The Monomeric GTPase Rab35 Regulates Phagocytic Cup Formation and Phagosomal Maturation in Entamoeba histolytica.

One of the hallmarks of amoebic colitis is the detection of Entamoeba histolytica (Eh) trophozoites with ingested erythrocytes. Therefore, erythrophagocytosis is traditionally considered as one of the most important criteria to identify the pathogenic behavior of the amoebic trophozoites. Phagocytosis is an essential process for the proliferation and virulence of this parasite. Phagocytic cargo, upon internalization, follows a defined trafficking route to amoebic lysosomal degradation machinery. Here, we demonstrated the role of EhRab35 in the early and late phases of erythrophagocytosis by the amoeba. EhRab35 showed large vacuolar as well as punctate vesicular localization. The spatiotemporal dynamics of vacuolar EhRab35 and its exchange with soluble cytosolic pool were monitored by fluorescence recovery after photobleaching experiments. Using extensive microscopy and biochemical methods, we demonstrated that upon incubation with RBCs EhRab35 is recruited to the site of phagocytic cups as well as to the nascent phagosomes that harbor Gal/GalNAc lectin and actin. Overexpression of a dominant negative mutant of EhRab35 reduced phagocytic cup formation and thereby reduced RBC internalization, suggesting a potential role of the Rab GTPase in the cup formation. Furthermore, we also performed a phagosomal maturation assay and observed that the activated form of EhRab35 significantly increased the rate of RBC degradation. Interestingly, this mutant also significantly enhanced the number of acidic compartments in the trophozoites. Taken together, our results suggest that EhRab35 is involved in the initial stage of phagocytosis as well as in the phagolysosomal biogenesis in E. histolytica and thus contributes to the pathogenicity of the parasite.

A case of amebiasis with negative serologic markers that caused intra-abdominal abscess.

A 23-year-old Japanese woman presented with abdominal distention following fever, diarrhea, and abdominal pain during a stay in Taiwan. Serology for the detection of amebic-antibodies and stool microscopic examination were both negative. A computed tomography scan showed a 13 cm diameter abscess spreading from the lower abdominal wall to the pelvic retroperitoneal space. Needle aspiration of the abscess was done under computed tomography guidance, and microscopy of the aspirated fluid revealed trophozoites of Entamoeba. The patient was diagnosed as amebiasis with negative serologic markers that caused intra-abdominal abscess. Intravenous metronidazole treatment for two weeks did not result in any improvement of the abscess. After irrigation and drainage of the abscess, her symptoms resolved. This case report highlights that amebiasis should be considered when indicated by patient history, including travelers returning from endemic areas, and that further evaluation is necessary for diagnosis, even if the serology and stool test are negative.

Efficacy of aspiration in amebic liver abscess.

BACKGROUND: Amebic liver abscess (ALA) is a common and serious problem in our country. There are only a few controlled trials on the efficacy and advantages of combination therapy with percutaneous needle aspiration and pharmacotherapy, over pharmacotherapy alone for amebic liver abscess. MATERIAL AND METHODS: This study was conducted to compare the efficacy of two different treatment modalities i.e. drug treatment alone vs. drug treatment and aspiration of abscess cavity in patients with small (up to 5 cm) and large (5 cm to 10 cm) size ALA. This is one of the largest single center, prospective, randomized studies comparing the efficacy of aspiration in ALA. RESULTS: (i) Mean body temperature, liver tenderness, total leukocyte count (TLC), serum alanine aminotransferase (ALT) and liver span were significantly decreased in the aspiration group on days 8 and 15 as compared to non-aspiration group especially in large abscess (5 cm to 10 cm). (ii) Abscess cavity maximum diameter decreased significantly in aspiration group on days 8 and 15, and 1 month & 3 months in large abscess (5cm to 10 cm). CONCLUSIONS: (i) Needle aspiration along with metronidazole hastens clinical improvement especially in large (5 cm up to 10 cm) cavities in patients with ALA. (ii) Aspiration is safe and no major complications occurred. (iii) Hence, combination therapy should be the first choice especially in large ALA (5 cm to 10 cm).

Prevalence of intestinal parasites and molecular characterization of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica in the village of Fortín Mbororé (Puerto Iguazú, Misiones, Argentina).

BACKGROUND: Intestinal parasites (IPs) are widely distributed worldwide and are one of the major contributors to gastrointestinal disease. Their prevalence is associated with poor access to water, sanitation and hygiene (WASH). The objective of this study was to identify the prevalence of IPs, including soil-transmitted helminths (STH), and their relation to socioeconomic characteristics, as well as a first approach to molecularly characterize the types of Giardia intestinalis, Blastocystis spp. and Entamoeba histolytica present in an indigenous community from Puerto Iguazú, Misiones, Argentina. METHODS: A cross-sectional study was conducted in the rural settlement of Fortin Mbororé between January and March 2018. Socioeconomic variables, household characteristics, and stool and blood samples were collected. Standard coprological techniques were used to analyze stool samples, and a complete hemogram was performed on the blood samples. Giardia intestinalis microscopy-positive samples were genetically typed by the ß-giardin (bg) gene. Molecular identification of Blastocystis spp. subtypes and E. histolytica were carried out by amplification and sequencing of a partial fragment of the small subunit ribosomal RNA gene (SSU rDNA). RESULTS: The overall prevalence of IPs was 92.7%, with 72.0% specifically for hookworm. IPs were significantly more prevalent in preschool- and school-age children (P < 0.05). No formal education (P = 0.035), the presence of unimproved floors (P = 0.001) and overcrowding (P = 0.005) were significantly associated with IP infection. Hookworm was associated with anemia (P = 0.019). Molecular characterization revealed the presence of E. histolytica sub-assemblages AII (12.5%), AIII (87.5%) and BIV (100%); one case of sub-assemblage D for G. intestinalis; and the presence of subtypes ST1 (14.8%), ST2 (14.8%) and ST3 (70.4%) of Blastocystis spp. CONCLUSIONS: Protozoans detected in this study are transmitted mainly through water contaminated with fecal matter, evidencing the need to improve the quality of water and sanitation for the inhabitants of Fortín Mbororé. Molecular characterization showed that domestic animals can be implicated in the zoonotic transmission of G. intestinalis and Blastocystis spp. to humans. A hyperendemic area for STH was found, with hookworm prevalence greater than 50%. Therefore, improvements in WASH as well as mass deworming programs need to be implemented in this area to control and decrease the prevalence of IPs in general and STH in particular.

Application of real-time polymerase chain reaction in detection of Entamoeba histolytica in pus aspirates of liver abscess patients.

Entamoeba histolytica is the second major cause of liver abscess disease in humans, particularly in developing countries. Recently, DNA molecular-based methods have been employed to enhance the detection of E. histolytica in either pus or stool specimens. In this study, the results of real-time polymerase chain reaction (PCR) to detect E. histolytica DNA in pus from liver abscess cases were compared with those of indirect hemagglutination assay on the corresponding serum samples. Bacterial cultures were also performed on the pus samples for the diagnosis of pyogenic liver abscess. The real-time PCR detected E. histolytica DNA in 23 of 30 (76.7%) pus samples, when compared with 14 of 30 (46.7%) serum samples in which anti-Entamoeba antibodies were detected by indirect hemagglutination assay and 4 of 30 (13.3%) pus samples that showed bacterial infection by culture. The use of real-time PCR is a promising detection method for diagnosis and epidemiology assessment of amoebic liver abscess.

Seroprevalence of antibodies against Chlamydia trachomatis and enteropathogens and distance to the nearest water source among young children in the Amhara Region of Ethiopia.

The transmission of trachoma, caused by repeat infections with Chlamydia trachomatis, and many enteropathogens are linked to water quantity. We hypothesized that children living further from a water source would have higher exposure to C. trachomatis and enteric pathogens as determined by antibody responses. We used a multiplex bead assay to measure IgG antibody responses to C. trachomatis, Giardia intestinalis, Cryptosporidium parvum, Entamoeba histolytica, Salmonella enterica, Campylobacter jejuni, enterotoxigenic Escherichia coli (ETEC) and Vibrio cholerae in eluted dried blood spots collected from 2267 children ages 0-9 years in 40 communities in rural Ethiopia in 2016. Linear distance from the child's house to the nearest water source was calculated. We derived seroprevalence cutoffs using external negative control populations, if available, or by fitting finite mixture models. We used targeted maximum likelihood estimation to estimate differences in seroprevalence according to distance to the nearest water source. Seroprevalence among 1-9-year-olds was 43% for C. trachomatis, 28% for S. enterica, 70% for E. histolytica, 54% for G. intestinalis, 96% for C. jejuni, 76% for ETEC and 94% for C. parvum. Seroprevalence increased with age for all pathogens. Median distance to the nearest water source was 473 meters (IQR 268, 719). Children living furthest from a water source had a 12% (95% CI: 2.6, 21.6) higher seroprevalence of S. enterica and a 12.7% (95% CI: 2.9, 22.6) higher seroprevalence of G. intestinalis compared to children living nearest. Seroprevalence for C. trachomatis and enteropathogens was high, with marked increases for most enteropathogens in the first two years of life. Children living further from a water source had higher seroprevalence of S. enterica and G. intestinalis indicating that improving access to water in the Ethiopia's Amhara region may reduce exposure to these enteropathogens in young children.

Correlation of interferon-gamma production by peripheral blood mononuclear cells with childhood malnutrition and susceptibility to amebiasis.

The contribution of interferon-gamma (IFN-gamma) to immunity from amebiasis was assessed in a three-year prospective study of children 2-5 years of age in an urban slum of Dhaka, Bangladesh. IFN-gamma produced by peripheral blood mononuclear cells stimulated with soluble amebic antigen was measured upon enrollment. Thirty-one of the 209 enrolled children had Entamoeba histolytica-associated diarrhea. Children who produced higher than the median level of IFN-gamma (median = 580 pg/mL) had longer survival without E. histolytica diarrhea/dysentery (log rank test P = 0.03) and a reduction in the risk of E. histolytica diarrhea/dysentery by more than half (Cox proportional hazard regression = 0.45, P = 0.04). When adjusted for stunting, the association between IFN-gamma and the time to the first episode of E. histolytica-associated diarrhea remained marginally significant (Cox proportional hazard regression = 0.49, P = 0.07). We conclude that production of IFN-gamma is linked to nutritional status and predicts future susceptibility to symptomatic amebiasis.

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.

Identification of a parasite-specific cytoplasmic 67 kDa Entamoeba histolytica antigen recognized by patient sera and monoclonal antibodies.

To avoid false positive reactions in tests for anti-ameba antibodies, we wanted to identify parasite-specific component(s). Amebiasis patient sera recognized an antigen of 67 kDa by immunoblotting in an active E. histolytica fraction obtained by ion exchange chromatography. Monoclonal antibodies against the fraction were made. Antibody 3G2 reacted with three antigenic components of 67, 40 and 25 kDa and in the immunocytology with an epitope located in the cytoplasm of E. histolytica trophozoites. ELISAs using the isolated parasite fraction and monoclonal antibody 3G2 (to assay inhibition of binding) were capable of distinguishing specific reactivity in sera from amebiasis patients.

Amebiasis in Nicaragua: class specific serum antibody responses.

With the aid of the indirect hemagglutination (IHA) test and IgG ELISA the antibody profile against E. histolytica in León, Nicaragua was investigated in 562 sera from individuals belonging to various age groups. The highest reactivity was invariably recorded in the age group 6-15 years where 48% were seropositive. Several sera reactive by either one of IHA and IgG ELISA were negative by the other test. The main reason for this seems to be reactivity in different Ig classes. Treatment with 2-mercaptoethanol reduced the titre level in 63 of the 66 sera tested. Immunofluorescence using an anti-IgM conjugate showed that 26 of 43 sera contained specific IgM-antibodies, indicating that also unspecific reactions are involved in the IHA test. A comparison was made between class-specific reactivity in three population groups: healthy residents, healthy cyst carriers and patients with recent or acute liver abscess. No significant difference in the prevalence of reactions above the diagnostic significance level was recorded between cyst carriers and healthy residents. However, among the cyst carriers 33% had IgA and/or IgM antibodies but no demonstrable specific IgG. Most patients with recent and all with acute liver abscess reacted significantly above the diagnostic limit in all three tests.

Recombinant expression, purification and biochemical characterization of a superoxide dismutase from Entamoeba histolytica.

A recombinant iron-containing superoxide dismutase (recFeSOD) of Entamoeba histolytica was produced in a prokaryotic expression system. Purified recFeSOD was found to be enzymatically active as determined by (i) inhibition of ferri-cytochrome c reduction, (ii) dismutation of superoxide anions generated by human neutrophils and (iii) inhibition of nitroblue tetrazolium reduction. The enzymatic properties of recFeSOD were similar to those of the native protein in trophozoite extracts. In an ELISA using recFeSOD as antigen, 96% of sera from patients having invasive amebiasis were reactive whereas none of the healthy controls or of patients suffering from malaria, bacterial or viral infections were reactive. Only sera of Toxoplasma-, Leishmania- or Trypanosoma-infected individuals exhibited partial cross-reactivity to recFeSOD.

Comparative evaluation of somatic & excretory-secretory antigens of Entamoeba histolytica in serodiagnosis of human amoebiasis by ELISA.

The excretory-secretory antigens collected from the axenic culture medium and conventional somatic antigen prepared from the whole trophozoites of E. histolytica were compared for their efficacy in serodiagnosis of amoebiasis. A total of 280 sera collected from different clinically proven cases of amoebiasis and healthy subjects were analysed against both the antigens in ELISA. Both antigens showed a 100 per cent correlation in amoebic liver abscess cases, patients infected with enteropathogens other than amoeba and healthy subjects. However, excretory-secretory antigens showed slightly higher detection rate in patients suffering from acute amoebic dysentery and asymptomatic cyst passers groups. These results clearly suggested the use of excretory-secretory antigens by ELISA for serodiagnosis of amoebiasis due to its better or equal sensitivity, specificity and easier preparation compare to conventional antigen. The use of excretory-secretory antigen in serodiagnosis will not only help in performing more tests utilizing the same chemicals, but also save the cost, time and troubles for importing the foreign chemicals required for cultivation of E. histolytica.

A study of serum antibody levels against Entamoeba histolytica in patients with different amoebic zymodemes.

The present study has looked into relative agreement between Clinical diagnosis, Indirect fluorescent Antibody Test and Iso-enzyme grouping in 35 patients with E histolytica infection. Thirty Caucasians, who had never travelled to Tropical Countries were chosen as negative control subjects. There was no remarkable difference in the mean amoebic antibody levels observed with the control subjects and patients harbouring amoebic zymodeme I or III or IV. Whereas, comparative analysis revealed a significant difference (P less than 0.01) between the mean reciprocal antibody titres observed with patients harbouring Isoenzyme group II amoebae and control subjects. The hypothesis that invasive amoebiasis and infection with E. histolytica zymodeme II were positively related was supported IFAT was found more sensitive than isoenzyme grouping in the detection of invasive amoebiasis, but the latter method appeared more reliable in differentiating dysenteric from asymptomatic intestinal amoebiasis.

[3 cases of hematogenous lung abscess of amebic origin]. / Tres casos de absceso pulmonar hematógeno de origen amibiano.

Invasive amebiasis is a very serious health problem in Mexico as it is presumably related to the presence of virulent strains of Entamoeba histolytica and poor hygienic and sanitary conditions; other factors related to invasive amebiasis are undernutrition, alcoholism, and homosexuality. We present three patients with pulmonary amebic hematogenous abscess. Clinically all patients had the typical "chocolate" exudate. The three patients had pulmonary consolidations by chest roentgenogram; one of them had multiple opacities with air fluid level, and the others had an isolated opacity with air fluid level. The ultrasound and hepatogammagram were negative for diaphragmatic communication in all; in one of them the pneumoperitoneum was negative for diaphragmatic communication. The transthoracic needle biopsy of the lesions was positive to ameba in two patients. The serologic tests were positive in all. We treated the patients with metronidazole and emetine during 10 days; since the clinical picture and the radiologic findings did not remit completely, we gave a second course of metronidazole during 10 days more and achieved complete resolution.

Regulation of serum-responsive transmembrane kinase EhTMKB1-9 by an unsaturated lipid, oleic acid in protistan parasite Entamoeba histolytica.

Transmembrane kinases of Entamoeba histolytica are known to play a wide range of roles from virulence, phagocytosis, and proliferation to stress response. Transmembrane kinase EhTMKB1-9 is thought to be involved in early proliferative response and it was originally identified as a serum inducible gene. Ability to stimulate EhTMKB1 expression of serum starved cells resides in unsaturated fatty acids associated with albumin fraction of serum and the mechanism of stimulation follows activation of EhTMKB1-9 promoter. Gel shift assay showed the presence of proteins that bind to the specific site of EhTMKB1-9 upstream region and the concentration of these protein(s) go down on serum starvation, but level of binding protein(s) go up on serum or fatty acid replenishment. This increase in concentration of binding molecule(s) is due to new synthesis rather than activation of existing molecule(s) as a protein synthesis inhibitor blocked enhanced level of gel shifted material on replenishment. The stimulating activity resides in the fatty acyl chain, but not in the head group. Moreover, the fatty acid initiates signaling through class I PI3 kinases that result in activation of EhTMKB1-9 expression. These results suggest a novel mechanism of gene regulation in E. histolytica, and unsaturated fatty acids as potential new signaling molecules.

Seroprevalence of Entamoeba histolytica infection among men who have sex with men in Sydney, Australia.

A retrospective analysis was undertaken to determine the seroprevalence of Entamoeba histolytica infection in Sydney, Australia. Using an enzyme-linked immunosorbent assay, 429 high risk human immunodeficiency virus (HIV)-infected men who have sex with men (MSM), 446 low risk HIV-uninfected MSM, and 456 HIV-uninfected controls were assessed. Seroprevalence rates were 5.13% for the high risk HIV-infected MSM group, 0.22% for the low risk HIV-uninfected MSM group, and 0.44% for the control group. We found that high risk HIV-infected MSM have a significantly greater seroprevalence of E. histolytica with a relative risk of 22.87, when compared with low risk HIV-uninfected MSM and 11.69 when compared with controls. These findings show that in Sydney, sexually active HIV-infected MSM are at greater risk of developing amoebic disease caused by E. histolytica than HIV-uninfected MSM and the general population.