Evidence of Submicroscopic Plasmodium knowlesi Mono-Infection in Remote Indigenous Communities in Kelantan, Peninsular Malaysia.

Malaysia has maintained zero cases of indigenous human malaria since 2018. However, zoonotic malaria is still prevalent in underdeveloped areas and hard-to-reach populations. This study aimed to determine the prevalence of malaria among remote indigenous communities in Peninsular Malaysia. A cross-sectional survey was conducted in six settlements in Kelantan state, from June to October 2019. Blood samples were tested for malaria using microscopy and nested polymerase chain reaction (nPCR) targeting the Plasmodium cytochrome c oxidase subunit III (cox3) gene. Of the 1,954 individuals who appeared healthy, no malaria parasites were found using microscopy. However, nPCR revealed seven cases of Plasmodium knowlesi mono-infection (0.4%), and six out of seven infections were in the group of 19 to 40 years old (P = 0.026). No human malaria species were detected by nPCR. Analysis of the DNA sequences also showed high similarity that reflects common ancestry to other P. knowlesi isolates. These findings indicate low submicroscopic P. knowlesi infections among indigenous communities in Malaysia, requiring PCR-based surveillance to support malaria control activities in the country.

Naturally acquired antibody response to Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Peninsular Malaysia.

Malaria remains a public health problem in many parts of the world. In Malaysia, the significant progress towards the national elimination programme and effective disease notification on malaria has resulted in zero indigenous human malaria cases since 2018. However, the country still needs to determine the extent of malaria exposure and transmission patterns, particularly in high-risk populations. In this study, a serological method was used to measure transmission levels of Plasmodium falciparum and Plasmodium vivax among indigenous Orang Asli communities in Kelantan, Peninsular Malaysia. A community-based cross-sectional survey was conducted in three Orang Asli communities (i.e., Pos Bihai, Pos Gob, and Pos Kuala Betis) in Kelantan from June to July 2019. Antibody responses to malaria were assessed by enzyme-linked immunosorbent assay (ELISA) using two P. falciparum (PfAMA-1 and PfMSP-119) and two P. vivax (PvAMA-1 and PvMSP-119) antigens. Age-adjusted antibody responses were analysed using a reversible catalytic model to calculate seroconversion rates (SCRs). Multiple logistic regression was used to investigate factors associated with malaria exposure. The overall malaria seroprevalence was 38.8% for PfAMA-1, 36.4% for PfMSP-119, 2.2% for PvAMA-1, and 9.3% for PvMSP-119. Between study areas, the proportion of seropositivity for any P. falciparum and P. vivax antigens was significantly highest in Pos Kuala Betis with 34.7% (p < 0.001) and 13.6% (p < 0.001), respectively. For all parasite antigens except for PvAMA-1, the proportion of seropositive individuals significantly increased with age (all p < 0.001). Based on the SCR, there was a higher level of P. falciparum transmission than P. vivax in the study area. Multivariate regression analyses showed that living in Pos Kuala Betis was associated with both P. falciparum (adjusted odds ratio [aOR] 5.6, p < 0.001) and P. vivax (aOR 2.1, p < 0.001) seropositivities. Significant associations were also found between age and seropositivity to P. falciparum and P. vivax antigens. Analysis of community-based serological data helps describe the level of transmission, heterogeneity, and factors associated with malaria exposure among indigenous communities in Peninsular Malaysia. This approach could be an important adjunct tool for malaria monitoring and surveillance in low malaria transmission settings in the country.

Comparison of Two Serological Assays in Detecting Strongyloides Infection in Immunocompromised Patients.

Strongyloides infection may develop into fatal hyperinfection and dissemination syndrome in immunocompromised hosts. Despite suboptimal specificity issues, the detection of IgG antibodies by ELISA has been central in the serodiagnosis of Strongyloides infection. Recently, an IgG4-based lateral-flow test (SsRapid) using recombinant NIE (rNIE) protein with good diagnostic performance has been reported. This study assessed the result concordance between a commercial IgG-ELISA and the SsRapid. Additionally, we determined the Strongyloides seroprevalence and its association with clinical manifestations. Immunocompromised patients (N = 200) were from Hospital Canselor Tuanku Muhriz, Kuala Lumpur, Malaysia, and were diagnosed with HIV/AIDS, hematological malignancy, and solid organ cancers. Their plasma samples were tested using a commercial IgG-ELISA and SsRapid. A fair concordance (κ = 0.27-0.33; P < 0.05) among the tests was demonstrated. The SsRapid exhibited a significantly higher (P < 0.05) seroprevalence (10.5% [21/200]) compared with IgG-ELISA (7.5% [15/200]). After adsorption with rNIE, all SsRapid-positive samples tested negative with the rapid test, thus showing binding specificity. There was no significant association with clinical manifestations. This study revealed that SsRapid is a useful diagnostic tool for Strongyloides infection, and there is a notable seroprevalence among the immunocompromised patients.

Malaria Cases in a Tertiary Hospital in Kuala Lumpur, Malaysia: A 16-Year (2005-2020) Retrospective Review.

While there has been a tremendous decline in malaria disease burden in the remote parts of Malaysia, little is known about malaria incidence in its urban localities. This study aimed to analyze trends of malaria cases in urban Kuala Lumpur, Malaysia. All suspected cases presented to a university hospital in Kuala Lumpur from January 2005 to December 2020 were examined by microscopy. Infection status was analyzed using descriptive statistics and curve estimation analysis. Of 3105 blood films examined, 92 (3%) were microscopically confirmed malaria cases. Plasmodium vivax infections accounted for the majority (36.9%) of all malaria cases. Nearly half (47.8%) of cases were found among foreign cases (p < 0.001). The majority of foreign cases were male (86.4%) and came from Southeast Asian countries (65.9%). The curve estimation analysis showed significant decreases in malaria cases due to P. vivax (R2 = 0.598; p < 0.001) and Plasmodium falciparum (R2 = 0.298, p = 0.029), but increases for Plasmodium knowlesi (R2 = 0.325, p = 0.021) during the 16 years. This study showed that malaria incidence in urban Kuala Lumpur is low and has remained stable since 2005. However, P. knowlesi has played a significant role in the increase in overall malaria in the area, highlighting the importance of continued vigilance and improved surveillance.

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.

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.

First molecular identification of Entamoeba moshkovskii in Malaysia.

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.

Giardiasis among different tribes of Orang Asli in Malaysia: highlighting the presence of other family members infected with Giardia intestinalis as a main risk factor.

The flagellate protozoan parasite, Giardia intestinalis, is widely distributed throughout the world with a high prevalence in developing countries in the tropics and subtropics, including Malaysia. Approximately 200 million people are infected with the parasite globally, with 500,000 new cases reported annually. This cross-sectional study was conducted among three tribes of Orang Asli communities in Selangor, Perak and Pahang states of Malaysia. The main objective was to determine the prevalence of and risk factors for giardiasis. Stool samples were collected from 500 individuals aged between 2 and 74 years (males=219, females=281). The samples were examined with formalin-ether sedimentation and trichrome staining techniques. Socioeconomic data were collected through a pre-tested questionnaire. The overall prevalence of giardiasis was 20.0% with the highest prevalence in the Proto-Malays (33.3%) followed by Negritos (20.1%) and Senois (10.4%). The positive cases showed a decrease with increasing age and most of the positive cases were observed in individuals less than 24 years old. Males had significantly higher prevalence than females (χ(2)=5.283, P=0.022). Logistic regression analysis of the overall population studied and the Senoi tribe confirmed that being a child aged less than 15 years, being male, the consumption of raw vegetables and the presence of other family members infected with G. intestinalis were the main risk factors for giardiasis. The presence of other family members infected with G. intestinalis was the only risk factor highlighted in the Proto-Malay and Negrito tribes. Diarrhoea was significantly associated with giardiasis. However, the cause and effect relationship has yet to be determined. Thus, screening family members and treating the infected individuals are the main strategies that should be adopted by the public health authority in combating this infection in Orang Asli communities as well as health education regarding good personal and food hygiene practises.