Burden and Epidemiology of Human Intestinal Giardia duodenalis Infection in Colombia: A Systematic Review

The genus Giardia is a unicellular protozoan able to parasitize both humans and animals. Cysts of Giardia can be found in soil samples, aquatic environments, food, and any surface that gets in contact with the feces of parasitized animals. The aim of this systematic review was to analyze the burden and epidemiology of Giardia infection in Colombia summarizing recent scientific reports and existing knowledge and to identify knowledge gaps that may be addressed in future investigations. This work follows the guidelines established by “Preferred Reporting Items for Systematic Reviews and Meta-Analyzes” (PRISMA). Published scientific literature from 1 January 2010 to 18 September 2022 was searched in six electronic scientific databases using the search terms: “Giardia” OR “Giardiasis” AND “Colombia”. Twenty-three scientific articles were performed in 22 departments of Colombia at rural, urban, and a combination of rural and urban contexts. The prevalence of Giardia in the Colombian population was between 0.9 and 48.1% when the samples were analyzed with classical microscopy; the range of Giardia prevalence was even bigger (4.2–100%) when qPCR and nested PCR were used. The dominant Giardia assemblages found in Colombia were A and B, and most frequent subassemblages were AII, BIII, and BIV.


Introduction
Giardia duodenalis (syn. Giardia intestinalis, Giardia lamblia) is a cosmopolitan flagellated, microscopic protozoan parasite [1][2][3] able to infect a great diversity of domestic and wild animals [4][5][6]. Giardia spp. cysts are capable of maintaining their viability for a long time outside their host [7]. Eight Giardia spp. variants or genotypes, identified with the letters A-H, have been recognized to date [8,9]. Two of them, specifically the A and B genotypes, are frequently found in humans and in many animal species including cats, dogs, sheep, chickens, horses, pigs, and cows [10,11]. More recently, novel genotypes E and F have also been found in humans in Australia and Slovenia [12,13]. Parasite transmission among people generally occurs by the fecal-oral route i.e., consuming water or food [1,14].
The Giardiasis was, for a long time, an underrated and under-attended disease despite the large number of cases worldwide, probably because most people are asymptomatic or only present diarrhea as the most notable symptom [15]. Citizens living in developing countries, such as those located in the Caribbean and Latin America, with deficient water sanitary supply services and inadequate wastewater treatments, are particularly exposed

Inclusion and Exclusion Criteria
The inclusion criteria, applied to full-texts for assessing their eligibility, were: (a) original article focusing on the identification of Giardia spp. in Colombia, (b) article published from 1 January 2010 to 18 September 2022, (c) article written in English or Spanish, (d) study limited to human beings, (e) cross-sectional study, (f) article published in peer-reviewed journals in the Scimago Quartiles database.
The exclusion criteria, applied to full-texts for assessing their eligibility, were: (a) article published in a non-peer-reviewed source, (b) review of the literature or meta-analyses, (c) retrospective study, (d) short communication, (e) study with a score below three points based on the Joanna Briggs Institute (JBI) tool [36].

Selection of Studies
The identified articles were compiled using the Mendeley Desktop Reference Management System 1.19.8 and any duplicates were removed. Subsequently, two authors (YSG and CF) independently screened titles and abstracts. Irrelevant titles were removed. A third author (YCR) made a final decision when the two researchers had differing opinions. Inclusion and exclusion criteria were applied to full-texts to assess their eligibility; two authors (YSG and CF) independently analyzed the full-texts and only those that met all criteria were finally selected. Disagreements between the two researchers were resolved through consultation with a third author (YCR).

Data Extraction and Analysis
Article-level data were extracted from each selected paper; subsequently, they were summarized and tabulated in an abstraction-analysis matrix developed in MS Excel ® (Microsoft for Windows). The summarized information was organized in columns with the following subjects: (a) Reference, (b) Quartile, (c) Location, (d) Rural/Urban, (e) Collection period, (f) Study population/Group studied, (g) Age, (h) Feces samples, (i) Sample size, (j) Number of replicas, (k) Concentration method, (l) Detection method, (m) Giardia spp. genes, (n) Assemblages or subassemblages, (o) Prevalence (microscopy), (p) 95% confidence interval (microscopy), (q) Prevalence (molecular detection), (r) 95% confidence interval (molecular detection) and finally, (s) Study quality based on JBI tool.

Quality Assessment
The quality of the included studies was assessed with standardized critical appraisal instruments from the JBI [36]. The checklist consists of nine items, each one with four options (yes, no, unclear, and not applicable). The JBI score rating system divides the studies in two groups i.e., high quality studies (scores between 7 and 9), moderate quality studies (scores between 4 and 6).
Two researchers (YSG and CF) worked independently to analyze the selected material and, disagreements were resolved through consultation with a third author (YCR). The quality assessment results are presented in Table S2.

Literature Search
A total of 739 publications were recorded in the identification phase. Duplicates were removed and the remaining 630 articles were screened for title and abstract pertinence. Only 36 full-text articles have passed the screening of title and abstract phase. Hence, their eligibility was assessed based on inclusion and exclusion criteria. Finally, 23 articles were included in this systematic review . The PRISMA Statement flow diagram, composed of four phases (identification, screening, eligibility, and inclusion) is shown in Figure 1. removed and the remaining 630 articles were screened for title and abstract pertinence Only 36 full-text articles have passed the screening of title and abstract phase. Hence, their eligibility was assessed based on inclusion and exclusion criteria. Finally, 23 articles were included in this systematic review . The PRISMA Statement flow diagram, composed of four phases (identification, screening, eligibility, and inclusion) is shown in

Characteristics of Included Studies
General characteristics of the selected articles are summarized in Table 1.

Molecular Characteristics of the Selected Studies
The main characteristics of the applied experimental methodologi in Table 2.

Molecular Characteristics of the Selected Studies
The main characteristics of the applied experimental methodologies can be reviewed in Table 2.  In all the studies, fecal samples were analyzed to identify Giardia. Most authors did not report the number of fecal replicas used in their analysis, only Arias et al. [37], Villafañe-Ferrer and Pinilla-Pérez [45], and Muñoz Salas et al. [58] indicated that the experiments were performed in triplicate.
Extraction and detection of Giardia spp. was carried out by means of commercial kits such as (1) [49], and (4) AccuPrept Stool Genomic DNA kit (BioNeer Corp., Munpyeong-seo, Republic of Korea) [58], that were convenient and rapid methods to isolate total DNA from fresh or frozen stool samples.

Reported Prevalence of Giardiasis
The size of studied groups ranged between 23 [50] and 649 [55] individuals. The estimated prevalence of Giardia based on molecular identification methods was generally higher than the prevalence obtained with microscopy methods.

Epidemiology of Giardia in Colombia
A large variety of factors such as geographic-climatic conditions, unequal distribution of resources, unfavorable socioeconomic indicators, and inadequate sanitary indica-

Epidemiology of Giardia in Colombia
A large variety of factors such as geographic-climatic conditions, unequal distribution of resources, unfavorable socioeconomic indicators, and inadequate sanitary indicators influence the transmission of parasitic diseases, particularly Giardiasis, among the Colombian individuals [52,60]. People living in rural areas and children are usually the individuals most exposed to health risks derived from Giardia infection [51]. In this sense, Peña-Quistial et al. [56] studied children belonging to disadvantaged migrant populations in the mountain area of the Valle del Cauca and pointed out that the lack of drinking water and a sewage system could be the main detonators of parasitic diseases. Hernández et al. [52] indicated that all the children in their study group, in the Department of Cundinamarca at 100 km from Bogota, were found to be infected by parasites; the microscopic examination revealed that Giardia was the most prevalent protozoa (39.1%); the molecular analysis, conducted on a total of 14 Giardia positive samples, allowed the authors to identify the presence of subassemblages AI, AII (the most frequent subassemblages), BIII, BIV, BIII/BIV, and a mixed subassemblage AII + BIII. Also, the results of Sánchez et al. [46], who studied the prevalence of intestinal parasites among indigenous children from the Colombian Amazon basin, go in the same direction; the authors attributed the contamination of public water and close contact with domestica and wild animals in the Amazon region with the presence of AI Giardia subassemblage.
Ramírez et al. [44], through specific molecular markers, identified in a reliable manner the assemblage B in a great proportion of Cundinamarca children and the assemblage A in a few samples; subsequently, the subassemblages were described as AI, AII, BIII, and BIV.
Fecal samples, collected from children or teenagers living in central and southwest Colombian regions, were analyzed by Avendaño et al. [50], who allocated the Giardia assemblages principally to B and to a lesser extent A; the authors suggested a basic transmission among the children attending educational establishments and individuals from urban areas. Also, Muñoz Salas et al. [58] reported that around 13% of schoolchildren, between two and ten years of age, in the department of Atlántico, were infected by Giardia protozoa, but the genotypes A and B did not show an association with their nutritional status.
Higuera et al. [55] collected and analyzed 649 stool samples from adults and children in different regions of Colombia and showed the different performances between molecular analysis and microscopic examination; using molecular detection by PCR, 43.1% of samples tested positive for Giardia while through microscopy only a quarter of the samples (25.4%) were assessed for Giardia. Citizens living in the Caribbean region were more exposed to parasitic infection, particularly in the Bolivar department that registered the highest prevalence of Giardia equivalent to 89.5% of the analyzed samples. Finally, the main assemblages identified were BIV and BIII; in some cases, assemblages AII, D, and G were encountered.
Villalba-Vizcaíno et al. [49], looking to obtain frequency and circulating genotypes of Giardia in the Colombia Caribbean coast, analyzed fecal samples of citizens from Cartagena de Indias and Santa Marta; all the samples from Santa Marta were molecularly characterized as assemblage A of Giardia, while in Cartagena the presence of assemblages A and B has been confirmed.
An interesting study case of Espinosa Aranzales et al. [47] reported a low prevalence of pathogenic intestinal parasites such as Giardia in pregnant women in the districts of Bogota; nevertheless, the authors highlighted the need for educational campaigns aimed at the poorest and most marginalized groups in the capital city to disrupt transmission routes for prevalent parasites.
Giardiasis, defined by the World Health Organization (WHO) as a neglected tropical disease, represents a serious concern in public health not only for Colombians but for all people living in Latin America [61]. Annually, approximately 200 million people are estimated to develop Giardiasis symptoms in the developing countries [62]. Studies conducted in Mexico have shown that humans, living in rural areas, are mostly infected by Giardia assemblage A [63,64]; while, as reported by Lebbad et al. [65] and Minvielle et al. [66], in Nicaragua and Argentina the assemblage B was dominant.
Similar to Colombia, the Giardia assemblages A, B, and A + B were dominant in Cuba; these specific assemblages were detected in groups of children living in La Habana and rural inhabitants in the central region of the country [67][68][69][70]. Brazilian and Colombian indigenous groups, with poor sanitation and unsafe water, are particularly exposed to health risks [71] and specifically to Giardia infection [72]; according to Coelho et al. [73], the rate of Giardiasis in the adults and children belonging to Amazonian communities ranging from 44.8 to 52.9%. Köster et al. [74], who investigated the Giardia prevalence among the indigenous of the Brazilian Amazonian region, have typified the assemblages as A, B, and A + B, while the dominant subassemblages were AII, AIII, BIII, and BIV; these results coincide with those reported by Sanchez et al. [46], who studied rural people in the Colombia Amazonia. According to Merchán Garzón et al. [19], the prevalence Giardia spp. reached up to 63% of individuals in the indigenous and black communities of Colombia.

Microscopic vs Molecular Methods
Most reports on intestinal protozoan pathogens such as Giardia among Colombian population have focused exclusively on microscopic detection [51]. However, many authors have shown substantial differences in detection rates using molecular methods, which also allow to identify cryptic species and their genotypes [46,47,52,54,55,58]. There are clear benefits and additional value in using complementary molecular techniques for molecular epidemiological studies [46,55]; specific marker may help to improve knowledge of the transmission dynamics of intestinal parasites and to establish better prevention campaigns [81].
Classical microscopy is used routinely for Giardia cyst detection in water, food, stool, and tissue samples [82,83]. Classical microscopy technique is considered the gold standard for the diagnosis of Giardiasis. However, this technique is subject to subjective interpretation by the observer [84], in addition, the sensitivity is low when only one sample is analyzed, particularly if there is a low density of parasites or if the excretion of Giardia cysts is intermittent [85]. Despite this, the sensitivity can be increased if other diagnostic techniques are added [86,87]. Nevertheless, the microscopy remains the mainstay of Giardia diagnosis despite its limitations, specially in the developing countries located in Latin America, Asia, and Sub-Saharan Africa with low or middle economic resources and poor access to health facilities [33,76,88].
For the routine medical diagnosis of Giardiasis, it is recommended to combine traditional microscopy with a stool concentration method, thus increasing sensitivity. Immunological and molecular methods are recommended as complementary tests to the traditional microscopy technique [33].
The detection of pathogenic enteroparasites based on DNA analysis offers greater sensitivity and robustness, in addition to enabling the identification and/or characterization of genetic variants. The information obtained is very useful for epidemiological surveillance in the event of an outbreak [89].
PCR-based techniques are powerful molecular tools that make it possible to obtain numerous copies of a desired DNA fragment for the detection of a molecular target or for its subsequent characterization. PCR-based assays have been widely adopted for the detection of Giardia in various environments [90][91][92]. These techniques are adaptable and allow the automated processing of large numbers of samples in a short time [93]. Phylogenetic analysis of the 18S rDNA gene of Giardia might reveal significant intraspecies diversity, and, at the same time, highlight the danger of zoonoses from specific assemblages [94]. These techniques require their own standardization, expensive instrumentation, and consumables.
The PCR classifies the genetic variants of Giardia [33,95]; the genes most used for this purpose are the small subunit (SSU) encoding ribosomal RNA, glutamate dehydrogenase (gdh), triosaphosphate isomerase genes (tpi), and ß-giardin (bg-a protein in the adhesive disc of Giardia) [33].
Nested PCR is widely used in the detection of Giardia [96][97][98][99]. It is characterized using two sets of primers. The first set binds to sequences slightly outside the target DNA, then this amplified fraction serves as the basis for the second set of primers, this variant of the technique allows increasing sensitivity [91,100].
Polymorphic length restriction fragment-based PCR (PCR-RFLP) uses specific primer sets for the selective amplification of different regions of the genome. The amplification product is then subjected to enzymatic digestion to characterize and classify Giardia genetic variants based on the number and size of fragments produced [101,102]. This technique is also frequently used in Giardia characterization studies.
Quantitative PCR (qPCR) has had a wide field of application in the detection and quantification of pathogens in environmental and clinical samples [103][104][105]. This variant of conventional PCR offers the advantage of being able to follow the amplification process in real time and thus being able to calculate the number of copies along the process.
The qPCR is widely used in the detection of pathogens due to its great sensitivity and savings in time and effort, in addition there are different variants for this technique such as Molecular Beacon probes, Taqman probes, Scorpion probes, FRET probes, and intercalating dyes such as SYBR Green, that alone or in combination with other techniques have been used for the detection, quantification, and characterization of Giardia [92,[106][107][108][109].
The choice of the most appropriate technique depends on the objective of the investigation and the type of sample analyzed. The detailed understanding of the foundation of the molecular tool provides the opportunity to make pertinent adjustments when necessary [89].

Burden and Perspective to Colombia
Colombia has faced numerous barriers in improving healthcare for its citizens due to both its topography with wide-ranging landscapes and socioeconomic inequity [110]. The internal armed conflict, lasted over fifty years, produced one of the largest internally displaced populations in the world [111]; it has been an amplifier of social inequalities limiting public health access especially for the weakest groups in society such as the indigenous, farmers, and children. The Colombian health system has been able to react to these difficulties and taken giant steps to ensure better access to public health and adequate medical care for all citizens [112]; nevertheless, the high burden of numerous neglected tropical diseases such as Giardiasis negatively affect the lives of people with low incomes.
Most of the selected studies of this systematic review were developed in the central region and in the north of the country [38][39][40]42,43,45,49,53,55,[57][58][59]; only a few studies have been conducted on southern populations [46,48,54,56]. Census or high-quality research could make up for this data gap by providing reliable values for taking decisions and any eventually encountered, specific Giardia assemblages and subassemblages.
In addition, efforts to ensure safe drinking water, sufficient sanitation, and sewage systems in the poorest departments of the country such as Amazonas, Putumayo, Vaupés, and Caquetá are promptly required. Low levels of schooling and a significant food risk can increase the consequences of gastrointestinal infections in the most vulnerable groups in society. Valid measures of food security could form a key component to protect citizens from gastrointestinal diseases, especially Giardia infection. The Colombian healthcare system needs a multidisciplinary management to eradicate Giardia spp. infection. Microbiology analysis such classical microscopy and molecular techniques should be integrated with projects in the fields of human and social sciences [34]. Educational and awareness campaigns could be key elements to educate Colombian individuals in correct sanitary hygiene and prevent the Giardia spp. infection and distribution of other tropical diseases.

Conclusions
The prevalence of human Giardiasis between 2006 and 2022 in 22 departments of central and western Colombia was 0.9-48.1% when using classical microscopy and 4.2-87.0% using PCR. Study areas included urban and rural, but due to differences found between the different publications, it was not possible to generalize the type of area associated with the highest prevalence. Apparently, the difference becomes less noticeable when conditions of poverty and deficiencies in public services prevail in urban settlements.
In Colombia, assemblages A and B are present in humans. Two outliers of assemblages D and G are also reported, but further studies are needed to confirm the information. The predominant subassemblages were AII, BIII, and BIV.