Hydatid disease is not endemic in Malaysia; however, its migrant workers originate from neighboring countries where the disease is prevalent. Thus, this study was aimed at investigating the seroprevalence of hydatid disease among the workers. A total of 479 migrant workers were screened for hydatid disease. The sociodemographic information was collected, and serum samples were tested with a rapid dipstick test for hydatid disease called Hyd Rapid™. The present study showed that 13.6% of the migrant workers were found to be seropositive for hydatid disease. The highest seroprevalence was seen among Indian workers (29.41%), followed by Myanmarese (21.43%), Bangladeshis (14.92%), Nepalese (10.68%), and Indonesian (10.66%). This is the first study that highlights the likely presence of hydatid disease among the migrant workers in Malaysia, which may be of interest to the health authorities.
A broad range of cell lines with characteristic features are used as bio-factories to produce recombinant proteins for basic research and therapeutic purposes. Genetic engineering strategies have been used to manipulate the genome of mammalian cells, insects, and yeasts for heterologous expression. One reason is that the glycosylation pattern of the expression hosts differs somehow from mammalian cells, which may cause immunogenic reactions upon administration in humans. CRISPR-Cas9 is a simple, efficient, and versatile genome engineering tool that can be programmed to precisely make double-stranded breaks at the desired loci. Compared to the classical genome editing methods, a CRISPR-Cas9 system is an ideal tool, providing the opportunity to integrate or delete genes from the target organisms. Besides broadened applications, limited studies have used CRISPR-Cas9 for editing the endogenous pathways in expression systems for biopharmaceutical applications. In the present review, we discuss the use of CRISPR-Cas9 in expression systems to improve host cell lines, increase product yield, and humanize glycosylation pathways by targeting intrinsic genes.
Strongyloidiasis is a mysterious yet important parasitic disease that is hard to diagnose. While microscopic examination remains a "controversial" gold standard method, improved diagnosis is achieved through confirmatory assays with serological and/or molecular diagnostic approaches. In the current serodiagnosis of strongyloidiasis, recombinant proteins have been adopted in place of the use of native parasite antigens, although the availability of diagnostically potential proteins are still limited. Here, we introduce a novel Strongyloides recombinant protein that is uniquely attached to two different short peptide tags as a potential diagnostic biomarker for serodiagnosis of strongyloidiasis, namely lysine (7K) and aspartic acid (7D). The work presented focus on improving the yield and purity of the previously unexpressed recombinant protein. Preliminary diagnostic evaluation of the recombinant favors Ss3a7K protein owing to its higher antigenicity performance with 80% sensitivity and 100% specificity, respectively.
Amoebiasis, caused by Entamoeba histolytica, is one of the leading parasitic infections in the world. This study was aimed at profiling antigenic membrane proteins of a virulent variant of E. histolytica HM-1:IMSS. The membrane proteins were extracted using ProteoExtract® kit (Merck, Darmstadt, Germany) or conventional method, separated using OFFGEL 3100 fractionator (Agilent Technologies, Santa Clara, California), followed by SDS-PAGE and Western blot analysis. Selected antigenic membrane proteins were identified using LC-ESI-MS/MS. Subsequently, the proteins were classified according to their biological processes and predictions were made on membrane and membrane-associated proteins. When the proteins were probed with pooled sera from amoebic liver abscess (ALA) patients, 10 and 15 antigenic proteins with molecular weights 25 to 200 kDa were identified using the ProteoExtract® kit and conventional method, respectively. LC-ESI-MS/MS identified 13 antigenic proteins, and both extraction methods predicted six of them as membrane and membrane-associated proteins. The topmost biological processes which comprised of six proteins were involved in cellular processes.. These antigenic membrane proteins merit further investigations as potential candidates for vaccine studies.
This study investigated the applications of recombinant monoclonal antibodies (rmAbs) produced against two recombinant filarial proteins of diagnostic value. Ab5B and Ab3A were produced against recombinant BmSXP, and Ab4 and Ab4-fragment crystallizable (Fc) against recombinant BmR1. Ab5B and Ab4-Fc were found to be useful as quality control (QC) reagents for two commercial rapid test kits, such as Brugia RapidTM and BLF Rapid® (Reszon Diagnostics International Sdn. Bhd., 47600 Subang Jaya, Selangor, Malaysia), respectively. The two rmAbs reacted positively with the corresponding recombinant proteins lined on the nitrocellulose strips of the cassette tests, thus may replace or reduce the need for patient serum samples as positive controls for QC of the commercial kits. They were also successfully conjugated to gold nanoparticles and reacted positively with the test lines containing the corresponding recombinant proteins when directly applied to the cassette tests. The gold-conjugated reagents can be used to confirm the antigenicity of test lines after the storage of the rapid tests for a prolonged period or under unfavorable conditions. Furthermore, Ab5B and Ab3A were shown to be able to capture the target recombinant proteins through immunoaffinity purification, enabling their use for applications that need very highly purified proteins. In conclusion, this study demonstrated several potential uses of rmAb proteins produced against recombinant filarial proteins.
Infection by Toxoplasma gondii is prevalent worldwide. The parasite can infect a broad spectrum of vertebrate hosts, but infection of fetuses and immunocompromised patients is of particular concern. Easy-to-perform, robust, and highly sensitive and specific methods to detect Toxoplasma infection are important for the treatment and management of patients. Rapid diagnostic methods that do not sacrifice the accuracy of the assay and give reproducible results in a short time are highly desirable. In this context, rapid diagnostic tests (RDTs), especially with point-of-care (POC) features, are promising diagnostic methods in clinical microbiology laboratories, especially in areas with minimal laboratory facilities. More advanced methods using microfluidics and sensor technology will be the future trend. In this review, we discuss serological and molecular-based rapid diagnostic tests for detecting Toxoplasma infection in humans as well as animals.
Toxocariasis is a human infection primarily caused by larvae of Toxocara canis from dogs, and also by T. cati from cats. Children have a more significant risk of acquiring the infection due to their closer contact with pets, and greater chances of ingesting soil. Diagnosis of toxocariasis is based on clinical, epidemiological, and serological data. Indirect IgG ELISA is a widely used serodiagnostic method for toxocariasis, with native T. canis TES most commonly used as the antigen. Western blots, using the same antigen, can be used to confirm positive ELISA findings to reduce false-positive results. Improvements in Toxocara serodiagnosis include the use of recombinant TES antigens, simpler and more rapid assay formats, and IgG4 subclass detection. Also, incorporation of recombinant T. cati TES protein increases the diagnostic sensitivity. Development of antigen detection tests using polyclonal and monoclonal antibodies, nanobodies, or aptamers can complement the antibody detection assays, and enhance the effectiveness of the serodiagnosis.
Toxoplasma gondii is the etiologic agent of toxoplasmosis, a disease which can lead to morbidity and mortality of the fetus and immunocompromised individuals. Due to the limited effectiveness or side effects of existing drugs, the search for better drug candidates is still ongoing. In this study, we performed structure-based screening of potential dual-targets inhibitors of active sites of T. gondii drug targets such as uracil phosphoribosyltransferase (UPRTase) and adenosine kinase (AK). First screening of virtual compounds from the National Cancer Institute (NCI) was performed via molecular docking. Subsequently, the hit compounds were tested in-vitro for anti- T. gondii effect using cell viability assay with Vero cells as host to determine cytotoxicity effects and drug selectivities. Clindamycin, as positive control, showed a selectivity index (SI) of 10.9, thus compounds with SI > 10.9 specifically target T. gondii proliferation with no significant effect on the host cells. Good anti- T. gondii effects were observed with NSC77468 (7-ethoxy-4-methyl-6,7-dihydro-5H-thiopyrano[2,3-d]pyrimidin-2-amine) which showed SI values of 25. This study showed that in-silico selection can serve as an effective way to discover potentially potent and selective compounds against T. gondii.
The influx of low skilled migrant workers to Malaysia from low socio-economic countries where gastrointestinal parasitic infections are prevalent has raised concerns about transmission to the local population. Three methods for detection (serology, microscopy and molecular techniques) were utilized to identify Entamoeba infections amongst the targeted cohort and determine risk factors associated with infection. Serological screening of 484 migrant workers from five working sectors in Peninsular Malaysia using IgG4 ELISA based on the rPPDK antigen showed an overall seroprevalence of 7.4% (n = 36; CL95 = 5.3-10.1%) with only one factor statistically associated with seropositivity of anti-amoebic antibodies, i.e. years of residence in Malaysia (χ2 1 = 4.007, p = 0.045). Microscopic examination of 388 faecal samples for protozoan cysts and trophozoites showed a slightly higher prevalence (11.6%; n=45; CL95: 8.4-14.8%). Meanwhile, amplification of the 16S rDNA gene detected two species i.e. Entamoeba dispar (23/388; 5.9%; CL95: 3.6-8.3%) and E. histolytica (11/388; 2.8%; CL95: 1.2-4.5%) and mixed infections with both parasites in only three samples (3/388; 0.8%; CL95: 0.2-2.2%). Entamoeba dispar infection was significantly associated with those employed in food and domestic services (χ2 4 = 12.879, p = 0.012). However, none of the factors affected the prevalence of E. histolytica infection. Despite the low prevalence of E. histolytica in faecal samples of the study cohort, the presence of this pathogenic parasite still poses potential public health risks and calls for tighter control strategies based on better availability of chemotherapeutic treatment and accessibility to appropriate health education.
This cross-sectional study aimed to determine the prevalence and risk factors of S. stercoralis infection among 1142 Orang Asli primary schoolchildren in six different states of Peninsular Malaysia. Fecal samples were examined using direct smear, formalin-ether sedimentation (FES), agar plate culture (APC) and PCR techniques. Overall, 15.8% of the children were found to be infected with S. stercoralis. The prevalence was 0.2, 1.3, 15.2 and 13.7% by direct smear, FES, APC and PCR, respectively. Multivariate analysis showed that an age of >10 years, being male, belonging to a Proto-Malay tribe, belonging to the Senoi tribe, indiscriminate defecation, using an unimproved water source for drinking water and not wearing shoes when outside were the significant risk factors of infection among these children. In conclusion, we provide new evidence on the occurrence of S. stercoralis in Malaysia to show that there is a relatively high prevalence of infection among Orang Asli schoolchildren. Therefore, the use of specific methods for detecting S. stercoralis should be considered when screening these children for intestinal parasites. Moreover, prevention and control measures specific to S. stercoralis should be integrated into the intestinal parasitic infections control programme in Malaysia.
The geographical distribution of tuberculosis (TB) overlaps with various parasitic infections. Uncovering the characteristics of coinfecting parasites that potentially affect the host susceptibility to TB is pertinent as it may provide input to current TB therapeutic and prophylactic measures. The present study was aimed at examining the types of parasitic infections in TB patients and healthy TB contacts (HC) in Orang Asli, Malaysian aborigines, who dwelled in the co-endemic areas. Stool and serum samples were collected from Orang Asli who fulfilled the selection criteria and provided written informed consents. Selected parasitic infections in the two study groups were determined by stool examination and commercial serum antibody immunoassays. The prevalence of parasitic infections in TB and HC participants were 100% (n = 82) and 94.6% (n = 55) respectively. The parasitic infections comprised toxocariasis, trichuriasis, amoebiasis, toxoplasmosis, hookworm infection, ascariasis, strongyloidiasis, and brugian filariasis, in decreasing order of prevalence. Overall, helminth or protozoa infection did not show any significant association with the study groups. However, when the species of the parasite was considered, individuals exposed to trichuriasis and toxoplasmosis showed significant odds reduction (odds ratio (OR) 0.338; 95% confidence interval (CI) 0.166, 0.688) and odds increment (OR 2.193; 95% CI 1.051, 4.576) to have active pulmonary TB, respectively. In conclusion, trichuriasis and toxoplasmosis may have distinct negative and positive associations respectively with the increase of host susceptibility to TB.
Lymphatic filariasis is a mosquito-borne parasitic disease responsible for morbidity and disability that affects 1.2 billion people worldwide, mainly the poor communities. Currently, filarial antigen testing is the method of choice for the detection of bancroftian filariasis, and to date, there are two commonly used tests. In the present study, a recently reported recombinant monoclonal antibody (5B) specific to BmSXP filarial antigen was used in developing an ELISA for the detection of circulating filarial antigen in sera of patients with bancroftian filariasis. The performance of the ELISA was evaluated using 124 serum samples. The ELISA was positive with all sera from microfilaremic bancroftian filariasis patients (n = 34). It also showed 100% diagnostic specificity when tested with sera from 50 healthy individuals and 40 patients with other parasitic diseases. The developed assay using the novel 5B recombinant monoclonal antibody could potentially be a promising alternative antigen detection test for bancroftian filariasis.
The conventional method of detecting Strongyloides stercoralis in fecal samples has poor diagnostic sensitivity. Detection of Strongyloides-specific antibodies increases the sensitivity; however, most tests are ELISAs that use parasite extract which may cross-react with the sera of other helminth infections. To improve the serological diagnosis of strongyloidiasis, this study aimed at developing a sensitive and specific lateral flow rapid dipstick test. Two recombinant proteins, recombinant NIE (rNIE) and recombinant Ss1a (rSs1a), were used in preparing the dipstick, with gold-conjugated antihuman IgG4 as detector reagent. In parallel, the corresponding ELISA was performed. Both assays demonstrated diagnostic sensitivity of 91.3% (21/23) when tested with serum samples of patients with Strongyloides infection, and 100% specificity with 82 sera of asymptomatic (healthy) and those with other parasitic infections. The ELISA and dipstick test results were positively correlated to each other (r = 0.6114, P = 0.0019). The developed lateral flow dipstick test may improve the serodiagnosis of strongyloidiasis and merit further validation studies.
Strongyloidiasis is a major neglected tropical disease with the potential of causing lifelong infection and mortality. One of the ways for effective control of this disease is developing improved diagnostics, particularly using serological approaches. A serological test can achieve high diagnostic sensitivity and specificity, has the potential for point-of-care translation, and can be used as a screening tool for early detection. More research is needed to find clinically important antibody biomarkers for early disease detection, mapping, and epidemiological surveillance. This article summarizes human strongyloidiasis and the available diagnostic tools for the disease, focusing on describing the current antibody assays for strongyloidiasis. Finally, prospects of developing a more effective serodiagnostic tool for strongyloidiasis are discussed.
Cystic echinococcosis (CE) is a neglected zoonotic disease with a worldwide distribution and is a major public health problem in some areas. Diagnosis of CE is mainly based on clinical symptoms, imaging and serological testing, however, improvement in serodiagnosis is still needed. This study was aimed at detecting circulating Echinococcus antigen in CE patients using a lateral flow dipstick (LFD) assay. Three types of hydatid antigens i.e. hydatid cyst fluid (HCF), native antigen B (nAgB) and recombinant antigen B (rAgB) were prepared and polyclonal rabbit antiserum was raised against each antigen. Purified IgG fractions were prepared and a portion was conjugated to gold nanoparticles. After a series of optimizations, a final antigen detection LFD assay was developed using a combination of anti-nAgB-IgG and gold-conjugated anti-HCF-IgG. Evaluation of the assay showed that 27 out of 35 (77%) serum samples from CE patients gave positive results. Meanwhile, the test showed a diagnostic specificity of 82% when tested with sera from 38 healthy individuals and 13 patients with other parasitic diseases. In conclusion, the antigen detection LFD assay seemed to be useful for diagnosis of CE and possibly for post-treatment follow-up, and merit further evaluation studies. We foresee that it may improve serodiagnosis of CE when used in tandem with an antibody detection test.
Amoebiasis, an enteric protozoan disease caused by Entamoeba histolytica, is a public health problem in many developing countries, causing up to 100,000 fatal cases annually. Detection of the pathogenic E. histolytica and its differentiation from the non-pathogenic Entamoeba spp. play a crucial role in the clinical management of patients. Laboratory diagnosis of intestinal amoebiasis in developing countries still relies on labour-intensive and insensitive methods involving staining of stool sample and microscopy. Newer and more sensitive methods include a variety of antigen detection ELISAs and rapid tests; however, their diagnostic sensitivity and specificity seem to vary between studies, and some tests do not distinguish among the Entamoeba species. Molecular detection techniques are highly sensitive and specific and isothermal amplification approaches may be developed into field-applicable tests; however, cost is still a barrier for their use as a routine laboratory test method in most endemic areas. Laboratory diagnosis of extraintestinal amoebiasis faces challenges of lack of definitive detection of current infection and commercially available point-of-care tests. For both types of amoebiasis, there is still a need for highly sensitive and specific tests that are rapid and cost-effective for use in developing countries where the disease is prevalent. In recent years, new molecules of diagnostic value are being discovered and new tests developed. The advances in 'omics' technologies are enabling discoveries of new biomarkers that may help distinguish between different infection stages.
At the end phase of the Global Programme to Eliminate Lymphatic Filariasis, antibody testing may have a role in decision-making for bancroftian filariasis-endemic areas. This study evaluated the diagnostic performance of BLF Rapid™, a prototype immunochromatographic IgG4-based test using BmSXP recombinant protein, for detection of bancroftian filariasis. The test was evaluated using 258 serum samples, comprising 96 samples tested at Universiti Sains Malaysia (in-house) and 162 samples tested independently at three international laboratories in the USA and India, and two laboratories in Malaysia. The independent testing involved 99 samples from Wuchereria bancrofti microfilaria or antigen positive individuals and 63 samples from people who were healthy or had other infections. The in-house evaluation showed 100% diagnostic sensitivity and specificity. The independent evaluations showed a diagnostic sensitivity of 84-100% and 100% specificity (excluding non-lymphatic filarial infections). BLF Rapid has potential as a surveillance diagnostic tool to make "Transmission Assessment Survey"-stopping decisions and conduct post-elimination surveillance.
Entamoeba histolytica is a protozoan parasite that causes amebiasis and poses a significant health risk for populations in endemic areas. The molecular mechanisms involved in the pathogenesis and regulation of the parasite are not well characterized. We aimed to identify and quantify the differentially abundant membrane proteins by comparing the membrane proteins of virulent and avirulent variants of E. histolytica HM-1:IMSS, and to investigate the potential associations among the differentially abundant membrane proteins. We performed quantitative proteomics analysis using isobaric tags for relative and absolute quantitation labeling, in combination with two mass spectrometry instruments, that is, nano-liquid chromatography (nanoLC)-matrix-assisted laser desorption/ionization-mass spectrometry/mass spectrometry and nanoLC-electrospray ionization tandem mass spectrometry. Overall, 37 membrane proteins were found to be differentially abundant, whereby 19 and 18 membrane proteins of the virulent variant of E. histolytica increased and decreased in abundance, respectively. Proteins that were differentially abundant include Rho family GTPase, calreticulin, a 70-kDa heat shock protein, and hypothetical proteins. Analysis by Protein ANalysis THrough Evolutionary Relationships database revealed that the differentially abundant membrane proteins were mainly involved in catalytic activities (29.7%) and metabolic processes (32.4%). Differentially abundant membrane proteins that were found to be involved mainly in the catalytic activities and the metabolic processes were highlighted together with their putative roles in relation to the virulence. Further investigations should be performed to elucidate the roles of these proteins in E. histolytica pathogenesis.
Entamoeba histolytica membrane proteins are important players toward the pathogenesis of amoebiasis, but the roles of most of the proteins are not fully understood. Since efficient protein extraction method is crucial for a successful MS analysis, three extractions methods are evaluated for the use in studying the membrane proteome of E. histolytica: Two commercial kits (ProteoExtract from Calbiochem and ProteoPrep from Sigma), and a conventional laboratory method. The results show that ProteoExtract and the conventional method gave higher protein yields compared to ProteoPrep. LC-ESI-MS/MS identifies 456, 482, and 551 membrane fraction proteins extracted using ProteoExtract, ProteoPrep, and a conventional method, respectively. In silico analysis predicts 108 (21%), 235 (45%), and 177 (34%) membrane proteins from the extracts of ProteoExtract, ProteoPrep, and the conventional method, respectively. Furthermore, analysis of the cytosolic and membrane fractions shows the highest selectivity of the membrane proteins using the ProteoPrep extraction kit. Overall, this study reports 828 E. histolytica membrane fraction proteins that include 249 predicted membrane proteins. The data are available via ProteomeXchange with identifier PXD010171.