Jonathan I. Ravdin

Real-Time PCR for Detection and Differentiation of Entamoeba histolytica and Entamoeba dispar in Fecal Samples

ABSTRACT 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: an update

Purpose of review Over the past decade, since it was formally recognized that Entamoeba histolytica and Entamoeba dispar were two distinct species, studies in this field have made dramatic in-roads into the understanding of E. histolytica and the pathogenesis of invasive amoebiasis. Over the same period it has also become clear that the true incidence of E. histolytica infection, particularly in vulnerable populations such as low socioeconomic children, is exceedingly high. Understanding the epidemiology, pathophysiology, and the molecular and genetic biology of the organism will not only lead to improved diagnostic and treatment options but, ultimately, to the development of a safe and efficacious vaccine. Recent findings The recent advances in the genetic and molecular sciences have increased our understanding of the mechanisms that make E. histolytica unique among enteric protozoa in causing invasive disease. In addition, host factors, which predispose individuals or populations to infection or disease, are beginning to be elucidated. New diagnostic tools specific to E. histolytica are being exploited by clinicians and researchers to identify and treat patients as well as to add to the knowledge of the epidemiology and natural history of this infection. The ultimate goal - eradication of disease - is theoretically feasible since humans and primates are the only reservoirs of E. histolytica. Many talented and dedicated individuals are pursuing the development of an effective and safe amoebiasis vaccine. Summary E. histolytica remains an important pathogen in many populations of the world and although there has been substantial progress into understanding the disease major challenges still exist.

Diagnosis of amoebic colitis by antigen capture ELISA in patients presenting with acute diarrhoea in Cairo Egypt.

We studied 84 consecutive patients presenting with acute diarrhoea (less than 1 week in duration) at an outpatient tropical medicine clinic in Cairo, Egypt. The diagnosis of amoebic colitis was established by the presence of Entamoeba histolytica galactose‐inhibitable lectin antigen and the presence of occult blood in stool. Controls were 182 healthy regional people and 64 patients complaining of prolonged diarrhoea lasting more than 1 week. Entamoeba histolytica infection was found more frequently in patients with acute diarrhoea (57.1%) than in healthy controls (21.4%) or patients with prolonged diarrhoea (25%) (P < 0.001). There was a higher prevalence of Entamoeba dispar infection in the two control groups (24.2 and 20.3%, respectively, P=0.004 and 0.061) compared with those with acute diarrhoea (8.3%). Of the 84 patients with acute diarrhoea 32 had amoebic colitis (38%), and of these, 31 (97%) had at least one positive assay for serum amoebic antibodies (P < 0.001 compared with control groups). In summary, as determined by antigen‐detection enzyme‐linked immunosorbent assay, there is an unexpectedly high prevalence of amoebic colitis among patients presenting with acute diarrhoea to a tropical disease clinic in Cairo, Egypt.

Genotyping of Entamoeba Species in South Africa: Diversity, Stability, and Transmission Patterns within Families

Using a recently described polymerase chain reaction-based DNA typing method for Entamoeba histolytica and E. dispar, we investigated the genetic diversity of these species in a geographically restricted region of South Africa. Patterns were stable over time in the same infection, and, with few exceptions, infected family members carried the same strain. However, both species exhibited remarkable variation, with no 2 family groups being infected with the same strain of E. histolytica. Mixed infections were rare. The results indicate that this typing method will be useful in identifying epidemiological linkage between infections.

Intestinal Antilectin Immunoglobulin A Antibody Response and Immunity to Entamoeba dispar Infection following Cure of Amebic Liver Abscess

ABSTRACT We followed 93 subjects with amebic liver abscess (ALA) and 963 close associate controls at 3-month intervals for 36 months to characterize intestinal and humoral antibody responses to the amebic galactose-inhibitable lectin and to determine whether immunity developed to Entamoeba histolytica or Entamoeba dispar infection following cure of ALA. We found that ALA subjects had a higher prevalence and level of intestinal antilectin immunoglobulin A (IgA) and serum anti-LC3 (cysteine-rich recombinant lectin protein) IgA and IgG antibodies, P < 0.01 and P < 0.05, respectively, compared to controls. The intestinal antilectin IgA antibody response was sustained over a longer time period in ALA subjects (71.8% remained positive at 18 months and 52.6% at 36 months, P < 0.001 compared to 17.6% and 10.3% of controls, respectively). ALA subjects were highly immune to E. dispar infection throughout the study (0% infected at 6 and 36 months, compared to 6.5% and 4.9% of control subjects, respectively, P < 0.05). Upon entry into the study, 6.3% of ALA subjects were infected with E. histolytica; the incidence of new E. histolytica infections in controls (as determined by culture) was too low (1.4%) to determine whether ALA subjects exhibited immunity to new infections. We found that stool cultures every 3 months markedly underestimated the occurrence of new E. histolytica infections, as 15.3% of controls seroconverted after 12 months of follow-up. Unfortunately, under the field conditions present in Durban, South Africa, enzyme-linked immunosorbent assay for detection of lectin antigen in stool yielded unreliable results. In summary, subjects cured of ALA exhibited sustained mucosal IgA antibody responses to the amebic galactose-inhibitable lectin and a high level of immunity to E. dispar infection. Determination of immunity to E. histolytica following cure of ALA will require the use of more sensitive and reliable diagnostic methods.

Identification of the Entamoeba histolytica Galactose-Inhibitable Lectin Epitopes Recognized by Human Immunoglobulin A Antibodies following Cure of Amebic Liver Abscess

ABSTRACT Immunity to Entamoeba species intestinal infection is associated with the presence of intestinal IgA antibodies against the parasites galactose-inhibitable adherence lectin. We determined the epitope specificity of serum and intestinal antilectin IgA antibodies by enzyme-linked immunosorbent assay using overlapping fragments of a recombinant portion of the lectin heavy subunit, designated LC3. These findings were correlated with the effects of epitope-specific murine antilectin immunoglobulin A (IgA) monoclonal antibodies (MAbs) on amebic in vitro galactose-specific adherence. LC3 is a highly antigenic and immunogenic cysteine-rich protein (amino acids [aa] 758 to 1150) that includes the lectins carbohydrate binding domain. The study subjects, from Durban, South Africa, were recently cured of amebic liver abscess (ALA) with or without concurrent Entamoeba histolytica intestinal infection or were infection free 1 year after cure. We also studied seropositive subjects that were infected with E. histolytica, disease free, and asymptomatic. Serum anti-LC3 IgA antibodies from all study groups exclusively recognized the third (aa 868 to 944) and the seventh (aa 1114 to 1134) LC3 epitopes regardless of clinical status; epitope 6 (aa 1070 to 1114) was also recognized by serum anti-LC3 IgG antibodies. However, IgG antibody recognition of epitope 6 but not 3 or 7 was lost 1 year following cure of ALA. We produced 14 murine anti-LC3 IgA MAbs which collectively recognized five of the seven LC3 epitopes. The majority of the murine MAbs recognized the first epitope (aa 758 to 826), which was not recognized by human IgA antibodies. Interestingly, adherence of E. histolytica trophozoites to CHO cells was inhibited by MAbs against epitopes 1, 3, 4 (aa 944 to 987), and 6 (P < 0.01). The LC3 epitopes recognized by human IgA antibodies (3 and 7) were further characterized by use of overlapping synthetic peptides. We identified four peptides (aa 891 to 903, 918 to 936, 1114 to 1134, and 1128 to 1150) that in linear or cyclized form were recognized by pooled intestinal IgA antibodies and serum IgG antibodies from subjects with ALA and asymptomatic, seropositive infected subjects. This study identifies the lectin epitopes to be studied in an amebiasis subunit vaccine designed to elicit mucosal immunity mimicking that of humans cured of ALA.

Adherence-Inhibitory Intestinal Immunoglobulin A Antibody Response in Baboons Elicited by Use of a Synthetic Intranasal Lectin-Based Amebiasis Subunit Vaccine

ABSTRACT We designed an amebiasis subunit vaccine that is constructed by using four peptide epitopes of the galactose-inhibitable lectin heavy subunit that were recognized by intestinal secretory immunoglobulin A (IgA) antibodies from immune human subjects. These epitopes are contained in the region encompassing amino acids 758 to 1134 of the lectin heavy subunit, designated LC3. Baboons (Papio anubis) are natural hosts for Entamoeba histolytica; naturally infected baboons raised in captivity possess serum IgA antibodies to the same four LC3 epitopes as humans. Uninfected, seronegative baboons received four intranasal immunizations at 7-day intervals with the synthetic peptide vaccine (400, 800, or 1,600 μg per nostril) with cholera toxin (20 μg) as the adjuvant. As determined by an enzyme-linked immunosorbent assay (ELISA), each dose of the peptide vaccine elicited antipeptide serum IgA and IgG and intestinal IgA antibody responses in all six immunized baboons by day 28, 7 days after the last immunization (P, <0.01 for each dose compared to the cholera toxin control). The peptide vaccine elicited serum IgG and intestinal IgA antibodies that recognized purified recombinant LC3 protein (P, <0.008 and 0.02, respectively) and native lectin protein (P < 0.01). In addition, an indirect immunofluorescence assay with whole trophozoites (P < 0.01) and Western blot analysis confirmed that serum IgG antibodies from vaccinated baboons recognized native lectin protein on the surfaces of axenic E. histolytica trophozoites or from solubilized amebae. All four synthetic peptides were immunogenic; the vaccine elicited dose- and time-dependent responses, as determined by ELISA optical density readings indicating the production of serum and intestinal antibodies (P, <0.02 for antipeptide and antilectin antibodies). As a positive control, intranasal immunization with purified recombinant LC3 protein with cholera toxin as the adjuvant elicited a serum anti-LC3 IgA and IgG antibody response (P, 0.05 and <0.0001, respectively); however, no intestinal anti-LC3 IgA antibody response was observed (P = 0.4). Of interest, serum IgA and IgG antibodies elicited by the recombinant LC3 vaccine did not recognize any of the four putatively protective LC3 peptide epitopes. Both serum and fecal antibodies elicited by the peptide vaccine exhibited neutralizing activity, as determined by their dose-dependent inhibition of the galactose-specific adherence of E. histolytica trophozoites to Chinese hamster ovary cells in vitro (P, <0.001 for each group of antibodies compared to the control). In summary, a lectin-based intranasal polylysine-linked synthetic peptide vaccine was effective in eliciting an adherence-inhibitory, intestinal antilectin IgA antibody response in baboons. Future studies with the baboon model will determine vaccine efficacy against asymptomatic E. histolytica intestinal infection.

Mucosal Immunity to Asymptomatic Entamoeba histolytica and Entamoeba dispar Infection Is Associated with a Peak Intestinal Anti-Lectin Immunoglobulin A Antibody Response

ABSTRACT We monitored 93 subjects cured of amebic liver abscess (ALA) and 963 close associate controls in Durban, South Africa, and determined by enzyme-linked immunosorbent assay that the intestinal immunoglobulin A (IgA) antibody response to the Entamoeba histolytica galactose-inhibitable adherence lectin is most accurately represented by a complex pattern of transitory peaks. One or more intestinal anti-lectin IgA antibody peaks occurred in 85.9% of ALA subjects over 36 months compared to 41.6% of controls (P < 0.0001). ALA subjects exhibited a greater number of anti-lectin IgA antibody peaks (P < 0.0001) than controls. In addition, their peak optical density values were higher (peak numbers 1 to 3, P < 0.003), peaks were of longer duration (for peaks 1 and 2, P ≤ 0.0054), and there was a shorter time interval between peaks (between 1 and 2 or 2 and 3, P ≤ 0.0106) than observed for control subjects. A prior E. histolytica infection was associated with the occurrence of an anti-lectin IgA antibody peak (79.1%, P < 0.0001) more so than for Entamoeba dispar infection (57.2%, P < 0.001). The annual number of anti-lectin IgA antibody peaks in ALA subjects was 0.71 per year, compared to just 0.22 in controls (P<0.0001), indicating a higher rate of exposure to the parasite than previously appreciated. Anti-lectin IgA antibody peaks were of higher amplitude following a E. histolytica infection compared to E. dispar (P = 0.01) and, for either, were of greater height in ALA subjects than controls (P < 0.01). ALA subjects demonstrated greater clearance of amebic infection after an anti-lectin IgA antibody peak compared to controls, and only 14.3% remained with a positive culture after the peak, compared to 38.9% in controls (P = 0.035). In summary, this prospective controlled longitudinal study elucidated the dynamic nature of the human intestinal IgA antibody response to E. histolytica and E. dispar infection and revealed that ALA subjects exhibit heightened intestinal anti-lectin IgA antibody peaks that are associated with clearance of E. histolytica and E. dispar infection.

[35] Galactose-specific adhesion mechanisms of Entamoeba histolytica: Model for study of enteric pathogens

Publisher Summary This chapter discusses the evidence indicating that adhesion of Entamoeba histolytica is mediated by a galactose-inhibitable lectin and presents a method used to analyze the subunit structure of the adhesion protein. These investigations serve as a model to approach lectin–carbohydrate interactions of other pathogenic microorganisms. Adhesion to intestinal mucins, epithelium, and neutrophils is essential to the pathogenicity of E. histolytica . Initial efforts to characterize the nature of amebic adhesion focused on cell–cell interactions between amebic trophozoites and various target cells. The rosetting assay is described in the chapter in which Chinese hamster ovary (CHO) cells are bound by E. histolytica trophozoites and rosettes quantified in a hemacytometer chamber. Using the assay adhesion and contact-dependent cytolysis of CHO cells by trophozoites is shown to be completely blocked by millimolar concentrations of galactose and N -acetylgalactosamine (GalNAc), but not by other oligosaccharides, including N -acetylglucosamine, glucose, or mannose. In addition to adhesion, cell-contact dependent cytolysis has also been shown to be dependent on the galactose-inhibitable lectin using two different strategies. In the first, galactose or GalNAc is shown to inhibit amebic lysis of CHO cells completely. In the second approach, amebic binding and cytolysis of CHO cell glycosylation mutants are compared using cell-sorter methodology.

Natural Infection of Baboons by Entamoeba Histolytica Elicits Anti-Gal Lectin Heavy Subunit Iga and Igg Antibodies with Shared Epitope Specificity to That of Humans

Non-human primates, such as baboons (Papio hamadryas anubis), are natural hosts for Entamoeba species; infections can be asymptomatic or result in invasive lethal disease. It was sought to determine whether following natural infection by Entamoeba. histolytica, baboon anti-amebic antibodies recognized native Gallectin, a recombinant portion of the lectin heavy subunit (designated LC3) and specific heavy subunit epitopes; we compared the specificity of anti-amebic antibodies from baboons to that of humans following asymptomatic E. histolytica infection or cure of amebic liver abscess (ALA). Female baboons (n=54), aged one to three years of age and living in captivity were screened for infection by real time PCR. E. histolytica infection was found in 37 baboons and was associated with serum anti-LC3 IgG (73%) and anti-LC3 IgA (46%) or intestinal anti-Gal-Lectin IgA antibody responses (49%), p<0.021 for each compared to that observed with baboons having an E. dispar infection (n=10) or uninfected baboons (n=7). The ELISA OD reading for anti-LC3 or anti-lectin antibodies correlated strongly with the presence of a PCR CT value indicative of E. histolytica infection. In humans with asymptomatic E. histolytica infection or those recently cured of ALA, 63% and 57% had serum anti- LC3 IgA and 65% and 57% had serum anti-LC3 IgG antibodies respectively. Epitope- specific synthetic peptides were used as capture antigens in ELISA; for baboons that possessed anti-LC3 and anti-lectin antibodies, 74% had anti-peptide IgG or IgA antibodies, compared to 86% of asymptomatic humans and 92% of ALA subjects(P>0.05).