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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 Tests

Needle aspiration

  • Needle aspiration is used to differentiate between amoebic and pyogenic liver abscess
  • The ultrasound and CT are used to guide percutaneous aspiration and drainage.
  • The aspirated fluid is odorless unless secondarily infected
  • The pathognomonic of the aspirate is the reddish-brown anchovy paste appearance, which indicates that the abscess has been present for weeks.
  • The trophozoites are present only in the wall of the abscess. Rest of abscess is composed of lysed leukocytes.
Laboratory Method Findings
Microscopy
  • Microscopic techniques include 1. Wet preparation 2. Concentration 3. Permanently stained smears[1]
  • Less reliable method than culture or antigen detection test[2][3]
  • Minimum three stool samples are examined for ova and parasites within 10days of sample collection as organisms are excreted intermittently[4]
  • Presence of RBCs in trophozoites is diagnostic of Entamoeba histolytica
  • The specificity of the test is low as the trophozoites containing RBCs are not present in all cases and E dispar may also contain RBCs in trophozoites[5][6]
  • Freezing a fresh fecal specimen at -20°C before extraction of DNA will not effect the sensitivity of the molecular assays.[7][8][9]
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
  • This is an important method for identification and recovery of Entamoeba species
Culture Methods
  • The specimens for the culture of E histolytica include fecal specimens, rectal biopsy specimens or liver abscess aspirates.
  • There are two culture techniques. They are xenic and axenic systems.[10]
  • Culture of E histolytica is less sensitive than microscopy as a detection method.
  • Parasite cultures are expensive, labor intensive and difficult to maintain in the diagnostic laboratory.

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
  • Isoenzyme analysis is useful in the differentiation of Entamoeba species.[11][12][13]
  • There are three isoenzymes (zymodyme) for E histolytica and one for E dispar.
  • This techniques helps in differentiation of E histolytica and E dispar and is considered gold standard for diagnosing amoebic infection prior to development of newer DNA-based techniques.

Disadvantages

  • Difficult to perform test, time-consuming procedure, and not always successful.
  • The isoenzyme analysis is negative for many microscopy positive stool samples[5][14][15][6]
Antibody Detection Tests
  • Antibody detection tests are useful in the case of amoebic liver abscess as patients do not have detectable parasites in feces.
  • The sensitivity of the test is 100% in patients with amoebic liver abscess. The techniques include enzyme-linked immunosorbent assay (ELISA), indirect hemagglutination (IHA), latex agglutination, immunoelectrophoresis, counterimmunoelectrophoresis (CIE), the amebic gel diffusion test, immunodiffusion, complement fixation, and indirect immunofluorescence assay (IFA).

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 amebiasis 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
  • The antigen detection tests use monoclonal antibodies against the Gal/GalNAc-specific lectin of E. histolytica or against serine-rich antigen of E. histolytica.
  • This test is useful in differentiation of disease causing E histolytica as there are antigenic differences in the lectins of E histolytica and E dispar.
  • The test has good sensitivity and specificity for the detection of E histolytica antigen in stool specimens of patients with amoebic colitis and asymptomatic intestinal infection.[23][14][15]
  • Antigen detection using ELISA is is both rapid and technically simple to perform. Hence used in developing countries where amoebiasis is most prevalent.

Disadvantage

  • Antigens detected are denatured by fixation of the stool sample. hence the test is limited to frozen or fresh samples.
Immunochromatographic Assays
  • The triage parasite panel (TPP) is used for antigen detection of antigens specific for E. histolytica/E. dispar, Giardia lamblia, and Cryptosporidium parvum simultaneously.[24][25]
  • The antigens specific for these organisms are used by using specific antibodies and immobilized on a membrane.
  • This test is highly sensitive and specific.
  • This test can be performed within 15 min with fresh or frozen, unfixed human fecal specimens.

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 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
  • This is the method of choice in developed countries for epidemiological and clinical studies.[36][37][38][39]
  • This method helps in identifying Entamoeba histolytica in various clinical specimens like liver abscess aspirate, feces, and tissues.[2]
  • PCR is used for the detection and differentiation of various Entamoeba species.
  • 18SrDNA is used as a target for the differentiation of E histolytica and E dispar.
Real-Time PCR
Microarray Development
Typing Methods

References

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  2. 2.0 2.1 2.2 Tanyuksel M, Petri WA (2003). "Laboratory diagnosis of amebiasis". Clin Microbiol Rev. 16 (4): 713–29. PMC 207118. PMID 14557296.
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  30. https://www.cdc.gov/dpdx/reference.html Accessed on February 10, 2017
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