These data should be corroborated and expanded in future epidemiological studies targeting simultaneously human, animal, and environmental (water) samples to improve our understanding of the epidemiology of giardiasis in Iran.

Data availability

The data that support the findings of this study are available from the First author upon reasonable request.

Abbreviations

aOR:

Adjusted odds ratio

bg :

Beta-giardin

BLAST:

Basic local alignment search tool

COVID-19:

Coronavirus Disease 2019

DNA:

Deoxyribonucleic acid

gdh :

Glutamate dehydrogenase

HIV:

Human immunodeficiency virus

IRSHUMS:

Iranshahr University of Medical Sciences

ML:

Maximum likelihood

MLST:

Multilocus sequence typing

PCR:

Polymerase chain reaction

ssu :

rRNA Small subunit ribosomal RNA

tpi :

Triosephosphate isomerase

95% CI:

95% Confidence interval

References

  1. Ryan U, Cacciò SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43(12–13):943–56. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijpara.2013.06.001.

    Article  CAS  PubMed  Google Scholar 

  2. Ryan UM, Feng Y, Fayer R, Xiao L. Taxonomy and molecular epidemiology of Cryptosporidium and Giardia - a 50 year perspective (1971–2021). Int J Parasitol. 2021;51:13–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijpara.2021.08.007.

    Article  CAS  Google Scholar 

  3. Kirk MD, Pires SM, Black RE, Caipo M, Crump JA, Devleesschauwer B, et al. World Health Organization estimates of the Global and Regional Disease Burden of 22 foodborne bacterial, protozoal, and viral diseases, 2010: a data synthesis. PLoS Med. 2015;12(12):e1001921. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pmed.1001921.

    Article  PubMed  PubMed Central  Google Scholar 

  4. Fletcher SM, Stark D, Harkness J, Ellis J. Enteric protozoa in the developed world: a public health perspective. Clin Microbiol Rev. 2012;25(3):420–49. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/CMR.05038-11.

    Article  PubMed  PubMed Central  Google Scholar 

  5. Mmbaga BT, Houpt ER. Cryptosporidium and Giardia infections in chilaren: A review. Pediatr Clin North Am. 2017;64(4):837–50. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.pcl.2017.03.014.

    Article  PubMed  Google Scholar 

  6. Squire SA, Ryan U. Cryptosporidium and Giardia in Africa: current and future challenges. Parasit Vectors. 2017;10(1):195. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13071-017-2111-y.

    Article  PubMed  PubMed Central  Google Scholar 

  7. Dougherty M, Bartelt LA. Giardia and growth impairment in children in high-prevalence settings: consequence or co-incidence? Curr Opin Infect Dis. 2022;35(5):417–23. https://doiorg.publicaciones.saludcastillayleon.es/10.1097/qco.0000000000000877.

    Article  PubMed  PubMed Central  Google Scholar 

  8. Einarsson E, Ma’ayeh S, Svärd SG. An up-date on Giardia and giardiasis. Curr Opin Microbiol. 2016;34:47–52. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mib.2016.07.019.

    Article  PubMed  Google Scholar 

  9. Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium join the ‘Negldiseasesseases Initiative’. Trends Parasitol. 2006;22(5):203–8. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.pt.2006.02.015.

    Article  CAS  PubMed  Google Scholar 

  10. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013;382(9888):209–22. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/s0140-6736(13)60844-2.

    Article  PubMed  Google Scholar 

  11. Platts-Mills JA, Babji S, Bodhidatta L, Gratz J, Haque R, Havt A, et al. Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED). Lancet Glob Health. 2015;3(9):e564–75. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S2214-109X(15)00151-5.

    Article  PubMed  PubMed Central  Google Scholar 

  12. Rogawski ET, Bartelt LA, Platts-Mills JA, Seidman JC, Samie A, Havt A, Network MAL-ED, Investigators et al. 2017. Determinants and impact of Giardia infection in the first 2 years of life in the MAL-ED birth cohort. J Pediatric Infect Dis Soc. 2017;6(2):153–60. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/jpids/piw082

  13. Singer SM, Angelova VV, DeLeon H, Miskovsky E. What’s eating you? An update on Giardia, the microbiome and the immune response. Curr Opin Microbiol. 2020;58:87–92. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mib.2020.09.006.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Donowitz JR, Pu Z, Lin Y, Alam M, Ferdous T, Shama T, et al. Small intestine bacterial overgrowth in Bangladeshi infants is associated with growth stunting in a longitudinal cohort. Am J Gastroenterol. 2022;117(1):167–75. https://doiorg.publicaciones.saludcastillayleon.es/10.14309/ajg.0000000000001535.

    Article  PubMed  Google Scholar 

  15. Adam RD. Giardia duodenalis: Biology and pathogenesis. Clin Microbiol Rev. 2021;34(4):e0002419. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/cmr.00024-19.

    Article  CAS  PubMed  Google Scholar 

  16. Ma JY, Li MY, Qi ZZ, Fu M, Sun TF, Elsheikha HM, et al. Waterborne protozoan outbreaks: an update on the global, regional, and national prevalence from 2017 to 2020 and sources of contamination. Sci Total Environ. 2022;806(Pt 2):150562. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.scitotenv.2021.150562.

    Article  CAS  PubMed  Google Scholar 

  17. Ryan U, Hijjawi N, Feng Y, Xiao L. Giardia: an under-reported foodborne parasite. Int J Parasitol. 2019;49(1):1–11. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijpara.2018.07.003.

    Article  PubMed  Google Scholar 

  18. Weiss JB, van Keulen H, Nash TE. Classification of subgroups of Giardia lamblia based upon ribosomal RNA gene sequence using the polymerase chain reaction. Mol Biochem Parasitol. 1992;54(1):73–86. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/0166-6851(92)90096-3.

    Article  CAS  PubMed  Google Scholar 

  19. Hopkins RM, Meloni BP, Groth DM, Wetherall JD, Reynoldson JA, Thompson RC. Ribosomal RNA sequencing reveals differences between the genotypes of Giardia isolates recovered from humans and dogs living in the same locality. J Parasitol. 1997;83(1):44–51. https://doiorg.publicaciones.saludcastillayleon.es/10.2307/3284315.

    Article  CAS  PubMed  Google Scholar 

  20. Cai W, Ryan U, Xiao L, Feng Y. Zoonotic giardiasis: an update. Parasitol Res. 2021;120(12):4199–218. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00436-021-07325-2.

    Article  PubMed  Google Scholar 

  21. Dixon BR. GiDuodenalisenalis in humans and animtransmissionission and disease. Res Vet Sci. 2021;135:283–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.rvsc.2020.09.034.

    Article  CAS  PubMed  Google Scholar 

  22. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24(1):110–40. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/cmr.00033-10.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Ryan U, Zahedi A. Molecular epidemiology of giardiasis from a veterinary perspective. Adv Parasitol. 2019;106:209–54. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/bs.apar.2019.07.002.

    Article  PubMed  Google Scholar 

  24. Seabolt MH, Konstantinidis KT, Roellig DM. Hidden diversity within common protozoan parasites as revealed by a novel genotyping scheme. Appl Environ Microbiol. 2021;87(6):e02275–20. https://doiorg.publicaciones.saludcastillayleon.es/10.1128/aem.02275-20.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Haque R, Mondal D, Karim A, Molla IH, Rahim A, Faruque AS, et al. Prospective case-control study of the association between common enteric protozoal parasites and diarrhea in Bangladesh. Clin Infect Dis. 2009;48(9):1191–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1086/597580.

    Article  CAS  PubMed  Google Scholar 

  26. Messa A Jr, Köster PC, Garrine M, Gilchrist C, Bartelt LA, Nhampossa T, et al. Molecular diversity of Giardia Duodenalis in children under 5 years from the Manhiça district, Southern Mozambique enrolled in a matched case-control study on the aetiology of diarrhoea. PLoS Negl Trop Dis. 2021;15(1):e0008987. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pntd.0008987.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Köster PC, Malheiros AF, Shaw JJ, Balasegaram S, Prendergast A, Lucaccioni H, et al. Multilocus genotyping of Giardia Duodenalis in mostly asymptomatic indigenous people from the Tapirapé tribe, Brazilian Amazon. Pathogens. 2021;10(2):206. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/pathogens10020206.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Mohebali M, Keshavarz H, Abbaszadeh Afshar MJ, Hanafi-Bojd AA, Hassanpour G. Spatial distribution of common pathogenic human intestinal protozoa in Iran: a systematic review. Iran J Public Health. 2021;50(1):69–82. https://doiorg.publicaciones.saludcastillayleon.es/10.18502/ijph.v50i1.5073.

    Article  PubMed  PubMed Central  Google Scholar 

  29. Kiani H, Haghighi A, Rostami A, Azargashb E, Tabaei SJ, et al. Prevalence, risk factors and symptoms associated to intestinal parasite infections among patients with gastrointestinal disorders in Nahavand, western Iran. Rev Inst Med Trop Sao Paulo. 2016;58:42. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/s1678-9946201658042.

    Article  PubMed  PubMed Central  Google Scholar 

  30. Karami S, Beiromvand M, Kohansal K. Prevalence of intestinal parasitic infections among diarrheic patients from Ahvaz, southwestern Iran. Infect Disord Drug Targets. 2021;21(6):e170721188043. https://doiorg.publicaciones.saludcastillayleon.es/10.2174/1871526520999201116202411.

    Article  CAS  PubMed  Google Scholar 

  31. Abbasi E, van Belkum A, Ghaznavi-Rad E. Common etiological agents in adult patients with gastroenteritis from Central Iran. Microb Drug Resist. 2022;28(11):1043–55. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/mdr.2021.0177.

    Article  CAS  PubMed  Google Scholar 

  32. Mamishi S, Eghbali AN, Rezaei N, Abolhassani H, Parvaneh N, Aghamohammadi A. A single center 14 years study of infectious complications leading to hospitalization of patients with primary antibody deficiencies. Braz J Infect Dis. 2010;14(4):351–5. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S1413-8670(10)70074-X.

    Article  PubMed  Google Scholar 

  33. Parvaneh L, Sharifi N, Azizi G, Abolhassani H, Sharifi L, Mohebbi A, et al. Infectious etiology of chronic diarrhea in patients with primary immunodeficiency diseases. Eur Ann Allergy Clin Immunol. 2019;51(1):32–7. https://doiorg.publicaciones.saludcastillayleon.es/10.23822/eurannaci.1764-1489.77.

    Article  CAS  PubMed  Google Scholar 

  34. Zali MR, Mehr AJ, Rezaian M, Meamar AR, Vaziri S, Mohraz M. Prevalence of intestinal parasitic pathogens among HIV-positive individuals in Iran. Jpn J Infect Dis. 2004;57(6):268–70.

    Article  PubMed  Google Scholar 

  35. Agholi M, Hatam GR, Motazedian MH. HIV/AIDS-associated opportunistic protozoal diarrhea. AIDS Res Hum Retroviruses. 2013;29(1):35–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1089/aid.2012.0119.

    Article  PubMed  PubMed Central  Google Scholar 

  36. Masoumi-Asl H, Khanaliha K, Bokharaei-Salim F, Esteghamati A, Kalantari S, Hosseinyrad M. Enteric opportunistic infection and the impact of antiretroviral therapy among HIV/AIDS patients from Tehran, Iran. Iran J Public Health. 2019;48(4):730–9.

    PubMed  PubMed Central  Google Scholar 

  37. Pestechian N, Rasekh H, Rostami-Nejad M, Yousofi HA, Hosseini-Safa A. Molecular identification of Giardia lamblia; is there any correlation between diarrhea and genotyping in Iranian population? Gastroenterol Hepatol Bed Bench. 2014;7(3):168–72.

    PubMed  PubMed Central  Google Scholar 

  38. Viesy S, Abdi J, Haghani K, Valizadeh R, Mirzaei A. Giardia lamblia subtypes and their relationship with clinical symptoms in patients with giardiasis. Infect Disord Drug Targets. 2020;20(3):396–400. https://doiorg.publicaciones.saludcastillayleon.es/10.2174/1871526519666190314094437.

    Article  CAS  PubMed  Google Scholar 

  39. Rafiei A, Roointan ES, Samarbafzadeh AR, Shayesteh AA, Shamsizadeh A, Pourmahdi Borujeni M. Investigation of possible correlation between Giardia duodenalis genotypes and clinical symptoms in southwest of Iran. Iran J Parasitol. 2013;8(3):389–95.

    PubMed  PubMed Central  Google Scholar 

  40. Kashinahanji M, Haghighi A, Bahrami F, Fallah M, Saidijam M, Matini M, et al. Giardia lamblia assemblages A and B isolated from symptomatic and asymptomatic persons in Hamadan, west of Iran. J Parasit Dis. 2019;43(4):616–23. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12639-019-01139-x.

    Article  PubMed  PubMed Central  Google Scholar 

  41. Hatam-Nahavandi K, Carmena D, Rezaeian M, Mirjalali H, Mohammad Rahimi H, Badri M, et al. Gastrointestinal parasites of domestic mammalian hosts in Southeastern Iran. Vet Sci. 2023;10(4):261. https://doiorg.publicaciones.saludcastillayleon.es/10.3390/vetsci10040261.

    Article  PubMed  PubMed Central  Google Scholar 

  42. Census of the Islamic Republic of Iran. The Statistical Center of Iran. 2016. https://www.amar.org.ir/english. Accessed 1 January 2024.

  43. Lwanga SK, Lemeshow D. Sample size determination in health studies: A practical manual. World Health Organization. 1991. https://apps.who.int/iris/handle/10665/40062. Accessed 1 January 2024.

  44. Teimouri A, Alimi R, Farsi S, Mikaeili F. Intestinal parasitic infections among patients referred to hospitals affiliated to Shiraz University of Medical Sciences, southern Iran: a retrospective study in pre- and post-COVID-19 pandemic. Environ Sci Pollut Res. 2022;29(24):36911–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11356-021-18192-w.

    Article  CAS  Google Scholar 

  45. World Health Organization. Bench aids for the diagnosis of intestinal parasites. World Health Organization. 1994. http://apps.who.int/iris/bitstream/handle/10665/37323/9789241544764_eng.pdf?sequence=1. Accessed 1 January 2024.

  46. Hatam-Nahavandi K, Mohebali M, Mahvi AH, Keshavarz H, Najafian HR, Mirjalali H, et al. Microscopic and molecular detection of Cryptosporidium andersoni and Cryptosporidium Xiaoi in wastewater samples of Tehran province, Iran. Iran J Parasitol. 2016;11(4):499–506.

    PubMed  PubMed Central  Google Scholar 

  47. Fallah E, Mahdavi-poor B, Jamali R, Hatam-Nahavandi K, Asgharzadeh M. Molecular characterization of Cryptosporidium isolates from cattle in a slaughterhouse in Tabriz, northwestern Iran. J Biol Sci. 2008;8(3):639–43. https://doiorg.publicaciones.saludcastillayleon.es/10.3923/jbs.2008.639.643.

    Article  CAS  Google Scholar 

  48. Park YJ, Cho HC, Jang DH, Park J, Choi KS. Multilocus genotyping of Giardia Duodenalis in pre-weaned calves with diarrhea in the Republic of Korea. PLoS ONE. 2023;18(1):e0279533. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0279533.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Cacciò SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 2002;32(8):1023–30. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/s0020-7519(02)00068-1.

    Article  PubMed  Google Scholar 

  50. Lalle M, Pozio E, Gapelli G, Bruschi F, Crotti D, Caccio SM. Genetic heterogeneity at the beta-giardin locus among human and animal isolates of Giardia Duodenalis and identification of potentially zoonotic subgenotypes. Int J Parasitol. 2005;35(2):207–21. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.ijpara.2004.10.022.

    Article  CAS  PubMed  Google Scholar 

  51. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia Duodenalis. Emerg Infect Dis. 2003;9(11):1444–52. https://doiorg.publicaciones.saludcastillayleon.es/10.3201/eid0911.030084.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Tamura K, Stecher G, Kumar S. MEGA11: Molecular Evolutionary Genetics Analysis Version 11. Mol Biol Evol. 2021;38(7):3022–27. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/molbev/msab120.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  53. Hosmer DW Jr, Lemeshow S, Sturdivant RX. Applied logistic regression. New York: Wiley; 2013.

    Book  Google Scholar 

  54. Rayani M, Zasmy Unyah N, Hatam G. Molecular identification of Giardia Duodenalis isolates from Fars Province, Iran. Iran J Parasitol. 2014;9(1):70–8.

    CAS  PubMed  PubMed Central  Google Scholar 

  55. Abasian L, Bazyar A, Shirbazo S, Sayehmiri K, Ahmad N. Prevalence of Giardia lamblia in Iran: a systematic review and meta-analysis study. Res Med. 2013;36(5):111–16.

    Google Scholar 

  56. Nkrumah B, Nguah SB. Giardia lamblia: a major parasitic cause of childhood diarrhoea in patients attending a district hospital in Ghana. Parasit Vectors. 2011;4:163. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1756-3305-4-163.

    Article  PubMed  PubMed Central  Google Scholar 

  57. Hatam-Nahavandi K, Mohebali M, Mahvi AH, Keshavarz H, Khanaliha K, Tarighi F, et al. Evaluation of Cryptosporidium oocyst and Giardia cyst removal efficiency from urban and slaughterhouse wastewater treatment plants and assessment of cyst viability in wastewater effluent samples from Tehran. Iran J Water Reuse Desal. 2015;5(3):372–90. https://doiorg.publicaciones.saludcastillayleon.es/10.2166/wrd.2015.108.

    Article  CAS  Google Scholar 

  58. Alasvand-Javadi R, Kazemi F, Fallahizadeh S, Arjmand R. The prevalence of intestinal parasitic infections in Ahvaz, Southwest of Iran, during 2007–2017. Iran J Public Health. 2019;48(11):2070–3.

    PubMed  PubMed Central  Google Scholar 

  59. Nasiri V, Esmailnia K, Karim G, Nasir M, Akhavan O. Intestinal parasitic infections among inhabitants of Karaj City, Tehran province, Iran in 2006–2008. Korean J Parasitol. 2009;47(3):265–8. https://doiorg.publicaciones.saludcastillayleon.es/10.3347/kjp.2009.47.3.265.

    Article  PubMed  PubMed Central  Google Scholar 

  60. Mahmoudi MR, Mahdavi F, Ashrafi K, Forghanparast K, Rahmati B, Mirzaei A, et al. Report of Giardia assemblages and giardiasis in residents of Guilan Province-Iran. Parasitol Res. 2020;119(3):1083–91. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00436-019-06595-1.

    Article  CAS  PubMed  Google Scholar 

  61. Ayatollahi J, Elahi M, Sharifyazdi M, Shahcheraghi SH. Prevalence of intestinal parasites in the investigated samples in the Central Laboratory and Shahid Sadoughi Hospital Laboratory of Yazd, Iran. J Shahid Sadoughi Univ Med Sci. 2017;25(12):931–39.

    Google Scholar 

  62. Kia E, Hosseini M, Nilforoushan M, Meamar A, Rezaeian M. Study of intestinal protozoan parasites in rural inhabitants of Mazandaran Province, Northern Iran. Iran J Parasitol. 2008;3(1):21–5.

    Google Scholar 

  63. Mowlavi GR, Mirahmadi H, Rezaeian M, Kia E, Ebrahimi-Daryani M, Rokni MB, et al. Prevalence of intestinal parasites in tribal parts of Khuzestan Province during 2005-07. Govaresh. 2008;12(4):219–27.

    Google Scholar 

  64. Abbaszadeh Afshar MJ, Barkhori Mehni M, Rezaeian M, Mohebali M, Baigi V, Amiri S, et al. Prevalence and associated risk factors of human intestinal parasitic infections: a population-based study in the southeast of Kerman province, southeastern Iran. BMC Infect Dis. 2020;20(1):12. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12879-019-4730-8.

    Article  PubMed  PubMed Central  Google Scholar 

  65. Sarkari B, Hosseini G, Motazedian MH, Fararouei M, Moshfe A. Prevalence and risk factors of intestinal protozoan infections: a population-based study in rural areas of Boyer-Ahmad district, Southwestern Iran. BMC Infect Dis. 2016;16(1):703. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s12879-016-2047-4.

    Article  PubMed  PubMed Central  Google Scholar 

  66. Babakhani M, Safari R, Rajati F, Salimi S, Omidian Doost A. Prevalence and risk factors associated with intestinal parasitic infections among school children in Gashky, West of Iran. Int J Pediatr. 2017:5(7):5263–73. https://doiorg.publicaciones.saludcastillayleon.es/10.22038/ijp.2017.23173.1949

  67. Rafiei A, Baghlaninezhad R, Köster PC, Bailo B, Hernández de Mingo M, Carmena D, et al. Multilocus genotyping of Giardia Duodenalis in Southwestern Iran. A community survey. PLoS ONE. 2020;15(2):e0228317. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0228317.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  68. Haghighi A, Salimi-Khorashad A, Nazemalhosseini-Mojarad E, Kazemi B, Rostami-Nejad M, Rasti S. Frequency of enteric protozoan parasites among patients with gastrointestinal complaints in medical centers of Zahedan, Iran. Trans R Soc Trop Med Hyg. 2009;103(5):452–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.trstmh.2008.11.004.

    Article  PubMed  Google Scholar 

  69. Barkhori Mahni M, Rezaeian M, Kia EB, Raeisi A, Khanaliha K, Tarighi F, et al. Prevalence of intestinal parasitic infections in Jiroft, Kerman Province, Iran. Iran J Parasitol. 2016;11(2):232–8.

    PubMed  PubMed Central  Google Scholar 

  70. Siyadatpanah A, Sharif M, Daryani A, Sarvi S, Kohansal MH, Barzegari S, et al. Spatial distribution of Giardia lamblia infection among general population in Mazandaran Province, north of Iran. J Parasit Dis. 2018;42(2):171–6. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12639-018-0976-0.

    Article  PubMed  PubMed Central  Google Scholar 

  71. Choy SH, Al-Mekhlafi HM, Mahdy MA, Nasr NN, Sulaiman M, Lim YA, et al. Prevalence and associated risk factors of Giardia infection among indigenous communities in rural Malaysia. Sci Rep. 2014;4:6909. https://doiorg.publicaciones.saludcastillayleon.es/10.1038/srep06909.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  72. Naz A, Nawaz Z, Rasool MH, Zahoor MA. Cross-sectional epidemiological investigations of Giardia lamblia in children in Pakistan. Sao Paulo Med J. 2018;136(5):449–53. https://doiorg.publicaciones.saludcastillayleon.es/10.1590/1516-3180.2018.0350060918.

    Article  PubMed  PubMed Central  Google Scholar 

  73. Raso G, Luginbühl A, Adjoua CA, Tian-Bi NT, Silué KD, Matthys B, et al. Multiple parasite infections and their relationship to self-reported morbidity in a community of rural Côte d’Ivoire. Int J Epidemiol. 2004;33(5):1092–102. https://doiorg.publicaciones.saludcastillayleon.es/10.1093/ije/dyh241.

    Article  PubMed  Google Scholar 

  74. Escobedo AA, Almirall P, Alfonso M, Salazar Y, Avila I, Cimerman S, et al. Hospitalization of Cuban children for giardiasis: a retrospective study in a paediatric hospital in Havana. Ann Trop Med Parasitol. 2011;105(1):47–56. https://doiorg.publicaciones.saludcastillayleon.es/10.1179/136485911X12899838413420.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  75. Ngui R, Ishak S, Chuen CS, Mahmud R, Lim YAL. Prevalence and risk factors of intestinal parasitism in rural and remote West Malaysia. PLoS Negl Trop Dis. 2011;5(3):e974. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pntd.0000974.

    Article  PubMed  PubMed Central  Google Scholar 

  76. Samie A, Tanih NF, Seisa I, Seheri M, Mphahlele J, ElBakri A, et al. Prevalence and genetic characterization of Giardia lamblia in relation to diarrhea in Limpopo and Gauteng provinces, South Africa. Parasite Epidemiol Control. 2020;9:e00140. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.parepi.2020.e00140.

    Article  PubMed  PubMed Central  Google Scholar 

  77. Bodhidatta L, McDaniel P, Sornsakrin S, Srijan A, Serichantalergs O, Mason CJ. Case–control study of diarrheal disease etiology in a remote rural area in Western Thailand. Am J Trop Med Hyg. 2010;83(5):1106–9. https://doiorg.publicaciones.saludcastillayleon.es/10.4269/ajtmh.2010.10-0367.

    Article  PubMed  PubMed Central  Google Scholar 

  78. Veenemans J, Mank T, Ottenhof M, Baidjoe A, Mbugi EV, Demir AY, et al. Protection against diarrhea associated with Giardia Intestinalis is lost with multi-nutrient supplementation: a study in Tanzanian children. PLoS Negl Trop Dis. 2011;5(6):e1158. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pntd.0001158.

    Article  PubMed  PubMed Central  Google Scholar 

  79. Ignatius R, Gahutu JB, Klotz C, Steininger C, Shyirambere C, Lyng M, et al. High prevalence of Giardia Duodenalis assemblage B infection and association with underweight in Rwandan children. PLoS Negl Trop Dis. 2012;6(6):e1677. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pntd.0001677.

    Article  PubMed  PubMed Central  Google Scholar 

  80. Helmy MM, Abdel-Fattah HS, Rashed L. Real-time PCR/RFLP assay to detect Giardia Intestinalis genotypes in human isolates with diarrhoea in Egypt. J Parasitol. 2009;95(4):1000–4. https://doiorg.publicaciones.saludcastillayleon.es/10.1645/ge-1670.1.

    Article  CAS  PubMed  Google Scholar 

  81. Bartelt LA, Platts-Mills JA, Giardia. A pathogen or commensal for children in high-prevalence settings? Curr Opin Infect Dis. 2016;29:502–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1097/QCO.0000000000000293.

    Article  PubMed  PubMed Central  Google Scholar 

  82. Fallahizadeh S, Feiz-Haddad MH, Kazemi F, Afrisham R. Prevalence of intestinal parasitic infections in Shush County, Southwest of Iran during 2014–2016. Int J Infect. 2017;4(3):e14588. https://doiorg.publicaciones.saludcastillayleon.es/10.5812/iji.14588.

    Article  Google Scholar 

  83. Belete YA, Kassa TY, Baye MF. Prevalence of intestinal parasite infections and associated risk factors among patients of Jimma health center requested for stool examination, Jimma, Ethiopia. PLoS ONE. 2021;16(2):e0247063. https://doiorg.publicaciones.saludcastillayleon.es/10.1371/journal.pone.0247063.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  84. Badparva E, Kheirandish F, Ebrahimzade F. Prevalence of intestinal parasites in Lorestan Province, West of Iran. Asian Pac J Trop Dis. 2014;4:S728–32. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/S2222-1808(14)60716-7.

    Article  Google Scholar 

  85. Kasaei R, Carmena D, Jelowdar A, Beiromvand M. Molecular genotyping of Giardia Duodenalis in children from Behbahan, southwestern Iran. Parasitol Res. 2018;117(5):1425–31. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s00436-018-5826-6.

    Article  PubMed  Google Scholar 

  86. Shobha M, Bithika D, Bhavesh S. The prevalence of intestinal parasitic infections in the urban slums of a city in Western India. J Infect Public Health. 2013;6(2):142–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.jiph.2012.11.004.

    Article  PubMed  Google Scholar 

  87. Sadeghi H, Bakht M, Saghafi H, Shahsavari T. Prevalence of intestinal parasites in a population in Eghbalieh city from Qazvin Province, Iran. J Parasit Dis. 2015;39(2):126–9. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s12639-013-0366-6.

    Article  CAS  PubMed  Google Scholar 

  88. Matthys B, Bobieva M, Karimova G, Mengliboeva Z, Jean-Richard V, Hoimnazarova M, et al. Prevalence and risk factors of helminths and intestinal protozoa infections among children from primary schools in western Tajikistan. Parasit Vectors. 2011;4:195. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1756-3305-4-195.

    Article  PubMed  PubMed Central  Google Scholar 

  89. Odoi A, Martin SW, Michel P, Holt J, Middleton D, Wilson J. Geographical and temporal distribution of human giardiasis in Ontario, Canada. Int J Health Geogr. 2003;2(1):5. https://doiorg.publicaciones.saludcastillayleon.es/10.1186/1476-072x-2-5.

    Article  PubMed  PubMed Central  Google Scholar 

  90. Yoder JS, Harral C, Beach M. Giardiasis surveillance - United States, 2006–2008. MMWR Surveill Summ. 2010;59(6):15–25.

    PubMed  Google Scholar 

  91. Sepahvand A, Hosseini-Safa A, Yousofi HA, Tajedini MH, Pahlavan Gharehbabah R, Pestehchian N. Genotype characteristics of Giardia Duodenalis in patients using high resolution melting analysis technique in Khorramabad, Iran. Iran J Parasitol. 2020;15(2):204–13.

    PubMed  PubMed Central  Google Scholar 

  92. Shahnazi M, Naghizadeh F, Hajialilo E, Alizadeh SA, Saraei M, Alipour M. Investigation of Giardia Intestinalis genotypes among the food handlers of Qazvin, Iran. Iran J Parasitol. 2019;14(4):534–41.

    PubMed  PubMed Central  Google Scholar 

  93. Nooshadokht M, Kalantari-Khandani B, Sharifi I, Kamyabi H, Liyanage NPM, Lagenaur LA, et al. Stool antigen immunodetection for diagnosis of Giardia duodenalis infection in human subjects with HIV and cancer. J Microbiol Methods. 2017;141:35–41. https://doiorg.publicaciones.saludcastillayleon.es/10.1016/j.mimet.2017.07.004.

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  94. Hatam-Nahavandi K, Mohebali M, Mahvi AH, Keshavarz H, Mirjalali H, Rezaei S, et al. Subtype analysis of Giardia Duodenalis isolates from municipal and domestic raw wastewaters in Iran. Environ Sci Pollut Res. 2017;24(14):12740–7. https://doiorg.publicaciones.saludcastillayleon.es/10.1007/s11356-016-6316-y/.

    Article  CAS  Google Scholar 

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Acknowledgements

The authors are grateful to all laboratory staff from Clinical Reference Laboratory, Iran, and Khatam Hospitals, Iranshahr, Iran for their valuable collaboration.

Funding

This work was supported by a grant from the Office of Vice-Chancellor for Research of Iranshahr University of Medical Sciences (9900039).

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Authors and Affiliations

  1. Department of Parasitology and Mycology, School of Medicine, Sistan and Baluchestan, Iranshahr University of Medical Sciences, Iranshahr, Iran

    Kareem Hatam-Nahavandi

  2. Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran

    Ehsan Ahmadpour

  3. Department of Parasitology and Mycology, School of Public health, Tehran University of Medical Sciences, Tehran, Iran

    Mostafa Rezaeian

  4. Foodborne and Waterborne Diseases Research Center, Research Institute for Gastroenterology and Liver Diseases, Shahid Beheshti University of Medical Sciences, Tehran, Iran

    Hanieh Mohammad Rahimi & Hamed Mirjalali

  5. Department of Parasitology and Mycology, School of Medicine, Iran University of Medical Sciences, Tehran, Iran

    Ahmadreza Meamar

  6. Medical Microbiology Research Center, Qazvin University of Medical Sciences, Qazvin, Iran

    Milad Badri & Aida Vafae Eslahi

  7. Department of Microbiology, School of Medicine, Sistan and Baluchestan, Iranshahr University of Medical Sciences, Iranshahr, Iran

    Hossein-Ali Rahdar

  8. Department of Pathology, School of Medicine, Iranshahr University of Medical Sciences, Iranshahr, Iran

    Solmaz Sepahi

  9. Parasitology Reference and Research Laboratory, Spanish National Centre for Microbiology, Health Institute Carlos III, Majadahonda, Spain

    David Carmena

  10. CIBERINFEC, ISCIII – CIBER Infectious Diseases, Health Institute Carlos III, Madrid, Spain

    David Carmena

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  1. Kareem Hatam-Nahavandi
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Contributions

Study concept and design: KHN, EA, MR, DC, AM. Sample collection and acquisition of data: KHN, HMR, SS, EA, HM. Statistical analysis and interpretation of data: KHN, HMR, DC, EA. Writing-original draft preparation: KHN, MB, AVE, MB. Writing-review and editing: KHN, DC, EA, AVE, MB. Critical revision of the manuscript for important intellectual content: DC. Supervision: KHN. Project administration: KHN, EA, HAR. Funding acquisition: KHN. Final approval of the version: all authors.

Corresponding authors

Correspondence to Ehsan Ahmadpour, Hamed Mirjalali or David Carmena.

Ethics declarations

Ethics approval and consent to participate

This study was carried out according to the principles of the Declaration of Helsinki, the ARRIVE guidelines and the 3Rs. The research protocol was approved by the Research Ethics Committee of IRSHUMS (protocol code IR.IRSHUMS.REC.1400.002, 06.03.2021). All human isolates were collected as part of a standard clinical investigation of anonymous patients with clinical manifestations. Signed and informed consent forms were not required.

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The authors declare no competing interests.

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Hatam-Nahavandi, K., Ahmadpour, E., Rezaeian, M. et al. Occurrence and assemblage distribution of Giardia Duodenalis in symptomatic and asymptomatic patients in southeastern Iran (2019–2022). Gut Pathog 16, 68 (2024). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13099-024-00666-0

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Keywords

Gut Pathogens

ISSN: 1757-4749