Current Tropical Medicine Reports https://doi.org/10.1007/s40475-018-0149-3

TROPICAL DISEASES IN COLOMBIA (A RESTREPO, SECTION EDITOR)

Cryptosporidiosis in Colombia: a Systematic Review Ana Luz Galván-Díaz 1

# Springer International Publishing AG, part of Springer Nature 2018

Abstract Purpose of Review Cryptosporidium is an intestinal apicomplexan recently classified as a gregarine. It is associated with diarrhea both in immunocompromised and immunocompetent patients. Direct person-to-person, zoonotic, and waterborne are the principal transmission routes. Despite the significant impact on public health of cryptosporidiosis, especially in developing countries, there are few studies on this parasitic disease. This systematic review will focus on the cryptosporidiosis situation in Colombia, describing the prevalence data on humans, other animals, and environmental sources. Species and subtypes, risk factors, and diagnosis techniques used in these studies are summarized. Recent Findings Six electronic databases were searched for articles in Spanish, English, or Portuguese, using the keywords Cryptosporidium, cryptosporidiosis, intestinal parasites, and Colombia. Out of 569 studies identified in the electronic search, 42 articles were eligible for inclusion in the systematic review. The prevalence of cryptosporidiosis in humans, animals, and water sources was 7.8, 20.4, and 38.9% respectively. Seroepidemiologic studies described a seropositivity prevalence rate of 83.3% in humans and 53.3% in adult cattle. Staining techniques were the most frequent method used in the parasite detection. Circulating species in Colombia included C. parvum and C. hominis both in humans and animals; C. felis and C. viatorum in humans; and C. muris and C. bovis in felines and bovines, respectively. Summary Cryptosporidium prevalence data in Colombia indicate a high risk of infection for this protozoon and reinforce the importance of its routine surveillance among humans, animals, and waterbodies. More genetic studies on circulating species and subtypes are required in order to characterize the transmission dynamics of cryptosporidiosis in the country, including host range, reservoirs, and infection sources. Keywords Cryptosporidium . Prevalence . Species . Subtypes . Colombia

Introduction Cryptosporidium is a ubiquitous enteric protozoan that infect a broad range of vertebrates (humans, domestic and wild animals) [1]. Environmental-resistant oocyst can be acquired through a variety of transmission routes including direct contact with infected persons (person-to-person transmission), contact with animals (zoonotic transmission), and ingestion of contaminated food (foodborne transmission) or water (waterborne transmission) [2••, 3]. Cryptosporidium is the most This article is part of the Topical Collection on Tropical Diseases in Colombia * Ana Luz Galván-Díaz [email protected] 1

Escuela de Microbiología, Grupo de Microbiología ambiental, Universidad de Antioquia, Building 5-410, Medellín, Colombia

frequently identified protozoa in waterborne enteric outbreaks worldwide [4••]. Although initially associated with chronic diarrhea in AIDS and other immunocompromised patients, now it is widely accepted as a leading cause of childhood diarrhea and malnutrition, especially in low-income countries [5]. Several issues are of major concern in the control of cryptosporidiosis, including the high oocyst resistance to water disinfection treatments, the absence of an effective drug treatment, and the lack of a vaccine [3].

History Edward Tyzzer (1875–1965) originally described the genus Cryptosporidium in 1907. He found the parasite in the gastric glands of common mice (Mus musculus), and proposed the specie Cryptosporidium muris (Tyzzer, 1907) [6]. He placed it in the coccidian family Asporocystidae, whose members lack

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sporocysts in the oocyst (i.e., naked sporozoites) and what he thought possessed similar lifecycle [2••]. Later in 1912, Tyzzer identified Cryptosporidium parvum from the small intestine of laboratory mice. This new specie had oocysts smaller than those of C. muris [6, 7]. It is important to highlight Tyzzer’s work, given that most of what we currently know about the biology and lifecycle of Cryptosporidium comes from these early investigations. Cryptosporidium was first recognized as a potential cause of diarrhea in turkeys in 1955 [7]. For almost 70 years, there was little interest on this parasite, with only sporadic cases in vertebrate animals in the decade of 70s. The first cases in humans were reported until 1976. During the early 1980s, with the onset of the HIV/AIDS pandemic, Cryptosporidium emerged as one of the major causes of chronic diarrhea in these patients, often with a fatal outcome and becoming one of the most frequent opportunistic protozoa [7]. At the beginning of the 90s, a massive waterborne outbreak of cryptosporidiosis in Milwaukee (Wisconsin, USA) triggered concerns about Cryptosporidium as a significant waterborne pathogen [8]. All these data confirm the clinical significance and widespread distribution of cryptosporidiosis, recognizing its impact on human and animal health.

Taxonomy The genus Cryptosporidium belong to the phylum Apicomplexa and is composed of protozoan that infect epithelial cells in the microvillus border of the gastrointestinal tract of vertebrates [2••, 3]. Cryptosporidium taxonomy has been subject of debate, since this parasite possess features of both the coccidia and gregarines [2••, 4••]. Although it was previously included within the coccidia subclass, several characteristics distinguish it from this group of parasites. These include (1) an epicellular location and the presence of a feeding organelle (both shared with gregarines); (2) the production of two types of oocysts, thick-walled and thin-walled, the latter associated with autoinfection cycle; (3) small-size oocysts without sporocyst, micropyle, and polar granules; (4) lack of host specificity; and (5) resistance to anticoccidial drugs [2••, 4••, 9••]. In relation to gregarines, the similarities with Cryptosporidium have been supported with microscopic, molecular, genomic, and biochemical data [4••]. Molecular analysis of 18S rRNA and β-tubulin gene shows that Cryptosporidium is closely related to gregarines, also sharing (1) lack of apicoplast, (2) extracellular development with the presence of a gamont-like extracellular stage, (3) syzygy reproduction, and (4) myzocytosis nutrition, among others [9••, 10]. These findings influenced the reclassification of Cryptosporidium from subclass Coccidia, class Coccidiomorphea to a new subclass, Cryptogregaria, within class Gregarinomorphea [4••].

Cryptosporidium species are defined by oocyst morphometric characteristics: host specificity and molecular data [11]. So far, there are 31 valid species, which have been found in mammals, birds, amphibians, and reptiles [12]. According to its frequency, the human infective species are divided into two groups: major species (C. hominis and C. parvum) and minor species (C. meleagridis, C. cuniculus, C. felis, C. canis, C. muris, C. suis, and C. ubiquitum) [13]. There are more than 40 genotypes of Cryptospordium, which are not a valid taxonomic category [1]. This term is used as a temporary or partial descriptor of those isolates with undefined taxonomic status and 18S rRNA gene sequences different from those previously published. Eventually, in the future, many of these genotypes will reach the category of specie.

Lifecycle Cryptosporidium species have a monoxenous lifecycle completed within the gastrointestinal tract (mainly stomach and small intestine) of a single host [1]. Thick-walled sporulated oocysts are the infective and environmental-resistant stage of this protozoan. Once ingested, oocysts release motil sporozoites in the small intestine, where the enterocyte host apical membrane encapsulates them, forming an extra-cytoplasmic parasitophorous vacuole (epicellular) [14, 15•]. Actin filaments of the cell reorganize at the base of the vacuole, forming an electrodense band, known as a feeder organelle [16]. Excystation is associated with pH and temperature variations, bile salts, and pancreatic enzymes. Parasite molecules like serine and cysteine proteases, aminopeptidases, phospholipase A2, and heat shock proteins also stimulate the sporozoite liberation [16]. The internalized sporozoite matures to trophozoite, and the parasite undergoes multiple rounds of asexual multiplication (schizogony or merogony). Merogony I produce 6 to 8 merozoites, which can infect adjacent epithelial cells either developing to another trophozoite or into a type 2 meront (4 merozoites) [14, 15•, 16]. Upon infection of another cell, type II merozoites can enter in a sexual phase of the cycle, transforming themselves into gametocytes, a female macrogamont and a male microgamont. Microgamonts then release microgametes that fertilize a macrogamont. The resulting diploid zygote differentiates into four haploid sporozoites, and an oocyst wall is developed around them. Two types of oocyst are produced: thin-walled oocysts associated with the autoinfection cycle and thick-walled oocysts, excreted into the environment to facilitate parasite transmission to a new host [16]. Cryptosporidium is not an obligate epicellular parasite. In vitro studies describe a complete successful lifecycle free of cells in this protozoan, with the trophozoites as the dominant stage [9••, 10]. Novel extracellular stages like gamonts and gigantic gamont-like stage are also described. Their exact

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role is unknown. Some authors suggest that they could transform into merozoites and trophozoites, increasing the number of this form without going through a sexual phase [9••]. Cryptosporidium extracellular multiplication in biofilms and aquatic environments with the production of infective oocyst has been confirmed, exacerbating the waterborne transmission of the parasite [4••, 9••].

Clinical Manifestations Cryptosporidiosis clinical manifestations and severity depends on host factors such as age, nutritional and immune status, and parasite features, especially the virulence of the strain that causes the infection [15•, 17, 18]. Cryptosporidium is mainly found in the small intestine of immunocompetent hosts, but it can be detected throughout the gastrointestinal tract and even the respiratory tract (in cases of severe immunocompromised hosts) [17]. Self-limiting diarrhea occurs in immunocompetent individuals (9 to 15 days); however, the infection may be chronic and life-threatening to those that are immunosuppressed. Diarrhea is usually voluminous and watery, sometimes mucous but rarely bloody. Abdominal pain, nausea, vomiting, anorexia, weight loss, fever, and fatigue may be also present. A progression to a malabsorption syndrome can occur due to a decreased absorptive surface, contributing to the wasting syndrome seen in AIDS patients. Cryptosporidiosis pathology is associated with: 1—villus atrophy; 2—crypt hyperplasia; 3—leukocyte infiltration of the lamina propria; 4—chloride (Clˉ) hypersecretion; 5—glucose, D-xylose, and B12 vitamin malabsorption; and 6—reduced barrier function and downregulation of innate immune mechanisms such as defensin expression [17, 19]. Extraintestinal cryptosporidiosis has been described mainly in the hepatobiliary tract and pancreas and less often in lungs [17, 20]. These localizations probably are extensions of a primary intestinal infection.

Laboratory Diagnosis Cryptosporidium should be investigated in patients with acute, persistent, or chronic diarrhea, especially immunocompromised individuals and children. Differential diagnosis is usually made with other infectious agents such as Giardia intestinalis, Cyclospora, Cystoisospora, microsporidia, norovirus, rotavirus, Campylobacter, Salmonella, and Shigella, among others [17]. Several techniques have been developed for the diagnosis of Cryptosporidium. Current laboratory methods rely on microscopic examination of fecal samples for detecting the parasite oocysts by tinctorial (acidfast), fluorescent (auramine phenol), or immunofluorescent stains [19]. Modified acid-alcohol-resistant stain (Ziehl Neelsen modified or of Kinyoun) is the most widely used

[3]. Unfortunately, staining methods had a low sensitivity, so the evaluation of serial samples is required, and they cannot differentiate between the different species of this genus, since oocysts are morphologically indistinguishable. Antigen detection by ELISA and rapid immunochromatographic (strip) tests are available [3]. For epidemiological studies, serological tests are commonly used [5]. Molecular methods had greater sensitivity and specificity than microscopic traditional diagnostics. PCR-based procedures are increasingly used, and several platforms for rapid detection and characterization of microbial pathogens, like the FilmArray Gastrointestinal Panel (Biofire Diagnostics, Salt Lake City, UT, USA) and the xTAG Gastrointestinal Pathogen Panel (Luminex, Austin, TX, USA), include Cryptosporidium [21].

Epidemiology Cryptosporidiosis has a wide distribution, with cases in both developed and developing countries [7]. Prevalence data in humans vary according to the geographical area, climate (it is more common in tropical climates), and season (high frequency are described during the rainy season in many tropical countries), among others [7]. It is considered a neglected tropical disease [22], with a significant impact on public health, due to its high frequency in children from regions with low economic resources, the deleterious effect on cognitive functions and child growth, and the persistence and recurrence of such infections [23]. A prevalence of 2.2% (ranging from 0.26 to 22%) has been reported in symptomatic immunocompetent population from developed countries, being more frequent in children under 5 years old and young adults [24]. In developing countries, there is a prevalence of 6.1% (ranging from 1.4 to 41%), mainly in children under 1 year old [24]. According to the Global Enteric Multicenter Study (GEMS), Cryptosporidium is one of the main causes of persistent diarrhea in children in developing settings of sub-Saharan Africa and parts of Asia [3]. It was the second-leading pathogen causing life-threatening diarrhea in children under 2 years of age [14]. Parasite surveillance in AIDS individuals with diarrhea report a prevalence of 24% (ranging from 8.7 to 48%) in patients from developing countries and 14% (ranging from 6 to 70%) in high-income countries [24]. Cryptosporidium species have emerged as major waterborne pathogens causing gastroenteritis in humans. Outbreaks have been reported worldwide, which have been attributed to a combination of disinfectant resistance of oocysts and water treatment deficiencies [25]. World Health Organization (WHO) included Cryptosporidium in the list of drinking water contaminant candidate lists and drinking water contaminant list of the U.S. Environmental Protection Agency—EPA [25]. Increasing vigilance and improvement in the water monitoring of the public water supply are required

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to reduce the parasite water transmission. Application of antiretroviral therapy and implementation of new methods of treatment of drinking water (including ultraviolet radiation and ozone) have contributed to reduce the impact of cryptosporidiosis in the developed world; on the contrary, this protozoan disease still represent a public health problem in third world countries.

Treatment Currently, antiparasitic treatment options for cryptosporidiosis are limited and there is no effective drug for this protozoa [15•, 26]. Nitazoxanide is the only U.S. Food and Drug Administration (FDA) approved for treatment of Cryptosporidium in children older than 1 year. Unfortunately, this medicine has limited or no efficacy in compromised or malnourished hosts, including AIDS and transplanted patients [15•, 26]. Other drugs reported to have some effect in case series include paromomycin, rifamycin, azithromycin, spiramycin, HIV protease inhibitors, and bovine anti-cryptosporidium immunoglobulin [15•, 26]. However, all were ineffective in controlled trials in AIDS patients. In these patients, treatment relies on improvement of the symptoms, since complete clearance of the parasite is unlikely unless the underlying immune deficiency is resolved.

Epidemiology of Cryptosporidium in Colombia Methods Search Strategy and Study Selection A systematic review was performed following an electronic search of Medline-Pubmed, Scielo, Elsevier, Scopus, Embase, and Lilacs databases. Spanish, English, and Portuguese articles were evaluated using the terms “Cryptosporidium,” “Cryptosporidiosis,” “Intestinal parasites,” and “Colombia.” An exhaustive literature review was guaranteed by the application of 15 search strategies, resulting from the combination of the selected keywords. Study reproducibility was achieved following the identification, screening, eligibility, and inclusion phases of the PRISMA (Preferred Reporting Items for Systematic reviews and Meta-Analyses) guidelines. After removing the duplicate records, publications screening was carried out with the following inclusion criteria: (i) original articles and (ii) study population, sample and diagnosis technique specified. Exclusion criteria applied were (i) reviews, editorials, national epidemiological surveys, letters to the editors, and abstracts from conferences; (ii) articles evaluating the

same population; and (iii) articles with no clear methodology or results. Data Extraction and Information Analysis Original data were retrieved independently from the eligible publications by two investigators. Full text of the relevant articles was assessed. The following information was extracted from each study (PRISMA third phase): title, surname of first author, year of publication, state or region of the study, study population (human, animals, or environmental), sample size, frequency of positive samples, diagnosis technique, and source of the sample evaluated. During data extraction, a quality methodological and editorial evaluation of the systematized publications was done. Cryptosporidium prevalence was estimated in the total human and animal population. For subgroup analysis, we categorized global data. In the case of humans, specific prevalence was determined for HIV and other immunocompromised adult patients, children, and general population. For animals, prevalence was established separately for bovines, cats, dogs, and monkeys. Finally, water frequency contamination was also calculated.

Results and Discussion Five hundred and sixty-nine studies published between 1985 and 2018 were identified using the search strategies described above. After reading the abstracts, titles, and full-text articles, 42 studies were selected for the systematic review. Information describing the studies analyzed is summarized in Table 1. Twenty-three studies focused on humans, 13 on animals, 5 on environmental samples, and one on the diagnosis test evaluation applied to samples from different sources. Human studies analyzed 7066 individuals. Using direct diagnosis techniques, 5288 stool samples were evaluated for the parasite presence, establishing a Cryptosporidium prevalence of 7.8%, with children and immunocompromised patients being the most affected population (Table 2). Although symptoms and risk factors were assessed, a few investigations found a statistical association with the Cryptosporidium infection. These variables included the presence of diarrhea, fever, chronic malnutrition, and children attending day-care centers and with severe HIV stage infection and contact with animals, among others. One seroepidemiological study was performed on general population of the Andean region (1778 subjects), with a seroprevalence of 83.3%, which indicates the high level of exposition to this protozoan, and suggests endemicity in the studied area. Animal cryptosporidiosis studies in Colombia evaluated 1845 samples. Cryptosporidium prevalence by direct diagnosis techniques was 20.4% (Table 2), with cattle and calves as the most frequently studied and infected population, confirming it as an important host for this protozoan and a

Curr Trop Med Rep Table 1

List of 42 studies included in the final analysis

Author

Year

Studied region

Study design

Study population

Reference

Ángel V.

1985

Medellín

Descriptive

[27]

Zaraza C. Soto M.

1994 1997

Bucaramanga Medellín

Descriptive Case report

Patients with diarrheal disease (All age groups) All age groups Adult HIV/AIDS patient

[28] [29]

Vergara C.

2000

Descriptive

General population

[30]

Humans

Flórez A.

2001

Multicentric (Four city capitals of the Andean region) Bogotá

Descriptive

HIV patients

[31]

Botero J.

2003

Medellín

Descriptive

[32]

Arzuza O. Botero J.

2003 2004

Cartagena Medellín

Descriptive Descriptive

Immunocompromised patients (acute lymphoid leukemia, chronic myeloid leukemia, HIV, other immunodeficiencies) HIV patients HIV patients

[33] [34]

Carreño M.

2005

Bucaramanga

Descriptive

Children with and without cancer

[35]

Navarro L. Rivera O. De Arango M. Siuffi M. De la Ossa N.

2006 2006 2006 2006 2007

Descriptive Case report Descriptive Descriptive Descriptive

HIV patients AIDS patient Children HIV children General population

[36] [37] [38] [39] [40]

Velasco C.

2011

Descriptive

HIV/AIDS children

[41]

Vélez D. Bayona M.

2011 2011

Descriptive Descriptive

Children Children

[42] [43]

Lucero T. Agudelo S. Isaza J.

2015 2015 2015

Medellín Popayán Arauca Cali Multicentric (Barranquilla district and nearby municipalities) Multicentric (Cali and other municipalities from Colombian southwest) Cali Multicentric (Sabana Center area from Cundinamarca). Florencia Neiva Medellín

Descriptive Descriptive Genomic study

[44] [45] [46•]

Montúfar F.

2016

Medellín

Descriptive

Children HIV patients C. parvum and C. hominis isolates HIV patients

Sánchez A.

2017

Descriptive

Children

[48]

Gaviria L. Animals Vergara C.

2017

Multicentric (several municipalities from Amazonas) Caldono (Cauca)

Descriptive

Children

[49]

Viability and infectivity assays

[50]

Vergara C. Santín M. Rodríguez E. Avendaño C.

2001 2006 2009 2010

Descriptive Descriptive Descriptive Descriptive

Bovine isolates from Colombia and Spain Adult cattle Cats Dogs Calves

[51] [52] [53] [54]

Montaño J.

2012

Descriptive

Calves

[55]

Hernández N. Ocampo R. Pardo D.

2012 2012 2012

Descriptive Descriptive Descriptive

Calves Calves Calves

[56] [57] [58]

Montoya C. Cadavid D. Pulido M.

2013 2014 2014

Descriptive Descriptive Descriptive

Red howler monkeys Calves Adult cattle and calves

[59] [60] [61]

2000 Cundinamarca Bogotá Tunja (Boyacá) Multicentric (Ubaté-Chinquinquirá valley) Multicentric (Chinquinquirá valley) Bogotá Manizales Multicentric (several municipalities from Bogota savanna) Jericó (Antioquia) Belmira (Antioquia) Multicentric (Boyaca)

[47]

Curr Trop Med Rep Table 1 (continued) Author

Year

Studied region

Study design

Study population

Reference

Avendaño C.

2018

Multicentric (central area of Colombia)

Descriptive

Calves

[62]

Water samples Alarcón M.

2005

Bogotá

Descriptive

[63]

Campos C.

2008

Bogotá

Descriptive

Surface water from Bogota river and drinking water plants Surface water from Bogota river

Rodríguez D.

2012

Descriptive

Triviño J.

2016

Multicentric (northeast of Antioquia) Quindío

Lora F.

2016

Quindío

Descriptive

Descriptive

[64]

Supply water and wastewater from dairy farms Surface, treated and network water Raw and drinkable water in three different water treatment plants

[65]

Stool samples from humans, calves, dogs and rabbits; water samples

[68]

[66] [67]

Humans, animals and water samples Ocampo R.

2011

Manizales

Diagnosis test evaluation

reservoir for human infection. The most frequent variables significantly associated with the infection were diarrhea and age (under 21 days of birth). Similar to human population, a high seroprevalence was observed on cattle from different farms from the central area of Colombia. These animals were also evaluated for the presence of the parasite in the stool, but none of them was positive, which can be explained by the age of the population, since Cryptosporidium is more frequent in young calves. Dogs, cats, and monkeys were analyzed, with prevalence data of 17.4, 13, and 14.3%, respectively. These results indicate that both domestic and wild animals are infected with Cryptosporidium, contributing to the oocyst environmental contamination and facilitating the transmission cycle of this parasite.

Table 2 Cryptosporidium prevalence in humans, animals, and water samples from Colombiaa

Population Humans Total human population General population Adult immunocompromised patientsb Childrenc Animals Total animal population Cattle and calves Cats Dogs Monkeys Water sources a

In relation to water, a Cryptosporidium frequency of 38.9% was founded (116 positive samples out of 298 evaluated) (Table 2). These included surface water from rivers, wastewater, raw, and treated water from different drinking water treatment plants. The high level of Cryptosporidium water contamination in the regions studied favors a greater exposure to the parasite for both animals and humans, since they are used for agricultural and domestic activities. In only one study the oocyst viability was determined, with one positive sample in a treated water sample from a DWTP (drinking water treatment plant), which indicates the need to implement the search of this protozoa in the quality control of water and improve the treatment protocols applied to its elimination in water used for human consumption.

Total (n)

Positive samples (n)

Prevalence (%)

5288 2861 915 1512

412 23 91 298

7.8 0.73 9.9 19.7

1845 1646 46 132 21 298

377 345 6 23 3 116

20.4 21 13.0 17.4 14.3 38.9

Seroepidemiological studies were not included in this table

b

HIV, oncologic diseases, hematological disorders, and immunodeficiencies

c

Both immunocompetent and immunocompromised children were included in this category

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Two case reports in HIV/AIDS patients were included in this review. Patients lived in Medellin (Antioquia State) and Popayán (Cauca State). Both showed intestinal manifestations, mainly chronic diarrhea, and had a CD4 count bellow 50 cells/μL. Additional respiratory symptoms were present in one of the patients, reflecting the dissemination potential of Cryptosporidium in severe immunocompromised patients. Finally, there were two analytical investigations on Cryptosporidium in Colombia. One of them evaluated the viability and infectivity of two Cryptosporidium bovine isolates from Colombia and Spain, in order to perform a comparative analysis of phenotypic variability between isolates from different geographic locations. In the second study, nextgeneration technology for the analysis of genomic DNA and RNA was applied for the generation of a new reference genome sequence of a Colombian C. hominis isolate and a new genome annotation of the C. parvum Iowa reference genome. This investigation demonstrates the access to high-throughput sequencing technologies and experience in the use of bioinformatic tools in Colombia, promoting the development of new research in the genomics of this apicomplexan. In silico analyses could be useful in the designing of new molecular diagnosis tests, novel antigenic targets as possible candidates

Table 3

for vaccines, and/or new drugs, a crucial aspect in the management of cryptosporidiosis. Detection techniques used in the evaluation of cryptosporidiosis in Colombia varied according to the sample and population analyzed (Table 3). Stain protocols were used in almost all the studies in humans and animals, with the modified Ziehl Neelsen stain as the most frequent. Other procedures include Ziehl Neelsen and Heine staining. Antigen detection by immunologic methods was applied in studies on humans (three studies), animals (two studies), and water samples (four studies). ELISA, immunofluorescence, and immunochromatography assays were used. Molecular methods, including conventional and real-time PCR, were described in eight publications, with animal samples the most frequently analyzed by these techniques (four studies). Two studies in humans and two in water samples included PCR. DNA sequence targets commonly used included the 18S rRNA gene, Cryptosporidium oocyst wall protein (COWP) gene, heat shock protein (HSP70) gene, and 60 kDa glycoprotein (GP60) gene. Some protocols had a previous concentration method, like formol ether for stool samples and filtration or flocculation for water samples. Since most of

Cryptosporidium diagnosis techniques used in different studies in Colombia

Technique

Samples

Target

Number of publications

Staining methods Ziehl Neelsen Modified Ziehl Neelsen

Human and animal stool Human and animal stool

Oocyst Oocyst

3 25

Human and animal stool

Oocyst

2

Human and animal stool Human stool Human stool and water samples Human and animal sera Animal sera

Antigens Antigens Antigens

3 1 5

Immunoglobulins Immunoglobulins

2 1

Human and animal stool; water samples

18S rRNA COWP GP60 HSP70

6

Human stool and water samples Human stool

DNA J-like protein COWP

2

Heine Immunologic methods ELISA Immunochromatography Immunofluorescence ELISA Western blot Molecular methods Conventional PCR

Real-time PCR Next-generation sequencing technology

1

Species/subtypes

C. hominis (IdA19, IaA12R8, IeA11G3T3) C. parvum (IIcA5G3c, IIaA15G2R1, IIaA16G2R1, IIaA17G4R1, IIaA18G5R1, IIaA19G6R1, IIaA20G5R1, IIaA20G6R1, IIaA20G7R1) C. viatorum C. bovis C. felis C. muris

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the prevalence studies in Colombia relied on staining techniques, which are the less sensitive detection methods, published data probably underestimate the real frequency of this protozoa. Data on Cryptosporidium species distribution in Colombia reveals that C. parvum and C. hominis are the most frequent circulating species in animals, humans, and water samples (Table 3). One study detected the presence of C. viatorum among indigenous children, a globally distributed pathogenic species of Cryptosporidium that has been mainly reported in humans. This is the first record in human population from Colombia. Other Cryptosporidium circulating species are described in Table 3. In relation to Cryptosporidium subtype information, a few studies included the analysis of the genetic variation of the species circulating in Colombia. Only C. hominis and C. parvum subtypes were evaluated. Three subtypes of C. hominis (IdA19, IaA12R8 and IeA11G3T3) and one for C. parvum (IIcA5G3c) were described for human isolates. A higher genetic diversity of C. parvum was observed in animals, with eight subtypes identified in calves (IIaA15G2R1, IIaA16G2R1, IIaA17G4R1, IIaA18G5R1, IIaA19G6R1, IIaA20G5R1, IIaA20G6R1, IIaA20G7R1). In Colombia, molecular data on Cryptosporidium genetic variability are scarce, and more information could be useful for a better understanding of the epidemiology of cryptosporidiosis in different geographical regions of the country, including the genetic structure of its species, transmission dynamics, susceptible host and reservoirs, and the identification of infectious sources, mainly in outbreaks. Regarding geographic distribution of Cryptosporidium in Colombia, it should be pointed out that 36 of the 42 studies analyzed in this review were carried out in cities and provinces of the Andean region, one of the six natural regions of the country, which is the most populated and has the majority of urban centers of Colombia. Industry, agriculture, and livestock activities are major activities in this region. Five studies were conducted in the Caribbean (two studies), Orinoquía (one study), and Amazon region (two studies), all of them focused on human population. Although less populated, some provinces of these regions have sociodemographic characteristics like poverty, deficient public services, and poor hygienic sanitary conditions that could be associated with a high risk of Cryptosporidium and other intestinal parasite infections; therefore, more investigations about the current situation of cryptosporidiosis are essential in these areas.

Conclusion The impact of cryptosporidiosis in Colombian population, including HIV/AIDS patients and children, is probably underestimated. One reason could be that diagnostic protocols for infectious diarrhea do not include the screening of the

parasite, and when searched, low sensitive techniques are used. High prevalence on animals, mainly livestock, suggests the parasite importance as a source of economic losses due to the disease and confirms these hosts as reservoirs for environmental contamination and human infection. More studies on the prevalence, clinical, immunological, and sociodemographic profile of this infection are required in the country. New data can influence the application of better diagnosis strategies and the implementation of appropriate prevention and control practices for this parasite. Funding Information The author gratefully acknowledges the Committee for the Research Development (Comité para el Desarrollo de la Investigación-CODI) of the Universidad de Antioquia by supporting this investigation through the Fund for the First Professor Project grant (Code 2015-7020).

Compliance with Ethical Standards Conflict of Interest interest.

The author declares that she has no conflict of

Human and Animal Rights and Informed Consent This article does not contain any studies with human or animal subjects performed by any of the authors.

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