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Molecular characterization of Cryptosporidium spp. and Giardia duodenalis in children in Egypt

  • 1, 2,
  • 1,
  • 2,
  • 1,
  • 3,
  • 2, 4,
  • 4 and
  • 4Email author
Parasites & Vectors201811:403

https://doi.org/10.1186/s13071-018-2981-7

  • Received: 9 March 2018
  • Accepted: 27 June 2018
  • Published:

Abstract

Background

The transmission of Cryptosporidium spp. and Giardia duodenalis into humans varies according to species/genotypes of the pathogens. Although infections with both parasites are recorded in Egypt, few data are available on the distribution of Cryptosporidium species and G. duodenalis genotypes. The present study assessed the occurrence and genetic diversity of Cryptosporidium spp. and G. duodenalis in Egyptian children.

Methods

In the present study, 585 fecal specimens were collected from children eight years old and younger in three provinces (El-Dakahlia, El-Gharbia and Damietta) during March 2015 to April 2016. PCR-RFLP analysis of the small subunit rRNA gene and sequence analysis of the 60 kDa glycoprotein gene were used to detect and subtype Cryptosporidium spp., respectively, whereas PCR and sequence analyses of the triose phosphate isomerase, glutamate dehydrogenase and β-giardin genes were used to detect and genotype Giardia duodenalis.

Results

The overall infection rates of Cryptosporidium spp. and G. duodenalis were 1.4% and 11.3%, respectively. The Cryptosporidium species identified included C. hominis and C. parvum, each with three subtype families. The C. hominis subtypes were IbA6G3 (n = 2), IdA17 (n = 1), IdA24 (n = 1) and IfA14G1R5 (n = 1), while C. parvum subtypes were IIdA20G1 (n = 1), IIaA15G2R1 (n = 1), and IIcA5G3a (n = 1). The G. duodenalis identified included both assemblages A (n = 31) and B (n = 34). All G. duodenalis assemblage A belonged to the anthroponotic sub-assemblage AII, while a high genetic heterogeneity was seen within assemblage B.

Conclusions

Data from this study are useful in our understanding of the genetic diversity of Cryptosporidium spp. and G. duodenalis in Egypt and the potential importance of anthroponotic transmission in the epidemiology of both pathogens.

Keywords

  • Cryptosporidium
  • Giardia duodenalis
  • Children
  • Egypt
  • Epidemiology
  • Subtypes

Background

Diarrhea is a worldwide public health issue, responsible for 2.3 billion sicknesses and 1.3 million deaths in 2015. It is the second most important cause of death among children under 5 years of age [1]. Most of the deaths are recorded in developing countries, particularly African countries. Various gastrointestinal pathogens, including bacteria, viruses and parasites cause diarrhea. Among the latter, Cryptosporidium spp. and Giardia duodenalis are common etiological agents in humans and animals globally [2, 3]. Cryptosporidium is second only to rotavirus in causing diarrhea and death in children in developing countries, responsible for 2.9 million cases annually in children aged < 24 months in the sub-Saharan Africa [4, 5]. Similarly, G. duodenalis is responsible for ~280 million cases of intestinal diseases per year worldwide [6]. Cryptosporidium spp. and G. duodenalis are transmitted in humans through the fecal-oral route, either directly by person-to-person transmission or contact with infected animals or indirectly via food-borne or water-borne transmission following ingestion of contaminated food or water [2, 3].

Currently, over 30 Cryptosporidium species have been recognized, but humans are mostly infected with C. parvum and C. hominis [7] with the former mostly transmitted anthroponotically while the latter can be transmitted either anthroponotically or zoonotically [8]. Similarly, among the eight established G. duodenalis genotypes (frequently referred as assemblages) identified using molecular tools, assemblages A and B are responsible for most human infections. Between them, assemblage A is also commonly seen in animals and thus could be responsible for zoonotic G. duodenalis infection [8, 9].

It has been noted that some subtype families of C. parvum are more frequently found in certain host species, such as IIa in cattle, IIc in humans, and IId in sheep and goats. While all three subtype families of C. parvum can infect humans, their distribution in humans differs geographically and socioeconomically, probably as a result of differences in the importance of various transmission routes [8]. Similarly, host adaptation also occurs within G. duodenalis assemblage A, with AI subtypes being more commonly found in domestic animals, AII subtypes mostly in humans, and AIII subtypes almost exclusively in wild ruminants [8, 9]. Thus, molecular characterizations of Cryptosporidium spp. and G. duodenalis at species and subtype levels are helpful in improving our understanding of cryptosporidiosis and giardiasis epidemiology [7].

Compared with other countries, few data exist on the occurrence of Cryptosporidium and G. duodenalis genotypes and subtypes in humans in Egypt. Previous microscopic and serologic studies had shown a common occurrence of Cryptosporidium spp. and G. duodenalis in humans in the country [1012]. Only a few studies have examined the molecular characteristics of Cryptosporidium spp. and G. duodenalis in a small number of human clinical specimens [1318]. The current study was conducted to collect data on the distribution of Cryptosporidium and G. duodenalis genotypes and subtypes in kindergarten age children (≤ 8 years) in order to improve our understanding of the transmission of these parasites in Egypt.

Methods

Specimen collection

This study was conducted during March 2015 to April 2016 in El-Dakahlia, El-Gharbia, and Damietta provinces, Egypt (Fig. 1). Fresh stool specimens were collected monthly from 585 different children in 18 childcare centers, who ranged 2 to 8 years in age (median age: 4 years). These specimens were collected individually in sterile plastic cups and transported to the laboratory in coolers. Information on the age, gender, diarrhea and health status, animal contact and residency, was recorded from parents or guardians. Specimens were preserved in 70% ethanol and kept at 4 °C to prevent DNA deterioration prior to DNA extraction at the Centers for Disease Control and Prevention, Atlanta, GA, USA. No microscopy of pathogens was conducted during the study. Informed consent was obtained from the parents or guardians of the study children.
Fig. 1
Fig. 1

Map of Egypt showing the locations of study sites: El-Dakahlia, El-Gharbia and Damietta provinces

DNA extraction

Stored stool specimens were washed twice with distilled water by centrifugation to remove ethanol. DNA was extracted from washed fecal materials using the FastDNA SPIN Kit for Soil (MP Biomedicals, Irvine, CA, USA) and manufacturer-recommended procedures. DNA was eluted in 100 μl molecular grade water and stored at -20 °C prior to molecular analyses.

Cryptosporidium detection, genotyping and subtyping

All specimens were examined for Cryptosporidium spp. using a nested polymerase chain reaction (PCR) targeting a 834 bp fragment of the small subunit rRNA (SSU rRNA) gene [19]. C. parvum- and C. hominis-positive specimens were further analyzed by a nested PCR targeting a 850 bp fragment of the 60 kDa glycoprotein (gp60) gene [20]. Each analysis was conducted in duplicate, using C. baileyi and C. parvum DNA as the positive control for SSU rRNA and gp60 PCR, respectively, and reagent-grade water as the negative control. Cryptosporidium species in the positive specimens were identified by RFLP analysis of the secondary SSU rRNA PCR products using restriction enzymes SspI (New England BioLabs, Ipswich, MA, USA) and VspI (Promega, Madison, WI, USA) as described [19]. C. hominis and C. parvum subtypes were identified by bidirectional DNA sequence analysis of the secondary PCR products of the gp60 gene [20].

Giardia detection, genotyping and subtyping

All 585 specimens were analyzed for G. duodenalis using nested-PCR assays targeting 3 genetic loci, including triose phosphate isomerase (tpi) [21], beta-giardin (bg) [22] and glutamate dehydrogenase (gdh) [23] genes. Specimens were identified as G. duodenalis-positive when the expected PCR product was obtained from at minimum one of the three loci. G. duodenalis genotypes and subtypes were identified by bidirectional DNA sequence analysis of the secondary PCR products.

DNA sequence analyses

All positive secondary PCR products generated in the study were purified using Montage PCR filters (Millipore, Bedford, MA, USA) and sequenced in both directions on an ABI 3130 Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). Nucleotide sequences generated were edited and assembled using the ChromasPro software (www.technelysium.com.au/ChromasPro.html). They were aligned against each other and reference sequences [7, 9] using ClustalX software (http://www.clustal.org/) to identify Cryptosporidium subtypes and G. duodenalis assemblages and subtypes. Multilocus genotypes (MLGs) of G. duodenalis assemblage A were identified based on nucleotide sequences at the tpi, bg, and gdh loci, using the established nomenclature system [9].

Statistical analysis

The Chi-square test was used to compare Cryptosporidium and G. duodenalis infection rates between age groups (≤ 3 to 8 years), gender (boys and girls), residency (urban and rural), and children with and without gastrointestinal symptoms (diarrhea and abdominal pain) or animal contact (with and without). The relationship between age and diarrhea was assessed using the nonparametric Kendall’s tau_b and Spearman’s rho tests. The statistical analysis was performed using the SPSS software version 20.0 (IBM, Armonk, NY, USA). Differences were considered significant at P < 0.05.

Results

Occurrence of Cryptosporidium spp. and G. duodenalis

Of the 585 fecal specimens examined in this study from kindergarten children, 8 (1.4%) and 66 (11.3%) were positive for Cryptosporidium spp. and G. duodenalis, respectively. No concurrence of the two pathogens was detected in any of the specimens.

By age, the highest rates of Cryptosporidium (2.7%) and G. duodenalis (14.2%) infections were detected in children of age ≤ 3 years and 4 years, respectively; neither Cryptosporidium nor G. duodenalis were detected in children of 8 years in age (Table 1). The infection rates of both protozoans were similar between girls and boys (1.0% and 1.7% for Cryptosporidium and 11.1% and 11.5% for G. duodenalis, respectively) (χ2 = 0.460, P = 0.49 and χ2 = 0.011, P = 0.91, respectively).
Table 1

Occurrence of Cryptosporidium spp. and Giardia duodenalis in children by age, gender, diarrhea or abdominal pain occurrence, animal contact, residency and locality

Variable

No. of samples

No. of positive (%)

Cryptosporidium spp.

95% confidence interval

Giardia duodenalis

95% confidence interval

Lower limit

Upper limit

Lower limit

Upper limit

Age

 ≤ 3 years

74

2 (2.7)

-0.009

0.063

7 (9.5)

0.028

0.161

 4 years

141

3 (2.1)

-0.002

0.044

20 (14.2)

0.084

0.199

 5 years

190

1 (0.5)

-0.005

0.015

26 (13.7)

0.088

0.185

 6 years

136

2 (1.5)

-0.005

0.035

10 (7.4)

0.030

0.117

 7years

27

0 (0.0)

0.000

0.000

3 (11.1)

-0.007

0.229

 8 yeas

17

0 (0.0)

0.000

0.000

0 (0.0)

0.000

0.000

Gender

 Female

289

3 (1.0)

-0.001

0.021

32 (11.1)

0.074

0.147

 Male

296

5 (1.7)

0.002

0.031

34 (11.5)

0.078

0.151

Diarrhea occurrence

 Yes

89

2 (2.3)

-0.008

0.054

17 (19.1)a

0.109

0.272

 No

496

6 (1.2)

0.002

0.021

49 (9.9)a

0.72

0.125

Abdominal pain

 Yes

351

7 (2.0)

0.005

0.034

38 (10.8)

0.075

0.140

 No

234

1 (0.4)

-0.004

0.012

28 (12.0)

0.078

0.161

Animal contact

 With

257

3 (1.2)

-0.001

0.025

27 (10.5)

0.067

0.142

 Without

328

5 (1.5)

0.001

0.028

39 (11.9)

0.083

0.154

Residency

 Rural

332

5 (1.5)

0.001

0.028

40 (12.1)

0.085

0.156

 Urban

253

3 (1.2)

-0.001

0.025

26 (10.3)

0.065

0.140

Locality

 El-Dakahlia

272

5 (1.8)

0.002

0.033

31 (11.4)

0.076

0.151

 El-Gharbia

189

2 (1.1)

-0.003

0.025

24 (12.7)

0.079

0.174

 Damietta

124

1 (0.8)

-0.007

0.023

11 (8.9)

0.038

0.139

aThe difference between the two groups is significant

Cryptosporidium infection rate was 2.3% and 1.2 % in children with and without diarrhea, respectively (χ2 = 0.576, P = 0.44). In contrast, the infection rate of G. duodenalis was significantly higher in diarrheic children (19.1%) than in non-diarrheic ones (9.9%) (χ2 = 6.149, P = 0.01). There was also an insignificantly higher occurrence of Cryptosporidium spp. in children with abdominal pain (2.0%) than those without it (0.4%) (χ2 = 2.612, P = 0.10). In contrast, G. duodenalis infection rates were similar between the two groups (10.8% and 12.0%, respectively; χ2 = 0.134, P = 0.71). The infection rates of Cryptosporidium and G. duodenalis were similar between children with (1.2% and 10.5%, respectively) and without (1.5% and 11.9%, respectively) animal contact (χ2 = 0.146, P = 0.92 and χ2 = 0.128, P = 0.93, respectively). In addition, children in rural areas had Cryptosporidium and G. duodenalis infection rates (1.5% and 12.1%, respectively) similar to those in urban areas (1.2% and 10.3%, respectively; χ2 = 0.091, P = 0.76 and χ2 = 0.339, P = 0.56, respectively; Table 1). The infection rate of Cryptosporidium spp. in El-Dakahlia (1.8%) was higher than in El-Gharbia (1.1%) and Damietta (0.8%). In contrast, the infection rate of G. duodenalis was higher in El-Dakahlia (11.4%) and El-Gharbia (12.7%) than in Damietta (8.9%; Table 1).

There was a significant negative correlation between age and diarrhea (correlation coefficient was -0.115 and -0.127. by Kendall’s tau_b and Spearman’s rho tests, respectively; P = 0.002 in both tests).

Cryptosporidium species and subtypes

The RFLP analysis of the SSU rRNA PCR products identified the presence of C. hominis in five specimens and C. parvum in three specimens (Table 2). Three subtype families were identified within C. hominis and C. parvum each by gp60 sequence analysis. The C. hominis subtypes families included Ib (in two specimens), Id (in two specimens) and If (in one specimen), while the C. parvum subtypes families included IIa, IIc, and IId (in one specimen each). There were two subtypes (IdA17 and IdA24) in the subtype family Id and one subtype each in subtype families Ib (IbA6G3 in two specimens) and If (IfA14G1R5 in one specimen). The C. parvum subtypes detected included IIaA15G2R1, IIdA20G1 and IIcA5G3a (in one specimen each).
Table 2

Characteristics of eight Cryptosporidium-positive children

Cryptosporidium spp.

Subtypes

Age (years)

Gender

Diarrhea occurrence

Abdominal pain occurrence

Animal contact

Residency

Cryptosporidium hominis

IbA6G3a

4

Female

No

Yes

No

Urban

IbA6G3a

4

Male

No

Yes

No

Urban

IdA17

2

Male

No

Yes

No

Urban

IdA24

5

Male

No

Yes

No

Rural

IfA14G1R5

3.5

Female

No

No

No

Rural

Cryptosporidium parvum

IIaA15G2R1

5.5

Male

Yes

Yes

Yes

Rural

IIdA20G1a

3

Male

Yes

Yes

Yes

Rural

IIcA5G3a

6

Female

No

Yes

Yes

Rural

aNew subtype identified in humans in Egypt

Giardia duodenalis genotypes and subtypes

Of the 66 G. duodenalis-positive specimens, 56 were positive in tpi PCR, 48 in gdh PCR, and 55 in bg PCR. Among them, 31 (47.0%) had assemblage A and 34 (51.5%) had assemblage B, with one specimen (1.5%) being positive for both assemblages A and B (Table 3). The latter was indicated by the identification of assemblage B at the tpi and gdh loci but assemblage A at the bg locus. There were mostly no double peaks in the chromatograms generated from the study. Assemblage A was identified in 28 specimens based on tpi and bg sequence analyses but in 25 specimens by gdh sequence analysis. In contrast, assemblage B was found in 28, 23 and 27 specimens at the tpi, gdh and bg loci, respectively (Table 3). The relative distribution of G. duodenalis assemblages A and B was similar among three provinces (Table 4); assemblage A was detected in 14, 11 and 6 specimens from El-Dakahlia, El-Gharbia and Damietta provinces, respectively, whereas, assemblage B was detected in 16, 13 and 5 specimens, respectively.
Table 3

Distribution of G. duodenalis assemblages in children from different kindergartens at the tpi, gdh and bg loci

Study areaa

No. of samples

No. of positive (%)

Number of positive

tpi

gdh

bg

Assemblage A (n)

Assemblage B (n)

Assemblage A (n)

Assemblage B (n)

Assemblage A (n)

Assemblage B (n)

El-Dakahlia

K1

34

2 (5.9)

0

2

0

1

0

1

K2

39

8 (20.5)

5

3

3

3

4

3

K3

33

3 (9.1)

2

0

3

0

2

0

K4

31

3 (9.7)

1

2

1

2

1

2

K5

22

2 (9.1)

2

0

1

0

1

0

K6

35

6 (17.1)

2

4

2

3

3

2

K7

37

2 (5.4)

0

2

0

2

0

2

K8

41

5 (12.2)

1

3

1

3

1

4

El-Gharbia

K1

27

3 (11.1)

2

1

1

1

1

1

K2

30

5 (16.7)

2

2

2

2

2

3

K3

34

5 (14.7)

4

1

3

1

4

1

K4

29

2 (6.9)

0

2

0

1

0

1

K5

32

4 (12.5)

1

2

1

0

1

2

K6

37

5 (13.5)

2

2

2

1

2

2

Damietta

K1

22

3 (13.6)

1

0

2

1

2

1

K2

28

4 (14.3)

1

1

1

2

2

1

K3

38

4 (10.5)

2

1

2

0

2

1

K4

36

0 (0.0)

0

0

0

0

0

0

Total

 

585

66 (11.3)

28

28

25

23

28

27

aK, kindergarten

Table 4

Distribution of Cryptosporidium species and subtypes and Giardia duodenalis assemblages by locality

Province

Cryptosporidium spp.

Giardia duodenalis

Species (n)

Subtypes (n)

Assemblage A

Assemblage B

Assemblages A+B

El-Dakahlia

C. parvum (2)

IIaA15G2R1 (1); IIcA5G3a (1)

14

16

1

C. hominis (3)

IbA6G3 (1); IdA17 (1); IdA24 (1)

   

El-Gharbia

C. parvum (1)

IIdA20G1 (1)

11

13

0

C. hominis (1)

IbA6G3 (1)

   

Damietta

C. hominis (1)

IfA14G1R5 (1)

6

5

0

Total

C. parvum (3)

IIdA20G1 (1); IIaA15G2R1 (1); IIcA5G3a (1)

31/66 (47.0%)

34/66 (51.5%)

1/66 (1.5%)

C. hominis (5)

IbA6G3 (2); IdA17 (1); IdA24 (1); IfA14G1R5 (1)

   

Multilocus genotypes (MLGs) of G. duodenalis

Sequence analysis of the three genetic loci showed only limited genetic diversity in assemblage A. All identified subtypes were belonged to sub-assemblage AII. Therefore, at the tpi locus, all assemblage A sequences were identical to the A2 subtype sequence (U57897) in GenBank (Table 5). Similarly, at the gdh locus, all 25 assemblage A sequences obtained were identical to the A2 subtype sequence (AY178737) in GenBank, while at the bg locus, 22 were identical to the A3 subtype (AY072724), 4 were identical to the A2 subtype (AY072723), and 2 belonged to a new subtype A9 (MG746615). Among the assemblage A specimens, 4 and 18 specimens had MLGs AII-1 and AII-9, respectively. In addition, one new MLG AII-15 was identified in one specimen (Table 5). In contrast, each of the 20 MLGs of assemblage B was identified in only one specimen.
Table 5

Multilocus sequence types of Giardia duodenalis assemblage A in children, Egypt

MLGs

Sequence type (GenBank ID)

No. positive

Specimen ID

tpi

gdh

bg

AII-1

A2 ( U57897)

A2 (AY178737)

A2 (FJ560582)

4

43567, 43664, 43968, 44106

AII-9

A2 (U57897)

A2 (AY178737)

A3 (AY072724)

18

43509, 43524, 43547, 43574,43581,43608, 43618, 43632, 43700, 43899, 43907, 43956, 44038, 44042, 44046, 44069, 44116, 44170

AII-15

A2 ( U57897)

A2 (AY178737)

A9a (MG746615)

1

43969

Ba (MG787952)

Ba (MG746609)

A3 (AY072724)

1

43642

A2 (U57897)

A3 (AY072724)

1

43532

A2 (U57897)

A9a (MG746615)

1

43936

A2 (AY178737)

A3 (AY072724)

1

44067

A2 (U57897)

3

43503, 43607, 43894

A2 (AY178737)

1

43569

A3 (AY072724)

1

44095

aNew sequence type identified in the study

Much higher genetic diversity was seen in assemblage B (Additional file 1: Table S1). Of the 28 specimens that were positive for assemblage B at the tpi locus, 14 had generated sequences identical to either KX668322 (n = 3), JF918523 (n = 2), KT948107 (n = 2), KT948111 (n = 2), AB781127 (n = 1), AY368163 (n = 1), JF918519 (n = 1), KY696816 (n = 1) or KX468984 (n = 1), while 14 specimens generated sequences of one of the 10 new types (MG787950–MG787959). Similarly, of the 23 specimens that were positive for assemblage B at the gdh locus, 14 had sequences identical to either KY696804 (n = 4), KM190714 (n = 3), KP687771 (n = 3), U362955 (n = 2), EF507654 (n = 1) or KP687770 (n = 1), while the remaining nine specimens produced sequences of one of the eight new types (MG746604–MG746611). At the bg locus, 24 specimens generated sequences identical to either KU504732 (n = 6), KY696836 (n = 5), JF918485 (n = 3), KU504720 (n = 2), KU504707 (n = 2), MF169196 (n = 2), AB480877 (n = 1), KT948086 (n = 1), KU504731 (n = 1) or KY483962 (n = 1), whereas three specimens yielded sequences that belonged to one of the three new subtypes (MG746612–MG746614). Altogether, 44 specimens were successfully subtyped at all three genetic loci, forming 3 MLGs of assemblage A and 20 MLGs of assemblage B.

Discussion

In the present study, the overall infection rates of Cryptosporidium spp. and G. duodenalis in children were 1.4 and 11.3%, respectively. Earlier studies based on microscopy had recorded 5.6–60.2% and 17.6–25.0% infection rates of Cryptosporidium spp. and G. duodenalis in Egyptian children, respectively [2427]. A previous molecular analysis of fecal specimens from Egyptian children produced 49.1% and 21% infection rate for Cryptosporidium spp. and G. duodenalis, respectively [13, 17]. In the neighboring Lebanon, infection rates of 10.4% and 28.5% were reported in school children for Cryptosporidium spp. and G. duodenalis, respectively [28]. Similar low Cryptosporidium occurrence (1.6–2.0%) was observed in children in China [29, 30]. The low occurrence of Cryptosporidium spp. in this study might be due to the older age of children enrolled in this study. In developing countries, children under two years have the highest occurrence of Cryptosporidium spp. [4, 31]. In addition, children participating in the study were healthy kindergartners rather than in-patients and outpatients in most previous studies. As expected, children with diarrhea had higher occurrence of both Cryptosporidium spp. and G. duodenalis in this and earlier studies [28]. These are also supported by results of the nonparametric analysis of the negative correlation between age and occurrence diarrhea in this study.

In our study, we identified only C. hominis and C. parvum in children. This is similar to results of other studies in Egypt [13, 14, 32]. Moreover, the more common occurrence of C. hominis in children in this and other African studies suggests that anthroponotic transmission is important in cryptosporidiosis epidemiology in this area, although the occurrence of zoonotic infections could not be fully excluded [1315, 28, 3235]. This is also supported by the identification of IIcA5G3a in C. parvum, which is considered a human-adapted C. parvum subtype [8]. In contrast, previous studies in the neighboring Mideast countries had shown a dominance of the zoonotic IIa and IId subtypes of C. parvum in children, which were only identified in two of the eight cryptosporidiosis cases in this study [3640]. The insignificant associations between cryptosporidiosis occurrence and animal contact or rural residency in this study also support the importance of anthroponotic transmission in Cryptosporidium spp. in Egyptian children.

Although Cryptosporidium spp. were detected in only a few specimens in the study, we recorded seven subtypes in six families, including Ib, Id and If subtype families of C. hominis and IIa, IIc, and IId subtype families of C. parvum. This indicates that the transmission of Cryptosporidium in the study area is intensive. It has been reported that subtype families Ia, Ib, Id and Ie are common in children in developing countries [8, 31]. Nevertheless, the IbA6G3, IdA17, IdA24, and IfA14G1R5 identified in this study are rare subtypes within these common C. hominis subtype families [8, 31], indicating that C. hominis transmission in Egypt is probably autochthonous in nature.

The genotypes (assemblages of similar sequence types identified by multilocus molecular characterization) of G. duodenalis in infected children from the three provinces in this study belonged to assemblages A and B. This agrees with the findings of a recent study of G. duodenalis in children in Egypt [18]. The assemblages E and C reported in a few Egyptian children in previous studies [16, 17] were not detected in the present study. The equal occurrence of assemblages A and B in the present study is in discordance with observations in previous Egyptian studies, which showed a dominance of assemblage B in children [1618]. Globally, assemblage B is more common than assemblage A in humans [7]. As assemblage B is much less frequently detected in animals [2], G. duodenalis transmission in Egyptian children appears to be mostly anthroponotic. This is also supported by the identification of assemblage A isolates in the study as the sub-assemblage AII, which is preferentially found in humans [7].

In this study, a much higher genetic diversity was observed in assemblage B than in assemblage A. Similar observations were made in previous studies [2]. This could be due to the more frequent occurrence of genetic recombination among assemblage A isolates, as assemblage B is known to have much higher allelic sequence heterozygosity than assemblage A. The existence of highly genetic variations among isolates of assemblage B has led to the inability of categorizing assemblage B isolates into well-defined specific sub-assemblages [9]. Comparative genomics rather than current MLG analysis might be needed for better characterization of assemblage B isolates [41].

Conclusions

Giardiasis is apparently common, and cryptosporidiosis remains to be a problem in kindergarten age children in Egypt. The dominance of C. hominis and common occurrence of G. duodenalis assemblage B and sub-assemblage AII in clinical specimens showcases the important role of anthroponotic transmission in disease epidemiology, although the occurrence of zoonotic infections could not be totally ruled out. Improved sanitation and hygiene and other intervention measures such as better health communication and the provision of clean and safe drinking water should be implemented to reduce the occurrence of cryptosporidiosis and giardiasis and minimize the impact of diarrhea on pediatric health in the country.

Abbreviations

bg

β-giardin

gdh

Glutamate dehydrogenase

MLGs: 

Multilocus genotypes

RFLP: 

Restriction fragment length polymorphism

SSU rRNA

Small subunit ribosomal ribonucleic acid

tpi

Triose phosphate isomerase

Declarations

Acknowledgements

The authors would like to thank Matthew H. Seabolt, Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, CDC for technical assistance during this study. The findings and conclusions in this report are those of the authors and do not necessarily represent the views of the Centers for Disease Control and Prevention.

Funding

This work was supported by the Ministry of Higher Education in Egypt and Centers for Disease Control and Prevention.

Availability of data and materials

The dataset supporting the conclusions of this article is included within the article. Representative nucleotide sequences generated in this study were deposited into the GenBank database under accession numbers MG746604-MG746617 and MG787950-MG787959.

Authors’ contributions

DN and LX conceived and designed the study. DN and NA collected the specimens. DN performed the experiments. DN and LX analyzed the data and prepared the manuscript. AE, DR, AM, NA, YW and YF helped with the study design, data collection and manuscript preparation. All authors read and approved the final manuscript.

Ethics approval and consent to participate

Informed consent was obtained from the parents or guardians prior to specimen collection. The fieldwork was approved by the ethics committee of the Faculty of Veterinary Medicine, Mansoura University, Egypt.

Consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

Publisher’s Note

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Authors’ Affiliations

(1)
Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt
(2)
Division of Foodborne, Waterborne, and Environmental Diseases, National Center for Emerging and Zoonotic Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, GA 30329, USA
(3)
Department of Poultry Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, 35516, Egypt
(4)
Key Laboratory of Zoonosis of Ministry of Agriculture, College of Veterinary Medicine, South China Agricultural University, Guangzhou, 510642, China

References

  1. GBD 2015 Disease and Injury Incidence and Prevalence Collaborators. Global, regional, and national incidence, prevalence, and years lived with disability for 310 diseases and injuries, 1990–2015: a systematic analysis for the Global Burden of Disease Study 2015. Lancet. 2016;388:1545–602.View ArticleGoogle Scholar
  2. Ryan U, Caccio SM. Zoonotic potential of Giardia. Int J Parasitol. 2013;43:943–56.View ArticlePubMedGoogle Scholar
  3. Checkley W, White AC Jr, Jaganath D, Arrowood MJ, Chalmers RM, Chen XM, et al. A review of the global burden, novel diagnostics, therapeutics, and vaccine targets for Cryptosporidium. Lancet Infect Dis. 2015;15:85–94.View ArticlePubMedGoogle Scholar
  4. Kotloff KL, Nataro JP, Blackwelder WC, Nasrin D, Farag TH, Panchalingam S, et al. Burden and aetiology of diarrhoeal disease in infants and young children in developing countries (the Global Enteric Multicenter Study, GEMS): a prospective, case-control study. Lancet. 2013;382:209–22.View ArticlePubMedGoogle Scholar
  5. Sow SO, Muhsen K, Nasrin D, Blackwelder WC, Wu Y, Farag TH, et al. The burden of Cryptosporidium diarrheal disease among children < 24 months of age in moderate/high mortality regions of Sub-Saharan Africa and South Asia, utilizing data from the Global Enteric Multicenter Study (GEMS). PLoS Negl Trop Dis. 2016;10:e0004729.View ArticlePubMedPubMed CentralGoogle Scholar
  6. Einarsson E, Ma’ayeh S, Svard SG. An up-date on Giardia and giardiasis. Curr Opin Microbiol. 2016;34:47–52.Google Scholar
  7. Xiao L, Feng Y. Molecular epidemiologic tools for waterborne pathogens Cryptosporidium spp. and Giardia duodenalis. Food Waterborne Parasitol. 2017;8–9:14–32.View ArticleGoogle Scholar
  8. Xiao L. Molecular epidemiology of cryptosporidiosis: an update. Exp Parasitol. 2010;124:80–9.View ArticlePubMedGoogle Scholar
  9. Feng Y, Xiao L. Zoonotic potential and molecular epidemiology of Giardia species and giardiasis. Clin Microbiol Rev. 2011;24:110–40.View ArticlePubMedPubMed CentralGoogle Scholar
  10. El-Moamly AA, El-Sweify MA. ImmunoCard STAT! cartridge antigen detection assay compared to microplate enzyme immunoassay and modified Kinyoun’s acid-fast staining technique for detection of Cryptosporidium in fecal specimens. Parasitol Res. 2012;110:1037–41.Google Scholar
  11. Antonios SN, Tolba OA, Othman AA, Saad MA. A preliminary study on the prevalence of parasitic infections in immunocompromised children. J Egypt Soc Parasitol. 2010;40:617–30.PubMedGoogle Scholar
  12. Hassanein SM, Abd-El-Latif MM, Hassanin OM, Abd-El-Latif LM, Ramadan NI. Cryptosporidium gastroenteritis in Egyptian children with acute lymphoblastic leukemia: magnitude of the problem. Infection. 2012;40:279–84.View ArticlePubMedGoogle Scholar
  13. Helmy YA, Krucken J, Nockler K, von Samson-Himmelstjerna G, Zessin KH. Molecular epidemiology of Cryptosporidium in livestock animals and humans in the Ismailia province of Egypt. Vet Parasitol. 2013;193:15–24.View ArticlePubMedGoogle Scholar
  14. El-Badry AA, Al-Antably AS, Hassan MA, Hanafy NA, Abu-Sarea EY. Molecular seasonal, age and gender distributions of Cryptosporidium in diarrhoeic Egyptians: distinct endemicity. Eur J Clin Microbiol Infect Dis. 2015;34:2447–53.View ArticlePubMedGoogle Scholar
  15. Ibrahim MA, Abdel-Ghany AE, Abdel-Latef GK, Abdel-Aziz SA, Aboelhadid SM. Epidemiology and public health significance of Cryptosporidium isolated from cattle, buffaloes, and humans in Egypt. Parasitol Res. 2016;115:2439–48.View ArticlePubMedGoogle Scholar
  16. Soliman RH, Fuentes I, Rubio JM. Identification of a novel Assemblage B subgenotype and a zoonotic Assemblage C in human isolates of Giardia intestinalis in Egypt. Parasitol Int. 2011;60:507–11.View ArticlePubMedGoogle Scholar
  17. Helmy YA, Klotz C, Wilking H, Krucken J, Nockler K, Von Samson-Himmelstjerna G, et al. Epidemiology of Giardia duodenalis infection in ruminant livestock and children in the Ismailia province of Egypt: insights by genetic characterization. Parasit Vectors. 2014;7:321.View ArticlePubMedPubMed CentralGoogle Scholar
  18. Ismail MA, El-Akkad DM, Rizk EM, El-Askary HM, El-Badry AA. Molecular seasonality of Giardia lamblia in a cohort of Egyptian children: a circannual pattern. Parasitol Res. 2016;115:4221–7.View ArticlePubMedGoogle Scholar
  19. Xiao L, Morgan UM, Limor J, Escalante A, Arrowood M, Shulaw W, et al. Genetic diversity within Cryptosporidium parvum and related Cryptosporidium species. Appl Environ Microbiol. 1999;65:3386–91.PubMedPubMed CentralGoogle Scholar
  20. Alves M, Xiao L, Sulaiman I, Lal AA, Matos O, Antunes F. Subgenotype analysis of Cryptosporidium isolates from humans, cattle, and zoo ruminants in Portugal. J Clin Microbiol. 2003;41:2744–7.View ArticlePubMedPubMed CentralGoogle Scholar
  21. Sulaiman IM, Fayer R, Bern C, Gilman RH, Trout JM, Schantz PM, et al. Triosephosphate isomerase gene characterization and potential zoonotic transmission of Giardia duodenalis. Emerg Infect Dis. 2003;9:1444–52.View ArticlePubMedPubMed CentralGoogle Scholar
  22. Caccio SM, De Giacomo M, Pozio E. Sequence analysis of the beta-giardin gene and development of a polymerase chain reaction-restriction fragment length polymorphism assay to genotype Giardia duodenalis cysts from human faecal samples. Int J Parasitol. 2002;32:1023–30.View ArticlePubMedGoogle Scholar
  23. Read CM, Monis PT, Thompson RC. Discrimination of all genotypes of Giardia duodenalis at the glutamate dehydrogenase locus using PCR-RFLP. Infect Genet Evol. 2004;4:125–30.View ArticlePubMedGoogle Scholar
  24. Rizk H, Soliman M. Coccidiosis among malnourished children in Mansoura, Dakahlia Governorate, Egypt. J Egypt Soc Parasitol. 2001;31:877–86.PubMedGoogle Scholar
  25. Abdel-Hafeez EH, Ahmad AK, Ali BA, Moslam FA. Opportunistic parasites among immunosuppressed children in Minia District, Egypt. Korean J Parasitol. 2012;50:57–62.View ArticlePubMedPubMed CentralGoogle Scholar
  26. Abdel-Messih IA, Wierzba TF, Abu-Elyazeed R, Ibrahim AF, Ahmed SF, Kamal K, et al. Diarrhea associated with Cryptosporidium parvum among young children of the Nile River delta in Egypt. J Trop Pediatr. 2005;51:154–9.View ArticlePubMedGoogle Scholar
  27. El-Mohamady H, Abdel-Messih IA, Youssef FG, Said M, Farag H, Shaheen HI, et al. Enteric pathogens associated with diarrhea in children in Fayoum, Egypt. Diagn Microbiol Infect Dis. 2006;56:1–5.View ArticlePubMedGoogle Scholar
  28. Osman M, El Safadi D, Cian A, Benamrouz S, Nourrisson C, Poirier P, et al. Prevalence and risk factors for intestinal protozoan infections with Cryptosporidium, Giardia, Blastocystis and Dientamoeba among schoolchildren in Tripoli, Lebanon. PLoS Negl Trop Dis. 2016;10:e0004496.View ArticlePubMedPubMed CentralGoogle Scholar
  29. Feng Y, Wang L, Duan L, Gomez-Puerta LA, Zhang L, Zhao X, et al. Extended outbreak of cryptosporidiosis in a pediatric hospital, China. Emerg Infect Dis. 2012;18:312–4.View ArticlePubMedPubMed CentralGoogle Scholar
  30. Wang T, Fan Y, Koehler AV, Ma G, Li T, Hu M, et al. First survey of Cryptosporidium, Giardia and Enterocytozoon in diarrhoeic children from Wuhan, China. Infect Genet Evol. 2017;51:127–31.View ArticlePubMedGoogle Scholar
  31. Squire SA, Ryan U. Cryptosporidium and Giardia in Africa: current and future challenges. Parasit Vectors. 2017;10:195.View ArticlePubMedPubMed CentralGoogle Scholar
  32. Abd El Kader NM, Blanco MA, Ali-Tammam M, Abd El Ghaffar Ael R, Osman A, El Sheikh N, et al. Detection of Cryptosporidium parvum and Cryptosporidium hominis in human patients in Cairo, Egypt. Parasitol Res. 2012;110:161–6.View ArticlePubMedGoogle Scholar
  33. Ayinmode AB, Fagbemi BO, Xiao L. Molecular characterization of Cryptosporidium in children in Oyo State, Nigeria: implications for infection sources. Parasitol Res. 2012;110:479–81.View ArticlePubMedGoogle Scholar
  34. Abu Samra N, Jori F, Caccio SM, Frean J, Poonsamy B, Thompson PN. Cryptosporidium genotypes in children and calves living at the wildlife or livestock interface of the Kruger National Park, South Africa. Onderstepoort J Vet Res. 2016;83:e1–7.View ArticlePubMedGoogle Scholar
  35. Eibach D, Krumkamp R, Al-Emran HM, Sarpong N, Hagen RM, Adu-Sarkodie Y, et al. Molecular characterization of Cryptosporidium spp. among children in rural Ghana. PLoS Negl Trop Dis. 2015;9:e0003551.View ArticlePubMedPubMed CentralGoogle Scholar
  36. Al-Braiken FA, Amin A, Beeching NJ, Hommel M, Hart CA. Detection of Cryptosporidium amongst diarrhoeic and asymptomatic children in Jeddah, Saudi Arabia. Ann Trop Med Parasitol. 2003;97:505–10.View ArticlePubMedGoogle Scholar
  37. Hijjawi N, Ng J, Yang R, Atoum MF, Ryan U. Identification of rare and novel Cryptosporidium GP60 subtypes in human isolates from Jordan. Exp Parasitol. 2010;125:161–4.View ArticlePubMedGoogle Scholar
  38. Alyousefi NA, Mahdy MA, Lim YA, Xiao L, Mahmud R. First molecular characterization of Cryptosporidium in Yemen. Parasitology. 2013;140:729–34.View ArticlePubMedGoogle Scholar
  39. Sulaiman IM, Hira PR, Zhou L, Al-Ali FM, Al-Shelahi FA, Shweiki HM, et al. Unique endemicity of cryptosporidiosis in children in Kuwait. J Clin Microbiol. 2005;43:2805–9.View ArticlePubMedPubMed CentralGoogle Scholar
  40. Iqbal J, Khalid N, Hira PR. Cryptosporidiosis in Kuwaiti children: association of clinical characteristics with Cryptosporidium species and subtypes. J Med Microbiol. 2011;60:647–52.View ArticlePubMedGoogle Scholar
  41. Wielinga C, Thompson RC, Monis P, Ryan U. Identification of polymorphic genes for use in assemblage B genotyping assays through comparative genomics of multiple assemblage B Giardia duodenalis isolates. Mol Biochem Parasitol. 2015;201:1–4.View ArticlePubMedGoogle Scholar

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