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Concept: Entamoeba histolytica

168

BACKGROUND: Six species of the genus Entamoeba, i.e., E. histolytica, E. dispar, E. moshkovskii, E. polecki, E. coli, and E. hartmanii can be found in human stools. Among these, only E. histolytica is considered to be pathogenic, causing intestinal and extra-intestinal disease, but it is morphologically identical to E. dispar and E. moshkovskii. In general, E. polecki, E. coli, and E. hartmanii can be differentiated morphologically from E. histolytica, but some of their diagnostic morphologic features may overlap creating issues for the differential diagnosis. Moreover, the previous inability to differentiate among Entamoeba species has limited epidemiologic information on E histolytica. The objective of this study was to develop a rapid, high-throughput screening method using Luminex technique for the simultaneous detection and differentiation of Entamoeba species. METHODS: PCR amplification was performed with biotinylated Entamoeba sp 18S rRNA gene primers, designed to amplify a fragment ranging from 382 to 429 bp of the Entamoeba spp studied. Regions of this fragment that could differentiate among E. histolytica, E. moshkovskii, E. dispar, E. hartmanii and E. coli were selected to design hybridization probes to link to Luminex beads. The assay was standardized with cloned DNA samples of each species and evaluated with 24 DNA extracts from samples obtained from individuals diagnosed with these amebas in their stools. RESULTS: Using this approach we were able to correctly identify E. histoltyica, E. dispar, E hartmanni, E. coli and E. moshkovskii in all specimens studied. From twenty four samples tested by microscopy, PCR/DNA Sequencing and real-time PCR, 100% agreed with PCR-Luminex assay for identification of E. dispar, E. moshkovskii, E. hartmanni, E. histolytica, and E. coli. CONCLUSION: These results show that this method could be used in the diagnostic detection of Entamoeba spp in fecal samples. This diagnostic test was useful to clearly distinguish E histolytica from other species and also to strengthen epidemiologic data on Entamoeba spp.

Concepts: Polymerase chain reaction, Biology, Entamoeba coli, Ribosomal RNA, Molecular biology, DNA, Entamoeba, Entamoeba histolytica

167

We have recently resurrected the name Entamoeba nuttalli Castellani, 1908 for a potentially virulent ameba isolate, P19-061405, obtained from a rhesus macaque in Kathmandu, Nepal. The ameba was morphologically indistinguishable from E. histolytica/E. dispar/E. moshkovskii, but located phylogenetically between E. histolytica and E. dispar. To evaluate the prevalence of E. nuttalli infection in wild rhesus macaques, 112 fecal samples were collected in four locations of the Kathmandu Valley. PCR analysis of DNA extracted from the feces showed positive rates of E. nuttalli, E. dispar, E. histolytica and E. moshkovskii of 51%, 12%, 0% and 0%, respectively. A total of 14 E. nuttalli isolates were obtained from four locations, of which 6 were established as axenic cultures. The sequences of the serine-rich protein gene of E. nuttalli isolates differed among four locations although no differences were found in the composition of sequence motifs. Isoenzyme pattern was analyzed in 8 isolates obtained from three locations. In hexokinase, the mobility of the slower migrating band was located between E. histolytica and E. dispar regardless of the culture conditions. These results demonstrate that E. nuttalli is high prevalent in wild rhesus macaques in Nepal. Rhesus macaques appear to be one of the natural hosts and heterogeneity of the serine-rich protein gene might be useful for geographical typing of isolates.

Concepts: DNA, Nepal, Entamoeba, Cercopithecine monkeys, Entamoeba histolytica, Primate, Macaque, Rhesus Macaque

167

In this study, Entamoeba histolytica had high prevalence and unusual presentation by affecting high proportion of infants under 1 year; severe clinical manifestations, and laboratory findings that were known to be usually encountered in invasive amebiasis as significant leukocytosis for age, neutrophilic leukocytosis for age, and positive C-reactive protein were found among more than 50% of admitted Saudi infants and children with E. histolytica infection in our locality. E. histolytica can be a re-emerging serious infection when it finds favorable environmental conditions and host factors which are mainly attributed to inadequate breastfeeding in this study. This may occur in any other area of the world with the same risk factors, so we must be ready to tackle it with effective and more powerful preventive measures.

Concepts: Medical statistics, Parasites, Amoebozoa, Dysentery, Amoebic dysentery, Amoebiasis, Entamoeba, Entamoeba histolytica

159

Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.

Concepts: Dysentery, Amoebiasis, Entamoeba, DNA, Amoebic dysentery, Amoebozoa, Liver abscess, Entamoeba histolytica

83

The disease severity of Entamoeba histolytica infection ranges from asymptomatic to life-threatening. Recent human and animal data implicate the gut microbiome as a modifier of E. histolytica virulence. Here we have explored the association of the microbiome with susceptibility to amebiasis in infants and in the mouse model of amebic colitis. Dysbiosis occurred symptomatic E. histolytica infection in children, as evidenced by a lower Shannon diversity index of the gut microbiota. To test if dysbiosis was a cause of susceptibility, wild type C57BL/6 mice (which are innately resistant to E. histiolytica infection) were treated with antibiotics prior to cecal challenge with E. histolytica. Compared with untreated mice, antibiotic pre-treated mice had more severe colitis and delayed clearance of E. histolytica. Gut IL-25 and mucus protein Muc2, both shown to provide innate immunity in the mouse model of amebic colitis, were lower in antibiotic pre-treated mice. Moreover, dysbiotic mice had fewer cecal neutrophils and myeloperoxidase activity. Paradoxically, the neutrophil chemoattractant chemokines CXCL1 and CXCL2, as well as IL-1β, were higher in the colon of mice with antibiotic-induced dysbiosis. Neutrophils from antibiotic pre-treated mice had diminished surface expression of the chemokine receptor CXCR2, potentially explaining their inability to migrate to the site of infection. Blockade of CXCR2 increased susceptibility of control non-antibiotic treated mice to amebiasis. In conclusion, dysbiosis increased the severity of amebic colitis due to decreased neutrophil recruitment to the gut, which was due in part to decreased surface expression on neutrophils of CXCR2.

Concepts: Shannon index, Innate immune system, Gut flora, Gene, Bacteria, Immune system, Entamoeba histolytica, Chemokine

41

The parasite Entamoeba histolytica kills human cells resulting in ulceration, inflammation and invasion of the colonic epithelium. We used the cytotoxic properties of ameba to select a genome-wide RNAi library to reveal novel host factors that control susceptibility to amebic killing. We identified 281 candidate susceptibility genes and bioinformatics analyses revealed that ion transporters were significantly enriched among susceptibility genes. Potassium (K(+)) channels were the most common transporter identified. Their importance was further supported by colon biopsy of humans with amebiasis that demonstrated suppressed K(+) channel expression. Inhibition of human K(+) channels by genetic silencing, pharmacologic inhibitors and with excess K(+) protected diverse cell types from E. histolytica-induced death. Contact with E. histolytica parasites triggered K(+) channel activation and K(+) efflux by intestinal epithelial cells, which preceded cell killing. Specific inhibition of Ca(2+)-dependent K(+) channels was highly effective in preventing amebic cytotoxicity in intestinal epithelial cells and macrophages. Blockade of K(+) efflux also inhibited caspase-1 activation, IL-1β secretion and pyroptotic death in THP-1 macrophages. We concluded that K(+) channels are host mediators of amebic cytotoxicity in multiple cells types and of inflammasome activation in macrophages.

Concepts: Chromosome, Human, DNA, Gene expression, Bacteria, Epithelium, Entamoeba histolytica, Cell

28

Protozoan parasites infect and kill millions of people worldwide every year, particularly in developing countries where access to clean fresh water is limited. Among the most common are intestinal parasites, including Giardia lamblia and Entamoeba histolytica. These parasites wreak havoc on the epithelium lining the small intestines (G. lamblia) and colon (E. histolytica) causing giardiasis and amebiasis, respectively. In addition, there are less common but far more deadly pathogens such as Naegleria fowleri that thrive in warm waters and infect the central nervous systems of their victims via the nasal passages. Despite their prevalence and associated high mortality rates, there remains an unmet need to identify more effective therapeutics for people infected with these opportunistic parasites. To address this unmet need, we have surveyed plants and traditional herbal medicines known throughout the world to identify novel antiparasitic agents with activity against G. lamblia, E. histolytica, and N. fowleri. Herein, we report Larrea tridentata, known as creosote bush, as a novel source for secondary metabolites that display antiparasitic activity against all three pathogens. This report also characterizes the lignan compound classes, nordihydroguairetic acid and demethoxyisoguaiacin, as novel antiparasitic lead agents to further develop more effective drug therapy options for millions of people worldwide.

Concepts: Large intestine, Central nervous system, Bacteria, Nervous system, Giardia lamblia, Protozoa, Entamoeba histolytica, Parasites

28

Amoebic liver abscess (ALA) is the most common extra intestinal manifestation of invasive amoebiasis caused by Entamoeba histolytica. The lack of early and accurate diagnostic test to differentiate various causes of liver abscess necessitates more reliable laboratory diagnostic test. The present study was conducted to assess the applicability of Loop-Mediated Isothermal Amplification (LAMP) assay for detection of E. histolytica in ALA cases. Fifty patients (n = 50) with clinical suspicion of ALA were enrolled in the study. All the clinical samples were subjected to conventional PCR assay. LAMP assay was standardized and the results were compared with that of PCR assay. Out of fifty pus samples thirty-six (72 %, 36/50) were positive for E. histolytica with conventional PCR assay and forty-one (82 %, 41/50) were positive by LAMP assay. Thus, five additional positive cases, missed by conventional PCR were positive with LAMP assay. Apart from rapidity, operational simplicity of LAMP assay high specificity and sensitivity, one-step amplification, higher yield and immediate visual detection may serve as a better diagnostic tool for diagnosis of ALA.

Concepts: Entamoeba, Diagnosis, Amoebic dysentery, Type I and type II errors, Amoebozoa, Liver abscess, Sensitivity and specificity, Entamoeba histolytica

28

The ability to detect intra-species polymorphism in Entamoeba histolytica and Entamoeba dispar is an important tool for studying geographic distribution and transmission echanisms. E. dispar and E. histolytica share the same mechanism for transmission among human hosts, and so after differentiation between these species we studied the intra-species variation and distribution of E. dispar strains obtained from cyst passers, specifically from African students and Tunisian food handlers. We analyzed the polymorphic region of the chitinase protein gene in 13 individuals infected with E. dispar, of which 9 were from Tunisia and 4 from other African countries. We identified 7 different chitinase patterns in Tunisians while the 4 isolates from other countries each had a distinct pattern. Two of the patterns we found have been reported in studies from Mexico and India, possibly indicating worldwide spread of certain strains.

Concepts: Entamoeba dispar, Africa, Parasites, Amoebozoa, Entamoeba, Entamoeba histolytica

28

Acetate kinase (ACK) catalyzes the reversible synthesis of acetyl phosphate by transfer of the γ-phosphate of ATP to acetate. Here we report the first biochemical and kinetic characterization of a eukaryotic ACK, that from the protist Entamoeba histolytica. Our characterization revealed that this protist ACK is the only known member of the ASKHA structural superfamily, which includes acetate kinase, hexokinase, and other sugar kinases, to utilize inorganic pyrophosphate (PP(i))/inorganic phosphate (P(i)) as the sole phosphoryl donor/acceptor. Detection of ACK activity in E. histolytica cell extracts in the direction of acetate/PP(i) formation but not in the direction of acetyl phosphate/P(i) formation suggests that the physiological direction of the reaction is toward acetate/PP(i) production. Kinetic parameters determined for each direction of the reaction are consistent with this observation. The E. histolytica PP(i)-forming ACK follows a sequential mechanism, supporting a direct in-line phosphoryl transfer mechanism as previously reported for the well-characterized Methanosarcina thermophila ATP-dependent ACK. Characterizations of enzyme variants altered in the putative acetate/acetyl phosphate binding pocket suggested that acetyl phosphate binding is not mediated solely through a hydrophobic interaction but also through the phosphoryl group, as for the M. thermophila ACK. However, there are key differences in the roles of certain active site residues between the two enzymes. The absence of known ACK partner enzymes raises the possibility that ACK is part of a novel pathway in Entamoeba.

Concepts: Amoebozoa, DNA, Pyrophosphate, Metabolism, Entamoeba histolytica, Phosphate, Enzyme, Adenosine triphosphate