Molecular Prevalence and Subtypes Distribution of Blastocystis spp. in Humans of Latin America: A Systematic Review

Blastocystis spp. are among the few enteric parasites with a prevalence that can reach up to approximately 80% in communities of developing countries. This systematic review updates and summarizes available literature on the molecular prevalence and subtype distribution of Blastocystis spp. in Latin American people. This work follows the PRISMA (Preferred Reporting Items for Systematic Reviews and Meta-Analyses) guidelines. The literature revised covers from 1 January 2015 to 6 October 2023 in seven different scientific databases, and the material was selected through inclusion and exclusion criteria. According to data found in the 36 selected articles, the prevalence of Blastocystis spp. in Latin America ranged between 5.8% (Bolivian rural communities) and 94.0% (Colombian general public). Generally, genomic DNA was extracted from approximately 200 mg fecal sediments using commercial kits, such as the QIAamp Stool Mini Kit (QIAGEN, Hilden, Germany) or the Norgen Stool DNA Isolation Kit (Norgen Biotek Corporation, Thorold, ON, Canada). Subtype-specific primers (such as the couple of primers BhRDr–RD5) developed from unique sequences of the SSU rRNA gene were applied to Blastocystis subtyping. Ten specific subtypes (STs) were found as well as various mixed infections, and the most circulating Blastocystis STs were in the order ST3, ST1, ST2, and ST4. The most recent data about Blastocystis spp. molecular epidemiology and the STs in communities of Latin America are limited to studies from specific countries. Novel scientific data from the other countries are required to obtain a complete picture and truly understand the distribution and prevalence of Blastocystis spp. and the STs.

Blastocystis spp.are among the few enteric parasites with a prevalence that frequently exceeds 5% in citizens of industrialized countries [25] and can reach up to approximately 80% in developing countries [26,27].The parasite transmission among humans generally occurs via the fecal-oral route, and may be either direct (i.e., person-to-person or zoonotic) or indirect (i.e., foodborne and waterborne) [28,29].
People living in rural contexts or developing countries, such as those located in the Caribbean and Latin America, with deficient water sanitary supply services, inadequate wastewater treatments, and close animal contact, are particularly exposed to health risks derived from a Blastocystis spp.infection [30].Additionally, favorable climatic conditions, i.e., high humidity and warm temperature, increase the chance of transmission of Blastocystis spp. in tropical areas [31,32].The Blastocystis spp.-infected patients are, in many cases, asymptomatic or generally experience mild symptoms, such as diarrhea, abdominal pain, flatulence, bloating, constipation, and skin lesions [33].However, specific groups of individuals, i.e., children, elderly people, and patients with anemia or irritable bowel syndrome, could experiment serious Blastocystosis symptoms [16,34,35].
The occurrence of Blastocystis spp.has been observed in various water sources [36,37], including rivers [38], lakes [39], streams [40], sewage [41], surface water [42], stored water [43], tap water [44], and water tanks [45].Outbreaks of Blastocystosis in poor communities have been linked to drinking water contaminated with fecal matter and poor sanitation services [40,46].Scientific reports and evidence about the occurrence and prevalence of parasites in water sources have given the international community a boost to adopt safety strategies, such as the safe drinking water, sanitation, and hygiene programs (WASH) of the World Health Organization (WHO) [47].
Despite the recent efforts of the scientific community, governments, and international health organizations to detect and typify Blastocystis spp. in the Caribbean and Latin American countries, the epidemiological-molecular characterization and spatial distribution are not yet fully clarified.This systematic review updates and summarizes available literature on the molecular prevalence and subtype distribution of Blastocystis spp. in individuals of Latin America.

Materials and Methods
The systematic review was performed according to the guidelines set forth in the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) and the checklist of Moher et al. [48] (Supplementary File S1).

Inclusion Criteria
The inclusion criteria, applied to full texts for assessing their eligibility, were: (a) original article focusing on molecular identification of Blastocystis spp. in Latin American humans, (b) article published from 1 January 2015 to 6 October 2023, (c) article written in English and/or Spanish, (d) study limited to humans, (e) cross-sectional study, and (f) article published in peer-reviewed journals inserted in the Scimago Journal Ranking (SJR) database.

Exclusion Criteria
The exclusion criteria, applied to full texts for assessing their eligibility, were: (a) abstract not associated with the full article, (b) article published in non-peer-reviewed source, (c) article not written in English or Spanish, (d) review papers of literature or meta-analyses, (e) retrospective studies, (f) short communication, (g) letters to the editor, and (h) studies with ≤3 points based on the Joanna Briggs Institute (JBI) tool [49].

Selection of Studies
The Mendeley Desktop Reference Management System 1.19.8 software was used to compile the identified articles and to remove the duplicates.Subsequently, articles were independently screened for title and abstract pertinence by two authors (Y.S.-G.and J.E.B.).Irrelevant titles were removed.Disagreements between the two researchers were resolved through consultation with a third author (C.F.).Inclusion and exclusion criteria were applied to full texts to assess their eligibility.Two authors (Y.S.-G.and J.E.B.) independently analyzed the full-text papers and only those that met all criteria were finally selected.

Quality Assessment
The quality of the included studies was assessed with standardized critical appraisal instruments from the JBI for prevalence [49]; the tool, composed by nine questions with four answer options (yes, no, unclear, and not applicable), assists in assessing the trustworthiness, relevance, and results of published scientific articles.
Based on a quality score rating system, the papers were divided into two categories: high-quality study (score 7-9) and moderate-quality study (score 4-6).
The nine questions (Q) of the JBI instrument are: (Q1) Was the sample frame appropriate to address the target population?(Q2) Were study participants sampled in an appropriate way? (Q3) Was the sample size adequate?(Q4) Were the study subjects and the setting described in detail?(Q5) Was the data analysis conducted with sufficient coverage of the identified sample?(Q6) Were valid methods used for the identification of the condition?(Q7) Was the condition measured in a standard, reliable way for all participants?(Q8) Was there appropriate statistical analysis?(Q9) Was the response rate adequate and, if not, was the low response rate managed appropriately?Two researchers (Y.S.-G.and J.E.B.) worked independently, and disagreements were resolved through consultation with a third author (C.F.) (Supplementary File S2).

Literature Search
The PRISMA Statement flow diagram that indicates the four phases of the literature search (identification, screening, eligibility, and inclusion) is shown in Figure 1.During the identification phase, a total of 948 publications were recorded.Duplicated articles were automatically removed via a bibliographic management software and the remaining 374 papers were screened for title and abstract pertinence.
priate to address the target population?(Q2) Were study participants sampled in an appropriate way? (Q3) Was the sample size adequate?(Q4) Were the study subjects and the setting described in detail?(Q5) Was the data analysis conducted with sufficient coverage of the identified sample?(Q6) Were valid methods used for the identification of the condition?(Q7) Was the condition measured in a standard, reliable way for all participants?(Q8) Was there appropriate statistical analysis?(Q9) Was the response rate adequate and, if not, was the low response rate managed appropriately?Two researchers (Y.S.-G.and J.E.B.) worked independently, and disagreements were resolved through consultation with a third author (C.F.) (Supplementary File S2).

Literature Search
The PRISMA Statement flow diagram that indicates the four phases of the literature search (identification, screening, eligibility, and inclusion) is shown in Figure 1.During the identification phase, a total of 948 publications were recorded.Duplicated articles were automatically removed via a bibliographic management software and the remaining 374 papers were screened for title and abstract pertinence.
The eligibility of 229 full-text articles that passed the title and abstract screening phase was assessed based on preset inclusion and exclusion criteria.Finally, 36 articles  were included in this systematic review.The eligibility of 229 full-text articles that passed the title and abstract screening phase was assessed based on preset inclusion and exclusion criteria.Finally, 36 articles  were included in this systematic review.

Molecular Characteristics of the Selected Articles
The main characteristics of the applied experimental methodologies are shown in Table 2.
Subtype-specific primers developed from unique sequences of the SSU rRNA gene were applied to Blastocystis subtyping.For instance, the BhRDr primer was designed to be combined with the RD5 primer, and the expected size of the PCR product is 600 bp.This set of primers was used in 21 of the 36 selected studies .Other sets of primers, such as F1/R1 (1100 bp SSU rRNA, barcoding region) [53,54,56,80] [75][76][77]82], PCR-RFLP [56], multi-parallel qPCR [82], and semi/nested PCR [69], were used to amplify small segments of DNA.

Epidemiology of Blastocystis spp. in Latin America
Blastocystis spp.comprise a genus of single-celled parasites that present a cosmopolitan distribution and colonize an estimated 1 to 2 billion people worldwide, many of them living in developing countries [19].Epidemiological studies at a molecular level have clearly demonstrated that humans can be colonized by one or more Blastocystis STs, some of which are commonly found in non-human hosts [71].
Brazil is a megadiverse country that possesses six terrestrial biomes and many regional differences in social, economic, and cultural characteristics [86].A large percentage of municipalities are not equipped with efficient sewage treatment systems and waste management sites [87].These structural gaps can amplify the diffusion of tropical neglected diseases, including those caused by many Blastocystis STs, especially in the poor and vulnerable demographic groups [88].The Blastocystis STs identified in Brazil were

Epidemiology of Blastocystis spp. in Latin America
Blastocystis spp.comprise a genus of single-celled parasites that present a cosmopolitan distribution and colonize an estimated 1 to 2 billion people worldwide, many of them living in developing countries [19].Epidemiological studies at a molecular level have clearly demonstrated that humans can be colonized by one or more Blastocystis STs, some of which are commonly found in non-human hosts [71].
Brazil is a megadiverse country that possesses six terrestrial biomes and many regional differences in social, economic, and cultural characteristics [86].A large percentage of municipalities are not equipped with efficient sewage treatment systems and waste management sites [87].These structural gaps can amplify the diffusion of tropical neglected diseases, including those caused by many Blastocystis STs, especially in the poor and vulnerable demographic groups [88].The Blastocystis STs identified in Brazil were ST1-ST4 and ST6-ST9, with the most prevalent being ST1, ST2, and ST3 [55][56][57][58][61][62][63]; furthermore, some data indicate mixed infections, such as ST1 + ST3 [59,60,64,65].The prevalence rate varied from 7.5% (patients-laboratory) [60] to 71.4% (schoolchildren) [56].Various studies about the prevalence of Blastocystis spp. in many Brazilian contexts were performed in the last eight years.Barbosa et al. [61] indicated an overall prevalence of intestinal parasitic infections above 50.0%and a wide range of Blastocystis spp.STs (ST1, ST2, ST3, ST4, and ST8) among the people who belong to a rural community in Rio de Janeiro.Blastocystis spp.STs, isolated by Barbosa et al. [64] in a carioca urban community, were genetically highly divergent, with ST3 being the most common among the participants, followed by ST1, ST2, and ST4.A mixed infection (ST1 + ST3) was detected in a few cases.David et al. [65] revealed large genetic variation of Blastocystis spp., with ST1 and ST3 being predominant, among asymptomatic people belonging to two small fishing villages along the Tietê river (São Paulo).According to Oishi et al. [60], Blastocystis spp., with a prevalence of 38.7%, were the most frequently parasites found among schoolchildren in the surrounding urban area of Curitiba.The molecular typification indicated various STs, in the order of prevalence ST3, ST1, and ST2, and a mixed infection of ST1 + ST3. de Melo et al. [57] indicated that approximately one-third of patients with diabetes mellitus in the Goias State were hosts of Blastocystis spp., and phylogenetic analyses revealed six STs, i.e., ST1, ST2, ST3, ST6, ST7, and ST8.Other studies also reported the great genetic variety of Blastocystis spp. in Brazil, indicating, at the same time, that the enteric parasites still represent a serious health concern principally due to educational deficits, poor socioeconomic rank, and inadequate sanitary conditions [55,56,62].
Reports from Argentina indicated that (1) approximately two-thirds (57.3%) of indigenous people living in the rural settlement of Puerto Iguazú (Misiones) were hosts of Blastocystis spp., and the parasite transmission occurred mainly through direct contact with fecal matter and contaminated water [50]; in addition, (2) one-quarter (24.8%) of patients attending the University Hospital of Cordoba City were infected with Blastocystis spp.[51].In both Argentinian studies, subtypes ST1, ST2, and ST3 were found, with the latter being the most common among symptomatic and asymptomatic people [50,51].
Molecular studies, performed in rural contexts in Bolivia, showed high prevalence of intestinal parasites among children and teenagers and, at the same time, pointed out the risk of zoonotic pathogen transmission.In particular, the cross-sectional parasitological survey realized by Aruni Chura et al. [52] evidenced three Blastocystis spp.STs (more specifically, ST1, ST2, and ST3) among schoolchildren from ecological zones in the Department of La Paz.Macchioni et al. [53] suggested that contaminated drinking water, a lack of basic sanitary services, and close contact with animals could increase the transmission of Blastocystis ST2 and ST9 (isolated on very few occasions) among children living in rural settlements of the Chaco region.
Tourists that visit Latin America or other developing countries may acquire Blastocystis STs during their stay, as demonstrated by van Hattem et al. [108].
As has been shown, the main routes of Blastocystis infection are: anthroponotic transmission, contaminated food and water, as well as close contact with animals [28,[109][110][111][112][113].The presence of Blastocystis spp.cysts, reported in water environments worldwide (rivers, lakes, streams, and lagoons), indicates fecal contamination of the water resources by humans or animals [11,38].Since Blastocystis spp.and their STs (especially ST1 and ST3) were found in animals, including dogs, rats, cows, monkeys, and chickens, zoonotic pathways pose a serious concern for health systems in Latin American countries [74,76,114].
In spite of the fact that molecular epidemiological studies about Blastocystis spp.and their STs have been conducted in the last years in several Latin American countries, more studies are required to clarify the circulating STs in this continent.

Protocols for Blastocystis spp. Molecular Analysis
A wide range of approaches based on DNA techniques are available for detection, investigation, and surveillance of pathogenic enteroparasites [115].PCR techniques are flexible, adaptable, and allow the automated processing of large numbers of samples in a short time [116].
The protocol to develop a descriptive study on molecular parasitology in a specific population group must be conducted under the ethical principles and approval of a bioethics committee.
The first step in carrying out a descriptive study on Blastocystis spp.prevalence is to establish the sample size representative of the target population.The minimum sample size (n) could be estimated through the following equation, as described by Oishi et al. [60]: where N is the study population, α is the sample standard deviation (usually set at 0.5), Z corresponds to the confidence level (for a 95% level of confidence, the critical value of Z is 1.96), e is the marginal error (usually set at 5%), and γ is the expected prevalence (usually set based on the intestinal parasite prevalence rate in close regions).The recruited voluntary participants should be knowledgeable about the methodology and the benefits of the study, sign an informed consent form [117,118], and answer a survey that includes sociodemographic patterns (age, sex, socioeconomic data, health affiliation, and level of instruction), hygienic condition of the dwellings (sanitary services and potable water, and presence of domestic animals), and behavioral aspects (personal hygiene and hand washing, and consuming raw or half-cooked meat).Furthermore, each participant must receive basic instructions for a correct fecal sample collection in a plastic container.
The recollected stool samples are generally stored in refrigerated boxes and transported to the laboratory.Prior to the laboratory processing analysis, macroscopic examination of all stool samples evaluates their consistency and the presence of mucus [55].Each sample could be divided into two aliquots for different uses: (1) the first aliquot is immediately frozen at −20 • C for extracting genomic DNA, and (2) the second fecal aliquot is used for parasite concentration processing (thought common techniques, such as Ritchie methods and Kato-Katz) or to direct microscopic examination.Additionally, the samples that test positive for Blastocystis under microscopic observation are destined for genomic analysis.
For Blastocystis detection and molecular subtyping, the genomic DNA is generally subjected to PCR [51,53], qPCR [75,76], or nested PCR [69,119].A fragment of about 600 bp from the SSU rDNA gene could be amplified using the BhRDr primer combined with the RD5 primer, according to standard protocol [50,52,55].
The PCR products are usually purified with ExoSap and sequenced by the Sanger method for determining the nucleotide sequences [72].The obtained sequences are compared to those of Blastocystis STs previously deposited in GenBank using the BLAST application (www.ncbi.nlm.nih.gov/BLAST,accessed on 17 January 2024).Blastocystis sequences could be submitted to the Blastocystis 18S database (http://pubmlst.org/blastocystis/,accessed on 17 January 2024) for subtype confirmation [58,60].

Limitations
Approximately one-third of Latin American countries have published studies on the Blastocystis spp.molecular epidemiology in their territories during the last nine years.The lack of data from various regions, as well as the peculiarities of each selected study and differences in populations, sampling strategies, and molecular methods (concentration, extraction, and detection) for Blastocystis spp.and the STs, do not allow for a more detailed analysis or the completion of a meta-analysis.

Conclusions
The data found in the selected articles indicate that the prevalence of Blastocystis spp. in Latin American populations has a significant variation, ranging between 5.8% and 94.0%.PCR techniques are frequently implemented to detect Blastocystis STs circulating in Latin America.Ten STs were reported and, in particular, the first three STs (ST1, ST2, and ST3) are widely diffused in Latin America, while ST5 and ST16 were reported in very few studies.The most recent data on Blastocystis spp.molecular epidemiology and STs in communities of Latin America are limited to studies from specific countries, including Argentina, Bolivia, Brazil, Chile, Colombia, Ecuador, Honduras, Mexico, Panama, and Peru.Further studies from other countries are required to obtain a complete picture and truly understand the distribution and prevalence of Blastocystis spp.and their STs.

Table 1 .
General characteristics of the selected studies published.
NR: not reported.

Table 2 .
Molecular characteristics of the selected studies in the systematic review.