Laboratory Studies
Laboratory diagnosis of amebiasis is made by demonstrating the organism or by employing immunologic techniques. [62, 63, 64, 65, 66]
Findings from basic blood tests may include the following:
-
Leukocytosis without eosinophilia (80% of patients)
-
Elevated alkaline phosphatase level (80%)
-
Elevated transaminase levels
-
Mildly elevated bilirubin level
-
Reduced albumin level
-
Mild anemia
-
Elevated erythrocyte sedimentation rate (ESR)
Other laboratory studies employed for diagnosis include microscopy, culture, serologic testing, and polymerase chain reaction (PCR) assay.
Microscopy
Laboratory diagnosis of intestinal amoebiasis in developing countries still relies on labor-intensive and insensitive methods involving staining and microscopic examination of fresh stool smears for trophozoites that contain ingested red blood cells (see the image below). [67] The presence of intracytoplasmic RBCs in trophozoites is diagnostic of E histolytica infection, although some studies have demonstrated the same phenomenon with E dispar. [18, 44]
Trichrome stain of Entamoeba histolytica trophozoites in amebiasis. Two diagnostic characteristics are observed. Two trophozoites have ingested erythrocytes, and all 3 have nuclei with small, centrally located karyosomes.
Examination of a single stool sample has a sensitivity of only 33-50%; however, examination of 3 stool samples over no more than 10 days can improve the detection rate to 85-95%. It should be kept in mind that routine microscopy cannot be relied on to distinguish the pathogenic E histolytica from the nonpathogenic E dispar, E bangladeshi, and E moshkovskii.
Stool leukocytes may be found, but in fewer numbers than in shigellosis.
Stool examination findings in patients with amebic liver abscess are usually negative. Repeated stool sampling in patients with proven amebic liver abscess is positive in 8-40% of cases. Identification of the parasite in a liver abscess aspirate is only 20% sensitive.
The World Health Organization (WHO) recommends that intestinal amebiasis be diagnosed with an E histolytica–specific test, thus rendering the classic ova-and-parasite stool examination obsolete.
Culture
Cultures can be performed either with fecal or rectal biopsy specimens or with liver abscess aspirates. [18] Culture has a success rate of 50-70%, but it is technically difficult. Overall, culture is less sensitive than microscopy.
Xenic cultivation, first introduced in 1925, is defined as the growth of the parasite in the presence of an undefined flora. This technique still is in use today, using modified Locke-egg media. Axenic cultivation, first achieved in 1961, involves growing the parasite in the absence of any other metabolizing cells. Only a few strains of E dispar have been reported to be viable in axenic cultures.
Antigen detection
Enzyme-linked immunosorbent assay (ELISA) is used to detect antigens from E histolytica in stool samples. Several kits are commercially available. [18]
Antigen-based ELISA kits using monoclonal antibodies against the galactose/N -acetylgalactosamine (GAL/GalNAc)–specific lectin of E histolytica (E histolytica II, TechLab, Blacksburg, VA) yield an overall sensitivity of 71-100% and a specificity of 93-100%. One study showed a much lower sensitivity (14.2%). In patients with amebic liver abscess, serum and liver aspirate antigen detection using the same kit was shown to yield a sensitivity of 96% in serum and 100% in liver aspirate.
Other stool detection kits use monoclonal antibodies against the serine-rich antigen of E histolytica (Optimum S; Merlin Diagnostika, Bornheim-Hersel, Germany) or against other specific antigens (Entamoeba CELISA-PATH, Cellabs, Brookvale, Australia; ProSpecT EIA, Remle Inc, Lenexa, KY).
No specific antigen tests are available for the detection of E dispar and E moshkovskii from clinical samples.
Polymerase Chain Reaction
Real-time PCR is considerably more sensitive than stool antigen detection. [68] Gastrointestinal panels such as the xTAG Gastrointestinal Pathogen Panel (Luminex, Austin, TX) and the BioFire FilmArrray GI Panel (BioFire Diagnostics, Salt Lake City, UT) are currently approved by FDA for the detection of E. histolytica in stool samples. [69, 70]
Antibody detection
Serum antibodies against amebae are present in 70-90% of individuals with symptomatic intestinal E histolytica infection. [18] Antiamebic antibodies are present in as many as 99% of individuals with liver abscess who have been symptomatic for longer than 1 week. Serologic examination should be repeated 1 week later in those with negative test on presentation. However, serologic tests do not distinguish new from past infection, because the seropositivity persists for years after an acute infection.
ELISA, the assay most commonly used worldwide, measures the presence of serum antilectin antibodies (immunoglobulin G [IgG]). The galactose lectin antigen is present in the serum of 75% of subjects with amebic liver abscess and may be particularly useful in patients presenting acutely, before an IgG antiamebic antibody response occurs. The sensitivity of ELISA for detection of antibodies to E histolytica in patients with amebic liver abscess is 97.9%, and its specificity is 94.8%. False-negative results can occur within the first 7-10 days after infection.
Immunofluorescent assay (IFA) is also rapid, reliable, and reproducible. In the setting of amebic liver abscess, the sensitivity and specificity of IFA were shown to be 93.6% and 96.7%, respectively.
Indirect hemagglutination assay (IHA) detects antibody specific for E histolytica. The antigen used in IHA consists of a crude extract of axenically cultured organisms. Antibody titers of more than 1:256 to the 170-kd subunit of the galactose-inhibitable adherence lectin are noted in approximately 95% of patients with extraintestinal amebiasis, 70% of patients with active intestinal infection, and 10% of asymptomatic individuals.
IHA is very specific (99.1%), but it is less sensitive than ELISA. It is not useful in differentiating acute infection from previous infection, because high titers may persist for years after successful treatment. False-positive reactions at titers higher than 1:256 are rare. ELISA has replaced IHA in most laboratories. [71]
Immunoelectrophoresis, counterimmunoelectrophoresis (CIE), and immunodiffusion (ID) use the precipitation property of antigen-antibody complexes in agar. CIE is time-consuming but has a sensitivity of 100% in invasive amebiasis. ID is simple to perform and thus ideal for laboratories that only rarely perform amebic serology; however, it requires a minimum of 24 hours, compared with 2 hours for IHA or ELISA. ID is slightly less sensitive than IHA and ELISA but is equally specific. Complement fixation (CF) is less sensitive than other techniques.
Although detection of immunoglobulin M (IgM) antibodies specific for E histolytica has been reported, sensitivity in patients with current invasive disease is only about 64%.
Polymerase chain reaction assay
A wide variety of PCR-based methods targeting different genes, including a small-subunit rRNA gene (18S rDNA), a 30-kd antigen gene, a serine-rich protein gene, a chitinase gene, a hemolysin gene, and extrachromosomal circular DNA, have been described for the detection and differentiation of E histolytica, E dispar, and E moshkovskii. [18]
Sensitivities can vary according to sampling and the specific target gene used. Field studies that directly compared PCR with stool culture or antigen-detection tests for the diagnosis of E histolytica infection found these methods to be comparably effective. PCR assay also can be used for detection of E histolytica in liver aspirates for the diagnosis of amoebic liver abscess. [72]
PCR-based tests have been strongly endorsed by the WHO. However, application of PCR-based methods in routine diagnosis still is very limited [73, 74, 75] ; the generation of nonspecific DNA fragments from environmental and clinical samples often leads to false-positive results.
Loop-mediated isothermal amplification assay
The loop-mediated isothermal amplification (LAMP) assay has been applied to the detection of E histolytica in cases of hepatic amebiasis. A study that compared this test with PCR testing in 50 patients with clinical suspicion of amebic liver abscess found that LAMP assay detected five additional abscesses that were missed by PCR assay. [76] The rapidity, operational simplicity, high specificity and sensitivity, and high yield of LAMP assay suggest that it may prove to be a better diagnostic tool than PCR assay for diagnosis of hepatic amebiasis.
Radiography, Ultrasonography, CT, and MRI
Chest radiography may reveal an elevated right hemidiaphragm and a right-side pleural effusion in patients with amebic liver abscess.
On ultrasonograms, amebic liver abscesses usually appear as a solitary homogenous hypoechoic round lesion in the posterosuperior aspect of the right lobe of the liver (70-80% of cases), though multiple abscesses may occur in some patients. In an ultrasonographic evaluation of 212 patients, 34 (16%) had multiple abscesses, 75 (35%) had an abscess in the left lobe, and the remaining 103 (49%) had a solitary abscess in the right lobe.
CT plays an important role in the diagnosis of amebic colitis by demonstrating the presence of colitis with the typical involvement of the cecum and rectum. [77] Both ultrasonography and CT scanning are sensitive but nonspecific for amebic liver abscess. Ultrasonography is preferred for the evaluation of amebic liver abscess because of its low cost, rapidity, and lack of adverse effects. CT may be slightly more sensitive than ultrasonography. In cerebral amebiasis, CT shows irregular lesions without a surrounding capsule or enhancement.
On CT scans with intravenous (IV) contrast, amebic liver abscess can appear as a rounded, low-attenuation lesion with an enhancing rim. Furthermore, the abscess may be homogenous or septated, with or without observable fluid levels.
Magnetic resonance imaging (MRI) reveals high signal intensity on T2-weighted images. Perilesional edema and enhancement of rim are noted after injection of gadolinium (86% of cases).
Complete resolution of liver abscess may take as long as 2 years. Repeat imaging is not indicated if the patient is otherwise doing well.
Liver Aspiration
Ultrasound- or CT-guided needle aspiration of the liver should be performed when a diagnosis must be established very rapidly; pyogenic liver abscess can present and appear similarly to amebic liver abscess.
Liver abscess aspirate is usually an odorless thick yellow-brown liquid classically referred to as “anchovy paste.” This liquid lacks white blood cells (WBCs) as a result of lysis by the parasite. Amebae are visualized in the abscess fluid in a minority of patients with amebic liver abscess (see the image below). Liver aspiration is indicated only if abscesses are large (> 12 cm), abscess rupture is imminent, medical therapy has failed, or abscesses are present in the left lobe.
Entamoeba histolytica in liver aspirate, trichrome stain. Image courtesy of Centers for Disease Control and Prevention.
The aspirate can be sent for microscopy, culture, antigen detection, and PCR, where available. A Gram stain should also be performed if a pyogenic etiology is suspected clinically.
Lower GI Endoscopy
Rectosigmoidoscopy and colonoscopy with biopsy or scraping at the margin of a colonic mucosal ulcer provide valuable materials for diagnostic information in intestinal amebiasis. Tissue can be sent for microscopic evaluation, culture, and PCR assay, where available.
Indications for endoscopy in suspected intestinal amebiasis include the following:
-
Stool examination findings are negative, but serum antibody test findings are positive
-
Stool examination findings are negative, but immediate diagnosis is required
-
Stool examination and antibody test results are negative, but amebiasis is strongly suspected
-
Evaluation of chronic intestinal syndromes or mass lesions is desired
Fulminant colitis is a relative contraindication to colonoscopy, because it increases the risk for intestinal perforation.
On endoscopic examination, small mucosal ulcers covered with yellowish exudates are observed. The mucosal lining between the ulcers appears normal. The mucosa resembles that seen in inflammatory bowel disease (IBD). Biopsy results and a scraping of ulcer edge may reveal trophozoites. Ameboma (a carcinomalike annular lesion) can also be seen, usually in the cecum and ascending colon. [4, 78]
Rectosigmoidoscopy and colonoscopy should be considered before steroids are used in patients with suspected IBD. In a multivariate analysis, the best combination of findings for predicting amebic colitis was the combination of cecal lesions, multiple lesions, and exudates. [79]
Histologic Findings
The intestinal biopsy specimen should be taken from the edge of ulcers and evaluated for motile trophozoites.
Histopathologic findings may include nonspecific mucosal thickening, multiple discrete ulcers separated by regions of normal-appearing colonic mucosa, diffusely inflamed and edematous mucosa, necrosis, or wall perforation.
Gross pathology of intestinal ulcers due to amebiasis. Image courtesy of Centers for Disease Control and Prevention.
Amebic invasion through the mucosa and into submucosal tissues is the hallmark of amebic colitis; lateral extension through the submucosal tissues gives rise to the classic flask-shaped ulcer of amebic colitis.
Histopathology of amebiasis. Image courtesy of Centers for Disease Control and Prevention.
Histopathology of typical flask-shaped ulcer of intestinal amebiasis. Image courtesy of Centers for Disease Control and Prevention.
Different chemical stains can be used, including periodic acid−Schiff stain, which makes E histolytica appear magenta in color.
Trichrome stain of Entamoeba histolytica cyst in amebiasis. Each cyst has 4 nuclei with characteristically centrally located karyosomes. Cysts measure 12-15 mm.
-
Trichrome stain of Entamoeba histolytica trophozoites in amebiasis. Two diagnostic characteristics are observed. Two trophozoites have ingested erythrocytes, and all 3 have nuclei with small, centrally located karyosomes.
-
Trichrome stain of Entamoeba histolytica cyst in amebiasis. Each cyst has 4 nuclei with characteristically centrally located karyosomes. Cysts measure 12-15 mm.
-
Entamoeba histolytica trophozoite. Image courtesy of Centers for Disease Control and Prevention.
-
Entamoeba histolytica cyst. Image courtesy of Centers for Disease Control and Prevention.
-
Gross pathology of intestinal ulcers due to amebiasis. Image courtesy of Centers for Disease Control and Prevention.
-
Histopathology of typical flask-shaped ulcer of intestinal amebiasis. Image courtesy of Centers for Disease Control and Prevention.
-
Entamoeba histolytica in liver aspirate, trichrome stain. Image courtesy of Centers for Disease Control and Prevention.
-
Histopathology of amebiasis. Image courtesy of Centers for Disease Control and Prevention.
-
Life cycle of amebiasis (Entamoeba histolytica). Courtesy of the Centers for Disease Control and Prevention (CDC).
