Microscopy detection and molecular characterisation of Giardia duodenalis infection in outpatients seeking medical care in Egypt.

Introduction: Giardiosis remains one of the most prevalent enteric parasitic infections globally. Earlier molecular-based studies conducted in Egypt have primarily focused on paediatric clinical populations and most were based on single genotyping markers. As a result, there is limited information on the frequency and genetic diversity of G. duodenalis infections in individuals of all age groups. Methods: Individual stool samples (n = 460) from outpatients seeking medical care were collected during January-December 2021 in Kafr El-Sheikh governorate, northern Egypt. Initial screening for the presence of G. duodenalis was conducted by coprological examination. Microscopy-positive samples were further confirmed by real-time PCR. A multilocus sequence typing approach targeted amplification of the glutamate dehydrogenase (gdh), beta-giardin (bg), and triose phosphate isomerase (tpi) genes was used for genotyping purposes. A standardised epidemiological questionnaire was used to gather basic sociodemographic and clinical features of the recruited patients. Results: Giardia duodenalis cysts were observed in 5.4% (25/460, 95% CI: 3.6-7.9) of the stool samples examined by conventional microscopy. The infection was more frequent in children under the age of 10 years and in individuals presenting with diarrhoea but without reaching statistical significance. Stool samples collected during the winter period were more likely to harbour G. duodenalis. All 25 microscopy-positive samples were confirmed by real-time PCR, but genotyping data was only available for 56.0% (14/25) of the isolates. Sequence analyses revealed the presence of assemblages A (78.6%, 11/14) and B (21.4%, 3/14). All assemblage A isolates were identified as sub-assemblage AII, whereas the three assemblage B sequences belonged to the sub-assemblage BIII. Patients with giardiosis presenting with diarrhoea were more frequently infected by the assemblage A of the parasite. Conclusion: This is one of the largest epidemiological studies evaluating G. duodenalis infection in individuals of all age groups in Egypt. Our molecular data suggest that G. duodenalis infections in the surveyed population are primarily of anthropic origin. However, because assemblages A and B are zoonotic, some of the infections identified can have an animal origin. Additional investigations targeting animal (domestic and free-living) and environmental (water) samples are warranted to better understand the epidemiology of giardiosis in Egypt.

Significance of a common 65 kDa antigen in the experimental fasciolosis and toxoplasmosis.

In the current study, cross-reaction between two important zoonotic parasites; extracellular helminthes Fasciola gigantica and intracellular protozoa Toxoplasma gondii was proved by enzyme linked immunosorbent assay. Five antigens were used to identify and compare the cross-binding activities in the prepared antisera. Two F. gigantica antigens; adult flukes (FgA) and eggs (FgEA) were used to detect IgG in T. gondii naturally infected human sera (TgIHS) and experimentally infected sera of sheep (TgISS), mice (TgIMS) and rats (TgIRS). Three types of T. gondii antigens; RH (TgRHA), local sheep isolate (TgLA) and ME49 isolate (TgMEA) were used to detect cross binding activities in F. gigantica experimental infected rabbit sera (FgIRS) and F. gigantica naturally infected bovine sera (FgIBS). The cross-binding activities in the prepared antisera were strongly directed towards FgA and TgLA rather than the other antigens. The characterization of the five antigens using SDS-PAGE showed 4 common bands of FgA and TgLA; 165, 97, 76, and 65 kDa. While two common bands were observed between TgRHA, TgMEA and FgA; 165, and 65 kDa. Whereas, two common bands found between three types of T. gondii antigens and FgEA were identified; 165 and 65 kDa. The immunogenic cross-reactive bands between FgA and TgLA with F. gigantica infected bovine sera were identified by immunoblot. In FgA, the common immunogenic bands were 165, 65 and 14 kDa. While in TgLA, common immunogenic bands were 165 and 65 kDa. Whereas, the common immunogenic band between FgA and TgLA identified with T. gondii experimentally infected sheep sera was 65 kDa. The current research proves cross reaction between F. gigantica and T. gondii. One common band of 65 kDa showed broad immunogenic cross-reactivity with the developed antisera raising the prospect of being putative common immunodiagnostic candidate of both infections.

Development of 116 kDa Fraction for Detecting Experimental Toxoplasma gondii Infections in Mice.

BACKGROUND: Serological diagnosis of Toxoplasma gondii infection using crude antigens may not be more accurate. To increase the diagnostic potency of antigens, isolation of their immunogenic fractions could be useful. The current research adopted to obtain an affinity isolated fraction from RH strain using CNBr Sepharose 4B column coupled with infected mice sera helping in detection of IgM and IgG of toxoplasmosis due to RH strain and other strains. METHODS: The isolated fraction was characterized by SDS-PAGE. Moreover, the diagnostic potency of the fraction was assessed by indirect ELISA in mice experimentally infected with RH strain and two other local strains; one of sheep origin and the other of human origin. RESULTS: The fraction was found to be consisted of a single band of 116 kDa compared with 17 bands ranged from 116 to 16 kDa associated with crude extract. The fraction proved potent diagnostic potentials of acute and chronic mice toxoplasmosis. Where it was detected both IgM and IgG antibodies as early as two days and as late as 2 months post experimental infection with any of the three strains. The level of detected IgM and IgG by RH fraction was higher in mice infected with RH strain than with local strains except IgM due to sheep strain parasite. CONCLUSIONS: The 116 kDa fraction of T. gondii tachyzoites can be considered as a candidate in improving of serodiagnosisof Toxoplasma infections.