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Fulltext Whole-genome sequencing to establish relapse or re-infection with Mycobacterium tuberculosis: a retrospective observational study

Bryant JM, Harris SR, Parkhill J, Dawson R, Diacon AH, van Helden P, et al.

Lancet Respir Med, 2013 Dec;1(10):786-92.
PMID: 24461758 DOI: 10.1016/S2213-2600(13)70231-5

BACKGROUND: Recurrence of tuberculosis after treatment makes management difficult and is a key factor for determining treatment efficacy. Two processes can cause recurrence: relapse of the primary infection or re-infection with an exogenous strain. Although re-infection can and does occur, its importance to tuberculosis epidemiology and its biological basis is still debated. We used whole-genome sequencing-which is more accurate than conventional typing used to date-to assess the frequency of recurrence and to gain insight into the biological basis of re-infection.
METHODS: We assessed patients from the REMoxTB trial-a randomised controlled trial of tuberculosis treatment that enrolled previously untreated participants with Mycobacterium tuberculosis infection from Malaysia, South Africa, and Thailand. We did whole-genome sequencing and mycobacterial interspersed repetitive unit-variable number of tandem repeat (MIRU-VNTR) typing of pairs of isolates taken by sputum sampling: one from before treatment and another from either the end of failed treatment at 17 weeks or later or from a recurrent infection. We compared the number and location of SNPs between isolates collected at baseline and recurrence.
FINDINGS: We assessed 47 pairs of isolates. Whole-genome sequencing identified 33 cases with little genetic distance (0-6 SNPs) between strains, deemed relapses, and three cases for which the genetic distance ranged from 1306 to 1419 SNPs, deemed re-infections. Six cases of relapse and six cases of mixed infection were classified differently by whole-genome sequencing and MIRU-VNTR. We detected five single positive isolates (positive culture followed by at least two negative cultures) without clinical evidence of disease.
INTERPRETATION: Whole-genome sequencing enables the differentiation of relapse and re-infection cases with greater resolution than do genotyping methods used at present, such as MIRU-VNTR, and provides insights into the biology of recurrence. The additional clarity provided by whole-genome sequencing might have a role in defining endpoints for clinical trials.
FUNDING: Wellcome Trust, European Union, Medical Research Council, Global Alliance for TB Drug Development, European and Developing Country Clinical Trials Partnership.

Matched MeSH terms: DNA, Bacterial/genetics*; Mycobacterium tuberculosis/genetics*; Tuberculosis/genetics*
Quantitative Proteomics Analysis Revealed Compromised Chicken Dendritic Cells Function at Early Stage of Very Virulent Infectious Bursal Disease Virus Infection

Yasmin AR, Omar AR, Farhanah MI, Hiscox AJ, Yeap SK

Avian Dis, 2019 06 01;63(2):275-288.
PMID: 31251527 DOI: 10.1637/11936-072418-Reg.1

Chicken dendritic cells (DCs) have been demonstrated to be susceptible to infectious bursal disease virus (IBDV), a causative agent of acute and immunosuppressed disease in young chicks known as infectious bursal disease. Further functional characterization of IBDV-infected DCs of chickens is required to provide a better understanding on the influence of the virus on chicken bone marrow-derived dendritic cells (BM-DCs) following very virulent (vv) IBDV infection. Membrane proteins of BM-DCs were extracted and the proteins were further denatured and reduced before performing labeling with isobaric tags for relative and absolute quantitation. The differential expression protein profiles were identified and quantified using liquid chromatography coupled with tandem mass spectrometry, and later validated using flow cytometry and real-time reverse transcriptase PCR. The analysis has identified 134 differentially regulated proteins from a total of 283 proteins (cutoff values of ≤0.67, ≥1.5, and ProtScore >1.3 at 95% confidence interval), which produced high-yield membrane fractions. The entry of vvIBDV into the plasma membrane of BM-DCs was observed at 3 hr postinfection by the disruption of several important protein molecule functions, namely apoptosis, RNA/DNA/protein synthesis, and transport and cellular organization, without the activation of proteins associated with signaling. At the later stage of infection, vvIBDV induced expression of several proteins, namely CD200 receptor 1-A, integrin alpha-5, HSP-90, cathepsin, lysosomal-associated membrane protein, and Ras-related proteins, which play crucial roles in signaling, apoptosis, stress response, and antigen processing as well as in secretion of danger-associated proteins. These findings collectively indicated that the chicken DCs are expressing various receptors regarded as potential targets for pathogen interaction during viral infection. Therefore, fundamental study of the interaction of DCs and IBDV will provide valuable information in understanding the role of professional antigen-presenting cells in chickens and their molecular interactions during IBDV infection and vaccination.

Matched MeSH terms: Poultry Diseases/genetics; Birnaviridae Infections/genetics; Avian Proteins/genetics*
Mitochondrial abnormalities in oculopharyngeal muscular dystrophy

Wong KT, Dick D, Anderson JR

Neuromuscul Disord, 1996 May;6(3):163-6.
PMID: 8784803

This report describes a 56-yr-old man with a dominantly inherited disorder affecting four generations and characterized by bilateral ptosis and dysphagia. Muscle biopsy showed only minor light microscopic abnormalities but electron microscopy revealed fibres containing paracrystalline mitochondrial inclusions. Southern analysis of mitochondrial DNA obtained from muscle did not reveal mitochondrial gene deletions. An extensive search eventually identified the characteristic intranuclear filaments of oculopharyngeal muscular dystrophy (OPMD). Abnormal mitochondria are non-specific epiphenomena in OPMD but a potential source of confusion with a late-onset mitochondrial cytopathy. This case further emphasizes the necessity for a diligent search for the diagnostic intranuclear filaments when oculopharyngeal muscular dystrophy is suspected clinically.

Matched MeSH terms: Blepharoptosis/genetics; Deglutition Disorders/genetics; Muscular Dystrophies/genetics
Potential of dihydropyrimidine dehydrogenase genotypes in personalizing 5-fluorouracil therapy among colorectal cancer patients

Teh LK, Hamzah S, Hashim H, Bannur Z, Zakaria ZA, Hasbullani Z, et al.

Ther Drug Monit, 2013 Oct;35(5):624-30.
PMID: 23942539 DOI: 10.1097/FTD.0b013e318290acd2

Dihydropyrimidine dehydrogenase (DPD) is a pyrimidine catabolic enzyme involved in the initial and rate-limiting step of the catabolic pathway of toxic metabolites of 5-fluorouracil (5-FU). Several studies have reported that deficiency of DPD and polymorphisms of its gene are related to 5-FU toxicities and death. Association between serum concentration of 5-FU and its related toxicity has also been previously demonstrated. Hence, this study aims to understand the role of DPYD variants in serum level of 5-FU and the risk of developing toxicity to prevent adverse reactions and maximize therapy outcome for personalized medicine.

Matched MeSH terms: Polymorphism, Genetic/genetics; Colorectal Neoplasms/genetics*; Dihydrouracil Dehydrogenase (NADP)/genetics*
Fulltext Is tissue still the issue in detecting molecular alterations in lung cancer?

Liam CK, Mallawathantri S, Fong KM

Respirology, 2020 09;25(9):933-943.
PMID: 32335992 DOI: 10.1111/resp.13823

Molecular biomarker testing of advanced-stage NSCLC is now considered standard of care and part of the diagnostic algorithm to identify subsets of patients for molecular-targeted treatment. Tumour tissue biopsy is essential for an accurate initial diagnosis, determination of the histological subtype and for molecular testing. With the increasing use of small biopsies and cytological specimens for diagnosis and the need to identify an increasing number of predictive biomarkers, proper management of the limited amount of sampling materials available is important. Many patients with advanced NSCLC do not have enough tissue for molecular testing and/or do not have a biopsy-amenable lesion and/or do not want to go through a repeat biopsy given the potential risks. Molecular testing can be difficult or impossible if the sparse material from very small biopsy specimens has already been exhausted for routine diagnostic purposes. A limited diagnostic workup is recommended to preserve sufficient tissue for biomarker testing. In addition, tumour biopsies are limited by tumour heterogeneity, particularly in the setting of disease resistance, and thus may yield false-negative results. Hence, there have been considerable efforts to determine if liquid biopsy in which molecular alterations can be non-invasively identified in plasma cell-free ctDNA, a potential surrogate for the entire tumour genome, can overcome the issues with tissue biopsies and replace the need for the latter.

Matched MeSH terms: Carcinoma, Non-Small-Cell Lung/genetics*; Lung Neoplasms/genetics*; Biomarkers, Tumor/genetics
Fulltext Early Pleistocene enamel proteome from Dmanisi resolves Stephanorhinus phylogeny

Cappellini E, Welker F, Pandolfi L, Ramos-Madrigal J, Samodova D, Rüther PL, et al.

Nature, 2019 10;574(7776):103-107.
PMID: 31511700 DOI: 10.1038/s41586-019-1555-y

The sequencing of ancient DNA has enabled the reconstruction of speciation, migration and admixture events for extinct taxa1. However, the irreversible post-mortem degradation2 of ancient DNA has so far limited its recovery-outside permafrost areas-to specimens that are not older than approximately 0.5 million years (Myr)3. By contrast, tandem mass spectrometry has enabled the sequencing of approximately 1.5-Myr-old collagen type I4, and suggested the presence of protein residues in fossils of the Cretaceous period5-although with limited phylogenetic use6. In the absence of molecular evidence, the speciation of several extinct species of the Early and Middle Pleistocene epoch remains contentious. Here we address the phylogenetic relationships of the Eurasian Rhinocerotidae of the Pleistocene epoch7-9, using the proteome of dental enamel from a Stephanorhinus tooth that is approximately 1.77-Myr old, recovered from the archaeological site of Dmanisi (South Caucasus, Georgia)10. Molecular phylogenetic analyses place this Stephanorhinus as a sister group to the clade formed by the woolly rhinoceros (Coelodonta antiquitatis) and Merck's rhinoceros (Stephanorhinus kirchbergensis). We show that Coelodonta evolved from an early Stephanorhinus lineage, and that this latter genus includes at least two distinct evolutionary lines. The genus Stephanorhinus is therefore currently paraphyletic, and its systematic revision is needed. We demonstrate that sequencing the proteome of Early Pleistocene dental enamel overcomes the limitations of phylogenetic inference based on ancient collagen or DNA. Our approach also provides additional information about the sex and taxonomic assignment of other specimens from Dmanisi. Our findings reveal that proteomic investigation of ancient dental enamel-which is the hardest tissue in vertebrates11, and is highly abundant in the fossil record-can push the reconstruction of molecular evolution further back into the Early Pleistocene epoch, beyond the currently known limits of ancient DNA preservation.

Matched MeSH terms: Perissodactyla/genetics*; Phosphorylation/genetics; Proteome/genetics*
A variant e13a3 BCR-ABL1 fusion transcript in refractory adult B-cell acute lymphoblastic leukemia achieving complete remission with CAR-Tcell therapy

Phan CL, Tan SN, Tan SM, Kadir SSSA, Ramli NLM, Lim TO, et al.

Cancer Genet, 2021 01;250-251:20-24.
PMID: 33220656 DOI: 10.1016/j.cancergen.2020.11.003

Acute lymphoblastic leukemia (ALL) cases with e13a3 fusion transcripts are extremely rare. We report a 24-year-old male with Ph-positive (Ph+) ALL with an aberrant e13a3 fusion transcript treated with CD19-specific chimeric antigen receptor T-cell (CAR-T) therapy. He developed refractory disease post-chemotherapy induction, andreceived allogeneic hematopoietic stem cell transplantation (allo-HSCT) after salvage with imatinib in combination with chemotherapy regimen. Unfortunately, the patient relapsed after +90 days post-transplant. He was consented to CAR-T therapy trial and achieved complete remission, highlighting the efficacy of CAR-T treatment in relapsed-refractory B-ALL irrespective of the underlying genetic drivers in leukemia cells .

Matched MeSH terms: RNA, Messenger/genetics*; Leukemia, B-Cell/genetics*; Fusion Proteins, bcr-abl/genetics*
Toll-like receptor 4 Asp299Gly and Thr399Ile polymorphisms and typhoid susceptibility in Asian Malay population in Malaysia

Bhuvanendran S, Hussin HM, Meran LP, Anthony AA, Zhang L, Burch LH, et al.

Microbes Infect, 2011 Sep;13(10):844-51.
PMID: 21612766 DOI: 10.1016/j.micinf.2011.04.007

Typhoid fever is a major health problem with frequent outbreaks in Kelantan, Malaysia. Prevalence of TLR4 gene polymorphisms varies with ethnic groups (0-20%) and predisposean individual to gram-negative infections. The prevalence rate of TLR4 Asp299Gly and Thr399lle polymorphisms in the Malay population or the influence of these on typhoid fever susceptibility is not yet reported. 250 normal and 304 susceptible Malay individuals were investigated for these polymorphisms using allele-specific PCR and analysed for its association with typhoid fever susceptibility. The total prevalence of polymorphisms in the normal population was 4.8% in comparison to 12.5% in the susceptible population (p = 0.002). An increased frequency of both polymorphisms was observed in the susceptible population (p

Matched MeSH terms: Typhoid Fever/genetics*; Amino Acid Substitution/genetics; Toll-Like Receptor 4/genetics*
Fulltext Plasmodium falciparum isolate with histidine-rich protein 2 gene deletion from Nyala City, Western Sudan

Boush MA, Djibrine MA, Mussa A, Talib M, Maki A, Mohammed A, et al.

Sci Rep, 2020 07 30;10(1):12822.
PMID: 32733079 DOI: 10.1038/s41598-020-69756-8

In remote areas of malaria-endemic countries, rapid diagnostic tests (RDTs) have dramatically improved parasitological confirmation of suspected malaria cases, especially when skilled microscopists are not available. This study was designed to determine the frequency of Plasmodium falciparum isolates with histidine-rich protein 2 (pfhrp2) gene deletion as one of the possible factors contributing to the failure of PfHRP2-based RDTs in detecting malaria. A total of 300 blood samples were collected from several health centres in Nyala City, Western Sudan. The performance of PfHRP2-based RDTs in relation to microscopy was examined and the PCR-confirmed samples were investigated for the presence of pfhrp2 gene. A total of 113 out of 300 patients were P. falciparum positive by microscopy. Among them, 93.81% (106 out of 113) were positives by the PfHRP2 RDTs. Seven isolates were identified as false negative on the basis of the RDTs results. Only one isolate (0.9%; 1/113) potentially has pfhrp2 gene deletion. The sensitivity and specificity of PfHRP2-based RDTs were 93.81% and 100%, respectively. The results provide insights into the pfhrp2 gene deletion amongst P. falciparum population from Sudan. However, further studies with a large and systematic collection from different geographical settings across the country are needed.

Matched MeSH terms: Antigens, Protozoan/genetics*; Plasmodium falciparum/genetics*; Protozoan Proteins/genetics*
First molecular genotyping of insensitive acetylcholinesterase associated with malathion resistance in Culex quinquefasciatus Say populations in Malaysia

Low VL, Chen CD, Lim PE, Lee HL, Lim YA, Tan TK, et al.

Pest Manag Sci, 2013 Dec;69(12):1362-8.
PMID: 23404830 DOI: 10.1002/ps.3512

Given that there is limited available information on the insensitive acetylcholinesterase in insect species in Malaysia, the present study aims to detect the presence of G119S mutation in the acetylcholinesterase gene of Culex quinquefasciatus from 14 residential areas across 13 states and a federal territory in Malaysia.

Matched MeSH terms: Acetylcholinesterase/genetics*; Culex/genetics; Insect Proteins/genetics*
IGFBP-2 in cervical cancer development

Kaur G, Balasubramaniam SD, Lee YJ

Exp Mol Pathol, 2020 04;113:104362.
PMID: 31870856 DOI: 10.1016/j.yexmp.2019.104362

OBJECTIVE: Increased expression of insulin-like growth factor binding protein 2, IGFBP-2, is associated with many cancers, though its role in cervical cancer is unclear. The aim of this study was to investigate the expression of IGFBP-2 protein and the transcriptomics profile of genes involved in the IGF signaling pathway during cervical cancer development.
DESIGN: Immunohistochemical expression of IGFBP-2 protein was semi-quantitatively assessed in tissue microarrays containing 9 normal cervix, 10 low grade cervical intraepithelial neoplasia (LGCIN), 10 high grade cervical intraepithelial neoplasia (HGCIN) and 42 squamous cell carcinoma (SCC) cases. The gene expression profiles of IGFBP-2, IGF-1, IGF-1R, PTEN, MDM2, AKT1 and TP53 were determined in three cervical tissue samples each from normal cervix, human papillomavirus (HPV)-infected LGCIN, HGCIN and SCC, using Human Transcriptome Array 2.0.
RESULTS: IGFBP-2 protein was highly expressed in the cytoplasm of SCC cells compared to normal cervix (p = .013). The expression was not significantly associated with CIN grade or SCC stage. Transcriptomics profiling demonstrated upregulation of IGFBP-2 and TP53 in HGCIN and SCC compared to normal cervix. IGF-1, IGF-1R and PTEN genes were downregulated in all histological groups. IGF-1 gene was significantly downregulated in SCC (p = .031), while PTEN gene was significantly downregulated in HGCIN (p = .012), compared to normal cervix. MDM2 and AKT1 genes were downregulated in LGCIN and HGCIN, while upregulated in SCC.
CONCLUSION: In cervical carcinogenesis, IGFBP-2 appears to play an oncogenic role, probably through an IGF-independent mechanism.

Matched MeSH terms: Uterine Cervical Neoplasms/genetics; Cervical Intraepithelial Neoplasia/genetics; Transcriptome/genetics
Biological properties of TW01 cells expressing latent membrane protein-1 gene of EBV-derived from nasopharyngeal carcinoma cells at different stages of malignancy

Tan EL, Sam CK

Exp Oncol, 2007 Sep;29(3):166-74.
PMID: 18004239

Epstein-Barr virus (EBV), a human gammaherpesvirus is intimately associated with nasopharyngeal carcinoma (NPC), with the incidence of the virus detected in malignant tissues being close to 100% in NPC endemic areas. The viral latent gene, latent membrane protein 1 (LMP1), has all the typical characteristics of an oncogene and extensive studies have shown beyond doubt its abilities in cellular transformation giving rise to malignant phenotypes. The present study compares the gene sequence and biological properties of LMP1 gene derived from two patients with different stages of NPC--one presented with dysplastic, pre-malignant lesion and the other with malignant lesion.

Matched MeSH terms: Herpesvirus 4, Human/genetics*; Neoplasm Invasiveness/genetics; Viral Matrix Proteins/genetics*
Aldosterone-Producing Adenomas: Histopathology-Genotype Correlation and Identification of a Novel CACNA1D Mutation

Tan GC, Negro G, Pinggera A, Tizen Laim NMS, Mohamed Rose I, Ceral J, et al.

Hypertension, 2017 07;70(1):129-136.
PMID: 28584016 DOI: 10.1161/HYPERTENSIONAHA.117.09057

Mutations in KCNJ5, ATP1A1, ATP2B3, CACNA1D, and CTNNB1 are thought to cause the excessive autonomous aldosterone secretion of aldosterone-producing adenomas (APAs). The histopathology of KCNJ5 mutant APAs, the most common and largest, has been thoroughly investigated and shown to have a zona fasciculata-like composition. This study aims to characterize the histopathologic spectrum of the other genotypes and document the proliferation rate of the different sized APAs. Adrenals from 39 primary aldosteronism patients were immunohistochemically stained for CYP11B2 to confirm diagnosis of an APA. Twenty-eight adenomas had sufficient material for further analysis and were target sequenced at hot spots in the 5 causal genes. Ten adenomas had a KCNJ5 mutation (35.7%), 7 adenomas had an ATP1A1 mutation (25%), and 4 adenomas had a CACNA1D mutation (14.3%). One novel mutation in exon 28 of CACNA1D (V1153G) was identified. The mutation caused a hyperpolarizing shift of the voltage-dependent activation and inactivation and slowed the channel's inactivation kinetics. Immunohistochemical stainings of CYP17A1 as a zona fasciculata cell marker and Ki67 as a proliferation marker were used. KCNJ5 mutant adenomas showed a strong expression of CYP17A1, whereas ATP1A1/CACNA1D mutant adenomas had a predominantly negative expression (P value =1.20×10-4). ATP1A1/CACNA1D mutant adenomas had twice the nuclei with intense staining of Ki67 than KCNJ5 mutant adenomas (0.7% [0.5%-1.9%] versus 0.4% [0.3%-0.7%]; P value =0.04). Further, 3 adenomas with either an ATP1A1 mutation or a CACNA1D mutation had >30% nuclei with moderate Ki67 staining. In summary, similar to KCNJ5 mutant APAs, ATP1A1 and CACNA1D mutant adenomas have a seemingly specific histopathologic phenotype.

Matched MeSH terms: Sodium-Potassium-Exchanging ATPase/genetics*; Calcium Channels, L-Type/genetics*; G Protein-Coupled Inwardly-Rectifying Potassium Channels/genetics*
Misidentification of Burkholderia pseudomallei as Burkholderia cepacia by the VITEK 2 system

Zong Z, Wang X, Deng Y, Zhou T

J Med Microbiol, 2012 Oct;61(Pt 10):1483-1484.
PMID: 22820689 DOI: 10.1099/jmm.0.041525-0

A previously healthy Chinese male returned from working in the Malaysian jungle with a fever. A blood culture grew Gram-negative bacilli that were initially identified as Burkholderia cepacia by the VITEK 2 system but were subsequently found to be Burkholderia pseudomallei by partial sequencing of the 16S rRNA gene. The identification of B. pseudomallei using commercially available automated systems is problematic and clinicians in non-endemic areas should be aware of the possibility of melioidosis in patients with a relevant travel history and blood cultures growing Burkholderia spp.

Matched MeSH terms: DNA, Bacterial/genetics; RNA, Bacterial/genetics; RNA, Ribosomal, 16S/genetics
Intrabacterial proton-dependent CagA transport system in Helicobacter pylori

Wu H, Nakano T, Daikoku E, Morita C, Kohno T, Lian HH, et al.

J Med Microbiol, 2005 Dec;54(Pt 12):1117-1125.
PMID: 16278423 DOI: 10.1099/jmm.0.46158-0

Helicobacter pylori CagA modifies the signalling of host cells and causes gastric diseases. Although CagA is injected into gastric epithelial cells through the type IV secretion machinery, it remains unclear how CagA is transported towards the machinery in the bacterial cytoplasm. In this study, it was determined that the proton-dependent intracytoplasmic transport system correlates with the priming of CagA secretion from H. pylori. The cytotoxicity of neutral-pH- and acidic-pH-treated H. pylori was examined in the AGS cell line. The amount of phosphorylated CagA in AGS cells incubated with acidic-pH- and neutral-pH-treated H. pylori was determined by enzyme immunoassay and Western blot. The production of CagA and adherence of the treated bacteria were examined by enzyme immunoassay and light microscopy, respectively. To clarify how CagA is transported towards the inner membrane of the treated bacteria, the localization of CagA was analysed by immunoelectron microscopy. The proportion of hummingbird cells in the AGS cell line rapidly increased following the inoculation of acidic-pH-treated H. pylori but increased more slowly with neutral-pH-treated H. pylori, and the phenomenon correlated with the amount of phosphorylated CagA in AGS cells. CagA was densely localized near the inner membrane in the acidic-pH-treated bacterial cytoplasm, but this localization was not observed in the neutral-pH-treated bacterial cytoplasm, suggesting that CagA shifts from the centre to the peripheral portion of the cytoplasm as a result of an extracellular decrease in pH. This phenomenon depended on the presence of UreI, a proton-dependent urea channel, but not on the presence of urea. The pH treatments did not enhance CagA production or the adherence of the bacterium to AGS cells. The authors propose that H. pylori possesses a proton-dependent intracytoplasmic transport system that probably accelerates priming for CagA injection.

Matched MeSH terms: Antigens, Bacterial/genetics; Bacterial Proteins/genetics; Helicobacter pylori/genetics
Development of a thermostabilized, one-step, nested, tetraplex PCR assay for simultaneous identification and differentiation of Entamoeba species, Entamoeba histolytica and Entamoeba dispar from stool samples

Foo PC, Chan YY, See Too WC, Tan ZN, Wong WK, Lalitha P, et al.

J Med Microbiol, 2012 Sep;61(Pt 9):1219-1225.
PMID: 22556327 DOI: 10.1099/jmm.0.044552-0

Entamoeba histolytica is the only Entamoeba species that causes amoebiasis in humans. Approximately 50 million people are infected, with 100, 000 deaths annually in endemic countries. Molecular diagnosis of Entamoeba histolytica is important to differentiate it from the morphologically identical Entamoeba dispar to avoid unnecessary medication. Conventional molecular diagnostic tests require trained personnel, cold-chain transportation and/or are storage-dependent, which make them user-unfriendly. The aim of this study was to develop a thermostabilized, one-step, nested, tetraplex PCR assay for the detection of Entamoeba histolytica, Entamoeba dispar and Entamoeba species in cold-chain-free and ready-to-use form. The PCR test was designed based on the Entamoeba small subunit rRNA (SSU-rRNA) gene, which detects the presence of any Entamoeba species, and simultaneously can be used to differentiate Entamoeba histolytica from Entamoeba dispar. In addition, a pair of primers was designed to serve as an internal amplification control to help identify inhibitors in the samples. All PCR reagents together with the designed primers were thermostabilized by lyophilization and were stable at 24 °C for at least 6 months. The limit of detection of the tetraplex PCR was found to be 39 pg DNA or 1000 cells for Entamoeba histolytica and 78 pg DNA or 1000 cells for Entamoeba dispar, and the specificity was 100 %. In conclusion, this cold-chain-free, thermostabilized, one-step, nested, multiplex PCR assay was found to be efficacious in differentiating Entamoeba histolytica from other non-pathogenic Entamoeba species.

Matched MeSH terms: Entamoeba/genetics*; Entamoeba histolytica/genetics*; DNA, Protozoan/genetics
A simplified multiplex PCR assay for fast and easy discrimination of globally distributed staphylococcal cassette chromosome mec types in meticillin-resistant Staphylococcus aureus

Ghaznavi-Rad E, Nor Shamsudin M, Sekawi Z, van Belkum A, Neela V

J Med Microbiol, 2010 Oct;59(Pt 10):1135-1139.
PMID: 20616192 DOI: 10.1099/jmm.0.021956-0

A multiplex PCR assay was developed for the identification of major types and subtypes of staphylococcal cassette chromosome mec (SCCmec) in meticillin-resistant Staphylococcus aureus (MRSA) strains. The method uses a novel 9 valent multiplex PCR plus two primer pairs for S. aureus identification and detection of meticillin resistance. All 389 clinical MRSA isolates from Malaysia and 18 European isolates from the Harmony collection harbouring different SCCmec types that we tested were correctly characterized by our PCR assay. SCCmec type III and V were by far the most common types among both hospital- and community-acquired Malaysian MRSA isolates, with an apparent emergence of MRSA harbouring the IVh type.

Matched MeSH terms: DNA, Bacterial/genetics*; DNA Primers/genetics; Methicillin-Resistant Staphylococcus aureus/genetics*
Regional and accelerated molecular evolution in group I snake venom gland phospholipase A2 isozymes

Chuman Y, Nobuhisa I, Ogawa T, Deshimaru M, Chijiwa T, Tan NH, et al.

Toxicon, 2000 Mar;38(3):449-62.
PMID: 10669032

In accordance with detection of a few phospholipase A2 (PLA2) isozyme genes by Southern blot analysis, only two cDNAs, named NnkPLA-I , and NnkPLA-II, encoding group I PLA2s, NnkPLA-I and NnkPLA-II, respectively, were isolated from the venom gland cDNA library of Elapinae Naja naja kaouthia of Malaysia. NnkPLA-I and NnkPLA-II showed four amino acid substitutions, all of which were brought about by single nucleotide substitution. No existence of clones encoding CM-II and CM-III, PLA2 isozymes which had been isolated from the venom of N. naja kaouthia of Thailand, in Malaysian N. naja kaouthia venom gland cDNA library was verified by dot blot hybridization analysis with particular probes. NnkPLA-I and NnkPLA-II differed from CM-II and CM-III with four and two amino acid substitutions, respectively, suggesting that their molecular evolution is regional. The comparison of NnkPLA-I, NnkPLA-II and cDNAs encoding other group I snake venom gland PLA2s indicated that the 5'- and 3'-untranslated regions are more conserved than the mature protein-coding region and that the number of nucleotide substitutions per nonsynonymous site is almost equal to that per synonymous site in the protein-coding region, suggesting that accelerated evolution has occurred in group I venom gland PLA2s possibly to acquire new physiological functions.

Matched MeSH terms: Isoenzymes/genetics; Phospholipases A/genetics; Snake Venoms/genetics
Fulltext A real-time loop-mediated isothermal amplification assay for rapid detection of Shigella species

Liew PS, Teh CS, Lau YL, Thong KL

Trop Biomed, 2014 Dec;31(4):709-20.
PMID: 25776596 MyJurnal

Shigellosis is a foodborne illness caused by the genus Shigella and is an important global health issue. The development of effective techniques for rapid detection of this pathogen is essential for breaking the chain of transmission. Therefore, we have developed a novel loop-mediated isothermal amplification (LAMP) assay targeting the invasion plasmid antigen H (ipaH) gene to rapidly detect Shigella species. This assay could be performed in 90 min at an optimal temperature of 64ºC, with endpoint results visualized directly. Notably, the method was found to be more sensitive than conventional PCR. Indeed, the detection limit for the LAMP assay on pure bacterial cultures was 5.9 x 10(5) CFU/ml, while PCR displayed a limit of 5.9 x 10(7) CFU/ml. In spiked lettuce samples, the sensitivity of the LAMP assay was 3.6 x 10(4) CFU/g, whereas PCR was 3.6 x 10(5) CFU/g. Overall, the assay accurately identified 32 Shigella spp. with one enteroinvasive Escherichia coli displaying positive reaction while the remaining 32 non-Shigella strains tested were negative.

Matched MeSH terms: Antigens, Bacterial/genetics; Bacterial Proteins/genetics; Shigella/genetics
Fulltext Low efficacy of delthamethrin-treated net against Singapore Aedes aegypti is associated with kdr-type resistance

Pang SC, Chiang LP, Tan CH, Vythilingam I, Lam-Phua SG, Ng LC

Trop Biomed, 2015 Mar;32(1):140-50.
PMID: 25801264 MyJurnal

There has been a worldwide surge in the number and severity of dengue in the past decades. In Singapore, relentless vector control efforts have been put in to control the disease since the 1960's. Space spraying, fogging, chemical treatment and source reduction are some commonly used methodologies for controlling its vectors, particularly Aedes aegypti. Here, as we explored the use of a commercially available delthamethrin-treated net as an alternative strategy and the efficacy of the treated net was found to be limited. Through bioassays and molecular studies, the failure of the treated net to render high mortality rate was found to be associated with the knockdown resistance (kdr) mutation. This is the first report of kdr- mutations in Singapore's Ae. aegypti. At least one point mutation, either homozygous or heterozygous, at amino acid residue V1016G of DIIS6 or F1269C of DIIIS6 was detected in 93% of field strains of Ae. aegypti. Various permutations of wild type and mutant amino acids of the four alleles were found to result in varying degree of survival rate among local field Ae. aegypti when exposed to the deltamethrin treated net. Together with the association of higher survival rate with the presence of both V1016G and F1269C, the data suggest the role of these mutations in the resistance to the deltamethrin. The high prevalence of these mutations were confirmed in a country wide survey where 70% and 72% of the 201 Ae. aegypti analysed possessed the mutations at residues 1016 and 1269 respectively. The highest mutated frequency combination was found to be heterozygous alleles (VG/FC) at both residues 1016 and 1269 (37.8%), followed by homozygous mutation at allele 1269 (24.4%) and homozygous mutation at allele 1016 (22.9%). The kdr- type of resistance among the vector is likely to undermine the effectiveness of pyrethroids treated materials against these mosquitoes.

Matched MeSH terms: Aedes/genetics*; Sodium Channels/genetics*; Mutant Proteins/genetics

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