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Fulltext Synthesis, characterization and biological properties of copper(II) and zinc(II) complexes against blood-stage Plasmodium falciparum

Lai, Jing-Wei, Ng, Chew-Hee, Lim, Yvonne Ai-Lian, Mohd Jamil Maah

Malaysian Journal of Medicine and Health Sciences, 2019;15(102):1-1.
MyJurnal

Introduction: The spread of multidrug-resistant malaria parasite – Plasmodium sp. to commercially available antimalarial drugs, i.e. artemisinin-based combination therapies (ACTs) and chloroquine (CQ), has become a global treat to eliminate malaria. To limit the impact of antimalarial drug resistance, a new potent and affordable alternative is urgently needed. A number of metal-based compounds (metallodrugs) have been found active against Plasmodium falciparum, the species that causes potentially fatal cerebral malaria, as they are ease in ligand grafting of multi-functional groups. Ferroquine (FQ) is one of the metalloantimalarial drugs that is currently undergoing clinical trials. Methods: In this study, a series of ternary copper(II) and zinc(II) complexes – Cu(phen)(edda) 1, Zn(phen)(edda) 2, [Cu(phen)(cdmg)] NO3 3 and [Zn(phen)(c-dmg)]NO3 4 were synthesized and characterized by the following tests: Fourier transformed infrared (FTIR), CHN elemental analysis, UV-Vis spectroscopy, molar conductivity and magnetic susceptibility measurements. Results: In vitro hemolytic and antimalarial assays using SYBR Green I dye were done to determine the biological properties of these complexes. Preliminary biological evaluation demonstrated that all the complexes 1, 2, 3 and 4 exhibit toxicity against the sensitive blood-stage Plasmodium falciparum 3D7 with IC50 in μM range. Conclusion: Thus, metal complex is a potentially viable candidate as antimalarial drug to overcome the emergence of drug resistance.
Heterologous expression of Plasmodium vivax apical membrane antigen 1 (PvAMA1) for binding peptide selection

Chew CH, Lim YAL, Chua KH

PeerJ, 2017;5:e3794.
PMID: 28929019 DOI: 10.7717/peerj.3794

BACKGROUND: Plasmodium is an obligate intracellular parasite. Apical membrane antigen 1 (AMA1) is the most prominent and well characterized malarial surface antigen that is essential for parasite-host cell invasion, i.e., for sporozoite to invade and replicate within hepatocytes in the liver stage and merozoite to penetrate and replicate within erythrocytes in the blood stage. AMA1 has long served as a potent antimalarial drug target and is a pivotal vaccine candidate. A good understanding of the structure and molecular function of this Plasmodium protein, particularly its involvement in host-cell adhesion and invasion, is of great interest and hence it offers an attractive target for the development of novel therapeutics. The present study aims to heterologous express recombinant Plasmodium AMA1 ectodomain of P. vivax (rPvAMA1) for the selection of binding peptides.
METHODS: The rPvAMA1 protein was heterologous expressed using a tag-free Profinity eXact(TM) system and codon optimized BL21-Codon Plus (DE3)-RIL Escherichia coli strain and further refolded by dialysis for renaturation. Binding peptides toward refolded rPvAMA1 were panned using a Ph.D.-12 random phage display library.
RESULTS: The rPvAMA1 was successfully expressed and refolded with three phage-displayed dodecapeptides designated as PdV1 (DLTFTVNPLSKA), PdV2 (WHWSWWNPNQLT), and PdV3 (TSVSYINNRHNL) with affinity towards rPvAMA1 identified. All of them exhibited positive binding signal to rPvAMA1 in both direct phage assays, i.e., phage ELISA binding assay and Western blot binding assay.
DISCUSSION: Phage display technology enables the mapping of protein-protein interactions based on a simple principle that a library of phage particles displaying peptides is used and the phage clones that bind to the target protein are selected and identified. The binding sites of each selected peptides toward PvAMA1 (Protein Data Bank, PDB ID: 1W8K) were in silico predicted using CABS-dock web server. In this case, the binding peptides provide a valuable starting point for the development of peptidomimetic as antimalarial antagonists directed at PvAMA1.
Fulltext Expression of Ascaris lumbricoides putative virulence-associated genes when infecting a human host

Mohd-Shaharuddin N, Lim YAL, Ngui R, Nathan S

Parasit Vectors, 2021 Mar 23;14(1):176.
PMID: 33757548 DOI: 10.1186/s13071-021-04680-y

BACKGROUND: Ascaris lumbricoides is the most common causative agent of soil-transmitted helminth infections worldwide, with an estimated 450 million people infected with this nematode globally. It is suggested that helminths are capable of evading and manipulating the host immune system through the release of a spectrum of worm proteins which underpins their long-term survival in the host. We hypothesise that the worm overexpresses these proteins when infecting adults compared to children to cirvumvent the more robust defence mechanisms of adults. However, little is known about the parasite's genes and encoded proteins involved during A. lumbricoides infection. Hence, this study was conducted to assess the expression profile of putative virulence-associated genes during an active infection of adults and children.
METHODS: In this study, quantitative PCR was performed to evaluate the expression profile of putative virulence-associated genes in A. lumbricoides isolated from infected children and adults. The study was initiated by collecting adult worms expelled from adults and children following anthelminthic treatment. High-quality RNA was successfully extracted from each of six adult worms expelled by three adults and three children, respectively. Eleven putative homologues of helminth virulence-associated genes reported in previous studies were selected, primers were designed and specific amplicons of A. lumbricoides genes were noted. The expression profiles of these putative virulence-associated genes in A. lumbricoides from infected adults were compared to those in A. lumbricoides from infected children.
RESULTS: The putative virulence-associated genes VENOM, CADHERIN and PEBP were significantly upregulated at 166-fold, 13-fold and fivefold, respectively, in adults compared to children. Conversely, the transcription of ABA-1 (fourfold), CATH-L (threefold) and INTEGRIN (twofold) was significantly suppressed in A. lumbricoides from infected adults.
CONCLUSIONS: On the basis of the expression profile of the putative virulence-associated genes, we propose that the encoded proteins have potential roles in evasion mechanisms, which could guide the development of therapeutic interventions.
Fulltext Ascaris lumbricoides harbors a distinct gut microbiota profile from its human host: Preliminary insights

Muslim A, Aazmi S, Er YX, Shahrizal S, Lim YAL

Food Waterborne Parasitol, 2024 Mar;34:e00223.
PMID: 38323096 DOI: 10.1016/j.fawpar.2024.e00223

In indigenous populations where soil-transmitted helminths (STH) infections are endemic, STH parasites (i.e., Ascaris lumbricoides, Trichuris trichiura, hookworms) often co-exist and co-evolve with the gut microbiota of their human hosts. The association between STH infections and the gut microbiota of the colonized human hosts has been established, but few studies explored the gut microbiota of the parasites. This preliminary study aimed to characterize the microbiota of the STH parasite for further understanding the STH parasite-host relationship. The gut microbial genomic DNA from four adult A. lumbricoides worms recovered from a six-year-old indigenous Negrito boy living in an STH-endemic village in Perak, Peninsular Malaysia was extracted and sequenced for the V3-V4 region of the 16S rRNA. The microbiota profiles of these worms were characterized and compared with the gut microbiota of their human host, including the profiles from four STH-positive and three STH-negative individuals from the same tribe and village. The gut microbial structure of A. lumbricoides was found to be differed significantly from their human host. The worms contained lower gut bacterial abundance and diversity than human. This difference was evident in the beta diversity analysis which showed a clear separation between the two sample types. While both Firmicutes (52.3%) and Bacteroidetes (36.6%) are the predominant phyla followed by Proteobacteria (7.2%) in the human gut, the microbiota of Ascaris gut is highly dominated by Firmicutes, constituting 84.2% relative abundance (mainly from the genus Clostridium), followed by Proteobacteria (11.1%), Tenericutes (1.8%) and Bacteroidetes (1.5%). The parasites were also found to alter the microbial structure of the human gut following infection based on the relatively higher bacterial abundance in STH-positive versus STH-negative participants. Further studies with a greater number of Ascaris adults and human hosts are needed to confirm the gut microbiota profiles.
Fulltext Loop-mediated isothermal amplification assay for detection of generic and verocytotoxin-producing Escherichia coli among indigenous individuals in Malaysia

Teh CS, Chua KH, Lim YA, Lee SC, Thong KL

ScientificWorldJournal, 2014;2014:457839.
PMID: 24967435 DOI: 10.1155/2014/457839

We have successfully developed a Loop-mediated isothermal amplification (LAMP) assay that could specifically detect generic Escherichia coli (E. coli). This assay was tested on 85 bacterial strains and successfully identified 54 E. coli strains (average threshold time, Tt = 21.26). The sensitivity of this assay was evaluated on serial dilutions of bacterial cultures and spiked faeces. The assay could detect 10(2) CFU/mL for bacterial culture with Tt = 33.30 while the detection limit for spiked faeces was 10(3) CFU/mL (Tt = 31.12). We have also detected 46 generic E. coli from 50 faecal samples obtained from indigenous individuals with 16% of the positive samples being verocytotoxin-producing E. coli (VTEC) positive. VT1/VT2 allele was present in one faecal sample while the ratio of VT1 to VT2 was 6 : 1. Overall, our study had demonstrated high risk of VTEC infection among the indigenous community and most of the asymptomatic infection occurred among those aged below 15 years. The role of asymptomatic human carriers as a source of dissemination should not be underestimated. Large scale screening of the VTEC infection among indigenous populations and the potential contamination sources will be possible and easy with the aid of this newly developed rapid and simple LAMP assay.
Fulltext Neglected tropical diseases among two indigenous subtribes in peninsular Malaysia: highlighting differences and co-infection of helminthiasis and sarcocystosis

Lee SC, Ngui R, Tan TK, Muhammad Aidil R, Lim YA

PLoS One, 2014;9(9):e107980.
PMID: 25248116 DOI: 10.1371/journal.pone.0107980

Soil-transmitted helminth (STH) infections have been documented among these minority groups since 1938. However the prevalence of STH is still high among these communities. Most studies tend to consider the Orang Asli (indigenous) as a homogenous group. In contrary, different subtribes have their own cultural practices. To understand this variation better, we studied the prevalence and associated factors of STH and other gut parasitic infections among two common subtribes (i.e. Temuan and Temiar). Results showed that the prevalence of the overall STH infections was higher in the Temuan subtribe (53.2% of 171) compared to the Temiar subtribe (52.7% of 98). Trichuris trichiura (46.2%) was the most prevalent parasite in the Temuan subtribe, followed by Ascaris spp. (25.7%) and hookworm (4.1%). In contrast, Ascaris spp. (39.8%) was more prevalent among the Temiar subtribe, preceded by T. trichiura (35.7%) and finally hookworm (8.3%). There were also co-infections of helminthiasis and intestinal protozoa among both Temuan and Temiar subtribes with rates being three times higher among the Temiar compared to Temuan. The most common co-infection was with Entamoeba histolytica/dispar/moshkovskii (n = 24; 24.5%, 16.0-33.0), followed by Giardia spp. (n = 3; 3.1%, -0.3-6.5). In Temuan, STH infection individuals were also infected with Entamoeba histolytica/dispar/moshkovskii (n = 11; 6.4%, 5.0-13.8), Cryptosporidium spp. (n = 3, 1.8%, -0.2-3.8) and Giardia spp. (n = 2, 1.2%, -0.4-2.8). In comparison, there was no Cryptosporidium spp. detected among the Temiar. However, it was interesting to note that there was an occurrence of co-infection of intestinal helminthiasis and sarcocystosis (intestinal) in a Temiar individual. The last report of sarcocystosis (muscular) among the Orang Asli was in 1978. The present study highlighted the importance of understanding the variation of infections amongst the different Orang Asli subtribes. It is vital to note these differences and use this knowledge to customise effective control measures for the various subtribes.
Fulltext Development of High Resolution Melting Analysis for the Diagnosis of Human Malaria

Chua KH, Lim SC, Ng CC, Lee PC, Lim YA, Lau TP, et al.

Sci Rep, 2015;5:15671.
PMID: 26507008 DOI: 10.1038/srep15671

Molecular detection has overcome limitations of microscopic examination by providing greater sensitivity and specificity in Plasmodium species detection. The objective of the present study was to develop a quantitative real-time polymerase chain reaction coupled with high-resolution melting (qRT-PCR-HRM) assay for rapid, accurate and simultaneous detection of all five human Plasmodium spp. A pair of primers targeted the 18S SSU rRNA gene of the Plasmodium spp. was designed for qRT-PCR-HRM assay development. Analytical sensitivity and specificity of the assay were evaluated. Samples collected from 229 malaria suspected patients recruited from Sabah, Malaysia were screened using the assay and results were compared with data obtained using PlasmoNex(TM), a hexaplex PCR system. The qRT-PCR-HRM assay was able to detect and discriminate the five Plasmodium spp. with lowest detection limits of 1-100 copy numbers without nonspecific amplifications. The detection of Plasmodium spp. in clinical samples using this assay also achieved 100% concordance with that obtained using PlasmoNex(TM). This indicated that the diagnostic sensitivity and specificity of this assay in Plasmodium spp. detection is comparable with those of PlasmoNex(TM). The qRT-PCR-HRM assay is simple, produces results in two hours and enables high-throughput screening. Thus, it is an alternative method for rapid and accurate malaria diagnosis.
Molecular characterization of Trichuris species isolated from humans, dogs and cats in a rural community in Peninsular Malaysia

Mohd-Shaharuddin N, Lim YAL, Hassan NA, Nathan S, Ngui R

Acta Trop, 2019 Feb;190:269-272.
PMID: 30500371 DOI: 10.1016/j.actatropica.2018.11.026

Trichuris trichiura (whipworm) are soil-transmitted helminths (STHs) that causing trichuriasis in human. Trichuris vulpis, a canine whipworm has also been reported occasionally in humans. However, an overlapping dimension in the morphology and due to limited external characters between both species may lead to the potential for misidentification. Although there has been an extensive study on the distribution of whipworm in both human and animal hosts, little is known about the molecular epidemiology of Trichuris species in both hosts. To investigate to characterize the whipworm species and to determine the genetic relationship between species infecting both humans and animals, we sequenced the small subunit ribosomal RNA (SSU rRNA) regions of Trichuris egg isolated from humans, dogs and cats in a rural community in Malaysia. A total of 524 fresh fecal samples were collected from humans and animals. The overall prevalence of Trichuris was 59.9% as determined by microscopy examination. The molecular analysis showed that 98.7% were identified as T. trichiura in the human fecal sample. Interestingly, 1.3% were identified as T. vulpis. As for animal fecal sample, 56.8% and 43.2% were identified as T. trichiura and T. vulpis, respectively. Phylogenetic and sequence analysis demonstrated that T. trichiura isolates were genetically distinct from T. vulpis isolates from both hosts. This finding implies that companion animals can be a reservoir and mechanical transmitter for T. trichiura infection in human and also highlighting the possible zoonotic potential of T. vulpis. This finding may also suggest that cross-transmission between humans and animal hosts in sympatric setting may be a source of infection in both hosts. More studies are needed to better understand the transmission dynamic and public health significance of Trichuris infection in both hosts.
Vertical infestation of the dengue vectors Aedes aegypti and Aedes albopictus in apartments in Kuala Lumpur, Malaysia

Roslan MA, Shafie A, Ngui R, Lim YA, Sulaiman WY

J Am Mosq Control Assoc, 2013 Dec;29(4):328-36.
PMID: 24551965

Dengue is a serious public health problem in Malaysia. The aim of this study was to compare the vertical infestation of Aedes population in 2 apartments in Kuala Lumpur with different status of dengue incidence (i.e., high-dengue-incidence area and area with no reported dengue cases). The study was also conducted to assess the relationship between environmental factors such as rainfall, temperature, and humidity and Aedes population that may influence Aedes infestation. Surveillance with a mosquito larvae trapping device was conducted for 28 continuous weeks (January to July 2012) in Vista Angkasa (VA) and Inderaloka (IL) apartments located in Kuala Lumpur, Malaysia. The results indicated that both Aedes spp. could be found from ground to higher floor levels of the apartments, with Aedes aegypti being more predominant than Ae. albopictus. Data based on mixed and single breeding of Aedes spp. on different floors did not show any significant difference. Both rainfall (R3; i.e., the amount of rainfall collected during the previous 3 wk before the surveillance period began) and RH data showed significant relationship with the number of Aedes larvae collected in VA and IL. No significant difference was found between the numbers of Aedes larvae in both study areas as well as maximum and minimum temperatures. Results also indicated adaptations of Ae. aegypti to the ecosystem at each elevation of high-rise buildings, with Ae. albopictus staying inside of apartment units.
Mosquito Biology and Mosquito-Borne Disease Awareness Among Island Communities In Malaysia

Shafie A, Roslan MA, Ngui R, Lim YA, Sulaiman WY

J Am Mosq Control Assoc, 2016 Dec;32(4):273-281.
PMID: 28206867 DOI: 10.2987/16-6604.1

Mosquito-borne diseases have been increasing at an alarming rate over the past decades. In Malaysia, one finds several important mosquito-borne diseases such as Japanese encephalitis, dengue, malaria, and chikungunya. Mosquito surveillance and control programs are the most effective way of detecting and controlling mosquito-borne diseases, but these programs are less effective without an aware and well-informed general public. In 2014 we used a questionnaire to evaluate the extent of awareness of basic mosquito biology and mosquito-borne diseases in 6 villages, Kampung Masjid, Kampung Teluk Gedung, Kampung Teluk Dalam, Kampung Ujung Kelawai, Kampung Sungai Pinang Besar, and Kampung Sungai Pinang Kechil on Pangkor Island, Malaysia. A total of 1,012 individuals responded to the questionnaire, consisting of 790 Malay (78.1%), 164 Chinese (16.2%), and 58 Indian (5.7%). More than 60% (Malay = 73.7%, Chinese = 64.0%, Indian = 79.3%) of the respondents were familiar with basic mosquito biology and practiced personal protection against mosquito bites, and the association was statistically significant (P = 0.02). However, the majority of the respondents had limited knowledge on mosquito-borne diseases, and this varied significantly among the 3 ethnic groups (P = 0.0001). Our recommendations are to improve and intensify public health education outreach programs to the island residents and to encourage community participation in vector control programs.
High Seroprevalence Against Typhus Group and Spotted Fever Group Rickettsiae in Rural Indigenous Populations of Peninsular Malaysia

Tappe D, Gross Y, Ngui R, Rauch J, Tay ST, Lim YAL

Vector Borne Zoonotic Dis, 2019 05;19(5):323-327.
PMID: 30540546 DOI: 10.1089/vbz.2018.2391

Rickettsioses of the typhus group (TG) and spotted fever group (SFG) are emerging bacterial infections worldwide, especially in the tropics. Only a few studies on these pathogens and their respective clinical diseases have been conducted in Malaysia. Here, we performed a seroprevalence study among 544 healthy, afebrile indigenous people (Orang Asli) from peninsular Malaysia for TG and SFG rickettsioses in nine rural and peri-urban settlements. The study population encompassed children, adolescents, and adults. The overall seroprevalence of rickettsiosis in the Orang Asli was 48.5%, with 27.9% seroprevalence against TG rickettsiae and 20.6% seroprevalence against SFG rickettsiae. In 7.9% of the study participants, antibodies against both rickettsial groups were found. The highest seropositivity rates against TG and SRG rickettsiae were detected in young children and adults. Overall, there were no gender differences. Seroprevalences were similar among inhabitants of different settlements, except for two localities. More studies are needed to shed more light on the ecology and risk factors for TG and SFG rickettsioses in Malaysia.
Fulltext Nutritional status, hemoglobin level and their associations with soil-transmitted helminth infections between Negritos (indigenous) from the inland jungle village and resettlement at town peripheries

Muslim A, Lim YA, Mohd Sofian S, Shaari SA, Mohd Zain Z

PLoS One, 2021;16(1):e0245377.
PMID: 33439889 DOI: 10.1371/journal.pone.0245377

This study compared the current nutritional status, hemoglobin levels and their associations with soil-transmitted helminth (STH) infections between two categories of Negritos (indigenous): (i) Inland Jungle Villages (IJV) (ii) and Resettlement Plan Scheme (RPS) near town peripheries, decades after redevelopment and demarginalization. A total of 416 Negritos (IJV: 149; RPS: 267) was included for nutritional profiling based on anthropometric analysis. However, only 196 (IJV: 64; RPS: 132) individuals consented to blood taking for the hemoglobin (Hb) measurements. Subsequently, the association of undernutrition and anemia with STH infections were determined based on univariate and multivariate logistic regression analyses. The overall prevalence of stunting, wasting, and underweight amongst children and adolescents (n = 343) were 45.8%, 42.3% and 59.1%, respectively. In adults (n = 73), the prevalence of underweight was low (6.8%) but overweight and obese was prominent (26.0%). For anemia (n = 196), an overall prevalence rate of 68.4% were observed with 80% and 70.4% of children aged 2-6 y/o and aged 7-12 y/o, respectively being anemic. Comparatively, the prevalence of underweight (WAZ) was significantly higher in the RPS versus the IJV (P = 0.03) In the IJV, children aged ≤ 6 y/o and having STH poly-parasitism were associated with underweight (P = 0.01) and moderate-severe T. trichiura infection was associated with anemia. Whilst in the RPS, underweight was highly associated with only T. trichiura infection (P = 0.04). Wasting was significantly associated with young children aged ≤10 in both IJV (P = 0.004) and RPS (P = 0.02). Despite efforts in improving provision of facilities and amenities among the indigenous, this study highlighted a high magnitude of nutritional issues among the Negritos especially those in the RPS and their likely association with STH infections and decades of demarginalization. Joint nutritional intervention strategies with mass anti-helminthic treatment are imperative and urgently needed to reduce the undernutrition problems especially among indigenous children.
Fulltext Updates on malaria incidence and profile in Malaysia from 2013 to 2017

Hussin N, Lim YA, Goh PP, William T, Jelip J, Mudin RN

Malar J, 2020 Jan 31;19(1):55.
PMID: 32005228 DOI: 10.1186/s12936-020-3135-x

BACKGROUND: To date, most of the recent publications on malaria in Malaysia were conducted in Sabah, East Malaysia focusing on the emergence of Plasmodium knowlesi. This analysis aims to describe the incidence, mortality and case fatality rate of malaria caused by all Plasmodium species between Peninsular Malaysia and East Malaysia (Sabah and Sarawak) over a 5-year period (2013-2017).
METHODS: This is a secondary data review of all diagnosed and reported malaria confirmed cases notified to the Ministry of Health, Malaysia between January 2013 and December 2017.
RESULTS: From 2013 to 2017, a total of 16,500 malaria cases were notified in Malaysia. The cases were mainly contributed from Sabah (7150; 43.3%) and Sarawak (5684; 34.4%). Majority of the patients were male (13,552; 82.1%). The most common age group in Peninsular Malaysia was 20 to 29 years (1286; 35.1%), while Sabah and Sarawak reported highest number of malaria cases in age group of 30 to 39 years (2776; 21.6%). The top two races with malaria in Sabah and Sarawak were Bumiputera Sabah (5613; 43.7%) and Bumiputera Sarawak (4512; 35.1%), whereas other ethnic group (1232; 33.6%) and Malays (1025; 28.0%) were the two most common races in Peninsular Malaysia. Plasmodium knowlesi was the commonest species in Sabah and Sarawak (9902; 77.1%), while there were more Plasmodium vivax cases (1548; 42.2%) in Peninsular Malaysia. The overall average incidence rate, mortality rate and case fatality rates for malaria from 2013 to 2017 in Malaysia were 0.106/1000, 0.030/100,000 and 0.27%, respectively. Sarawak reported the highest average incidence rate of 0.420/1000 population followed by Sabah (0.383/1000). Other states in Peninsular Malaysia reported below the national average incidence rate with less than 0.100/1000.
CONCLUSIONS: There were different trends and characteristics of notified malaria cases in Peninsular Malaysia and Sabah and Sarawak. They provide useful information to modify current prevention and control measures so that they are customised to the peculiarities of disease patterns in the two regions in order to successfully achieve the pre-elimination of human-only species in the near future.
Over two decades of Plasmodium knowlesi infections in Sarawak: Trend and forecast

Ooi CH, Bujang MA, Tg Abu Bakar Sidik TMI, Ngui R, Lim YA

Acta Trop, 2017 Dec;176:83-90.
PMID: 28768148 DOI: 10.1016/j.actatropica.2017.07.027

Malaria is still of great public health concern, especially in Malaysian Borneo. The aim of this study was to determine the trends of P. knowlesi infection in Sarawak, Malaysia and to forecast the incidence of P. knowlesi until the year 2040. Data on P. knowlesi malaria cases from 1992 to the year 2014 were obtained from the Sarawak Health Department, Malaysia. ARIMA model was applied to forecast the future incidence of P. knowlesi infection. The data for the whole of Sarawak and subsequently the selected six districts which have high incidence rates of P. knowlesi infection were analyzed. Results of the analysis showed that there was an increasing trend of P. knowlesi cases from the year 1992-2014 (p<0.001). The trend in the incidence started to increase in the year 2008 (p=0.029). The incidence rate per 100,000 populations was between 4.15 in the year 1992 and 42.03 in the year 2014. High incidence of P. knowlesi infections has been detected in the districts adjacent to each other within the interior region of Sarawak. The forecasted incidence and incidence rate per 100,000 populations in the year 2020 were 1229 and 44.04, respectively, while those in the year 2040 were 2056 and 62.91, respectively. The forecasted incidence showed an upward trend highlighting an urgent need to draw up strategic and holistic prevention plans to limit further the increase in P. knowlesi morbidity and mortality in Sarawak. It is imperative that these measures are customized taking into consideration the challenges faced in the interior areas of Sarawak and the behavior of the main vector of P. knowlesi (i.e., An. latens) in Sarawak.
Development and initial evaluation of a lateral flow dipstick test for antigen detection of Entamoeba histolytica in stool sample

Saidin S, Yunus MH, Othman N, Lim YA, Mohamed Z, Zakaria NZ, et al.

Pathog Glob Health, 2017 May;111(3):128-136.
PMID: 28335696 DOI: 10.1080/20477724.2017.1300421

Entamoeba histolytica infection remains a public health concern in developing countries. Early diagnosis of amoebiasis can avoid disease complications, thus this study was aimed at developing a test that can rapidly detect the parasite antigens in stool samples. Rabbits were individually immunized with recombinant pyruvate phosphate dikinase (rPPDK) and E. histolytica excretory-secretory antigens to produce polyclonal antibodies. A rapid dipstick test was produced using anti-rPPDK PAb lined on the dipstick as capture reagent and anti-EhESA PAb conjugated to colloidal gold as the detector reagent. Using E. histolytica-spiked in stool sample of a healthy individual, the detection limit of the dipstick test was found to be 1000 cells ml-1. Meanwhile when rPPDK was spiked in the stool sample, the minimum concentration detected by the dipstick test was 0.1 μg ml-1. The performances of the dipstick, commercial Techlab E. histolytica II enzyme-linked immunosorbent assays (ELISA) and real-time PCR were compared using 70 stool samples from patients infected with Entamoeba species (n = 45) and other intestinal pathogens (n = 25). When compared to real-time PCR, the diagnostic sensitivity of the dipstick for detection of E. histolytica was 65.4% (n = 17/26); while the diagnostic specificity when tested with stool samples containing other intestinal pathogens was 92% (23/25). In contrast, Techlab E. histolytica II ELISA detected 19.2% (5/26) of the E. histolytica-positive samples as compared to real-time PCR. The lateral flow dipstick test produced in this study enabled rapid detection of E. histolytica, thus it showed good potential to be further developed into a diagnostic tool for intestinal amoebiasis.
Fulltext Neglected Intestinal Parasites, Malnutrition and Associated Key Factors: A Population Based Cross-Sectional Study among Indigenous Communities in Sarawak, Malaysia

Rajoo Y, Ambu S, Lim YA, Rajoo K, Tey SC, Lu CW, et al.

PLoS One, 2017;12(1):e0170174.
PMID: 28095446 DOI: 10.1371/journal.pone.0170174

Intestinal parasitic infections (IPIs) have been recognized as one of the most significant causes of illness among disadvantaged communities. Many studies have been conducted on the prevalence of IPIs in Malaysia. However, these studies mostly focused on the indigenous groups in Peninsular Malaysia. The present study was conducted to provide the current baseline data on prevalence of IPIs, anaemia, malnutrition and associated risk factors among the indigenous communities in Sarawak, situation at northwest Borneo island of Malaysia. A cross sectional study was conducted among the longhouses communities. Stool samples were obtained and examined for the presence of IPIs using microscopy technique. Haemoglobin measurement was done using a portable haemoglobin analyzer. Malnutrition (i.e., stunting, underweight and wasting) was assessed using the WHO Anthro software. Statistical analysis was carried out using SPSS software. A total of 341participants took part in this study. The overall prevalence of IPIs was 57.5%. Multivariate analysis indicated that the absence of toilets (OR = 1.6; 95% CI = 1.1-2.7; p = 0.002) and close contact with animals (OR = 1.8; 95% CI = 1.3-2.9; p = 0.027) as significant predictors for IPIs. The incidence of anaemia was 36.4%. The incidence of underweight, wasting and stunting were 22.2%, 5.6% and 35.4%, respectively. Multivariate analysis demonstrated that low level of parental education attainment (OR = 1.9; 95% CI = 1.2-3.0; p = 0.006) was identified as significant predictor for anaemia. The incidence of wasting was significantly associated with mild anaemia (OR = 1.2; 95% CI = 0.9-1.7; p = 0.024). Low household income was identified as significant predictor for stunting (OR = 2.1; 95% CI = 9.8-22.2; p = 0.001) and underweight (OR = 1.9; 95% CI = 5.6-18.7; p = 0.037), respectively. Essentially, the present study highlighted that intestinal parasitic infections, anaemia and malnutrition are still prevalent among rural indigenous community in Sarawak. Improvement of socioeconomic status, periodic mass deworming, iron supplementation and health education program should be included in the control and prevention of public health strategies.
Temporal variation in diversity and community structure of preimaginal blackflies (Diptera: Simuliidae) in a tropical forest reserve in Malaysia

Pavitra SP, Low VL, Tan TK, Lim YAL, Ya'cob Z

Acta Trop, 2020 Feb;202:105275.
PMID: 31747545 DOI: 10.1016/j.actatropica.2019.105275

Blackflies (Diptera: Simuliidae) are ecologically and medically important insects but they have been understudied in Malaysia. Accordingly, a study on the temporal variation in diversity and community structure of preimaginal blackflies was conducted for the first time in Malaysia. A total of 865 preimaginal blackflies were collected in 120 samplings from five streams across three monsoon seasons from February 2018 until January 2019. Ten species were recorded and most frequently collected species were Simulium cheongi, Simulium vanluni and Simulium jeffreyi. Relatively common species were Simulium roslihashimi, Simulium tani complex and Simulium trangense. No significant changes of rainfall was observed between three monsoon seasons as well as the seasons with species and physiochemical parameters except acidity (pH) (P 20%) indicated that S. vanluni and S. jeffreyi were commonly associated with wider, deeper and fast-flowing streams with low conductivity and larger streambed particle. In contrast, S. cheongi was associated with smaller, slower and small streambed particle. This first extensive bimonthly study has uncovered the species community structure as well as the changes of stream physicochemical parameters over time although they were not greatly and significantly influenced by the monsoon seasons. Species distribution, richness and abundance, however, were highly determined by the stream width, depth and velocity, therefore, were vital in shaping diversity and community structure of preimaginal blackflies.
Genetic diversity and differentiation in the blackflies Simulium cheongi, Simulium jeffreyi and Simulium vanluni (Diptera: Simuliidae) in Peninsular Malaysia

Pavitra SP, Ya'cob Z, Tan TK, Lim YAL, Low VL

Acta Trop, 2020 May;205:105415.
PMID: 32088275 DOI: 10.1016/j.actatropica.2020.105415

The population genetic structures of S. vanluni, S. cheongi and S. jeffreyi were determined from mitochondria-encoded sequences of cytochrome c oxidase subunits I (COI) across different states in Peninsular Malaysia. High levels of genetic diversity and genetic differentiation were observed among three species. All three species revealed an intermediate level of gene flow among the populations. Negative values of Fu's Fs and low values of Raggedness index supported the hypothesis of population expansion in S. vanluni, S. cheongi and S. jeffreyi.
Seroprevalence of Nipah Virus Infection in Peninsular Malaysia

Yong MY, Lee SC, Ngui R, Lim YA, Phipps ME, Chang LY

J Infect Dis, 2020 05 11;221(Suppl 4):S370-S374.
PMID: 32392323 DOI: 10.1093/infdis/jiaa085

Nipah virus (NiV) outbreak occurred in Malaysia in 1998. The natural host reservoir for NiV is Pteropus bats, which are commonly found throughout Malaysia. Humans become infected when NiV spills over from the reservoir species. In this study, NiV serosurveillance in Peninsular Malaysia, particularly among the indigenous population, was performed. The collected samples were tested for presence of NiV antibodies using a comparative indirect enzyme-linked immunosorbent assay based on the recombinant NiV nucleocapsid (rNiV-N) protein. We found that 10.73% of the participants recruited in this study had antibodies against rNiV-N, suggesting possible exposure to NiV.
A novel clade of bat-associated Bartonella detected in the bat fly Leptocyclopodia ferrari (Diptera: Nycteribiidae) parasitizing Cynopterus brachyotis (Chiroptera: Pteropodidae)

Low VL, Tan TK, Tohiran KA, Lim YAL, AbuBakar S, Nasir DM

Vet Microbiol, 2022 Jan;264:109284.
PMID: 34826649 DOI: 10.1016/j.vetmic.2021.109284

Bartonelloses are emerging infectious diseases that are common in humans and animals worldwide. Several Bartonella species associated with companion animals such as Bartonella henselae and Bartonella rochalimae are species with zoonotic implications and have become a global concern. Other Bartonella species associated with wild animals, however, remain underappreciated particularly in the developing regions of the world. To explore further on this neglected bacterial agent, Leptocyclopodia ferrari (Nycteribiidae) bat flies collected from Cynopterus brachyotis (Pteropodidae), an endemic fruit bat species in Southeast Asia, were molecularly examined for the presence of Bartonella. Both 16 S-23 S ribosomal RNA intergenic spacer region and citrate synthase gene sequences exhibited less than 95 % similarity to all previously reported Bartonella spp. Further phylogenetic analysis revealed a novel clade of this Bartonella sp. with high bootstrap support. The vectorial capacity of bat flies in transmitting this novel pathogen merits further investigation.

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