Browsing by Subject "ASSAY"

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  • Vidilaseris, Keni; Kellosalo, Juho; Goldman, Adrian (2018)
    Membrane-bound pyrophosphatases (mPPases) are homodimeric integral membrane proteins that hydrolyse pyrophosphate into orthophosphates coupled to the active transport of protons or sodium ions across membranes. They occur in bacteria, archaea, plants, and protist parasites. As they are essential in protist parasites and there are no homologous proteins in animals and humans, these enzymes represent an excellent drug target for treating protistal diseases. Experimental screening to find drug candidates is an important step to discover new hit compounds. For that, a cheap, simple, and robust assay is needed. Here we report the application of the molybdenum blue reaction method for a medium throughput microplate activity assay of the hyperthermophilic bacterium Thermotoga maritima mPPase and the possible application of the assay to screen inhibitors of membrane-bound pyrophosphatases.
  • Cecchetti, Cristina; Strauss, Jannik; Stohrer, Claudia; Naylor, Claire; Pryor, Edward; Hobbs, Jeanette; Tanley, Simon; Goldman, Adrian; Byrne, Bernadette (2021)
    Membrane proteins have a range of crucial biological functions and are the target of about 60% of all prescribed drugs. For most studies, they need to be extracted out of the lipid-bilayer, e.g. by detergent solubilisation, leading to the loss of native lipids, which may disturb important protein-lipid/bilayer interactions and thus functional and structural integrity. Relipidation of membrane proteins has proven extremely successful for studying challenging targets, but the identification of suitable lipids can be expensive and laborious. Therefore, we developed a screen to aid the high-throughput identification of beneficial lipids. The screen covers a large lipid space and was designed to be suitable for a range of stability assessment methods. Here, we demonstrate its use as a tool for identifying stabilising lipids for three membrane proteins: a bacterial pyrophosphatase (Tm-PPase), a fungal purine transporter (UapA) and a human GPCR (A(2A)R). A(2A)R is stabilised by cholesteryl hemisuccinate, a lipid well known to stabilise GPCRs, validating the approach. Additionally, our screen also identified a range of new lipids which stabilised our test proteins, providing a starting point for further investigation and demonstrating its value as a novel tool for membrane protein research. The pre-dispensed screen will be made commercially available to the scientific community in future and has a number of potential applications in the field.
  • Hepojoki, Satu; Nurmi, Visa; Vaheri, Antti; Hedman, Klaus; Vapalahti, Olli; Hepojoki, Jussi (2014)
  • Kahma, Helinä; Aurinsalo, Laura; Neuvonen, Mikko; Katajamäki, Jani; Paludetto, Marie-Noelle; Viinamäki, Jenni; Launiainen, Terhi; Filppula, Anne M.; Tornio, Aleksi; Niemi, Mikko; Backman, Janne T. (2021)
    We developed an in vitro high-throughput cocktail assay with nine major drug-metabolizing CYP enzymes, optimized for screening of time-dependent inhibition. The method was applied to determine the selectivity of the time-dependent CYP2C8 inhibitors gemfibrozil 1-O-beta-glucuronide and clopidogrel acyl-beta-D-glucuronide. In vitro incubations with CYP selective probe substrates and pooled human liver microsomes were conducted in 96-well plates with automated liquid handler techniques and metabolite concentrations were measured with quantitative UHPLC-MS/MS analysis. After determination of inter-substrate interactions and Km values for each reaction, probe substrates were divided into cocktails I (tacrine/CYP1A2, bupropion/CYP2B6, amodiaquine/CYP2C8, tolbutamide/CYP2C9 and midazolam/CYP3A4/5) and II (coumarin/CYP2A6, S-mephenytoin/CYP2C19, dextromethorphan/CYP2D6 and astemizole/CYP2J2). Time-dependent inhibitors (furafylline/CYP1A2, selegiline/ CYP2A6, clopidogrel/CYP2B6, gemfibrozil 1-O-beta-glucuronide/CYP2C8, tienilic acid/CYP2C9, ticlopidine/ CYP2C19, paroxetine/CYP2D6 and ritonavir/CYP3A) and direct inhibitor (terfenadine/CYP2J2) showed similar inhibition with single substrate and cocktail methods. Established time-dependent inhibitors caused IC50 fold shifts ranging from 2.2 to 30 with the cocktail method. Under time-dependent inhibition conditions, gemfibrozil 1-O-beta-glucuronide was a strong (90% inhibition) and selective (<< 20% inhibition of other CYPs) inhibitor of CYP2C8 at concentrations ranging from 60 to 300 mu M, while the selectivity of clopidogrel acyl-beta-D-glucuronide was limited at concentrations above its IC80 for CYP2C8. The time-dependent IC50 values of these glucuronides for CYP2C8 were 8.1 and 38 mu M, respectively. In conclusion, a reliable cocktail method including the nine most important drug-metabolizing CYP enzymes was developed, optimized and validated for detecting timedependent inhibition. Moreover, gemfibrozil 1-O-beta-glucuronide was established as a selective inhibitor of CYP2C8 for use as a diagnostic inhibitor in in vitro studies.
  • Arte, Elisa; Huang, Xin; Nordlund, Emilia; Katina, Kati (2019)
    The effect of three combinations of bioprocessing methods by lactic acid fermentation, cell wall hydrolyzing enzymes and phytase on the biochemical (protein, fat, carbohydrate composition) and technofunctional properties (protein solubility, emulsifying and foaming properties) of wheat bran protein isolates were evaluated. The bioprocessing increased the protein (up to 80%) and fat content (up to 22.8%) in the isolates due to the degradation of starch and soluble pentosans. Additional proteins, globulin 3A and 3C, chitinase, beta-amylase and LMW glutenins, were identified from the electrophoretic pattern of the protein isolate bioprocessed with added enzymes. Generally, the bioprocessed protein isolate had lower protein solubility and stronger net charge in pH below 7, when compared to the protein isolate made without bioprocessing. The emulsifying properties of the protein isolates were not affected by bioprocessing. However, the foaming stability of the protein isolates was nearly doubled by bioprocessing with cell wall hydrolyzing enzymes and phytase.
  • Ala-Houhala, M.; Koukila-Kahkola, P.; Antikainen, Jenni; Valve, J.; Kirveskari, J.; Anttila, V. -J. (2018)
    Objectives: To assess the clinical use of panfungal PCR for diagnosis of invasive fungal diseases (IFDs). We focused on the deep tissue samples. Methods: We first described the design of panfungal PCR, which is in clinical use at Helsinki University Hospital. Next we retrospectively evaluated the results of 307 fungal PCR tests performed from 2013 to 2015. Samples were taken from normally sterile tissues and fluids. The patient population was nonselected. We classified the likelihood of IFD according to the criteria of the European Organization for Research and Treatment of Cancer/Invasive Fungal Infections Cooperative Group and the National Institute of Allergy and Infectious Diseases Mycoses Study Group (EORTC/MSG), comparing the fungal PCR results to the likelihood of IFD along with culture and microscopy results. Results: There were 48 positive (16%) and 259 negative (84%) PCR results. The sensitivity and specificity of PCR for diagnosing IFDs were 60.5% and 91.7%, respectively, while the negative predictive value and positive predictive value were 93.4% and 54.2%, respectively. The concordance between the PCR and the culture results was 86% and 87% between PCR and microscopy, respectively. Of the 48 patients with positive PCR results, 23 had a proven or probable IFD. Conclusions: Fungal PCR can be useful for diagnosing IFDs in deep tissue samples. It is beneficial to combine fungal PCR with culture and microscopy. M. Ala-Houhala, Clin Microbiol Infect 2018;24:301 (C) 2017 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
  • Becker, Anna; Schalin-Jäntti, Camilla; Itkonen, Outi (2021)
    Context: Patients with serotonin-secreting neuroendocrine neoplasms (NENs) have increased serum 5-hydroxyindoleacetic acid (5HIAA) concentrations. Serum 5HIAA thus serves as a biomarker in NEN. Objective: To evaluate an improved tandem mass spectrometric serum 5HIAA assay for diagnosis and follow-up of NEN in a clinical cohort. Design: A retrospective study during 2016-2018 at the Diagnostic Center and Department of Endocrinology at Helsinki University Hospital, Finland. Methods: Detailed patient data was obtained from 116 patients. Serum 5HIAA was analyzed by 2 different liquid chromatography with tandem mass spectrometry (LC-MS/MS) assays with samples prepared either by protein precipitation or solid phase extraction. Twenty-four-hour urine 5HIAA samples (n = 33) were analyzed by amperometric LC, and the results were compared. Specificity and sensitivity were calculated by receiver operating characteristic (ROC) analysis. Results: We achieved 5 to 10 000 nmol/L linearity and Conclusion: Serum 5HIAA by LC-MS/MS after protein precipitation performs equally well for the diagnosis of NEN as urinary 5HIAA LC assay. The outcome and sensitivity for serum and 24-h urine assays are convergent. Due to much more reliable and convenient sampling, we recommend serum instead of 24-h urine 5HIAA for diagnosis and follow-up of NEN patients.
  • Guryanov, Ivan; Korzhikov-Vlakh, Viktor; Bhattacharya, Madhushree; Biondi, Barbara; Masiero, Giulia; Formaggio, Fernando; Tennikova, Tatiana; Urtti, Arto (2021)
    The design of efficient vascular endothelial growth factor (VEGF) inhibitors is a high-priority research area aimed at the treatment of pathological angiogenesis. Among other compounds, v114* has been identified as a potent VEGF-binding peptide. In order to improve the affinity to VEGF, we built a conformational constrain in its structure. To this aim, C-alpha-tetrasubstituted amino acid Aib was introduced into the N-terminal tail, peptide loop, or C-terminal helix. NMR studies confirmed the stabilization of the helical conformation in proximity to the Aib residue. We found that the induction of the N-terminal helical structure or stabilization of the C-terminal helix can noticeably increase the peptide affinity to the VEGF. These peptides efficiently inhibited VEGF-stimulated cell proliferation as well. The insertion of the non-proteinogenic Aib residue significantly enhanced the stability of the peptides in the vitreous environment. Thus, these Aib-containing peptides are promising candidates for the design of VEGF inhibitors with improved properties.
  • Gong, BS; Li, D; Kusko, R; Novoradovskaya, N; Zhang, YF; Wang, SZ; Pabon-Pena, C; Zhang, ZH; Lai, K; Cai, WS; LoCoco, JS; Lader, E; Richmond, TA; Mittal, VK; Liu, LC; Johann, DJ; Willey, JC; Bushel, PR; Yu, Y; Xu, C; Chen, GC; Burgess, D; Cawley, S; Giorda, K; Haseley, N; Qiu, FJ; Wilkins, K; Arib, H; Attwooll, C; Babson, K; Bao, LL; Bao, WJ; Lucas, AB; Best, H; Bhandari, A; Bisgin, H; Blackburn, J; Blomquist, TM; Boardman, L; Burgher, B; Butler, DJ; Chang, CJ; Chaubey, A; Chen, T; Chierici, M; Chin, CR; Close, D; Conroy, J; Coleman, JC; Craig, DJ; Crawford, E; del Pozo, A; Deveson, IW; Duncan, D; Eterovic, AK; Fan, XH; Foox, J; Furlanello, C; Ghosal, A; Glenn, S; Guan, MJ; Haag, C; Hang, XY; Happe, S; Hennigan, B; Hipp, J; Hong, HX; Horvath, K; Hu, JH; Hung, LY; Jarosz, M; Kerkhof, J; Kipp, B; Kreil, DP; Lapunzina, P; Li, P; Li, QZ; Li, WH; Li, ZG; Liang, Y; Liu, SQ; Liu, ZC; Ma, C; Marella, N; Martin-Arenas, R; Megherbi, DB; Meng, QC; Mieczkowski, PA; Morrison, T; Muzny, D; Ning, BT; Parsons, BL; Paweletz, CP; Pirooznia, M; Qu, WB; Raymond, A; Rindler, P; Ringler, R; Sadikovic, B; Scherer, A; Schulze, E; Sebra, R; Shaknovich, R; Shi, Q; Shi, TL; Silla-Castro, JC; Smith, M; Lopez, MS; Song, P; Stetson, D; Strahl, M; Stuart, A; Supplee, J; Szankasi, P; Tan, HW; Tang, LY; Tao, YH; Thakkar, S; Thierry-Mieg, D; Thierry-Mieg, J; Thodima, VJ; Thomas, D; Tichy, B; Tom, N; Garcia, EV; Verma, S; Walker, K; Wang, C; Wang, JW; Wang, YX; Wen, ZN; Wirta, V; Wu, LH; Xiao, CL; Xiao, WZ; Xu, SB; Yang, M; Ying, JM; Yip, SH; Zhang, GL; Zhang, S; Zhao, MR; Zheng, YT; Zhou, XY; Mason, CE; Mercer, T; Tong, WD; Shi, LM; Jones, W; Xu, JS (2021)
    BackgroundTargeted sequencing using oncopanels requires comprehensive assessments of accuracy and detection sensitivity to ensure analytical validity. By employing reference materials characterized by the U.S. Food and Drug Administration-led SEquence Quality Control project phase2 (SEQC2) effort, we perform a cross-platform multi-lab evaluation of eight Pan-Cancer panels to assess best practices for oncopanel sequencing.ResultsAll panels demonstrate high sensitivity across targeted high-confidence coding regions and variant types for the variants previously verified to have variant allele frequency (VAF) in the 5-20% range. Sensitivity is reduced by utilizing VAF thresholds due to inherent variability in VAF measurements. Enforcing a VAF threshold for reporting has a positive impact on reducing false positive calls. Importantly, the false positive rate is found to be significantly higher outside the high-confidence coding regions, resulting in lower reproducibility. Thus, region restriction and VAF thresholds lead to low relative technical variability in estimating promising biomarkers and tumor mutational burden.ConclusionThis comprehensive study provides actionable guidelines for oncopanel sequencing and clear evidence that supports a simplified approach to assess the analytical performance of oncopanels. It will facilitate the rapid implementation, validation, and quality control of oncopanels in clinical use.
  • Erra, Elina O.; Korhonen, Essi M.; Voutilainen, Liina; Huhtamo, Eili; Vapalahti, Olli; Kantele, Anu (2013)
  • Ritari, Jarmo; Hultman, Jenni; Fingerroos, Rita; Tarkkanen, Jussi; Pullat, Janne; Paulin, Lars; Kivi, Niina; Auvinen, Petri; Auvinen, Eeva (2012)
  • Wang, Linping; Saarela, Jani; Poque, Sylvain; Valkonen, Jari P. T. (2020)
    The class 1 ribonuclease III (RNase III) encoded by Sweet potato chlorotic stunt virus (CSR3) suppresses RNA silencing in plant cells and thereby counters the host antiviral response by cleaving host small interfering RNAs, which are indispensable components of the plant RNA interference (RNAi) pathway. The synergy between sweet potato chlorotic stunt virus and sweet potato feathery mottle virus can reduce crop yields by 90%. Inhibitors of CSR3 might prove efficacious to counter this viral threat, yet no screen has been carried out to identify such inhibitors. Here, we report a novel high-throughput screening (HTS) assay based on fluorescence resonance energy transfer (FRET) for identifying inhibitors of CSR3. For monitoring CSR3 activity via HTS, we used a small interfering RNA substrate that was labelled with a FRET-compatible dye. The optimized HTS assay yielded 109 potential inhibitors of CSR3 out of 6,620 compounds tested from different small-molecule libraries. The three best inhibitor candidates were validated with a dose-response assay. In addition, a parallel screen of the selected candidates was carried out for a similar class 1 RNase III enzyme from Escherichia coli (EcR3), and this screen yielded a different set of inhibitors. Thus, our results show that the CSR3 and EcR3 enzymes were inhibited by distinct types of molecules, indicating that this HTS assay could be widely applied in drug discovery of class 1 RNase III enzymes.
  • Chen, Jason; Verni, Christopher C.; Jouppila, Annukka; Lassila, Riitta; Diamond, Scott L. (2018)
    Heparin proteoglycans (HEP-PGs) carry standard heparin-mediated anticoagulant properties as well as novel antiplatelet functions, a combination that may be significant for targeting multiple pathways in a single therapy. Recent work developing semisynthetic HEP-PG mimetics has shown promising results also in vivo, however flow conditions in vitro that replicate in vivo hemodynamics have not been reported. In this work, we present several assays (platelet calcium mobilization, aggregometry, microfluidic tests at venous and arterial hemodynamics) to characterize specific mechanistic effects of dual antiplatelet and anticoagulant (APAC) constructs as mimetics of HEP-PGs. Three APACs with different conjugation levels of heparin chains (CL10, CL18, HICL) were shown to decrease platelet deposition to collagen surfaces in PPACK-treated whole blood at venous shear rate (200 s(-1)). FXIIa-inhibited whole blood (CTI: corn trypsin inhibitor, 40 mu g/mL) perfused over collagen/tissue factor showed reduced both platelet and fibrin deposition when treated with APACs. IC50 values for platelet and fibrin inhibition were calculated for each molecule at venous shear rate. Increasing the shear rate to arterial flows (1000 s(-1)) and using APAC as the sole anticoagulant, resulted in a more potent antiplatelet effect of APAC, suggesting an added effect on von Willebrand Factor (vWF) function. Additionally, APAC caused an inhibition of calcium mobilization specific to thrombin and collagen stimulation and a dose-dependent reduction in collagen-mediated platelet aggregation. Understanding the sensitivity of APAC activity to shear rate, platelet signaling and procoagulant pathways is important for applications in which APAC administration may have beneficial therapeutic effects.
  • Gursoy, Oguz; Munsch-Alatossava, Patricia; Ertan, Kubra; Yilmaz, Yusuf; Alatossava, Tapani (2017)
    Continuous nitrogen gas (N-2) flushing extends the shelf life of raw milk (RM) during cold storage. The effect of N-2 treatment on the total antioxidant capacity (TAC) and ascorbic acid (AA) content of RM was determined during cold storage. TAC of RM or deproteinized RM was determined by ABTS and DPPH methods, while L(+)-AA content of RM was determined chromatographically on days 0, 4 and 7 during storage at 6 +/- 1 degrees C. With the ABTS method, the TAC of RM decreased from 472.33 +/- 16.70 to 369.47 +/- 62.06 mu M TEAC while it reduced from 13.30 +/- 0.84 to 8.20 +/- 0.66 mu M TEAC with DPPH method during cold storage. TAC of RM determined with ABTS method decreased after 4 day-storage; however, they remained statistically similar for N-2-treated samples during 7 day-storage. The AA content of RM ranged from 14.06 to 10.76 mg/L during storage but N-2-treatment did not influence AA content significantly. Deproteinization reduced TAC values of milk samples significantly, and the reduction with the ABTS method was about 47.50 % for control samples cold-stored for four days, while it was 11.67 % for N-2-treated deproteinized RM. In conclusion, N-2-flushing through the headspace of milk containing vessels showed a significant protective effect on the antioxidant components of RM during cold storage.
  • Jarvinen, Erkka; Sjöstedt, Noora; Koenderink, Jan B.; Kidron, Heidi; Finel, Moshe (2019)
    Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans-3 '-hydroxycotinine (OH-cotinine) glucuronides. To this end, we tested their transport by multidrug resistance-associated proteins 1 (MRP1, ABCC1) and MRP3-6 (ABCC3-6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P-gp, ABCB1) that are expressed in the apical membranes of these cells. ATP-dependent transport of these glucuronides was evaluated in inside-out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP-dependent transport was observed only for OH-cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K-m value of 895 mu mol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 mu mol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH-cotinine inhibited MRP2-4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.
  • Luukinen, Bruno Vincent; Vuento, Risto; Hirvonen, Jari Juhani (2020)
    In areas of low tuberculosis (TB) prevalence, laboratory diagnosis of TB may essentially cover non-tuberculous mycobacteria (NTM) in addition to Mycobacterium tuberculosis (MTB). In this study, a semi-automated PCR workflow distinguishing MTB and NTM (Anyplex (TM) MTB/NTMe, Seegene) and subsequently detecting MTB isoniazid/rifampicin resistance (Allplex (TM) MTB/MDRe, Seegene) was evaluated for replacing smear microscopy of acid-fast bacilli as the rapid screening method for TB. With 279 clinical samples, 47 cultures positive for MTB and 76 for NTM, the Anyplex (TM) MTB/NTMe assay and smear microscopy showed equal sensitivities (49.6% vs 50.8%, respectively) but Anyplex (TM) MTB/NTMe was more sensitive for MTB (63.8% vs 25.6%) than for NTM (40.8% vs 64.5%). Allplex (TM) MTB/MDRe showed a slightly higher sensitivity of 68.1% for MTB (32/47 positive, n = 222). Antibiotic resistance profiles were correctly identified for all MTB isolates (one MDR isolate). Specificity was 100% for both assays. Anyplex (TM) MTB/NTMe detected all the 18 NTM species present in the study. The analytical performance of the evaluated high-throughput workflow was relatively weak compared to culture but potentially adequate as a rapid screening method analogous to smear microscopy with additional differentiation between TB, MDR-TB, and NTM.
  • Ljungkvist, Marcus; Strandberg, Karin; Berntorp, Erik; Chaireti, Roza; Holme, Pål Andre; Larsen, Ole Halfdan; Lassila, Riitta; Jouppila, Annukka; Szanto, Timea; Zetterberg, Eva (2019)
    Introduction The thrombin generation assay-calibrated automated thrombogram (TGA-CAT) method is used to measure the overall coagulation capacity in plasma. However, the method is still considered to be a research tool, mainly because of its' lack of standardization. Aim Our study aimed to further raise the standardization level for the TGA-CAT method by evaluating a detailed standardization protocol and three reference plasmas' (RP)s ability to normalize results. Methods Six Nordic centres participated in the study, and with input from all centres a detailed laboratory standardization protocol based on the TGA-CAT manual of the manufacturer was established. Three types of plasma, hypo-,normal and hypercoagulable plasma were assessed. Three commercial lyophilized RPs were used for normalization of data. All samples were aliquoted at the Malmo centre and sent frozen at -20C to participating centres. Results Before normalization, all results under all testing conditions showed inter-laboratory coefficient of variability of 10% or lower except for endogenous thrombin potential (12%) and peak (14%) in hypo-plasma with 1 pmol/L tissue factor as starting agent. Successful normalization, improving variability in results, was obtained with two of the three evaluated RPs (HemosIL RP and Affinity RP). Conclusion With our standardization concept, we were able to produce TGA-CAT results as robust as standard coagulation assays used in the routine laboratories. Normalization with HemosIL RP may be considered in populations with low or unknown coagulability, while when analysing plasma samples from populations where hypercoagulability is known or suspected, normalization with Affinity RP may be preferred.
  • Hyytia, Heidi; Ristiniemi, Noora; Laitinen, Päivi; Lovgren, Timo; Pettersson, Kim (2014)
  • Cuadros, Juan; Perez-Tanoira, Ramon; Prieto-Perez, Laura; Martin-Martin, Ines; Berzosa, Pedro; Gonzalez, Vicenta; Tisiano, Gebre; Balcha, Seble; Manuel Ramos, Jose; Gorgolas, Miguel (2015)
    Background In up to one third of the hospitals in some rural areas of Africa, laboratory services in malaria diagnosis are limited to microscopy by thin film, as no capability to perform thick film exists (gold standard in terms of sensitivity for malaria diagnosis). A new rapid molecular malaria diagnostic test called Loop-mediated isothermal DNA amplification (LAMP) has been recently validated in clinical trials showing exceptional sensitivity and specificity features. It could be a reliable diagnostic tool to be implemented without special equipment or training. Objective The objective of this proof of concept study was to confirm the feasibility of using LAMP technique for diagnosis of malaria in a rural Ethiopian hospital with limited resources. Methodology/Principal Findings This study was carried out in Gambo General Hospital, West Arsi Province (Ethiopia), from November 1st to December 31st 2013. A total of 162 patients with a non-focal febrile syndrome were investigated. The diagnostic capability (sensitivity, specificity, positive predictive and negative predictive values) of rapid malaria tests and microscopy by thin film was evaluated in comparison with LAMP. Eleven (6.79%) out of the 162 patients with fever and suspected malaria, tested positive for LAMP, 3 (1.85%) for rapid malaria tests and none of the eleven cases was detected by thin film microscopy. Conclusions/Significance LAMP can be performed in basic rural laboratories without the need for specialized infrastructure and it may set a reliable tool for malaria control to detect a low level parasitemia.
  • Karthik, Govindasamy-Muralidharan; Rantalainen, Mattias; Stalhammar, Gustav; Lovrot, John; Ullah, Ikram; Alkodsi, Amjad; Ma, Ran; Wedlund, Lena; Lindberg, Johan; Frisell, Jan; Bergh, Jonas; Hartman, Johan (2017)
    Background: Transcriptomic profiling of breast tumors provides opportunity for subtyping and molecular-based patient stratification. In diagnostic applications the specimen profiled should be representative of the expression profile of the whole tumor and ideally capture properties of the most aggressive part of the tumor. However, breast cancers commonly exhibit intra-tumor heterogeneity at molecular, genomic and in phenotypic level, which can arise during tumor evolution. Currently it is not established to what extent a random sampling approach may influence molecular breast cancer diagnostics. Methods: In this study we applied RNA-sequencing to quantify gene expression in 43 pieces (2-5 pieces per tumor) from 12 breast tumors (Cohort 1). We determined molecular subtype and transcriptomic grade for all tumor pieces and analysed to what extent pieces originating from the same tumors are concordant or discordant with each other. Additionally, we validated our finding in an independent cohort consisting of 19 pieces (2-6 pieces per tumor) from 6 breast tumors (Cohort 2) profiled using microarray technique. Exome sequencing was also performed on this cohort, to investigate the extent of intra-tumor genomic heterogeneity versus the intra-tumor molecular subtype classifications. Results: Molecular subtyping was consistent in 11 out of 12 tumors and transcriptomic grade assignments were consistent in 11 out of 12 tumors as well. Molecular subtype predictions revealed consistent subtypes in four out of six patients in this cohort 2. Interestingly, we observed extensive intra-tumor genomic heterogeneity in these tumor pieces but not in their molecular subtype classifications. Conclusions: Our results suggest that macroscopic intra-tumoral transcriptomic heterogeneity is limited and unlikely to have an impact on molecular diagnostics for most patients.