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RESEARCH ARTICLE
New perspectives for fascioliasis in Upper
Egypt’s new endemic region:
Sociodemographic characteristics and
phylogenetic analysis of Fasciola in humans,
animals, and lymnaeid vectors
Alzahraa Abdelraouf AhmadID
1
*, Haidi Karam-Allah Ramadan
2
, Waleed Attia Hassan
2
,
Mohammed Ageeli Hakami
3
, Enas Abdelhameed Mahmoud Huseein
1
, Sara Abdel-
Aal Mohamed
4
, Adnan Ahmed Mohamed
2
, Nahed Ahmed Elossily
1
1Faculty of Medicine, Department of Medical Parasitology, Assiut University, Assiut, Egypt, 2Faculty of
Medicine, Department of Tropical Medicine and Gastroenterology, Assiut University, Egypt, 3Department of
Clinical Laboratory Sciences, College of Applied Medical Sciences, Al-Quwayiyah, Shaqra University,
Riyadh, Saudi Arabia, 4Faculty of Veterinary medicine, Department of Parasitology, Assiut University,
Assiut, Egypt
*zahraaabdelraouf@aun.edu.eg
Abstract
Background
Fascioliasis is a significant vector-borne disease that has emerged in numerous tropical and
subtropical countries causing severe health problems. Egypt is one of the fascioliasis
endemic regions; however, the current situation in Upper Egypt is understudied, with only
sporadic human cases or outbreaks. This study aims to highlight the sociodemographic
characteristics of human fascioliasis in a newly emerged endemic area in Upper Egypt,
along with risk factors analysis and the molecular characteristics of the fasciolid population
in humans, animals, and lymnaeid snails.
Methodology/Principal findings
The study reported Fasciola infection in patients and their close relatives by analyzing the
risk of human infection. Morphological and molecular characterization was performed on
lymnaeid snails. Multigene sequencing was also used to characterize fasciolids from human
cases, cattle, and pooled snail samples. The study identified asymptomatic Fasciola infec-
tion among family members and identified the presence of peridomestic animals as a signifi-
cant risk factor for infection. This is the first genetic evidence that Radix auricularia exists as
the snail intermediate host in Egypt.
Conclusions/Significance
This study revealed that Assiut Governorate in Upper Egypt is a high-risk area for human
fascioliasis that requires additional control measures. Fasciola hepatica was the main
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OPEN ACCESS
Citation: Ahmad AA, Ramadan HK-A, Hassan WA,
Hakami MA, Huseein EAM, Mohamed SA-A, et al.
(2022) New perspectives for fascioliasis in Upper
Egypt’s new endemic region: Sociodemographic
characteristics and phylogenetic analysis of
Fasciola in humans, animals, and lymnaeid vectors.
PLoS Negl Trop Dis 16(12): e0011000. https://doi.
org/10.1371/journal.pntd.0011000
Editor: Krystyna Cwiklinski, University of Liverpool,
UNITED KINGDOM
Received: July 15, 2022
Accepted: December 1, 2022
Published: December 28, 2022
Copyright: ©2022 Ahmad et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information
files.
Funding: The authors received no specific funding
for this work.
Competing interests: The authors have declared
that no competing interests exist.
causative agent infecting humans and snail vectors in this newly emerged endemic area. In
addition, this is the first report of R.auricularia as the snail intermediate host transmitting
fascioliasis in Upper Egypt. Further research is required to clarify the widespread distribution
of Fasciola in Egypt’s various animal hosts. This provides insight into the mode of transmis-
sion, epidemiological criteria, and genetic diversity of fasciolid populations in Upper Egypt.
Author summary
Human fascioliasis is a freshwater snail-transmitted disease that is widely distributed in
many tropical and subtropical countries including Egypt. Recently, Upper Egypt reported
sporadic cases of human fascioliasis and a few outbreaks establishing a new endemic area.
This study was conducted to get information on the sociodemographic characteristics of
the infected patients and possible risk factors of infection in this recently emerged
endemic region. Further, this study tried to describe lymnaeid intermediate host snails
involved in infection transmission morphologically and by molecular analysis. Analysis of
the phylogenetic relationships between natural fasciolid populations was performed in
humans, animals, and snail isolates by multilocus sequencing. Results indicated that
asymptomatic fascioliasis should be screened in areas at risk of infection. Also, younger
age groups and the presence of peri domestic animals were significantly associated with
an increased risk of infection. This is the first molecular proof of the existence of Radix
auricularia snails causing fascioliasis in Egypt. Results showed that Upper Egypt may be
an area at risk for human fascioliasis that needs additional control measures.
Introduction
Fascioliasis is an important food-borne zoonotic disease that poses a significant risk to
humans. Fasciolosis is one of the neglected tropical diseases (NTDs) listed by the World Health
Organization (WHO) [1]. In recent decades, the increase in the incidence of human fascioliasis
and its outbreaks have introduced an emerging or re-emerging disease rather than a global
zoonotic health problem [2]. Two major species, Fasciola hepatica, and F.gigantica are widely
distributed and infect both domestic animals and humans [3]. The intermediate hosts of the
two digenean species belonging to the family Lymnaeidae are different [4]. Infection with fasci-
oliasis is caused by eating contaminated aquatic plants containing metacercariae or by drink-
ing contaminated water [5]. Fasciola is primarily limited to the liver, resulting in hepatic
lesions, fibrosis, and chronic inflammation of the bile ducts [6]. However, most infections are
subclinical [5].
The diagnosis of infection in humans depends on a combination of different methods such
as coprological examination to detect Fasciola eggs in the stool, indirect detection of anti-Fas-
ciola antibodies or coproantigens by serological tests and imaging techniques such as abdomi-
nal ultrasonography (US) and/ or computed tomography (CT) [7,8].
It was necessary to emphasize the connections between the lymnaeid vector species and
Fasciola’s transmission pattern. In numerous regions, the distribution of F.hepatica among
humans parallels its intermediate host snail, Lymneaea [5]. Several factors influence snail dis-
tribution in the environment, including the type of water collection, population dynamics,
temperature thresholds, seasonality, and susceptibility to fascioliasis. Therefore, the identifica-
tion of the snail vector responsible for disease transmission serves as an effective disease
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indicator for differentiating between potential disease scenarios and patterns and conse-
quently, for determining the most effective control strategies [4]. Identification of lymnaeid
snails is difficult due to their general uniformity and few morpho-anatomical characteristics,
and sometimes even experts are unable to determine the precise species of the specimens [9].
Therefore, DNA sequencing data was recently used to help in determining the proper classifi-
cation of snail specimens [10].
In temperate zones of Europe and Oceania, F.hepatica is the dominant species, and the
only species in the Americas, whereas F.gigantica is widespread in Africa and Asia [11]. How-
ever, molecular studies throughout Africa, including Egypt, confirmed the existence of F.
hepatica in multiple countries [12–14]. In addition, the geographical coexistence of both spe-
cies has been extensively documented in many parts of Africa [14–17]. Several global studies
have been conducted, including in Egypt, where both Fasciola spp. coexist and have docu-
mented the emergence of hybrids (or ‘intermediate forms’) of Fasciola [14,18–21]. Conse-
quently, it is difficult to distinguish Fasciola spp. based on morphological, clinical,
coprological or immunological criteria, and the presence of hybrids further complicates the
matter [22]. Recent molecular studies based on DNA analysis are now the most reliable
method for identifying and characterizing the genetic makeup of morphologically similar fas-
ciolids. The two digenean species and the intermediate form of Fasciola can be distinguished
precisely by analyzing the ribosomal ITS1, ITS2, and 28S rRNA genes, as well as the mitochon-
drial (NADI) and (COXI) genes [14,23–26].
Notably, ribosomal DNA (rDNA) has demonstrated its accuracy in the identification of
Fasciola spp. due to the presence of conserved and variable regions and high copy numbers in
these genes [27], whereas mitochondrial DNA (mtDNA) sequences exhibit higher mutation
rates, lack of recombination, and maternal inheritance, therefore they are utilized as biomark-
ers for phylogenetic studies and genetic variability [28].
The phylogenetic classification of Fasciola spp. in Africa, including Egypt, particularly in
livestock, has been extensively studied; however, there are very few studies describing the
genetic structure of liver flukes that cause human fascioliasis. Human fascioliasis in Egypt was
sporadic until the last three decades, when it became endemic, ranging from hypo- to hyperen-
demic [29], as observed in some villages in the Nile Delta [30]. However, the prevalence of
human infections in Upper Egypt is likely underestimated, with only sporadic cases and few
reports of outbreaks [31–33].
The present study aims to describe the risk factor analysis, sociodemographic and molecu-
lar characteristics of the fasciolid population in humans in a newly emerged endemic region in
Assiut Governorate, Upper Egypt. Also, the identity of the lymnaeid intermediate host snails
involved in this endemic area was examined by morphometric and molecular tools. We ana-
lyzed, for the first time in Upper Egypt, the phylogenetic relationships between natural fascio-
lid populations, in humans, animals, and snail vectors by multilocus sequencing.
Materials and methods
Ethics statement
Before contributing to the study, all participants gave their informed written consent. The
study was approved by the Ethical Committee of institutional review board of the Faculty of
Medicine, Assiut University.
Study setting
This study was conducted on patients and their family members recruited from the outpatient
clinic of Al-Rajhi Liver University Hospital, Assiut University. They were residents of the
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Manfalout neighborhood of Assiut Governorate, Upper Egypt, which experienced an outbreak
of fascioliasis in 2018 and became endemic for the disease. Assiut Governorate, on the western
bank of the Nile, is the study area for the participants who were recruited, and is located 350
kilometers (230 miles) south of Cairo, the capital, with geographic coordinates 27˚190N 30˚
580E. It is primarily a rural community, where farming is the primary occupation of the resi-
dents and agricultural and pastoral activities are prevalent.
Study population
Participants in the study were divided into two groups; the first group consisted of 74 patients
recruited for the study during the period from August to October 2020 and exhibiting clinical
symptoms suggestive of fascioliasis, including fever, jaundice, abdominal pain, and hepato-
megaly. The second group consisted of asymptomatic close relatives of the patients (n = 65)
who shared the same feeding habits and environmental conditions as the patients.
Diagnosis of human fascioliasis
Each participant donated two blood samples (3 ml each) for the following laboratory screening
tests.
• Complete blood count (CBC) with eosinophil % and absolute count.
• Indirect hemagglutination test (IHT) using Distomatose Fumouze kit (Laboratoires
Fumouze Diagnostic, Levallois Perret, France) to detect anti-Fasciola antibody titer (a
titer 1/320 was considered positive).
Individuals with high eosinophilia and a positive antibody titer underwent additional tests
to assess their condition and confirm the diagnosis, including;
• Ultrasound of the abdomen (US) and/or CT.
• Endoscopic retrograde cholangiopancreatography (ERCP) was performed on patients diag-
nosed with obstructive jaundice by abdominal ultrasound and/or CT.
•Fasciola eggs were detected using a simple direct smear and formalin ether sedimentation
technique [34] on stool samples. The presence of other intestinal parasites that may cross-
react with fascioliasis or mask its symptoms was also investigated.
Assessment of the risk factors for Fasciola infection
Consistent with similar previous studies, a detailed clinical history was recorded for all partici-
pants regarding sociodemographic characteristics and potential sources of infection based on
the characteristics of the participants and the study locality [35,36].
Malacological study
1. Snail collections and identification. Over the course of three months, a total of 600
lymnaeid snails (n = 200/month) were collected for the study. They were taken from public
water reservoirs and irrigation canals in the Manfalout neighborhood of Assiut Governorate
in Upper Egypt, where no permits were required. Local veterinarians reported foci of animal
fascioliasis infection, which were used to select sampling locations. Lymnaeids were collected
as described by Relf et al. [37] and were initially distinguished from other cohabiting freshwa-
ter snails based on the morphology of their shells, which featured dextral conical shells, trian-
gular tentacles, and darkly pigmented eyes. After washing the snails onsite, they were
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immediately placed in moist, well-ventilated plastic containers for transport to the Parasitology
laboratory. The collected snails were identified taxonomically using reference keys [38,39].
2. Detection and identification of fasciolid larval stages. The snails were washed with
dechlorinated water. Each snail was then evaluated for Fasciola infections using two distinct
techniques: i) testing for cercarial shedding [40]; ii) method of crushing [41]. All the observed
stages were separated using a pipette for further research.
Molecular study
1. Fasciola and snail samples. Three adult flukes were collected from human patients
with dilated common bile duct (CBD) and extracted by ERCP. Two flukes were collected from
cattle’s infected livers from the same locality. Two pooled samples contained head–foot tissue
parts of infected lymnaeid snails and two pooled samples contained head–foot tissue parts of
noninfected lymnaeid snails that served as controls for molecular sequencing. Before DNA
extraction, the lateral margins of the posterior portion of each fluke, excluding the uterus and
ootype, were used. The collected snails were processed according to Kulsantiwong et al. for
DNA extraction [42]. The manufacturer’s instructions for the DNeasy Blood & Tissue Kit
(Qiagen, USA) were followed for DNA extraction, and an Epoch Spectrophotometer was used
to estimate DNA concentration.
2. PCR amplification and sequencing. For the characterization of fasciolid flukes and
lymnaeid snails, PCR amplification of the rDNA ITS1 and mtDNA NADI and COXI genes
was performed with the same reaction volumes and cycling conditions as previously reported
by Itagaki et al. [20]. Ultraclean DNA purification kit (Qiagen, USA) was used to purify PCR
products according to the manufacturer’s instructions. At the SolGent sequencing facility,
sequencing reactions were performed with a BigDye Terminator v3.1 Cycle Sequencing Kit
(Applied Biosystems, Foster City, CA, United States) according to the manufacturer’s instruc-
tions (SolGent Co., Ltd, South Korea). Each marker’s accuracy was confirmed through bidirec-
tional sequencing. An ABI 3130 Genetic Analyzer was used to analyze the sequencing data
(Applied Biosystems, Foster City, CA, United States).
3. Sequence analysis. GenBank Blast https://blast.ncbi.nlm.nih.gov/Blast.cgi) identified
the similarity between the two sequences. The sequences were aligned with each other and ref-
erence sequences using http://www.megasoftware.net with the default settings of MUSCLE in
MEGA X.0.[43]. The COX1 and NAD1 mitochondrial gene sequences were translated into
amino acids to detect the possibility of pseudogene amplification. The evolutionary relation-
ship between ribosomal ITS1 and mitochondrial genes was determined by constructing phylo-
genetic trees using maximum likelihood (ML) and the neighbor-joining distance method with
a confidence level of 1000 bootstrap replicates. As the outgroup, the nucleotide sequences of
Fascioloides magna (EF534991, EF535001, and GU599871) were used to root phylogenetic
trees.
4. Haplotype analysis. DnaSP 5.1 software [44] was utilized to calculate the diversity indi-
ces (Haplotype (Hd) and nucleotide (π) diversities) between the present isolates of F.hepatica
and F.gigantica, and the reference flukes.
Statistical analysis
Participants’ demographic data and relevant risk factors were analyzed by IBM-SPSS 24.0
(IBM-SPSS Inc., Chicago, IL, USA). Means, standard deviations, median, interquartile range
(IQR), frequency, and percentages were calculated for descriptive data. Significance tests: The
Chi-square test was used to compare group frequencies. Post-hoc tests were calculated using
Bonferroni corrections for pairwise comparisons between the study groups. One-way ANOVA
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was used to test for differences in means of data with normal distribution, while the Kruskal–Wal-
lis non- parametric test was used to compare differences in the median of data without normal
distribution. Subsequently, the association between variables was examined using multivariate
logistic regression analysis. A p-value was considered significant when <0.05.
Results
Demographics and risk factor analysis
This study included a total of 139 participants (68 male and 71 female). Seventy-four had acute
or chronic fascioliasis, while the remaining 65 were asymptomatic relatives. The participants’
demographic characteristics and disease-related information are summarized in Table 1.
Table 1. Baseline characteristics and disease-related data of the studied population showing the relationship between determinants of infection and fascioliasis.
Total (n = 139) Cases
(I)
(n = 74)
Positive Relatives (II)
(n = 20)
Negative Relatives (III)
(n = 45)
P-value
Age/year
(mean ±SD)
29.52 ±1.2 26.82 ±1.3 27.60 ±1.4 34.80 ±1.7 = 0.028
P-value I vs. II = 0.847 II vs. III = 0.045 I vs. III = 0.009
Sex (n, %) = 0.211
• Male 68 (48.9%) 32 (43.2%) 13 (65%) 23 (51.1%)
• Female 71 (51.1%) 42 (56.8%) 7 (35%) 22 (48.9%)
Occupation (n,%) = 0.011
• Housewife 36 (25.9%) 25 (33.8%) 3 (5%) 8 (17.8%)
• Student 51 (36.7%) 32 (43.2%) 7 (35%) 12 (26.7%)
• Farmer 27 (19.4%) 8 (10.8%) 5 (25%) 14 (31.1%)
• Skilled 10 (7.2%) 0 (0%) 3 (15%) 7 (15.6%)
• Professional 15 (10.8%) 9 (12.2%) 2 (10%) 4 (8.9%)
Education (n,%) = 0.045
• Illiterate 36 (25.9%) 26 (35.1%) 2 (10%) 8 (17.8%)
• Basic 75 (54%) 37 (50%) 14 (70%) 24 (53.3%)
• University 28 (20.1%) 11 (14.9%) 4 (20%) 13 (28.9%)
Residence (n,%) = 0.009
• Rural 89 (64%) 52 (70.3%) 16 (80%) 21 (46.7%)
• Urban 50 (36%) 22 (29.7%) 4 (20%) 24 (53.3%)
Piped Water supplies (n,%) 107 (77%) 58 (78.4%) 12 (60%) 37 (82.2%) = 0.133
Eating raw vegetables
(n,%)
112 (80.6%) 63 (85.1%) 15 (75%) 34 (75.6%) = 0.182
Peridomestic animals (n,%) 97 (69.8%) 60 (81.1%) 16 (80%) 21 (46.7%) <0.001
Eosinophils percent
(Med ±IQR)
14 (1–72) 35 (18) 8 (3) 3 (1) <0.001$
Cases with high eosinophilia (n,%) 89 (64%) 67 (90.5%) 19 (29.2%) 3 (4.6%) <0.001
P-value I vs. II <0.001 II vs. III = 0.051 I vs. III <0.001
Hepatic focal lesions (n,%) 34 (24.5%) 26 (35.1%) 8 (40%) 0 (0%) <0.001
ANOVA test was used to compare the mean difference between groups
Post-hoc test was used for pairwise comparison with Bonferroni correction
Chi-square test was used to compare frequency between groups
$
Kruskal Wallis test was used to compare the median difference between groups
Med ±IQR: Median ±Interquartile range.
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High mean eosinophilia 21.92 ±17.3, positive anti-Fasciola antibody titer (936.1 ±387.2),
and hepatic focal lesions (HFLs) in US confirmed the diagnosis (24.5%). Moreover, in 9.46%
of patients, the stool examination was positive.
In order to detect possible asymptomatic Fasciola infection, the relatives of patients were
screened for anti-Fasciola antibodies and eosinophilia in their sera using IHT and CBC. Anti-
Fasciola antibodies were detected in 20/65 (30.8%) asymptomatic relatives and elevated eosin-
ophilia (>6%) was detected in 19/65 (29.2%). However, the US examination revealed FHLs in
8 of these relatives. The analysis of variance revealed that the prevalence of fascioliasis was sig-
nificantly higher among young students, particularly those with primary education. Those liv-
ing in rural areas had higher prevalence rates. Significantly more peridomestic animals were
found near patients and their positive relatives. Other factors, including the consumption of
raw vegetables such as watercress, coriander, and lettuce, and the use of piped water supplies,
did not differ significantly.
The relationship between Fasciola infection and the sociodemographic characteristics of
the participants is depicted in Table 2. The risk of infection was significantly related to the age
of the participants (younger populations have a higher risk of infection) (P= 0.007) and the
proximity of farm animals to human dwellings (P<0.001). The odds of association between
animal breedings and the risk of Fasciola was 5.89 (CI 95%: 2.196–15.827), indicating that ani-
mals play a significant role in disease transmission in this area.
Morphological identification of lymnaeid snails and fasciolid
developmental stages
Six hundred lymnaeid snails from Manfalout locality, Assiut, Upper Egypt, were collected,
identified, and examined for the presence of Fasciola larvae. Lymnaeid snails were distin-
guished morphologically as Radix auricularia based on their elongated, thin, nonoperculated,
somewhat fragile shell with a large dextral shell opening and sharp apex. There were four
whorls, with the final one having a leftward convex angle. The surface of the shell appeared
smooth, thin, and brown with distinct ribs. The aperture was large and shaped like an ear, with
a curved columella in the center. The outer lip’s lower portion was rounded (Fig 1A).
Twenty-nine percentage of the collected snails were infected with various Fasciola develop-
mental stages, including sporocysts, rediae, and cercariae. Fig 1B depicts the microscopic fea-
tures of these stages.
Table 2. Logistic regression of the risk factors for fascioliasis infection.
Item Pvalue Odds ratio Confidence Interval 95%
Lower level Upper level
Age 0.0070.961 0.934 0.989
Sex 0.539 1.4 0.476 4.134
Occupation 0.293 0.798 0.524 1.215
Education 0.723 0.890 0.467 1.697
Residence 0.198 0.545 0.216 1.372
Eating raw vegetables 0.715 0.821 0.284 2.371
Animals in predomestics 0.0005.896 2.196 15.827
Water treatment 0.840 0.894 0.301 2.656
Significant P- value.
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Molecular analysis of Fasciola adult based on ribosomal ITS-1
The nucleotide sequences of Fasciola specimens have been deposited in GenBank under acces-
sion numbers as shown in Table 3. The ITS-1 sequences included the small ribosomal subunit
RNA gene, a partial sequence of the 18S, ITS1, and 5.8S rRNA genes, and a total of 591–617
base pairs.
Alignment analysis using online NCBI Blast confirmed the conservation of the 18S and
5.8S rDNA within Fasciola sp. and revealed that ITS-1 sequences of human samples
(MW248564, MW248568, and MW248573) were 100% identical to those of F.hepatica which
were previously published, while those of animal samples with accession No. (MW219535 and
MW219536) were 97–100% identical to those of F.gigantica reference sequences from differ-
ent countries. The presence of seven polymorphic sites indicative of the occurrence of both F.
hepatica and F.gigantica inferred from multiple alignments of ITS-1 sequences with Fasciola
spp. sequences available in GenBank to infer the annotation. No nucleotide deletions were
observed between the two Fasciola species. The primary difference between F.hepatica and F.
gigantica was a single-base substitution: A >T at the nucleotide site of 469. T >A at the nucle-
otide site of 297, C >T at positions 79 and 103, and T >C at the sites 207, 391, and 489
(Table 3).
F.hepatica and F.gigantica isolates from Assiut, Upper Egypt were compared to the most
closely related reference isolates using the ML method to generate a phylogenetic tree. The
ITS-1 phylogenetic tree revealed that Fasciola flukes were distributed into two main clades of
pure F.hepatica and F.gigantica, except for a hybrid isolate of F.gigantica from Vietnam
(MN970008), which was merged into the F.hepatica clade. It was demonstrated that the main
clade of F.hepatica included isolates from humans of the present study and those obtained
from various animals from different countries with a bootstrap confidence level of 69%. The F.
gigantica clade consisted of two main clusters that were further divided into terminal subclus-
ters involving F.gigantica isolates removed from cattle in this study (MW219535 and
MW219536) with the same species retrieved from different hosts (97% bootstrap value)
Fig 1. A. Shell of R.Auricularia collected snails from Assiut, Upper Egypt. A1: Dorsal view; A2: Ventral view. B.
Light micrograph of Fasciola larval stages collected from R.auricularia snails. B1: Mother redia showing the
pharynx (ph.), collar (c.), and a caudal papilliform process ×40. B2: Daughter redia showing the developing cercariae
(arrowheads) and a posterior process (post.p.) ×40. B3: Magnification of anterior portion showing the pharynx (ph.),
collar (c.), and birth pore x100. B4:Fasciola spp. Cercaria ×100.
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(Fig 2). There was no geographic or host segregation in both F.hepatica and F.gigantica
sequences. The neighbor-joining tree (NJ) yielded similar results, confirming the same topol-
ogy structure of the phylogenetic trees with high bootstrap confidence levels (S1 Fig).
Molecular analysis of lymnaeid snails
The ITS-1 molecular analyses of Radix spp. resulted in DNA fragments of approximately 720
bp in length, which were deposited in GenBank under accession no (MW248565, MW248569,
MW248575, and MW248576). Alignment analysis using online NCBI Blast showed that the
study specimens shared the highest similarities with R.auricularia from other countries
(89.36% with HG932017, JF922878 from Germany and Spain, respectively, 88.63% with
JX193589 from Netherlands, and 83.96% identity with R.auricularia MN194260 from India).
It was noted that only one isolate of R.natalensis (HQ283257) from France shared 87.85%
identity with our samples.The obtained sequences contained a portion of the small ribosomal
subunit, the entire ITS-1 sequence, and a portion of the 5.8S sequence.
The sequences are grouped into two primary clusters in the phylogenetic tree based on NJ
analysis (Fig 3). The first cluster included the GenBank reference sequences of R.auricularia,
our samples, and R.natalensis with a bootstrap confidence level of 58%. While those of R.pere-
gra and R.ampla sequences gathered into a separate cluster with a bootstrap confidence level
of 100%. In the same vein, the ML method analysis was identical to the NJ tree, which revealed
two significant clusters (S2 Fig).
Table 3. Comparison of the ITS-1 sequences of F.hepatica and F.gigantica of the present study with those from different hosts and geographical localities in
GenBank.
Species Locality
Accession no
Host Nucleotide sites of ITS1 region
79 103 207 297 391 469 489
F.hepatica FH4 ITS1 Assiut (MW248564) Human T T C A C T C
F.hepatica FH3 ITS1 Assiut (MW248571) Human T T C A C T C
F.hepatica FH5 ITS1 Assiut (MW248568) Human T T C A C T C
F.gigantica ITS1 Assiut (MW219536) Cattle C C T T T A T
F.gigantica ITS1 Assiut (MW219535) Cattle C T T T T A T
F.hepatica ITS1 Egypt (LC076196) Sheep T T C A C T C
F.hepatica ITS1 Iran (GQ925431) Human T T C A C T C
F.hepatica ITS1 India (KX198629) Sheep T T C A C T C
F.hepatica ITS1 Japan (AB514847) Cattle T T C A C T C
F.gigantica ITS1 Viet Nam (MN970008) N.A T T C A C T C
F.hepatica ITS1 Australia (MF678648) Sheep T T C A C T C
F.hepatica isolate ABC-02 France (JF294999) N.A T T C A C T C
F.hepatica isolate Bca2 Argentina (MG201869) Buffalo T T C A C T C
F.hepatica ITS1 Egypt Egypt (KP099942) N.A T T C A C T C
F.hepatica ITS1 Tunisia (JF423939) Equine - - C A C T C
F.gigantica ITS1 Chad (MK321606) Sheep - - T T T A T
F.gigantica ITS1 Kenya (KP760871) Connochaetes taurinus - - - T T A T
F.gigantica ITS1 Chad (MK321641) Cattle - - - T T A T
F.gigantica ITS1 Zimbabwe(MW046874) Radix natalensis - - - T T A T
F.hepatica ITS1 Egypt (KP215281) Human - - - A C T C
N.A = not available
https://doi.org/10.1371/journal.pntd.0011000.t003
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Fig 2. The phylogenetic relationships based on the ribosomal ITS-1 analysis: Including specimens collected from
humans and cattles in Upper Egypt and other representative isolates in the GenBank database from different
localities. Maximum likelihood method and Kimura 2-parameter model were used withthe percentage of bootstrap
confidence level (1000 replicates) This analysis involved 21 nucleotide sequences with a total of 420 positions in the
final dataset.
https://doi.org/10.1371/journal.pntd.0011000.g002
Fig 3. The phylogenetic relationships of Radix spp. based on ITS- 1 region: For lymnaeid specimens collected in this study and other representative
isolates in the GenBank database from different localities. Phylogenetic trees were constructed using MEGA X.0 with bootstrap values of 1000 replicates set
for NJ method and Tamura-Nei model.
https://doi.org/10.1371/journal.pntd.0011000.g003
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Mitochondrial gene analysis
After trimming the sequences, amplification of the COXI mitochondrial gene produced DNA
products (421–430 bp) that were specific to F.gigantica and F.hepatica, respectively. However,
NADI sequences generated approximately (578–581 bp). At Blastn, the studied samples exhib-
ited a high degree of similarity, ranging from 97% to 100%, with previously published
sequences of F.gigantica and F.hepatica recovered from various localities and stored in the
GenBank database. The alignment of COXI mtDNA nucleotide sequences with reference
sequences generated 7 haplotypes (4 haplotypes named FgCOI-H for F.gigantica and 3 haplo-
types named FhCOI-H for F.hepatica). For F.hepatica and F. gigantica, the mitochondrial
NADI gene generated 9 haplotypes (3 FhNDI-H and 6 FgNDI-H, respectively). Tables 4and 5
illustrate the haplotype numbers, sample accession numbers, and geographic distribution.
Furthermore, the evolutionary relationship between the studied isolates and reference
strains from other countries based on mitochondrial NADI gene analysis was determined
using ML and NJ phylogenetic trees (Figs 4and S3) in MEGA X.0, which revealed nearly iden-
tical evolutionary tree structures. ML analysis showed that Fasciola isolates of the present
study from humans, animals, and snails were grouped into two main clades of F.hepatica and
F.gigantica. The sequences belonging to the F.hepatica clade showed only 5 DNA variable
sites segregated on 3 haplotypes where all Egyptian F.hepatica isolates clustered into a distinct
haplotype (designated as Fh-NDIH1) with a high bootstrap value >90%. While those of F.
gigantica showed more genetic diversity at this marker with 18 DNA variable sites producing 6
haplotypes. The alignment clustered one F.gigantica isolate of the present study with an Egyp-
tian isolate (LC076211, [47]) into a distinct haplotype (FgNDI-H2) while the other isolate gen-
erated a separate FgNDI-H6 Haplotype (Table 4).
In accordance with the findings of NADI gene, the obtained partial sequences of the COXI
gene revealed nearly the same evolutionary history of F.hepatica and F.gigantica groups (Figs
5and S4). In addition, F.hepatica isolates of the present study revealed low genetic diversity
with only 2 segregating sites indicating 3 haplotypes that include F.hepatica human isolate
(MW217461) in a separate haplotype (FhCOI-H3) while the remaining of F.hepatica isolates
were clustered with an Egyptian isolate (AB553828), reported by Amer et al. [14] as Fh-
COIH1 haplotype.
Regarding to F.gigantica group, 4 haplotypes demonstrating 13 DNA segregating sites were
recorded. Phylogenetic analysis showed that Egyptian isolates of the F.gigantica group clus-
tered (bootstrap value 66%) with other sequences from Egypt, Iran, and Mauritania forming a
distinct haplotype (FgCOI-H1) (Table 5)
Haplotype analysis
The diversity indices of mitochondrial genes including the haplotype diversity (Hd), nucleo-
tide diversity (Pi), and the number of segregating sites (S) were calculated in both F.hepatica
and F.gigantica groups to compare our isolates with reference sequences. There was low
genetic diversity in F.hepatica isolates compared with reference sequences of both markers.
However, the diversity was higher in F.gigantica, group, especially for the NADI gene
(Table 6).
Discussion
Fascioliasis remains one of the most significant NTDs, particularly in endemic nations like
Egypt [47]. In Egypt, fasciolosis is endemic in the Nile Delta (Lower Egypt) [31,58] and causes
serious human and animal health problems [47]. However, the situation in Upper Egypt is
poorly understood, and precise infection estimates are lacking. Human fascioliasis reports
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from Upper Egypt were infrequent until 2015, and the incidence of human cases is on the rise
in Assiut Governorate, Upper Egypt [31,32], with a recent outbreak in 2018 [33]. Determining
the snail infection rate, the epidemiological criteria, the risk factors, and a prior genetic charac-
terization have become fundamental requirements for preventing further spread or outbreak
of this devastating disease.
Table 4. Comparison of the NADI sequences of Fasciola spp. from Upper Egypt with those from different geographical localities.
Species Country Host Haplotypes Accession number Reference
F.hepatica Iran
Poland
Egypt
Armenia
Human
Sheep
Sheep
Cattle
FhNDI-H1 GQ175362
KR422395
LC076257
MG972401
[45]
[46]
[47]
[48]
Egypt
Egypt
Egypt
Egypt
Egypt
Human
Human
Human
Snail
Snail
FhNDI-H1 MW218441
MW218442
MW218443
MW218444
MW218445
—
-
-
-
Spain Sheep FhNDI-H2 KF111680 [49]
Brazil Cattle FhNDI-H3 MK838728 [50]
F.gigantica Iran Cattle FgNDI-H1 KX021288 [45]
Egypt
Egypt
Cattle
Sheep
FgNDI-H2 MW218440
LC076211
-
[47]
Nigeria Bos_indicus FgNDI-H3 LC142768 [51]
Ghana Cattle FgNDI-H4 MF490247 [52]
Maghreb Sheep FgNDI-H5 MN913874 [53]
EgyptCattle FgNDI-H6 MW209692 -
= Sequences of the present study
https://doi.org/10.1371/journal.pntd.0011000.t004
Table 5. Comparison of the COXI sequences of Fasciola spp. from Upper Egypt with those from different geographical localities.
Species Country Host Haplotypes Accession number Reference
F.hepatica
Egypt
Human
Human
Snail
Snail
FhCOI-H1 MW217462
MW217468
MW217463
MW217465
-
Egypt Buffalo FhCOI-H1 AB553828 [14]
Iran
Poland
Iran
Ecuador
Sheep
Bison_bonasus
Goat
Cattle
FhCOI-H2 MN527599
KR422385
MK447990
LC273058
-
Egypthuman FhCOI-H3 MW217461 -
F.gigantica Egypt
Egypt
Cattle
Cattle
FgCOI-H1 MW217470
MW217466
-
-
Egypt
Iran
Mauritania
Buffalo
Cattle
Sheep
FgCOI-H1 AB553784
KX021275
HQ857101
[14]
[54]
[55]
India Bos_frontalis FgCOI-H2 KX656877 [56]
Viet Nam Cattle FgCOI-H3 MF287791 -
Turkey Buffalo FgCOI-H4 KY613945 [57]
= Sequences of the present study
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In this newly emerged endemic region in Upper Egypt, the sociodemographic characteris-
tics of the studied sample likely explain the emergence and describe the distribution of fascioli-
asis and the risk factors for human infection. Younger age groups had a higher infection rate,
representing a significant risk factor, according to the current findings. A significant finding of
this study is the high prevalence of fascioliasis infection among elementary school children.
Previous studies conducted in Egypt and other hyperendemic regions reported this observa-
tion [29,36,59–62]. They might become infected by eating uncooked contaminated plants
while helping their parents in farming or by drinking contaminated water while playing in
drainage canals [63,64]. This observation has been reported in other studies conducted in sev-
eral South American and Middle Eastern countries [32,65].
The results regarding the distribution of fascioliasis by gender are frequently inconsistent
[66]. The present study showed no correlation between gender and infection; both males
and females were infected. Several European regions reported a similar finding [3]. In other
Fig 4. ML tree based on partial sequence of NADI gene constructed by MEGA X.0. [43]: showing phylogenetic relationships of Egyptian Fasciola isolates
obtained in this study (marked with Asterix) compared to reference NAD1 sequences in the GenBank database. The percentage of bootstrap replicates (1000
replicates) is shown next to the branches The evolutionary history was inferred by using the Maximum Likelihood method and Tamura-Nei model.
https://doi.org/10.1371/journal.pntd.0011000.g004
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hyperendemic regions, however, females were considered the commonly infected group
[29,60].
In addition, residents of rural areas with nearby domestic animals such as cattle, sheep, etc.,
had a higher infection rate than those living in urban areas with no peridomestic animals. In
similar hyperendemic regions of Egypt, researchers such as Curtale et al. reported that the
Fig 5. Phylogenetic tree based on partial sequence of COXI gene constructed using ML method and Tamura-Nei model using MEGA X: the Egyptian
Fasciola isolates of the study were marked with Asterixcompared to reference COXI sequences in the GenBank database. The bootstrap confidence level
inferred from 1000 replicates is taken next to the branches.
https://doi.org/10.1371/journal.pntd.0011000.g005
Table 6. Diversity indices of mitochondrial DNA subunits in relation to F.gigantica and F.hepatica groups.
No of haplotypes Haplotype diversity
(Hd)
Variance of haplotype diversity Nucleotide diversity
(Pi)
Number of segregating sites (S)
NAD1 F.
hepatica
3 0.345 0.02967±0.172 0.0020 5
F.gigantica 6 0.952 0.00912±0.096 0.0134 18
COX1 F.
hepatica
3 0.644 0.01025±0.10 0.00199 2
F.
gigantica
4 0.643 0.03390±0.184 0.01090 13
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proximity of farm animals to farmers’ dwellings posed a risk [67–69]. A recent study revealed
that contact with domestic animals and the presence of animal dwellings in the households of
Egyptian farmers posed a risk of Fasciola transmission to consumable vegetables, utensils, and
drinking water supplies, and subsequently to the farmers’ wives and children [3]. This can
explain the significant association between peridomestic animal presence and infection risk in
the present study.
This study emphasizes the importance of eosinophilic count and anti-Fasciola antibody
screening in the families of Fasciola-infected patients. As documented previously, a single case
of fascioliasis may indicate a familial or local outbreak [70]. Members of the same family are at
risk of contracting the infection because they share the same eating habits or infection sources,
such as contaminated food or water [62]. These patients may continue to be asymptomatic;
consequently, eosinophilic count and serological screening of their sera are necessary to pre-
vent loss of diagnosis in asymptomatic cases. Herein, 30.8% (20/65) of the asymptomatic rela-
tives screened were positive for anti-Fasciola antibodies with elevated eosinophilia (>6%). In
addition, eight of them (8/20) exhibited radiological evidence of fascioliasis (HFLs) on ultra-
sound and/or CT. Similar observations were previously reported in Egypt [71], Spain [72],
Eastern Anatolia [70], and Peru [73]. These findings validate the utility of serological-based
tests and radiological methods as dependable diagnostic tools in community-based surveys for
fascioliasis, particularly in the early stages of infection when diagnostic eggs are absent from
the stool, and clinical abnormalities have not yet developed [70].
Similar to other vector-borne parasitic diseases, fascioliasis is transmitted via freshwater
bodies and lymnaeid snails, which serve as Fasciola’s major global intermediate hosts [39,74].
Consequently, it is essential to regularly update the information, faunal list, and snail distribu-
tion, especially as new endemic foci emerge [4,75]. At present, Egypt is a home to five species
of lymnaeids, including Galba truncatula,Galba schirazensis,Lymnaea stagnalis,Pseudosucci-
nea columella, and Radix natalensis [75,76]. Radix natalensis was the chief vector for Fasciola
in Qena, Upper Egypt [77], and is the specific vector for F.gigantica in Africa [9].
The collected snails in this study were identified as R.auricularia based on the morphologi-
cal characteristics of their shells. The typical form of R.auricularia is distinguished by its ear-
shaped shell and greatly expanded body whorl [78]. However, the proportions and shapes of
its shells vary widely owing to the plasticity of shell morphology affected by environmental
pressures and ontogeny [79]. On the other hand, most of Radix spp. expressed a relative simi-
larity in their morphological criteria in response to adaptations to shared environments.
Therefore, accurate identification between species is extremely difficult [80]. Thus, molecular
approaches to speciation are the most reliable [81]. The sequencing of lymnaeid DNA began at
the end of the 19th century, and its advanced application revealed the problems in the species
differentiation posed by conventional phenotypic malacological studies. Therefore, definitive
characterization is only achievable through marker sequencing [10].
Molecular analyses of Radix spp. confirmed the presence of R.auricularia in the study area.
Our sequences were grouped with R.auricularia reference sequences from different countries
through sequencing and phylogenetic analysis. Notably, Egyptian Radix isolates have a sepa-
rate position on the phylogenetic trees (Fig 3), which is supported by a high bootstrap value
reflecting the prominent nucleotide differences from reference sequences. Such variations may
be attributed to the genetic heterogeneity of Radix spp. in this newly emerged area in Egypt
which may indicate the occurrence of unique and uniform evolutionary events with the intro-
duction of this snail into novel geographic localities. The presence of geographically discrete
clusters of R.auricularia isolates has been previously reported [80,82].
In addition, a European isolate of R.natalensis (HQ283257) was clustered in close related-
ness with our isolates in the same R.auricularia group. Such interesting finding may indicate a
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potential Old-World origin along with genetic overlap with R.natalensis populations widely
distributed in Egypt. In consistent with our results, Lawton et al. reported that UK and Eur-
asian isolates of R.Auricularia were clustered with the African R.natalensis and the Asian R.
rubiginosa owing to the wide distribution of this species allover continents [80]. Furthermore,
Bargues et al. indicated that changes in the phylogenetic topology and the occurrence of inter-
relatedness between clades could be confounded by the type of genetic marker used for analy-
sis [10]. Therefore, some researchers illustrated the usefulness of mitochondrial genes such as
COXI gene over nuclear genes in differentiating lymnaeid snails and in providing a compre-
hensive biogeographical perspective [80,82].
To the best of our knowledge, no molecular studies on R.auricularia have been conducted
in Egypt to date. This is presumed to be the first set of data on the molecular identification and
phylogenetic analysis of this snail species in Egypt based on rDNA analysis methods. Due to
the lack of recent records, it is believed that this medically significant snail species has become
extinct in Egypt [75,83]. Notably, R.auricularia was last reported in the country in 1968 [84].
The repopulation and spread of this snail species in the area under study may be the result of
climate change and inadequate control measures [32].
Our findings have shown that the current Egyptian snail isolate serves efficiently as an
intermediate host for Fasciola, as 29% of the collected snails were found to be naturally
infected with different larval stages of Fasciola (Fig 1). The infection by F.hepatica haplotypes
was confirmed through a molecular examination of snail tissues using mtDNA markers NADI
and COXI. This highlights the role of the identified snails in the emergence and distribution of
fascioliasis infection in humans and animals, which parallels its distribution in snail hosts [85].
This association was explained by the spatial distribution of snail hosts and their habitats in
relation to Fasciola prevalence and intensity of infection [86]. R.auricularia likely played a sig-
nificant role in the introduction, expansion, and genetic diversity of the Fasciola population in
the study area.
Modern molecular techniques have been widely applied to the characterization of Fasciola
isolates exhibiting overlapping distribution in several African and Asian nations [6,47,87].
DNA sequencing provides clear differentiation between the two Fasciola spp. and the interme-
diate forms (hybrid and introgressed) based on multiple markers, including ribosomal and
mitochondrial markers [25,88]. To examine the population genetic structure of Fasciola spp.
collected from Fasciola-positive patients, animals, and snail hosts, molecular analysis was con-
ducted using genomic ITS-1 and mitochondrial (NADI and COXI) gene markers that have
been demonstrated to be reliable for the identification of Fasciola species [21].
Sequence analysis of the ITS-1 gene confirmed the co-occurrence of F.hepatica and F.
gigantica in Upper Egypt, corroborating a previous finding [89,90]. Through molecular char-
acterization, F.hepatica has been identified in human and snail isolates while F.gigantica has
been detected in bovine isolates. Furthermore, F.hepatica infection in human samples was
previously identified genetically using rDNA ITS markers in Upper Egypt [91] and Iran [92].
These reports demonstrate the remarkable adaptability of F.hepatica flukes to human hosts. In
contrast, a previous study conducted in Vietnam recovered a ‘pure’ F.gigantica isolate from
nine patients using nuclear ribosomal ITS1 and ITS2 markers [19].
This study established that F.hepatica was the dominant species infecting humans and
snails while F.gigantica is the isolated strain from cattle in the same endemic area [90]. These
findings contradict the recent findings of Omar et al., who reported that F.hepatica was pre-
dominant in cattle [90]. Previous research conducted in Egypt revealed that F.gigantica is the
predominant species found in cattle and buffaloes [14]. Earlier reports from elsewhere in the
world indicated that Fasciola spp. exhibited a wide variation in relation to the type of host. F.
hepatica was the predominant species in cattle in certain African nations, such as Algeria and
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South Africa [17,93], whereas F.gigantica is more prevalent in Zimbabwe and certain Asian
nations, such as Thailand [94] and Vietnam [19]. This variation in Fasciola species distribution
may be attributable to environmental, snail susceptibility, and other host-related factors. Due
to the limited number of samples collected from animal hosts and the lack of host variability, it
was not possible to determine the predominant Fasciola species inhabiting different animal
hosts in the studied area.
mtDNA is maternally inherited and traces the maternal lineage of single species or hybrid
forms. This is due to the high ratio of copies and rapid mutation rate, which produce sequence
differences among closely related species or subspecies [95]. As revealed by the molecular anal-
ysis of the mt COXI and NADI genes, Fasciola isolates from the current study shared a high
degree of similarity, up to 100%, with previously published sequences of F.gigantica and F.
hepatica retrieved from various geographic regions and stored in the GenBank database. Based
on haplotype analysis of mitochondrial genes, the snail intermediate host harbours the same F.
hepatica haplotype infecting humans in the studied area. Overall, the haplotype diversity of
our sequences compared to reference sequences was lower in F.hepatica group than F.gigan-
tica group. This is consistent with a previous report on bovine fascioliasis in Egypt that showed
a higher genetic diversity of F.gigantica than F.hepatica [14]. In contrast to our results, Amer
et al. reported a higher haplotype diversity of the mitochondrial NADI gene in F.hepatica than
in F.gigantica [47]. Also, numerous researchers have formerly described the heterogeneous
nature of the F.hepatica genome [96–98]. This disparity is primarily attributable to differences
in the number of both species of flukes characterized in those studies [47].
Noteworthy, our isolates, whether F.hepatica or F.gigantica belonged to one or two haplo-
types that revealed the low genetic variation in the studied samples. This could be attributed to
the small sample size, limited host distribution, and the restricted study area (the area of Fas-
ciola outbreak). Therefore, further research is needed on a wide geographic area including dif-
ferent types of animal hosts and different lymnaeid species.
Limitations and conclusions
In conclusion, the present study reported significant findings regarding the epidemiological
overview of fascioliasis in a newly established focus in Upper Egypt. Several factors, such as the
relatively small sample size and the lack of data regarding the effects of climate change, season-
ality, and the spatial distribution of the snail intermediate host, could limit the scope of the
study regarding the transmission pattern of the disease. In addition, the study emphasized the
significance of screening asymptomatic relatives in the neighborhood of suspected fascioliasis
cases to avoid underestimating the number of human cases and identifying the risk factors of
disease transmission. This study is the first molecular evidence of the existence of R.auricu-
laria in Egypt as the snail intermediate host in a newly emerged endemic area in Assiut Gover-
norate, Upper Egypt, to our knowledge. The findings indicated that the governorate of Assiut
can be considered a high-risk area for human populations, and health care policies must take
fascioliasis into account. Inferred from the data presented here is the presence of F.hepatica as
the chief Fasciola spp. infecting human and snail intermediate host in this new endemic
region. Further systematic research is required to clarify the genetic diversity and nationwide
distribution of Fasciola in different hosts and field-collected snails in Egypt using morphologi-
cal and molecular approaches. Also, extended studies are required to clarify the faunistic com-
position of lymnaeid snails in Upper Egypt using multiple nuclear and mitochondrial genes.
This study can be viewed as an initial step toward comprehending the epidemiological situa-
tion of human fasciolosis and the genetic diversity of the Fasciola population in a newly
emerged endemic focus in Upper Egypt.
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Supporting information
S1 Fig. Neighbor-Joining phylogenetic tree based on the ribosomal ITS-1 analysis of Egyp-
tian isolates of Fasciola spp. and other representative isolates in the GenBank database
from different localities. The evolutionary distances were computed using the Tamura 3-
parameter method. The tree is drawn to scale.
(TIF)
S2 Fig. Molecular phylogenetic analysis of Radix spp. using the ML method based on the
Tamura 3-parameter model. The tree is drawn to scale, with bootstrap value next to
branches.
(TIF)
S3 Fig. Neighbor-Joining Phylogenetic tree: Showing the evolutionary relationships
between Egyptian isolates of Fasciola spp. and references sequences based on the mito-
chonderial NADI gene analysis. The percentage of replicate trees in which the associated taxa
clustered together in the bootstrap test (1000 replicates) are shown next to the branches. The
tree is drawn to scale.
(TIF)
S4 Fig. Neighbor-Joining phylogenetic tree showing the evolutionary relationships
between Egyptian isolates of Fasciola spp. and references sequences based on the mito-
chondrial COXI gene analysis using the Tamura 3-parameter method with bootstrap value
next to branches (1000 replicates).
(TIF)
Acknowledgments
The authors would like to express their gratitude to Prof. Mohammad El-Taher Abdel- Rah-
man, professor of Tropical medicine and Gastroenterology, Faculty of Medicine, Assiut Uni-
versity for his great effort and contributions to this work.
Author Contributions
Conceptualization: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia
Hassan, Nahed Ahmed Elossily.
Data curation: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia
Hassan, Enas Abdelhameed Mahmoud Huseein, Sara Abdel-Aal Mohamed, Nahed Ahmed
Elossily.
Formal analysis: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia
Hassan, Adnan Ahmed Mohamed, Nahed Ahmed Elossily.
Investigation: Alzahraa Abdelraouf Ahmad, Mohammed Ageeli Hakami, Enas Abdelhameed
Mahmoud Huseein, Sara Abdel-Aal Mohamed, Nahed Ahmed Elossily.
Methodology: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia Has-
san, Enas Abdelhameed Mahmoud Huseein, Sara Abdel-Aal Mohamed, Nahed Ahmed
Elossily.
Resources: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia Hassan,
Enas Abdelhameed Mahmoud Huseein, Sara Abdel-Aal Mohamed, Adnan Ahmed
Mohamed, Nahed Ahmed Elossily.
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Fascioliasis in Upper Egypt: Epidemiological study and phylogenetic analysis
PLOS Neglected Tropical Diseases | https://doi.org/10.1371/journal.pntd.0011000 December 28, 2022 18 / 24
Software: Alzahraa Abdelraouf Ahmad, Mohammed Ageeli Hakami, Nahed Ahmed Elossily.
Supervision: Alzahraa Abdelraouf Ahmad.
Validation: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia Hassan,
Mohammed Ageeli Hakami, Sara Abdel-Aal Mohamed, Adnan Ahmed Mohamed, Nahed
Ahmed Elossily.
Visualization: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan, Waleed Attia Has-
san, Sara Abdel-Aal Mohamed, Adnan Ahmed Mohamed, Nahed Ahmed Elossily.
Writing – original draft: Alzahraa Abdelraouf Ahmad, Enas Abdelhameed Mahmoud
Huseein, Nahed Ahmed Elossily.
Writing – review & editing: Alzahraa Abdelraouf Ahmad, Haidi Karam-Allah Ramadan,
Mohammed Ageeli Hakami.
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