Target identification and intervention strategies against amebiasis.

Entamoeba histolytica is the etiological agent of amebiasis, which is an endemic parasitic disease in developing countries and is the cause of approximately 70,000 deaths annually. E. histolytica trophozoites usually reside in the colon as a non-pathogenic commensal in most infected individuals (90% of infected individuals are asymptomatic). For unknown reasons, these trophozoites can become virulent and invasive, cause amebic dysentery, and migrate to the liver where they cause hepatocellular damage. Amebiasis is usually treated either by amebicides which are classified as (a) luminal and are active against the luminal forms of the parasite, (b) tissue and are effective against those parasites that have invaded tissues, and (c) mixed and are effective against the luminal forms of the parasite and those forms which invaded the host's tissues. Of the amebicides, the luminal amebicide, metronidazole (MTZ), is the most widely used drug to treat amebiasis. Although well tolerated, concerns about its adverse effects and the possible emergence of MTZ-resistant strains of E. histolytica have led to the development of new therapeutic strategies against amebiasis. These strategies include improving the potency of existing amebicides, discovering new uses for approved drugs (repurposing of existing drugs), drug rediscovery, vaccination, drug targeting of essential E. histolytica components, and the use of probiotics and bioactive natural products. This review examines each of these strategies in the light of the current knowledge on the gut microbiota of patients with amebiasis.

Identification of free-living amoebae isolated from tap water in Istanbul, Turkey.

Free-living amoebae (FLA) are widely spread in the environment and also known to cause rare but often serious infections. The present work focuses on a local survey on FLA. It is essential to know the prevalence and distribution of these microorganisms in order to get infections caused by them under control. In this study, FLA isolated from domestic tap water samples from homes of contact lens wearers were identified by morphology and by 18S rRNA gene sequence analysis. Morphological analysis and partial sequencing of the 18S rDNA revealed the presence of Acanthamoeba genotype T4 and Vermamoeba vermiformis in the investigated tap water samples. Naegleria fowleri, Balamuthia mandrillaris, and Sappinia spp. were not detected during this study. It was shown that species of FLA known to cause eye infections in humans are widely distributed in tap water in Istanbul, Turkey. Contact lens wearers should be aware of the risk of contamination from tap water and strictly apply stringent contact lens hygiene. With this study, we established Acanthamoeba genotype T4 and Vermamoeba vermiformis as contaminants of tap water in Istanbul.

Protective immunity against Naegleria fowleri infection on mice immunized with the rNfa1 protein using mucosal adjuvants.

The free-living amoeba, Naegleria fowleri, causes a fatal disease called primary amoebic meningoencephalitis (PAM) in humans and experimental animals. Of the pathogenic mechanism of N. fowleri concerning host tissue invasion, the adherence of amoeba to hose cells is the most important. We previously cloned the nfa1 gene from N. fowleri. The protein displayed immunolocalization in the pseudopodia, especially the food-cups structure, and was related to the contact-dependent mechanism of the amoebic pathogenicity in N. fowleri infection. The cholera toxin B subunit (CTB) and Escherichia coli heat-labile enterotoxin B subunit (LTB) have been used as potent mucosal adjuvants via the parenteral route of immunization in most cases. In this study, to examine the effect of protective immunity of the Nfa1 protein for N. fowleri infection with enhancement by CTB or LTB adjuvants, intranasally immunized BALB/c mice were infected with N. fowleri trophozoites for the development of PAM. The mean time to death of mice immunized with the Nfa1 protein using LTB or CTB adjuvant was prolonged by 5 or 8 days in comparison with that of the control mice. In particular, the survival rate of mice immunized with Nfa1 plus CTB was 100% during the experimental period. The serum IgG levels were significantly increased in mice immunized with Nfa1 protein plus CTB or LTB adjuvants. These results suggest that the Nfa1 protein, with CTB or LTB adjuvants, induces strong protective immunity in mice with PAM due to N. fowleri infection.

Factors affecting the use of anti-amoebiasis protective measures among Taiwan immigrants returning to amoebiasis-endemic regions.

OBJECTIVE: To investigate the predictors of use of anti-amoebiasis protective measures (AAPMs) among Taiwan immigrants returning to their country of origin, using the Health Belief Model (HBM) to guide the investigation. DESIGN: Cross-sectional study. METHODS: Between March and May 2011, all permanent immigrants originating from amoebiasis-endemic countries who received services at the immigrant service centres in Taipei or Tainan and who reported that they had returned to their country of origin within the past five years were enrolled in the study. A structured questionnaire containing questions on sociodemographic characteristics and items related to the constructs of the HBM was used as the data collection instrument. RESULTS: Complete information was collected from 384 immigrants, with a response rate of 80% (384/480). The mean age of the subjects was 38.4 years (standard deviation 10.6 years). The majority (70%) of participants did not receive travel information through a pretravel consultation, and more than 17% reported that they did not use measures to prevent amoebiasis. Multiple regression analyses revealed that Chinese proficiency, pretravel consultation and lower barriers to using protective measures were significantly associated with the use of AAPMs during return trips to country of origin (R(2) = 0.45; F = 77.5; P < 0.001). CONCLUSION: The HBM significantly predicted the use of AAPMs in this study. A high proportion of immigrants did not use appropriate AAPMs when they returned to their country of origin. Educational approaches should be targeted at immigrants originating from amoebiasis-endemic regions who return to their country of origin.

The immune response induced by DNA vaccine expressing nfa1 gene against Naegleria fowleri.

The pathogenic free-living amoeba, Naegleria fowleri, causes fatal primary amoebic meningoencephalitis in experimental animals and in humans. The nfa1 gene that was cloned from N. fowleri is located on pseudopodia, especially amoebic food cups and plays an important role in the pathogenesis of N. fowleri. In this study, we constructed and characterized retroviral vector and lentiviral vector systems for nfa1 DNA vaccination in mice. We constructed the retroviral vector (pQCXIN) and the lentiviral vector (pCDH) cloned with the egfp-nfa1 gene. The expression of nfa1 gene in Chinese hamster ovary cell and human primary nasal epithelial cell transfected with the pQCXIN/egfp-nfa1 vector or pCDH/egfp-nfa1 vector was observed by fluorescent microscopy and Western blotting analysis. Our viral vector systems effectively delivered the nfa1 gene to the target cells and expressed the Nfa1 protein within the target cells. To evaluate immune responses of nfa1-vaccinated mice, BALB/c mice were intranasally vaccinated with viral particles of each retro- or lentiviral vector expressing nfa1 gene. DNA vaccination using viral vectors expressing nfa1 significantly stimulated the production of Nfa1-specific IgG subclass, as well as IgG levels. In particular, both levels of IgG2a (Th1) and IgG1 (Th2) were significantly increased in mice vaccinated with viral vectors. These results show the nfa1-vaccination induce efficiently Th1 type, as well as Th2 type immune responses. This is the first report to construct viral vector systems and to evaluate immune responses as DNA vaccination in N. fowleri infection. Furthermore, these results suggest that nfal vaccination may be an effective method for treatment of N. fowleri infection.

Prevalence of acanthamoeba from tap water in rio grande do Sul, Brazil.

A total of 136 samples of tap water were collected from state and municipal schools between March and November 2009. The samples were filtered through cellulose nitrate membranes that were seeded at non-nutrient agar 1.5% containing an overlayer of Escherichia coli suspension. Thirty-one (22.79%) tap water samples investigated were found positive for free-living amoebae (FLA). From these, 13 presented as FLA that seems to belong to the genus Acanthamoeba. All samples of FLA were cloned and identified as belonging to the genus Acanthamoeba by the morphology of cysts and trophozoites and by PCR using genus-specific primers that amplify the ASA.S1 region of 18S rDNA gene. Physiological tests of thermotolerance and osmotolerance were used to evaluate the pathogenicity of the isolates. The sequencing analysis by comparing the sequences submitted to GenBank, showed genotype distribution into groups T2, T2/T6, T6, and T4. In tests of thermotolerance and osmotolerance, 50% of the isolates had a low pathogenic potential. The results indicated the presence of Acanthamoeba in tap water in Rio Grande do Sul, Brazil, revealing its importance and the need for more epidemiological studies to determine their distribution in the environment and its pathogenic potential.

Review of zoonotic amebiasis: Epidemiology, clinical signs, diagnosis, treatment, prevention and control.

Amebiasis is a disease caused by the protozoan parasite Entamoeba histolytica, which mainly shows symptoms of acute diarrhea, dysentery, amebic colitis, and amebic liver abscesses. As the fourth leading parasitic cause of human mortality, E. histolytica mainly infect children in developing countries, transmitted by food and water contamination. In the majority of infected individuals, Entamoeba sp. asymptomatically colonizes the large intestine and self-limiting, while in others, the parasite breaches the mucosal epithelial barrier to cause amebic colitis and can disseminate to soft organs to cause abscesses. Metronidazole (MTZ) is the recommended and most widely used drug for treating the invasive amebiasis. No amebiasis vaccine has been approved for human clinical trials to date, but many recent vaccine development studies hold promise. For the prevention and control of amebiasis, improvement of water purification systems and hygiene practices could decrease disease incidence. In this review, we focus on the epidemiology, transmission, clinical signs, pathogenesis, diagnosis, treatment, prevention and control of the zoonotic amebiasis.

Balamuthia mandrillaris transmitted through organ transplantation --- Mississippi, 2009.

On December 14, 2009, a physician in Mississippi contacted CDC to report possible transplant-transmitted encephalitis in two kidney transplant recipients who shared the same organ donor. Histopathologic testing of donor autopsy brain tissue at CDC showed amebae, and subsequent testing of specimens from the donor and the two kidney recipients confirmed transmission by transplantation of Balamuthia granulomatous amebic encephalitis (GAE), a rare disease caused by Balamuthia mandrillaris, a free-living ameba found in soil. One kidney recipient, a woman aged 31 years, died; the other recipient, a man aged 27 years, survived with neurologic sequelae. Recipients of the heart and liver from the same donor received preemptive therapy and have shown no signs of infection. The donor, a previously healthy boy aged 4 years, was presumed to have died from acute disseminated encephalomyelitis (ADEM), an autoimmune neurologic disease, after infection with influenza A. An investigation was conducted by the state health departments in Mississippi, Kentucky, Florida, and Alabama and CDC to characterize the cases, elucidate possible exposures in the donor, and develop recommendations for early detection and prevention. This is the first reported transmission of Balamuthia by organ transplantation. Clinicians should be aware of Balamuthia infection as a potentially fatal cause of encephalitis. Organ procurement organizations (OPOs) and transplant centers should be aware of the potential for Balamuthia infection in donors with encephalitis of uncertain etiology, and OPOs should communicate this elevated risk for infection to transplant centers so they can make an informed risk assessment in the decision to accept an organ.

Acanthamoeba spp. in domestic tap water in houses of contact lens wearers in the metropolitan area of Mexico City.

A survey was carried out in the metropolitan area of Mexico City to determine the presence of Acanthamoeba in the tap water of houses of contact lens wearers. Water samples were taken from the mains water entry, bathroom sinks and storage containers (roof tanks, cisterns) of 27 houses; and from the solution contained in the contact lens cases. Samples were filtered and cultured onto NNE medium. The isolates were identified based on their morphological features and pathogenicity. Total and fecal coliforms, water temperature, pH, dissolved oxygen and residual free-chlorine were measured by standard methods. Forty five isolates of Acanthamoeba from 200 water samples were obtained. The highest number of amoebae was isolated from cisterns and roof tanks. Most Acanthamoeba isolates were non-pathogenic, however, their presence in tap water is a potential hazard since some species can cause Acanthamoeba keratitis and granulomatous amoebic encephalitis.

Detection and surveillance of waterborne protozoan parasites.

The majority of the world's population still live without access to healthy water and the contamination of drinking water with protozoan pathogens poses a serious threat to millions of people in the developing world. Even in the developed world periodic outbreaks of diarrhoeal diseases are caused by the protozoan parasites Cryptosporidium sp., Giardia duodenalis and Entamoeba histolytica. Thus, surveillance of drinking water is imperative to minimize such contaminations and ensure continuous supplies of healthy water world-wide. This article reviews the progress in technology for detection and surveillance of these important waterborne parasites.

[Screening the asymptomatic traveler returning from the tropics]. / Bilan de santé chez le voyageur asymptomatique de retour des tropiques.

Travelers to developing countries may be exposed to several infectious pathogens sometimes leading to complications after a long latency period. Screening of asymptomatic returning travelers is only justified in case of specific risks during travel or after a long tropical stay (> 3 months). Based mainly on the history taking of risk patterns, some occult infections have to be looked for: tuberculosis, sexual transmitted infections and some parasitic conditions (schistosomiasis, amebiasis, strongyloidosis,...). Post-travel screening is most effective about 3 months after return, and may be even more beneficial if associated with screening of major western morbidities and preventive counseling for future travels.

Kinetic characterization of methionine gamma-lyases from the enteric protozoan parasite Entamoeba histolytica against physiological substrates and trifluoromethionine, a promising lead compound against amoebiasis.

Methionine gamma-lyase (MGL) (EC 4.4.1.11), which is present in certain lineages of bacteria, plants, and protozoa but missing in mammals, catalyzes the single-step degradation of sulfur-containing amino acids (SAAs) to alpha-keto acids, ammonia, and thiol compounds. In contrast to other organisms possessing MGL, anaerobic parasitic protists, namely Entamoeba histolytica and Trichomonas vaginalis, harbor a pair of MGL isozymes. The enteric protozoon En. histolytica shows various unique aspects in its metabolism, particularly degradation of SAAs. Trifluoromethionine (TFM), a halogenated analog of Met, has been exploited as a therapeutic agent against cancer as well as against infections by protozoan organisms and periodontal bacteria. However, its mechanism of action remains poorly understood. In addition, the physiological significance of the presence of two MGL isozymes in these protists remains unclear. In this study, we compared kinetic parameters of the wild-type and mutants, engineered by site-directed mutagenesis, of the two MGL isotypes from En. histolytica (EhMGL1 and EhMGL2) for various potential substrates and TFM. Intracellular concentrations of l-Met and l-Cys suggested that these SAAs are predominantly metabolized by EhMGL1, not by EhMGL2. It is unlikely that O-acetyl-l-serine is decomposed by EhMGLs, given the kinetic parameters of cysteine synthase reported previously. Comparison of the wild-type and mutants revealed that the contributions of several amino acids implicated in catalysis differ between the two isozymes, and that the degradation of TFM is less sensitive to alterations of these residues than is the degradation of physiological substrates. These results support the use of TFM to target MGL.

Combating Acanthamoeba spp. cysts: what are the options?

Acanthamoeba spp. are protist pathogens and causative agents of serious infections including keratitis and granulomatous amoebic encephalitis. Its ability to convert into dormant and highly resistant cysts form limits effectiveness of available therapeutic agents and presents a pivotal challenge for drug development. During the cyst stage, Acanthamoeba is protected by the presence of hardy cyst walls, comprised primarily of carbohydrates and cyst-specific proteins, hence synthesis inhibition and/or degradation of cyst walls is of major interest. This review focuses on targeting of Acanthamoeba cysts by identifying viable therapeutic targets.

Eradication of amebiasis in a large institution for adults with mental retardation in Taiwan.

OBJECTIVE: Investigation of a program to eradicate amebiasis using consecutive intensive mass screenings followed by medication in a large institute for adults with mental retardation in Taiwan. DESIGN: Prospective cohort study, with 3 years of follow-up. SETTING: A large, 450-bed institution for adults with mental retardation located in southern Taiwan. PARTICIPANTS: All 443 adults with mental retardation in the institution, who have various motor and/or mental handicaps, were included in this study. INTERVENTIONS: A total of 7 consecutive intensive mass screenings for amebiasis for all residents (performed in March, August and November 2001, March and August 2002, January 2003, and May 2004). Infected patients were treated using the standard protocol of the Center for Disease Control of Taiwan. RESULTS: Enzyme immunoassay testing was used for the amebiasis screening, with the rapid detection of the specific antigen for Entamoeba histolytica in human fecal specimens confirmed by microscopic examination. The serial prevalence and cumulative incidence were then calculated. The prevalence of amebic infection declined in serial screenings, but new infections and reinfections were detected in 5 of 6 follow-up screenings. The prevalence was 10.8% at the beginning of the program and then gradually reduced, falling to 6.3%, 3.6%, 2.7%, 3.4%, and 2.2%. Finally, no more positive cases were identified in the last screening (May 2004). The cumulative incidence rate stabilized at around 40% by the fifth screening. Of the 179 infected patients, 120 had primary infections, with 59 cases of multiple amebic infections. CONCLUSIONS: Active surveillance with intensive mass screening is an effective method of identifying asymptomatic and latent cases of amebiasis in areas where it is endemic, such as an institution for adults with mental retardation.

Pathogenic and opportunistic free-living amoebae: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri, and Sappinia diploidea.

Among the many genera of free-living amoebae that exist in nature, members of only four genera have an association with human disease: Acanthamoeba spp., Balamuthia mandrillaris, Naegleria fowleri and Sappinia diploidea. Acanthamoeba spp. and B. mandrillaris are opportunistic pathogens causing infections of the central nervous system, lungs, sinuses and skin, mostly in immunocompromised humans. Balamuthia is also associated with disease in immunocompetent children, and Acanthamoeba spp. cause a sight-threatening infection, Acanthamoeba keratitis, mostly in contact-lens wearers. Of more than 30 species of Naegleria, only one species, N. fowleri, causes an acute and fulminating meningoencephalitis in immunocompetent children and young adults. In addition to human infections, Acanthamoeba, Balamuthia and Naegleria can cause central nervous system infections in animals. Because only one human case of encephalitis caused by Sappinia diploidea is known, generalizations about the organism as an agent of disease are premature. In this review we summarize what is known of these free-living amoebae, focusing on their biology, ecology, types of disease and diagnostic methods. We also discuss the clinical profiles, mechanisms of pathogenesis, pathophysiology, immunology, antimicrobial sensitivity and molecular characteristics of these amoebae.

[Challenge of developing anti-parasite vaccines in the tropics]. / Le défi de la vaccination antiparasitaire sous les tropiques.

Much effort has been devoted to developing anti-parasite vaccines. The main obstacles to overcome involve problems in cultivating parasites, variability in antigens of cultivated parasites, insufficient immune responses that do not provide full protection, lack of animal models and difficulty in evaluating immune protection acquired naturally or after vaccination in populations living in endemic areas. Numerous clinical trials have been conducted and several parasite antigens, in particular against malaria, have been tested in endemic areas. Up to now no candidate vaccine has shown sufficient, long-term efficacy to justify its inclusion in public health program. However trials using anti-parasite vaccination under both experimental and field conditions clearly demonstrate that a certain level of clinical immunity against malaria, bilharziasis, and leishmaniasis.

Contact lens care solution killing efficacy against Acanthamoeba castellanii by in vitro testing and live-imaging.

In the past decade there has been an increased incidence of Acanthamoeba keratitis, particularly in contact lens wearers. The aim of this study was to utilize in vitro killing assays and to establish a novel, time-lapse, live-cell imaging methodology to demonstrate the efficacy of contact lens care solutions in eradicating Acanthamoeba castellanii (A. castellanii) trophozoites and cysts. Standard qualitative and quantitative in vitro assays were performed along with novel time-lapse imaging coupled with fluorescent dye staining that signals cell death. Quantitative data obtained demonstrated that 3% non-ophthalmic hydrogen peroxide demonstrated the highest percent killing at 87.4% corresponding to a 4.4 log kill. The other contact lens care solutions which showed a 72.9 to 29.2% killing which was consistent with 4.3-2.8 log reduction in trophozoite viability. Both analytical approaches revealed that polyquaternium/PHMB-based was the least efficacious in terms of trophicidal activity. The cysticidal activity of the solutions was much less than activity against trophozoites and frequently was not detected. Live-imaging provided a novel visual endpoint for characterizing the trophocidal activity of the care solutions. All solutions caused rapid rounding or pseudocyst formation of the trophozoites, reduced motility and the appearance of different morphotypes. Polyquaternium/alexidine-based and peroxide-based lens care system induced the most visible damage indicated by significant accumulation of debris from ruptured cells. Polyquaternium/PHMB-based was the least effective showing rounding of the cells but minimal death. These observations are in keeping with care solution biocides having prominent activity at the plasma membrane of Acanthamoeba.

Serological investigation to identify risk factors for post-flood infectious diseases: a longitudinal survey among people displaced by Typhoon Morakot in Taiwan.

OBJECTIVES: After Typhoon Morakot struck Taiwan in 2009, thousands of Taiwanese citizens were displaced to shelters for several weeks. Others were placed in urban communities where they had family members. This study aimed to investigate serological status in both groups and identify risk factors associated with seroconversion of infectious diseases. DESIGN: A longitudinal survey. SETTING: All experimental and clinical investigations were performed in a tertiary teaching hospital. PARTICIPANTS: A total of 288 displaced persons (96 males and 192 females) were recruited and complete follow-up data through two rounds of sampling were collected. The average age was 58.42 years (range 31-87 years). INTERVENTIONS: First, serum specimens were collected between December 2009 and January 2010, 4-5 months after the typhoon. The second round of specimen collection was carried out after 6 months. PRIMARY AND SECONDARY OUTCOME MEASURES: The primary outcome measured was serological status of vaccine-preventable droplet-borne infectious diseases (ie, measles, mumps, rubella) and water-borne diseases (ie, amoebiasis and leptospirosis). The secondary outcome was identification of risk factors for seroconversion using univariate and multivariate analyses. RESULTS: Complete data were available for all 288 displaced persons (114 from the shelter group; 174 from the community group). Seroconversion of Entamoeba histolytica was observed in 128 (44.4%) participants, with a significantly higher rate in the shelter group than in the community group (56.1% vs 36.8%; p=0.001). There were 10 cases of rubella seroconversion. After adjusting for medical history, hypertension and hyperlipidaemia, shelter stay was associated with higher risk for seroconversion (OR=2.055, 95% CI 1.251 to 3.374; p=0.004). Amoebiasis was more evident in the shelter group, although the manifestations were mild. CONCLUSIONS: Our results suggested that (1) a clean water supply is essential postdisaster, especially in crowded shelters, and (2) vaccination programmes should be extended to populations at higher risk for post-disaster displacement or to those with weakened immune status.