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Editor-In-Chief: C. Michael Gibson, M.S., M.D. [1];Associate Editor(s)-in-Chief: Yamuna Kondapally, M.B.B.S[2]

Overview

Other diagnostic studies include microscopic techniques, culture methods, isoenzyme analysis, antibody detection tests, antigen detection tests, immunochromatographic assays and DNA based diagnostic tests.[1][2][3][4][4]

Other Diagnostic Tests

Needle aspiration

Laboratory Method Findings
Microscopy
Wet (Saline) preparation
  • Very insensitive method (<10%) Procedure
  • The sample is a fresh specimen that should be examined within 1 hr of collection
  • The test is positive when RBCs in trophozoites are detected
  • It is not used in patients without acute dysentery as trophozoites will not contain RBCs
 Concentration Technique
  • It is helpful in detecting cysts in the stool sample in asymptomatic carriers
 Permanently stained smears
Culture Methods

Disadvantage

  • Culture is not recommended as the routine diagnostic test due to overgrowth of other organisms like bacteria and fungi in the culture media.
Isoenzyme analysis

Disadvantages

  • Difficult to perform test, time-consuming procedure, and not always successful.
  • The isoenzyme analysis is negative for many microscopy positive stool samples.[6][14][15][7]
Antibody Detection Tests

ELISA

  • Useful in the diagnosis of asymptomatic and symptomatic amoebiasis after fecal examination.
  • Useful in amoebic liver abscess and in the evaluation of intestinal and extraintestinal infections where organisms cannot be detected in feces but amoebiasis is suspected.[16]
  • Easy to perform in the clinical laboratory.
  • The presence of IgM antibodies indicates current infection and IgG antibodies persists for years after E histolytica infection.[17]
  • Presence of antilectin antibodies are frequently used for the diagnosis of patients with amoebic liver abscess.[18]
  • The sensitivity is 95%. It is a useful test for the diagnostic clinical laboratory as this test does not have cross reaction with other non-Entamoeba histolytica species.[19][20][21][22][2]
Antigen Detection Tests

Disadvantage

  • Antigens detected are denatured by fixation of the stool sample. Hence the test is limited to frozen or fresh samples.
Immunochromatographic Assays

Disadvantage

  • This test does not differentiate between E histolytica and E dispar. Hence not a method of choice for the diagnostic laboratory.
  • Stool samples are transported to the laboratory as soon as possible as the test is performed on the fresh or fresh-frozen unpreserved stool samples.[26][27]

DNA-Based Diagnostic Tests

  • These tests are limited to developed countries in research and clinical laboratories.
  • Fecal specimens for DNA analysis may be preserved by refrigeration or in a formalin, SAF or PVA fixative. Formalin preserves cysts, and PVA and SAF preserves trophozoites and cysts in wet mounts.[28][29]
  • The sample medium which is used to transport amoebic DNA is ethanol and the reagent used for the preservation of fecal samples is 10% buffered formalin solution.[30]
  • The following are the methods used for DNA extraction from fecal samples.
Laboratory Methods Findings
Manual Methods

QIAamp tissue kit

  • QIAamp tissue kit spin columns are used for isolation of DNA using 2% polyvinylpolypyrrolidone and the purification of DNA from microscopy positive samples which improve sensitivity of the PCR. This is the most widely used method for the DNA extraction.[31][32]
  • Other kits which are used for DNA extraction include the genomic DNA Prep Plus kit and the XTRAX DNA extraction kit.[33][34]
Automated Methods

MagNA Pure LC DNA isolation kit

  • With this kit genomic DNA is lysed from organisms in buffer containing guanidine isothiocyanate and the lysed DNA binds to magnetic glass particles in chaotropic conditions.[35]
  • The magnetic particles are washed to remove impurities and unbound substances.
  • The washed DNA is removed from the magnetic particles under the conditions of elevated temperature and low salt concentration.
  • This method is used for DNA extraction from microsporidia in fecal specimens.
  • This method is not routinely used for DNA extraction.
Conventional PCR
Real-Time PCR
Microarray Development
Typing Methods
  • The nested PCR performed on DNA extracted from liver and stool samples helps in identifying the polymorphism exhibited in the SREHP gene. This is used for the genetic differentiation between strains of E histolytica which cause intestinal or liver disease.[50]
  • The strain specific gene (SSG), is also used to differentiate various strains of the parasite.[51][44]
  • Other DNA markers of E histolytica include the chitinase gene, as a marker for studying E dispar.

[52][53]

References

  1. ↑ 1.0 1.1 Huston CD, Haque R, Petri WA (1999). "Molecular-based diagnosis of Entamoeba histolytica infection". Expert Rev Mol Med. 1999: 1–11. doi:doi:10.1017/S1462399499000599 Check |doi= value (help). PMID 14987356.
  2. ↑ 2.0 2.1 2.2 2.3 Tanyuksel M, Petri WA (2003). "Laboratory diagnosis of amebiasis". Clin Microbiol Rev. 16 (4): 713–29. PMC 207118. PMID 14557296.
  3. ↑ 3.0 3.1 Krogstad DJ, Spencer HC, Healy GR, Gleason NN, Sexton DJ, Herron CA (1978). "Amebiasis: epidemiologic studies in the United States, 1971-1974". Ann Intern Med. 88 (1): 89–97. PMID 619763.
  4. ↑ 4.0 4.1 4.2 Clark CG, Diamond LS (2002). "Methods for cultivation of luminal parasitic protists of clinical importance". Clin Microbiol Rev. 15 (3): 329–41. PMC 118080. PMID 12097242.
  5. ↑ Li E, Stanley SL (1996). "Protozoa. Amebiasis". Gastroenterol Clin North Am. 25 (3): 471–92. PMID 8863036.
  6. ↑ 6.0 6.1 González-Ruiz A, Haque R, Aguirre A, Castañón G, Hall A, Guhl F; et al. (1994). "Value of microscopy in the diagnosis of dysentery associated with invasive Entamoeba histolytica". J Clin Pathol. 47 (3): 236–9. PMC 501902. PMID 8163695.
  7. ↑ 7.0 7.1 Strachan WD, Chiodini PL, Spice WM, Moody AH, Ackers JP (1988). "Immunological differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica". Lancet. 1 (8585): 561–3. PMID 2894495.
  8. ↑ Fotedar R, Stark D, Beebe N, Marriott D, Ellis J, Harkness J (2007). "PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia". J Clin Microbiol. 45 (3): 1035–7. doi:10.1128/JCM.02144-06. PMC 1829108. PMID 17229864.
  9. ↑ Lebbad M, Svärd SG (2005). "PCR differentiation of Entamoeba histolytica and Entamoeba dispar from patients with amoeba infection initially diagnosed by microscopy". Scand J Infect Dis. 37 (9): 680–5. doi:10.1080/00365540510037812. PMID 16126570.
  10. ↑ Núñez YO, Fernández MA, Torres-Núñez D, Silva JA, Montano I, Maestre JL; et al. (2001). "Multiplex polymerase chain reaction amplification and differentiation of Entamoeba histolytica and Entamoeba dispar DNA from stool samples". Am J Trop Med Hyg. 64 (5–6): 293–7. PMID 11463120.
  11. ↑ Sargeaunt PG, Jackson TF, Wiffen S, Bhojnani R, Williams JE, Felmingham D; et al. (1987). "The reliability of Entamoeba histolytica zymodemes in clinical laboratory diagnosis". Arch Invest Med (Mex). 18 (2): 69–75. PMID 2889435.
  12. ↑ Blanc D, Sargeaunt PG (1991). "Entamoeba histolytica zymodemes: exhibition of gamma and delta bands only of glucose phosphate isomerase and phosphoglucomutase may be influenced by starch content in the medium". Exp Parasitol. 72 (1): 87–90. PMID 1825198.
  13. ↑ Jackson TF, Suparsad S (1997). "Zymodeme stability of Entamoeba histolytica and E. dispar". Arch Med Res. 28 Spec No: 304–5. PMID 9033111.
  14. ↑ 14.0 14.1 Haque R, Faruque AS, Hahn P, Lyerly DM, Petri WA (1997). "Entamoeba histolytica and Entamoeba dispar infection in children in Bangladesh". J Infect Dis. 175 (3): 734–6. PMID 9041357.
  15. ↑ 15.0 15.1 Haque R, Neville LM, Hahn P, Petri WA (1995). "Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits". J Clin Microbiol. 33 (10): 2558–61. PMC 228528. PMID 8567882.
  16. ↑ Rosenblatt JE, Sloan LM, Bestrom JE (1995). "Evaluation of an enzyme-linked immunoassay for the detection in serum of antibodies to Entamoeba histolytica". Diagn Microbiol Infect Dis. 22 (3): 275–8. PMID 8565416.
  17. ↑ Abd-Alla MD, Jackson TG, Ravdin JI (1998). "Serum [[IgM]] antibody response to the galactose-inhibitable adherence lectin of [[Entameoba histolytica]]". Am J Trop Med Hyg. 59 (3): 431–4. PMID 9749639. URL–wikilink conflict (help)
  18. ↑ Ravdin JI, Jackson TF, Petri WA, Murphy CF, Ungar BL, Gathiram V; et al. (1990). "Association of serum antibodies to adherence lectin with invasive amebiasis and asymptomatic infection with pathogenic Entamoeba histolytica". J Infect Dis. 162 (3): 768–72. PMID 2388003.
  19. ↑ Braga LL, Mendonca Y, Paiva CA, Sales A, Cavalcante AL, Mann BJ (1998). "Seropositivity for and intestinal colonization with Entamoeba histolytica and entamoeba dispar in individuals in northeastern Brazil". J Clin Microbiol. 36 (10): 3044–5. PMC 105108. PMID 9738064.
  20. ↑ Goncalves ML, da Silva VL, de Andrade CM, Reinhard K, da Rocha GC, Le Bailly M; et al. (2004). "Amoebiasis distribution in the past: first steps using an immunoassay technique". Trans R Soc Trop Med Hyg. 98 (2): 88–91. PMID 14964807.
  21. ↑ Grundy MS (1982). "Preliminary observations using a multi-layer ELISA method for the detection of Entamoeba histolytica trophozoite antigens in stool samples". Trans R Soc Trop Med Hyg. 76 (3): 396–400. PMID 6287685.
  22. ↑ Shamsuzzaman SM, Haque R, Hasin SK, Hashiguchi Y (2000). "Evaluation of indirect fluorescent antibody test and enzyme-linked immunosorbent assay for diagnosis of hepatic amebiasis in Bangladesh". J Parasitol. 86 (3): 611–5. doi:10.1645/0022-3395(2000)086[0611:EOIFAT]2.0.CO;2. PMID 10864262.
  23. ↑ Haque R, Ali IK, Akther S, Petri WA (1998). "Comparison of PCR, isoenzyme analysis, and antigen detection for diagnosis of Entamoeba histolytica infection". J Clin Microbiol. 36 (2): 449–52. PMC 104557. PMID 9466756.
  24. ↑ Leiva B, Lebbad M, Winiecka-Krusnell J, Altamirano I, Tellez A, Linder E (2006). "Overdiagnosis of Entamoeba histolytica and Entamoeba dispar in Nicaragua: a microscopic, triage parasite panel and PCR study". Arch Med Res. 37 (4): 529–34. doi:10.1016/j.arcmed.2005.10.009. PMID 16624654.
  25. ↑ Pillai DR, Kain KC (1999). "Immunochromatographic strip-based detection of Entamoeba histolytica-E. dispar and Giardia lamblia coproantigen". J Clin Microbiol. 37 (9): 3017–9. PMC 85440. PMID 10449494.
  26. ↑ Garcia LS, Shimizu RY, Bernard CN (2000). "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. 38 (9): 3337–40. PMC 87383. PMID 10970380.
  27. ↑ Sharp SE, Suarez CA, Duran Y, Poppiti RJ (2001). "Evaluation of the Triage Micro Parasite Panel for detection of Giardia lamblia, Entamoeba histolytica/Entamoeba dispar, and Cryptosporidium parvum in patient stool specimens". J Clin Microbiol. 39 (1): 332–4. doi:10.1128/JCM.39.1.332-334.2001. PMC 87724. PMID 11136793.
  28. ↑ Ramos F, Zurabian R, Morán P, Ramiro M, Gómez A, Clark CG; et al. (1999). "The effect of formalin fixation on the polymerase chain reaction characterization of Entamoeba histolytica". Trans R Soc Trop Med Hyg. 93 (3): 335–6. PMID 10492774.
  29. ↑ 29.0 29.1 Troll H, Marti H, Weiss N (1997). "Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR". J Clin Microbiol. 35 (7): 1701–5. PMC 229825. PMID 9196177.
  30. ↑ https://www.cdc.gov/dpdx/reference.html Accessed on February 10, 2017
  31. ↑ 31.0 31.1 Verweij JJ, Blotkamp J, Brienen EA, Aguirre A, Polderman AM (2000). "Differentiation of Entamoeba histolytica and Entamoeba dispar cysts using polymerase chain reaction on DNA isolated from faeces with spin columns". Eur J Clin Microbiol Infect Dis. 19 (5): 358–61. PMID 10898137.
  32. ↑ Verweij JJ, van Lieshout L, Blotkamp C, Brienen EA, van Duivenvoorden S, van Esbroeck M; et al. (2000). "Differentiation of Entamoeba histolytica and Entamoeba dispar using PCR-SHELA and comparison of antibody response". Arch Med Res. 31 (4 Suppl): S44–6. PMID 11070218.
  33. ↑ Evangelopoulos A, Spanakos G, Patsoula E, Vakalis N, Legakis N (2000). "A nested, multiplex, PCR assay for the simultaneous detection and differentiation of Entamoeba histolytica and Entamoeba dispar in faeces". Ann Trop Med Parasitol. 94 (3): 233–40. PMID 10884867.
  34. ↑ Myjak P, Kur J, Pietkiewicz H (1997). "Usefulness of new DNA extraction procedure for PCR technique in species identification of Entamoeba isolates". Wiad Parazytol. 43 (2): 163–70. PMID 9374591.
  35. ↑ Wolk DM, Schneider SK, Wengenack NL, Sloan LM, Rosenblatt JE (2002). "Real-time PCR method for detection of Encephalitozoon intestinalis from stool specimens". J Clin Microbiol. 40 (11): 3922–8. PMC 139654. PMID 12409353.
  36. ↑ Calderaro A, Gorrini C, Bommezzadri S, Piccolo G, Dettori G, Chezzi C (2006). "Entamoeba histolytica and Entamoeba dispar: comparison of two PCR assays for diagnosis in a non-endemic setting". Trans R Soc Trop Med Hyg. 100 (5): 450–7. doi:10.1016/j.trstmh.2005.07.015. PMID 16274714.
  37. ↑ Hamzah Z, Petmitr S, Mungthin M, Leelayoova S, Chavalitshewinkoon-Petmitr P (2006). "Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay". J Clin Microbiol. 44 (9): 3196–200. doi:10.1128/JCM.00778-06. PMC 1594701. PMID 16954247.
  38. ↑ Haque R, Petri WA (2006). "Diagnosis of amebiasis in Bangladesh". Arch Med Res. 37 (2): 273–6. doi:10.1016/j.arcmed.2005.09.001. PMID 16380331.
  39. ↑ 39.0 39.1 Zaki M, Meelu P, Sun W, Clark CG (2002). "Simultaneous differentiation and typing of Entamoeba histolytica and Entamoeba dispar". J Clin Microbiol. 40 (4): 1271–6. PMC 140395. PMID 11923344.
  40. ↑ Clark CG, Diamond LS (1991). "Ribosomal RNA genes of 'pathogenic' and 'nonpathogenic' Entamoeba histolytica are distinct". Mol Biochem Parasitol. 49 (2): 297–302. PMID 1685555.
  41. ↑ Clark CG, Diamond LS (1992). "Differentiation of pathogenic Entamoeba histolytica from other intestinal protozoa by riboprinting". Arch Med Res. 23 (2): 15–6. PMID 1364099.
  42. ↑ Cruz-Reyes JA, Spice WM, Rehman T, Gisborne E, Ackers JP (1992). "Ribosomal DNA sequences in the differentiation of pathogenic and non-pathogenic isolates of Entamoeba histolytica". Parasitology. 104 ( Pt 2): 239–46. PMID 1594290.
  43. ↑ Que X, Reed SL (1991). "Nucleotide sequence of a small subunit ribosomal RNA (16S-like rRNA) gene from Entamoeba histolytica: differentiation of pathogenic from nonpathogenic isolates". Nucleic Acids Res. 19 (19): 5438. PMC 328914. PMID 1923831.
  44. ↑ 44.0 44.1 Clark CG, Diamond LS (1993). "Entamoeba histolytica: a method for isolate identification". Exp Parasitol. 77 (4): 450–5. doi:10.1006/expr.1993.1105. PMID 8253158.
  45. ↑ 45.0 45.1 Clark CG, Diamond LS (1997). "Intraspecific variation and phylogenetic relationships in the genus Entamoeba as revealed by riboprinting". J Eukaryot Microbiol. 44 (2): 142–54. PMID 9109261.
  46. ↑ Heckendorn F, N'Goran EK, Felger I, Vounatsou P, Yapi A, Oettli A; et al. (2002). "Species-specific field testing of Entamoeba spp. in an area of high endemicity". Trans R Soc Trop Med Hyg. 96 (5): 521–8. PMID 12474480.
  47. ↑ Klein D (2002). "Quantification using real-time PCR technology: applications and limitations". Trends Mol Med. 8 (6): 257–60. PMID 12067606.
  48. ↑ Blessmann J, Buss H, Nu PA, Dinh BT, Ngo QT, Van AL; et al. (2002). "Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples". J Clin Microbiol. 40 (12): 4413–7. PMC 154634. PMID 12454128.
  49. ↑ Wang Z, Vora GJ, Stenger DA (2004). "Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray". J Clin Microbiol. 42 (7): 3262–71. doi:10.1128/JCM.42.7.3262-3271.2004. PMC 446233. PMID 15243091.
  50. ↑ Ayeh-Kumi PF, Ali IM, Lockhart LA, Gilchrist CA, Petri WA, Haque R (2001). "Entamoeba histolytica: genetic diversity of clinical isolates from Bangladesh as demonstrated by polymorphisms in the serine-rich gene". Exp Parasitol. 99 (2): 80–8. doi:10.1006/expr.2001.4652. PMID 11748961.
  51. ↑ Burch DJ, Li E, Reed S, Jackson TF, Stanley SL (1991). "Isolation of a strain-specific Entamoeba histolytica cDNA clone". J Clin Microbiol. 29 (4): 696–701. PMC 269855. PMID 1890170.
  52. ↑ Haghighi A, Kobayashi S, Takeuchi T, Masuda G, Nozaki T (2002). "Remarkable genetic polymorphism among Entamoeba histolytica isolates from a limited geographic area". J Clin Microbiol. 40 (11): 4081–90. PMC 139687. PMID 12409379.
  53. ↑ de la Vega H, Specht CA, Semino CE, Robbins PW, Eichinger D, Caplivski D; et al. (1997). "Cloning and expression of chitinases of Entamoebae". Mol Biochem Parasitol. 85 (2): 139–47. PMID 9106188.
  54. ↑ Pinheiro SM, Maciel RF, Morais MA, Aca IS, Carvalho LB, Coimbra MR (2005). "Genetic characterization of Entamoeba dispar isolates in Northeast Brazil". Acta Trop. 94 (1): 35–40. doi:10.1016/j.actatropica.2005.01.012. PMID 15777694.
  55. ↑ Zaki M, Clark CG (2001). "Isolation and characterization of polymorphic DNA from Entamoeba histolytica". J Clin Microbiol. 39 (3): 897–905. doi:10.1128/JCM.39.3.897-905.2001. PMC 87847. PMID 11230401.
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