LUMINEX®: a new technology for the simultaneous identification of five Entamoeba spp. commonly found in human stools

Particle and Fibre Toxicology

Alexandre Januário Da Silva³  José Mauro Peralta¹  Regina Helena Saramago Peralta⁴  Rebecca Bandea³  Kakali Bandyopadhyay³  Helena Lúcia Carneiro Santos² 

[1] Instituto de Microbiologia da Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil;LAPSA, Instituto Oswaldo Cruz, Fundação Oswaldo Cruz, Rio de Janeiro, Brazil;Center for Global Health, Division of Parasitic Diseases and Malaria, Centers for Disease Control and Prevention, Atlanta, GA, USA;Departamento de Patologia, Universidade Federal Fluminense, Niterói, RJ, Brazil

关键词: Multiplex assay;    Molecular diagnostics;    PCR;    18SrRNA;    Entamoeba;   

Others  :  1228034
DOI  :  10.1186/1756-3305-6-69

 received in 2012-09-12, accepted in 2013-02-21,  发布年份 2013

【 摘 要 】

Background

Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species.

Methods

PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools.

Results

Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli.

Conclusion

These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

【 授权许可】

   
2013 Santos et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

【 参考文献 】

• [1]Clark CG, Diamond LS: Ribosomal RNA genes of ‘pathogenic’ and ‘nonpathogenic’ Entamoeba histolytica are distinct. Mol Biochem Parasitol 1991, 49:297-302.
• [2]Leber AL, Novak SM: Intestinal and urogenital amebae, flagellates, and ciliates. In Manual of Clinical Microbiology. Volume 2. 10th edition. Edited by Murray PR, Baron EJ, Pfaller MA, Tenover FC, Yolken RH. Washington, DC: ASM Press; 2011::1391-1405.
• [3]Petri WA Jr, Singh U: Diagnosis and management of amebiasis. Clin Infect Dis 1999, 29:1117-1125.
• [4]Ximenez C, Moran P, Rojas L, Valadez A, Gomez A: Reassessment of the epidemiology of amebiasis: state of the art. Infect Genet Evol 2009, 9:1023-1032.
• [5]Blessmann J, Van Linh P, Nu PA, Thi HD, Muller-Myhsok B, Buss H, Tannich E: Epidemiology of amebiasis in a region of high incidence of amebic liver abscess in central Vietnam. AmJTrop Med Hyg 2002, 66:578-583.
• [6]Santos HL, Peralta RSH, Macedo HW, Barreto MG, Peralta JM: Comparison of multiplex-PCR and antigen detection for differential diagnosis of Entamoeba histolytica. Braz J Infect Dis 2007, 11:365-370.
• [7]Haque R, Petri WA: Diagnosis of Amebiasis in Bangladesh. Arch Med Res 2006, 37:273-276.
• [8]Ali IK, Clarck CG, Petri WAJR: Molecular Epidemiology of Amebiasis. Infect Genet Evol 2008, 8:698-707.
• [9]Garcia LS: Intestinal Protozoa: Amebae. In Diagnostic Medical Parasitology. 5th edition. Washington, D.C: ASM PRESS; 2007:6-28.
• [10]Santos HL, Bandea R, Martins LA, Macedo HW, Peralta RSH, Peralta JM, Ndubuisi MI, Da Silva AJ: Differential identification of Entamoeba spp. based on the analysis of 18S rRNA. Parasitol Res 2010, 106:883-888.
• [11]Haque R, Neville LM, Hahn P, Petri WA Jr: Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits. J Clin Microbiol 1995, 33:2558-2561.
• [12]Pillai DR, Kain KC: Immunochromatographic strip-based detection of Entamoeba histolytica-E. dispar and Giardia lamblia coproantigen. J Clin Microbiol 1999, 37:3017-3029.
• [13]Garcia LS, Shimizu RY, Bernard CN: Detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum antigens in human fecal specimens using the triage parasite panel enzyme immunoassay. J Clin Microbiol 2000, 38:3337-3340.
• [14]Acuna-Soto R, Samuelson J, De Girolami P, Zarate L, Millan-Velasco F, Schoolnick G, Wirth D: Application of the polymerase chain reaction to the epidemiology of pathogenic and nonpathogenic Entamoeba histolytica. AmJTrop Med Hyg 1993, 48:58-70.
• [15]Aguirre A, Molina S, Blotkamp C, Verweij J, Vinuesa T, Valls ME, Guhl F, Polderman A, Jimenez MT, Corachan M, Gonzalez-Ruiz MA, Frame IA, Warhurst D: Diagnosis of Entamoeba histolytica and Entamoeba dispar in clinical specimens by PCR-SHELA. Arch Med Res 1997, 28:282-284.
• [16]Britten D, Wilson SM, McNerney R, Moody AH, Chiodini PL, Ackers JP: Detection and differentiation of Entamoeba histolytica and E. dispar using an improved colorimetric polymerase chain reaction method. Arch Med Res 1997, 28:279-281.
• [17]Evangelopoulos A, Spanakos G, Patsoula E, Vakalis N, Legakis N: A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces. Ann Trop Med Parasitol 2000, 94:233-240.
• [18]Gonin P, Trude L: Detection and differentiation of Entamoeba histolytica and Entamoeba dispar isolates in clinical samples by PCR and enzyme-linked immunosorbent assay. J Clin Microbiol 2003, 41:237-241.
• [19]Mirelman D, Nuchamowitz Y, Stolarsky T: Comparison of use of enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes for simultaneous detection of Entamoeba histolytica and E. dispar. J Clin Microbiol 1997, 35:2405-2407.
• [20]Qvarnstrom Y, James C, Xayavong M, Holloway BP, Visvesvara GS, Sriram R, Da Silva AJ: Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis. J Clin Microbiol 2005, 43:5491-5497.
• [21]Verweij JJ, Blange RA, Templeton K, Schinkel J, Brienen EA, van Rooyen MA, van Lieshout L, Polderman AM: Simultaneous detection of Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum in fecal samples by using multiplex real-time PCR. J Clin Microbiol 2004, 42:1220-1223.
• [22]Beck DL, Dogan N, Maro V, Sam N, Shao J, Houpt ER: High prevalence of Entamoeba moshkovskii in a Tanzanian HIV population. Acta Trop 2008, 107:48-49.
• [23]Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J: Laboratory diagnostic techniques for Entamoeba species. Clin Microbiol Rev 2007, 20:511-532.
• [24]Khairnar K, Parija SC, Palaniappan R: Diagnosis of intestinal amoebiasis by using nested polymerase chain reaction-restriction fragment length polymorphism assay. J Gastroenterol 2007, 42:631-640.
• [25]Ali IK, Hossain MB, Roy S, Ayeh-Kumi PF, Petri WA Jr, Haque R, Clark CG: Entamoeba moshkovskii infections in children, Bangladesh. Emerg Infect Dis 2003, 9:580-584.
• [26]Hamzah Z, Petmitr S, Mungthin M, Leelayoova S, Chavalitshewinkoon-Petmitr P: Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay. J Clin Microbiol 2006, 44:3196-3200.
• [27]Kebede A, Verweij JJ, Endeshaw T, Messele T, Tasew G, Petros B, Polderman AM: The use of real-time PCR to identify Entamoeba histolytica and E. dispar infections in prisoners and primary-school children in Ethiopia. Ann Trop Med Parasitol 2004, 98:43-48.
• [28]Verweij JJ, Laeijendecker D, Brienen EA, van Lieshout L, Polderman AM: Detection and identification of Entamoeba species in stool samples by a reverse line hybridization assay. J Clin Microbiol 2003, 41:5041-5045.
• [29]Bandyopadhyay K, Kellar KL, Moura I, Casaqui Carollo MC, Graczyk TC, Slemenda S, Johnston SP, Da Silva AJ: Rapid microsphere assay for identification of Cryptosporidium hominis and Cryptosporidium parvum in stool and environmental samples. J Clin Microbiol 2007, 45:2835-2840.
• [30]Boving MK, Pedersen LN, Moller JK: Eight-plex PCR and liquid-array detection of bacterial and viral pathogens in cerebrospinal fluid from patients with suspected meningitis. J Clin Microbiol 2009, 47:908-913.
• [31]Das S, Brown TM, Kellar KL, Holloway BP, Morrison CJ: DNA probes for the rapid identification of medically important Candida species using a multianalyte profiling system. FEMS Immunol Med Microbiol 2006, 46:244-250.
• [32]Diaz MR, Fell JW: Use of a suspension array for rapid identification of the varieties and genotypes of the Cryptococcus neoformans species complex. J Clin Microbiol 2005, 43:3662-3672.
• [33]Dunbar SA: Applications of Luminex xMAP technology for rapid, high-throughput multiplexed nucleic acid detection. Clin Chim Acta 2006, 363:71-82.
• [34]Dunbar SA, Vander Zee CA, Oliver KG, Karem KL, Jacobson JW: Quantitative, multiplexed detection of bacterial pathogens: DNA and protein applications of the Luminex LabMAP system. J Microbiol Methods 2003, 53:245-252.
• [35]Fitzgerald C, Collins M, Van Duyne S, Mikoleit M, Brown T, Fields P: Multiplex, bead-based suspension array for molecular determination of common Salmonella serogroups. J Clin Microbiol 2007, 45:3323-3334.
• [36]Mahony J, Chong S, Merante F, Yaghoubian S, Sinha T, Lisle C, Janeczko R: Development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex PCR and a fluid microbead-based assay. J Clin Microbiol 2007, 45:2965-2970.
• [37]McNamara DT, Kasehagen LJ, Grimberg BT, Cole-Tobian J, Collins WE, Zimmerman PA: Diagnosing infection levels of four human malaria parasite species by a polymerase chain reaction/ligase detection reaction fluorescent microsphere-based assay. AmJTrop Med Hyg 2006, 74:413-421.
• [38]Page BT, Kurtzman CP: Rapid identification of Candida species and other clinically important yeast species by flow cytometry. J Clin Microbiol 2005, 43:4507-4514.
• [39]Schmitt M, Bravo IG, Snijders PJ, Gissmann L, Pawlita M, Waterboer T: Bead-based multiplex genotyping of human papillomaviruses. J Clin Microbiol 2006, 44:504-512.
• [40]Taniuchi M, Verweij JJ, Noor Z, Sobuz SU, Lieshout L, Petri WA, Haque R, Houpt E: High throughput multiplex PCR and probes-based detection with Luminex beads for seven intestinal parasites. AmJTrop Med Hyg 2011, 84:332-337.
• [41]Da Silva AJ, Bornay-Llinares FJ, Moura IN, Slemenda SB, Tuttle JL, Pieniazek NJ: Fast and reliable extraction of protozoan parasite DNA from fecal specimens. Mol Diagn 1999, 4:57-64.
• [42]Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997, 25:3389-3402.
• [43]Cowan LS, Diem L, Brake MC, Crawford JT: Transfer of a Mycobacterium tuberculosis genotyping method, Spoligotyping, from a reverse line-blot hybridization, membrane-based assay to the Luminex multianalyte profiling system. J Clin Microbiol 2004, 42:474-477.
• [44]Diaz MR, Fell JW: High-throughput detection of pathogenic yeasts of the genus Trichosporon. J Clin Microbiol 2004, 42:3696-3706.
• [45]Etienne KA, Kano R, Balajee SA: Development and validation of a microsphere-based Luminex assay for rapid identification of clinically relevant aspergilli. J Clin Microbiol 2009, 47:1096-1100.