High prevalence of infection with Entamoeba dispar, but not E. histolytica, in captive macaques

A total of 268 nonhuman primates (20 species) kept in the Primate Research Institute, Kyoto University, Japan, were surveyed for intestinal amebas. Total positive rates as based on the presence of cysts in the stool following formalin-ether sedimentation were as follows: Entamoeba histolytica/E. dispar, 53%; E. coli, 34%; E. hartmanni, 34%; Iodamoeba buetschlii, 25%; Endolimax nana, 8%; and E. chattoni, 3%. Positive rates were higher in Old World monkeys and lower in New World monkeys. All the 141 E. histolytica/E. dispar-positive animals were Macaca monkeys. The E. histolytica/E. dispar-positive samples were analyzed by polymerase chain reaction (PCR) for identification of E. histolytica and E. dispar. E. dispar DNA was detected in 137 samples, whereas no E. histolytica DNA was seen. Zymodeme analysis and reactivity to monoclonal antibodies of cultured trophozoites also supported the presence of E. dispar and the absence of E. histolytica. When the sera of 93 macaques were examined by an indirect fluorescent antibody test, only 3 animals proved to be positive for E. histolytica, showing the lowest titer. These results demonstrate that infection with E. dispar, but not E. histolytica, is predominant in macaques. Received: 23 March 2000 / Accepted: 3 July 2000     

Entamoeba histolytica and Entamoeba dispar infections in cynomolgus monkeys imported into Japan for research

Three hundred and three stool samples of cynomolgus monkeys (Macaca fascicularis) imported from China and the Philippines were examined for Entamoeba histolytica/Entamoeba dispar infections. Microscopy detected E. histolytica/E. dispar cysts in 41 samples. Positive rates were higher in the monkeys from China (37.5%) than in the monkeys from the Philippines (3.7%). PCR analysis of 25 samples successfully cultured from the cysts demonstrated that 24 were E. dispar, one of the samples from China was E. histolytica. The one sample was also identified as E. histolytica by an antigen detection kit, although the monkey was asymptomatic and serology was negative. To our knowledge, this is the first report of E. histolytica isolation from cynomolgus monkeys based on the discrimination between E. histolytica and E. dispar.     

Preparation of a monoclonal antibody specific for Entamoeba dispar and its ability to distinguish E. dispar from E. histolytica.

A monoclonal antibody (MAb), MAb ED17 (immunoglobulin G2a [IgG2a]), prepared against trophozoites of Entamoeba dispar SAW1734RclAR cultured monoxenically with Crithidia fasciculata, reacted with 25 of 26 isolates of E. dispar by an indirect fluorescent-antibody test. In contrast, the MAb failed to react with any of 20 isolates of E. histolytica or other enteric protozoan parasites. Western blot (immunoblot) analysis showed that the molecular mass of the E. dispar antigen recognized by the MAb was 160 kDa under reduced conditions. Immunoelectron microscopy revealed that the antigen was mainly located on digested C. fasciculata, but not on undigested organisms. Double staining with a mixture of MAb ED17 and MAb 4G6 (an IgG1 MAb which reacts exclusively with E. histolytica), followed by incubation with a mixture of fluorescein isothiocyanate-labeled anti-mouse IgG2a and tetramethylrhodamine isothiocyanate-labeled anti-mouse IgG1 antibodies, simultaneously identified mixed populations of E. dispar and E. histolytica. This method may prove to be useful for the accurate identification of E. dispar and E. histolytica, even in mixed infections.     

DNA sequences corresponding to the ariel gene family of Entamoeba histolytica are not present in E. dispar

For the identification of quantitative genetic differences between pathogenic Entamoeba histolytica and the closely related but nonpathogenic species E. dispar, a set of 68 independent probes that had previously been isolated from an E. histolytica cDNA library were hybridized to total genomic DNA of both amoeba species. Besides ehcp5, the sequence that codes for cysteine proteinase 5 and has recently been shown to be missing in E. dispar, only one of the probes exclusively reacted with E. histolytica DNA, whereas the remainder hybridized to DNA of both species. Sequence analysis revealed that the specific probe represents a copy of the multicopy ariel gene family, which has 80% sequence identity to srehp, the gene encoding a serine-rich E. histolytica membrane protein. In contrast to ariel, srehp is present in both amoeba species, suggesting that the ariel gene product might have a particular function in E. histolytica. Received: 1 February 1999 / Accepted: 16 February 1999     

HLA characterization in adult asymptomatic cyst passers of Entamoeba histolytica/E. dispar

The present work aimed at studying the possible association of HLA antigens with Entamoeba histolytica/E. dispar asymptomatic infection in a Mexican mestizo population. A case-control design was selected for evaluation of the role of genetic markers in parasite infection. For this purpose the HLA-A, HLA-B, and HLA-DR profiles of a population of asymptomatic E. histolytica/E. dispar adult cyst passers (cases) and a corresponding nonparasitized adult group (controls) followed for 12 months were identified. Entamoeba species were identified through zymodeme patterns and/or amplification of species-specific DNA sequences. A healthy, nonparasitized group of individuals was included as a control. Our results show that apparently, no specific HLA marker is associated with the asymptomatic cyst passers' condition. These findings have to be added to previous results in which, in contrast to a demonstrated association between HLA-DR3 and amebic liver abscess in Mexican mestizo adults and infants, no significant association with amebic rectocolitis was found. Received: 18 March 1999 / Accepted: 16 April 1999     

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.     

Identification of Entamoeba histolytica and E. dispar cysts in stool by polymerase chain reaction

 An attempt to identify cysts of Entamoeba histolytica and E. dispar in human stool was conducted by polymerase chain reaction (PCR) using two sets of primers (p11 plus p12 and p13 plus p14) specific for either species of ameba. The cysts in stool specimens obtained from 12 infected individuals were concentrated, freeze-thawed, and treated with Triton X-100 before their examination by PCR. The results of PCR on the cysts were generally consistent with data obtained by PCR on ameba trophozoites hatched from the cysts, by zymodeme analysis, and by enzyme-linked immunosorbent assay (ELISA) and with clinical findings. This PCR was negative for the stool containing large numbers of cysts of either E. coli, E. hartmanni, or Giardia lamblia as well as for the stool specimens obtained from uninfected individuals. The ameba cyst in stool processed using the present method was effective for the PCR analysis even after 1 month of storage at 4°C. The present PCR was sensitive enough to detect ten cysts of either of the amebae. Received: 20 February 1996 / Accepted: 16 July 1996     

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.     

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.     

Differences in cap formation between invasive Entamoeba histolytica and non-invasive Entamoeba dispar

The rapid redistribution of surface antigen-antibody complexes in trophozoites of the human protozoan parasite Entamoeba histolytica, in a process known as capping, has been considered as a means of the parasite to evade the host immune response. So far, capping has been documented in the invasive E. histolytica, whereas the mobility of surface components in the non-invasive Entamoeba dispar is not known. E. dispar does not induce liver lesions in rodent experimental models, in contrast to the liver abscesses produced by E. histolytica in the same animal model. We have therefore analyzed the mobility of surface receptors to the lectin concanavalin A and of Rab11, a membrane-associated protein, in both species of Entamoebae by confocal fluorescence microscopy and transmission and scanning electron microscopy. The great majority of E. histolytica trophozoites became morphologically polarized through the formation of well-defined caps at the posterior pole of the parasite. Actin colocalized with the lectin caps. Antibodies against the membrane protein Rab 11 also produced capping. In striking contrast, in E. dispar, the mobility of concanavalin A surface receptors was restricted to the formation of irregular surface patches that did no progress to constitute well-defined caps. Also, anti-Rab 11 antibodies did not result in capping in E. dispar. Whether the failure of E. dispar to efficiently mobilize surface molecules in response to lectin or antibodies as shown in the present results is related to its non-invasive character represents an interesting hypothesis requiring further analysis.     

The distribution of Entamoeba histolytica and Entamoeba dispar in northern, central, and southern Iran

The present study was carried out from August 1999 through February 2002 in order to determine the prevalence of Entamoeba histolytica and Entamoeba dispar in three different climatic regions of Iran by using a PCR-RFLP method. A total of 16,592 stool samples were randomly collected from different age-groups in central, northern, and southern Iran in both urban and rural areas. The samples were examined by direct and formalin-ether concentration methods. A total of 226 samples were positive for E. histolytica/E. dispar cysts. Of these, 101 isolates were cultured and maintained successfully in Robinsons medium and were identified by the PCR-RFLP method. The study showed that 92.1% of isolates were E. dispar and 7.9% were E. histolytica or mixed infections. The ratio of E. histolytica to E. dispar was higher in southern regions (tropical and subtropical) than in the other two regions. This study demonstrated that E. dispar is the predominant species found among cyst passers in Iran.     

Differentiation of Entamoeba histolytica from E. dispar facilitated by monoclonal antibodies against a 150-kDa surface antigen

Murine monoclonal antibodies (mAbs) were produced against an n-octyl-β-D-glucopyranoside- extracted fraction of trophozoites of Entamoeba histolytica HM-1:IMSS. Four of the mAbs were reactive with a 150-kDa surface antigen characterized by Western-immunoblot analysis under nonreducing conditions. When the reactivity of the four mAbs with nine reference strains of E. histolytica was examined by an indirect fluorescence antibody test, two of the mAbs (EH3015 and EH3023) were found to react with all nine strains and the other two mAbs (EH3056 and EH3126) reacted with seven strains. The four mAbs did not react with any E. dispar reference strain or with other enteric protozoan parasites. The reactivity of EH3015 and EH3023 with numerous isolates of E. histolytica and E. dispar collected in our laboratories was also examined. The 2 mAbs reacted with all of the 37 E. histolytica isolates tested but did not react with any of the 33 isolates of E. dispar. These results indicate that common antigenic epitopes of E. histolytica are on the 150-kDa surface molecule and that mAbs can distinguish between E. histolytica and E. dispar. Received: 12 October 1996 / Accepted 13 December 1996     

Altered lipid parameters in patients infected with Entamoeba histolytica, Entamoeba dispar and Giardia lamblia

Information on the effect of parasitic infections on lipid parameters is scarce. Certain parasites induce significant changes in lipid parameters, as demonstrated by the fact that substitution of lipid/cholesterol for serum in axenic culture medium (in vitro) and in experimental models (in vivo) supports vigorous growth of Entamoeba histolytica. Thus, significant changes in lipid parameters may be induced in an infected host. Blood samples are obtained from intestinal amoebiasis patients passing E. histolytica (n=8), E. dispar (n=15) or Giardia lamblia (n=9) cysts, or diagnosed with amoebic liver abscess (ALA; n=50) and from apparently normal healthy individuals (control group; n=30). Levels of total serum cholesterol, high-density lipoprotein and low-density lipoprotein are assessed using commercial kits. E. histolytica and E. dispar isolates are differentiated by hexokinase isoenzyme electrophoresis and by enzyme-linked immunosorbent assay (ELISA; Techlab) tests. Results show that E. histolytica, E. dispar and G. lamblia cyst passers had significantly lower levels of total serum cholesterol (73.42 +/- 2.24 mg/dL), compared to levels in ALA cases (101 +/- 2.85 mg/dL) and in controls (166.26 +/- 2.02 mg/dL). Further study of a greater number of cases is needed to explore the relevance of this finding.     

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.     

Entamoeba histolytica and Entamoeba dispar utilize externalized phosphatidylserine for recognition and phagocytosis of erythrocytes

Amebic erythrophagocytosis is characteristic of invasive amebiasis, and mutants deficient in erythrocyte ingestion are avirulent. We sought to understand the molecular mechanisms underlying erythrocyte phagocytosis by Entamoeba histolytica. Following adherence to amebae, erythrocytes became round and crenulated, and phosphatidylserine (PS) was exposed on their outer membrane leaflets. These changes were similar to the effects of calcium treatment on erythrocytes, which we utilized to separate ameba-induced exposure of erythrocyte PS from the process of phagocytosis. The adherence and phagocytosis of calcium-treated erythrocytes were less inhibited by galactose than were those of healthy erythrocytes, suggesting the existence of an amebic coreceptor specific for PS. To test whether PS was recognized by amebae, calcium-treated cells were incubated with annexin V prior to adherence to or ingestion by E. histolytica. Annexin V blocked both adherence (50% +/- 12% inhibition; P < 0.05) and phagocytosis (65% +/- 10%; P < 0.05), providing evidence that at least one galactose-independent coreceptor was involved in the adherence and ingestion of red blood cells. The coreceptor was inhibited by phospho-l-serine and to a lesser extent by phospho-d-serine but not by phospho-l-threonine, which is consistent with the coreceptor functioning in the adherence and ingestion of erythrocytes via recognition of PS. We expanded our investigations to the highly related but noninvasive parasite Entamoeba dispar and demonstrated that it was deficient in red-blood-cell adherence, induction of PS exposure, and phagocytosis. These findings establish phosphatidylserine involvement in erythrophagocytosis by amebae and suggest the existence of a PS receptor on the surfaces of both E. histolytica and E. dispar.     

An improved colorimetric PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in feces.

The epidemiological implications of the recent separation of "Entamoeba histolytica" into two separate species, pathogenic E. histolytica sensu stricto and commensal E. dispar, will not become apparent without methods of distinguishing between them which are applicable to large numbers of specimens. We have modified a PCR-based method to produce such a technique which may be completed in 1 day while still identifying 10(-1) E. histolytica and 1 to 10 E. dispar trophozoites per g of feces when present separately and 10 E. histolytica and 100 E. dispar trophozoites per g in the presence of 10(6) trophozoites per g of the other species. Applied to fecal specimens from 18 patients from which E. histolytica or E. dispar had been grown and identified to the species level by hexokinase isoenzyme analysis, the method in every case yielded the correct result. Positive and negative results are easily distinguished by eye, and we are now applying this technique to a large-scale epidemiological study of amebiasis in the eastern Mediterranean region.     

Detection and genotyping of Entamoeba histolytica, Entamoeba dispar, Giardia lamblia, and Cryptosporidium parvum by oligonucleotide microarray

Entamoeba histolytica, Giardia lamblia, and Cryptosporidium parvum are the most frequently identified protozoan parasites causing waterborne disease outbreaks. The morbidity and mortality associated with these intestinal parasitic infections warrant the development of rapid and accurate detection and genotyping methods to aid public health efforts aimed at preventing and controlling outbreaks. In this study, we describe the development of an oligonucleotide microarray capable of detecting and discriminating between E. histolytica, Entamoeba dispar, G. lamblia assemblages A and B, and C. parvum types 1 and 2 in a single assay. Unique hybridization patterns for each selected protozoan were generated by amplifying six to eight diagnostic sequences/organism by multiplex PCR; fluorescent labeling of the amplicons via primer extension; and subsequent hybridization to a set of genus-, species-, and subtype-specific covalently immobilized oligonucleotide probes. The profile-based specificity of this methodology not only permitted for the unequivocal identification of the six targeted species and subtypes, but also demonstrated its potential in identifying related species such as Cryptosporidium meleagridis and Cryptosporidium muris. In addition, sensitivity assays demonstrated lower detection limits of five trophozoites of G. lamblia. Taken together, the specificity and sensitivity of the microarray-based approach suggest that this methodology may provide a promising tool to detect and genotype protozoa from clinical and environmental samples.     

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.     

Seropositivity for and Intestinal Colonization with Entamoeba histolytica and Entamoeba dispar in Individuals in Northeastern Brazil

In a slum community in northeastern Brazil 20% of a sample population was colonized with Entamoeba histolytica or Entamoeba dispar and 10.6% was colonized with E. histolytica alone. No correlation between seropositivity for anti-GalNAc lectin antibody and colonization was found. These results suggest that colonization does not necessarily produce immunity to reinfection.     

A new method for isolation and differentiation of native Entamoeba histolytica and E. dispar cysts from fecal samples

A new method for the purification of protozoan cysts from feces was established, allowing the isolation of native cysts. The procedure consists of two sucrose-density gradients and enzymatic digestion of cellulose particles by cellulase and can be accomplished in a few hours. The cyst fractions were differentiated into Entamoeba histolytica and E. dispar using the DNA probes P145 and B133 in a dot-blot test. Received: 7 March 1997 / Accepted: 24 March 1997