Naegleria fowleri is a free-living amoeba, known as a causative agent for a fatal disease of the central nervous system (CNS) in man such as Primary amoebic meningoencephalitis (PAM). Factors contributing to its pathogenicity and its distribution in the environment have been investigated by previous researchers. In case of its pathogenicity, several enzymes such as phospolipase A and sphingomyelinase, have been proposed to probably act as aggressors in promoting PAM but no study so far have been conducted to investigate the presence of proteinase enzyme in this amoeba although a 56kDa cystein proteinase enzyme has been identified in Entamoeba histolytica as an important contributing factor in the amoeba's virulence. In this preliminary study, a pathogenic amoeba, Naegleria fowleri (strain NF3) was examined for the presence of proteinases. Samples of enzymes in this amoeba were analysed by electrophoresis using SDS-PAGE-gelatin gels. The results showed that this amoeba possesses at least two high molecular weight proteinases on gelatin gels; their apparent molecular weights are approximately 128 kDa and approximately 170 kDa. Band of approximately 128 kDa enzyme is membrane-associated and its activity is higher at alkaline pH compared with lower pH; at lower pH, its activity is greatly stimulated by DTT. The approximately 170 kDa band enzyme appears to be inactivated at pH 8.0, at lower ph its activity is higher and DTT-dependance. The activity of this enzyme is partially inhibited by inhibitor E-64 but markedly inhibited to antipain suggesting it belongs to the cysteine proteinase group.
Intestinal protozoa are increasingly being studied because of their association with acute and chronic diarrhoea in immunocompromised as well as immunocompetent patients. Various community outbreaks due to contamination of water or food with these protozoa have further highlighted their importance in public health. Among these important pathogens are Giardia duodenalis, Entamoeba histolytica, Cryptosporidium parvum, Cyclospora cayetanensis, Isospora belli, and microsporidia. Except for the cyst-forming G. duodenalis and E. histolytica, the others are intracellular and form spores which are passed out with the faeces. These organisms are also found in various animals and birds and zoonotic transmission is thought to occur. These infections are distributed worldwide, with a higher prevalence in developing compared to developed countries. However, the relative importance of zoonotic infections especially in developing countries has not been studied in detail. The prevalence rates are generally higher in immunodeficient compared to immunocompetent patients. Higher prevalence rates are also seen in rural compared to urban communities. Most studies on prevalence have been carried out in developed countries where the laboratory and other health infrastructure are more accessible than those in developing countries. This relative inadequacy of laboratory diagnosis can affect accurate estimates of the prevalence of these infections in developing countries. However, reports of these infections in travellers and workers returning from developing countries can provide some indication of the extent of these problems. Most studies on prevalence of amoebiasis in developing countries were based on morphological identification of the parasite in faecal smears. As the pathogenic E. histolytica is morphologically indistinguishable from that of non-pathogenic E. dispar, estimates of amoebiasis may not be accurate. The epidemiology of human microsporidia infections is not completely understood. Two species, Enterocytozoon bieneusi and Encephalitozoon intestinalis, are associated with gastrointestinal disease in humans and it is believed that human to human as well as animal to human infections occur. However, the importance of zoonotic infections has not been fully characterised. G. duodenalis cysts, microsporidia and Cryptosporidium oocysts have been detected in various ground water resources, but their role in community outbreaks and maintenance of the infection has not been fully characterised. The taxonomic classification and pathogenic potential of B. hominis are still controversial. While considered by many as yeast, fungi or protozoon, recent sequence analysis of the complete SSUrRNA gene has placed it within an informal group, the stramenopiles. This review covers recent published data on these zoonotic infections and examines their public health importance in Asian countries.
Is Acanthamoeba sp. normally found in the eyes? A study was carried out to establish the possibility of Acanthamoeba sp. as a part of the normal conjunctival flora. Conjunctiva swabbing were carried out in 286 healthy Orang Asli school children using sterile cotton swab. The swab was then inoculated onto non-nutrient agar (NN-A). Heat killed Escherichia coli that was used as food source for the growth of the amoebae was pipetted onto and away from the smear. The plates were incubated at 30 degrees C and examined daily using an inverted microscope for 14 days. Morphology of the trophozoites and cysts of the amoebae were used as the taxonomic criteria for identification. Positive-controls and negative-controls were done to check for the consistency of the technique used and monitoring of contamination respectively. None of the conjunctiva swab cultured was positive for Acanthamoeba sp. This finding may indicate that Acanthamoeba sp. is not part of normal conjunctival flora or conjunctiva swab is an insensitive technique to isolate the organism. However, a more extensive research is needed to investigate these possibilities.
Entamoeba histolytica (E. histolytica), the causative agent of amoebiasis, is still a global public health problem that cannot be controlled, especially in tropical and subtropical countries. This study was conducted to obtain information about the incidence of Entamoeba histolytica/dispar/ moshkovskii complex infection and the factors that influence it. The prevalence of infection with the Entamoeba histolytica/dispar/moshkovskii complex and the factors that influence it in people living on the smallest and outermost island of Indonesia, Sabang Island, Aceh Province. This study involved 335 respondents aged >= 10 years. Respondents were selected by non-probability sampling technique. Interviews and observations were conducted to identify risk factors. The Entamoeba histolytica/dispar/ moshkovskii complex was identified by direct examination, concentration, and Whitley's trichrome staining techniques. A Chi-Square test was performed to analyze the correlation of risk factors with the incidence of infection. The prevalence of infection with the Entamoeba histolytica/dispar/ moshkovskii complex in the people of Sabang Island was 26.6% (89/335). Source and adequacy of clean water correlated with the incidence of Entamoeba histolytica/dispar/moshkovskii complex infection. Demographic variables are not correlated with the incidence of infection. However, the group of women aged > 61 years, unemployed, unmarried, and earning less than the regional minimum wage tend to be more likely to be found with Entamoeba histolytica/dispar/moshkovskii complex infections. Thus it can be concluded that the prevalence of infection with the Entamoeba histolytica/dispar/moshkovskii complex on Sabang Island is in the high category. The prevalence of E. histolytica as the causative agent of amoebiasis cannot be explained with certainty because the two identical non-pathogenic Entamoeba species cannot be distinguished by microscopic identification. Sources and adequacy of clean water correlate with the incidence of Entamoeba histolytica/dispar/moshkovskii complex infection in the people of Sabang Island.
Entamoeba histolytica is the parasite responsible for amoebiasis, which can result in amoebic colitis or amoebic liver abscess. Metronidazole has been the conventional treatment for intestinal amoebiasis, but concerns regarding resistance have emerged due to the identification of resistance pathways in E. histolytica. This study investigates a novel anti-amoebic approach targeting the CDP-choline pathway. Inhibition studies were conducted using potential choline kinase (CK) inhibitors to inhibit the EhCK enzyme, and RNA interference was employed to knock down the EhCK gene. Km and Vmax of purified EhCK and hCKa2 proteins were determined by pyruvate kinase-lactate dehydrogenase (PK-LDH) coupled assay. The IC50 values for EhCK and hCKa2 were determined with several commercial CK inhibitors. Selected inhibitors were incubated with E. histolytica trophozoites for 48 hours to determine the EC50 for each inhibitor. Silencing of gene encoding EhCK was carried out using duplex siRNA and the gene expression level was measured by real-time qPCR. Based on the IC50 values, three of the inhibitors, namely CK37, flavopiridol and H-89 were more potent against EhCK than hCKa2. Trophozoites growth inhibition showed that only HDTAB, H-89 and control drug metronidazole could penetrate and induce cell death after 48-hour incubation. siRNA concentration of 10 µg/mL was used for the transfection of positive control GAPDH, EhCK, and non-targeting GFP siRNAs. RNAi experiment concluded with positive control GAPDH downregulated by 99% while the level of EhCK mRNA was downregulated by 47%. In this study, potential inhibitors of EhCK and siRNA have been identified, paving the way for further refinement and testing to enhance their potency against EhCK while sparing hCK. The utilization of these specific inhibitors and siRNA targeting EhCK represents a novel approach to impede the growth of E. histolytica by disrupting its phospholipid synthesis pathway.
Fulminant necrotising amoebic colitis is a complication of untreated amoebiasis. This is seen in mainly low-income countries. It has a high mortality rate and is difficult to diagnose. We present an extremely rare case of fulminant necrotising amoebic colitis that caused diagnostic confusion in mimicking an acute abdomen, presumably caused by a perforated duodenal ulcer.
A hospital based retrospective study of amoebiasis was carried out for a ten-year period at the University Hospital, Kuala Lumpur. Of the 51 cases traced, 30 (59%) had amoebic dysentery, 20 (39%) were amoebic liver abscess (ALA) and one patient had both conditions. Entameoba histolytica trophozoites were identified in 13 (43%) of the amoebic dysenteric stools and 9 (30%) from biopsy. Of the 20 (39%) ALA cases, only one showed parasites in the stool and biopsy. Majority of the patients with dysentery were Malays while Chinese comprised 40% with ALA. Males predominated overall with a male female ratio of 3:1, while for ALA it was 9:1. Most of ALA were single (71.4%) and were localised in the right lobe. The majority of the patients were unemployed. Eighty three percent (83%) of the patients presented with diarrhoea or dysentery followed by abdominal pain while those with ALA had fever, chills, rigors and pain in the right hypochondrium. Eighty percent of the ALA cases showed hepatomegaly. All patients responded to treatment with metronidazole.
Amebiasis is one of the major causes of diarrhea in the developing countries and it can present with a wide range of gastrointestinal symptoms depending on the phase of infection. We described a case of 50 year-old male patient who presented with abdominal pain, diarrhea and vomiting. After right hemicolectomy for appendicular abscess with tumour over the ileum, histopathological examinations revealed numerous trophozoites of Entamoeba histolytica in a background of inflammations (Figure 1). Following resection of the ameboma, he received intravenous metronidazole treatment for total of two weeks duration.
Acanthamoeba, one of free-living amoebae (FLA), remains a high risk of direct contact with this protozoan parasite which is ubiquitous in nature and man-made environment. This pathogenic FLA can cause sight-threatening amoebic keratitis (AK) and fatal granulomatous amoebic encephalitis (GAE) though these cases may not commonly be reported in our clinical settings. Acanthamoeba has been detected from different environmental sources namely; soil, water, hot-spring, swimming pool, air-conditioner, or contact lens storage cases. The identification of Acanthamoeba is based on morphological appearance and molecular techniques using PCR and DNA sequencing for clinico-epidemiological purposes. Recent treatments have long been ineffective against Acanthamoeba cyst, novel anti-Acanthamoeba agents have therefore been extensively investigated. There are efforts to utilize synthetic chemicals, lead compounds from medicinal plant extracts, and animal products to combat Acanthamoeba infection. Applied nanotechnology, an advanced technology, has shown to enhance the anti-Acanthamoeba activity in the encapsulated nanoparticles leading to new therapeutic options. This review attempts to provide an overview of the available data and studies on the occurrence of pathogenic Acanthamoeba among the Association of Southeast Asian Nations (ASEAN) members with the aim of identifying some potential contributing factors such as distribution, demographic profile of the patients, possible source of the parasite, mode of transmission and treatment. Further, this review attempts to provide future direction for prevention and control of the Acanthamoeba infection.
Amebiasis is a frequently occurring parasitic infection in South East Asia. We present a case of a 54-year-old man with right lower quadrant abdominal pain that persisted for longer than 1 year. He had been diagnosed with inflammatory bowel disease in Indonesia. His abdominal pain persisted, despite therapy, and he visited Malaysia for transnational medical advice. Abdominal ultrasound showed fatty liver, gallbladder polyps, and a small left renal stone. Colonoscopy showed multiple ulcers in the cecum and a histopathological examination confirmed amebic infection of the cecum. The colonic ulcers subsided after anti-amebic treatment. This case highlights the need to consider the differential diagnosis of amebic colitis in patients presenting with manifestations of inflammatory bowel disease, especially in patients who live in or have traveled to endemic areas.
The indirect hemagglutination test was used to study antibody titers to Entamoeba histolytica in different Malaysian populations. Eighty-seven percent of Orang Asli (western Malaysian aborigines) adults and 79% of Orang Asli children with acute amebic dysentery were seropositive. However, significantly fewer children (39%) with amebic dysentery had high titer responses (titer greater than or equal to 1:1,280) than did adults with amebic dysentery (76%). No correlation between proctoscopic severity and amebic titer was found. Forty-four percent of asymptomatic family members were seroresponders. Satak, an Orang Asli village located near towns, had significantly more seroresponders (32%) than did the isolated, deep jungle village, Belatim (4%).
Independent evaluations of XEh Rapid®, an IgG4-based rapid dipstick test, were performed to assess its diagnostic performance to detect amebic liver abscess (ALA) using 405 samples at seven laboratories in four countries. The test showed high diagnostic specificity (97-100%) when tested with samples from healthy individuals (n = 100) and patients with other diseases (n = 151). The diagnostic sensitivity was tested with a total of 154 samples, and the results were variable. It was high in three laboratories (89-94%), and moderate (72%) and low (38%) in two other laboratories. Challenges and issues faced in the evaluation process are discussed. Nevertheless, XEh Rapid is promising to be developed into a point-of-care test in particular for resource-limited settings, and thus merits further confirmation of its diagnostic sensitivity.
Acanthamoeba, a genus that contains at least 24 species of free-living protozoa, is ubiquitous in nature. Successful treatment of Acanthamoeba infections is always very difficult and not always effective. More effective drugs must be developed, and medicinal plants may have a pivotal part in the future of drug discovery. Our research focused on investigating the in vitro anti- acanthamoebic potential of Leea indica and its constituent gallic acid in different concentrations. Water and butanol fractions exhibited significant amoebicidal activity against trophozoites and cysts. Gallic acid (100 µg/mL) revealed 83% inhibition of trophozoites and 69% inhibition of cysts. The butanol fraction induced apoptosis in trophozoites, which was observed using tunnel assay. The cytotoxicity of the fractions and gallic acid was investigated against MRC-5 and no adverse effects were observed. Gallic acid was successfully loaded within poly (D,L-lactide-co-glycolide) (PLGA) nanoparticles with 82.86% encapsulation efficiency, while gallic acid showed 98.24% in vitro release at 48 hours. Moreover, the gallic acid encapsulated in the PLGA nanoparticles exhibited 90% inhibition against trophozoites. In addition, gallic acid encapsulated nanoparticles showed reduced cytotoxicity towards MRC-5 compared to gallic acid, which evidenced that natural product nanoencapsulation in polymeric nanoparticles could play an important role in the delivery of natural products.
Herein, we report green synthesized nanoparticles based on stabilization by plant gums, loaded with citrus fruits flavonoids Hesperidin (HDN) and Naringin (NRG) as novel antimicrobial agents against brain-eating amoebae and multi-drug resistant bacteria. Nanoparticles were thoroughly characterized by using zetasizer, zeta potential, atomic force microscopy, ultravoilet-visible and Fourier transform-infrared spectroscopic techniques. The size of these spherical nanoparticles was found to be in the range of 100-225 nm. The antiamoebic effects of these green synthesized Silver and Gold nanoparticles loaded with HDN and NRG were tested against Acanthamoeba castellanii and Naegleria fowleri, while antibacterial effects were evaluated against methicillin-resistant Staphylococcus aureus (MRSA) and neuropathogenic Escherichia coli K1. Amoebicidal assays revealed that HDN loaded Silver nanoparticles stabilized by gum acacia (GA-AgNPs-HDN) quantitatively abolished amoeba viability by 100%, while NRG loaded Gold nanoparticles stabilized by gum tragacanth (GT-AuNPs-NRG) significantly reduced the viability of A. castellanii and N. fowleri at 50 µg per mL. Furthermore, these nanoparticles inhibited the encystation and excystation by more than 85%, as well as GA-AgNPs-HDN only completely obliterated amoeba-mediated host cells cytopathogenicity. Whereas, GA-AgNPs-HDN exhibited significant bactericidal effects against MRSA and E. coli K1 and reduced bacterial-mediated host cells cytotoxicity. Notably, when tested against human cells, these nanoparticles showed minimal (23%) cytotoxicity at even higher concentration of 100 µg per mL as compared to 50 µg per mL used for antimicrobial assays. Hence, these novel nanoparticles formulations hold potential as therapeutic agents against infections caused by brain-eating amoebae, as well as multi-drug resistant bacteria, and recommend a step forward in drug development.
The term 'tropical' refers to the region of the Earth lying between the Tropic of Cancer and the Tropic of Capricorn. Located between these equatorial parallels demarcating the Torrid Zone are several underdeveloped and developing countries: Thailand, the Philippines, Malaysia, Singapore, Indonesia, southern India, Sri Lanka, Brazil, Cuba, Ethiopia, Sudan and Nigeria, to name but a few considered to be 'tropical'. The climate in most of these countries is characterized by high temperatures and high humidity. The tropical climate and general state of socio-economic underdevelopment in such countries provide an ideal environment for pathogenic organisms, their vectors and intermediate hosts to flourish. Furthermore, the cultural habits and educational background of the people living in such countries expose them to pathogens and, when these people become infected, they readily become reservoirs for, or carriers of, those organisms. Ultimately, the adverse socioeconomic conditions of underdeveloped countries impede attempts to eradicate or control tropical diseases.