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Fulltext Zika virus and its potential re-emergence in Malaysia

Sam JI, Chan YF, Vythilingam I, Wan Sulaiman WY

Med J Malaysia, 2016 04;71(2):66-8.
PMID: 27326944 MyJurnal

Zika virus (ZIKV) has re-emerged to cause explosive epidemics in the Pacific and Latin America, and appears to be associated with severe neurological complications including microcephaly in babies. ZIKV is transmitted to humans by Aedes mosquitoes, principally Ae. aegypti, and there is historical evidence of ZIKV circulation in Southeast Asia. It is therefore clear that Malaysia is at risk of similar outbreaks. Local and international guidelines are available for surveillance, diagnostics, and management of exposed and infected individuals. ZIKV is the latest arbovirus to have spread globally beyond its initial restricted niche, and is unlikely to be the last. Innovative new methods for surveillance and control of vectors are needed to target mosquito-borne diseases as a whole.
Fulltext Modified technique of Wolbachia removal from Malaysian Aedes albopictus

Joanne S, Vythilingam I, Yugavathy N, Doss JI

Asian Pac J Trop Biomed, 2014 Jul;4(7):557-60.
PMID: 25183276 DOI: 10.12980/APJTB.4.2014APJTB-2014-0020

To develop an artificial and modified Wolbachia removal technique using tetracycline from naturally Wolbachia infected Aedes albopictus (Ae. albopictus) so as to be able to produce generations of Wolbachia free offsprings.
Fulltext Bionomics of Anopheles latens in Kapit, Sarawak, Malaysian Borneo in relation to the transmission of zoonotic simian malaria parasite Plasmodium knowlesi

Tan CH, Vythilingam I, Matusop A, Chan ST, Singh B

Malar J, 2008;7:52.
PMID: 18377652 DOI: 10.1186/1475-2875-7-52

A large focus of human infections with Plasmodium knowlesi, a simian parasite naturally found in long-tailed and pig-tailed macaques was discovered in the Kapit Division of Sarawak, Malaysian Borneo. A study was initiated to identify the vectors of malaria, to elucidate where transmission is taking place and to understand the bionomics of the vectors in Kapit.
Natural transmission of Plasmodium knowlesi to humans by Anopheles latens in Sarawak, Malaysia

Vythilingam I, Tan CH, Asmad M, Chan ST, Lee KS, Singh B

Trans R Soc Trop Med Hyg, 2006 Nov;100(11):1087-8.
PMID: 16725166

Four species of malaria parasites are known to infect humans. A fifth species, Plasmodium knowlesi, has been reported to infect humans in Malaysian Borneo. Here we report for the first time the incrimination of Anopheles latens as the vector of P. knowlesi among humans and monkeys in Sarawak, Malaysia.
The impact of development and malaria control activities on its vectors in the Kinabatangan area of Sabah, East Malaysia

Vythilingam I, Chan ST, Shanmugratnam C, Tanrang H, Chooi KH

Acta Trop, 2005 Oct;96(1):24-30.
PMID: 16076459

A study was carried out from July 2001 until January 2003 in the Kinabatangan area of Sabah, part of Borneo island, where malaria used to be mesoendemic. Vector surveys determined that Plasmodium falciparum was the predominant species and Anopheles balabacensis the primary vector. Malaria cases have dropped drastically over the years but P. falciparum is still predominant. In the present study, Anopheles donaldi was the predominant species and was positive for sporozoites. Although An. balabacensis was present, none were infective. An. donaldi bite more outdoors than indoors and have a peak biting time from 18:00 to 19:00 h when most people are still out of their homes. An integrated malaria control programme along with area development has helped in the control of malaria and its vector.
Fulltext A highly sensitive, nested polymerase chain reaction based method using simple dna extraction to detect malaria sporozoites in mosquitos

Vythilingam I, Nitiavathy K, Yi P, Bakotee B, Hugo B, Singh B, et al.

Southeast Asian J. Trop. Med. Public Health, 1999 Dec;30(4):631-5.
PMID: 10928352

Dried Anopheles farauti mosquitos caught in Solomon Islands in 1990 were examined for malaria sporozoites by ELISA and nested polymerase chain reaction (PCR). Only heads and thoraces were used. Plasmodium genus-specific nested PCR amplifications were carried out on all samples. Of the 402 pools of mosquitos that were processed, 30 were positive for malaria. Nest 1 products of positive samples were subjected to further PCR amplifications with species-specific primers for P. falciparum and P. vivax. Twenty pools were positive for P. vivax by PCR while only 7 were positive by ELISA. For P. falciparum 2 pools were positive by both ELISA and PCR, and one of these was a pool which was positive for P. vivax by PCR and ELISA. Thus the sensitivity of PCR for P. vivax was 100% while the specificity was 96.7%. For P. falciparum the sensitivity and specificity were 100%. The PCR technique is highly sensitive and can be used on dried mosquitos which makes it a valuable tool for determining sporozoite rates of mosquitos, even in remote areas.
Fulltext Bionomics of important mosquito vectors in Malaysia

Vythilingam I, Chiang GL, Lee HL, Singh KI

Southeast Asian J. Trop. Med. Public Health, 1992 Dec;23(4):587-603.
PMID: 1363679
Single-dose diethylcarbamazine in the control of periodic brugian filariasis in Peninsular Malaysia

Hakim SL, Vythilingam I, Marzukhi MI, Mak JW

Trans R Soc Trop Med Hyg, 1995 11 1;89(6):686-9.
PMID: 8594697

The study compared the effectiveness of a single dose of diethylcarbamazine (DEC) (6mg/kg) with the standard regimen of 6 doses (total 36 mg/kg) in mass chemotherapy for the control of brugian filariasis. Mass chemotherapy with single-dose DEC was instituted in one area and standard dose in the other and treatment was repeated after one year. Parasitological surveys were conducted before, and 3, 7 and 12 months after treatment. Pretreatment characteristics were not significantly different between the 2 treatment areas. There was a significant reduction in microfilaraemia prevalence rate from 24.7% to 14.7% at 12 months and to 6.8% at 19 months in the single dose area and from 22.8% to 9.6% at 12 months and to 2.7% at 19 months with the standard dose. Maximum reduction was at 7 months after treatment with both regimens. There was also significant progressive reduction in mean microfilarial density from 4.39 +/- 20.37 to 0.89 +/- 4.16 per 60 microL in the single-dose area and from 4.43 +/- 17.31 to 0.75 +/- per 60 microL in the standard dose area. There was a greater reduction of both microfilarial prevalence and density using the standard regimen but it was not statistically significant. Thus, a single dose of DEC is as effective as the standard dose in controlling periodic brugian filariasis.
Fulltext Studies on adult mosquito vectors of Japanese encephalitis in a pig farm in Selangor, Malaysia

Vythilingam I, Mahadevan S, Zaridah MZ, Ong KK, Abdullah G, Ong YF

Southeast Asian J. Trop. Med. Public Health, 1994 Jun;25(2):383-6.
PMID: 7855662

Mosquito collections were carried out for a period of one year from January to December 1992 in a pig farm in Sungai Pelek, Selangor, Malaysia. A total of 41,022 mosquitos belonging to 52 species and 20 genera were collected. Culex tritaeniorhynchus and Cx. gelidus, the important vectors, comprised 63% of all mosquitos collected. Both these species were collected in large numbers during the wet months of May and December. The other predominant species in that area were Cx. fuscocephala, Cx. quinquefasciatus, Cx. sitiens, Aedes butleri, and Armigeres subalbatus.
Fulltext Anopheles donaldi incriminated as a vector of periodic Brugia malayi in Grik, Perak, Malaysia

Vythilingam I, Hakim SL, Chan ST, Mak JW

Southeast Asian J. Trop. Med. Public Health, 1996 Sep;27(3):637-41.
PMID: 9185284

Studies were carried out to observe the species composition of mosquitos and to determine the vectors responsible for the transmission of filariasis in Grik, Perak, Malaysia. A total of 2,155 mosquitos belonging to 7 genera and 30 species were collected. Anopheles donaldi comprised 24.1% of the collection. Twelve out of 519 An. donaldi were infected with L3 larvae of Brugia malayi. The peak biting time was around 23.00-24.00 hours. The infective bites per month ranged from 0 to 6.3.
Isolation of Japanese encephalitis virus from mosquitoes collected in Sabak Bernam, Selangor, Malaysia in 1992

Vythilingam I, Oda K, Chew TK, Mahadevan S, Vijayamalar B, Morita K, et al.

J Am Mosq Control Assoc, 1995 Mar;11(1):94-8.
PMID: 7616198

Detection and isolation of Japanese encephalitis (JE) virus were attempted from female mosquitoes collected in Kampong Pasir Panjang, Sabak Bernam, Selangor, from May to November 1992. A total of 7,400 mosquitoes consisting of 12 species in 148 pools were processed and inoculated into Aedes albopictus clone C6/36 cell cultures. Of these, 26 pools showed the presence of viral antigens in the infected C6/36 cells by specific immunoperoxidase staining using an anti-JE virus polyclonal antibody. Presence of JE virus genome was confirmed in the infected culture fluid for 16 pools by using reverse transcriptase-polymerase chain reaction and JE virus-specific primers. Of these, 3 pools were from Culex tritaeniorhynchus, 4 from Culex vishnui, 3 from Culex bitaeniorhynchus, 2 from Culex sitiens, one from Aedes species, and 3 from Culex species. Isolation of JE virus from Cx. sitiens, Cx. bitaeniorhynchus, and Aedes sp. (Aedes butleri and Ae. albopictus) is reported for the first time in Malaysia.
The prevalence of Anopheles (Diptera: Culicidae) mosquitoes in Sekong Province, Lao PDR in relation to malaria transmission

Vythilingam I, Phetsouvanh R, Keokenchanh K, Yengmala V, Vanisaveth V, Phompida S, et al.

Trop Med Int Health, 2003 Jun;8(6):525-35.
PMID: 12791058

A longitudinal study was conducted on the prevalence of Anopheles in three malaria endemic villages in Sekong province, in the southern region of Lao PDR, from August 2000 to October 2001. All night, human landing collections took place in August and October 2000 and April and October 2001, and blood smears were taken for malaria parasites during the same period. Mosquitoes were tested for sporozoite antigen using enzyme-linked immunosorbent assay. In August 2000 (wet season) and April 2001 (dry season) the ovaries of the mosquitoes were examined for parity. A total of 16 species of Anopheles were caught in the study sites of which An. dirus A, An. maculatus sl and An. jeyporiensis were positive for sporozoites. The entomological inoculation rate (EIR) ranged from 0.06 to 0.25. There was a good correlation between EIR and vectorial capacity in the wet season, especially in Pai Mai where the prevalence of malaria was also high during the wet seasons (11.8 and 10.53). An. dirus A showed ambivalence in their choice of feeding as approximately 50% attacked man indoors and an equal proportion outdoors. An dirus A was the main vector in Pai Mai. The parous rate did not significantly differ between the wet and dry season, although it was higher in the dry season. In Takaio the parasite prevalence ranged from 8.7% (dry season) to 37.1% (wet season) and An. jeyporiensis was the vector, and the risk of infection was 0.85 in the dry season while 0.99 in the wet season. In Toumgno An. maculatus sl was the vector and infection was found only in August and October 2000. However, malaria prevalence ranged from 9.69 to 20.4% and was equally high in the dry season. Cattle were also present close to the houses in all the villages and this might be a contributory factor in the prevalence of malaria.
Histopathology of Brugia pahangi and Plasmodium berghei ANKA co-infection in the Gerbil (Meriones unguiculatus)

Junaid OQ, Wong KT, Khaw LT, Mahmud R, Vythilingam I

Trop Biomed, 2018 Dec 01;35(4):981-998.
PMID: 33601846

Co-infection with multiple different parasites is a common phenomenon in both human and animals. Among parasites that frequently co-infect the same hosts, are the filarial worms and malaria parasites. Despite this, the mechanisms underlying the interactions between these parasites is still relatively unexplored with very few studies available on the resulting pathologies due to co-infection by filarial nematodes and malaria parasites. Hence, this study investigated the histopathological effect of Brugia pahangi and Plasmodium berghei ANKA (PbA) infections in gerbil host. Gerbils grouped into B. pahangi-infected, PbA-infected, B. pahangi and PbA-coinfected, and uninfected control, were necropsied at different time points of post PbA infections. Brugia pahangi infections in the gerbils were first initiated by subcutaneous inoculation of 50 infective larvae, while PbA infections were done by intraperitoneal injection of 106 parasitized red blood cells after 70 days patent period of B. pahangi. Organs such as the lungs, kidneys, spleen, heart and liver were harvested aseptically at the point of necropsy. There was significant hepatosplenomegaly observed in both PbA-infected only and coinfected gerbils. The spleen, liver and lungs were heavily pigmented. Both B. pahangi and PbA infections (mono and coinfections) resulted in pulmonary edema, while glomerulonephritis was associated with PbA infections. The presence of both parasites induced extramedullary hematopoiesis in the spleen and liver. These findings suggest that the pathologies associated with coinfected gerbils were synergistically induced by both B. pahangi and PbA infections.
Fulltext Within-population genetic diversity and population structure of Plasmodium knowlesi merozoite surface protein 1 gene from geographically distinct regions of Malaysia and Thailand

Ahmed MA, Chu KB, Vythilingam I, Quan FS

Malar J, 2018 Nov 29;17(1):442.
PMID: 30497496 DOI: 10.1186/s12936-018-2583-z

BACKGROUND: The C-terminal 42 kDa domain of Plasmodium knowlesi merozoite surface protein 1 (PkMSP1) is a potential asexual blood-stage vaccine candidate, however, only a limited number of clinical isolates have been analysed from Malaysia and no inter-country comparative diversity study has been conducted. In the present study, nucleotide diversity, haplotypes and natural selection levels of pkmsp1 in clinical samples from geographically distinct regions of Malaysia and Thailand were investigated. The overall population structure of the parasite from the region was determined.
METHODS: Eleven full-length pkmsp1 sequences obtained from clinical isolates of Malaysia along with the H-strain were downloaded from the database for domain wise characterization of pkmsp1 gene. Additionally, 76 pkmsp-142 sequences from Thailand and Malaysia were downloaded from the database for intra and inter-population analysis. DnaSP 5.10 and MEGA 5.0 software were used to determine genetic diversity, polymorphism, haplotypes and natural selection. Genealogical relationships were determined using haplotype network tree in NETWORK software v5.0. Population genetic differentiation index (FST) of parasites were analysed using Arlequin v3.5.
RESULTS: Sequence analysis of 11 full-length pkmsp1 sequences along with the H-strain identified 477 (8.4%) polymorphic sites, of which 107 were singleton sites. The overall diversity observed in the full-length genes were high in comparison to its ortholog pvmsp1 and the 4 variable domains showed extensive size variations. The nucleotide diversity was low towards the pkmsp1-42 compared to the conserved domains. The 19 kDa domain was less diverse and completely conserved among isolates from Malaysian Borneo. The nucleotide diversity of isolates from Peninsular Malaysia and Thailand were higher than Malaysian Borneo. Network analysis of pkmsp1-42 haplotypes showed geographical clustering of the isolates from Malaysian Borneo and grouping of isolates from Peninsular Malaysia and Thailand. Population differentiation analysis indicated high FST values between parasite populations originating from Malaysian Borneo, Peninsular Malaysia and Thailand attributing to geographical distance. Moderate genetic differentiation was observed for parasite populations from Thailand and Peninsular Malaysia. Evidence of population expansion and purifying selection were observed in all conserved domains with strongest selection within the pkmsp1-42 domain.
CONCLUSIONS: This study is the first to report on inter country genetic diversity and population structure of P. knowlesi based on msp1. Strong evidence of negative selection was observed in the 42 kDa domain, indicating functional constrains. Geographical clustering of P. knowlesi and moderate to high genetic differentiation values between populations identified in this study highlights the importance of further evaluation using larger number of clinical samples from Southeast Asian countries.
Effect of Brugia pahangi co-infection with Plasmodium berghei ANKA in gerbils (Meriones unguiculatus)

Junaid OQ, Vythilingam I, Khaw LT, Sivanandam S, Mahmud R

Parasitol Res, 2020 Apr;119(4):1301-1315.
PMID: 32179986 DOI: 10.1007/s00436-020-06632-4

Malaria and lymphatic filariasis (LF) are two leading and common mosquito-borne parasitic diseases worldwide. These two diseases are co-endemic in many tropical and sub-tropical regions and are known to share vectors. The interactions between malaria and filarial parasites are poorly understood. Thus, this study aimed at establishing the interactions that occur between Brugia pahangi and Plasmodium berghei ANKA (PbA) co-infection in gerbils. Briefly, the gerbils were matched according to age, sex, and weight and grouped into filarial-only infection, PbA-only infection, co-infection, and control group. The parasitemia, survival and clinical assessment of the gerbils were monitored for a period of 30 days post Plasmodium infection. The immune responses of gerbils to both mono and co-infection were monitored. Findings show that co-infected gerbils have higher survival rate than PbA-infected gerbils. Food and water consumption were significantly reduced in both PbA-infected and co-infected gerbils, although loss of body weight, hypothermia, and anemia were less severe in co-infected gerbils. Plasmodium-infected gerbils also suffered hypoglycemia, which was not observed in co-infected gerbils. Furthermore, gerbil cytokine responses to co-infection were significantly higher than PbA-only-infected gerbils, which is being suggested as a factor for their increased longevity. Co-infected gerbils had significantly elicited interleukin-4, interferon-gamma, and tumor necrotic factor at early stage of infection than PbA-infected gerbils. Findings from this study suggest that B. pahangi infection protect against severe anemia and hypoglycemia, which are manifestations of PbA infection.
Fulltext Evaluation of sticky traps for adult Aedes mosquitoes in Malaysia: a potential monitoring and surveillance tool for the efficacy of control strategies

Roslan MA, Ngui R, Vythilingam I, Sulaiman WYW

J Vector Ecol, 2017 12;42(2):298-307.
PMID: 29125255 DOI: 10.1111/jvec.12270

The present study compared the performance of sticky traps in order to identify the most effective and practical trap for capturing Aedes aegypti and Aedes albopictus mosquitoes. Three phases were conducted in the study, with Phase 1 evaluating the five prototypes (Models A, B, C, D, and E) of sticky trap release-and-recapture using two groups of mosquito release numbers (five and 50) that were released in each replicate. Similarly, Phase 2 compared the performance between Model E and the classical ovitrap that had been modified (sticky ovitrap), using five and 50 mosquito release numbers. Further assessment of both traps was carried out in Phase 3, in which both traps were installed in nine sampling grids. Results from Phase 1 showed that Model E was the trap that recaptured higher numbers of mosquitoes when compared to Models A, B, C, and D. Further assessment between Model E and the modified sticky ovitrap (known as Model F) found that Model F outperformed Model E in both Phases 2 and 3. Thus, Model F was selected as the most effective and practical sticky trap, which could serve as an alternative tool for monitoring and controlling dengue vectors in Malaysia.
Fulltext Community surveillance of Aedes albopictus associated with Wolbachia detection in low-rise residential areas in Selangor, Malaysia

Roslan MA, Ngui R, Vythilingam I, Wan Sulaiman WY

J Vector Ecol, 2022 Dec;47(2):142-152.
PMID: 36314668 DOI: 10.52707/1081-1710-47.2.142

The study assessed the distribution of Malaysian Ae. albopictus adults associated with Wolbachia detection in low-rise residential areas using a modified sticky ovitrap (MSO). The relationship between Ae. albopictus and climatological parameters were also determined. Fifty-two weeks of surveillance using 273 MSOs were conducted in four installation areas of eleven sampling sites. Specimens were subjected to PCR using wsp-specific primers for Wolbachia detection. The relationship between climatological parameters and Ae. albopictus captured were analyzed using Spearman rank correlation coefficient test. The majority of Ae. albopictus were captured in residential houses (87%), followed by playgrounds or parks (11.5%), guardhouses (1%), and community halls (0.5%). Most of the specimens (92%) were superinfected with wAlbA and wAlbB strains. A positive correlation with no significant association was found for rainfall (r = 0.015, P = 0.072), relative humidity (r = 0.005, P = 0.526), minimum temperature (r = 0.005, P = 0.516), and mean temperature (r = 0.003, P = 0.689). MSO effectively captured a high number of Ae. albopictus that was determined to be the predominant mosquito species found in low-rise residential areas. The adult collection is not only influenced by climatological parameters but also by other factors, including environmental conditions and general sanitation status.
Fulltext Phylogenetic analysis of simian Plasmodium spp. infecting Anopheles balabacensis Baisas in Sabah, Malaysia

Chua TH, Manin BO, Daim S, Vythilingam I, Drakeley C

PLoS Negl Trop Dis, 2017 Oct;11(10):e0005991.
PMID: 28968395 DOI: 10.1371/journal.pntd.0005991

BACKGROUND: Anopheles balabacensis of the Leucospyrus group has been confirmed as the primary knowlesi malaria vector in Sabah, Malaysian Borneo for some time now. Presently, knowlesi malaria is the only zoonotic simian malaria in Malaysia with a high prevalence recorded in the states of Sabah and Sarawak.
METHODOLOGY/PRINCIPAL FINDINGS: Anopheles spp. were sampled using human landing catch (HLC) method at Paradason village in Kudat district of Sabah. The collected Anopheles were identified morphologically and then subjected to total DNA extraction and polymerase chain reaction (PCR) to detect Plasmodium parasites in the mosquitoes. Identification of Plasmodium spp. was confirmed by sequencing the SSU rRNA gene with species specific primers. MEGA4 software was then used to analyse the SSU rRNA sequences and bulid the phylogenetic tree for inferring the relationship between simian malaria parasites in Sabah. PCR results showed that only 1.61% (23/1,425) of the screened An. balabacensis were infected with one or two of the five simian Plasmodium spp. found in Sabah, viz. Plasmodium coatneyi, P. inui, P. fieldi, P. cynomolgi and P. knowlesi. Sequence analysis of SSU rRNA of Plasmodium isolates showed high percentage of identity within the same Plasmodium sp. group. The phylogenetic tree based on the consensus sequences of P. knowlesi showed 99.7%-100.0% nucleotide identity among the isolates from An. balabacensis, human patients and a long-tailed macaque from the same locality.
CONCLUSIONS/SIGNIFICANCE: This is the first study showing high molecular identity between the P. knowlesi isolates from An. balabacensis, human patients and a long-tailed macaque in Sabah. The other common simian Plasmodium spp. found in long-tailed macaques and also detected in An. balabacensis were P. coatneyi, P. inui, P. fieldi and P. cynomolgi. The high percentage identity of nucleotide sequences between the P. knowlesi isolates from the long-tailed macaque, An. balabacensis and human patients suggests a close genetic relationship between the parasites from these hosts.
Detection of Persistent Chikungunya Virus RNA but not Infectious Virus in Experimental Vertical Transmission in Aedes aegypti from Malaysia

Wong HV, Vythilingam I, Sulaiman WY, Lulla A, Merits A, Chan YF, et al.

Am J Trop Med Hyg, 2016 Jan;94(1):182-6.
PMID: 26598564 DOI: 10.4269/ajtmh.15-0318

Vertical transmission may contribute to the maintenance of arthropod-borne viruses, but its existence in chikungunya virus (CHIKV) is unclear. Experimental vertical transmission of infectious clones of CHIKV in Aedes aegypti mosquitoes from Malaysia was investigated. Eggs and adult progeny from the second gonotrophic cycles of infected parental mosquitoes were tested. Using polymerase chain reaction (PCR), 56.3% of pooled eggs and 10% of adult progeny had detectable CHIKV RNA, but no samples had detectable infectious virus by plaque assay. Transfected CHIKV RNA from PCR-positive eggs did not yield infectious virus in BHK-21 cells. Thus, vertical transmission of viable CHIKV was not demonstrated. Noninfectious CHIKV RNA persists in eggs and progeny of infected Ae. aegypti, but the mechanism and significance are unknown. There is insufficient evidence to conclude that vertical transmission exists in CHIKV, as positive results reported in previous studies were almost exclusively based only on viral RNA detection.
Fulltext Insecticide resistance and its underlying mechanisms in field populations of Aedes aegypti adults (Diptera: Culicidae) in Singapore

Koou SY, Chong CS, Vythilingam I, Lee CY, Ng LC

Parasit Vectors, 2014;7:471.
PMID: 25301032 DOI: 10.1186/s13071-014-0471-0

In Singapore, dose-response bioassays of Aedes aegypti (L.) adults have been conducted, but the mechanisms underlying resistance to insecticides remain unclear. In this study, we evaluated insecticide resistance and its underlying mechanism in field populations of Ae. aegypti adults.

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