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Journal of Clinical Microbiology, December 2006, p. 4569-4571, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01979-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Evaluation of Entamoeba histolytica Antigen and Antibody Point-of-Care Tests for the Rapid Diagnosis of Amebiasis{triangledown}

Megan Leo,1 Rashidul Haque,2 Mamun Kabir,2 Shantanu Roy,2 Rita Marie Lahlou,1 Dinesh Mondal,2 Egbert Tannich,3 and William A. Petri Jr.1*

University of Virginia, Charlottesville, Virginia,1 ICDDR,B, Dhaka, Bangladesh,2 Bernhard Nocht Institute for Tropical Medicine, Hamburg, Germany3

Received 24 September 2006/ Accepted 29 September 2006


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ABSTRACT
 
The bedside diagnosis of amebiasis could improve patient care. In Bangladesh and Vietnam, a novel and simple-to-use Entamoeba histolytica rapid antigen test had 97% sensitivity and 100% specificity compared to the results of a standard enzyme-linked immunosorbent assay antigen detection method, and a rapid antibody test had 89 to 100% sensitivity and 89 to 95% specificity.


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TEXT
 
In 1997, the World Health Organization recommended that improved diagnostic methods that are specific for Entamoeba histolytica and that use technologies that are appropriate for the developing world be developed (11, 33). Methods of diagnosis have improved in recent years, but a rapid, point-of-care test would be a more appropriate technology for use in the developing world (7, 10, 20, 21). Recently, real-time PCR has proven to be the most sensitive test for the detection of E. histolytica in stool compared to the sensitivities of the traditional nested PCR and enzyme-linked immunosorbent assay (ELISA) (29). However, real-time PCR is cumbersome for routine diagnosis because it requires expensive equipment and specialized personnel for analysis of the results. For this reason, antigen and antibody detection by ELISA is becoming the standard method for diagnosis of E. histolytica infection (13).

Rapid tests that use stool specimens have been developed for Giardia lamblia and Cryptosporidium parvum (6, 26). A rapid dipstick method for the detection of E. histolytica antibodies in serum has been evaluated and was found to have sensitivities and specificities similar to those of an ELISA method (32). No rapid test technology, however, has been introduced for the detection of E. histolytica antigen in stool. Here we report in our experience with a prototype E. histolytica rapid antigen test for use with stool specimens and a rapid antibody test for use with serum specimens. They use the immunochromatographic technology and are packaged for individual use, making them appropriate for point-of-care testing (2, 13, 15, 23, 27, 31).

Serum and stool samples. Stool and serum specimens were obtained from a cohort of children from an area of Dhaka, Bangladesh, where E. histolytica is endemic (14). The sample panel included monthly stool specimens from asymptomatic children and those with diarrhea. Of the 294 stool specimens, 67 (23%) were diarrheal specimens. Of the 337 serum samples tested from Bangladesh, 42 (12.5%) were from patients with confirmed cases of liver abscess. In Hue, Vietnam, 150 serum samples were collected from an area of endemicity for amebic liver abscess. Informed consent was obtained from the patients or the parents of children. The human experimental guidelines of the U.S. Department of Health and Human Services, the University of Virginia, and the Centre for Health and Population Research of the International Centre for Diarrheal Disease Research, Bangladesh (ICDDR,B), were followed in the conduct of this research in Bangladesh. The studies conducted in Vietnam received institutional review board approval through the Scientific Council of Education, Training and Ethics of Hue Medical School in Hue, Vietnam, and the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany.

E. histolytica rapid antigen test. The prototype E. histolytica rapid antigen test was supplied by TechLab, Inc. (Blacksburg, VA), and was specifically designed to identify E. histolytica-specific antigen in stool samples. For this study, 200 µl of stool sample was added to 500 µl of the diluent supplied with the test kit in a 1-ml tube. If the stool sample was formed and unable to be cleanly drawn up into a pipette, an approximately 200-µl equivalent sample was transferred to the tube with a wooden applicator. The samples were mixed by lightly vortexing them. They were then centrifuged at 1,500 x g for 2 min, and 500 µl of supernatant was resuspended in a new tube. Two drops of conjugate (60 µl), consisting of 7F4 antibody coupled to horseradish peroxidase (HRP) in a buffered solution, was added to the sample and the tube was again vortexed. The sample-conjugate mixture was incubated at room temperature for 15 min. A total of 400 µl of the sample was then added to a membrane through a sample well and was allowed to migrate over two antibody-striped lines: a control line and a test line. The control line bound to the conjugate regardless of the sample antigen content and indicated whether the test ran properly. The test line contained antibodies specific for E. histolytica lectin and trapped antigen-conjugate complexes if they were present in the sample. The sample was allowed to incubate at room temperature for 10 min. The reaction well was then washed with 500 µl of wash solution. Two drops of the substrate solution was added to the reaction well containing the HRP enzyme conjugate. The test was allowed to develop for 10 min before the sample was classified as positive or negative (Fig. 1). The total assay time was approximately 35 min.


Figure 1
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  		FIG. 1. Demonstration of E. histolytica-positive (+) and -negative (–) fecal specimens evaluated by the E. histolytica rapid antigen test. The left-hand lines on both tests are an internal control line to indicate proper test function. The right-hand line (visible only in the positive sample) is the test line, which indicates the positive-negative status of the sample.

E. histolytica rapid antibody test. The prototype E. histolytica rapid antibody test was supplied by TechLab, Inc., and was designed as a modified version of the protocol for the antigen test (see above) but used a reaction well flowthrough format. The 25 µl of serum sample was diluted with 225 µl of the buffer supplied with the test kit, and 250 µl of diluted sample was applied to the reaction well and allowed to completely absorb for 1 min. The reaction well was washed with 2 drops of membrane wash buffer, followed by a second wash after complete absorption. The membrane that it flowed through had two dots. The control dot was designed to bind to any anti-human immunoglobulin G (IgG), while the test dot consisted of an E. histolytica-specific recombinant protein, termed LecA, which captured E. histolytica-specific IgG. Anti-human IgG conjugated to HRP was added to the reaction well and was allowed to react for 1 min. The well was again washed twice with 2 drops of membrane wash solution. To determine whether bound human IgG was present, 2 drops of substrate solution was added, and the HRP enzyme converted the substrate to a blue color if it was present on the control dot or the test dot. The test was allowed to develop for 5 min, and 2 drops of a stop solution was added before the test was read.

E. histolytica antibody ELISA. The anti-lectin IgG ELISA procedure performed in Dhaka used a highly purified, native E. histolytica lectin as a detection antigen isolated by using monoclonal antibody chromatography (13). For the work in Vietnam, the antibody ELISA used a recombinant fragment of the E. histolytica Gal/GalNAc-specific adherence lectin, a recombinant 125-kDa E. histolytica surface protein, or an immunofluorescence assay format with whole E. histolytica trophozoites (23).

The E. histolytica rapid antigen test had a sensitivity of 97% and a specificity of 100% compared to the results of the E. histolytica II ELISA (Table 1 and Table 2). Seventy-seven percent of the stool samples were from asymptomatic patients. The E. histolytica rapid antibody test of 337 serum samples from Bangladesh had a sensitivity of 100% and a specificity of 95% compared to the results of the E. histolytica adherence lectin antibody ELISA (Table 3 and Table 4). When the E. histolytica rapid antibody test was evaluated with 150 serum samples from Hue, Vietnam, the sensitivity was 89% and the specificity was 92% (Table 5 and Table 6) (24). The differences in the results between the two sites are most likely due to differences in the serology tests that were used for comparison. In Bangladesh, the same antigen that is present in the rapid test was used, whereas in Hamburg, independent antigens were used. Accordingly, the results from Bangladesh were more concordant.


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  		TABLE 1. Evaluation of the E. histolytica rapid stool antigen test at the Bangladesh site


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  		TABLE 2. Summary of data for E. histolytica rapid stool antigen test at the Bangladesh site


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  		TABLE 3. Evaluation of the E. histolytica rapid serum antibody test at the Bangladesh site


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  		TABLE 4. Summary of data for E. histolytica rapid serum antibody test at the Bangladesh site


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  		TABLE 5. Evaluation of the E. histolytica rapid serum antibody test at the Hamburg clinical study site


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  		TABLE 6. Summary of data for E. histolytica rapid serum antibody test at the Hamburg clinical study site

The major conclusion of this study is that the prototype E. histolytica rapid antigen individual-use test and the prototype E. histolytica rapid antibody individual-use test can be reliably used for the routine diagnosis of intestinal and extraintestinal Entamoeba histolytica infections. It should be noted, however, that a negative antigen test result does not rule out amebiasis, as antigen detection is approximately 80% sensitive compared to the results of real-time PCR. Both rapid tests can be run at the bedside for individual patients, they do not require high-tech equipment for processing of the results, and analysis of the results can be accomplished by health care workers with very little training.


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ACKNOWLEDGMENTS
 
The study was conducted at the ICDDR,B Centre for Health and Population Research in Dhaka, Bangladesh, and the Bernhard Nocht Institute for Tropical Medicine in Hamburg, Germany, with the support of grants AI-058450 and AI-056872 from the National Institutes of Health and a New Opportunities grant from the Middle Atlantic Regional Center of Excellence in Biodefense and Emerging Infectious Diseases to W.A.P. R.H. is a Howard Hughes Medical Institute international research scholar.

The ELISAs and rapid tests were developed in collaboration with TechLab Inc. (Blacksburg, Virginia).

W. A. Petri, Jr., received royalties from a patent license agreement with TechLab for a diagnostic test for amebiasis. These royalties accrue to the American Society of Tropical Medicine and Hygiene without benefit to W. A. Petri, Jr.


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FOOTNOTES
 
* Corresponding author. Mailing address: University of Virginia Health System, P.O. Box 801340, Charlottesville, VA 22908-1340. Phone: (434) 924-5621. Fax: (434) 924-0075. E-mail: wap3g{at}virginia.edu. Back

{triangledown} Published ahead of print on 11 October 2006. Back


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Journal of Clinical Microbiology, December 2006, p. 4569-4571, Vol. 44, No. 12
0095-1137/06/$08.00+0     doi:10.1128/JCM.01979-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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