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  1. Fulltext Molecular phylogenetics of a recently isolated goat pox virus from Vietnam
    Pham TH, Rahaman NYA, Lila MAM, Lai HLT, Nguyen LT, Van Nguyen G, et al.
    BMC Vet Res, 2021 Mar 08;17(1):115.
    PMID: 33685458 DOI: 10.1186/s12917-021-02777-1
    BACKGROUND: After a decade of silence, an outbreak of the contagious and Asian endemic disease, goat pox re-emerged in North Vietnam affecting more than 1800 heads with a mortality rate of 6.5%. The inevitable impact of goat pox on hide quality, breeding, chevon and milk production has resulted in a significant economic losses to the developing goat industry of Vietnam. In the act of establishing an effective control of this devastating disease, tracing the source of re-emergence via a phylogenetic study was carried out to reveal their genetic relatedness. Either skin scab or papule from the six affected provinces were collected, cultured into Vero cells followed by restricted enzyme digestion of targeted P32 gene DNA encoding. The P32 gene was then cloned and transformed into E.coli competent cells for further sequencing.

    RESULTS: The isolated sequence is deposited into GenBank under Accession No. MN317561/VNUAGTP1. The phylogenetic tree revealed high similarity of nucleotide and amino acid sequences to references goat pox strains accounting for 99.6 and 99.3, respectively. The Vietnamese strain is clustered together with currently circulating goat pox virus in China, India and Pakistan which suggested the origin of South China.

    CONCLUSIONS: This Vietnam isolate is clustered together with other Asian goat pox strains indicating the dissemination of a common goat pox virus within this continent.

    Matched MeSH terms: Viral Proteins/genetics; Capripoxvirus/genetics
  2. Molecular characterization of chicken infectious anemia virus circulating in Argentina during 2007
    Craig MI, Rimondi A, Delamer M, Sansalone P, König G, Vagnozzi A, et al.
    Avian Dis, 2009 Sep;53(3):331-5.
    PMID: 19848068
    Chicken infectious anemia virus (CAV) is a worldwide-distributed infectious agent that affects commercial poultry. Although this agent was first detected in Argentina in 1994, no further studies on CAV in this country were reported after that. The recent increased occurrence of clinical cases of immunosuppression that could be caused by CAV has prompted this study. Our results confirmed that CAV is still circulating in commercial flocks in Argentina. Phylogenetic analysis focusing on the VP1 nucleotide sequence showed that all Argentinean isolates grouped together in a cluster, sharing a high similarity (> 97%) with genotype B reference strains. However, Argentinean isolates were distantly related to other strains commonly used for vaccination in this country, such as Del-Ros and Cux-1. Sequence analysis of predicted VP1 peptides showed that most of the Argentinean isolates have a glutamine residue at positions 139 and 144, suggesting that these isolates might have a reduced spread in cell culture compared with Cux-1. In addition, a particular amino acid substitution at position 290 is present in all studied Argentinean isolates, as well as in several VP1 sequences from Malaysia, Australia, and Japan isolates. Our results indicate that it is possible to typify CAV strains by comparison of VPI nucleotide sequences alone because the same tree topology was obtained when using the whole genome sequence. The molecular analysis of native strains sheds light into the epidemiology of CAV in Argentinean flocks. In addition, this analysis could be considered in future control strategies focused not only on breeders but on broilers and layer flocks.
    Matched MeSH terms: Chicken anemia virus/genetics*; Capsid Proteins/genetics
  3. Electrochemical DNA detection of hepatitis E virus genotype 3 using PbS quantum dot labelling
    Ngo DB, Chaibun T, Yin LS, Lertanantawong B, Surareungchai W
    Anal Bioanal Chem, 2021 Feb;413(4):1027-1037.
    PMID: 33236225 DOI: 10.1007/s00216-020-03061-1
    The aim of this study was to develop a highly specific electrochemical DNA sensor using functionalized lead sulphide (PbS) quantum dots for hepatitis E virus genotype 3 (HEV3) DNA target detection. Functionalized-PbS quantum dots (QDs) were used as an electrochemical label for the detection of HEV3-DNA target by the technique of square wave anodic stripping voltammetry (SWASV). The functionalized-PbS quantum dots were characterized by UV-vis, FTIR, XRD, TEM and zeta potential techniques. As-prepared, functionalized-PbS quantum dots have an average size of 4.15 ± 1.35 nm. The detection platform exhibited LOD and LOQ values of 1.23 fM and 2.11 fM, respectively. HEV3-DNA target spiked serum is also reported.Graphical abstract.
    Matched MeSH terms: DNA, Viral/genetics; Hepatitis E virus/genetics
  4. Lymphocyte predominant Hodgkin lymphoma, antigen-driven after all?
    Poppema S
    J Pathol, 2021 01;253(1):1-10.
    PMID: 33044742 DOI: 10.1002/path.5567
    Nodular lymphocyte predominant Hodgkin lymphoma (NLPHL) was suggested as an entity separate from other types of Hodgkin lymphoma 40 years ago and recognized in the WHO classification in 2001. Based on its relatively benign course with late distant relapses, relation with lymph node hyperplasia with progressively transformed germinal centers, presence of clonal immunoglobulin gene rearrangements with somatic hypermutations and ongoing mutations, and relation with a number of inherited defects affecting the immune system, it has been suspected that NLPHL might be antigen-driven. Recent evidence has shown that cases of IgD-positive NLPHL are associated with infection by Moraxella catarrhalis, a common bacterium in the upper respiratory tract and in lymph nodes. This review summarizes the evidence for NLPHL as a B-cell lymphoma involving follicular T-lymphocytes normally found in germinal centers, its molecular features and relation to inherited immune defects, and its relation and differential diagnosis from similar entities. Finally, it discusses the evidence that in many cases a watch and wait policy might be a viable initial management strategy. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.
    Matched MeSH terms: Antigens, Neoplasm/genetics; Hodgkin Disease/genetics
  5. PARK16 is associated with PD in the Malaysian population
    Gopalai AA, Ahmad-Annuar A, Li HH, Zhao Y, Lim SY, Tan AH, et al.
    Am J Med Genet B Neuropsychiatr Genet, 2016 09;171(6):839-47.
    PMID: 27174169 DOI: 10.1002/ajmg.b.32454
    PARK16 was identified as a risk factor for Parkinson's disease in a Japanese cohort; however, subsequent studies in the other populations including the Chinese, European, Caucasian, and Chilean have shown a protective role instead. To investigate this locus in our Malaysian cohort, 1,144 individuals were screened for five SNPs in the PARK16 locus and logistic regression analysis showed that the A allele of the rs947211 SNP reduced the risk of developing PD via a recessive model (Odds ratio 0.57, P-value 0.0003). Pooled analysis with other Asian studies showed that A allele of the rs947211 SNP decreased the risk of developing PD via a recessive model (Odds ratio 0.71, P-value 0.0001). In addition, when meta-analysis was performed with other Asian population, three SNPs (rs823128, rs823156, and rs11240572) reduced risk of developing PD via a dominant model. © 2016 Wiley Periodicals, Inc.
    Matched MeSH terms: Parkinson Disease/genetics*; Polymorphism, Single Nucleotide/genetics
  6. Fulltext Development of a luciferase/luciferin cell proliferation (XenoLuc) assay for real-time measurements of Gfp-Luc2-modified cells in a co-culture system
    Teow SY, Liew K, Che Mat MF, Marzuki M, Abdul Aziz N, Chu TL, et al.
    BMC Biotechnol, 2019 06 14;19(1):34.
    PMID: 31200673 DOI: 10.1186/s12896-019-0528-4
    BACKGROUND: In vitro modelling of cancer cells is becoming more complex due to prevailing evidence of intimate interactions between cancer cells and their surrounding stroma. A co-culture system which consists of more than one cell type is physiologically more relevant and thus, could serve as a useful model for various biological studies. An assay that specifically detects the phenotypic changes of cancer cells in a multi-cellular system is lacking for nasopharyngeal carcinoma (NPC).

    RESULTS: Here, we describe a luciferase/luciferin (XenoLuc) assay that could specifically measure changes in the proliferation of cancer cells in the co-culture system using two modified NPC patient-derived tumour xenograft (PDTXs) cells: Xeno284-gfp-luc2 and XenoB110-gfp-luc2. Through this assay, we are able to show that the growth of NPC xenograft cells in both two-dimensional (2D) and three-dimensional (3D) models was enhanced when co-cultured with normal human dermal fibroblasts (NHDFs). In addition, potential applications of this assay in in vitro drug or inhibitor screening experiments are also illustrated.

    CONCLUSIONS: XenoLuc assay is specific, sensitive, rapid and cost-effective for measuring the growth of luciferase-expressing cells in a co- or multiple-culture system. This assay may also be adapted for tumour microenvironment studies as well as drug screening experiments in more complex 3D co-culture systems.

    Matched MeSH terms: Luciferases/genetics; Green Fluorescent Proteins/genetics
  7. Methods for Predicting Warfarin Dose Requirements
    Saffian SM, Wright DF, Roberts RL, Duffull SB
    Ther Drug Monit, 2015 Aug;37(4):531-8.
    PMID: 25549208 DOI: 10.1097/FTD.0000000000000177
    The aim of this study was to compare the predictive performance of different warfarin dosing methods.
    Matched MeSH terms: Vitamin K Epoxide Reductases/genetics; Cytochrome P-450 CYP2C9/genetics
  8. Pandanus tectorius fruit extract promotes Hsp70 accumulation, immune-related genes expression and Vibrio parahaemolyticus tolerance in the white-leg shrimp Penaeus vannamei
    Anirudhan A, Okomoda VT, Mimi Iryani MT, Andriani Y, Abd Wahid ME, Tan MP, et al.
    Fish Shellfish Immunol, 2021 Feb;109:97-105.
    PMID: 33352338 DOI: 10.1016/j.fsi.2020.12.011
    Plants and herbal extracts are indispensable for controlling the spread of disease-causing bacteria, including those that infect aquatic organisms used in aquaculture. The use of plant or herbal extract is expected to be safe for aquatic animals and less harmful to the environment, as opposed to conventional therapeutic alternatives such as antibiotics that promote the occurrence of potential antibiotic-resistant bacteria when used improperly. The efficacy of Pandanus tectorius fruit extract in the regulation of Hsp70 expression, pro-phenoloxidase (ProPO), peroxinectin, penaeidin, crustin and transglutaminase, all immune peptides essential for Vibrio tolerance in white leg shrimp, Penaeus vannamei, was investigated in this study, which included the determination of the safety levels of the extract. Tolerance of shrimp against Vibrio parahaemolyticus, a pathogenic bacteria that causes Acute Hepatopancreas Necrosis Disease (AHPND), was assessed on the basis of median lethal dose challenge survival (LD50 = 106 cells/ml). Mortality was not observed 24 h after exposure of 0.5-6 g/L of the fruit extract, indicating that P. tectorius was not toxic to shrimp at these concentrations. A 24-h incubation of 2-6 g/L of the fruit extract increased shrimp tolerance to V. parahaemolyticus, with survival doubled when the maximum dose tested in this study was used. Concomitant with a rise in survival was the increase in immune-related proteins, with Hsp70, ProPO, peroxinectin, penaeidin, crustin and transglutaminase increased 10, 11, 11, 0.4, 8 and 13-fold respectively. Histological examination of the hepatopancreas and muscle tissues of Vibrio-infected shrimp primed with P. tectorius extract revealed reduced signs of histopathological degeneration, possibly due to the accumulation of Hsp70, a molecular chaperone crucial to cellular protein folding, tissue repair and immune response of living organisms, including Penaeid shrimp.
    Matched MeSH terms: Immunity, Innate/genetics*; Penaeidae/genetics
  9. Quantification of human PPARgamma1 gene expression by competitive PCR using an homologous internal standard
    Yaacob NS, Bakar RA, Norazmi MN
    Ann Clin Lab Sci, 2004;34(1):47-56.
    PMID: 15038667
    The polymerase chain reaction (PCR) is useful for amplifying specific mRNAs, particularly those present in low copy numbers. However, due to the exponential nature of the amplification process, PCR cannot readily be used to quantify gene expression. A competitive PCR technique was developed to address this shortcoming. An internal standard that is 100% homologous to, but shorter than, the target gene was constructed. The practicality of the method was demonstrated by determining the expression levels of a human transcription factor, peroxisome proliferator-activated receptor gamma 1 (hPPARgamma1) which is normally present in low copy numbers in selected cells. A mock system was used to test the accuracy and sensitivity of the method, which was subsequently used to determine the expression of this receptor in lipopolysaccharide (LPS)-activated monocytes, which are known to express hPPARgamma1 differentially during cellular activation. Densitometric analysis showed that the competitive PCR method reliably estimated the expression levels of hPPARgamma1 at the attomole (10(-18)) level in monocytes.
    Matched MeSH terms: Transcription Factors/genetics*; Receptors, Cytoplasmic and Nuclear/genetics*
  10. Overview of key molecular and pharmacological targets for diabetes and associated diseases
    Shahcheraghi SH, Aljabali AAA, Al Zoubi MS, Mishra V, Charbe NB, Haggag YA, et al.
    Life Sci, 2021 Aug 01;278:119632.
    PMID: 34019900 DOI: 10.1016/j.lfs.2021.119632
    Diabetes epidemiological quantities are demonstrating one of the most important communities' health worries. The essential diabetic difficulties are including cardiomyopathy, nephropathy, inflammation, and retinopathy. Despite developments in glucose decreasing treatments and drugs, these diabetic complications are still ineffectively reversed or prohibited. Several signaling and molecular pathways are vital targets in the new therapies of diabetes. This review assesses the newest researches about the key molecules and signaling pathways as targets of molecular pharmacology in diabetes and diseases related to it for better treatment based on molecular sciences. The disease is not cured by current pharmacological strategies for type 2 diabetes. While several drug combinations are accessible that can efficiently modulate glycemia and mitigate long-term complications, these agents do not reverse pathogenesis, and in practice, they are not established to modify the patient's specific molecular profiling. Therapeutic companies have benefited from human genetics. Genome exploration, which is agnostic to the information that exists, has revealed tens of loci that impact glycemic modulation. The physiological report has begun to examine subtypes of diseases, illustrate heterogeneity and propose biochemical therapeutic pathways.
    Matched MeSH terms: Diabetes Mellitus/genetics; Diabetes Complications/genetics
  11. Fulltext Sensitive Detection of Plasmodium vivax Using a High-Throughput, Colourimetric Loop Mediated Isothermal Amplification (HtLAMP) Platform: A Potential Novel Tool for Malaria Elimination
    Britton S, Cheng Q, Grigg MJ, Poole CB, Pasay C, William T, et al.
    PLoS Negl Trop Dis, 2016 Feb;10(2):e0004443.
    PMID: 26870958 DOI: 10.1371/journal.pntd.0004443
    INTRODUCTION: Plasmodium vivax malaria has a wide geographic distribution and poses challenges to malaria elimination that are likely to be greater than those of P. falciparum. Diagnostic tools for P. vivax infection in non-reference laboratory settings are limited to microscopy and rapid diagnostic tests but these are unreliable at low parasitemia. The development and validation of a high-throughput and sensitive assay for P. vivax is a priority.

    METHODS: A high-throughput LAMP assay targeting a P. vivax mitochondrial gene and deploying colorimetric detection in a 96-well plate format was developed and evaluated in the laboratory. Diagnostic accuracy was compared against microscopy, antigen detection tests and PCR and validated in samples from malaria patients and community controls in a district hospital setting in Sabah, Malaysia.

    RESULTS: The high throughput LAMP-P. vivax assay (HtLAMP-Pv) performed with an estimated limit of detection of 1.4 parasites/ μL. Assay primers demonstrated cross-reactivity with P. knowlesi but not with other Plasmodium spp. Field testing of HtLAMP-Pv was conducted using 149 samples from symptomatic malaria patients (64 P. vivax, 17 P. falciparum, 56 P. knowlesi, 7 P. malariae, 1 mixed P. knowlesi/P. vivax, with 4 excluded). When compared against multiplex PCR, HtLAMP-Pv demonstrated a sensitivity for P. vivax of 95% (95% CI 87-99%); 61/64), and specificity of 100% (95% CI 86-100%); 25/25) when P. knowlesi samples were excluded. HtLAMP-Pv testing of 112 samples from asymptomatic community controls, 7 of which had submicroscopic P. vivax infections by PCR, showed a sensitivity of 71% (95% CI 29-96%; 5/7) and specificity of 93% (95% CI87-97%; 98/105).

    CONCLUSION: This novel HtLAMP-P. vivax assay has the potential to be a useful field applicable molecular diagnostic test for P. vivax infection in elimination settings.

    Matched MeSH terms: Plasmodium/genetics; Plasmodium vivax/genetics
  12. Fulltext A Sensitive, Colorimetric, High-Throughput Loop-Mediated Isothermal Amplification Assay for the Detection of Plasmodium knowlesi
    Britton S, Cheng Q, Grigg MJ, William T, Anstey NM, McCarthy JS
    Am J Trop Med Hyg, 2016 07 06;95(1):120-2.
    PMID: 27162264 DOI: 10.4269/ajtmh.15-0670
    The simian parasite Plasmodium knowlesi is now the commonest cause of malaria in Malaysia and can rapidly cause severe and fatal malaria. However, microscopic misdiagnosis of Plasmodium species is common, rapid antigen detection tests remain insufficiently sensitive and confirmation of P. knowlesi requires polymerase chain reaction (PCR). Thus available point-of-care diagnostic tests are inadequate. This study reports the development of a simple, sensitive, colorimetric, high-throughput loop-mediated isothermal amplification assay (HtLAMP) diagnostic test using novel primers for the detection of P. knowlesi. This assay is able to detect 0.2 parasites/μL, and compared with PCR has a sensitivity of 96% for the detection of P. knowlesi, making it a potentially field-applicable point-of-care diagnostic tool.
    Matched MeSH terms: Plasmodium knowlesi/genetics; DNA Primers/genetics
  13. Fulltext Evaluation of BOX-PCR and ERIC-PCR as Molecular Typing Tools for Pathogenic Leptospira
    Bilung LM, Pui CF, Su'ut L, Apun K
    Dis Markers, 2018;2018:1351634.
    PMID: 30154937 DOI: 10.1155/2018/1351634
    In the last decades, leptospirosis had gained public health concern due to morbidity and mortality rates caused by pathogenic Leptospira. The need for rapid and robust molecular typing methods to differentiate this zoonotic pathogen is of utmost importance. Various studies had been conducted to determine the genetic relatedness of Leptospira isolates using molecular typing methods. In this study, 29 pathogenic Leptospira isolates from rat, soil, and water samples in Sarawak, Malaysia, were characterized using BOX-PCR and ERIC-PCR. The effectiveness of these two methods with regard to the ease of interpretation, reproducibility, typeability, and discriminatory power was also being evaluated. Using BOX-PCR, six clusters and 3 single isolates were defined at a genetic distance percentage of 11.2%. ERIC-PCR clustered the isolates into 6 clusters and 2 single isolates at a genetic distance percentage of 6.8%. Both BOX-PCR and ERIC-PCR produced comparable results though the discriminatory index for ERIC-PCR (0.826) was higher than that for BOX-PCR (0.809). From the constructed dendrogram, it could be summarized that the isolates in this study were highly heterogeneous and genetically diverse. The findings from this study indicated that there is no genetic relatedness among the pathogenic Leptospira isolates in relation to the locality, source, and identity, with some exceptions. Out of the 29 pathogenic Leptospira isolates studied, BOX-PCR and ERIC-PCR successfully discriminated 4 isolates (2 isolates each) into the same cluster in relation to sample sources, as well as 2 isolates into the same cluster in association with the sample locality. Future studies shall incorporate the use of other molecular typing methods to make a more thorough comparison on the genetic relatedness of pathogenic Leptospira.
    Matched MeSH terms: DNA, Bacterial/genetics*; Leptospira/genetics
  14. Cross-resistance and inheritance of resistance to Bacillus thuringiensis toxin Cry1Ac in diamondback moth (Plutella xylostella L) from lowland Malaysia
    Sayyed AH, Wright DJ
    Pest Manag Sci, 2001 May;57(5):413-21.
    PMID: 11374157
    A field population of Plutella xylostella from Malaysia (SERD4) was divided into five sub-populations and four were selected (G2-G5) with the Bacillus thuringiensis insecticidal crystal (Cry) toxins Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da. Bioassay at G6 gave resistance ratios of 88, 5, 2 and 3 for Cry1Ac, Cry1Ab, Cry1Ca and Cry1Da respectively compared with the unselected sub-population (UNSEL-SERD4). The Cry1Ac-selected population showed little cross-resistance to Cry1Ab, Cry1Ca and Cry1Da, (3-, 2- and 3-fold compared with UNSEL-SERD4), whereas the Cry1Ab-SEL sub-population showed marked cross-resistance to Cry1Ac (40-fold), much greater than Cry1Ab itself. In contrast, the Cry1Ca- and Cry1Da-SEL sub-population showed little if any cross-resistance to Cry1Ac and Cry1Ab. The mode of inheritance of resistance to Cry1Ac was examined in Cry1Ac-selected SERD4 by standard reciprocal crosses and back-crosses using a laboratory insecticide-susceptible population (ROTH). Logit regression analysis of F1 reciprocal crosses indicated that resistance to Cry1Ac was inherited as an incompletely dominant trait. At the highest dose of Cry1Ac tested, resistance was recessive, while at the lowest dose it was almost completely dominant. The F2 progeny from a back-cross of F1 progeny with ROTH were tested with a concentration of Cry1Ac that would kill 100% of ROTH. The mortality ranged between 50 and 95% in seven families of back-cross progeny, which indicated that more than one allele on separate loci were responsible for resistance to Cry1Ac.
    Matched MeSH terms: Insecticide Resistance/genetics; Moths/genetics*
  15. Fulltext Mutation-specific pathophysiological mechanisms define different neurodevelopmental disorders associated with SATB1 dysfunction
    den Hoed J, de Boer E, Voisin N, Dingemans AJM, Guex N, Wiel L, et al.
    Am J Hum Genet, 2021 02 04;108(2):346-356.
    PMID: 33513338 DOI: 10.1016/j.ajhg.2021.01.007
    Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.
    Matched MeSH terms: Matrix Attachment Region Binding Proteins/genetics*; Neurodevelopmental Disorders/genetics*
  16. Fulltext Development and evaluation of a Multiplex Polymerase Chain Reaction for the detection of Salmonella species
    Thong KL, Teh CS, Chua KH
    Trop Biomed, 2014 Dec;31(4):689-97.
    PMID: 25776594 MyJurnal
    The present study aims to develop a system which consists of four pairs of primers that specifically detects Salmonella spp., Salmonella serovar Typhi and Salmonella serovar Paratyphi A with an internal amplification control. The system, when applied in Polymerase Chain Reaction (PCR) under specific conditions, reaction mixture and cycling temperatures produced four bands; 784 bp, 496 bp, 332 bp and 187 bp. The DNA band 784 bp is present in all Salmonella spp., while the bands of 496 bp and 332 bp are only present in S. Paratyphi A and S. Typhi, respectively. An internal amplification control as indicated by the 187 bp shows the system is working in optimum condition in all the tests. This multiplex PCR was evaluated on 241 bacterial cultures and 691 naturally contaminated samples. Overall, this multiplex PCR detection system provides a single step for simultaneous detection of DNAs of Salmonella spp., S. Typhi and S. Paratyphi A.
    Matched MeSH terms: Salmonella/genetics; DNA Primers/genetics
  17. Fulltext Rapid detection and identification of pathogens in patients with continuous ambulatory peritoneal dialysis (CAPD) associated peritonitis by 16s rRNA gene sequencing
    Ahmadi SH, Neela V, Hamat RA, Goh BL, Syafinaz AN
    Trop Biomed, 2013 Dec;30(4):602-7.
    PMID: 24522129 MyJurnal
    Peritonitis still remains a serious complication with high rate of morbidity and mortality in patients on CAPD. Rapid and accurate identification of pathogens causing peritonitis in a CAPD patient is essential for early and optimal treatment. The aim of this study was to use 16S rRNA and ITS gene sequencing to identify common bacterial and fungal pathogens directly from the peritoneal fluid without culturing. Ninety one peritoneal fluids obtained from 91 different patients on CAPD suspected for peritonitis were investigated for etiological agents by 16S rRNA and ITS gene sequencing. Data obtained by molecular method was compared with the results obtained by culture method. Among the 45 patients confirmed for peritonitis based on international society of peritoneal dialysis (ISPD) guidelines, the etiological agents were identified in 37(82.2%) samples by culture method, while molecular method identified the etiological agents in 40(88.9%) samples. Despite the high potential application of the 16S rRNA and ITS gene sequencing in comparison to culture method to detect the vast majority of etiological agents directly from peritoneal fluids; it could not be used as a standalone test as it lacks sensitivity to identify some bacterial species due to high genetic similarity in some cases and inadequate database in Gene Bank. However, it could be used as a supplementary test to the culture method especially in the diagnosis of culture negative peritonitis.
    Matched MeSH terms: RNA, Ribosomal, 16S/genetics; DNA, Ribosomal Spacer/genetics
  18. Fulltext Recombinant proteins from new constructs of SAG1 and GRA7 sequences and their usefulness to detect acute toxoplasmosis
    Kotresha D, Poonam D, Muhammad Hafiznur Y, Saadatnia G, Nurulhasanah O, Sabariah O, et al.
    Trop Biomed, 2012 Mar;29(1):129-37.
    PMID: 22543613 MyJurnal
    In this study we have cloned unreported gene fragments of Toxoplasma gondii GRA7 and SAG1 and expressed the corresponding recombinant proteins, followed by evaluation of their usefulness for the serological diagnosis of toxoplasmosis. Both recombinant proteins were expressed efficiently in insoluble form, purified by single step Ni-NTA affinity chromatography and their antigenicity to detect toxoplasma specific IgG antibodies were determined by immunoblotting. A total of 60 serum samples from three groups of individuals based on their anti-toxoplasma antibody profiles were tested, namely (I) IgM+, IgG+ (n=20), (II) IgM-, IgG+ (n=20) and (III) IgM-, IgG- (n=20). Both recombinant proteins exhibited high sensitivity (100%) with sera from Group I. rGRA7 and rSAG1 reacted 40% and 80% respectively with Group II sera. The specificity of the recombinant proteins based on reactivities with Group III sera were 100% and 80% with rGRA7 and rSAG1 respectively. Thus rGRA7 was found to be better at discriminating probable acute from chronic phases of toxoplasmosis, and it also showed higher specificity.
    Matched MeSH terms: Recombinant Proteins/genetics; Toxoplasma/genetics
  19. Fulltext Acanthamoeba genotype T4 detected in naturally-infected feline corneas found to be in homology with those causing human keratitis
    Ithoi I, Mahmud R, Abdul Basher MH, Jali A, Abdulsalam AM, Ibrahim J, et al.
    Trop Biomed, 2013 Mar;30(1):131-40.
    PMID: 23665719 MyJurnal
    A total of 10 out of 65 cornea swab samples from cats with eye symptoms showed Acanthamoeba-like morphology after cultivation. By PCR and DNA sequencing of Acanthamoeba diagnostic fragment 3 (DF3), all 10 isolates from the positive samples were categorized into two homologous groups of AfC1 (PM1, PM2, PM3, PF6, KM7, KF8, KMK9) and AfC2 (PM4, PM5, KFK10) due to the presence of bases A(354) and G(354), respectively. Furthermore, DF3 of AfC1 and AfC2 showed 100% similarity with Genbank reference isolates with the accession numbers DQ087314, EU146073 and U07401, GU808323, which were Acanthamoeba castellanii strains genotype T4 originating from human keratitis. This finding suggests that A. castellani strains have the capability to infect cats and human under favorable conditions.
    Matched MeSH terms: DNA, Protozoan/genetics; Acanthamoeba castellanii/genetics*
  20. Fulltext Plasmodium falciparum 19 kDa of merozoite surface protein-1 (MSP-1(19)) expressed in Mycobacterium bovis bacille Calmette Guerin (BCG) is reactive with an inhibitory but not a blocking monoclonal antibody
    Nurul AA, Rapeah S, Norazmi MN
    Trop Biomed, 2010 Apr;27(1):60-7.
    PMID: 20562815
    Proteins on the surface of Plasmodium falciparum merozoites are good targets for vaccine development against malaria because they are accessible to antibodies in the plasma. The 19 kDa C-terminus of merozoite surface protein-1 (MSP-1(19)) has been shown to induce both inhibitory as well as blocking antibodies, the latter blocking the protective effects of the former. Inhibitory antibodies bind to MSP-1(19) and inhibit merozoite invasion of red blood cells (RBC) but the binding of blocking antibodies can prevent binding of inhibitory antibodies thereby allowing the parasite to invade RBC. We constructed a synthetic version of the MSP-1(19) of the P. falciparum using mycobacterium codon usage by assembly PCR. The synthetic MSP-1(19) was mutated at various sites to promote the production of inhibitory but not blocking antibodies as previously reported. The native and mutated MSP-1(19) were cloned and expressed in Mycobacterium bovis bacille Calmette-Guerin (BCG) and the expressions of the recombinant proteins were detected by specific monoclonal antibodies (mAbs) namely, 12.10 and 1E1 against MSP-1(19) using Western blotting. The mutated MSP-1(19) protein reacted with the inhibitory mAb, 12.10, but not the blocking mAb, 1E1, paving the way for the construction of a potential recombinant BCG (rBCG) blood stage vaccine against malaria.
    Matched MeSH terms: Mycobacterium bovis/genetics; Merozoite Surface Protein 1/genetics
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