In Vitro Cytopathogenic Activities of Acanthamoeba T3 and T4 Genotypes on HeLa Cell Monolayer.

Amoebic keratitis and encephalitis are mainly caused by free-living amoebae of the genus Acanthamoeba, which consists of both pathogenic and nonpathogenic species. The global distribution, amphizoic properties and the severity of the disease caused by Acanthamoeba species have inspired the scientific community to put more effort into the isolation of Acanthamoeba, besides exploring the direct and indirect parameters that could signify a pathogenic potential. Therefore, this study was performed to characterize the pathogenic potential of Acanthamoeba isolated from contact lens paraphernalia and water sources in Malaysia. Various methodologies were utilized to analyze the thermotolerance and osmotolerance, the secretion level of proteases and the cytopathic effect of trophozoites on the cell monolayer. In addition, the in vitro cytopathogenicity of these isolates was assessed using the LDH-release assay. A total of 14 Acanthamoeba isolates were classified as thermo- and osmotolerant and had presence of serine proteases with a molecular weight of 45-230 kDa. Four T4 genotypes isolated from contact lens paraphernalia recorded the presence of serine-type proteases of 107 kDa and 133 kDa. In contrast, all T3 genotypes isolated from environmental samples showed the presence of a 56 kDa proteolytic enzyme. Remarkably, eight T4 and a single T3 genotype isolates demonstrated a high adhesion percentage of greater than 90%. Moreover, the use of the HeLa cell monolayer showed that four T4 isolates and one T3 isolate achieved a cytopathic effect in the range of 44.9-59.4%, indicating an intermediate-to-high cytotoxicity level. Apart from that, the LDH-release assay revealed that three T4 isolates (CL5, CL54 and CL149) and one T3 isolate (SKA5-SK35) measured an exceptional toxicity level of higher than 40% compared to other isolates. In short, the presence of Acanthamoeba T3 and T4 genotypes with significant pathogenic potential in this study reiterates the essential need to reassess the functionality of other genotypes that were previously classified as nonpathogenic isolates in past research.

Acanthamoeba species isolated from marine water in Malaysia exhibit distinct genotypes and variable physiological properties.

The present study identifies the Acanthamoeba genotypes and their pathogenic potential in five marine waters in Malaysia. Fifty water samples were collected between January and May 2019. Physical parameters of water quality were measured in situ, whereas chemical and microbiological analyses were conducted in the laboratory. All samples had undergone filtration using nitrocellulose membrane and were tested for Acanthamoeba using cultivation and polymerase chain reaction by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using physiological tolerance tests. Thirty-six (72.0%) samples were positive for Acanthamoeba. Total coliforms (p = 0.013) and pH level (p = 0.023) displayed significant correlation with Acanthamoeba presence. Phylogenetic analysis showed that 27 samples belonged to genotype T4, four (T11), two (T18) and one from each genotype T5, T15 and T20. Thermo- and osmo-tolerance tests signified that three (8.3%) Acanthamoeba strains displayed highly pathogenic attributes. This study is the first investigation in Malaysia describing Acanthamoeba detection in marine water with molecular techniques and genotyping. The study outcomes revealed that the marine water in Malaysia could be an integral source of Acanthamoeba strains potentially pathogenic in humans. Thus, the potential risk of this water should be monitored routinely in each region.

In vitro effects of multi-purpose contact lens disinfecting solutions towards survivability of Acanthamoeba genotype T4 in Malaysia.

The incidence of Acanthamoeba keratitis has been increasing since the previous decades, especially among contact lens users. This infection is majorly caused by the use of ineffective contact lens disinfecting solution. Thus, this study was conducted to evaluate the in vitro effects of multi-purpose disinfecting solutions (MPDS) against Acanthamoeba trophozoites and cysts. Acanthamoeba genotype T4 isolated from contact lens paraphernalia and an environmental strains were propagated for trophozoite or cyst-containing culture and adjusted in final concentration of 1 × 105 cells/ml. Amoebicidal and cysticidal assays were conducted by incubating trophozoites and cysts with OPTI-FREE® Express®, ReNu® Fresh™, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive according to the manufacturer's minimum recommended disinfectant time (MMRDT) for up to 12 h at 30 °C. Trypan blue hemocytometer-based microscopic counts determined amoebicidal and cysticidal effects. The viability of Acanthamoeba trophozoites and cysts was confirmed by re-inoculated them in the 1.5% non-nutrient agar plates. It was found that none of the MPDS showed amoebicidal and cysticidal effects during the MMRDT. However, OPTI-FREE® Express® demonstrated a significant differences in average cell reduction for both stages within MMRDT. When subjected to 12 h exposure, both OPTI-FREE® Express® and ReNu® Fresh™ led to significant reduction in the number of trophozoite and cyst cells. Notably, Complete® Multi-Purpose Solution and AVIZOR Unica® Sensitive did appreciably improve the solution effectiveness towards trophozoite cells when incubated for 12 h. All MPDS were largely ineffective, with 100% survival of all isolates at MMRDT, while OPTI-FREE® Express® showed limited amoebicidal activity against the contact lens paraphernalia isolate, however, it was more against the environmental strains after 12 h incubation time. The commercially available MPDS employed in this research offered minimal effectiveness against the protozoa despite the contact time. Improvement or development of new solution should consider the adjustment of the appropriate disinfectant concentration, adequate exposure time or the incorporation of novel chemical elements, which are effective against Acanthamoeba for accelerated disinfecting and more reduction of potential exposure of contact lens users to Acanthamoeba keratitis.

Occurrence and molecular characterisation of Acanthamoeba isolated from recreational hot springs in Malaysia: evidence of pathogenic potential.

This study aimed to identify the Acanthamoeba genotypes and their pathogenic potential in five recreational hot springs in Peninsular Malaysia. Fifty water samples were collected between April and September 2018. Physical parameters of water quality were measured in situ while chemical and microbiological analyses were performed in the laboratory. All samples were filtered through the nitrocellulose membrane and tested for Acanthamoeba using both cultivation and polymerase chain reaction (PCR) by targeting the 18S ribosomal RNA gene. The pathogenic potential of all positive isolates was identified using thermo- and osmotolerance tests. Thirty-eight (76.0%) samples were positive for Acanthamoeba. Water temperature (P = 0.035), chemical oxygen demand (P = 0.026), sulphate (P = 0.002) and Escherichia coli (P < 0.001) were found to be significantly correlated with the presence of Acanthamoeba. Phylogenetic analysis revealed that 24 samples belonged to genotype T4, nine (T15), two (T3) and one from each genotype T5, T11 and T17. Thermo- and osmotolerance tests showed that 6 (15.79%) of the Acanthamoeba strains were highly pathogenic. The existence of Acanthamoeba in recreational hot springs should be considered as a health threat among the public especially for high-risk people. Periodic surveillance of hot spring waters and posting warning signs by health authorities is recommended to prevent disease related to pathogenic Acanthamoeba.

Different clinical outcomes of Entamoeba histolytica in Malaysia: does genetic diversity exist?

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.

Evaluation of formalin-ether sedimentation and trichrome staining techniques: its effectiveness in detecting Entamoeba histolytica/dispar/moshkovskii in stool samples.

This study was conducted to evaluate two routinely microscopic diagnostic methods in comparison with single-round PCR assay as the reference technique to detect Entamoeba histolytica/dispar/moshkovskii. Examination was performed on 500 stool samples obtained from Orang Asli communities in different states of Malaysia using formalin-ether sedimentation, trichrome staining and single-round PCR techniques. Ninety-three stool samples were detected E. histolytica/dispar/moshkovskii positive by routine microscopy, while single-round PCR detected 106 positive samples. Additional positives detected by PCR assay were eventually confirmed to be negative by both microscopic techniques. Detection rate of E. histolytica/dispar/moshkovskii was highest in combination techniques (18.6%), followed by trichrome staining (13.4%) and formalin-ether sedimentation (11.2%) techniques. Single-round PCR detected 21.2% of the stool samples. The sensitivity and specificity of formalin-ether sedimentation and trichrome staining techniques compared to the reference technique were 31.1% (95% CI: 29.0-36.0) and 94.2% (95% CI: 89.8-98.9), and 53.8% (95% CI: 46.0-76.2) and 97.5% (95% CI: 92.8-99.1), respectively. However, the sensitivity [59.4% (95% CI: 48.9-78.5)] of the method increased when both techniques were performed together, but the specificity decreased to 92.4% (95% CI: 81.0-98.0). The agreement between the reference technique, trichrome staining and combination techniques were statistically significant by Kappa statistics (trichrome staining: K = 0.592, p < 0.05; combination techniques: K = 0.543, p < 0.05). Hence, the combination technique is recommended to be used as a screening method in the diagnosis of E. histolytica/dispar/moshkovskii infections either for clinical or epidemiological study.

Prevalence and risk factors associated with Entamoeba histolytica/dispar/moshkovskii infection among three Orang Asli ethnic groups in Malaysia.

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.

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

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.