Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 105 CFU/ml, while PCR displayed a limit of 5.9 x 107 CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 104 CFU/g, whereas PCR was 3.6 x 105 CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Surface antigens are the most abundant proteins found on the surface of the parasite Toxoplasma gondii. Surface antigen 1 (SAG1) and Surface antigen 2 (SAG2) remain the most important and extensively studied surface proteins. These antigens have been identified to play a role in host cell invasion, immune modulation, virulence attenuation. Recombinant SAG1/2 was cloned and expressed in yeast Pichia pastoris. We describe here optimization of critical parameters involved in high yield expression of the recombinant SAG1/2. Our results suggest that recombinant SAG1/2 were best expressed at 30ºC, pH 6 and 1% methanol as the carbon source by X33 Pichia cells. Additional optimizations included the downstream process such as ammonium sulphate precipitation and dialysis. The fusion protein was purified using Ni-NTA purification system with 80% recovery. The purified protein was 100% specific and sensitive in detection of toxoplasmosis.

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

Malaria causes high global mortality and morbidity annually. Plasmodium knowlesi has been recognised as the fifth human Plasmodium sp. and its infection is widely distributed in Southeast Asia. Merozoite surface protein-119 (MSP-119) appears as a potential candidate for malaria blood stage vaccine as it could induce protective immunity. In this study, codon optimized P. knowlesi MSP-119 (pkMSP-119) was expressed and purified in yeast Pichia pastoris expression system. The purified recombinant protein was further evaluated using Western blot assay using knowlesi malaria, non-knowlesi human malaria, non-malarial parasitic infections and healthy serum samples (n = 50). The sensitivity of purified pkMSP-119 towards detection of knowlesi infection was as 28.6% (2/7). pkMSP-119 did not react with all nonmalarial parasitic infections and healthy donor sera, yet reacted with some non-knowlesi human malaria sera, therefore lead to a specificity of 86.0% (37/43).

A survey was undertaken to investigate the prevalence of intestinal and blood parasites among wild rats in urban area of Kuala Lumpur, Malaysia. A total of 137 stool and blood samples were collected from wild rats from Sentul and Chow Kit areas. Five species of rats were captured and supplied by Kuala Lumpur City Hall. The most common was Rattus rattus diardii (Malayan Black rat), 67%, followed by Rattus norvegicus (Norway rat), 10%, Rattus argentiventer (rice-field rat), 10%, Rattus tiomanicus (Malaysian field rat), 9% and Rattus exulans (Polynesian rat), 4%. Rattus rattus diardii is commonly known to live in human environment and they are normally identified as pests to human community. More male rats were captured (61%) compared to female (39%). Out of 137 samples, 81.8% samples were positive with intestinal parasites, with 86.2% from Sentul area and 78.5% from Chow Kit area. Six different parasites were detected. The most common intestinal helminth parasite detected was Nippostrongylus brasiliensis (80.3%), followed by Hymenolepis nana (23.4%), Capillaria hepatica (13.9%) and Hymenolepis diminuta (2.9%). Intestinal protozoan detected was Entamoeba histolytica/E. dispar (8.8%). Trypanosoma lewisi (1.5%) was the only blood parasite detected.

The Plasmodium knowlesi apical membrane antigen-1 (PkAMA-1) plays an important role in the invasion of the parasite into its host erythrocyte, and it has been regarded as a potential vaccine candidate against human knowlesi malaria. This study investigates genetic diversity and natural selection of the full length PkAMA-1 of P. knowlesi clinical isolates from Peninsular Malaysia. Blood samples were collected from P. knowlesi malaria patients from Peninsular Malaysia. The PkAMA-1 gene was amplified from DNA samples using PCR, cloned into a plasmid vector and sequenced. Results showed that nucleotide diversity of the full length PkAMA-1 from Peninsular Malaysia isolates (π: 0.006) was almost similar to that of Sarawak (π: 0.005) and Sabah (π: 0.004) isolates reported in other studies. Deeper analysis revealed Domain I (π: 0.007) in the PkAMA-1 had the highest diversity as compared to Domain II (π: 0.004) and Domain III (π: 0.003). Z-test indicated negative (purifying) selection of the gene. Combined alignment analysis at the amino acid level for the Peninsular Malaysia and Sarawak PkAMA-1 sequences revealed 34 polymorphic sites. Thirty-one of these sites were dimorphic, and 3 were trimorphic. The amino acid sequences could be categorised into 31 haplotypes. In the haplotype network, PkAMA-1 from Peninsular Malaysia and Sarawak were separated into two groups.

Circumsporozoite protein (CSP) is a sporozoite major surface protein of Plasmodium species. The protein showed promising protection level as a vaccine candidate against Plasmodium falciparum infection. There is a lack of studies on P. knowlesi CSP (PkCSP) as a vaccine candidate due to the high polymorphic characteristic of central repeat region. Recent studies showed the protein has a relatively conserved region at the C-terminal, which consists of T- and B-cell epitopes. This could be the target region for vaccine development against the pre-erythrocytic stage of the parasite. In this study, recombinant PkCSP was expressed using Escherichia coli system. Recombinant PkCSP was immunized in animal models and the antiserum was evaluated using immunoblot analysis. Results showed that PkCSP can be successfully expressed using the bacterial system. Endpoint titre of the antiserum were ranged up to 1:819200. Immunoblot analysis showed the antiserum recognized recombinant PkCSP but not total protein extract from P. knowlesi erythrocytic stage. In conclusion, PkCSP could elicit strong immune response in animal models. However, serum antibodies could not recognize protein from the parasite’s erythrocytic stage extract indicating it is not expressed at the erythrocytic stage. Therefore, PkCSP remains as a potential pre-erythrocytic vaccine candidate against P. knowlesi infection.

The LAMP assay, amplifies the target DNA rapidly, with 10-fold greater sensitivity than conventional PCR. The greater sensitivity also comes with greater risks of contamination. To overcome this issue, the current project includes either uracil DNA glycosylase (UDG) or a mineral oil overlay in the LAMP assay. Our results indicated that UDG or a mineral oil overlay can effectively prevent carryover contamination in the LAMP assay for the detection of human malaria. By incorporating these preventative methods, contamination can be eliminated and LAMP can potentially be used in the field; and point of care diagnosis for human malaria.

The focus of the current study was to disrupt the Toxo 5699 gene via CRISPR/Cas9 to evaluate the effects of gene disruption on the parasite lytic cycle. In the present work, a single plasmid expressing both the guide RNA and Cas9 nuclease together with a selectable marker of human dihydrofolate reductase (DHFR) was introduced into Toxoplasma gondii. Targeted disruption of the Toxo 5699 gene was carried out via the CRISPR/Cas9 system and confirmed by PCR, sequencing, and immunofluorescence microscopy. Disrupted and nondisrupted control parasites were allowed to invade HS27 cell monolayers and plaques were counted. The average number of plaques from three replicates per group was obtained between the disrupted and non-disrupted T. gondii RH strain and was compared using a onetailed t-test. It was observed that there was a significant decrease in number and size of plaque formation in the Toxo 5699 gene disrupted parasite line. This is an indication that the Toxo 5699 gene may play a role in the lytic cycle of the parasite, particularly during the replication phase and thus would be a novel target for disruption or silencing. The Toxo 5699 gene presented in the current work is an important part of the T. gondii lytic cycle, therefore meriting further inquiry into its potential as a target for further genetic-silencing or disruption studies.

Rickettsioses are a common health problem in many geographical areas, including rural areas in Southeast Asia. Co-infection of rickettsioses and malaria has been reported in Africa, where common reservoir and vectors are available. In this study, blood samples of Malaysian patients microscopically positive (n=148) and negative (n=88) for malaria parasites (Plasmodium knowlesi, Plasmodium malariae, Plasmodium falciparum, and Plasmodium vivax) were screened for the presence of rickettsial DNA, using PCR assays targeting specific genes. A partial fragment of rickettsial ompB gene was successfully amplified and sequenced from a patient microscopically positive for Plasmodium spp. and PCR-positive for P. vivax. BLAST analysis of the ompB sequence demonstrated the highest sequence similarity (99.7% similarity, 408/409nt) with Rickettsia sp. RF2125 (Genbank accession no. JX183538) and 91.4% (374/409 nt) similarity with Rickettsia felis URRWXCal2 (Genbank accession no. CP000053). This study reports rickettsial infection in a malaria patient for the first time in the Southeast Asia region.