Real-time PCR for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in fecal samples

A closed-tube, real-time PCR assay was developed for sensitive and specific detection and differentiation of the two closely related intestinal protozoan parasites Entamoeba histolytica and Entamoeba dispar directly from human feces. The assay is performed with the LightCycler system using fluorescence-labeled detection probes and primers amplifying a 310-bp fragment from the high-copy-number, ribosomal DNA-containing ameba episome. The assay was able to detect as little as 0.1 parasite per g of feces. The two pairs of primers used were specific for the respective ameba species, and results were not influenced by the presence of other Entamoeba species even when present in exceeding amounts. PCR was evaluated using several hundred stool samples from areas of amebiasis endemicity in Vietnam and South Africa, and results were compared with those of microscopy and ameba culture. PCR was found to be significantly more sensitive than microscopy or culture, as all samples positive by microscopy and 22 out of 25 (88%) samples positive by culture were also positive by PCR, but PCR revealed a considerable number of additional E. histolytica- or E. dispar-positive samples. Compared to culture and subsequent ameba differentiation by isoenzyme analysis, PCR was 100% specific for each of the two Entamoeba species. Interestingly, the comparison with PCR revealed that culture, in particular, underestimates E. histolytica infections. Given the high sensitivity and specificity of the developed PCR assay, the inability of microscopy to distinguish between the two ameba species, and the time it takes to culture and subsequently differentiate entamoebae by isoenzyme analysis, this assay is more suitable than microscopy or culture to correctly diagnose intestinal E. histolytica or E. dispar infection.     

Diagnostic methods for differentiation of Entamoeba histolytica and Entamoeba dispar in carriers: performance and clinical implications in a non-endemic setting

Unpreserved faecal samples, suspected to contain Entamoeba histolytica/Entamoeba dispar cysts or trophozoites on the basis of microscopic examination, and serum samples from 416 patients were collected in a prospective study to determine whether stool antigen assays and detection of antibodies in serum are reliable methods to distinguish between carriers of E. histolytica and E. dispar in comparison to the reference test: real-time PCR. In 283 patients (68%) DNA of E. histolytica or E. dispar was amplified by real-time PCR: 6 patients with amoebic colitis (2%), 19 carriers of E. histolytica (6.7%), and 258 carriers of E. dispar (91.2%). In 133 patients (31%) no DNA of E. histolytica or E. dispar could be amplified in the stool samples. This patient group was used as control for the evaluation of diagnostic tests. Using real-time PCR as a reference test, the sensitivity and specificity of (1) the Entamoeba test for the diagnosis of E. histolytica/E. dispar carrier were 59% and 98%, (2) E. histolytica II for the diagnosis of E. histolytica carrier was 71% and 100%, and (3) serology for the diagnosis of E. histolytica infection was 83.3% and 95.2%, respectively. Applied to carriers that did not originate from an endemic country the sensitivity of serology for E. histolytica infection was 90% and specificity was 98.8%. In comparison to real-time PCR the performances of Entamoeba test and E. histolytica II lacked sensitivity for a reliable diagnosis of E. histolytica/E. dispar infection in a non-endemic setting. In carriers of E. histolytica/E. dispar from non-endemic countries the high specificity of serology can be used to establish the diagnosis of E. histolytica infection if antibodies are present.     

Rapid diagnosis of Entamoeba infection by using Entamoeba and Entamoeba histolytica stool antigen detection kits.

Humans are infected by two morphologically identical species of Entamoeba: Entamoeba histolytica causes amebic colitis and liver abscess, and Entamoeba dispar is noninvasive. Several weeks of culture and isoenzyme (zymodeme) analysis are required to differentiate E. histolytica from E. dispar. Here we report a field trial of commercial antigen detection kits designed to rapidly detect and differentiate E. histolytica from E. dispar in stool specimens. Stool specimens from 202 patients with diarrhea were examined for E. histolytica and E. dispar by microscopy, culture, and antigen detection. Compared with culture, microscopic identification of the E. histolytica-E. dispar complex was 60% sensitive and 79% specific, while the screening antigen detection test for the E. histolytica-E. dispar complex was 80% sensitive and 99% specific. Differentiation of E. dispar from E. histolytica by the E. histolytica-specific test was 95% sensitive and 93% specific compared with zymodeme analysis. We conclude that the antigen detection test for the E. histolytica-E. dispar complex is more sensitive and specific than microscopy and that the E. histolytica-specific antigen detection test is as reliable and much more rapid than zymodeme analysis for the differentiation of E. histolytica from E. dispar.     

Laboratory diagnostic techniques for Entamoeba species

The genus Entamoeba contains many species, six of which (Entamoeba histolytica, Entamoeba dispar, Entamoeba moshkovskii, Entamoeba polecki, Entamoeba coli, and Entamoeba hartmanni) reside in the human intestinal lumen. Entamoeba histolytica is the causative agent of amebiasis and is considered a leading parasitic cause of death worldwide in humans. Although recent studies highlight the recovery of E. dispar and E. moshkovskii from patients with gastrointestinal symptoms, there is still no convincing evidence of a causal link between the presence of these two species and the symptoms of the host. New approaches to the identification of E. histolytica are based on detection of E. histolytica-specific antigen and DNA in stool and other clinical samples. Several molecular diagnostic tests, including conventional and real-time PCR, have been developed for the detection and differentiation of E. histolytica, E. dispar, and E. moshkovskii in clinical samples. The purpose of this review is to discuss different methods that exist for the identification of E. histolytica, E. dispar, and E. moshkovskii which are available to the clinical diagnostic laboratory. To address the need for a specific diagnostic test for amebiasis, a substantial amount of work has been carried out over the last decade in different parts of the world. The molecular diagnostic tests are increasingly being used for both clinical and research purposes. In order to minimize undue treatment of individuals infected with other species of Entamoeba such as E. dispar and E. moshkovskii, efforts have been made for specific diagnosis of E. histolytica infection and not to treat based simply on the microscopic examination of Entamoeba species in the stool. The incorporation of many new technologies into the diagnostic laboratory will lead to a better understanding of the public health problem and measures to control the disease.     

PCR detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from Sydney, Australia

This study investigated the presence of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii in stool samples from a patient population in Sydney, Australia. Stool samples were tested by microscopy and PCR. Five patients were found with E. histolytica infections, while E. dispar and E. moshkovskii were observed in 63 (70.8%) and 55 (61.8%) patients, respectively, by PCR. This is the first study in Australia using molecular techniques to determine the presence of E. histolytica, E. dispar, and E. moshkovskii.     

Evaluation of an immunochromatographic dip strip test for simultaneous detection of Cryptosporidium spp, Giardia duodenalis, and Entamoeba histolytica antigens in human faecal samples

Immunochromatographic (IC) tests may play an important role in the future diagnosis of parasitic diseases because of their speed and simplicity of use. A recently developed test to detect Cryptosporidium spp, Giardia duodenalis and Entamoeba histolytica was evaluated. Microscopy and PCR were the "gold standard" reference techniques and the results of this IC test were compared with those obtained with ELISA and IC single test for the three parasites. One hundred sixty stool samples were assayed. Using microscopy, 22 samples were diagnosed as positive for Cryptosporidium spp., 31 for Giardia duodenalis, 41 for Entamoeba histolytica/dispar, and 68 had a negative diagnosis for the three parasites. Results of IC tests show sensitivities of 70-72% for Cryptosporidium, 90-97% for Giardia and 62.5% for Entamoeba histolytica. Specificities were of 93.6-94.9%, >99% and 96.1%, respectively. In all diagnoses, agreement with microscopy and PCR was over 90%, except in the triple test and microscopy in E. histolytica detection that was 76.3%, due to the inability of microscopy to differentiate E. histolytica from nonpathogenic species such as E. dispar or E. moshkovskii. The triple stool immunoassays provide adequate sensitivities and specificities for use in outbreak situations, for screening proposals and for massive assays in endemic areas where a large number of samples must be analysed or as complementary test for individual diagnosis.     

Entamoeba histolytica and Entamoeba dispar: differentiation methods and implications

Entamoeba histolytica and Entamoeba dispar are two species morphologically identical (except hematophagous trophozoites) but one of them is pathogenic. Sensitive and specific molecular techniques which are able to distinguish E. histolytica from E. dispar have been developed recently. Detection of antigen in stool using the ELISA method is the diagnostic test method of choice for clinical use in the developing world. It is rapid and simple. Cultures for zymodeme analysis and PCR detection of the parasite remain research tools. Species identification is imperative both for improved clinical diagnosis and treatment and for planning control strategies.     

Comparison of stool antigen detection kits to PCR for diagnosis of amebiasis

The present study was conducted to compare two stool antigen detection kits with PCR for the diagnosis of Entamoeba histolytica infections by using fecal specimens submitted to the Department of Microbiology at St. Vincent's Hospital, Sydney, and the Institute of Medical and Veterinary Science, Adelaide, Australia. A total of 279 stool samples containing the E complex (E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii) were included in this study. The stool specimens were tested by using two commercially produced enzyme immunoassays (the Entamoeba CELISA PATH and TechLab E. histolytica II kits) to detect antigens of E. histolytica. DNA was extracted from all of the samples with a Qiagen DNA stool mini kit (Qiagen, Hilden, Germany), and a PCR targeting the small-subunit ribosomal DNA was performed on all of the samples. When PCR was used as a reference standard, the CELISA PATH kit showed 28% sensitivity and 100% specificity. The TechLab ELISA (enzyme-linked immunosorbent assay) kit did not prove to be useful in detecting E. histolytica, as it failed to identify any of the E. histolytica samples which were positive by PCR. With the TechLab kit, cross-reactivity was observed for three specimens, one of which was positive for both E. dispar and E. moshkovskii while the other two samples contained E. moshkovskii. Quantitative assessment of the PCR and ELISA results obtained showed that the ELISA kits were 1,000 to 10,000 times less sensitive, and our results show that the CELISA PATH kit and the TechLab ELISA are not useful for the detection of E. histolytica in stool samples from patients in geographical regions where this parasite is not endemic.     

Simultaneous differentiation and typing of Entamoeba histolytica and Entamoeba dispar

Sequences corresponding to some of the polymorphic loci previously reported from Entamoeba histolytica have been detected in Entamoeba dispar. Comparison of nucleotide sequences of two loci between E. dispar strain SAW760 and E. histolytica strain HM-1:IMSS revealed significant differences in both repeat and flanking regions. The tandem repeat units varied not only in sequence but also in number and arrangement between the two species at both the loci. Using the sequences obtained, primer pairs aimed at amplifying species-specific products were designed and tested on a variety of E. histolytica and E. dispar samples. Amplification results were in complete agreement with the original species classification in all cases, and the PCR products displayed discernible size and pattern variations among the isolates.     

Differential detection of Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii by a single-round PCR assay

A single-round PCR assay was developed for detection and differential diagnosis of the three Entamoeba species found in humans, Entamoeba moshkovskii, Entamoeba histolytica, and Entamoeba dispar, that are morphologically identical as both cysts and trophozoites. A conserved forward primer was derived from the middle of the small-subunit rRNA gene, and reverse primers were designed from signature sequences specific to each of these three Entamoeba species. PCR generates a 166-bp product with E. histolytica DNA, a 752-bp product with E. dispar DNA, and a 580-bp product with E. moshkovskii DNA. Thirty clinical specimens were examined, and the species present were successfully detected and differentiated using this assay. It was possible to detect as little as 10 pg of E. moshkovskii and E. histolytica DNA, while for E. dispar the sensitivity was about 20 pg of DNA. Testing with DNA from different pathogens, including bacteria and other protozoa, confirmed the high specificity of the assay. We propose the use of this PCR assay as an accurate, rapid, and effective diagnostic method for the detection and discrimination of these three morphologically indistinguishable Entamoeba species in both routine diagnosis of amoebiasis and epidemiological surveys.     

Amebiasis and comparison of microscopy to ELISA technique in detection of Entamoeba histolytica and Entamoeba dispar

The analysis of records of amoebal infection in various hospitals in Kilimanjaro indicated frequent occurrence of amebiasis. The population over the age of five years had higher rate of amoebal infection compared to less than that of a five-year-old population; however, both age groups had similar patterns of amebiasis during January 1999 to June 2001. To investigate misdiagnosis of amebiasis, 226 patients (passive cases) in three hospitals and 616 individuals (active cases) from three different localities in Kilimanjaro were examined. In passive cases, the prevalences of Entamoeba histolytica and Entamoeba dispar were 1% and 7.3%, respectively. Among active cases, 1% were infected with E. histolytica, and 15% were infected with E. dispar. There were no significant differences in amoebal infection between the male and female populations. A pool of 842 stool samples was used for diagnosis of E. histolytica and E. dispar by microscopic examination or ELISA kits. The microscopic examination indicated 8.7% amoebal infection; however, using ELISA as the gold standard, the prevalence of histolytica/dispar was 0.8% and 7.4%, respectively. This study indicated that E. dispar infection was 14.5 times more prevalent than E. histolytica infection.     

Immunochromatographic Strip-Based Detection of Entamoeba histolytica-E. dispar and Giardia lamblia Coproantigen

BIOSITE Triage was 68.3% sensitive and 100% specific for the detection of Entamoeba histolytica-E. dispar (n = 71) compared to Alexon-Trend's ProSpecT test (reference standard) using fresh-frozen stool. Neither test is able to distinguish E. histolytica from E. dispar. Triage was 83.3% sensitive and 100% specific compared to microscopy (formalin-ether concentrates and permanent stains) for the detection of Giardia lamblia.     

Simple differential detection of Entamoeba histolytica and Entamoeba dispar in fresh stool specimens by sodium acetate-acetic acid-formalin concentration and PCR.

Amoebiasis is caused by two distinct species, a pathogenic form (Entamoeba histolytica) and a nonpathogenic form (Entamoeba dispar), which are morphologically identical. Although the distinction between these two species is of great clinical importance, the methods developed for this purpose either are very time-consuming or involve laborious procedures for isolation of the DNA. We report here a simple PCR method starting with fresh stool specimen that allows for the sensitive and reliable distinction between E. histolytica and E. dispar. After initial concentration by the sodium acetate-acetic acid-formalin (SAF) method and digestion with proteinase K, a 0.88-kb sequence of the multicopy 16S rRNA gene served as a target for PCR amplification. The method starting with unpreserved specimens proved to be very sensitive and was not influenced by the quick exposure to SAF fixative during the initial concentration step. However, storage in SAF fixative prior to testing resulted in a decreased sensitivity within 2 days. The detection limit of the method was as low as one copy of the 16S rRNA gene. No cross-reactivity was observed with other common intestinal protozoa. Mixed infections involving both E. histolytica and E. dispar could easily be detected at a ratio of 1:10,000 by agarose gel electrophoresis or a DNA hybridization immunoassay.     

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.

A comparison of the use of three commercially available enzyme-linked immunosorbent assay-based kits and PCR amplification of rRNA genes to detect and differentiate Entamoeba histolytica from E. dispar was carried out. Only the Techlab kit did not cross-react with E. dispar antigens, but it was 100 times less sensitive than PCR in detection of and differentiation between the two types of Entamoeba.     

Diagnosis of amebic liver abscess and intestinal infection with the TechLab Entamoeba histolytica II antigen detection and antibody tests

A noninvasive diagnostic test for amebic liver abscess is needed, because amebic and bacterial abscesses appear identical on ultrasound or computer tomography and because it is rarely possible to identify Entamoeba histolytica in stool specimens from patients with amebic liver abscess. Here we report a method of detection in serum of circulating E. histolytica Gal/GalNAc lectin to diagnose amebic liver abscess, which was used in patients from Dhaka, Bangladesh. The TechLab E. histolytica II test (which differentiates the true pathogen E. histolytica from Entamoeba dispar) detected Gal/GalNAc lectin in the sera of 22 of 23 (96%) amebic liver abscess patients tested prior to treatment with the antiamebic drug metronidazole and 0 of 70 (0%) controls. After 1 week of treatment with metronidazole, 9 of 11 (82%) patients became serum lectin antigen negative. The sensitivity of the E. histolytica II antigen detection test for intestinal infection was also evaluated. Antigen detection identified E. histolytica infection in 50 samples from 1, 164 asymptomatic preschool children aged 2 to 5 years, including 16 of 16 (100%) culture-positive specimens. PCR analysis of stool specimens was used to confirm that most antigen-positive but culture-negative specimens were true-positive: PCR identified parasite DNA in 27 of 34 (79%) of the antigen-positive, culture-negative stool specimens. Antigen detection was a more sensitive test for infection than antilectin antibodies, which were detected in only 76 of 98 (78%) amebic liver abscess patients and in 26 of 50 (52%) patients with intestinal infection. We conclude that the TechLab E. histolytica II kit is a sensitive means to diagnose hepatic and intestinal amebiasis prior to the institution of metronidazole treatment.     

An unusual surface peroxiredoxin protects invasive Entamoeba histolytica from oxidant attack

Peroxiredoxins are an important class of antioxidant enzymes found from Archaea to humans, which reduce and thereby detoxify peroxides and peroxynitrites. The major thiol-containing surface antigen of the invasive ameba, Entamoeba histolytica, is a peroxiredoxin and is likely to be important during the transition from the anaerobic environment of the large intestine to human tissues. The closely related species, Entamoeba dispar, is incapable of invasion and more sensitive to hydrogen peroxide, yet also has a peroxiredoxin. We cloned and expressed the two active recombinant enzymes and found that their activity was similar by a fluorometric stopped-flow assay, giving a Km of <10 microM for hydrogen peroxide. Three monoclonal antibodies produced to recombinant E. histolytica peroxiredoxin cross-reacted with Entamoeba dispar.E. histolytica contains as much as 50 times more peroxiredoxin than E. dispar as demonstrated by a sensitive capture ELISA. In addition, the peroxiredoxin is present largely on the outer surface of the cell, in contrast to E. dispar. This unusual peroxiredoxin localizes to the site of parasite-host cell contact where it can effectively counteract oxidants generated by host cells, thus facilitating invasion.     

Comparison of a stool antigen detection kit and PCR for diagnosis of Entamoeba histolytica and Entamoeba dispar infections in asymptomatic cyst passers in Iran

The present study was conducted to compare stool antigen detection with PCR for the diagnosis of Entamoeba sp. infection in asymptomatic cyst passers from Iran. Entamoeba dispar and, in one case, E. moshkovskii were the Entamoeba spp. found in the amebic cyst passers. There was a 100% correlation between the results from the TechLab E. histolytica II stool antigen kit and those from nested PCR. We concluded that E. dispar is much more common in asymptomatic cyst passers in Iran and that antigen detection and PCR are comparable diagnostic modalities.     

Use of PCR amplification of tRNA gene-linked short tandem repeats for genotyping Entamoeba histolytica

We have developed a reliable method for PCR-based genotyping of Entamoeba histolytica based on variation in the numbers of short tandem repeats that are linked to tRNA genes in this species. Species-specific primer pairs were designed that differentiate E. histolytica from E. dispar as well as that reveal intraspecies PCR product length polymorphisms. The primers were tested with samples from different parts of the world, and DNA was extracted from cultured cells as well as liver abscess pus and feces by various methods. We now have the tools necessary to investigate a possible link between parasite genotype and the outcome of infection with Entamoeba histolytica, as well as other aspects of the organism's epidemiology.     

Comparison of real-time PCR protocols for differential laboratory diagnosis of amebiasis

Specific identification of Entamoeba spp. in clinical specimens is an important confirmatory diagnostic step in the management of patients who may be infected with Entamoeba histolytica, the species that causes clinical amebiasis. Distinct real-time PCR protocols have recently been published for identification of E. histolytica and differentiation from the morphologically identical nonpathogenic Entamoeba dispar. In this study, we compared three E. histolytica real-time PCR techniques published by December 2004. The limits of detection and efficiency of each real-time PCR assay were determined using DNA extracted from stool samples spiked with serially diluted cultured E. histolytica trophozoites. The ability of each assay to correctly distinguish E. histolytica from E. dispar was evaluated with DNA extracted from patients' stools and liver aspirates submitted for confirmatory diagnosis. Real-time PCR allowed quantitative analysis of the spiked stool samples, but major differences in detection limits and assay performance were observed among the evaluated tests. These results illustrate the usefulness of comparative evaluations of diagnostic assays.