Displaying publications 21 - 40 of 117 in total

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  1. Comparison of IgG4 assays using whole parasite extract and BmR1 recombinant antigen in determining antibody prevalence in brugian filariasis
    Noordin R, Wahyuni S, Mangali A, Huat LB, Yazdanbakhsh M, Sartono E
    Filaria journal, 2004 Aug 12;3(1):8.
    PMID: 15307892
    BACKGROUND: Brugia malayi is endemic in several Asian countries with the highest prevalence in Indonesia. Determination of prevalence of lymphatic filariasis by serology has been performed by various investigators using different kinds of antigen (either soluble worm antigen preparations or recombinant antigens). This investigation compared the data obtained from IgG4 assays using two different kinds of antigen in a study on prevalence of antibodies to B. malayi. METHODS: Serum samples from a transmigrant population and life long residents previously tested with IgG4 assay using soluble worm antigen (SWA-ELISA), were retested with an IgG4 assay that employs BmR1 recombinant antigen (BmR1 dipstick [Brugia Rapid trade mark ]). The results obtained with the two antigens were compared, using Pearson chi-square and McNemar test. RESULTS: There were similarities and differences in the results obtained using the two kinds of antigen (SWA and BmR1). Similarities included the observation that assays using both antigens demonstrated an increasing prevalence of IgG4 antibodies in the transmigrant population with increasing exposure to the infection, and by six years living in the area, antibody prevalence was similar to that of life-long residents. With regards to differences, of significance is the demonstration of similar antibody prevalence in adults and children by BmR1 dipstick whereas by SWA-ELISA the antibody prevalence in adults was higher than in children. CONCLUSIONS: Results and conclusions made from investigations of prevalence of anti-filarial IgG4 antibody in a population would be affected by the assay employed in the study.
  2. Fulltext Comparison of Two Serological Assays in Detecting Strongyloides Infection in Immunocompromised Patients
    Osman E, Amin NA, Noon TPM, Lahat SNH, Rosli MS, Sham SF, et al.
    Am J Trop Med Hyg, 2022 Jul 25;107(3):636-9.
    PMID: 35895335 DOI: 10.4269/ajtmh.22-0076
    Strongyloides infection may develop into fatal hyperinfection and dissemination syndrome in immunocompromised hosts. Despite suboptimal specificity issues, the detection of IgG antibodies by ELISA has been central in the serodiagnosis of Strongyloides infection. Recently, an IgG4-based lateral-flow test (SsRapid) using recombinant NIE (rNIE) protein with good diagnostic performance has been reported. This study assessed the result concordance between a commercial IgG-ELISA and the SsRapid. Additionally, we determined the Strongyloides seroprevalence and its association with clinical manifestations. Immunocompromised patients (N = 200) were from Hospital Canselor Tuanku Muhriz, Kuala Lumpur, Malaysia, and were diagnosed with HIV/AIDS, hematological malignancy, and solid organ cancers. Their plasma samples were tested using a commercial IgG-ELISA and SsRapid. A fair concordance (κ = 0.27-0.33; P < 0.05) among the tests was demonstrated. The SsRapid exhibited a significantly higher (P < 0.05) seroprevalence (10.5% [21/200]) compared with IgG-ELISA (7.5% [15/200]). After adsorption with rNIE, all SsRapid-positive samples tested negative with the rapid test, thus showing binding specificity. There was no significant association with clinical manifestations. This study revealed that SsRapid is a useful diagnostic tool for Strongyloides infection, and there is a notable seroprevalence among the immunocompromised patients.
  3. Comparison of protein-free defined media, and effect of L-cysteine and ascorbic acid supplementation on viability of axenic Entamoeba histolytica
    Wong WK, Tan ZN, Lim BH, Mohamed Z, Olivos-Garcia A, Noordin R
    Parasitol Res, 2011 Feb;108(2):425-30.
    PMID: 20922423 DOI: 10.1007/s00436-010-2083-8
    Entamoeba histolytica is the etiologic agent for amoebiasis. The excretory-secretory (ES) products of the trophozoites contain virulence factors and antigens useful for diagnostic applications. Contaminants from serum supplements and dead trophozoites impede analysis of ES. Therefore, a protein-free medium that can sustain maximum viability of E. histolytica trophozoites for the longest time duration will enable collection of contaminant-free and higher yield of ES products. In the present study, we compared the efficacy of four types of media in maintaining ≥ 95% trophozoite viability namely Roswell Memorial Park Institute (RPMI-1640), Dulbecco's Modified Eagle Medium (DMEM), phosphate-buffered saline for amoeba (PBS-A), and Hank's balanced salt solution (HBSS). Concurrently, the effect of adding L: -cysteine and ascorbic acid (C&A) to each medium on the parasite viability was also compared. DMEM and RPMI 1640 showed higher viabilities as compared to PBS-A and HBSS. Only RPMI 1640 showed no statistical difference with the control medium for the first 4 h, however the ≥ 95% viability was only maintained for the first 2 h. The other protein-free media showed differences from the serum- and vitamin-free TYI-S-33 control media even after 1 h of incubation. When supplemented with C&A, all media were found to sustain higher trophozoite viabilities than those without the supplements. HBSS-C&A, DMEM-C&A, and RPMI 1640-C&A demonstrated no difference (P>0.05) in parasite viabilities when compared with the control medium throughout the 8-h incubation period. DMEM-C&A showed an eightfold increment in time duration of sustaining ≥ 95% parasite viability, i.e. 8 h, as compared to DMEM alone. Both RPMI 1640-C&A and HBSS-C&A revealed fourfold and threefold increments (i.e., 8 and 6 h, respectively), whereas PBS-A-C&A showed only one fold improvement (i.e., 2 h) as compared to the respective media without C&A. Thus, C&A-supplemented DMEM or RPMI are recommended for collection of ES products.
  4. Comparison of two IgG4 assay formats (ELISA and rapid dipstick test) for detection of brugian filariasis
    Noordin R, Shenoy RK, Rahman RA
    Southeast Asian J. Trop. Med. Public Health, 2003 Dec;34(4):768-70.
    PMID: 15115085
    Brugia malayi infection is endemic in several Asian countries. Filaria-specific IgG4 antibody detection based on BmR1 recombinant antigen has been shown to be sensitive and specific for the diagnosis of brugian filariasis. Two formats of the test has been reported ie indirect ELISA (BE) and rapid dipstick test (BR). Since different test formats use different amounts of sample and reagents which may affect its sensitivity and specificity, this study was performed to compare these two test formats in the detection of B. malayi. A total of 264 blinded serum samples from India and Malaysia were employed. Group 1 comprised 164 samples from actively infected individuals and group 2 comprised 100 samples from filaria non-endemic areas. Sensitivity was 96.3% (158/164) and 90.8% (149/164) for rapid test and ELISA respectively; chi-square p=0.00. Both test formats demonstrated 100% specificity. Therefore the rapid test format was equally specific but more sensitive than the ELISA format. The ELISA format would be able to demonstrate decline in IgG4 titer post-treatment while the rapid test would be very useful for screening and diagnosis in the field.
  5. Delineation of BmSXP antibody V-gene usage from a lymphatic filariasis based immune scFv antibody library
    Rahumatullah A, Ahmad A, Noordin R, Lim TS
    Mol Immunol, 2015 Oct;67(2 Pt B):512-23.
    PMID: 26277276 DOI: 10.1016/j.molimm.2015.07.040
    Phage display technology is an important tool for antibody generation or selection. This study describes the development of a scFv library and the subsequent analysis of identified monoclonal antibodies against BmSXP, a recombinant antigen for lymphatic filariasis. The immune library was generated from blood of lymphatic filariasis infected individuals. A TA based intermediary cloning approach was used to increase cloning efficiency for the library construction process. A diverse immune scFv library of 10(8) was generated. Six unique monoclonal antibodies were identified from the 50 isolated clones against BmSXP. Analysis of the clones showed a bias for the IgHV3 and Vκ1 (45.5%) and IgHV2 and Vκ3 (27.3%) gene family. The most favored J segment for light chain is IgKJ1 (45.5%). The most favored D and J segment for heavy chain are IgHD6-13 (75%) and IgHJ3 (47.7%). The information may suggest a predisposition of certain V genes in antibody responses against lymphatic filariasis.
  6. Fulltext Detection of Entamoeba histolytica in experimentally induced amoebic liver abscess: comparison of three staining methods
    Ning TZ, Kin WW, Mustafa S, Ahmed A, Noordin R, Cheong TG, et al.
    Asian Pac J Trop Biomed, 2012 Jan;2(1):61-5.
    PMID: 23569836 DOI: 10.1016/S2221-1691(11)60191-3
    To compare the efficacy of three different tissue stains, namely haematoxylin and eosin (H&E), periodic-acid Schiff (PAS) and immunohistochemical (IHC) stains for detection of Entamoeba histolytica (E. histolytica) trophozoites in abscessed liver tissues of hamster.
  7. Fulltext Detection of Strongyloides stercoralis infection among cancer patients in a major hospital in Kelantan, Malaysia
    Zueter AM, Mohamed Z, Abdullah AD, Mohamad N, Arifin N, Othman N, et al.
    Singapore Med J, 2014 Jul;55(7):367-71.
    PMID: 25091885
    INTRODUCTION: Strongyloidiasis is one of the most commonly neglected but clinically important parasitic infections worldwide, especially among immunocompromised patients. Evidence of infection among immunocompromised patients in Malaysia is, however, lacking. In this study, microscopy, real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assays (ELISAs) were used to detect Strongyloides stercoralis (S. stercoralis) infection among cancer patients in a Malaysian hospital.

    METHODS: A total of 192 stool and serum samples were collected from cancer patients who were receiving chemotherapy with or without steroid treatment at a hospital in northeastern Malaysia. Stool samples were examined for S. stercoralis using parasitological methods and real-time PCR. Serology by ELISA was performed to detect parasite-specific immunoglobulin G (IgG), IgG4 and immunoglobulin E (IgE) antibodies. For comparison, IgG4- and IgG-ELISAs were also performed on the sera of 150 healthy individuals from the same area.

    RESULTS: Of the 192 samples examined, 1 (0.5%) sample was positive for S. stercoralis by microscopy, 3 (1.6%) by real-time PCR, 8 (4.2%) by IgG-ELISA, 6 (3.1%) by IgG4-ELISA, and none was positive by IgE-ELISA. In comparison, healthy blood donors had significantly lower prevalence of parasite-specific IgG (2.67%, p < 0.05) and IgG4 (2.67%, p < 0.05) responses.

    CONCLUSION: This study showed that laboratory testing may be considered as a diagnostic investigation for S. stercoralis among immunocompromised cancer patients.
  8. Detection of immunogenic parasite and host-specific proteins in the sera of active and chronic individuals infected with Toxoplasma gondii
    Yeng C, Osman E, Mohamed Z, Noordin R
    Electrophoresis, 2010 Dec;31(23-24):3843-9.
    PMID: 21080484 DOI: 10.1002/elps.201000038
    Toxoplasma gondii infection in pregnant women may result in abortion and foetal abnormalities, and may be life-threatening in immunocompromised hosts. To identify the potential infection markers of this disease, 2-DE and Western blot methods were employed to study the parasite circulating antigens and host-specific proteins in the sera of T. gondii-infected individuals. The comparisons were made between serum protein profiles of infected (n=31) and normal (n=10) subjects. Antigenic proteins were identified by immunoblotting using pooled sera and monoclonal anti-human IgM-HRP. Selected protein spots were characterised using mass spectrometry. Prominent differences were observed when serum samples of T. gondii-infected individuals and normal controls were compared. A significant up-regulation of host-specific proteins, α(2)-HS glycoprotein and α(1)-B glycoprotein, was also observed in the silver-stained gels of both active and chronic infections. However, only α(2)-HS glycoprotein and α(1)-B glycoprotein in the active infection showed immunoreactivity in Western blots. In addition, three spots of T. gondii proteins were detected, namely (i) hypothetical protein chrXII: 3984434-3 TGME 49, (ii) dual specificity protein phosphatase, catalytic domain TGME 49 and (iii) NADPH-cytochrome p450 reductase TGME 49. Thus, 2-DE approach followed by Western blotting has enabled the identification of five potential infection markers for the diagnosis of toxoplasmosis: three are parasite-specific proteins and two are host-specific proteins.
  9. Fulltext Detection of selected intestinal helminths and protozoa at Hospital Universiti Sains Malaysia using multiplex real-time PCR
    Basuni M, Mohamed Z, Ahmad M, Zakaria NZ, Noordin R
    Trop Biomed, 2012 Sep;29(3):434-42.
    PMID: 23018507
    Intestinal parasites are the causative agents of a number of important human infections in developing countries. The objective of this study was to determine the prevalence of selected helminths and protozoan infections among patients admitted with gastrointestinal disorders at Hospital Universiti Sains Malaysia, Kelantan, Malaysia using multiplex real-time PCR. In addition microscopic examination was also performed following direct smear, zinc sulphate concentration and Kato-Katz thick smear techniques; and the presence of protozoan parasites was confirmed using trichrome and acid-fast stains. Of the 225 faecal samples analysed, 26.2% were positive for intestinal parasites by the multiplex real-time PCR, while 5.3% were positive by microscopy. As compared to microscopy, the multiplex real-time PCR detected 5.8 and 4.5 times more positives for the selected helminth and protozoan infections respectively. Among the selected helminths detected in this study, hookworm was the most prevalent by real-time PCR, while Ascaris lumbricoides was detected the most by microscopy. Meanwhile, among the selected protozoa detected in this study, Entamoeba histolytica was the most prevalent by real-time PCR, however microscopy detected equal number of cases with E. histolytica and Giardia lamblia. This study showed that real-time PCR can be used to obtain a more accurate prevalence data on intestinal helminths and protozoa.
  10. Fulltext Development and evaluation of a sensitive and specific assay for diagnosis of human toxocariasis by use of three recombinant antigens (TES-26, TES-30USM, and TES-120)
    Mohamad S, Azmi NC, Noordin R
    J Clin Microbiol, 2009 Jun;47(6):1712-7.
    PMID: 19369434 DOI: 10.1128/JCM.00001-09
    Diagnosis of human toxocariasis currently relies on serologic tests that use Toxocara excretory-secretory (TES) antigen to detect immunoglobulin G (IgG) antibodies to the larvae. In general, however, these assays do not have adequate specificity for use in countries in which other soil-transmitted helminths are endemic. The use of recombinant antigens in these assays, however, is promising for improving the specificity of the diagnosis of toxocariasis. Toward this goal, we developed an IgG4 enzyme-linked immunosorbent assay (ELISA) involving three recombinant antigens: rTES-30USM (previously produced), rTES-26, and rTES-120. The latter two antigens were produced by reverse transcription-PCR cloning; subcloned into glutathione S-transferase (GST)-tagged and His-tagged prokaryotic expression vectors, respectively; and expressed in Escherichia coli. The recombinant proteins were subsequently purified by affinity chromatography using GST and His-Trap resins. The diagnostic potential of each purified recombinant antigen was tested with various immunoglobulin classes (IgG, IgM, and IgE) and IgG subclasses. The IgG4 ELISA was determined to have the highest specificity and was further evaluated using a panel of serum samples. The rTES-26 IgG4 ELISA showed 80.0% (24/30 samples positive) sensitivity, and both the rTES-30USM IgG4 ELISA and rTES-120 IgG4 ELISA had 93.0% (28/30) sensitivity. Combined use of rTES-120 and rTES-30 IgG4 ELISA for the diagnosis of toxocariasis provided 100% sensitivity. The specificities of rTES-26, rTES-30USM, and rTES-120 antigens were 96.2%, 93.9%, and 92.0%, respectively. These results indicate that the development of a diagnostic test using the three recombinant antigens will allow for more-accurate detection of toxocariasis.
  11. 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.
  12. Fulltext Development of Multiplexed Infectious Disease Lateral Flow Assays: Challenges and Opportunities
    Mohd Hanafiah K, Arifin N, Bustami Y, Noordin R, Garcia M, Anderson D
    Diagnostics (Basel), 2017 Sep 07;7(3).
    PMID: 28880218 DOI: 10.3390/diagnostics7030051
    Lateral flow assays (LFAs) are the mainstay of rapid point-of-care diagnostics, with the potential to enable early case management and transform the epidemiology of infectious disease. However, most LFAs only detect single biomarkers. Recognizing the complex nature of human disease, overlapping symptoms and states of co-infections, there is increasing demand for multiplexed systems that can detect multiple biomarkers simultaneously. Due to innate limitations in the design of traditional membrane-based LFAs, multiplexing is arguably limited to a small number of biomarkers. Here, we summarize the need for multiplexed LFA, key technical and operational challenges for multiplexing, inherent in the design and production of multiplexed LFAs, as well as emerging enabling technologies that may be able to address these challenges. We further identify important areas for research in efforts towards developing multiplexed LFAs for more impactful diagnosis of infectious diseases.
  13. Fulltext Development of a Quantitative Real-Time PCR Assay for Detection of Toxoplasma gondii in Brain Samples
    Berizi M, Babaie J, Fard-Esfahani P, Enshaeieh M, Noordin R, Saadatnia G, et al.
    Iran J Parasitol, 2022 1 28;16(4):621-630.
    PMID: 35082891 DOI: 10.18502/ijpa.v16i4.7875
    Background: Toxoplasmosis is a worldwide-distributed infection that can cause serious diseases, mainly in congenitally infected and immunodeficient individuals. PCR assays play an indispensable role in the detection of Toxoplasma gondii in different biological samples.

    Methods: This study was conducted in the Parasitology Department at Pasteur Institute of Iran (Tehran) during 2016-2018. We designed a highly sensitive quantitative real-time PCR (RT-qPCR) targeted REP-529, a noncoding repetitive DNA. We cloned the amplicon in a plasmid (pTZREP-529) and used it to generate the standard curve. The Toxoplasma RT-qPCR characteristics, i.e., detection limit, specificity, linear dynamic range, linearity, intra-, and inter-assay precisions, were determined. The detection limit of the assay was one plasmid copy number (PCN) per reaction (about 0.004 T. gondii genome), and the linear dynamic range was equal to 6 logs (1× 101 to 1× 107 PCN per reaction).

    Results: The assay showed no signal when genomic DNA of Plasmodium falciparum, Leishmania major, and Trichomonas vaginallis were used. The standard curve was drawn using dilutions of pTZREP-529 plasmid spiked with genomic DNA from a mouse brain, and test characteristics were shown unaffected. Applying the Toxoplasma RT-qPCR, we showed brain cysts were significantly decreased in mice vaccinated with GRA2 antigen of Toxoplasma formulated in Monophosphoryl Lipid A (MPL) adjuvant.

    Conclusion: We have developed a quantitative, specific, and highly sensitive PCR for detecting T. gondii in biological samples.

  14. Development of an Antigen Detection ELISA for Bancroftian Filariasis Using BmSXP-Specific Recombinant Monoclonal Antibody
    Rahumatullah A, Lim TS, Yunus MH, Noordin R
    Am J Trop Med Hyg, 2019 08;101(2):436-440.
    PMID: 31162018 DOI: 10.4269/ajtmh.19-0034
    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.
  15. Fulltext Development of an antigen-DNAzyme based probe for a direct antibody-antigen assay using the intrinsic DNAzyme activity of a daunomycin aptamer
    Omar N, Loh Q, Tye GJ, Choong YS, Noordin R, Glökler J, et al.
    Sensors (Basel), 2013;14(1):346-55.
    PMID: 24379042 DOI: 10.3390/s140100346
    G-Quadruplex (G-4) structures are formed when G-rich DNA sequences fold into intra- or intermolecular four-stranded structures in the presence of metal ions. G-4-hemin complexes are often effective peroxidase-mimicking DNAzymes that are applied in many detection systems. This work reports the application of a G-rich daunomycin-specific aptamer for the development of an antibody-antigen detection assay. We investigated the ability of the daunomycin aptamer to efficiently catalyze the hemin-dependent peroxidase activity independent of daunomycin. A reporter probe consisting of biotinylated antigen and daunomycin aptamer coupled to streptavidin gold nanoparticles was successfully used to generate a colorimetric readout. In conclusion, the daunomycin aptamer can function as a robust alternative DNAzyme for the development of colorimetric assays.
  16. Development of rapid gold nanoparticles based lateral flow assays for simultaneous detection of Shigella and Salmonella genera
    Yahaya ML, Zakaria ND, Noordin R, Abdul Razak K
    Biotechnol Appl Biochem, 2021 Oct;68(5):1095-1106.
    PMID: 32935878 DOI: 10.1002/bab.2029
    Salmonella and Shigella genera are common pathogens that contaminate foods and beverages. Lateral flow assays (LFA) are commonly used to detect these pathogens. However, most of the developed LFAs are for single detection. Simultaneous detection of pathogens is required to reduce cost and time. In this work, 40 nm gold nanoparticles (AuNPs) were synthesized using the seeding growth method as labeling agent. The AuNPs were characterized and conjugated with mouse anti-Gram negative endotoxin antibody. The nitrocellulose membrane HF135 was immobilized with anti-mouse IgG antibody as a control line and two separate test lines with either anti-Shigella or anti-Salmonella antibody, respectively. Color intensity of test lines was observed for positive samples. A milk sample was used as proof of concept to mimic actual contamination. The limit of detection of the LFA was 3.0 × 106 CFU/mL for multiplex detection of Shigella flexneri and Salmonella Typhi and for both single detections. The result was comparable with the enzyme-linked immunosorbent assay (ELISA) analysis. The produced LFA could differentiate between Shigella flexneri, Shigella boydii, Salmonella Enteritidis, and Salmonella Typhi. The developed LFA was able to identify Shigella flexneri and Salmonella Typhi with good sensitivity in milk samples, thus, beneficial to ensure the safety of food before entering the market.
  17. Fulltext Development of triplex real-time PCR and detection of Toxoplasma gondii DNA in infected mice tissues and spiked human samples
    Rahumatullah A, Khoo BY, Noordin R
    Trop Biomed, 2015 Jun;32(2):376-85.
    PMID: 26691266 MyJurnal
    Toxoplasma gondii is an important pathogen in veterinary and human medicine. In this study, a new multiplex TaqMan real-time PCR for detection of T. gondii DNA was developed. This assay consisted of new sets of primers and probes which targeted B1 gene and ITS-1 region of T. gondii, with Vibrio cholera gene as internal control. The B1 gene primers were designed to detect T. gondii RH strain, while the ITS-1 region primers detected most T. gondii strains. Specificity test using common protozoal and bacterial DNA revealed that the assay was very specific to T. gondii. Standard curves constructed using human body fluids spiked with T. gondii (RH and ME49 strains) showed that the sensitivity of the assay was one parasite, with R² value of 0.975 to 0.999 and efficiency of 97% to 99% for all types of samples. The assay performed on DNA extracted from tissues of mice infected with T. gondii showed that liver contained the highest parasite load for both strains of T. gondii. The multiplex real-time PCR developed in this study would be potentially useful for detection of T. gondii in human and animal samples.
  18. Fulltext Diagnostic Potential of an IgE-ELISA in Detecting Strongyloidiasis
    Ahmad H, Balachandra D, Arifin N, Nolan TJ, Lok JB, Hayat Khan A, et al.
    Am J Trop Med Hyg, 2020 12;103(6):2288-2293.
    PMID: 32996454 DOI: 10.4269/ajtmh.20-0265
    Strongyloides stercoralis infection is prevalent worldwide and can cause lifelong infection in immunocompetent individuals, and potentially death in immunosuppressed patients. The diagnosis is hindered by the low sensitivity of microscopic examination, thus making serology an important complementary test to improve the detection rate. However, there were reports that some Strongyloides-infected individuals were negative with specific IgG and IgG4 assays, and other helminth infections were positive with commercial Strongyloides IgG-ELISAs. Thus, there is a need to develop better serodiagnostic methods for strongyloidiasis. We investigated the diagnostic potential of IgE-ELISAs using Strongyloides larval lysate. Sera from two groups infected with Strongyloides served as the positive reference, that is, 1) positive by commercial IgG-ELISAs and IgG4 rapid test, and stool samples positive by microscopy and/or PCR (group IA; n = 20); and 2) negative by IgG-ELISAs and IgG4 rapid test, but stool samples were PCR positive (group IB sera; n = 11). Sera from another two groups served as negative reference (controls), that is, 1) infected with other parasites (group II; n = 73) and 2) healthy donors (group III; n = 22). Results showed a 100% diagnostic sensitivity in detecting sera from groups IA and IB. The latter group of individuals probably had early infection because their IgG and IgG4 assays were negative. The optical density values of group IB sera were also significantly lower than those of group IA (P < 0.003). The IgE-ELISA was 100% specific when tested against sera from groups II and III. This study highlights the diagnostic potential of IgE-ELISA using larval lysate to detect strongyloidiasis, especially those with probable early infection.
  19. Fulltext Diagnostics and the neglected tropical diseases roadmap: setting the agenda for 2030
    Souza AA, Ducker C, Argaw D, King JD, Solomon AW, Biamonte MA, et al.
    Trans R Soc Trop Med Hyg, 2021 01 28;115(2):129-135.
    PMID: 33169166 DOI: 10.1093/trstmh/traa118
    Accurate and reliable diagnostic tools are an essential requirement for neglected tropical diseases (NTDs) programmes. However, the NTD community has historically underinvested in the development and improvement of diagnostic tools, potentially undermining the successes achieved over the last 2 decades. Recognizing this, the WHO, in its newly released draft roadmap for NTD 2021-2030, has identified diagnostics as one of four priority areas requiring concerted action to reach the 2030 targets. As a result, WHO established a Diagnostics Technical Advisory Group (DTAG) to serve as the collaborative mechanism to drive progress in this area. Here, the purpose and role of the DTAG are described in the context of the challenges facing NTD programmes.
  20. Fulltext Diagnostics to support elimination of lymphatic filariasis-Development of two target product profiles
    Won KY, Gass K, Biamonte M, Dagne DA, Ducker C, Hanna C, et al.
    PLoS Negl Trop Dis, 2021 11;15(11):e0009968.
    PMID: 34780503 DOI: 10.1371/journal.pntd.0009968
    As lymphatic filariasis (LF) programs move closer to established targets for validation elimination of LF as a public health problem, diagnostic tools capable of supporting the needs of the programs are critical for success. Known limitations of existing diagnostic tools make it challenging to have confidence that program endpoints have been achieved. In 2019, the World Health Organization (WHO) established a Diagnostic Technical Advisory Group (DTAG) for Neglected Tropical Diseases tasked with prioritizing diagnostic needs including defining use-cases and target product profiles (TPPs) for needed tools. Subsequently, disease-specific DTAG subgroups, including one focused on LF, were established to develop TPPs and use-case analyses to be used by product developers. Here, we describe the development of two priority TPPs for LF diagnostics needed for making decisions for stopping mass drug administration (MDA) of a triple drug regimen and surveillance. Utilizing the WHO core TPP development process as the framework, the LF subgroup convened to discuss and determine attributes required for each use case. TPPs considered the following parameters: product use, design, performance, product configuration and cost, and access and equity. Version 1.0 TPPs for two use cases were published by WHO on 12 March 2021 within the WHO Global Observatory on Health Research and Development. A common TPP characteristic that emerged in both use cases was the need to identify new biomarkers that would allow for greater precision in program delivery. As LF diagnostic tests are rarely used for individual clinical diagnosis, it became apparent that reliance on population-based surveys for decision making requires consideration of test performance in the context of such surveys. In low prevalence settings, the number of false positive test results may lead to unnecessary continuation or resumption of MDA, thus wasting valuable resources and time. Therefore, highly specific diagnostic tools are paramount when used to measure low thresholds. The TPP process brought to the forefront the importance of linking use case, program platform and diagnostic performance characteristics when defining required criteria for diagnostic tools.
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