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.

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.

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.

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.

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).

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.

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.

Coinfections with Schistosoma haematobium, Necator americanus, and Entamoeba histolytica/Entamoeba dispar in children: chemokine and cytokine responses and changes after antiparasite treatment.

The effect of polyparasite infections on cytokine and chemokine responses as well as the effect of antiparasite treatment was studied in children without parasite infection (the G0 group), in children singly infected with Schistosoma haematobium (the G1 group), and in children multiply infected with S. haematobium/Schistosoma mansoni, Entamoeba histolytica/Entamoeba dispar, and Necator americanus (the G3+ group). Linear regression analysis disclosed a significant risk for coinfection with hookworm and Schistosoma species. Polyparasite infections detected in 23% of children before treatment were present in 5% at 15 months after treatment. Chemokine responses to S. mansoni adult worm antigen (SmAg) diminished after treatment for macrophage inflammatory chemokine (MIP)-1alpha/chemokine (C-C motif) ligand (CCL)-3 (among G3+ children, by a factor of 200 [95% confidence interval {CI}, 33-1111]) and for MIP-1beta/CCL-4 (among G3+ children, by a factor of 26 [95% CI, 6-117]) but were enhanced for thymus- and activation-regulated chemokine/CCL-17 (among G3+ children, by a factor of 10 [95% CI, 3-32]) (P < .001 for all). In response to E. histolytica antigen, interleukin (IL)-13 levels increased after treatment among G1 children by a factor of 138 (95% CI, 12-1569) and among G3+ children by a factor of 21 (95% CI, 7-64) (P < .001 for both). Cellular production of interferon (IFN)-gamma in response to SmAg decreased 4 weeks after treatment among G3+ children, whereas T helper cell type 2 (Th2) IL-13 production was enhanced among G1 and G3+ children. In summary, polyparasite infections with S. haematobium/S. mansoni, E. histolytica/E. dispar, and N. americanus generated prominent proinflammatory cytokine and chemokine responses, and, after antihelminth treatment, the inflammatory chemokine response lessened as the Th2 responsiveness in coinfected children increased.

Differences in complement-mediated killing of Entamoeba histolytica between men and women--an explanation for the increased susceptibility of men to invasive amebiasis?

Men are more than 7 times more likely to develop amebic liver abscess or amebic dysentery caused by Entamoeba histolytica than women. Because the complement system could play a key role in controlling amebiasis, we determined whether serum from men and women differ in the ability to kill amebic trophozoites. We found that serum from women was significantly more effective in killing E. histolytica trophozoites than serum from men, and this killing was complement dependent. Our results provide a possible explanation for the differential susceptibility of men and women to amebic liver abscess and amebic colitis.

Increased risk for Entamoeba histolytica infection and invasive amebiasis in HIV seropositive men who have sex with men in Taiwan.

BACKGROUND: Incidence of Entamoeba histolytica infection and clinical manifestations and treatment response of invasive amebiasis (IA) in HIV-infected patients have rarely been investigated before. METHODOLOGY/PRINCIPAL FINDINGS: At the National Taiwan University Hospital, medical records of HIV-infected patients who received a diagnosis of IA between 1994 and 2005 were reviewed. The incidence of amebiasis was investigated in serial blood and stool samples from 670 and 264 HIV-infected patients, respectively, using serological and specific amebic antigen assays. DNA extracted from stool samples containing E. histolytica were analyzed by PCR, sequenced, and compared. Sixty-four (5.8%) of 1,109 HIV-infected patients had 67 episodes of IA, and 89.1% of them were men having sex with men (MSM). The CD4 count at diagnosis of IA was significantly higher than that of the whole cohort (215 cells/microL vs. 96 cells/microL). Forty episodes (59.7%) were liver abscesses, 52 (77.6%) colitis, and 25 (37.3%) both liver abscesses and colitis. Fever resolved after 3.5 days of metronidazole therapy (range, 1-11 days). None of the patients died. The incidence of E. histolytica infection in MSM was higher than that in other risk groups assessed by serological assays (1.99 per 100 person-years [PY] vs. 0 per 100 PY; p<0.0001) and amebic antigen assays (3.16 per 100 PY vs. 0.68 per 100 PY; p = 0.12). In multiple logistic regression analysis, only MSM was significantly associated with acquisition of E. histolytica infection (adjusted odds ratio, 14.809; p = 0.01). Clustering of E. histolytica isolates by sequencing analyses from geographically-unrelated patients suggested person-to-person transmission. CONCLUSIONS/SIGNIFICANCE: HIV-infected MSM were at significantly higher risk of amebiasis than patients from other risk groups. Despite immunosuppression, amebic liver abscesses and colitis responded favorably to treatment.

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.

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.

Influence of human leukocyte antigen class II alleles on susceptibility to Entamoeba histolytica infection in Bangladeshi children.

BACKGROUND: The association of antibody responses with both innate and acquired immunity to amebiasis indicate that CD4+ T cells play a role in protection against Entamoeba histolytica infection. To test this hypothesis, we compared the genotype frequencies of human leukocyte antigen (HLA) class II alleles in a cohort of Bangladeshi children intensively monitored for E. histolytica infection for a 3-year period. METHODS: Using logistic regression, we calculated the odds of disease by genotype and by haplotype. RESULTS: The DQB1*0601 heterozygous and homozygous genotypes were found in 55% of E. histolytica-negative children but in only 34% of E. histolytica-positive children (overall odds ratio, 2.39; 95% confidence interval [CI], 1.26-4.54). Children who were heterozygous for the DQB1*0601/DRB1*1501 haplotype were 10.1 times (95% CI, 2.02-50.6) more likely to be both E. histolytica negative and serum anti-lectin immunoglobulin G negative at baseline. Other DQB1 and DRB1 alleles (DQB1*0202, DQB1*0301, and DRB1*0701) were not associated with any of the clinical outcomes related to amebiasis. CONCLUSION: A potential protective association was observed with the HLA class II allele DQB1*0601 and the heterozygous haplotype DQB1*0601/DRB1*1501. This association may explain why amebiasis does not occur in some children who are exposed to the parasite and implicates HLA class II-restricted immune responses in protection against E. histolytica infection.

Regulatory effects of IL-12 and IL-18 on Onchocerca volvulus- and Entamoeba histolytica-specific cellular reactivity and cytokine profiles.

In the present study, the cytokines interleukin (IL)-12 and IL-18 were evaluated for their capacity to modulate and to re-direct in vitro parasite antigen-specific cellular responsiveness in patients exposed to Onchocerca volvulus and Entamoeba histolytica infection. We found that IL-18 was highly capable of reducing parasite antigen-induced IL-10 production by PBMC. In contrast, addition or neutralization of IL-12, also in combination with IL-18 and the interferon-gamma-inducible chemokine IP-10 did not affect IL-10 production. Interestingly, the highest IL-10 levels were measured when IL-18 and IP-10 were both neutralized. Although having no effect on IL-10, IL-12 strongly promoted spontaneous and parasite antigen-driven IFN-gamma production by PBMC, whereas IL-18 was only moderately affecting IFN-gamma release by PBMC re-stimulated with E. histolytica- or O. volvulus-specific antigens. Both IL-12 and IL-18 diminished the cellular production of IL-13, and a synergistic effect was observed when the cytokines were combined. Likewise, neutralization of IL-12 enhanced Entamoeba and Onchocerca antigen-driven IL-13 production, but no further increase of IL-13 was observed, when anti-IL-12 and anti-IL-18 were used together. This study disclosed that IL-18 will significantly down-regulate parasite-specific IL-10 production, whereas IL-12 induced IFN-gamma and inhibited IL-13 production by PBMC from humans exposed to O. volvulus and E. histolytica. Such selective immune-regulatory capacity of IL-12 and IL-18 may comprise an important tool to re-direct polarized cytokine responses towards a balanced Th1/Th2 cytokine profile, which may prevent pathology and promote immunity against helminth and protozoan parasite infections.