Advertisement

Parasitology Research

, Volume 116, Issue 9, pp 2393–2406 | Cite as

Toxoplasma genotyping in congenital toxoplasmosis in Upper Egypt: evidence of type I strain

  • Hanan E. M. Eldeek
  • Alzahraa Abdel Raouf Ahmad
  • Mohamed Ahmed El-Mokhtar
  • Abdel Rahman M.M. Abdel Kader
  • Ahmad M. Mandour
  • Mahmoud Elhady M. Mounib
Original Paper

Abstract

Toxoplasma gondii has subpopulation structures in different geographical regions caused by less frequent sexual recombination, population sweeps, and biogeography. The majority of strains isolated in North America and Europe fall into one of three clonal lineages, referred to as types I, II, and III. So far, little is known about genetics of Toxoplasma strains in Africa. The present study aimed to determine the genotype of Toxoplasma strains obtained directly from trophoblastic/placental tissues of 29 complicated pregnant women using multilocus nested-PCR-RFLP technique depending on four independent genetic loci (5′ SAG2 and 3′ SAG2), SAG3, GRA6, and BTUB genes. All samples gave positive amplicons at 5′-3′ SAG2 and SAG3 genes. Meanwhile, no amplification products were observed in 12 (41.37%) and 10 (34.48%) samples with GRA6 and BTUB genes, respectively. The restriction pattern revealed the presence of genotype I in all samples, except one sample, which revealed atypical genotype with unusual restriction pattern at 3′ SAG2 gene. The negative amplifications in some samples could be due to presence of mutations or polymorphisms in the primer binding sites of these isolates, raising the possibility of mixed or recombinant genotypes. To the best of our knowledge, this is the first time to perform genotype analysis study based on Multiplex nPCR-RFLP technique for genetic characterization of T. gondii in Egypt. Besides, it is the first time to prove that the most prevalent strain of T. gondii, responsible for congenital toxoplasmosis in Upper Egypt, is the highly virulent type I. Atypical genotype was detected as well.

Keywords

Toxoplasma gondii Multilocus genotyping Congenital toxoplasmosis Type I strain Atypical strains 

Notes

Compliance with ethical standards

All study subjects were properly informed about the nature of the study, and informed written consents were obtained from all participants. Ethical clearance was obtained from the Ethical Committee, Faculty of Medicine, Assuit University.

References

  1. Abdoli A, Dalimi A, Soltanghoraee H, Ghaffarifar F (2017) Molecular detection and genotypic characterization of Toxoplasma gondii in paraffin-embedded fetoplacental tissues of women with recurrent spontaneous abortion. Int J Fertil Steril 10(4):327–336PubMedGoogle Scholar
  2. Abdel-Hameed DM, Hassanein OM (2008) Genotyping of Toxoplasma gondii strains from female patients with toxoplasmosis. J Egypt Soc Parasitol 38(2):511–520PubMedGoogle Scholar
  3. Ajzenberg D, Cogne N, Paris L, Bessieres MH, Thulliez P, Fillisetti D, Pelloux H, Marty P, Darde ML (2002) Genotype of 86 Toxoplasma gondii isolates associated with human congenital toxoplasmosis and correlation with clinical findings. J Infect Dis 186:684–689CrossRefPubMedGoogle Scholar
  4. Ajzenberg D, Dumetre A, Darde ML (2005) Multiplex PCR for typing strains of Toxoplasma gondii. J Clin Microbiol 43(4):1940–1943CrossRefPubMedPubMedCentralGoogle Scholar
  5. Ajzenberg D, Yera H, Marty P, Paris L, Dalle F, Menotti J, Aubert D, Franck J, Bessières MH, Quinio D, Pelloux H, Delhaes L, Desbois N, Thulliez P, Robert-Gangneux F, Kauffmann-Lacroix C, Pujol S, Rabodonirina M, Bougnoux ME, Cuisenier B, Duhamel C, Duong TH, Filisetti D, Flori P, Gay-Andrieu F, Pratlong F, Nevez G, Totet A, Carme B, Bonnabau H, Dardé ML, Villena I (2009) Genotype of 88 toxoplasma gondii isolates associated with toxoplasmosis in immunocompromised patients and correlation with clinical findings. J Infect Dis 199(8):1155–1167CrossRefPubMedGoogle Scholar
  6. Ajzenberg D, Collinet F, Mercier A, Vignoles P, Dardé ML (2010) Genotyping of Toxoplasma gondii isolates with 15 microsatellite markers in a single multiplex PCR assay. J Clin Microbiol 48(12):4641–4645CrossRefPubMedPubMedCentralGoogle Scholar
  7. Alghamdi J, Elamin MH, Alhabib S (2015) Prevalence and genotyping of Toxoplasma gondii among Saudi pregnant women in Saudi Arabia. Saudi Pharm J 82:1–7Google Scholar
  8. Al-Kappany YM, Rajendran C, Abu-Elwafa S, Hilali M, Su C, Dubey JP (2010) Genetic diversity of Toxoplasma gondii isolates in Egyptian feral cats reveals new genotypes. J Parasitol 96(6):1112–1114CrossRefPubMedGoogle Scholar
  9. Asgari Q, Fekri M, Monabati A, Kalantary M, Mohammadpour I, Motazedian MH, Sarkari B (2013) Molecular genotyping of Toxoplasma gondii in human spontaneous aborted fetuses in shiraz, southern Iran. Iranian J Publ Health 42(6):620–625Google Scholar
  10. Bachmeyer C, Mouchnino G, Thulliez P, Blum L (2006) Congenital toxoplasmosis from an HIV-infected woman as a result of reactivation. J Inf Secur 52:55–57Google Scholar
  11. Boughattas S, Ben-Abdallah R, Siala E, Souissi O, Aoun K, Bouratbine A (2010) Direct genotypic characterization of Toxoplasma gondii strains associated with congenital toxoplasmosis in Tunisia (North Africa). Am J Trop Med Hyg 82(6):1041–1046CrossRefPubMedPubMedCentralGoogle Scholar
  12. Boughattas S, Abdallah RB, Siala E, Aoun K, Bouratbine A (2011) An atypical strain associated with congenital toxoplasmosis in Tunisia. New Microbiol 34(4):413–416PubMedGoogle Scholar
  13. Boumahni B, Randrianivo H, Flodrops H, Kauffmann E, Sauve F, Chauvet O, Renouil M, Fourmaintraux A (2004) Maternal toxoplasmosis before conception and chorioretinitis in twin sisters. J of Gynecol Obst and Biol of Rep (Paris) 33:248–250CrossRefGoogle Scholar
  14. Burg JL, Grover CM, Pouletty P, Boothroyd JC (1989) Direct and sensitive detection of a pathogenic protozoan, Toxoplasma gondii, by polymerase chain reaction. J Clin Microbiol 27:1787–1792PubMedPubMedCentralGoogle Scholar
  15. Carneiro ACAV, Andrade GM, Costa JGL, Pinheiro BV, Vasconcelos-Santos DV, Ferreira AM, Su C, Januário JN, Vitor RWA (2013) Genetic characterization of Toxoplasma gondii revealed highly diverse genotypes for isolates from newborns with congenital toxoplasmosis in southeastern Brazil. J Clin Microbiol 51:901–907CrossRefPubMedPubMedCentralGoogle Scholar
  16. Coutermarsh-Ott SL, Doran JT, Campbell C, Williams TM, Lindsay DS (2016) Allen IC (2016) caspase-11 modulates inflammation and attenuates Toxoplasma gondii pathogenesis. Mediat Inflamm 2016:9848263CrossRefGoogle Scholar
  17. Cunha MM, Carneiro ACAV, Costa JGL, Vitor RWA (2016) Genotyping of Toxoplasma gondii directly from human and animal biological samples: from partial genotypes to a new genotype. J Parasitol 102(1):157–160CrossRefPubMedGoogle Scholar
  18. Dardé ML (2004) Genetic analysis of the diversity in Toxoplasma gondii. Ann Ist Super Sanità 40(1):57–63PubMedGoogle Scholar
  19. Darde ML (2008) Toxoplasma gondii, “new” genotypes and virulence. Parasite 15:366–371CrossRefPubMedGoogle Scholar
  20. de Oliveira Azevedo CT, do Brasil PE, Guida L, Lopes Moreira ME (2016) Performance of polymerase chain reaction analysis of the amniotic fluid of pregnant women for diagnosis of congenital toxoplasmosis: a systematic review and meta-analysis. PLoS One 7;11(4):e0149938CrossRefGoogle Scholar
  21. Delhaes L, Ajzenberg D, Sicot B, Bourgeot P, Dardé ML (2010) Severe congenital toxoplasmosis due to a Toxoplasma gondii strain with an atypical genotype: case report and review. Prenat Diagn 30:902–905CrossRefPubMedGoogle Scholar
  22. Demar M, Hommel D, Djossou F, Peneau C, Boukhari R, Louvel D, Bourbigot AM, Nasser V, Ajzenberg D, Darde ML, Carme B (2012) Acute toxoplasmosis in immunocompetent patients hospitalized in an intensive care unit in French Guiana. Clin Microbiol Infect 18(7):222–232CrossRefGoogle Scholar
  23. Dubey JP, Graham DH, Dahl E, Hilali M, El-Ghaysh A, Sreekumar C, Kwok OCH, Shen SK, Lehmann T (2003) Isolation and molecular characterization of Toxoplasma gondii from chickens and ducks from Egypt. Vet Parasitol 114:89–89CrossRefPubMedGoogle Scholar
  24. Dubey JP (2007) The history and life cycle of toxoplasma gondii. In toxoplasma gondii. The model apicomplexan. In: Weiss LM, Kim K (eds) Perspectives and methods. Academic Press, New York, pp 1–17Google Scholar
  25. Dubey JP (2010) Toxoplasmosis of animals and humans, 2nd edn. CRC Press, Boca Raton, 313 pGoogle Scholar
  26. Dubey JP, Lago EG, Gennari SM, Su C, Jones JL (2012) Toxoplasmosis in humans and animals in Brazil: high prevalence, high burden of disease, and epidemiology. Parasitology 10:1–50CrossRefGoogle Scholar
  27. Dubey JP, Ferreira LR, Alsaad M, Verma SK, Alves DA, Holland GN, McConkey GA (2016) Experimental toxoplasmosis in rats induced orally with eleven strains of Toxoplasma gondii of seven genotypes: tissue tropism, tissue cyst size, neural lesions, tissue cyst rupture without reactivation, and ocular lesions. PLoS One 11(5):e0156255CrossRefPubMedPubMedCentralGoogle Scholar
  28. El Bolaky HAE, Hassanain MEA, Younis AIH, Abu Sarea EY, El Fadaly HA, Abd El Wahab WM (2015) Bio-typing of Toxoplasma Gondii isolates from complicated pregnant Egyptian women. Merit Res J Med Sci 3(9):453–458Google Scholar
  29. Elbez-Rubinstein A, Ajzenberg D, Dardé ML, Cohen R, Dumètre A, Yera H, Gondon E, Janaud JC, Thulliez P (2009) Congenital toxoplasmosis and reinfection during pregnancy: case report, strain characterization, experimental model of reinfection, and review. J Infect Dis 199:280–285CrossRefPubMedGoogle Scholar
  30. Ferreira IMR, Vidal JE, De Mattos CCB, De Mattos LC, Qu D, Su C, Pereira-Chioccola VL (2011) Toxoplasma gondii isolates: Multilocus RFLP-PCR genotyping from human patients in Sa˜o Paulo state, Brazil identified distinct genotypes. Exp Parasit 129:190–195CrossRefPubMedGoogle Scholar
  31. Fuentes I, Rubio JM, Ramĺrez C, Alvar J (2001) Genotypic characterization of Toxoplasma gondii strains associated with human toxoplasmosis in Spain: direct analysis from clinical samples. J Clin Microbiol 39:1566–1570CrossRefPubMedPubMedCentralGoogle Scholar
  32. Gallego C, Saavedra-Matiz C, Gómez-Marín JE (2006) Direct genotyping of animal and human isolates of Toxoplasma gondii from Colombia (South America). Acta Trop 97(2):161–167CrossRefPubMedGoogle Scholar
  33. Garcia-Meric P, Franck J, Dumon H, Piarroux R (2010) Management of congenital toxoplasmosis in France: current data. Presse Med 39:530–538CrossRefPubMedGoogle Scholar
  34. Gontijo da Silva M, Clare Vinaud M, de Castro AM (2015) Prevalence of toxoplasmosis in pregnant women and vertical transmission of Toxoplasma gondii in patients from basic units of health from Gurupi, Tocantins, Brazil, from 2012 to 2014. PLoS One 10:e0141700CrossRefPubMedPubMedCentralGoogle Scholar
  35. Grigg ME, Boothroyd JC (2001) Rapid identification of virulent type I strains of the protozoan pathogen Toxoplasma gondii by PCR-restriction fragment length polymorphism analysis at the B1 gene. J Clin Microbiol 39(1):398–400CrossRefPubMedPubMedCentralGoogle Scholar
  36. Grigg ME, Sundar N (2009) Sexual recombination punctuated by outbreaks and clonal expansions predicts Toxoplasma gondii population genetics. Int J Parasitol 39:925–933CrossRefPubMedPubMedCentralGoogle Scholar
  37. Hassanain MEA, El Bolaky HAE, Younis HAI, Abu Sarea EY, Abd El-Razik KA, El Fadaly HA, Abd El Wahab WM (2015) Serological and molecular diagnosis of T. Gondii in complicated pregnant Egyptian women. Basic Res J Med Clin Sci 4(9):231–236Google Scholar
  38. Honore S, Couvelard A, Garin YJF, Bedel C, Henin D, Darde ML, Derouin F (2000) Genotyping of Toxoplasma gondii strains from immunocompromised patients. Pathol Biol (Paris) 48:541–547Google Scholar
  39. Howe DK, Honore S, Derouin F, Sibley DL (1997) Determination of genotypes of Toxoplasma gondii strains isolated from patients with toxoplasmosis. J Clin Microbiol 35:1411–1414PubMedPubMedCentralGoogle Scholar
  40. Ivović V, Vujanic M, Zivkovic T, Klun I, Djurkovic-Djakovic O (2012) Molecular detection and genotyping of Toxoplasma gondii from clinical samples. In: Toxoplasmosis-Recent Advances, (Edited by Djurković Djaković O), In Tech, Rijek, Croatia: 103–120. At: http://www.intechopen.com/books/toxoplasmosis-recent advances/molecular-detection-and-genotyping-of-toxoplasma-gondii-from-clinical-samples
  41. Jeffers V, Kamau ET, Srinivasan AR, Harper J, Sankaran P, Post SE, Varberg JM, Sullivan WJ Jr, Boyle JP (2017) TgPRELID, a mitochondrial protein linked to multidrug resistance in the parasite toxoplasma gondii. mSphere 1;2(1):e00229-16CrossRefGoogle Scholar
  42. Jung BK, Lee SE, Lim H, Cho J, Kim DG, Song H, Kim MJ, Shin EH, Chai JY (2015) Toxoplasma gondii B1 gene detection in feces of stray cats around Seoul, Korea and genotype analysis of two laboratory-passaged isolates. Korean J Parasitol 53:259–263CrossRefPubMedPubMedCentralGoogle Scholar
  43. Khan A, Su C, German M, Storch GA, Clifford D, Sibley LD (2005) Genotyping of Toxoplasma gondii strains from immunocompromised patients reveals high prevalence of type I strains. J Clin Microbiol 43:5881–5887CrossRefPubMedPubMedCentralGoogle Scholar
  44. Khan A, Dubey JP, Su C, Ajioka JW, Rosenthal BM, Sibley LD (2011) Genetic analyses of atypical Toxoplasma gondii strains reveal a fourth clonal lineage in North America. Int J Parasitol 41(6):645–655CrossRefPubMedPubMedCentralGoogle Scholar
  45. Kong QM, Lu SH, Tong QB, Lou D, Chen R, Zheng B, Kumagai T, Wen LY, Ohta N, Zhou XN (2012) Loop-mediated isothermal amplification (LAMP): early detection of Toxoplasma gondii infection in mice. Parasit Vect 3(5):2–7CrossRefGoogle Scholar
  46. Lau YL, Lee WC, Gudimella R, Zhang G, Ching XT, Razali R, Aziz F, Anwar A, Fong MY (2016) Deciphering the draft genome of Toxoplasma gondii RH strain. PLoS One 11(6):e0157901CrossRefPubMedPubMedCentralGoogle Scholar
  47. Lindsay DS, Dubey JP (2011) Toxoplasma gondii: the changing paradigm of congenital toxoplasmosis. Parasitology 138:1829–1831CrossRefPubMedGoogle Scholar
  48. Liu Q, Wang ZD, Huang SY, Zhu XQ (2015) Diagnosis of toxoplasmosis and typing of Toxoplasma gondii. Parasit Vectors 28(8):292–306CrossRefGoogle Scholar
  49. Mandour AM, Mounib MEM, Eldeek HEM, Ahmed AAR, Abdel Kader MAE (2017) Prevalence of congenital toxoplasmosis in pregnant women with complicated pregnancy outcomes in Assiut Governorate. EgyptJ Advanc Parasitol 1(4):1–8Google Scholar
  50. McLeod R, Boyer KM, Lee D, Mui E, Wroblewski K, Karrison T, Noble AG, Withers S, Swisher CN, Heydemann PT, Sautter M, Babiarz J, Rabiah P, Meier P, Grigg ME, Toxoplasmosis study group (2012) Prematurity and severity are associated with toxoplasma gondii alleles NCCCTS, 1981–2009. Clin Infect Dis 54:1595–1605CrossRefPubMedPubMedCentralGoogle Scholar
  51. Mercier A, Devillard S, Ngoubangoye B, Bonnabau H, Bañuls AL, Durand P, Salle B, Ajzenberg D, Dardé ML (2010) Additional haplogroups of Toxoplasma gondii out of Africa: population structure and mouse-virulence of strains from Gabon. PLoS Negl Trop Dis 4(11):e876CrossRefPubMedPubMedCentralGoogle Scholar
  52. Nassef NE, El-Ghaffara MMA, El-Nahasa NS, Hassanain MEA, Shams El-Din SA, Ammar AIM (2015) Sero-prevalence and genotyping of Toxoplasma gondii in Menoufia Governorate. Menoufia Med J 28:617–626Google Scholar
  53. Nowakowska D, Colón I, Remington JS, Grigg M, Golab E, Wilczynski J, Sibley LD (2006) Genotyping of Toxoplasma gondii by multiplex PCR and peptide-based serological testing of samples from infants in Poland diagnosed with congenital toxoplasmosis. J Clin Microbiol 44(4):1382–1389CrossRefPubMedPubMedCentralGoogle Scholar
  54. Pomares C, Ajzenberg D, Bornard L, Bernardin G, Hasseine L, Darde ML, Marty P (2011) Toxoplasmosis and horse meat, France. Emerg Infect Dis 17:1327–1328CrossRefPubMedPubMedCentralGoogle Scholar
  55. Regan L, Backos M, Rai R (2011) The investigation and treatment of couples with recurrent first-trimester and second trimester miscarriage. London: RCOG Press Green-top Guideline No (17) 1–18Google Scholar
  56. Remington JS, McLeod R, Wilson CB, Desmonts G (2011) Toxoplasmosis. Remington JS, Klein JO, Wilson CB, Nizet V, Maldonado YA eds. Infectious diseases of the fetus and newborn infant. Seventh edition. Philadelphia, PA: Elsevier Saunders, 918–1041Google Scholar
  57. Robert-Gangneux F, Dardé ML (2012) Epidemiology of and diagnostic strategies for toxoplasmosis. Clin Microbiol Rev 25(2):264–296CrossRefPubMedPubMedCentralGoogle Scholar
  58. Sarkari B, Khabisi SA (2015) Case report: severe congenital toxoplasmosis: A case Report and strain characterization. Case reports Infect Dis: e851085Google Scholar
  59. Sibley LD, Ajioka JW (2008) Population structure of Toxoplasma gondii: clonal expansion driven by infrequent recombination and selective sweeps. Ann Rev Microbiol 62:329–351CrossRefGoogle Scholar
  60. Sibley LD, Khan A, Ajioka JW, Rosenthal BM (2009) Genetic diversity of Toxoplasma gondii in animals and humans. Philos Trans R Soc Lond Ser B Biol Sci 364(1530):2749–2761CrossRefGoogle Scholar
  61. Silva LA, Reis-Cunha JL, Bartholomeu DC, Vítor RW (2017) Genetic polymorphisms and phenotypic profiles of sulfadiazine-resistant and sensitive toxoplasma gondii isolates obtained from newborns with congenital toxoplasmosis in Minas Gerais, Brazil. PLoS One 24;12(1):e0170689CrossRefGoogle Scholar
  62. Stajner T, Bobic B, Klun I, Nikolic A, Srbljanovic J, Uzelac A, Rajnpreht I, Djurkovic-Djakovic O (2016) Prenatal and early postnatal diagnosis of congenital toxoplasmosis in a setting with no systematic screening in pregnancy. Medicine (Baltimore) 95(9):e2979CrossRefGoogle Scholar
  63. Su C, Zhang X, Dubey JP (2006) Genotyping of toxoplasma gondii by multilocus PCR-RFLP markers: a high resolution and simple method for identification of parasites. Int J Parasitol 36:841–848CrossRefPubMedGoogle Scholar
  64. Su C, Shwab EK, Zhou P, Zhu XQ, Dubey JP (2010) Moving towards an integrated approach to molecular detection and identification of Toxoplasma gondii. Parasitology 137(1):1–11CrossRefPubMedGoogle Scholar
  65. Su C, Khan A, Zhou P, Majumdar D, Ajzenberg D, Dardé ML, Zhu XQ, Ajioka JW, Rosenthal BM, Dubey JP, Sibley LD (2012) Globally diverse Toxoplasma gondii isolates comprise six major clades originating from a small number of distinct ancestral lineages. Proc Natl Acad Sci U S A 109:5844–5849CrossRefPubMedPubMedCentralGoogle Scholar
  66. Tolba MM, El-Taweel HA, Khalil SS, Hazzah WA, Heshmat MG (2014) Genotype analysis of T. gondii strains associated with human infection in Egypt. Parasitol Res 113(4):1563–1569CrossRefPubMedGoogle Scholar
  67. Villard O, Cimon B, L’Ollivier C, Fricker-Hidalgo H, Godineau N, Houze S, Paris L, Pelloux H, Villena I, Candolfi E (2016) Serological diagnosis of Toxoplasma gondii infection. Diagn Microbiol Infect Dis 84(1):22–33CrossRefPubMedGoogle Scholar
  68. Wallon M, Peyron F, Cornu C, Vinault S, Abrahamowicz M, Kopp CB, Binquet C (2013) Congenital Toxoplasma infection: monthly prenatal screening decreases transmission rate and improves clinical outcome at age 3 years. Clin Infect Dis 56:1223–1231CrossRefPubMedGoogle Scholar
  69. Wang L, He LY, Meng DD, Chen ZW, Wen H, Fang GS, Luo QL, Huang KQ, Shen JL (2015) Sero-prevalence and genetic characterization of Toxoplasma gondii in cancer patients in Anhui Province, eastern China. Parasit Vectors 15(8):162–169CrossRefGoogle Scholar
  70. Weiss LM, Dubey JP (2009) Toxoplasmosis: a history of clinical observations. Int J Parasitol 39:895–901CrossRefPubMedPubMedCentralGoogle Scholar
  71. Wujcicka W, Wilczyński J, Nowakowska D (2014) Do the placental barrier, parasite genotype and toll-like receptor polymorphisms contribute to the course of primary infection with various Toxoplasma gondii genotypes in pregnant women? Eur J Clin Microbiol Infect Dis 33(5):703–709CrossRefPubMedGoogle Scholar
  72. Xiao J, Jones-Brando L, Talbot CCJr, Yolken RH (2011) Differential effects of three canonical Toxoplasma strains on gene expression in human neuroepithelial cells. Infect Immun 79(3):1363–1373Google Scholar
  73. Xiao J, Yolken RH (2015) Strain hypothesis of Toxoplasma gondii infection on the outcome of human diseases. Acta Physiol 213(4):828–845CrossRefGoogle Scholar
  74. Yamamoto M, Standley DM, Takashima S, Saiga H, Okuyama M, Kayama H, Kubo E, Ito H, Takaura M, Matsuda T, Soldati-Favre D, Takeda K (2009) A single polymorphic amino acid on Toxoplasma gondii kinase ROP16 determines the direct and strain-specific activation of Stat3. J Exp Med 206(12):2747–2760CrossRefPubMedPubMedCentralGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany 2017

Authors and Affiliations

  • Hanan E. M. Eldeek
    • 1
  • Alzahraa Abdel Raouf Ahmad
    • 1
  • Mohamed Ahmed El-Mokhtar
    • 2
  • Abdel Rahman M.M. Abdel Kader
    • 3
  • Ahmad M. Mandour
    • 1
  • Mahmoud Elhady M. Mounib
    • 1
  1. 1.Medical Parasitology Department, Faculty of MedicineAssiut UniversityAssiutEgypt
  2. 2.Medical Microbiology and Immunology Department, Faculty of MedicineAssiut UniversityAssiutEgypt
  3. 3.Obstetrics and Gynecology Department, Faculty of MedicineAssiut UniversityAssiutEgypt

Personalised recommendations