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).
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).
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.
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.
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.