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Fulltext Comparison of the Anaerocult A and the oil blocking methods for the in vitro cultivation of Entamoeba histolytica

Mohd Shah NA, Wan Abdul Wahab WN, Mohd Nawi SF, Mohd-Zain Z, Latif B, Suhaimi R

Malays J Pathol, 2015 Dec;37(3):271-4.
PMID: 26712674 MyJurnal

Entamoeba histolytica, the causative agent for human amoebiasis, is among the most deadly parasites, accounting for the second highest mortality rate among parasitic diseases. Because this parasite dwells in low oxygen tension, for its cultivation, microaerophilic conditions are required to mimick the human gut environment. Several methods developed for optimal growth environment are commercially available and some are conventionally modified in-house which include the Anaerocult A and oil blocking preparation methods. This study was undertaken to compare the reliability of the Anaerocult A and the oil blocking methods in generating anaerobic environment for cultivation of E. histolytica. The trophozoites of E. histolytica HM1: IMSS strains were axenically cultivated in TYI-S-33 medium in culture incubated anaerobically by using Anaerocult A (Merck) and mineral oil blocking method. The outcomes of both methods were determined by the minimum inhibitory concentration (MIC) of metronidazole against E. histolytica by giving a score to the growth pattern of the trophozoites. The reliability of both methods was assessed based on susceptibility testing of E. histolytica to metronidazole. The MIC obtained by both anaerobic condition methods was 6.25 ug/ ml, thus showing that oil-blocking method is comparable to the Anaerocult A method and therefore, considered as a reliable method for generating an anaerobic environment for the cultivation of E. histolytica.

Matched MeSH terms: Entamoeba histolytica*
Fulltext Infeksi entamoeba histolytica di kalangan kanak-kanak orang asli di Pos Lenjang, Pahang

Hartini Yusof, Mohamed Kamel Abd Ghani

Jurnal Sains Kesihatan Malaysia, 2009;7(1):59-64.
MyJurnal

Infeksi Entamoeba histolytica adalah tersebar di seluruh dunia dengan prevalens infeksi yang lebih tinggi di kalangan masyarakat terpinggir termasuk Orang Asli yang tinggal di kawasan tropika dan subtropika. Seramai 71 orang kanak-kanak Orang Asli dari Pos Lenjang, Pahang telah terlibat di dalam kajian ini. Bagi kajian yang lebih terperinci, kumpulan kanak-kanak ini telah dibahagikan menurut jantina dan umur. Sampel feses dikumpul dan setiap sampel diperiksa bagi pengenalpastian Entamoeba histolytica dengan menggunakan 3 jenis teknik diagnostik yang berbeza iaitu teknik apusan langsung, konsentrasi formalin-eter dan perwarnaan trikrom. Prevalens infeksi protozoa usus Entamoeba histolytica di kalangan kanak-kanak Orang Asli di Pos Lenjang, Pahang adalah tinggi iaitu 22.5%. Dari segi jantina, prevalens infeksi lebih tinggi di kalangan kanak-kanak perempuan (32.5%) berbanding kanak-kanak lelaki (9.7%). Infeksi juga didapati lebih kerap berlaku di kalangan kanak-kanak yang bersekolah (32.4%) berbanding kanak-kanak prasekolah (11.8%). Prevalens infeksi Entamoeba histolytica yang tinggi di kalangan kanak-kanak Orang Asli di Pos Lenjang, Pahang adalah berhubung kait dengan pelbagai faktor termasuk status sosioekonomi yang rendah, budaya, kekurangan kemudahan asas dan tahap pengetahuan mengenai penjagaan kesihatan serta kebersihan diri yang rendah.

Matched MeSH terms: Entamoeba; Entamoeba histolytica
Infeksi Entamoeba histolytica dan/atau Entamoeba dispar dalam kalangan kanak-kanak Orang Asli di Pos Sungai Rual, Jeli, Kelantan

Mariam Ahmad Zawawi, Mohamed Kamel Abd Ghani, Gopal G, Hidayatulfathi Othman, Hartini Yusof, Norhisham Haron

Sains Malaysiana, 2012;41:1095-1098.

entamoeba histolytica dan/atau entamoeba dispar merupakan protozoa usus yang mempunyai prevalens infeksi yang tinggi dalam kalangan masyarakat pedalaman terutamanya masyarakat Orang Asli. Ia tersebar secara meluas di kawasan tropika dan subtropika serta di negara membangun berbanding negara maju. Sebanyak 111 sampel feses kanak-kanak Orang Asli daripada suku kaum Jahai telah diterima dan disaring untuk entamoeba histolytica dan/atau entamoeba dispar menggunakan kaedah apusan langsung yang memberi hasil positif terhadap 43 sampel atau 38.7%. Oleh sebab amaun sampel yang diterima adalah sedikit, hanya 66 sampel feses sahaja yang dapat diperiksa menggunakan tiga jenis teknik diagnostik berbeza iaitu apusan langsung, kepekatan formalin-eter dan pewarnaan trikrom. Hasil kajian mendapati, prevalens yang tinggi bagi infeksi entamoeba histolytica dan/atau entamoeba dispar iaitu 50% menggunakan
ketiga-tiga teknik diagnosis. Prevalens infeksi yang tinggi juga turut ditunjukkan pada kanak-kanak perempuan iaitu 62.5% berbanding kanak-kanak lelaki 30.8% (p<0.05). Selain itu, daripada segi umur, kanak-kanak yang berumur 7-9 tahun adalah lebih terdedah kepada infeksi entamoeba histolytica dan/atau entamoeba dispar dengan prevalens 60.7% (p>0.05). Teknik pewarnaan trikrom menunjukkan pengesanan 100% ke atas infeksi entamoeba histolytica dan/atau entamoeba dispar, diikuti teknik kepekatan formalin-eter 78.8% dan apusan langsung 72.7%. Prevalens infeksi entamoeba histolytica dan/atau entamoeba dispar yang tinggi dalam kalangan kanak-kanak Orang Asli di Pos Sungai Rual ini berhubungkait dengan pelbagai faktor iaitu status sosioekonomi yang rendah, kekurangan pengetahuan tentang penjagaan kesihatan serta kebersihan diri yang rendah. Peningkatan prevalens infeksi dalam kajian ini juga menunjukkan pentingnya penggunaan teknik diagnostik yang lebih berkesan dalam pemeriksaan rutin bagi mendapatkan hasil diagnosis yang lebih tepat.

Matched MeSH terms: Entamoeba; Entamoeba histolytica
Oral parasitic protozoan Entamoeba gingivalis in periodontal disease patients, northeastern Thailand

Boonsuya A, Chitpitaklert P, Pechdee P, Srithongklang W, Thanchonnang C, La N, et al.

Trop Biomed, 2023 Dec 01;40(4):471-477.
PMID: 38308835 DOI: 10.47665/tb.40.4.013

Entamoeba gingivalis is present in the oral cavity of humans and is associated with periodontal disease. Consequently, this study aimed to comprehensively investigate the E. gingivalis infection and the associated risk factors among individuals suffering from periodontal conditions. A cross-sectional descriptive study was carried out within a cohort of periodontal patients. Dental plaque specimens were meticulously collected and subsequently subjected to thorough examination using the polymerase chain reaction (PCR)-based technique targeting the small subunit ribosomal RNA (SrRNA) gene of the organism. The occurrence of risk factors for E. gingivalis infection was analyzed by the chi-square test and binary logistic regression. Out of the 230 participants, 60 were clinically diagnosed with periodontitis, while 170 were afflicted with gingivitis. Out of the 230 patients, 25 (10.9%) tested positive for E. gingivalis infections. An in-depth analysis unveiled that a significant majority of infections were recorded within subgroups characterized by a marital status (15.45%), manifestation of periodontitis (25.00%), and concomitant presence of underlying disease (20.83%). Furthermore, the high risk factor associated with E. gingivalis infection was the female (ORadj = 13.65, 95% CI = 1.08-173.21), followed by periodontitis (ORadj = 3.30, 95% CI = 1.21-9.00), respectively. The study employs a molecular diagnostic approach to screen for E. gingivalis enrichment within a subset of periodontal patients with advancing disease. The findings emphasize the necessity for further research to elucidate the pathogenesis of E. gingivalis and advocate for vigilant surveillance within a substantial population of periodontal patients.

Matched MeSH terms: Entamoeba*
Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples

Foo PC, Chan YY, See Too WC, Tan ZN, Wong WK, Lalitha P, et al.

J Med Microbiol, 2012 Sep;61(Pt 9):1219-1225.
PMID: 22556327 DOI: 10.1099/jmm.0.044552-0

Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

Matched MeSH terms: Entamoeba/classification*; Entamoeba/genetics*; Entamoeba/isolation & purification; Entamoeba histolytica/classification*; Entamoeba histolytica/genetics*; Entamoeba histolytica/isolation & purification
Prevalence and risk factors for infection with Entamoeba histolytica/dispar/ moshkovskii complex in people living in the slightest and outermost islands of Indonesia

Junaidi -, Asmaruddin MS, Kurrohman T, Nurdin -, Khazanah W

Trop Biomed, 2023 Jun 01;40(2):160-164.
PMID: 37650401 DOI: 10.47665/tb.40.2.005

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.

Matched MeSH terms: Entamoeba histolytica*
First molecular identification of Entamoeba moshkovskii in Malaysia

Anuar TS, Al-Mekhlafi HM, Ghani MK, Azreen SN, Salleh FM, Ghazali N, et al.

Parasitology, 2012 Oct;139(12):1521-5.
PMID: 22939193 DOI: 10.1017/S0031182012001485

Entamoeba moshkovskii and Entamoeba dispar are microscopically indistinguishable from the pathogenic species Entamoeba histolytica. Although sporadic cases of human infection with E. moshkovskii have been reported, the amoeba is still considered primarily as a free-living amoeba. A cross-sectional study was carried out among Orang Asli communities in 3 different states of Peninsular Malaysia. Fecal samples were examined by formalin-ether sedimentation and trichrome staining techniques and then single-round PCR assay was used to detect E. moshkovskii. Out of 500 fecal samples examined microscopically, 93 (18·6%) samples were positive for E. histolytica/E. dispar/E. moshkovskii complex cysts and/or trophozoites. PCR products were detected in 106 fecal samples. E. moshkovskii isolates were detected in 13 (12·3%) fecal samples. Of the 13 E. moshkovskii-positive samples, 5 were of single isolation of E. moshkovskii, 6 were also positive for E. dispar, and only 2 samples were positive for E. dispar and E. histolytica. Moreover, 3 E. moshkovskii-positive samples were collected from symptomatic individuals while the remaining 10 samples were from asymptomatic subjects. This is the first report on the identification of E. moshkovskii in Malaysia. Further studies are needed to confirm the pathogenicity of E. moshkovskii infection and determine the epidemiology among Orang Asli communities in Malaysia.

Matched MeSH terms: Entamoeba/classification; Entamoeba/genetics*; Entamoeba/isolation & purification
Comparison of protein-free defined media, and effect of L-cysteine and ascorbic acid supplementation on viability of axenic Entamoeba histolytica

Wong WK, Tan ZN, Lim BH, Mohamed Z, Olivos-Garcia A, Noordin R

Parasitol Res, 2011 Feb;108(2):425-30.
PMID: 20922423 DOI: 10.1007/s00436-010-2083-8

Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.

Matched MeSH terms: Entamoeba histolytica/cytology; Entamoeba histolytica/drug effects*; Entamoeba histolytica/physiology
Fulltext Molecular detection of Entamoeba histolytica and Entamoeba dispar infection among wild rats in Kuala Lumpur, Malaysia

Lau YL, Jamaiah I, Rohela M, Fong MY, Siti CO, Siti FA

Trop Biomed, 2014 Dec;31(4):721-7.
PMID: 25776597 MyJurnal

Entamoeba histolytica infection is the third-greatest parasitic disease responsible for death in the world. Wild rats harbouring E. histolytica can be the possible reservoir hosts for human amoebiasis. There were numerous studies on prevalence of intestinal parasites among wild rats in Malaysia but none has reported E. histolytica. Rats were captured from Sentul and Chow Kit areas, Kuala Lumpur, Malaysia. The preserved stool samples were used for microscopy examination and molecular analysis. Out of 137 samples collected, 12 were positive for E. histolytica / E. dispar / E. moshkovskii microscopically. Two E. histolytica (1.4%), 1 E. dispar (0.7%) and 6 mixed infections of E. histolytica and E. dispar (4.3%) were detected using PCR. This is the first report of molecular detection of E. histolytica/dispar infection among wild rats in Malaysia. This study provides useful information about the potential risks of zoonotic agents and the importance of developing control measures to prevent zoonotic transmission.

Matched MeSH terms: Entamoeba/classification*; Entamoeba/genetics; Entamoeba/isolation & purification*
Fulltext Use of molecular tools to distinguish Entamoeba histolytica and Entamoeba dispar infection among the aborigines in Cameron Highlands

Noor Azian MY, Lokman Hakim S, Maslawaty MN

Trop Biomed, 2006 Jun;23(1):31-6.
PMID: 17041549 MyJurnal

Amoebiasis is an infectious diseased caused by parasitic one-celled protozoan called Entamoeba histolytica. Numerous protozoa also can inhabit the gastro-intestinal tract of human. Majority of these protozoa are non-pathogenic commensals or only causes disease under certain circumstances. Morphologically, E. histolytica, the invasive form, share the same characteristic with the nonpathogenic form, E. dispar. Both strains can be distinguished by using DNA identification. Many previous researches in Malaysia only reported infection with E. histolytica infection. Therefore in this study we tried to classify infection among the aborigines in Cameron Highland as true E. histolytica or E. dispar by Nested Polymerase Chain Reaction (Nested PCR) and Restriction enzyme (RE) digestion. Results showed that 31 samples were positive by microscopic examination, however of these 28 (13.2%) samples were positive for E. histolytica and 12 (5.6%) samples were positive for E. dispar by molecular tools.

Matched MeSH terms: Entamoeba/genetics*; Entamoeba histolytica/genetics*
Development of a thermostabilised triplex LAMP assay with dry-reagent four target lateral flow dipstick for detection of Entamoeba histolytica and non-pathogenic Entamoeba spp

Foo PC, Chan YY, Mohamed M, Wong WK, Nurul Najian AB, Lim BH

Anal Chim Acta, 2017 May 08;966:71-80.
PMID: 28372729 DOI: 10.1016/j.aca.2017.02.019

This study highlighted the development of a four target nitrocellulose-based nucleic acid lateral flow immunoassay biosensor in a dry-reagent strip format for interpretation of double-labelled double-stranded amplicons from thermostabilised triplex loop-mediated isothermal amplification assay. The DNA biosensor contained two test lines which captured biotin and texas red labelled amplicons; a LAMP internal amplification control line that captured digoxigenin labelled amplicon; and a chromatography control line that validated the functionality of the conjugated gold nanoparticles and membrane. The red lines on detection pad were generated when the gold nanoparticles conjugated antibody bound to the fluorescein labelled amplicons, and the capture agents bound to their specific hapten on the other 5' end of the double-stranded amplicon. The applicability of this DNA biosensor was demonstrated using amoebiasis-causing Entamoeba histolytica simultaneously with the non-pathogenic but morphologically identical Entamoeba dispar and Entamoeba moshkovskii. The biosensor detection limit was 10 E. histolytica trophozoites, and revealed 100% specificity when it was evaluated against 3 medically important Entamoeba species and 75 other pathogenic microorganisms. Heat stability test showed that the biosensor was stable for at least 181 days at ambient temperature. This ready-to-use and cold-chain-free biosensor facilitated the post-LAMP analysis based on visualisation of lines on strip instead of observation of amplicon patterns in agarose gel.

Matched MeSH terms: Entamoeba/isolation & purification*; Entamoeba histolytica/isolation & purification*
First detection and molecular identification of Entamoeba bovis from Japanese cattle

Matsubayashi M, Matsuura Y, Nukata S, Daizi Y, Shibahara T, Teramoto I, et al.

Parasitol Res, 2018 Jan;117(1):339-342.
PMID: 29185030 DOI: 10.1007/s00436-017-5689-2

Thus far, Entamoeba species have been classified based on morphology such as the number of nuclei in mature cysts and their hosts. Using recently developed molecular tools, ruminant Entamoeba spp. are currently classified into four species/genotypes: E. bovis and Entamoeba ribosomal lineages (RL) 1, 2, and 4. However, the distribution or pathogenicity of ruminant Entamoeba has not been well documented. In the present study, we examined a total of 25 fecal and seven environmental samples collected from six farms in Japan from 2016 to 2017 by the floatation method and PCR and sequencing analyses. Consequently, we detected Entamoeba cysts in 18 of 25 cattle samples and four of the seven environmental samples, including soil and drinking water, by microscopic examinations. In sequential examinations, Entamoeba-positive cattle were found to shed cysts without any clinical symptoms for more than 8 months. By PCR for molecular identification, isolates in ten cattle and one soil sample were successfully sequenced and formed a cluster of E. bovis, which was separated from those of other Entamoeba species/genotypes such as RL1-4 in phylogenetic analysis. To our knowledge, this is the first report about E. bovis in Japan, and our results may implicate that E. bovis is not pathogenic.

Matched MeSH terms: Entamoeba/genetics; Entamoeba/isolation & purification*; Entamoeba/pathogenicity
Fulltext Antigenic membrane proteins of virulent variant of Entamoeba histolytica HM-1:IMSS

Kumarasamy G, Abdus Sani AA, Olivos-García A, Noordin R, Othman N

Pathog Glob Health, 2020 09;114(6):333-342.
PMID: 32536281 DOI: 10.1080/20477724.2020.1780402

Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.

Matched MeSH terms: Entamoeba histolytica*
Fulltext Real-time PCR assay in differentiating Entamoeba histolytica, Entamoeba dispar, and Entamoeba moshkovskii infections in Orang Asli settlements in Malaysia

Lau YL, Anthony C, Fakhrurrazi SA, Ibrahim J, Ithoi I, Mahmud R

Parasit Vectors, 2013;6(1):250.
PMID: 23985047 DOI: 10.1186/1756-3305-6-250

Amebiasis caused by Entamoeba histolytica is the third leading cause of death worldwide. This pathogenic amoeba is morphologically indistinguishable from E. dispar and E. moshkovskii, the non-pathogenic species. Polymerase chain reaction is the current method of choice approved by World Health Organization. Real-time PCR is another attractive molecular method for diagnosis of infectious diseases as post-PCR analyses are eliminated and turnaround times are shorter. The present work aimed to compare the results of Entamoeba species identification using the real-time assay against the established nested PCR method.

Matched MeSH terms: Entamoeba/classification*; Entamoeba/genetics; Entamoeba/isolation & purification*
Fulltext Different clinical outcomes of Entamoeba histolytica in Malaysia: does genetic diversity exist?

Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK, Azreen SN, Salleh FM, Ghazali N, et al.

Korean J Parasitol, 2013 Apr;51(2):231-6.
PMID: 23710093 DOI: 10.3347/kjp.2013.51.2.231

The present study was conducted to investigate the clinical outcomes of Entamoeba histolytica infection in symptomatic and asymptomatic Orang Asli (aborigine) communities in Malaysia. Examination was performed on 500 stool samples obtained from Orang Asli communities in 3 different states using formalin-ether concentration, trichrome staining, and single-round PCR techniques. Out of 500 stool samples, single infection of E. histolytica, Entamoeba dispar, and Entamoeba moshkovskii was identified in 3.2%, 13.4%, and 1%, respectively. In addition, 10 samples had mixed infections with E. histolytica and E. dispar. Six samples containing E. dispar were also positive for E. moshkovskii, and only 2 samples had E. histolytica in association with E. dispar and E. moshkovskii. Seventeen E. histolytica-positive samples were from symptomatic subjects, whereas the remaining 11 samples came from asymptomatic subjects. These findings suggest a predominant distribution of pathogenic potential of E. histolytica strains in this community. Therefore, further studies on genotyping of E. histolytica is required, to find out association between E. histolytica genotype and the outcome of the infection.

Matched MeSH terms: Entamoeba/classification*; Entamoeba/isolation & purification; Entamoeba/pathogenicity*
Fulltext Prevalence and risk factors associated with Entamoeba histolytica/dispar/moshkovskii infection among three Orang Asli ethnic groups in Malaysia

Shahrul Anuar T, M Al-Mekhlafi H, Abdul Ghani MK, Osman E, Mohd Yasin A, Nordin A, et al.

PLoS One, 2012;7(10):e48165.
PMID: 23133561 DOI: 10.1371/journal.pone.0048165

BACKGROUND: Entamoeba histolytica/Entamoeba dispar/Entamoeba moshkovskii infection is still prevalent in rural Malaysia especially among Orang Asli communities. Currently, information on prevalence of this infection among different ethnic groups of Orang Asli is unavailable in Malaysia. To contribute to a better comprehension of the epidemiology of this infection, a cross-sectional study aimed at providing the first documented data on the prevalence and risk factors associated with E. histolytica/E. dispar/E. moshkovskii infection was carried out among three Orang Asli ethnic groups (Proto-Malay, Negrito, and Senoi) in selected villages in Negeri Sembilan, Perak, and Pahang states, Malaysia.
METHODS/FINDINGS: Faecal samples were examined by formalin-ether sedimentation and trichrome staining techniques. Of 500 individuals, 8.7% (13/150) of Proto-Malay, 29.5% (41/139) of Negrito, and 18.5% (39/211) of Senoi were positive for E. histolytica/E. dispar/E. moshkovskii, respectively. The prevalence of this infection showed an age-dependency relationship, with higher rates observed among those aged less than 15 years in all ethnic groups studied. Multivariate analysis confirmed that not washing hands after playing with soils or gardening and presence of other family members infected with E. histolytica/E. dispar/E. moshkovskii were significant risk factors of infection among all ethnic groups. However, eating with hands, the consumption of raw vegetables, and close contact with domestic animals were identified as significant risk factors in Senoi.
CONCLUSIONS: Essentially, the findings highlighted that E. histolytica/E. dispar/E. moshkovskii parasites are still prevalent in Malaysia. Further studies using molecular approaches to distinguish the morphologically identical species of pathogenic, E. histolytica from the non-pathogenic, E. dispar and E. moshkovskii are needed. The establishment of such data will be beneficial for the public health authorities in the planning and implementation of specific prevention and control strategies of this infection in different Orang Asli ethnic groups in Malaysia.

Matched MeSH terms: Entamoeba/metabolism*; Entamoeba histolytica/metabolism*
Analysis of Entamoeba histolytica Membrane Proteome Using Three Extraction Methods

Ujang J, Sani AAA, Lim BH, Noordin R, Othman N

Proteomics, 2018 12;18(23):e1700397.
PMID: 30284757 DOI: 10.1002/pmic.201700397

Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC-ESI-MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.

Matched MeSH terms: Entamoeba histolytica
Molecular epidemiology of amoebiasis in Malaysia: highlighting the different risk factors of Entamoeba histolytica and Entamoeba dispar infections among Orang Asli communities

Anuar TS, Al-Mekhlafi HM, Abdul Ghani MK, Abu Bakar E, Azreen SN, Salleh FM, et al.

Int J Parasitol, 2012 Dec;42(13-14):1165-75.
PMID: 23123168 DOI: 10.1016/j.ijpara.2012.10.003

Currently, species-specific information on Entamoeba infections is unavailable in Malaysia and is restricted worldwide due to the re-description of pathogenic Entamoeba histolytica and non-pathogenic Entamoeba dispar and Entamoeba moshkovskii. Therefore, this cross-sectional study was conducted to provide the first known documented data on the true prevalence of these three species in western Malaysia using a molecular method. Another aim of this study was to determine the association of potential risk factors associated with each Entamoeba sp. A total of 500 stool samples from three Orang Asli tribes were randomly collected. The overall prevalence of E. histolytica, E. dispar and E. moshkovskii determined by microscopy was 18.6% (93/500). Molecular analysis revealed that while most Entamoeba-positive individuals were infected with E. dispar (13.4%), followed by E. histolytica (3.2%) and E. moshkovskii (1.0%), the present findings show low prevalence rates of mixed infections with E. histolytica and E. dispar (2%), E. dispar and E. moshkovskii (1.2%) and association infections of E. histolytica, E. dispar and E. moshkovskii (0.4%). Logistical regression analysis indicates that the dynamics of the transmission of the three Entamoeba spp. was different. Of six statistically significant variables observed in the univariate analysis, three were retained as significant risk factors for E. histolytica infection in the logistical regression model. These factors were (i) not washing hands after playing with soil or gardening (Odds ratio (OR)=4.7; 95% confidence level (CI)=1.38, 16.14; P=0.013), (ii) indiscriminate defecation in the river or bush (OR=5.7; 95% CI=1.46, 21.95; P=0.012) and (iii) close contact with domestic animals (OR=5.4; 95% CI=1.36, 2.51; P=0.017). However, subjects with family members who were infected with E. histolytica/E. dispar/E. moshkovskii (OR=3.8; 95 CI=2.11, 6.86; P<0.001) and those who consumed raw vegetables (OR=1.8; 95% CI=1.01, 3.23; P=0.047) were more likely to be infected with E. dispar. On the other hand, no associated factor was identified with E. moshkovskii infection. Nevertheless, diarrhoea (P=0.002) and other gastroenteritis symptoms (P<0.001) were only associated with E. histolytica infection. The present study provides new insight into the distribution and risk factors of E. histolytica, E. dispar and E. moshkovskii infections among Orang Asli communities in Malaysia. Identifying the different risk factors of E. histolytica and E. dispar infections will help in the planning specific strategies in the control and prevention of each infection in the communities. Moreover, it emphasises the need for molecular methods to determine the species-specific prevalence of Entamoeba spp.

Matched MeSH terms: Entamoeba/classification*; Entamoeba/isolation & purification
Fulltext Public health and clinical importance of amoebiasis in Malaysia: a review

Tengku SA, Norhayati M

Trop Biomed, 2011 Aug;28(2):194-222.
PMID: 22041740 MyJurnal

Entamoeba histolytica, the causative agent of human amoebiasis remains a significant cause of morbidity and mortality in developing countries and is responsible for up to 100,000 deaths worldwide each year. Entamoeba dispar, morphologically indistinguishable from E. histolytica is more common in humans in many parts of the world. Similarly Entamoeba moshkovskii, which was long considered to be a free-living amoeba is also morphologically identical to E. histolytica and E. dispar, and is highly prevalent in some E. histolytica endemic countries. Humans are the host of infection and there would not appear to be other meaningful animal reservoirs of E. histolytica. Entamoeba. histolytica can be present in sewage and contaminated water. The infection is mainly transmitted via ingestion of water or food contaminated by faeces containing E. histolytica cysts. Clinical features of amoebiasis range from asymptomatic colonization to amoebic dysentery and invasive extraintestinal amoebiasis, which is manifested most commonly in the form of abscesses in liver and lungs. The epidemiology of amoebiasis has dramatically changed since the separation of E. histolytica and E. dispar species and the worldwide prevalence of these species has not been estimated until recently. Morever, E. moshkovskii, another morphologically indistinguishable human parasitic Entamoeba was not mentioned or considered as a contributor to the prevalence figures in endemic areas. Amoebiasis is still a major health problem especially in aboriginal settlements and amongst people living in remote area in Malaysia. However, until now there is only one data currently available to indicate the true prevalence and incidence of E. histolytica and E. dispar. Further studies are needed to determine the burden of E. histolytica, E. dispar and E. moshkovskii infections in Malaysia. In the present review, we briefly summarize all methods use in diagnosing Entamoeba species, ranging from microscopic identification to molecular detection such as culture and isoenzyme analysis, antibody detection tests, antigen detection tests, immunochromatographic assays, conventional PCR, real-time PCR and loop-mediated isothermal amplification (LAMP).

Matched MeSH terms: Entamoeba/classification; Entamoeba/isolation & purification*
Fulltext Identification of Entamoeba histolytica trophozoites in fresh stool sample: comparison of three staining techniques and study on the viability period of the trophozoites

Tan ZN, Wong WK, Nik Zairi Z, Abdullah B, Rahmah N, Zeehaida M, et al.

Trop Biomed, 2010 Apr;27(1):79-88.
PMID: 20562817 MyJurnal

Entamoeba histolytica causes about 50 million infections worldwide with a death rate of over 100,000 annually. In endemic developing countries where resources are limited, microscopic examinations based on Wheatley trichrome staining is commonly used for diagnosis of intestinal amoebiasis. Other than being a time-consuming method, it must be performed promptly after stool collection as trophozoites disintegrate rapidly in faeces. The aim of this study was to compare the efficacies of Eosin-Y, Wheatley trichrome and Iodine stains in delineating the diagnostic features of the parasite, and subsequently to determine the suitable microscopy observation period for detection of erythrophagocytic and non-erythrophagocytic trophozoites spiked in semi-solid stool sample. Wheatley trichrome staining technique was performed using the standard method while the other two techniques were performed on the slides by mixing the respective staining solution with the spiked stool sample. One million of axenically cultured non-erythrophagocytic E. histolytica and erythrophagocytic E. histolytica were separately spiked into 2 g of fresh semisolid faeces. Percentage viability of the trophozoites in the spiked stool sample was determined at 30 minute intervals for eight hours using the 0.4% Trypan blue exclusion method. The results showed that Eosin-Y and Wheatley trichrome stained the karyosome and chromatin granules better as compared to Iodine stain. The percentage viability of non-erythrophagocytic trophozoites decreased faster than the erythrophagocytic form in the first 5 hours and both dropped to ~10% in the 6th hour spiked sample. In conclusion, Eosin-Y staining technique was found to be the easiest to perform, most rapid and as accurate as the commonly used Wheatley trichrome technique; Eosin-Y stained slide sealed with DPX could also be kept as a permanent record. A period not exceeding 6 hours after stool collection was found to be the most suitable in order to obtain good microscopy results of viable trophozoites.

Matched MeSH terms: Entamoeba histolytica/cytology; Entamoeba histolytica/isolation & purification*

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