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Synergistic green extraction of nickel ions from electroplating waste via mixtures of chelating and organophosphorus carrier

Sulaiman RNR, Othman N

J Hazard Mater, 2017 Oct 15;340:77-84.
PMID: 28711835 DOI: 10.1016/j.jhazmat.2017.06.060

The discharge of electroplating waste containing nickel ions has led to environmental issues owing to the toxicity problem mainly to the aquatic organisms and humans. Liquid-liquid extraction offers a great potential treatment for nickel removal with several advantages of simple, high efficiency and high separation factor. In this study, a green synergistic liquid-liquid extraction of nickel ions from electroplating waste solution using chelating oxime (LIX63) and organophosphorus (D2EHPA) carriers individually as well as their synergistic mixture has been studied. The result demonstrated that about 83% of nickel ions have been successfully extracted via the mixture system of 0.08M LIX63 +0.02M D2EHPA with the maximum synergistic enhancement factor, Rmax of 29.56. Meanwhile, the back extraction study also revealed that HNO3 was the most suitable stripping agent while the diluent screening also showed that palm oil has high potential to be incorporated as a diluent in the green synergistic liquid-liquid extraction of nickel.
Fulltext Limitation of nutrients stimulates musty odor production by Streptomyces sp. isolated from a tropical environment

Shudirman S, Abang Kassim A, Shamsol Anuar NS, Utsumi M, Shimizu K, Muhammad Yuzir MA, et al.

J Gen Appl Microbiol, 2021 Jul 31;67(3):92-99.
PMID: 33642451 DOI: 10.2323/jgam.2020.08.001

Musty odor production by actinomycetes is usually related to the presence of geosmin and 2-methylisoborneol (2-MIB), which are synthesized by enzymes encoded by the geoA and tpc genes, respectively. Streptomyces spp. strain S10, which was isolated from a water reservoir in Malaysia, has the ability to produce geosmin when cultivated in a basal salt (BS) solid medium, but no 2-MIB production occurred during growth in BS medium. Strain S10 could produce higher levels of geosmin when the phosphate concentration was limited to 0.05 mg/L, with a yield of 17.53 ± 3.12 ✕ 105 ng/L, compared with growth in BS medium. Interestingly, 2-MIB production was suddenly detected when the nitrate concentration was limited to 1.0 mg/L, with a yield of 1.4 ± 0.11 ✕ 105 ng/L. Therefore, it was concluded that phosphate- and nitrate-limiting conditions could induce the initial production of geosmin and 2-MIB by strain S10. Furthermore, a positive amplicon of geoA was detected in strain S10, but no tpc amplicon was detected by PCR analysis. Draft genome sequence analysis showed that one open reading frame (ORF) contained a conserved motif of geosmin synthase with 95% identity with geoA in Streptomyces coelicolor A3 (2). In the case of the tpc genes, it was found that one ORF showed 23% identity to the known tpc gene in S. coelicolor A3(2), but strain S10 lacked one motif in the N-terminus.
Fulltext Evaluation of female Aedes aegypti proteome via LC-ESI-MS/MS using two protein extraction methods

Shettima A, Ishak IH, Abdul Rais SH, Abu Hasan H, Othman N

PeerJ, 2021;9:e10863.
PMID: 33717682 DOI: 10.7717/peerj.10863

Background: Proteomic analyses have broadened the horizons of vector control measures by identifying proteins associated with different biological and physiological processes and give further insight into the mosquitoes' biology, mechanism of insecticide resistance and pathogens-mosquitoes interaction. Female Ae. aegypti ingests human blood to acquire the requisite nutrients to make eggs. During blood ingestion, female mosquitoes transmit different pathogens. Therefore, this study aimed to determine the best protein extraction method for mass spectrometry analysis which will allow a better proteome profiling for female mosquitoes.
Methods: In this present study, two protein extractions methods were performed to analyze female Ae. aegyti proteome, via TCA acetone precipitation extraction method and a commercial protein extraction reagent CytoBusterTM. Then, protein identification was performed by LC-ESI-MS/MS and followed by functional protein annotation analysis.
Results: The CytoBusterTM reagent gave the highest protein yield with a mean of 475.90 µg compared to TCA acetone precipitation extraction showed 283.15 µg mean of protein. LC-ESI-MS/MS identified 1,290 and 890 proteins from the CytoBusterTM reagent and TCA acetone precipitation, respectively. When comparing the protein class categories in both methods, there were three additional categories for proteins identified using CytoBusterTM reagent. The proteins were related to scaffold/adaptor protein (PC00226), protein binding activity modulator (PC00095) and intercellular signal molecule (PC00207). In conclusion, the CytoBusterTM protein extraction reagent showed a better performance for the extraction of proteins in term of the protein yield, proteome coverage and extraction speed.
Fulltext Evaluation of Total Female and Male Aedes aegypti Proteomes Reveals Significant Predictive Protein-Protein Interactions, Functional Ontologies, and Differentially Abundant Proteins

Shettima A, Joseph S, Ishak IH, Abdul Raiz SH, Abu Hasan H, Othman N

Insects, 2021 Aug 20;12(8).
PMID: 34442320 DOI: 10.3390/insects12080752

Aedes aegypti is a significant vector for many tropical and subtropical flavivirus diseases. Only the female mosquito transmits pathogens, while the male plays a vital role in mating and species continuity. This study explored the total proteomes of females and males based on the physiological and genetic differences of female and male mosquitoes. Protein extracts from mosquitoes were analysed using LC-ESI-MS/MS for protein identification, protein interaction network analysis, functional ontology enrichment, and differential protein abundance analyses. Protein identification revealed 422 and 682 proteins exclusive to males and females, respectively, with 608 common proteins found in both sexes. The most significant PPIs (<1.0 × 10-16) were for common proteins, followed by proteins exclusive to females (<1.0 × 10-16) and males (1.58 × 10-12). Significant functional enrichments were observed in the biological process, molecular function, and cellular component for the male and female proteins. The abundance of the proteins differed, with one protein showing an increase (elongation factor 1 α, EF1α) and two showing reductions (actin family) in females versus males. Overall, the study verified the total proteomes differences between male and female Ae. aegypti based on protein identification and interactions, functional ontologies, and differentially abundant proteins. Some of the identified proteins merit further investigation to elucidate their roles in blocking viral transmission.
Fulltext Quantitative proteomics analysis of permethrin and temephos-resistant Ae. aegypti revealed diverse differentially expressed proteins associated with insecticide resistance from Penang Island, Malaysia

Shettima A, Ishak IH, Lau B, Abu Hasan H, Miswan N, Othman N

PLoS Negl Trop Dis, 2023 Sep;17(9):e0011604.
PMID: 37721966 DOI: 10.1371/journal.pntd.0011604

Synthetic insecticides are the primary vector control method used globally. However, the widespread use of insecticides is a major cause of insecticide-resistance in mosquitoes. Hence, this study aimed at elucidating permethrin and temephos-resistant protein expression profiles in Ae. aegypti using quantitative proteomics. In this study, we evaluated the susceptibility of Ae. aegypti from Penang Island dengue hotspot and non-hotspot against 0.75% permethrin and 31.25 mg/l temephos using WHO bioassay method. Protein extracts from the mosquitoes were then analysed using LC-ESI-MS/MS for protein identification and quantification via label-free quantitative proteomics (LFQ). Next, Perseus 1.6.14.0 statistical software was used to perform differential protein expression analysis using ANOVA and Student's t-test. The t-test selected proteins with≥2.0-fold change (FC) and ≥2 unique peptides for gene expression validation via qPCR. Finally, STRING software was used for functional ontology enrichment and protein-protein interactions (PPI). The WHO bioassay showed resistance with 28% and 53% mortalities in adult mosquitoes exposed to permethrin from the hotspot and non-hotspot areas. Meanwhile, the susceptibility of Ae. aegypti larvae revealed high resistance to temephos in hotspot and non-hotspot regions with 80% and 91% mortalities. The LFQ analyses revealed 501 and 557 (q-value <0.05) differentially expressed proteins in adults and larvae Ae. aegypti. The t-test showed 114 upregulated and 74 downregulated proteins in adult resistant versus laboratory strains exposed to permethrin. Meanwhile, 13 upregulated and 105 downregulated proteins were observed in larvae resistant versus laboratory strains exposed to temephos. The t-test revealed the upregulation of sodium/potassium-dependent ATPase β2 in adult permethrin resistant strain, H15 domain-containing protein, 60S ribosomal protein, and PB protein in larvae temephos resistant strain. The downregulation of troponin I, enolase phosphatase E1, glucosidase 2β was observed in adult permethrin resistant strain and tubulin β chain in larvae temephos resistant strain. Furthermore, the gene expression by qPCR revealed similar gene expression patterns in the above eight differentially expressed proteins. The PPI of differentially expressed proteins showed a p-value at <1.0 x 10-16 in permethrin and temephos resistant Ae. aegypti. Significantly enriched pathways in differentially expressed proteins revealed metabolic pathways, oxidative phosphorylation, carbon metabolism, biosynthesis of amino acids, glycolysis, and citrate cycle. In conclusion, this study has shown differentially expressed proteins and highlighted upregulated and downregulated proteins associated with insecticide resistance in Ae. aegypti. The validated differentially expressed proteins merit further investigation as a potential protein marker to monitor and predict insecticide resistance in field Ae. aegypti. The LC-MS/MS data were submitted into the MASSIVE database with identifier no: MSV000089259.
Lymphoma with superimposed tuberculosis and fungal infection mimicking parapharyngeal abscess complicated with recurrent neurocardiogenic syncope: a case report

Sharudin SN, Huda Al Firdas AN, Hitam S, Hamid Z, Nordin NJ, Othman N, et al.

Malays J Pathol, 2020 Aug;42(2):287-291.
PMID: 32860384

INTRODUCTION: Lymphoma of parapharyngeal space (PPS) is a rare condition. The clinical presentations may vary and often masquerades as infection or an inflammatory condition. A misdiagnosis will lead to a delay in treatment of the disease. Due to the complex anatomy of PPS, any attributed pressure from masses can lead to a life-threatening event such as cardiac syncope.
CASE REPORT: We report a rare case of PPS B-cell non-Hodgkin lymphoma with superimposed Tuberculosis (TB) and fungal infection that presents with several episodes of syncope and hemodynamic depression.
DISCUSSION: The clinical entities in PPS lesions syncope and its associated syndromes, pathophysiology, and differential diagnosis together with possible managements are further discussed.
Fulltext Genetic analyses favour an ancient and natural origin of elephants on Borneo

Sharma R, Goossens B, Heller R, Rasteiro R, Othman N, Bruford MW, et al.

Sci Rep, 2018 01 17;8(1):880.
PMID: 29343863 DOI: 10.1038/s41598-017-17042-5

The origin of the elephant on the island of Borneo remains elusive. Research has suggested two alternative hypotheses: the Bornean elephant stems either from a recent introduction in the 17th century or from an ancient colonization several hundreds of thousands years ago. Lack of elephant fossils has been interpreted as evidence for a very recent introduction, whereas mtDNA divergence from other Asian elephants has been argued to favor an ancient colonization. We investigated the demographic history of Bornean elephants using full-likelihood and approximate Bayesian computation analyses. Our results are at odds with both the recent and ancient colonization hypotheses, and favour a third intermediate scenario. We find that genetic data favour a scenario in which Bornean elephants experienced a bottleneck during the last glacial period, possibly as a consequence of the colonization of Borneo, and from which it has slowly recovered since. Altogether the data support a natural colonization of Bornean elephants at a time when large terrestrial mammals could colonise from the Sunda shelf when sea levels were much lower. Our results are important not only in understanding the unique history of the colonization of Borneo by elephants, but also for their long-term conservation.
Malignant paraganglioma of frontoethmoidal region

Sharma HS, Madhavan M, Othman NH, Muhamad M, Abdullah JM

Auris Nasus Larynx, 1999 Oct;26(4):487-93.
PMID: 10530746

Nonchromaffin paragangliomas are unusual tumours arising from widely distributed paraganglionic tissues probably of neural crest origin. In the head and neck region they are usually seen as carotid body or jugulotympanic tumours. Other rarely reported sites in the head and neck region are the orbit, nose and larynx. This report deals with a case of sinonasal paraganglioma which was initially treated with surgery and radiotherapy. Twenty two years later the tumour recurred and showed a rapid growth due to malignant transformation which we believe is late effect of radiotherapy. The clinical features, histopathology and role of radiotherapy in sinonasal paragangliomas together with a review of the medical literature have been discussed.
Teratocarcinosarcoma of the nasal cavity and ethmoid

Sharma HS, Abdullah JM, Othman NH, Muhamad M

J Laryngol Otol, 1998 Jul;112(7):682-6.
PMID: 9775307

Sinonasal teratocarcinosarcoma is very unusual malignant neoplasm histologically consisting of an epithelial element and one or more mesenchymal components. This is a report of teratocarcinosarcoma, in a 74-year-old male, involving the right nasal cavity and ethmoids with intracranial extension. The tumour was totally resected via the craniofacial approach and the patient was given post-operative chemotherapy. Extensive tumour necrosis, rapid growth and local destruction are the prominent features of this tumour. The clinical presentation, pathological features and clinical course of this rare malignancy are discussed with a review of the literature.
Fulltext Validation of target proteins of down-regulated miR-221-5p in HeLa cells treated with Polyalthia longifolia leaf extract using label-free quantitative proteomics approaches

Shanmugapriya, Othman N, Sasidharan S

3 Biotech, 2020 Sep;10(9):399.
PMID: 32850286 DOI: 10.1007/s13205-020-02396-x

The current study was conducted to validate the target proteins of down-regulated miR-221-5p in HeLa cells treated with P. longifolia leaf extract. The validation was done by label-free quantitative proteomics approaches, Gene Ontology (GO) and protein-protein interaction analyses after the cells transfected with miRNA mimics or miRNA inhibitor. The LC-ESI-MS/MS identified a total of 1061, 668, 564 and 940 proteins from untransfected and untreated HeLa cells, untransfected P. longifolia leaf extract-treated HeLa cells, miR-221-5p mimic-transfected P. longifolia leaf extract-treated HeLa cells and anti-miR-221-5p-transfected P. longifolia leaf extract-treated HeLa cells, respectively. The proteomic, GO and protein-protein interaction analyses showed that P. longifolia treatment regulated various protein expressions in HeLa cells, namely tropomyosin, PRKC apoptosis WT1 regulator protein (PAWR), alpha-enolase and beta-enolase, which induced apoptotic cell death after the down-regulation of miR-221-5p. Conclusively, this study showed P. longifolia leaf extract's vital contribution in regulating various protein expressions in HeLa cervical cancer cells to induce apoptotic cell death after downregulation miR-221-5p.
The Effect of H2S Pressure on the Formation of Multiple Corrosion Products on 316L Stainless Steel Surface

Shah M, Ayob MTM, Rosdan R, Yaakob N, Embong Z, Othman NK

ScientificWorldJournal, 2020;2020:3989563.
PMID: 32774180 DOI: 10.1155/2020/3989563

H2S gas when exposed to metal can be responsible for both general and localized corrosion, which depend on several parameters such as H2S concentration and the corrosion product layer formed. Therefore, the formation of passive film on 316L steel when exposed to H2S environment was investigated using several analysis methods such as FESEM and STEM/EDS analyses, which identified a sulfur species underneath the porous structure of the passive film. X-ray photoelectron spectroscopy analysis demonstrated that the first layer of CrO3 and Cr2O3 was dissolved, accelerated by the presence of H2S-Cl-. An FeS2 layer was formed by incorporation of Fe and sulfide; then, passivation by Mo took place by forming a MoO2 layer. NiO, Ni(OH)2, and NiS barriers are formed as final protection for 316L steel. Therefore, Ni and Mo play an important role as a dual barrier to maintain the stability of 316L steel in high pH2S environments. For safety concern, this paper is aimed to point out a few challenges dealing with high partial pressure of H2S and limitation of 316L steel under highly sour condition for the oil and gas production system.
Fulltext Whole gene transcriptomic analysis of PCB/biphenyl degrading Rhodococcus jostii RHA1

Sha'arani S, Hara H, Araie H, Suzuki I, Mohd Noor MJM, Akhir FNM, et al.

J Gen Appl Microbiol, 2019 Sep 14;65(4):173-179.
PMID: 30686798 DOI: 10.2323/jgam.2018.08.003

This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.
Removal efficiency of Gram-positive and Gram-negative bacteria using a natural coagulant during coagulation, flocculation, and sedimentation processes

Sha'arani S, Azizan SNF, Md Akhir FN, Muhammad Yuzir MA, Othman N, Zakaria Z, et al.

Water Sci Technol, 2019 Nov;80(9):1787-1795.
PMID: 32039910 DOI: 10.2166/wst.2019.433

Staphylococcus sp. as Gram-positive and Escherichia coli as Gram-negative are bacterial pathogens and can cause primary bloodstream infections and food poisoning. Coagulation, flocculation, and sedimentation processes could be a reliable treatment for bacterial removal because suspended, colloidal, and soluble particles can be removed. Chemical coagulants, such as alum, are commonly used. However, these chemical coagulants are not environmentally friendly. This present study evaluated the effectiveness of coagulation, flocculation, and sedimentation processes for removing Staphylococcus sp. and E. coli using diatomite with standard jar test equipment at different pH values. Staphylococcus sp. demonstrated 85.61% and 77.23% significant removal in diatomite and alum, respectively, at pH 5. At pH 7, the removal efficiency decreased to 79.41% and 64.13% for Staphylococcus sp. and E. coli, respectively. At pH 9, there was a decrease in Staphylococcus sp. after adding diatomite or alum compared with that of E. coli. The different removal efficiencies of the Gram-positive and Gram-negative bacteria could be owing to the membrane composition and different structures in the bacteria. This study indicates that diatomite has higher efficiency in removing bacteria at pH 5 and can be considered as a potential coagulant to replace alum for removing bacteria by the coagulation process.
Knowledge, perceived barriers and facilitators of medication error reporting: a quantitative survey in Malaysian primary care clinics

Samsiah A, Othman N, Jamshed S, Hassali MA

Int J Clin Pharm, 2020 Aug;42(4):1118-1127.
PMID: 32494990 DOI: 10.1007/s11096-020-01041-0

Background Medication errors are the most common types of medical errors that occur in health care organisations; however, these errors are largely underreported. Objective This study assessed knowledge on medication error reporting, perceived barriers to reporting medication errors, motivations for reporting medication errors and medication error reporting practices among various health care practitioners working at primary care clinics. Setting This study was conducted in 27 primary care clinics in Malaysia. Methods A self-administered survey was distributed to family medicine specialists, doctors, pharmacists, pharmacist assistants, nurses and assistant medical officers. Main outcome measures Health care practitioners' knowledge, perceived barriers and motivations for reporting medication errors. Results Of all respondents (N = 376), nurses represented 31.9% (n = 120), followed by doctors (n = 87, 23.1%), pharmacists (n = 63, 16.8%), assistant medical officers (n = 53, 14.1%), pharmacist assistants (n = 46, 12.2%) and family medicine specialists (n = 7, 1.9%). Of the survey respondents who had experience reporting medication errors, 56% (n = 62) had submitted medication error reports in the preceding 12 months. Results showed that 41.2% (n = 155) of respondents were classified as having good knowledge on medication error and medication error reporting. The mean score of knowledge was significantly higher among prescribers and pharmacists than nurses, pharmacist assistants and assistant medical officers (p
Fulltext Perceptions and attitudes towards medication error reporting in primary care clinics: a qualitative study in Malaysia

Samsiah A, Othman N, Jamshed S, Hassali MA

PLoS One, 2016;11(12):e0166114.
PMID: 27906960 DOI: 10.1371/journal.pone.0166114

OBJECTIVE: To explore and understand participants' perceptions and attitudes towards the reporting of medication errors (MEs).

METHODS: A qualitative study using in-depth interviews of 31 healthcare practitioners from nine publicly funded, primary care clinics in three states in peninsular Malaysia was conducted for this study. The participants included family medicine specialists, doctors, pharmacists, pharmacist assistants, nurses and assistant medical officers. The interviews were audiotaped and transcribed verbatim. Analysis of the data was guided by the framework approach.

RESULTS: Six themes and 28 codes were identified. Despite the availability of a reporting system, most of the participants agreed that MEs were underreported. The nature of the error plays an important role in determining the reporting. The reporting system, organisational factors, provider factors, reporter's burden and benefit of reporting also were identified.

CONCLUSIONS: Healthcare practitioners in primary care clinics understood the importance of reporting MEs to improve patient safety. Their perceptions and attitudes towards reporting of MEs were influenced by many factors which affect the decision-making process of whether or not to report. Although the process is complex, it primarily is determined by the severity of the outcome of the errors. The participants voluntarily report the errors if they are familiar with the reporting system, what error to report, when to report and what form to use.
Medication errors reported to the National Medication Error Reporting System in Malaysia: a 4-year retrospective review (2009 to 2012)

Samsiah A, Othman N, Jamshed S, Hassali MA, Wan-Mohaina WM

Eur J Clin Pharmacol, 2016 Dec;72(12):1515-1524.
PMID: 27637912

PURPOSE: Reporting and analysing the data on medication errors (MEs) is important and contributes to a better understanding of the error-prone environment. This study aims to examine the characteristics of errors submitted to the National Medication Error Reporting System (MERS) in Malaysia.
METHODS: A retrospective review of reports received from 1 January 2009 to 31 December 2012 was undertaken. Descriptive statistics method was applied.
RESULTS: A total of 17,357 MEs reported were reviewed. The majority of errors were from public-funded hospitals. Near misses were classified in 86.3 % of the errors. The majority of errors (98.1 %) had no harmful effects on the patients. Prescribing contributed to more than three-quarters of the overall errors (76.1 %). Pharmacists detected and reported the majority of errors (92.1 %). Cases of erroneous dosage or strength of medicine (30.75 %) were the leading type of error, whilst cardiovascular (25.4 %) was the most common category of drug found.
CONCLUSIONS: MERS provides rich information on the characteristics of reported MEs. Low contribution to reporting from healthcare facilities other than government hospitals and non-pharmacists requires further investigation. Thus, a feasible approach to promote MERS among healthcare providers in both public and private sectors needs to be formulated and strengthened. Preventive measures to minimise MEs should be directed to improve prescribing competency among the fallible prescribers identified.
Fulltext Evaluation of polymerase chain reaction (PCR) method and hybrid capture II (HCII) assay for the detection of human papillomavirus in cervical scrapings

Saini R, Shen TH, Othman NH, Santhanam J, Othman N, Tang TH

Med J Malaysia, 2007 Aug;62(3):206-9.
PMID: 18246908 MyJurnal

In order to investigate the reliability of detecting HPV DNA in cervical smears, we compared the performance of nested MY/GP PCR and FDA approved-Hybrid Capture II (HCII) using clinical cervical scrapings from 40 patients. It was found that PCR was more sensitive (81.8%) in comparison to HCII (36.4%) in detecting HPV although specificity of HCII was much higher (96.6%) than PCR (58.6%). The Negative Predictive Value (NPV) of both the techniques were quite similar but Positive Predictive Value (PPV) of HCII was much higher (80.0%) compared to PCR (42.9%). While the HCII method showed good specificity for HPV detection, its lack of sensitivity as compared to PCR may be a drawback for diagnostic use.
Fulltext Production of recombinant Entamoeba histolytica pyruvate phosphate dikinase and its application in a lateral flow dipstick test for amoebic liver abscess

Saidin S, Yunus MH, Zakaria ND, Razak KA, Huat LB, Othman N, et al.

BMC Infect Dis, 2014 Apr 04;14:182.
PMID: 24708664 DOI: 10.1186/1471-2334-14-182

BACKGROUND: Amoebic liver abscess (ALA) is the most common clinical manifestation of extraintestinal amoebiasis especially in developing countries, causing up to 100 000 fatal cases annually. Accurate and early diagnosis is important to prevent the disease complications, however its diagnosis still poses many challenges due to the limitations of the available detection tools. Pyruvate phosphate dikinase (PPDK), an excretory-secretory protein of E. histolytica, has been reported as a potential diagnostic marker for ALA, hence it may be exploited in the development of a new test for ALA.
METHODS: Recombinant PPDK (rPPDK) was expressed, purified and evaluated by Western blot. In parallel, recombinant galactose-and-N-acetyl-D-galactosamine inhibitable lectin (Gal/GalNAc lectin) was produced and tested similarly. The protein identity was confirmed by analysis using MALDI-TOF/TOF. A lateral flow dipstick (LFD) test using rPPDK was subsequently developed (rPPDK-LFD) and evaluated for serodiagnosis of ALA.
RESULTS: rPPDK was expressed as soluble protein after 4 hours of induction with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at 30°C. Purification using nickel-nitrilotriacetic acid (Ni-NTA) resin yielded 1.5 mg of rPPDK from 1 L of culture with estimated molecular mass of 98 kDa on SDS-PAGE. Western blots using sera from patients with ALA, healthy individuals and other diseases probed with anti-human IgG4-HRP showed the highest sensitivity (93.3%) and specificity (100%); as compared to blots using IgG and IgG1 as secondary antibodies. Moreover, rPPDK showed better specificity when compared to rGal/GalNAc lectin. In the development of the LFD test, the optimum amount of rPPDK was 0.625 μg per dipstick and the optimum working concentration of colloidal gold conjugated anti-human IgG4 was optical density (OD) 5 (1.7 μg of anti-human IgG4). Evaluation of rPPDK-LFD using ALA patients and controls serum samples showed 87% diagnostic sensitivity and 100% specificity.
CONCLUSION: The developed rPPDK-LFD showed good potential for rapid diagnosis of ALA, and merit further multicentre validation using larger number of serum samples.
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.
Update on laboratory diagnosis of amoebiasis

Saidin S, Othman N, Noordin R

Eur J Clin Microbiol Infect Dis, 2019 Jan;38(1):15-38.
PMID: 30255429 DOI: 10.1007/s10096-018-3379-3

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.

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