Current Tropical Medicine Reports (2018) 5:144–153

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

Published online: 19 May 2018

# 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 prin-
cipal 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 frequently identified protozoa in waterborne enteric outbreaks

Cryptosporidium is a ubiquitous enteric protozoan that infect
a broad range of vertebrates (humans, domestic and wild an-
imals) [1]. Environmental-resistant oocyst can be acquired
through a variety of transmission routes including direct con-
tact with infected persons (person-to-person transmission),
contact with animals (zoonotic transmission), and ingestion
of contaminated food (foodborne transmission) or water (wa-
terborne transmission) [2••, 3]. Cryptosporidium is the most
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 cryp-
tosporidiosis, including the high oocyst resistance to water
disinfection treatments, the absence of an effective drug treat-
ment, and the lack of a vaccine [3].

This article is part of the Topical Collection on Tropical Diseases History

in Colombia

Edward Tyzzer (1875–1965) originally described the genus

* Ana Luz Galván-Díaz
ana.galvan@udea.edu.co
Cryptosporidium in 1907. He found the parasite in the gastric
glands of common mice (Mus musculus), and proposed the
1
Escuela de Microbiología, Grupo de Microbiología ambiental, specie Cryptosporidium muris (Tyzzer, 1907) [6]. He placed it
Universidad de Antioquia, Building 5-410, Medellín, Colombia in the coccidian family Asporocystidae, whose members lack
Curr Trop Med Rep (2018) 5:144–153
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 inves-
tigations. 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 dis-
tribution 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 epithe-
lial 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 previ-
ously included within the coccidia subclass, several character-
istics distinguish it from this group of parasites. These include
(1) an epicellular location and the presence of a feeding or-
ganelle (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, mo-
lecular, genomic, and biochemical data [4••]. Molecular anal-
ysis 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 re-
production, 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••].
145
Cryptosporidium species are defined by oocyst morpho-
metric 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 taxo-
nomic 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 previous-
ly published. Eventually, in the future, many of these geno-
types will reach the category of specie.
Lifecycle
Cryptosporidium species have a monoxenous lifecycle com-
pleted 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 sporozo-
ites in the small intestine, where the enterocyte host apical
membrane encapsulates them, forming an extra-cytoplasmic
parasitophorous vacuole (epicellular) [14, 15•]. Actin fila-
ments 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, phospholi-
pase A2, and heat shock proteins also stimulate the sporozoite
liberation [16]. The internalized sporozoite matures to tropho-
zoite, and the parasite undergoes multiple rounds of asexual
multiplication (schizogony or merogony). Merogony I pro-
duce 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 sporo-
zoites, 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, excret-
ed 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 domi-
nant stage [9••, 10]. Novel extracellular stages like gamonts
and gigantic gamont-like stage are also described. Their exact
146
role is unknown. Some authors suggest that they could trans-
form 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 chron-
ic 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 progres-
sion 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) hypersecre-
tion; 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 immunocom-
promised 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 lab-
oratory methods rely on microscopic examination of fecal
samples for detecting the parasite oocysts by tinctorial (acid-
fast), fluorescent (auramine phenol), or immunofluorescent
stains [19]. Modified acid-alcohol-resistant stain (Ziehl
Neelsen modified or of Kinyoun) is the most widely used
Curr Trop Med Rep (2018) 5:144–153
[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 detec-
tion by ELISA and rapid immunochromatographic (strip) tests
are available [3]. For epidemiological studies, serological tests
are commonly used [5]. Molecular methods had greater sen-
sitivity and specificity than microscopic traditional diagnos-
tics. 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 coun-
tries), 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 infec-
tions [23]. A prevalence of 2.2% (ranging from 0.26 to 22%)
has been reported in symptomatic immunocompetent popula-
tion from developed countries, being more frequent in children
under 5 years old and young adults [24]. In developing coun-
tries, 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 di-
arrhea in children under 2 years of age [14]. Parasite surveil-
lance 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 water-
borne pathogens causing gastroenteritis in humans.
Outbreaks have been reported worldwide, which have been
attributed to a combination of disinfectant resistance of oo-
cysts 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
Curr Trop Med Rep (2018) 5:144–153
to reduce the parasite water transmission. Application of anti-
retroviral therapy and implementation of new methods of
treatment of drinking water (including ultraviolet radiation
and ozone) have contributed to reduce the impact of crypto-
sporidiosis in the developed world; on the contrary, this pro-
tozoan disease still represent a public health problem in third
world countries.
Treatment
Currently, antiparasitic treatment options for cryptosporid-
iosis 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 inhibi-
tors, and bovine anti-cryptosporidium immunoglobulin
[15•, 26]. However, all were ineffective in controlled trials
in AIDS patients. In these patients, treatment relies on im-
provement of the symptoms, since complete clearance of
the parasite is unlikely unless the underlying immune de-
ficiency 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 arti-
cles were evaluated using the terms “Cryptosporidium,”
“Cryptosporidiosis,” “Intestinal parasites,” and “Colombia.”
An exhaustive literature review was guaranteed by the appli-
cation of 15 search strategies, resulting from the combination
of the selected keywords. Study reproducibility was achieved
following the identification, screening, eligibility, and inclu-
sion phases of the PRISMA (Preferred Reporting Items for
Systematic reviews and Meta-Analyses) guidelines. After re-
moving the duplicate records, publications screening was car-
ried out with the following inclusion criteria: (i) original arti-
cles and (ii) study population, sample and diagnosis technique
specified. Exclusion criteria applied were (i) reviews, edito-
rials, national epidemiological surveys, letters to the editors,
and abstracts from conferences; (ii) articles evaluating the
147
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 extract-
ed 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 qual-
ity methodological and editorial evaluation of the systema-
tized 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 oth-
er immunocompromised adult patients, children, and general
population. For animals, prevalence was established separate-
ly for bovines, cats, dogs, and monkeys. Finally, water fre-
quency 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 evalua-
tion 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 chil-
dren 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 chil-
dren attending day-care centers and with severe HIV stage in-
fection and contact with animals, among others. One
seroepidemiological study was performed on general popula-
tion of the Andean region (1778 subjects), with a seropreva-
lence 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 diagno-
sis 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
148 Curr Trop Med Rep (2018) 5:144–153

Table 1 List of 42 studies included in the final analysis

Author Year Studied region Study design Study population Reference

Humans
Ángel V. 1985 Medellín Descriptive Patients with diarrheal disease [27]
(All age groups)
Zaraza C. 1994 Bucaramanga Descriptive All age groups [28]
Soto M. 1997 Medellín Case report Adult HIV/AIDS patient [29]
Vergara C. 2000 Multicentric (Four city Descriptive General population [30]
capitals of the Andean region)
Flórez A. 2001 Bogotá Descriptive HIV patients [31]
Botero J. 2003 Medellín Descriptive Immunocompromised patients [32]
(acute lymphoid leukemia,
chronic myeloid
leukemia, HIV, other
immunodeficiencies)
Arzuza O. 2003 Cartagena Descriptive HIV patients [33]
Botero J. 2004 Medellín Descriptive HIV patients [34]
Carreño M. 2005 Bucaramanga Descriptive Children with and without cancer [35]
Navarro L. 2006 Medellín Descriptive HIV patients [36]
Rivera O. 2006 Popayán Case report AIDS patient [37]
De Arango M. 2006 Arauca Descriptive Children [38]
Siuffi M. 2006 Cali Descriptive HIV children [39]
De la Ossa N. 2007 Multicentric (Barranquilla district Descriptive General population [40]
and nearby municipalities)
Velasco C. 2011 Multicentric (Cali and other Descriptive HIV/AIDS children [41]
municipalities from
Colombian southwest)
Vélez D. 2011 Cali Descriptive Children [42]
Bayona M. 2011 Multicentric (Sabana Center area Descriptive Children [43]
from Cundinamarca).
Lucero T. 2015 Florencia Descriptive Children [44]
Agudelo S. 2015 Neiva Descriptive HIV patients [45]
Isaza J. 2015 Medellín Genomic study C. parvum and [46•]
C. hominis isolates
Montúfar F. 2016 Medellín Descriptive HIV patients [47]
Sánchez A. 2017 Multicentric (several Descriptive Children [48]
municipalities from
Amazonas)
Gaviria L. 2017 Caldono (Cauca) Descriptive Children [49]
Animals
Vergara C. 2000 Viability and infectivity assays Bovine isolates from [50]
Colombia and Spain
Vergara C. 2001 Cundinamarca Descriptive Adult cattle [51]
Santín M. 2006 Bogotá Descriptive Cats [52]
Rodríguez E. 2009 Tunja (Boyacá) Descriptive Dogs [53]
Avendaño C. 2010 Multicentric Descriptive Calves [54]
(Ubaté-Chinquinquirá valley)
Montaño J. 2012 Multicentric Descriptive Calves [55]
(Chinquinquirá valley)
Hernández N. 2012 Bogotá Descriptive Calves [56]
Ocampo R. 2012 Manizales Descriptive Calves [57]
Pardo D. 2012 Multicentric (several Descriptive Calves [58]
municipalities from
Bogota savanna)
Montoya C. 2013 Jericó (Antioquia) Descriptive Red howler monkeys [59]
Cadavid D. 2014 Belmira (Antioquia) Descriptive Calves [60]
Pulido M. 2014 Multicentric (Boyaca) Descriptive Adult cattle and calves [61]
Curr Trop Med Rep (2018) 5:144–153 149

Table 1 (continued)

Author Year Studied region Study design Study population Reference

Avendaño C. 2018 Multicentric (central area Descriptive Calves [62]

of Colombia)
Water samples
Alarcón M. 2005 Bogotá Descriptive Surface water from Bogota river [63]
and drinking water plants
Campos C. 2008 Bogotá Descriptive Surface water from Bogota river [64]
Rodríguez D. 2012 Multicentric (northeast Descriptive Supply water and wastewater [65]
of Antioquia) from dairy farms
Triviño J. 2016 Quindío Descriptive Surface, treated and [66]
network water
Lora F. 2016 Quindío Descriptive Raw and drinkable water in three [67]
different water treatment plants
Humans, animals and water samples
Ocampo R. 2011 Manizales Diagnosis test evaluation Stool samples from humans, [68]
calves, dogs and rabbits;
water samples

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 infect-
ed with Cryptosporidium, contributing to the oocyst environ-
mental contamination and facilitating the transmission cycle
of this parasite.
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, wastewa-
ter, raw, and treated water from different drinking water treat-
ment plants. The high level of Cryptosporidium water con-
tamination 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.

Table 2 Cryptosporidium
prevalence in humans, animals, Population Total (n) Positive samples (n) Prevalence (%)
and water samples from
Colombiaa Humans
Total human population 5288 412 7.8
General population 2861 23 0.73
Adult immunocompromised patientsb 915 91 9.9
Childrenc 1512 298 19.7
Animals
Total animal population 1845 377 20.4
Cattle and calves 1646 345 21
Cats 46 6 13.0
Dogs 132 23 17.4
Monkeys 21 3 14.3
Water sources 298 116 38.9
a
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
150 Curr Trop Med Rep (2018) 5:144–153

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 manifesta-
tions, 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 iso-
lates from Colombia and Spain, in order to perform a compar-
ative analysis of phenotypic variability between isolates from
different geographic locations. In the second study, next-
generation technology for the analysis of genomic DNA and
RNA was applied for the generation of a new reference ge-
nome 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 bioin-
formatic 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
for vaccines, and/or new drugs, a crucial aspect in the man-
agement of cryptosporidiosis.
Detection techniques used in the evaluation of crypto-
sporidiosis 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 stain-
ing. Antigen detection by immunologic methods was ap-
plied in studies on humans (three studies), animals (two
studies), and water samples (four studies). ELISA, immu-
nofluorescence, 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 tar-
gets 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 concentra-
tion method, like formol ether for stool samples and fil-
tration or flocculation for water samples. Since most of

Table 3 Cryptosporidium diagnosis techniques used in different studies in Colombia

Technique Samples Target Number of Species/subtypes

publications

Staining methods
Ziehl Neelsen Human and animal stool Oocyst 3
Modified Ziehl Neelsen Human and animal stool Oocyst 25
Heine Human and animal stool Oocyst 2
Immunologic methods
ELISA Human and animal stool Antigens 3
Immunochromatography Human stool Antigens 1
Immunofluorescence Human stool and Antigens 5
water samples
ELISA Human and animal sera Immunoglobulins 2
Western blot Animal sera Immunoglobulins 1
Molecular methods
Conventional PCR Human and animal stool; 18S rRNA 6 C. hominis (IdA19, IaA12R8,
water samples COWP IeA11G3T3)
GP60 C. parvum (IIcA5G3c,
HSP70 IIaA15G2R1, IIaA16G2R1,
IIaA17G4R1, IIaA18G5R1,
IIaA19G6R1, IIaA20G5R1,
IIaA20G6R1, IIaA20G7R1)
C. viatorum
C. bovis
C. felis
C. muris
Real-time PCR Human stool and DNA J-like protein 2
water samples COWP
Next-generation sequencing technology Human stool 1
Curr Trop Med Rep (2018) 5:144–153
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 de-
scribed 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 sub-
types 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 vari-
ability 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, sus-
ceptible host and reservoirs, and the identification of infec-
tious 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 prov-
inces of these regions have sociodemographic characteristics
like poverty, deficient public services, and poor hygienic san-
itary 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, in-
cluding 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
151
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 prev-
alence, clinical, immunological, and sociodemographic profile
of this infection are required in the country. New data can in-
fluence 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 The author declares that she has no conflict of
interest.
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.
References
Papers of particular interest, published recently, have been
highlighted as:
• Of importance
•• Of major importance
1. Khan A, Shaik JS, Grigg ME. Genomics and molecular epidemiol-
ogy of Cryptosporidium species. Acta Trop. 2017:1–14. https://doi.
org/10.1016/j.actatropica.2017.10.023.
2.•• Thompson RCA, Koh WH, Clode PL. Cryptosporidium—what is
it? Food Waterborne Parasitol. 2016;4:54–61. https://doi.org/10.
1016/j.fawpar.2016.08.004. A very good review on the new
taxonomic data of Cryptosporidium.
3. Ryan U, Zahedi A, Paparini A. Cryptosporidium in humans and
animals—a one health approach to prophylaxis. Parasite Immunol.
2016;38(9):535–47.
4.•• Ryan U, Paparini A, Monis P, Hijjawi N. It’s official—
Cryptosporidium is a gregarine: what are the implications for the
water industry? Water Res. 2016;105:305–13. https://doi.org/10.
1016/j.watres.2016.09.013. Excellent review on the
implications of Cryptosporidium as a gregarine.
5. Checkley W, White AC, Jaganath D, Arrowood MJ, Chalmers RM,
Chen X-M, et al. A review of the global burden, novel diagnostics,
therapeutics, and vaccine targets for cryptosporidium. Lancet Infect
Dis. 2015;15(1):85–94. https://doi.org/10.1016/S1473-3099(14)
70772-8.
6. Tzipori S, Griffiths JK. Natural history and biology of
Cryptosporidium parvum. Adv Parasitol. 1998;40:5–36.
7. Tzipori S, Widmer G. A hundred-year retrospective on cryptospo-
ridiosis. Trends Parasitol. 2008;24:184–9. https://doi.org/10.1016/j.
pt.2008.01.002.
152
8. Mac Kenzie WR, Hoxie NJ, Proctor ME, Gradus MS, Blair KA,
Peterson DE, et al. A massive outbreak in Milwaukee of
Cryptosporidium infection transmitted through the public water
supply. N Engl J Med. 1994;331(3):161–7. https://doi.org/10.
1056/NEJM199407213310304.
9.•• Clode PL, Koh WH, Thompson RCA. Life without a host cell:
what is Cryptosporidium? Trends Parasitol. 2015;31(12):614–24.
https://doi.org/10.1016/j.pt.2015.08.005. Excellent review on
Cryptosporidium host cell-free life cycle.
10. Aldeyarbi HM, Karanis P. The ultra-structural similarities between
Cryptosporidium parvum and the gregarines. J Eukaryot Microbiol.
2016;63(1):79–85. https://doi.org/10.1111/jeu.12250.
11. Xiao L. Molecular epidemiology of cryptosporidiosis: an update.
Exp Parasitol. 2010;124(1):80–9. https://doi.org/10.1016/j.exppara.
2009.03.018.
12. Certad G, Viscogliosi E, Chabé M, Cacciò SM. Pathogenic mech-
anisms of Cryptosporidium and Giardia. Trends Parasitol.
2017;33(7):561–76. https://doi.org/10.1016/j.pt.2017.02.006.
13. Šlapeta J. Cryptosporidiosis and Cryptosporidium species in animals
and humans: a thirty colour rainbow? Int J Parasitol. 2013;43(12–13):
957–70. https://doi.org/10.1016/j.ijpara.2013.07.005.
14. Ryan U, Hijjawi N. New developments in Cryptosporidium re-
search. Int J Parasitol. 2015;45(6):367–73. https://doi.org/10.
1016/j.ijpara.2015.01.009.
15.• Manjunatha UH, Chao AT, Leong FJ, Diagana TT. Cryptosporidiosis
drug discovery: opportunities and challenges. ACS Infect Dis.
2016;2(8):530–7. https://doi.org/10.1021/acsinfecdis.6b00094.
Review on terapeutic alternatives for Cryptosporidium.
16. Lendner M, Daugschies A. Cryptosporidium infections: molecular
advances. Parasitology. 2014;141(11):1511–32. https://doi.org/10.
1017/S0031182014000237.
17. Chalmers RM, Davies AP. Minireview: clinical cryptosporidiosis.
Exp Parasitol. 2010;124(1):138–46. https://doi.org/10.1016/j.
exppara.2009.02.003.
18. Ryan U, Fayer R, Xiao L. Cryptosporidium species in humans
and animals: current understanding and research needs.
Parasitology. 2014;141(13):1667–85. https://doi.org/10.1017/
S0031182014001085.
19. Bouzid M, Hunter PR, Chalmers RM, Tyler KM. Cryptosporidium
pathogenicity and virulence. Clin Microbiol Rev. 2013;26(1):115–
34. https://doi.org/10.1017/S0031182009991132.
20. Sponseller JK, Griffiths JK, Tzipori S. The evolution of respiratory
cryptosporidiosis: evidence for transmission by inhalation. Clin
Microbiol Rev. 2014;27(3):575–86. https://doi.org/10.1128/CMR.
00115-13.
21. Freeman K, Mistry H, Tsertsvadze A, Royle P, McCarthy N,
Taylor-Phillips S, et al. Multiplex tests to identify gastrointestinal
bacteria, viruses and parasites in people with suspected infectious
gastroenteritis: a systematic review and economic analysis. Health
Technol Assess (Rockv). 2017;21(23):1–188. https://doi.org/10.
3310/hta21230.
22. Savioli L, Smith H, Thompson A. Giardia and Cryptosporidium
join the “neglected diseases initiative”. Trends Parasitol.
2006;22(5):203–8. https://doi.org/10.1016/j.pt.2006.02.015.
23. Siwila J, Phiri IGK, Enemark HL, Nchito M, Olsen A. Seasonal
prevalence and incidence of Cryptosporidium spp. and Giardia
duodenalis and associated diarrhoea in children attending pre-
school in Kafue, Zambia. Trans R Soc Trop Med Hyg.
2011;105(2):102–8. https://doi.org/10.1016/j.trstmh.2010.10.004.
24. Medema G, Teunis P, Blokker M, Deere D, DAvison A, Charles P,
Loret JF. WHO guidelines for drinking water quality.
Cryptosporidium. 2006. Available from: http://www.who.int/
water_sanitation_health/gdwqrevision/cryptodraft2.pdf.
25. Galván AL, Magnet A, Izquierdo F, Fernández Vadillo C, Peralta
RH, Angulo S, et al. A year-long study of Cryptosporidium species
and subtypes in recreational, drinking and wastewater from the
Curr Trop Med Rep (2018) 5:144–153
central area of Spain. Sci Total Environ. 2013;468–469:368–75.
https://doi.org/10.1016/j.scitotenv.2013.08.053.
26. Sparks H, Nair G, Castellanos-Gonzalez A, White AC. Treatment
of Cryptosporidium: what we know, gaps, and the way forward.
Curr Trop Med Rep. 2015;2(3):181–7. https://doi.org/10.1007/
s40475-015-0056-9.
27. Angel VE, Franco L, Jaramillo JC, Medina LA, Ochoa FL, Velez
AM, et al. Prevalencia de Cryptosporidium en muestras fecales
diarreicas en 6 laboratorios de Medellín. Biomedica. 1985;5(3–4):
53–61.
28. Zaraza C, Díaz L, Vargas L, Santos R, Sepúlveda J, Pinto M, et al.
Prevalencia de Cryptosporidium en muestras de materia fecal
llevadas a diez laboratorios del Area Metropolitana de
Bucaramanga, Colombia. Med UIS. 1994;8:2–5.
29. Soto M, Velasquez G, Cuervo C, Galvis MT, Botero D.
Criptosporidiosis respiratoria en un paciente con SIDA. Acta
Med Colomb. 1997;22(3):148–50.
30. Vergara-Castiblanco CA, Santos Nuñez S, Freire Santos F, Ares
Mazas E. La criptosporidiosis en la región andina de Colombia:
seroprevalencia y reconocimiento de antígenos. Rev Panam Salud
Publica. 2000;8(6):373–9.
31. Florez AC, Garcia DA, Moncada L, Beltran M. Prevalence of
microsporidia and other intestinal parasites in patients with HIV
infection, Bogota, 2001. Biomedica. 2003;23(3):274–82.
32. Botero JH, Castaño A, Montoya MN, Ocampo NE, Hurtado MI,
Lopera MM. A preliminary study of the prevalence of intestinal
parasites in immunocompromised patients with and without gastro-
intestinal manifestations. Rev Inst Med Trop Sao Paulo.
20 03 ;45(4 ): 197 –20 0. https ://doi.org /10.1590/S0036-
46652003000400004.
33. Arzuza O, Arroyo B, Villegas S, Rocha A, Díaz H. Infecciones
parasitarias intestinales en pacientes positivos para el virus de la
inmunodeficiencia humana (VIH) en la ciudad de Cartagena de
Indias, Colombia. Infectio. 2003;7(2):58–63.
34. Botero JH, Montoya MN, Vanegas AL, Díaz A, Navarro-i-Martínez
LBF, Izquierdo F, et al. Frequency of intestinal microsporidian in-
fections in HIV-positive patients, as diagnosis by quick hot gram
chromotrope staining and PCR. Biomedica. 2004;24(4):375–84.
35. Carreño M, Velasco CA, Rueda E. Prevalence of Cryptosporidium
spp. in children with cancer, younger than 13 years old [Spanish].
Colomb Med. 2005;36(2):6–9.
36. Navarro-i-Martinez L, da Silva AJ, Botero-Garces JH, Montoya-
Palacio MN, del Aguila C, Bornay-Llinares FJ. Cryptosporidiosis
in HIV-positive patients from Medellín, Colombia. J Eukaryot
Microbiol. 2006;53(Suppl. 1):S37–9.
37. Rivera O, Vásquez L. Cryptosporidium spp: Informe de un caso
clínico en Popayán, Cauca. Rev Colomb Gastroenterol. 2006;21(3):
225–9.
38. de Arango M, Rodríguez DA, Prada NE. Frecuencia de
Cryptosporidium spp en materia fecal de niños entre un mes y trece
años en un hospital local colombiano. Colomb Med. 2006;37(2):
121–5.
39. Siuffi M, Angulo M, Velasco CA, López P, Dueñas VH, Rojas C.
Relation between viral load and CD4 versus Cryptosporidium spp. in
feces of children with AIDS. Colomb Med. 2006;37(1):15–20. 6p
40. de la Ossa MN, Falconar A, Llinás Solano HJ, Romero Vivas CM.
Manifestaciones clínicas y factores de riesgo asociados a la
infección por Cryptosporidium en pacientes de Barranquilla y tres
municipios del Atlántico (Colombia). Rev Científica Salud
Uninorte. 2007;23(1):18–31.
41. Velasco CA, Méndez F, López P. Cryptosporidiosis in Colombian
children with HIV/AIDS infection. Colomb Med. 2011;42(4):418–29.
42. Velez-Velasquez DM, Velasco-Benitez C, Rojas C, Dueñas-Rivera D,
Neuta P. Cryptosporidium spp. en niños menores de 10 años de la
comuna 18 de Cali, Colombia. Rev Gastrohnup. 2011;13(2):S4–10.
Curr Trop Med Rep (2018) 5:144–153
43. Bayona M, Avendaño C, Amaya Á. Caracterización
epidemiológica de la criptosporidiosis en población infantil de la
región Sabana centro (Cundinamarca). Rev UDCA Act & Div
Cient. 2011;14(1):7–13.
44. Álvarez J, Bedoya L, Lucero-Garzón T. Parasitosis intestinal en
niños de 0 a 5 años de edad, inscritos en centros de atención integral
ubicados en las comunas sur y nororiental del municipio de
Florencia, Caquetá (Colombia). Biomedica. 2015;35(Supl. 4):74.
45. Agudelo-Gonzalez S, Murcia-Sanchez F, Salinas D, Osorio J.
Infecciones oportunistas en pacientes con VIH en el hospital
universitario de Neiva, Colombia. 2007-2012. Infectio.
2015;19(2):52–9.
46.• Isaza JP, Galván AL, Polanco V, Huang B, Matveyev AV, Serrano
MG, et al. Revisiting the reference genomes of human pathogenic
Cryptosporidium species: reannotation of C. parvum Iowa and a
new C. hominis reference. Sci Rep. 2015;5(1):16324. https://doi.
org/10.1038/srep16324. First genome description of a colombian
Cryptosporidium isolate.
47. Montúfar Andrade F, Quiroga A, Builes C, Saldarriaga C, Aguilar
C, Mesa M, et al. Epidemiology of human immunodeficiency virus
infection in inpatients in a teaching hospital of high complexity in
Medellin, Colombia. Infection. 2016;20(1):9–16. https://doi.org/
10.1016/j.infect.2015.05.004.
48. Sánchez A, Munoz M, Gómez N, Tabares J, Segura L, Salazar Á,
Restrepo C, Ruíz M, Reyes P, Qian Y, Xiao L, López MC, Ramírez
JD Molecular epidemiology of Giardia, Blastocystis and
Cryptosporidium among indigenous children from the Colombian
Amazon Basin. Front Microbiol. 2017;8:248. https://doi.org/10.
3389/fmicb.2017.00248.
49. Gaviria L, Soscue D, Campo-Polanco L, Cardona-Arias JG-DAL.
Prevalence of intestinal parasites, anemia and malnutrition among
the children of a Nasa indigenous reservation, Cauca-Colombia,
2015. Rev Fac Nac Salud Pública. 2017;35(3):390–9.
50. Vergara-Castiblanco CA, Freire-Santos F, Oteiza-López AM, Ares-
Mazás ME. Viability and infectivity of two cryptosporidium
parvum bovine isolates from different geographical location. Vet
Parasitol. 2000;89(4):261–7. https://doi.org/10.1016/S0304-
4017(00)00210-7.
51. Vergara-Castiblanco CA, Freire-Santos F, Castro-Hermida JA,
Ares-Mazás ME, Quílez-Cinca J. Serological response to
Cryptosporidium parvum in adult cattle from the Andean region
of Colombia. Parasitol Res. 2001;87(6):500–4. https://doi.org/10.
1007/s004360100375.
52. Santín M, Trout JM, Vecino JAC, Dubey JP, Fayer R.
Cryptosporidium, Giardia and Enterocytozoon bieneusi in cats
from Bogota (Colombia) and genotyping of isolates. Vet
Parasitol. 2006;141(3–4):334–9. https://doi.org/10.1016/j.vetpar.
2006.06.004.
53. Rodríguez E, Manrique-Abril F, Pulido M, Ospina-Díaz J.
Frequency of Cryptosporidium spp in canine from city of
Tunja—Colombia. Rev MVZ Cordoba. 2009;14(2):1697–704.
54. Avendaño C, Quílez J, Sánchez-Acedo C. Prevalencia de
Cryptosporidiumen en terneros en el Valle de Ubaté –
Chiquinquirá (Colombia). Rev UDCA Act & Div Cient.
2010;13(1):41–7.
153
55. Montaño JS, Avendaño C. Contribución al conocimiento de la
epidemiología d ela criptosporidiosis bovina en el Valle de
Chiquinquirá. Rev UDCA Act & Div Cient. 2012;15(2):391–8.
56. Hernández-Gallo N, Cortés-Vecino JA. Prevalencia y factores de
riesgo de Cryptosporidium spp. y Giardia spp. en terneros de
ganado lechero de la zona noroccidental de la Sabana de Bogotá.
Rev Salud Pública. 2012;14(1):169–81. https://doi.org/10.1590/
S0124-00642012000100014.
57. Ocampo RJ, Rivera FA, López GA, Álvarez ME, Cardozo LA,
Pérez JE. Primer reporte de Cryptosporidium parvum en terneros
holstein (Bos Taurus) de Manizales, Caldas, Colombia. Rev Med
Vet Zoo. 2012;59(3):159–64.
58. Pardo D, Oliver O. Identificación de agentes infecciosos asociados
con Diarrea Neonatal Bovina en la Sabana de Bogotá. Rev MVZ
Córdoba. 2012;17(3):3162–8.
59. Montoya C, Oyola N, Ocampo M, Polanco D, Ríos S, Molina P, et
al. Evaluation of intestinal parasitism in red howler monkeys
(Alouatta seniculus) under rehabilitation at the CAVR Ecosantafé,
Jericó, Colombia. Rev Lasallista Investig. 2013;10(2):25–34.
60. Cadavid-Betancur DA, Giraldo-Echeverri CA, Sierra-Bedoya S,
Montoya-Pino M, Chaparro-Gutierrez JJ, Restrepo-Botero JE, et
al. Bovine neonatal diarrhea in a herd in the northern Antioquia
(Colombia) highlands, a descriptive study. Vet y Zootec.
2014;8(2):120–9.
61. Pulido-Medellín M, Andrade-Becerra JR, Vivas R, Garcia-
Corredor J. Prevalence and possible risk factors for
Cryptosporidium spp oocyst excretion in dairy cattle in Boyacá,
Colombia. Rev Mex Cienc Pecu. 2014;15(3):357–64.
62. Avendaño C, Ramo A, Vergara-Castiblanco C, Sánchez-Acedo C,
Quílez J. Genetic uniqueness of Cryptosporidium parvum from
dairy calves in Colombia. Parasitol Res. 2018;117(5):1317–23.
https://doi.org/10.1007/s00436-018-5818-6.
63. Alarcón MA, Beltrán M, Cárdenas ML, Campos MC. Presence and
viability of Giardia spp. and Cryptosporidium spp. in drinking wa-
ter and wastewater in the high basin of Bogotá river. Biomedica.
2005;25(3):353–65.
64. Campos-Pinilla C, Cárdenas-Guzmán M, Guerrero-Cañizares A.
Performance of Faecal contamination indicators in different type
of waters from the Sabana of Bogotá ( Colombia). Univ Sci.
2008;13:103–8.
65. Rodríguez DC, Pino N, Peñuela G. Microbiological quality indica-
tors in waters of dairy farms: detection of pathogens by PCR in real
time. Sci Total Environ. 2012;427–428:314–8. https://doi.org/10.
1016/j.scitotenv.2012.03.052.
66. Triviño-Valencia J, Lora F, Zuluaga JD, Gomez-Marin JE.
Detection by PCR of pathogenic protozoa in raw and drinkable
water samples in Colombia. Parasitol Res. 2016;115(5):1789–97.
https://doi.org/10.1007/s00436-016-4917-5.
67. Lora-Suarez F, Rivera R, Triviño-Valencia J, Gomez-Marin JE.
Detection of protozoa in water samples by formalin/ether concen-
tration method. Water Res. 2016;100:377–81. https://doi.org/10.
1016/j.watres.2016.05.038.
68. Ocampo Gallego RJ, Cardozo Duque LA, Lopez Garnert GA,
Alvarez ME, Perez JE, Rivera Paez FA. Evaluation of molecular
and microscopic methods for detection of Cryptosporidium spp.
(Apicomplexa—Cryptosporidiidae). Biosalud. 2011;10(1):19–29.