Research Article
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Year 2023, Volume: 50 Issue: 2 - Cilt 50, Sayı 2, 194 - 202, 12.06.2023
https://doi.org/10.5798/dicletip.1313299

Abstract

References

  • 1.Basuni M, Mohamed Z, Ahmad M, Zakaria NZ,Noordin R. Detection of selected intestinalhelminths and protozoa at hospiatl universiti sainsmalaysia using multiplex real-time PCR. TropBiomed. 2012; 29(3):434-42.
  • 2.Zebardast N, Yeganeh F, Gharavi MJ, et al.Simultaneous detection and differentiation ofEntamoeba histolytica, E. dispar, E. moshkovskii,Giardia lamblia and Cryptosporidium spp. in humanfecal samples using multiplex PCR and Qpcr-MCA.Acta Trop. 2016; 162:233-8.
  • 3.Klassen MK, Neafie RC, Wear DJ, Meyers WM.Cryptosporidiosis, Isosporiasis, Cyclosporiasis,Sarcocystosis. Topics on the pathology of protozoanand invasive arthropod diseases, Defense Technical Information Center 2011; 13thsection, 1-19.
  • 4.Kwon JY, Seo JY, Kim TY, Lee H, Won Ju J. Firstidentification and genotyping of Enterocytozoonbieneusi and prevalence of Encephalitozoonintestinalis in patients with acute diarrhea in therepublic of Korea. Pathogens. 2021; 3;10(11):1424.
  • 5.Jerez LE, Nunez-Fernandez FA, Fraga Nodarse J, etal. Diagnosis of intestinal protozoan infections inpatients in Cuba by microscopy and molecularmethods: advantages and disadvantages. J MicrobiolMethods. 2020; 179: 106102.
  • 6.Halanova M, Valencakova A, Jarcuska P, et al.Screening of opportunistic Encephalitozoonintestinalis and Enterocytozoon bieneusi inimmunocompromised patients in Slovakia. Cent EurJ Public Health. 2019; 27(4):330-4.
  • 7.Polat E, Özdemi S, Sirekbasan S. The distributionof intestinal parasites in patients presenting to auniversity hospital in Istanbul: a seven yearretrospektive analysis. Turkiye Parazitol Derg.2020;4;44(3):139.
  • 8.Morio F, Valot S, Laude A, et al. Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis,Entamoeba histolytica and Entamoebadispar/Entamoeba moshkovskii from stoolspecimens: evidence for the limted performances ofmicroscopy-based approach for amoeba speciesidentification. Clin Microbiol Infect. 2018;24(11):1205-9.
  • 9.Won EJ, Kim SH, Kee SJ, et al. Multiplex Real-TimePCR assay targeting eight parasites customized tothe Korean population: potential use for detection in diarrheal stool samples from gastroenteritispatients. Los One. 2016; 18;11(11):e0166957.
  • 10.Hove RJ, van Esbroeck M, Vervoort T, et al.Molecular diagnostics of intestinal parasites inreturning travellers. Eur J Clin Microbiol Infect Dis.2009; 28:1045-53.
  • 11.Sow D, Parola P, Sylla K, et al. Performance ofreal-time polymerase chain reaction for thedetection of 20 gastrointestinal parasites in clinicalsamples from Senegal. Am J Trop Med Hyg.2017;97(1):173-182.
  • 12.Shi X, Liu X, Wang Q, et al. A multiplex real-timePCR panel assay for simultaneous detection anddifferentiation of 12 common swine viruses. J VirolMethods. 2016; 236:258-265.
  • 13.Köppel R, Ganeshan A, van Velsen F, Bucher T.Five pentaplex real-time PCR systems for theefficient determination of 20 genetically modifiedmaize traits in food. Eur Food ResTechnol. 2017;243 (2).
  • 14.Jiang C, Luo Y, Yuan Y, et al. Conventionaloctaplex PCR for the simultaneous identification ofeight mainstream closely related Dendrobiumspecies. Industrial Crops&Products. 2018; 112; 576-96.
  • 15.Leone A, Ripabelli G, Sammarco ML, Grasso GM.Detection of cryptosporidium spp. from humanfaeces by PCR-RFLP, cloning and sequencing.Parasitol Res. 2009; 104(3): 583-7.
  • 16.Taniuchi M, Verweij JJ, Sethabutr O, et al.Multiplex polymerase chain reaction method todetect Cyclospora, Cystoisopora, and Microsporidiain stool samples. Diagn Microbiol Infect Dis. 2011;71(4):386-90.
  • 17.Hamidinejet H, Moetamedi H, Alborzi A, HatamiA.Molecular detection of Sarcoystis species inslaughtered sheep by PCR-RFLP from South-western of Iran. J Parasitol Dis. 2014; 38(2):233-37.
  • 18.Clinical and Laboratory Standards Institute,webstore.ansi.org. (accessed date: 01.04.2022).
  • 19.Çelebi D, Çelebi Ö. Prevalence of Entamoebahistolytica, Giardia intestinalis and Cryptosporidium spp. Dicle Med J. 2021; 48(3): 425-30.
  • 20.Bustin SA, Benes V, Garson JA, et al. The MIQEguidelines: minimum information for publication ofQuantitative real-time PCR Experiments. ClinicalChemistry. 2009; 611-22.
  • 21.Wolday D, Genrecherkos T, Arefaine ZG, et al.Effect of co-infection with intestinal parasites onCOVID-19 severity: a prospective observation cohorstudy. Eclinical Medicine. 2021; 39; 101054.
  • 22.Frickmann H, Schwarz NG, RakotozandrindrainyR, May J, Hagen RM. PCR for enteric pathogens inhigh-prevalence settings. What does a positivesignal tell us? Infect Dis 2015; 47(7): 491-98.
  • 23.Nazeer JT, El Sayed Khalifa K, von Theien H, et al.Use of multiplex real-time PCR for detection ofcommon diarrhea causing protozoan parasites inEgypt. Parasitol Res. 2013; 112(2): 595-01.
  • 24.Basuni M, Muhi J, Othman N, et al. A pentaplexreal-time polymerase chain reaction assay fordetection of four species of soil-transmittedhelmints. Am J Trop Med Hyg. 2011; 84(2): 338-43.
  • 25.Mishra N, Ng J, Rakeman JL, et al. One steppentaplex real-time polymerase chain reactionassay for detection of zika, dengue, chikungunya,west nile viruses and a human housekeeping gene.Journal of Clinical Virology. 2019; 120: 44-50.
  • 26.Okolie CE, Wooldridge KG, Turner DP, CockayneA, James R. Development of a new pentaplex real-time PCR assay for the identificatio of poly-microbialspecimens containing Staphylococcus aureus andother staphylococci, wth simultaneous detection ofstaphylococcal virulence and methicillin resistancemarkers. Molecular and Cellular Probes. 2015; 29;144-50.
  • 27.Sow D, Parola P, Sylla K, et al. Performance ofreal-tiem polymerase chain reaction assays for thedetection of 20 gastrointestinal parasites in clinicalsamples from Senegal. Am J Trop Med Hyg. 2017;97(1): 173-82.
  • 28.Merdekios B, Pareyn M, Tadesse D, et al.Evalutaion of conventional and four real-time PCRmethods fort he detection of Leishmania on fieldcollected samples in Ethiopia. Plos Negl Trop Dis.2021; 15(1):e0008903.
  • 29.Mehraj V, Hatcher J, Akhtar S, Rafique G, Beg MA.Prevalence and factors associated with intestinalparasitic infection among children in an urban slumof Karachi. Plos One. 2008; 3(11): e3680.
  • 30.Cengiz Taş Z, Beyhan YE, Çiçek M, Yılmaz H.Intestinal and hepatic parasites determined in auniversity hospital parasitology laboratory. DicleMedical Journal. 2015; 42(3): 350-54.

Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR

Year 2023, Volume: 50 Issue: 2 - Cilt 50, Sayı 2, 194 - 202, 12.06.2023
https://doi.org/10.5798/dicletip.1313299

Abstract

Objective: Spore-forming intestinal parasites; Cryptosporidium spp., Cyclospora spp., Cytoisospora spp., Encephalitozoon spp., and Sarcoystis spp. are very common in immunocompromised patients, but these parasites are overlooked by healthcare proffesionals. It was aimed to develop a new pentaplex real-time PCR panel for the identification of spore-forming intestinal parasites in this study.
Methods: Primer-probes for pentaplex real-time PCR were designed using the “PrimerQuest Tool (Integrated DNA technologies, Coralville, USA) software program” and “Multiple sequence alignment use a computer software Primer Express™Software v3.0.1 Lience (ThermoFisher Scientific, Waltham, USA)”. The primer-probes designed in the study were spore-forming Cryptosporidium spp. (ATCC®87715™), Cyclospora spp. (ATCC®PRA-3000SD™), Cystoispora spp. (KF648871), Encephalitozoon spp. (FJ026010) and Sarcocystis spp. (ATCC®CCL-70) parasites were amplified with DNA isolates from the American Type Culture Collection (ATCC) and then these primer-probes were validated with 232 DNA samples obtained from the stools of the patient samples.
Results: It was found that Cycle Threshold (Ct) ±25.7, Standard curve (R2 ): ±0,993, and Efficiency (E): %96,1 according to the results of multiplex real-time PCR analysis. Similar results were found in pentaplex real-time PCR analysis of DNA isolates of stool samples. When the pentaplex real-time PCR results of DNA samples isolates from stool samples were compared with the positivie predictive value results of traditional methods, it was found that the pentaplex results were higher.
Conclusion: The new designed pentaplex real-time PCR panel can be used in the diagnosis of spore-forming intestinal parasites, which are very common in immunocompromised patients. Thus, the diagnosis of five different parasites can be made faster, more economically and faster with a single reaction.

References

  • 1.Basuni M, Mohamed Z, Ahmad M, Zakaria NZ,Noordin R. Detection of selected intestinalhelminths and protozoa at hospiatl universiti sainsmalaysia using multiplex real-time PCR. TropBiomed. 2012; 29(3):434-42.
  • 2.Zebardast N, Yeganeh F, Gharavi MJ, et al.Simultaneous detection and differentiation ofEntamoeba histolytica, E. dispar, E. moshkovskii,Giardia lamblia and Cryptosporidium spp. in humanfecal samples using multiplex PCR and Qpcr-MCA.Acta Trop. 2016; 162:233-8.
  • 3.Klassen MK, Neafie RC, Wear DJ, Meyers WM.Cryptosporidiosis, Isosporiasis, Cyclosporiasis,Sarcocystosis. Topics on the pathology of protozoanand invasive arthropod diseases, Defense Technical Information Center 2011; 13thsection, 1-19.
  • 4.Kwon JY, Seo JY, Kim TY, Lee H, Won Ju J. Firstidentification and genotyping of Enterocytozoonbieneusi and prevalence of Encephalitozoonintestinalis in patients with acute diarrhea in therepublic of Korea. Pathogens. 2021; 3;10(11):1424.
  • 5.Jerez LE, Nunez-Fernandez FA, Fraga Nodarse J, etal. Diagnosis of intestinal protozoan infections inpatients in Cuba by microscopy and molecularmethods: advantages and disadvantages. J MicrobiolMethods. 2020; 179: 106102.
  • 6.Halanova M, Valencakova A, Jarcuska P, et al.Screening of opportunistic Encephalitozoonintestinalis and Enterocytozoon bieneusi inimmunocompromised patients in Slovakia. Cent EurJ Public Health. 2019; 27(4):330-4.
  • 7.Polat E, Özdemi S, Sirekbasan S. The distributionof intestinal parasites in patients presenting to auniversity hospital in Istanbul: a seven yearretrospektive analysis. Turkiye Parazitol Derg.2020;4;44(3):139.
  • 8.Morio F, Valot S, Laude A, et al. Evaluation of a new multiplex PCR assay (ParaGENIE G-Amoeba Real-Time PCR kit) targeting Giardia intestinalis,Entamoeba histolytica and Entamoebadispar/Entamoeba moshkovskii from stoolspecimens: evidence for the limted performances ofmicroscopy-based approach for amoeba speciesidentification. Clin Microbiol Infect. 2018;24(11):1205-9.
  • 9.Won EJ, Kim SH, Kee SJ, et al. Multiplex Real-TimePCR assay targeting eight parasites customized tothe Korean population: potential use for detection in diarrheal stool samples from gastroenteritispatients. Los One. 2016; 18;11(11):e0166957.
  • 10.Hove RJ, van Esbroeck M, Vervoort T, et al.Molecular diagnostics of intestinal parasites inreturning travellers. Eur J Clin Microbiol Infect Dis.2009; 28:1045-53.
  • 11.Sow D, Parola P, Sylla K, et al. Performance ofreal-time polymerase chain reaction for thedetection of 20 gastrointestinal parasites in clinicalsamples from Senegal. Am J Trop Med Hyg.2017;97(1):173-182.
  • 12.Shi X, Liu X, Wang Q, et al. A multiplex real-timePCR panel assay for simultaneous detection anddifferentiation of 12 common swine viruses. J VirolMethods. 2016; 236:258-265.
  • 13.Köppel R, Ganeshan A, van Velsen F, Bucher T.Five pentaplex real-time PCR systems for theefficient determination of 20 genetically modifiedmaize traits in food. Eur Food ResTechnol. 2017;243 (2).
  • 14.Jiang C, Luo Y, Yuan Y, et al. Conventionaloctaplex PCR for the simultaneous identification ofeight mainstream closely related Dendrobiumspecies. Industrial Crops&Products. 2018; 112; 576-96.
  • 15.Leone A, Ripabelli G, Sammarco ML, Grasso GM.Detection of cryptosporidium spp. from humanfaeces by PCR-RFLP, cloning and sequencing.Parasitol Res. 2009; 104(3): 583-7.
  • 16.Taniuchi M, Verweij JJ, Sethabutr O, et al.Multiplex polymerase chain reaction method todetect Cyclospora, Cystoisopora, and Microsporidiain stool samples. Diagn Microbiol Infect Dis. 2011;71(4):386-90.
  • 17.Hamidinejet H, Moetamedi H, Alborzi A, HatamiA.Molecular detection of Sarcoystis species inslaughtered sheep by PCR-RFLP from South-western of Iran. J Parasitol Dis. 2014; 38(2):233-37.
  • 18.Clinical and Laboratory Standards Institute,webstore.ansi.org. (accessed date: 01.04.2022).
  • 19.Çelebi D, Çelebi Ö. Prevalence of Entamoebahistolytica, Giardia intestinalis and Cryptosporidium spp. Dicle Med J. 2021; 48(3): 425-30.
  • 20.Bustin SA, Benes V, Garson JA, et al. The MIQEguidelines: minimum information for publication ofQuantitative real-time PCR Experiments. ClinicalChemistry. 2009; 611-22.
  • 21.Wolday D, Genrecherkos T, Arefaine ZG, et al.Effect of co-infection with intestinal parasites onCOVID-19 severity: a prospective observation cohorstudy. Eclinical Medicine. 2021; 39; 101054.
  • 22.Frickmann H, Schwarz NG, RakotozandrindrainyR, May J, Hagen RM. PCR for enteric pathogens inhigh-prevalence settings. What does a positivesignal tell us? Infect Dis 2015; 47(7): 491-98.
  • 23.Nazeer JT, El Sayed Khalifa K, von Theien H, et al.Use of multiplex real-time PCR for detection ofcommon diarrhea causing protozoan parasites inEgypt. Parasitol Res. 2013; 112(2): 595-01.
  • 24.Basuni M, Muhi J, Othman N, et al. A pentaplexreal-time polymerase chain reaction assay fordetection of four species of soil-transmittedhelmints. Am J Trop Med Hyg. 2011; 84(2): 338-43.
  • 25.Mishra N, Ng J, Rakeman JL, et al. One steppentaplex real-time polymerase chain reactionassay for detection of zika, dengue, chikungunya,west nile viruses and a human housekeeping gene.Journal of Clinical Virology. 2019; 120: 44-50.
  • 26.Okolie CE, Wooldridge KG, Turner DP, CockayneA, James R. Development of a new pentaplex real-time PCR assay for the identificatio of poly-microbialspecimens containing Staphylococcus aureus andother staphylococci, wth simultaneous detection ofstaphylococcal virulence and methicillin resistancemarkers. Molecular and Cellular Probes. 2015; 29;144-50.
  • 27.Sow D, Parola P, Sylla K, et al. Performance ofreal-tiem polymerase chain reaction assays for thedetection of 20 gastrointestinal parasites in clinicalsamples from Senegal. Am J Trop Med Hyg. 2017;97(1): 173-82.
  • 28.Merdekios B, Pareyn M, Tadesse D, et al.Evalutaion of conventional and four real-time PCRmethods fort he detection of Leishmania on fieldcollected samples in Ethiopia. Plos Negl Trop Dis.2021; 15(1):e0008903.
  • 29.Mehraj V, Hatcher J, Akhtar S, Rafique G, Beg MA.Prevalence and factors associated with intestinalparasitic infection among children in an urban slumof Karachi. Plos One. 2008; 3(11): e3680.
  • 30.Cengiz Taş Z, Beyhan YE, Çiçek M, Yılmaz H.Intestinal and hepatic parasites determined in auniversity hospital parasitology laboratory. DicleMedical Journal. 2015; 42(3): 350-54.
There are 30 citations in total.

Details

Primary Language English
Subjects Medical Education
Journal Section Original Articles
Authors

Fadime Eroglu This is me

Publication Date June 12, 2023
Submission Date November 12, 2022
Published in Issue Year 2023 Volume: 50 Issue: 2 - Cilt 50, Sayı 2

Cite

APA Eroglu, F. (2023). Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR. Dicle Tıp Dergisi, 50(2), 194-202. https://doi.org/10.5798/dicletip.1313299
AMA Eroglu F. Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR. diclemedj. June 2023;50(2):194-202. doi:10.5798/dicletip.1313299
Chicago Eroglu, Fadime. “Identification of Spore-Forming Intestinal Parasites With Pentaplex Real-Time PCR”. Dicle Tıp Dergisi 50, no. 2 (June 2023): 194-202. https://doi.org/10.5798/dicletip.1313299.
EndNote Eroglu F (June 1, 2023) Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR. Dicle Tıp Dergisi 50 2 194–202.
IEEE F. Eroglu, “Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR”, diclemedj, vol. 50, no. 2, pp. 194–202, 2023, doi: 10.5798/dicletip.1313299.
ISNAD Eroglu, Fadime. “Identification of Spore-Forming Intestinal Parasites With Pentaplex Real-Time PCR”. Dicle Tıp Dergisi 50/2 (June 2023), 194-202. https://doi.org/10.5798/dicletip.1313299.
JAMA Eroglu F. Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR. diclemedj. 2023;50:194–202.
MLA Eroglu, Fadime. “Identification of Spore-Forming Intestinal Parasites With Pentaplex Real-Time PCR”. Dicle Tıp Dergisi, vol. 50, no. 2, 2023, pp. 194-02, doi:10.5798/dicletip.1313299.
Vancouver Eroglu F. Identification of Spore-Forming Intestinal Parasites with Pentaplex Real-Time PCR. diclemedj. 2023;50(2):194-202.