Review
Molecular Epidemiology of Giardia Infections in
the Genomic Era
Paul Capewell,1
Sarah Krumrie,2
Frank Katzer,3
Claire L. Alexander,4
and William Weir 2,
*
Giardia duodenalis is a major gastrointestinal parasite of humans and animals
across the globe. It is also of interest from an evolutionary perspective as it
possesses many features that are unique among the eukaryotes, including its
distinctive binucleate cell structure. While genomic analysis of a small number
of isolates has provided valuable insights, efforts to understand the epidemiology
of the disease and the population biology of the parasite have been limited by the
molecular tools currently available. We review these tools and assess the impact
of affordable and rapid genome sequencing systems increasingly being deployed
in diagnostic settings. While these technologies have direct implications for public
and veterinary health, they will also improve our understanding of the unique
biology of this fascinating parasite.
A Major Worldwide Pathogen
Giardia duodenalis (also known as Giardia intestinalis or Giardia lamblia) is one of the most common
gastrointestinal parasites in the world, causing an estimated 180 million infections annually [1].
Although giardiasis is treatable, the correct administration of therapeutics depends on accurately identifying
the parasite, either in an individual or within a community during an outbreak. Asymptomatic
infection can occur and may represent the majority of cases [2–5], although it should be noted that
apparently asymptomatic individuals can still display signs of mild malnutrition [6]. Patients with
overt clinical disease experience severe gastrointestinal disturbances for several weeks due to
trophozoites (see Glossary) attaching to the intestinal lining of the host, disrupting nutrient and
water uptake, and eliciting an immune response [7]. In rare cases, some patients can develop postinfection
complications that lead to long-term gastrointestinal disorders similar to irritable bowel
syndrome (IBS) [8]. These symptoms are linked to a loss of barrier function and dysbiosis of the
gut flora [9–11]. Infective cysts are shed into the environment by infected hosts where they can be
ingested by new individuals, maintaining transmission. Outbreaks are frequently associated with
contaminated water [12] or food sources [13].
Giardia Genetics
Giardia species are described as early divergent eukaryotes and lack common subcellular
structures such as mitochondria, a true Golgi complex, and peroxisomes [14]. However, the
identification of mitochondrial genes in the genome suggests that G. duodenalis and other
diplomonads once possessed these organelles but subsequently lost them [15]. In addition to
being amitochondriate, G. duodenalis is unusual in that it has two nuclei despite being unicellular.
This gives rise to an unusual ploidy throughout its life cycle in which the trophozoites cycle between
4N (2×2N) and 8N (4×2N) in vegetative growth, doubling to 16N (4×4N) during the transition
to cysts (Figure 1A) [16]. After excystation, the cell divides, without DNA replication, to create
four trophozoites, each with a ploidy of 4N. The relatively small genome (11.7 Mb) is distributed
across five chromosomes that feature few intergenic spaces, introns, or noncoding regions.
Promoters and untranslated regions are also minimized, leading to a highly condensed and efficient
genome [14]. This appears to be a distinctive trait of the diplomonads, and the genome is
Highlights
Research into Giardia duodenalis is
hampered by low-resolution genetic
markers, difficulties in sequencing
parasite material, and a unique biology.
Recent advances in sequencing technology
are leading to more detailed genome
sequences across more parasite strains,
including clinical isolates.
This new resource allows the design of
better tools to understand the biology
and molecular epidemiology of Giardia.
The expansion of genomics into
diagnostic settings benefits from
and contributes to these sequencing
efforts, supporting work to control the
parasite.
1
Institute of Biodiversity, Animal Health
and Comparative Medicine, College of
Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, G61
1QH, UK
2
School of Veterinary Medicine, College
of Medical, Veterinary and Life Sciences,
University of Glasgow, Glasgow, G61
1QH, UK
3
Moredun Research Institute, Pentlands
Science Park, Edinburgh, EH26 0PZ, UK
4
Scottish Parasitology Diagnostic and
Reference Laboratories, Glasgow, G31
2ER, UK
*Correspondence:
willie.weir@glasgow.ac.uk (W. Weir).
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© 2020 Elsevier Ltd. All rights reserved.
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even more compact in the closely related rodent parasite Giardia muris [17]. Although similar, the
genomes of each nucleus in an individual parasite are not identical and the differences between
the four different genomes are termed allelic sequence heterozygosity (ASH). The proportion
of heterozygous bases within a genome typically ranges between 0.25% and 0.74% for most
Giardia isolates [18–20] but can be extremely low (<0.01%) [9,11]. The majority of these heterozygous
sites contain two different bases but some feature three or four, capturing the diversity
present across all of the copies of the genome [20]. Regions of heterozygosity are not distributed
evenly throughout the genome and more typically occur in noncoding regions, as might be expected
with purifying selection acting on coding regions [20].
Genotyping G. duodenalis
Initial isozyme and 18S ssu-rRNA gene sequencing demonstrated that two broad groups of
G. duodenalis (eventually termed A and B) infected human patients. These were characterized as
assemblages to reflect the fact that the relationships between the groups were undefined [21–23].
Additional genetic data from animal-derived isolates, coupled with various biological differences,
allowed a further six distinct assemblages to be differentiated (C–H) (Table 1). Assemblages A and
B contain zoonotic isolates that can infect humans and animals, whereas assemblages C–H
show specificity to particular animal hosts (Table 1). However, isolates with molecular sequences
similar to assemblages C–H have been amplified from humans, suggesting that there are either
limits to current molecular typing tools or G. duodenalis may have a wider zoonotic potential
than first assumed [24–26]. As these molecular detections in humans are often from asymptomatic
individuals, it is unclear whether the DNA detected in the molecular screens represents
infection, carriage, or contamination. Antigen-capture assay, immunofluorescent antibody
testing (IFAT), direct microscopy, and ssu-rRNA qPCR are the standard methods for detecting
G. duodenalis, although many diagnostic laboratories rely on microscopy for detection due to
cost and established pipelines [27]. Microscopy can lack sensitivity when parasitemia is low
(which is common) or where expertise is lacking, indicating a switch to immunological assays
or qPCR in diagnostic settings may be preferable to assess accurately the presence of the
parasite [28]. However, it should be appreciated that although qPCR sensitively detects Giardia
nucleic acid, a positive test result does not necessarily confirm the presence of viable parasite
cells. Routine qPCR-based detection methods are also unsuitable for determining the relationships
between isolates as they do not provide detailed genetic information [29]. Over the years,
several molecular markers have been developed to create a multilocus sequence typing
(MLST) panel to investigate the molecular phylogeny of G. duodenalis. These involve targeted
PCR and subsequent sequencing of genes that are relatively stable but possess some degree
of variability for differentiating isolates. The most commonly used regions of the genome are the
loci encoding β-giardin (bg) [30], triosephosphate isomerase (tpi) [31], and glutamate dehydrogenase
(gdh) [32,33]. Of these three primary markers, tpi displays the most polymorphisms in
the currently sequenced population in terms of substitutions per nucleotide site (r] = 0.12) and
bg the least (π = 0.03), with gdh intermediate between the two (π = 0.06) [34]. There are also
some rarely used loci that are more difficult to amplify than the typical markers, including the
internal transcribed spacer (its1) and elongation factor 1 (ef1) [35,36]. In common with other
parasite species, PCR and sequencing of the ssu-rRNA region is also employed for
molecular genotyping [34]. Amplification success for this region can be higher than other
PCR targets due to being multicopy, making it highly sensitive and ideal for identification [37].
However, single-copy markers are still commonly employed due to the relatively low discriminatory
power of the ssu-rRNA region [34] and unusually high GC content that can lead to issues
with specificity [38,39]. Further analysis has shown that assemblage B isolates display greater
polymorphism than other strains at the commonly used marker sites, possessing higher ASH
within individual parasites and greater allelic diversity in the population [40,41]. Infections with
Glossary
18S ssu-rRNA gene: a highly
conserved gene encoding ribosomal
RNA and commonly used for
phylogenetic studies.
Allelic sequence heterozygosity
(ASH): genetic differences at a genetic
locus as assessed across the four
different genomes in an individual
Giardia isolate.
Cyst: an infective, environmental stage
of the parasite.
Diplomixis: a unique parasexual
recombination cycle that occurs
between two nuclei of a Giardia cell
during encystation.
Diplomonads: a group of flagellated
protozoa with double cells and two
nuclei.
Dysbiosis: disruption of the gut
microflora.
Excyzoite: a newly excysted Giardia
cell with 4×4N ploidy.
Gene conversion: transfer of genetic
material from an intact chromosomal
DNA sequence to a homologous
sequence which contains double-strand
breaks.
Inter-nuclei heterozygosity: the
degree of polymorphism between the
two nuclei of a Giardia cell, which is
typically lower than would be expected.
Isozyme (or isoenzyme): different
forms of the same enzyme that differ
in amino acid sequence and which
can be used as the basis for a typing
method.
Lateral gene transfer: the horizontal
movement of genetic material between
organisms, distinct from the vertical
transmission of DNA from parent to
offspring.
Linkage disequilibrium: the
nonrandom association of alleles at two
or more loci in a population.
Loss of heterozygosity (LOH): refers
to regions that display no heterozygous
sites in a genome.
Multilocus sequence typing (MLST):
a method used to characterize
individuals genetically based on the
sequence at a number of marker loci
distributed throughout the genome.
Parasexual recombination: a
process of genetic recombination,
utilized by some organisms, that does
not require the production and fusing of
haploid gametes.
Peroxisome: a membrane-bound
organelle, found in eukaryotic cells,
involved in oxidation and lipid
metabolism.
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multiple assemblages [42–44] or subassemblages [45] can also occur in humans and animals,
affecting estimates of heterozygosity. Furthermore, it appears that mixed genotypic infections
can affect infection dynamics, such as increasing cyst shedding [43]. However, it is unclear
how often mixed Giardia infections occur as they may only be detectable after subcloning
and sequencing at a depth able to detect low levels of ASH [40].
Current Methods Provide Insufficient Resolution
MLST approaches using targeted PCR with sequencing of amplicons have largely validated the
assemblage model, leading to the proposal that the assemblages should be formally redefined
as species [46]. However, as the current markers do not provide the resolution required to determine
accurately relationships between isolates beyond assemblages, their effectiveness in the
public health sphere has been limited. While putative subassemblages have been defined within
assemblage A (AI–III) [45], the framework describing substructuring within assemblage B appears
less robust [34]. There are also situations in which there is a lack of concurrence between the different
markers that may be due to their low discriminatory power [47,48]. For example, there is
only a single nucleotide difference that discriminates between the allele subtypes AI and AII within
the bg amplicon and two for the corresponding tpi subtypes [34]. These issues are clearly
demonstrated by recent high-resolution analysis that used an MLST consisting of six markers
to examine assemblage A isolates [45]. While delineation into three distinct subassemblages
was supported, individual markers were less stable and showed conflicting results when examined
in isolation. This was attributed to potential recombination within the population. Similar incongruities
were noted in a recent study of primate Giardia isolates; the current assemblage
model could not be reliably reproduced with ssu-rRNA data, likely due to the low resolution of
the marker [49]. There are also issues with the reliability of PCR assays targeting single-copy
genes and it is common for only one gene to amplify. Success rates vary from 11% to 91% across
the different markers, depending on the study [37]. Mixed infections and ASH can also make it
impossible to infer alleles from direct PCR sequencing and it is necessary to use laborious
transformation and cloning protocols before sequencing. These are often difficult and costly to
implement in a diagnostic setting. This lack of reliability is likely due in part to the large amounts
of contaminating DNA and inhibitors found in fecal material, compounded by the variable number
of G. duodenalis cysts present [50,51]. However, it is also likely that there is a degree of sequence
variability present in the genes affecting primer-binding sites and amplification success (Box 1
and Figure 2). It is therefore probable that a great deal of genetic diversity in the Giardia population
is being overlooked due to the specificity of the primers used and the difficulties in amplifying from
fecal material. As such, the currently available marker-based system for understanding the molecular
phylogeny of G. duodenalis is limited in scope and does not provide a high level of genetic
discrimination. This makes it virtually impossible to identify reliably transmission routes, reservoirs,
and relationships between strains, hampering public health efforts to control giardiasis.
Investigating the Epidemiology of Giardia Infections
The lack of high-resolution genotyping tools also limits the ability to answer fundamental biological
questions concerning the parasite, many of which have wider effects on understanding transmission
and controlling disease. For example, a large number of companion and livestock animals
are infected with G. duodenalis, including assemblages that can infect humans [52]. Although
in some cases disease manifestation can be severe, the typical clinical impact for animals appears
to be low and may often not be associated with clinical signs [53]. While companion animals
appear more likely to be infected with species-specific assemblages (C/D in dogs, F in cats),
they can also be infected with assemblages A and B. However, whether these actually pose a
zoonotic risk is inherently difficult to ascertain due to the low resolution of current markers. Several
studies have shown that animals and humans can share genotypes [54] and even subassemblages
Ploidy: the number of sets of
chromosomes within the cell of an
organism.
Trophozoite: the active ‘feeding’ form
of the parasite located in the small
intestine and responsible for pathology.
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[41,55–60], but incomplete MLSTs, in addition to the low resolution and incongruences between
markers, mean these data cannot be definitive. Indeed, many studies rely on the use of a single
marker, despite this being inadequate to group isolates reliably [34,40,49,61]. Instead, identifying
G. duodenalis transmission between humans and animals has been inferred using classical epidemiological
studies and indirect observations. For example, wide-scale vaccination of dogs in a deprived
community in Argentina led to a corresponding decrease in the prevalence of Giardia in the local children
[62]. Additionally, epidemiological analyses in India found a highly significant association between
the prevalence of G. duodenalis in humans, dog ownership, and the presence of a G. duodenalispositive
dog in the same household [36]. Similar links were found between dog ownership and
human infection with assemblage A in a UK setting [63]. However, no link has been found in other
communities [64] and it likely that the epidemiology and zoonotic risk of Giardia infections vary in
different locations. This diversity of epidemiological contexts underlines the need for novel
2 2N
2 4N
4 2N
4 2N
4 4N
4 4N
2 4N
Cyst Excyzoite
Trophozoite
Inter-nuclei
heterozygosity
Reduced
inter-nuclei
heterozygosity
i. ii.
(A) (B)
Trophozoite (2 4N)
Nuclear division and
DNA replication
Cytokinesis and
nuclear division
Cyst (4 4N) Cyst (4 4N)
Diplomixis
Cytokinesis and
nuclear division
Trophozoites (2 2N)
+
+
Or
A B
X Y
X A Y B
X B AY
A or B
X or Y
A B
X Y
Trophozoites (2 2N)Mammalian gut
×
×
×
×
×
×
×
×
×
×
×
×
TrendsTrendsininParasitologyParasitology
Figure 1. An Overview of the Giardia duodenalis Life Cycle. (A) In the mammalian intestinal tract, binucleate trophozoites cycle between 4N and 8N during vegetative
growth. Trophozoites swept into the large intestine differentiate into cysts and are released into the environment for direct transmission. During encystation, the two nuclei
divide and the DNA replicates, resulting in a ploidy of 16N. After activation in the mammalian stomach, cysts excyst in the intestine to release a 16N excyzoite with four
nuclei. This excyzoite divides twice without DNA replication, resulting in four trophozoites that begin the vegetative cycle in a new host. (B) G. duodenalis exhibits a unique
parasexual cycle (diplomixis) that may contribute to lower than expected heterozygosity between the two nuclei of the cell. When the 16N cyst is formed during
encystation, genetic exchange can occur between nuclei via homologous recombination. (i) Without diplomixis, inter-nucleus heterozygosity is maintained in the
daughter cells. Consequently, inter-nucleus heterozygosity will continue to increase and the genomes of the two nuclei will diverge. (ii) With occasional diplomixis,
regions of inter-nucleus heterozygosity can be transferred, reducing heterozygosity in some of the daughter cells and slowing the rate of divergence between the two
nuclei. In addition, the process can generate genotypes with new allele combinations, further emulating sexual recombination.
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high-resolution genotyping methods which can be applied to reveal the particular transmission
pathways in action in different areas.
Population Genetic Structure of Giardia
Another important aspect of G. duodenalis biology that cannot be resolved with the current molecular
tools is the role that sexual recombination plays in creating diversity in natural populations
of the parasite. Although seemingly an academic question, this issue is of practical importance as
the occurrence of genetic exchange in pathogen populations can have a significant impact on
disease epidemiology. For example, in asexual organisms only rare mutations at specific loci or
horizontal gene transfers can provide new genotypes that may lead to drug resistance or increased
virulence. Conversely, sexual organisms are able to produce new genotypes constantly
through meiosis and chromosomal reassortment, allowing alleles conferring a fitness advantage
to spread in the population. This in turn allows pathogens to adapt and exploit new conditions. In
diagnostic and public health settings, sexual recombination also affects the ability to track outbreaks
and identify transmission networks by disrupting the genotypes involved. Although
G. duodenalis has many of the genes for meiosis [65], sexual reproduction has never directly
been observed, and examination of linkage disequilibrium among isozymes suggests that
G. duodenalis is asexual [66]. There are hints that the parasite may not be completely asexual,
and sexual recombination may simply be a rare event [14,67]. In particular, extensive examination
of the genetics of G. duodenalis from a single household found evidence for the reassortment of
alleles between infections, suggesting sexual recombination [68]. Similar reassortment was
suggested by a high-resolution study of assemblage A isolates that also indicated possible
cross-assemblage recombination with assemblage E [45]. Horizontal gene transfer has also
been documented between assemblages A and B [69,70].
Table 1. Giardia duodenalis Host Assemblages
Giardia assemblage/
sub-assemblage
Host Proposed nomenclature
[46]
A I, II, III Humans, non-human primates, canines, felines, other
mammals
G. duodenalis
B Humans, non-human primates, canines, felines, other
mammals
Giardia enterica
C Canines Giardia canis
D Canines G. canis
E Livestock Giardia bovis
F Felines Giardia cati
G Rodents Giardia simondi
H Marine mammals
Box 1. G376 Primer Annealing Site Diversity
The diversity found within the three main genes used to genotype G. duodenalis (bg, tpi, and gdh) makes them useful to
differentiate assemblages and subtypes. The separate assemblages also display different diversities, allowing a degree of
substructuring to be observed. However, this diversity may also encompass the primer annealing sites, affecting the
amplification success rates for isolates and assemblages. For example, there are 1598 publicly available G. duodenalis sequences
that include the annealing site for the commonly used β-giardin primer G376. Within these sequences, there can
be up to eight polymorphisms compared with the primer sequence (see Figure 2 in main text). Assemblage A sequences
are the least likely to have polymorphisms, likely reflecting the fact that primers were initially designed using this assemblage.
Conversely, assemblages B–H are more likely to contain polymorphisms in the annealing site (most have at least
two), making them harder to amplify. In addition, as these public sequences are, by definition, the products of successful
reactions with optimal conditions for promoting amplification, it is reasonable to speculate that many sequences fail to amplify
due to polymorphisms in the primer-binding sites and other inhibiting factors.
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An alternative explanation for the low levels of variance and heterozygosity observed in
G. duodenalis is the utilization of a parasexual cycle during reproduction, similar to that in
many fungi [71]. A parasexual cycle differs from meiosis in that it involves the fusing of two diploid
parent cells rather than haploid gametes prior to genetic exchange. To return to a diploid state
there must be a reduction in chromosomal number after this process. Microscopic evidence
has shown that, within G. duodenalis cysts, nuclear fusion and genetic exchange can occur during
the transition from the 4x2N to the 4x4N stage (Figure 1B), although without the loss of chromosomes
[72]. This unique parasexual cycle could act to decrease heterozygosity within the
G. duodenalis population, reducing the negative effects associated with deleterious mutations
that accumulate in asexual eukaryotes [73]. It can also lead to the generation of new allele
combinations, emulating some of the benefits of true sexual recombination. However, as this is
essentially a form of self-fertilization (an extreme form of inbreeding), the system can only slow
down the accumulation of mutations rather than eliminate them completely. Additionally, a parasexual
cycle cannot explain the apparent recombination observed between Giardia isolates across
assemblages and subassemblages [45,68–70], nor incidences of lateral gene transfer from
bacteria and the host [14]. Alternatively, G. duodenalis may utilize large-scale gene conversion –
evidenced by long-range loss of heterozygosity (LOH) – achieved through homologous recombination
events to compensate for the build-up of deleterious mutations, similar to that described for
the asexual parasite Trypanosoma brucei gambiense [74]. It is currently unclear if the large regions of
homozygosity found in G. duodenalis genomes are due to loss of heterozygosity. Irrespective of the
method, it seems likely that some form of recombination occurs in G. duodenalis. Higher-resolution
markers or widespread sequencing will make it easier to understand how common the
0
10
20
30
40
50
60
70
80
90
100
0 1 2 3 4 5
secneuqesfoegatnecreP
Number of polymorphisms
A (n = 400)
B (n = 629)
C (n = 90)
D (n = 97)
E (n = 369)
F (n = 13)
Assemblage
TrendsTrendsininParasitologyParasitology
Figure 2. β-Giardin G376 Primer- Annealing Site Diversity among Assemblages. For this analysis, 1598 publicly
available Giardia duodenalis sequences were downloaded and the G376 primer-annealing site identified and aligned. The
number of differences between each sequenced site and the primer were calculated using the Levenstein distance. The data
are presented for each assemblage, showing that assemblage A sequences have few polymorphisms in the annealing site
compared with the published primer. By contrast, assemblages B–H have, for the most part, at least two and up to five
polymorphisms in total.
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phenomenon is and what the effects may be on the molecular epidemiology of Giardia infections.
This is further supported by a recent study that found evidence for recombination between assemblage
A subassemblages using six markers [45]. However, it is likely that infrequent recombination
would affect estimates of how related individuals are, complicating interpretation.
A Need for New Genotyping Tools
Together, these issues indicate a need for more robust tools for genotyping G. duodenalis to understand
better the molecular epidemiology of the disease and the biology of the parasite to improve
outbreak management. The recent publication of updated reference genomes for
G. duodenalis [75] and G. muris [17] provides more complete scaffolds to build upon and a
well characterized outgroup for comparison, contributing to these aims. However, the development
of new tools depends on collecting a large and diverse selection of sequenced isolates to
capture the diversity in the field more fully. Previously, sequencing of G. duodenalis samples
has been restricted due to the limitations of sampling from fecal material and the requirement
to adapt strains to axenic culture [14,19,20,67]. This adds significant cost, is labor intensive,
and introduces time delays to sequencing efforts. It also ensures that only culturable strains
can be sequenced, introducing potential bias, although assemblage-specific techniques may improve
axenic culture techniques in the future. Comparative genomic analysis may provide information
that would improve the axenic culture of specific assemblages, such as the recent
analysis of assemblages C and D that identified assemblage-specific genes [18]. Several clinical
isolates have recently been sequenced without axenic culture by concentrating cysts from clinical
samples [76]. This approach may have limited effectiveness in many situations due to the requirement
of a large number of starting cysts (often difficult to obtain in a diagnostic setting) and also
results in highly variable sequencing quality and coverage. Several new technologies, now
reaching maturity, may allow the rapid whole-genome sequencing (WGS) of G. duodenalis isolates
from the small amount of starting material available in the clinical diagnostic setting. Accurate
genomes that represent the individual assemblages would also assist in resequencing efforts in
samples with low starting material. For many years this was limited to assemblages A, B, and
E, although genomes for assemblages C and D have recently been added as a resource for
the community [18].
WGS of Giardia
Central to efforts to sequence G. duodenalis clinical isolates are affordable and relatively simple
sequencing platforms that can be inserted into diagnostic pathways with little disruption. These
technologies have led to the average cost of sequencing falling from US$1000 per Mb in 2009
to $0.01 in 2019. Costs are predicted to fall further with the drive to perform WGS routinely for
certain pathogens to generate epidemiological data and to assist the management of outbreaks.
Indeed, wider deployment of Illumina NovaSeq and third-generation long-read sequencing have
recently been used to update the G. duodenalis reference genome [75]. The long-term aim would
be user-friendly sequencing machines that could be deployed at the benchtop and used by nonspecialist
scientists in diagnostic laboratories. While we are still some way from this goal, and
even upcoming 'black box' technologies (such as Seeplex or Filmarray) only detect pathogens
rather than genotype them, on-site rapid sequencing has shown promise in improving the management
of bacterial and viral outbreaks by enhancing throughput, reproducibility, and sensitivity.
It has also led to a rapid expansion in the number of detectable genotypes and new strategies
to understand the molecular epidemiology of these diseases [77]. This in turn has led to an improvement
in identifying infectious agents and sources, tracking outbreaks and monitoring
drug-resistance markers in infected individuals who do not respond to treatment. We suggest
similar efforts should be made to build a substantial collection of sequenced samples from
multiple centers across the globe to capture the diversity of G. duodenalis in clinical, veterinary,
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and environmental samples, leading to better management of clusters/outbreaks, reservoirs, and
drug resistance. To avoid the bottleneck of adapting strains to culture, several approaches have
the potential to be developed to allow sequencing of isolates directly from fecal samples.
For example, researchers in a recent study used a combination of cytometric sorting and
single-cell whole-genome amplification to sequence assemblage C and D isolates from dogs,
neither of which have been successfully cultured [18]. This revealed numerous genes that
may be linked to host specificity and highlighted important differences in heterozygosity
between the assemblages. Another promising technology is exome capture, an approach
using biotinylated DNA or RNA ‘baits’ to capture DNA fragments from a target genome. This has
successfully been used to identify and sequence material with a large amount of contaminating
DNA, including enteric pathogens from faces [78].
Making Sense of the Genomic Data
If the issues of concentrating and purifying cysts to obtain sufficient quantity and quality of
G. duodenalis DNA for sequencing are overcome, the next concern that limits the development
of high-resolution genotyping markers becomes collating and analyzing the large amounts of genomic
data. A centralized global database will be required to develop a standardized set of
markers efficiently, either by adapting current resources like GiardiaDB [79] or developing a dedicated
system that receives MLST or other forms of sequence data. Several such databases have
emerged that collate data from bacterial and viral sources, such as Enterobase [80], PubMLST,
and the European Nucleotide Archive, that facilitate standards used for MLST genotyping. Similar
efforts have been established in the past for Giardia species, for example, ZOOPNET [54];
however, only now is the technology maturing sufficiently to meet the ambitions of the community
for research and clinical applications. While a diagnostic panel of SNPs would provide the highest
resolution for discriminating G. duodenalis genotypes, the most widely deployable output in the
first instance will be additional MLST loci that expand on the current markers to increase reliability
and resolution. These would ideally target genes or regions without indels that would cause
frame-shift mutations, making them more amenable to direct sequencing and avoid cloning
procedures. Direct sequencing is able to identify heterozygous positions across each of the
four genomes present in a single Giardia isolate, providing extra discriminatory information [40].
Indeed, it may be preferable to target heterozygous regions to identify potential recombination
events occurring between generations. Alternatively, if it is shown that LOH occurs in
G. duodenalis to reduce deleterious alleles, identifying and contrasting such regions would also
serve as a means to establish relationships between strains. In addition, a selection of genes
that are under a range of selection pressures would be ideal to provide different temporal
resolution. This may entail using markers with relatively high rates of mutation to track close
relationships, while using slower evolving genes to elucidate more ancestral relationships. The
power of an expanded MLST panel with higher discriminatory power has recently been
demonstrated using a combination of six markers, revealing evidence for recombination and
zoonotic transfer in assemblage A isolates [45]. However, increasing the number of markers
further, and improving reliability across all of the different assemblages, would allow the collective
effort of the Giardia community to quantify the degree of zoonotic transmission in different
epidemiological contexts and to identify environmental or animal reservoirs of infection.
The Application of Genomics to Clinical Isolates
The ability to link closely related G. duodenalis isolates within a short period of time would allow
potential outbreaks to be rapidly identified and effectively managed and could also be used to
identify drug-resistant strains. This approach is already in use for other pathogens, including
Mycobacterium tuberculosis, Salmonella spp. and Escherichia coli 0157 [81]. Due to selective
testing protocols largely based on patient travel history, there is potential for underdetection of
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8 Trends in Parasitology, Month 2020, Vol. xx, No. xx
the parasite in clinical samples and under-reporting of domestically acquired cases, an issue recently
highlighted in Scotland, UK [82]. Compared with other pathogens, limited resources are directed
towards Giardia surveillance activities and, for this reason, it may be hypothesized that
public health systems would lack the power to detect small-scale endemic outbreaks should
they occur. This is particularly the case if these outbreaks had low case numbers and were not
associated with a clear ‘point source’, such as a water contamination event. For these reasons,
having the capacity to detect outbreaks routinely as part of a clinical genomics laboratory service
would represent a major step forward for public health [12]. PCR and Sanger sequencing-based
MLST approaches have the benefit of being more easily inserted into current laboratory pathways
[83,84], are rapid, cost-effective, and are also more likely to be adopted in lower- to middle-income
countries that lack the capacity to perform large amounts of sequencing. As sequencing
technology reaches greater penetration, clinical diagnostic services could begin to incorporate
high-throughput sequencing into their pipelines while maintaining backwards compatibility with
established MLST systems [84]. The cooperation of low-, middle-, and higher-income countries
will not only be essential to identify both endemic outbreaks and reservoirs but also to distinguish
cases caused by ‘foreign’ genotypes of Giardia that have been imported through international
travel. Working in such a broadly collaborative manner will undoubtedly raise issues in the sharing
of public health data, the policies for which can vary widely between countries. Fortunately, efforts
such as the Global Alliance for Genomics and Health are working to facilitate such programs, and
their recommendations have been adopted by a number of health services worldwide [85].
Using Genomics to Understand the Biology of Giardia
Although the collection of large amounts of sequencing data and the development of more robust
sets of MLSTs will directly impact the management of giardiasis, these data will also contribute to
answering several long-standing questions concerning the biology of the parasite that have implications
for the disease. For example, large numbers of genomic or high-resolution MLST sequences
would reveal the degree to which allelic recombination occurs between generations of
parasites, especially if isolates were closely linked in terms of geographical location and time of
sampling. The differences between generations would also demonstrate whether recombination
was occurring between individuals or a parasexual cycle was being utilized [72]. Determining the
amount of recombination occurring in the field is important as it directly impacts our understanding
of transmission networks and the likelihood of positive mutations becoming fixed in the population.
These data would also confirm which assemblages are true species and no longer share
genetic information, and those that have host-specific adaptations which limit their zoonotic potential.
For example, in assemblage C isolates, several genes have been identified that are suggested
to be involved in host specificity [18]. Finally, the capacity to genotype large numbers of
isolates accurately has the potential to reveal associations between parasite genes and phenotypes.
This will allow forward genetic techniques to be used in G. duodenalis for the first time,
making it easier to link genotype to phenotype. Similarly, the ongoing refinement of single-cell genomics
and transcriptomics also provides a tool to examine important biological questions in
Giardia [86]. This would include identifying genes that distinguish between drug resistance and
treatment failure [87], and identifying genotypes involved in more severe sequelae [11]. These
approaches along with other advances in functional analysis will, to some extent, compensate
for the lack of a reverse genetic framework for Giardia which has stifled research in this area
[88]. Fortunately, in-roads are being made with the development of CRISPR/Cas9-mediated
gene knockdown protocols, although the capacity for complete knockout remains elusive
[89,90].
The current genetic contribution to drug resistance is unclear [91] and appears to be largely linked
to transcriptional changes mediated by epigenetic factors [87]. However, there are numerous
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Trends in Parasitology, Month 2020, Vol. xx, No. xx 9
polymorphisms in several of the genes believed to be involved [92]. This suggests that, if associating
polymorphisms are identified in these candidate genes, then there is the capacity to identify
and distinguish cases of true drug resistance from treatment failure for alternative reasons. The
application of WGS to clinical isolates would also reveal whether de novo positively selected
mutations, including LOH events, arise in vivo and what role they may play in drug resistance.
This knowledge would lead to an improvement in patient treatment by allowing alternative drug
regimens to be followed immediately rather than waiting for treatment failure. If an effective alternative
treatment can be utilized quickly in such cases, this would reduce the selective power of
the ineffective treatment and limit the spread of resistant genotypes, therefore benefiting wider
public health. To date, no allelic variants of genes have been identified that associate with different
clinical outcomes of G. duodenalis infection, despite symptoms ranging from asymptomatic
carriage to long-term IBS [11]. Preliminary data are largely ambiguous, with conflicting genotypes
associating with the development of symptoms [2,93]. Again, the use of a publicly available
pathogen database (or expanding current resources such as GiardiaDB) that integrates data
from forward genetic screens and association studies would facilitate the identification of the
genes involved. However, this would require a degree of clinical information being made available
alongside the genetic information, complicating data-sharing across jurisdictions. It will also be
important to determine the molecular profile of isolates from asymptomatic cases, raising further
ethical and logistical issues. The input that host and parasite genetics have in determining
outcome is important to establish as these asymptomatic cases may represent a large and
overlooked reservoir of infection for susceptible individuals, again impacting public health.
Concluding Remarks
In summary, there has been a long-held view that new genotyping markers are required for
G. duodenalis to address numerous issues (see Outstanding Questions). New sequencing
technologies based on genome capture and single-cell sequencing mean that it is now
possible to achieve these aims using clinical samples. However, successfully expanding the
MLST framework for G. duodenalis will require cooperation across the research, medical,
and veterinary communities to develop a consistent set of standards and methods to avoid
replicating effort and maximizing return. Efforts to develop a genotyping framework will also
benefit from establishing a centralized database to collate and process data to deliver tangible
outcomes that benefit public health. This does not necessarily require the generation of new
tools, as current resources such as GiardiaDB may be expanded to perform a wider role.
The routine application of WGS to clinical samples in the public health sphere would allow a
genomics-led approach to outbreak detection, which contrasts to the ‘response mode’ approach
currently taken where only large-scale outbreaks identified by other surveillance activities
are genetically characterized. Clinical genomics would also allow drug-resistant isolates to
be comprehensively genotyped, determining whether resistant lineages are circulating and
whether de novo, positively selected mutants play a role in this poorly understood phenomenon.
If successful, these approaches will greatly improve the global effort to reduce Giardia infections
effectively and minimize outbreaks, and also answer long-standing questions
concerning the biology of these unique eukaryotes.
Acknowledgments
The figures use art adapted from Servier Medical Art under a Creative Commons Attribution 3.0 Unported License
https://creativecommons.org/licenses/by/3.0/).
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