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Sample records for falciparum freshly isolates

  1. Low anticoagulant heparin disrupts Plasmodium falciparum rosettes in fresh clinical isolates.

    PubMed

    Leitgeb, Anna M; Blomqvist, Karin; Cho-Ngwa, Fidelis; Samje, Moses; Nde, Peter; Titanji, Vincent; Wahlgren, Mats

    2011-03-01

    The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria. PMID:21363975

  2. Low Anticoagulant Heparin Disrupts Plasmodium falciparum Rosettes in Fresh Clinical Isolates

    PubMed Central

    Leitgeb, Anna M.; Blomqvist, Karin; Cho-Ngwa, Fidelis; Samje, Moses; Nde, Peter; Titanji, Vincent; Wahlgren, Mats

    2011-01-01

    The binding of Plasmodium falciparum parasitized erythrocytes to uninfected erythrocytes (rosetting) is associated with severe malaria. The glycosaminoglycan heparan sulfate is an important receptor for rosetting. The related glycosaminoglycan heparin was previously used in treatment of severe malaria, although abandoned because of the occurrence of severe bleedings. Instead, low anticoagulant heparin (LAH) has been suggested for treatment. LAH has successfully been evaluated in safety studies and found to disrupt rosettes and cytoadherence in vitro and in vivo in animal models, but the effect of LAH on fresh parasite isolates has not been studied. Herein, we report that two different LAHs (DFX232 and Sevuparin) disrupt rosettes in the majority of fresh isolates from Cameroonian children with malaria. The rosette disruption effect was more pronounced in isolates from complicated cases than from mild cases. The data support LAH as adjunct therapy in severe malaria. PMID:21363975

  3. Base isolation: Fresh insight

    SciTech Connect

    Shustov, V.

    1993-07-15

    The objective of the research is a further development of the engineering concept of seismic isolation. Neglecting the transient stage of seismic loading results in a widespread misjudgement: The force of resistance associated with velocity is mostly conceived as a source of damping vibrations, though it is an active force at the same time, during an earthquake type excitation. For very pliant systems such as base isolated structures with relatively low bearing stiffness and with artificially added heavy damping mechanism, the so called `damping`` force may occur even the main pushing force at an earthquake. Thus, one of the two basic pillars of the common seismic isolation philosophy, namely, the doctrine of usefulness and necessity of a strong damping mechanism, is turning out to be a self-deception, sometimes even jeopardizing the safety of structures and discrediting the very idea of seismic isolation. There is a way out: breaking with damping dependancy.

  4. Molecular epidemiology of malaria in Cameroon. XXV. In vitro activity of fosmidomycin and its derivatives against fresh clinical isolates of Plasmodium falciparum and sequence analysis of 1-deoxy-D-xylulose 5-phosphate reductoisomerase.

    PubMed

    Tahar, Rachida; Basco, Leonardo K

    2007-08-01

    The in vitro activities of fosmidomycin derivatives, chloroquine, and pyrimethamine were assessed by the radioisotopic assay in clinical isolates of Plasmodium falciparum. In a series of experiments with RPMI 1640 medium-10% fetal bovine serum, the geometric mean 50% inhibitory concentrations (IC(50)s) (n = 34) for fosmidomycin and FR900098 were 301 nM and 118 nM, respectively. In another series of experiments, the geometric mean IC(50)s (n = 33) for fosmidomycin and TH II46 were 413 nM and 249 nM, respectively. The IC(50)s were 2-3 times lower with RPMI-10% fetal bovine serum than the IC(50)s obtained with RPMI-10% human serum. FR900098 and TH II46 were 2.6 and 1.7 times more potent, respectively, than fosmidomycin. There was no correlation between chloroquine or pyrimethamine and fosmidomycin, which suggested the absence of in vitro cross-resistance. Sequence analysis showed five amino acid substitutions, but their possible relationship with the response to fosmidomycin is not clear. Fosmidomycin derivatives are promising candidates for further development. PMID:17690389

  5. A cross strain Plasmodium falciparum microarray optimized for the transcriptome analysis of Plasmodium falciparum patient derived isolates.

    PubMed

    Subudhi, Amit Kumar; Boopathi, P A; Middha, Sheetal; Acharya, Jyoti; Rao, Sudha Narayana; Mugasimangalam, Raja C; Sirohi, Paramendra; Kochar, Sanjay K; Kochar, Dhanpat K; Das, Ashis

    2016-09-01

    Malarial parasite P. falciparum, an apicomplexan protozoan has a 23.3 MB nuclear genome and encodes ~ 5600 transcripts. The genetic diversity of the parasite within and across geographical zones is a challenge to gene expression studies which are essential for understanding of disease process, outcome and developing markers for diagnostics and prognostics. Here, we describe the strategy involved in designing a custom P. falciparum 15K array using the Agilent platform and Genotypic's Right Design methodology to study the transcriptome of Indian field isolates for which genome sequence information is limited. The array contains probes representing genome sequences of two distinct geographical isolates (i.e. 3D7 and HB3) and sub-telomeric var gene sequences of a third isolate (IT4) known to adhere in culture condition. Probes in the array have been selected based on their efficiency to detect transcripts through a 244K array experimentation. Array performance for the 15K array, was evaluated and validated using RNA materials from P. falciparum clinical isolates. A large percentage (91%) of the represented transcripts was detected from Indian P. falciparum patient isolates. Replicated probes and multiple probes representing the same gene showed perfect correlation between them suggesting good probe performance. Additional transcripts could be detected due to inclusion of unique probes representing HB3 strain transcripts. Variant surface antigen (VSA) transcripts were detected by optimized probes representing the VSA genes of three geographically distinct strains. The 15K cross strain P. falciparum array has shown good efficiency in detecting transcripts from P. falciparum parasite samples isolated from patients. The low parasite loads and presence of host RNA makes arrays a preferred platform for gene expression studies over RNA-Seq. PMID:27489776

  6. Dynamics in the Cytoadherence Phenotypes of Plasmodium falciparum Infected Erythrocytes Isolated during Pregnancy

    PubMed Central

    Doritchamou, Justin; Sossou-tchatcha, Sylvain; Cottrell, Gilles; Moussiliou, Azizath; Hounton Houngbeme, Christophe; Massougbodji, Achille; Deloron, Philippe; Ndam, Nicaise Tuikue

    2014-01-01

    Pregnant women become susceptible to malaria infection despite their acquired immunity to this disease from childhood. The placental sequestration of Plasmodium falciparum infected erythrocytes (IE) is the major feature of malaria during pregnancy, due to ability of these parasites to bind chondroitin sulfate A (CSA) in the placenta through the VAR2CSA protein that parasites express on the surface of IE. We collected parasites at different times of pregnancy and investigated the adhesion pattern of freshly collected isolates on the three well described host receptors (CSPG, CD36 and ICAM-1). Var genes transcription profile and VAR2CSA surface-expression were assessed in these isolates. Although adhesion of IE to CD36 and ICAM-1 was observed in some isolates, CSA-adhesion was the predominant binding feature in all isolates analyzed. Co-existence in the peripheral blood of several adhesion phenotypes in early pregnancy isolates was observed, a diversity that gradually tightens with gestational age in favour of the CSA-adhesion phenotype. Infections occurring in primigravidae were often by parasites that adhered more to CSA than those from multigravidae. Data from this study further emphasize the specificity of CSA adhesion and VAR2CSA expression by parasites responsible for pregnancy malaria, while drawing attention to the phenotypic complexity of infections occurring early in pregnancy as well as in multigravidae. PMID:24905223

  7. Improved In Vitro Culture of Plasmodium falciparum Permits Establishment of Clinical Isolates with Preserved Multiplication, Invasion and Rosetting Phenotypes

    PubMed Central

    Albrecht, Letusa; Ahmed Ismail, Hodan; Normark, Johan; Flaberg, Emilie; Szekely, Laszlo; Hultenby, Kjell; Persson, Kristina E. M.; Egwang, Thomas G.; Wahlgren, Mats

    2013-01-01

    To be able to robustly propagate P. falciparum at optimal conditions in vitro is of fundamental importance for genotypic and phenotypic studies of both established and fresh clinical isolates. Cryo-preserved P. falciparum isolates from Ugandan children with severe or uncomplicated malaria were investigated for parasite phenotypes under different in vitro growth conditions or studied directly from the peripheral blood. The parasite cultures showed a minimal loss of parasite-mass and preserved percentage of multiple infected pRBCs to that in peripheral blood, maintained adhesive phenotypes and good outgrowth and multiplication rates when grown in suspension and supplemented with gas. In contrast, abnormal and greatly fluctuating levels of multiple infections were observed during static growth conditions and outgrowth and multiplication rates were inferior. Serum, as compared to Albumax, was found necessary for optimal presentation of PfEMP1 at the pRBC surface and/or for binding of serum proteins (immunoglobulins). Optimal in vitro growth conditions of P. falciparum therefore include orbital shaking (50 rev/min), human serum (10%) and a fixed gas composition (5% O2, 5% CO2, 90% N2). We subsequently established 100% of 76 frozen patient isolates and found rosetting with schizont pRBCs in every isolate (>26% schizont rosetting rate). Rosetting during schizogony was often followed by invasion of the bound RBC as seen by regular and time-lapse microscopy as well as transmission electron microscopy. The peripheral parasitemia, the level of rosetting and the rate of multiplication correlated positively to one another for individual isolates. Rosetting was also more frequent with trophozoite and schizont pRBCs of children with severe versus uncomplicated malaria (p<0.002; p<0.004). The associations suggest that rosetting enhances the ability of the parasite to multiply within the human host. PMID:23894537

  8. Plasmodium falciparum Mating Patterns and Mosquito Infectivity of Natural Isolates of Gametocytes

    PubMed Central

    Morlais, Isabelle; Nsango, Sandrine E.; Toussile, Wilson; Abate, Luc; Annan, Zeinab; Tchioffo, Majoline T.; Cohuet, Anna; Awono-Ambene, Parfait H.; Fontenille, Didier; Rousset, François; Berry, Antoine

    2015-01-01

    Plasmodium falciparum infections in malaria endemic areas often harbor multiple clones of parasites. However, the transmission success of the different genotypes within the mosquito vector has remained elusive so far. The genetic diversity of malaria parasites was measured by using microsatellite markers in gametocyte isolates from 125 asymptomatic carriers. For a subset of 49 carriers, the dynamics of co-infecting genotypes was followed until their development within salivary glands. Also, individual oocysts from midguts infected with blood from 9 donors were genotyped to assess mating patterns. Multiplicity of infection (MOI) was high both in gametocyte isolates and sporozoite populations, reaching up to 10 genotypes. Gametocyte isolates with multiple genotypes gave rise to lower infection prevalence and intensity. Fluctuations of genotype number occurred during the development within the mosquito and sub-patent genotypes, not detected in gametocyte isolates, were identified in the vector salivary glands. The inbreeding coefficient Fis was positively correlated to the oocyst loads, suggesting that P. falciparum parasites use different reproductive strategies according to the genotypes present in the gametocyte isolate. The number of parasite clones within an infection affects the transmission success and the mosquito has an important role in maintaining P. falciparum genetic diversity. Our results emphasize the crucial importance of discriminating between the different genotypes within an infection when studying the A. gambiae natural resistance to P. falciparum, and the need to monitor parasite diversity in areas where malaria control interventions are implemented. PMID:25875840

  9. Pfcrt Gene in Plasmodium falciparum Field Isolates from Muzaffargarh, Pakistan

    PubMed Central

    Sahar, Sumrin; Tanveer, Akhtar; Ali, Akbar; Bilal, Hazrat; Muhammad Saleem, Rana

    2015-01-01

    Background: The aim of the study was to identify the prevalence of different species of Plasmodium and haplotypes of pfcrt in Plasmodium falciparum from the selected area. Methods: Overall, 10,372 blood films of suspected malarial patients were examined microscopically from rural health center Sinawan, district Muzaffargarh, Pakistan from November 2008 to November 2010. P. falciparum positive samples (both whole blood and FTA blood spotted cards) were used for DNA extraction. Nested PCR was used to amplify the pfcrt (codon 72–76) gene fragment. Sequencing was carried out to find the haplotypes in the amplified fragment of pfcrt gene. Result: Over all slide positivity rate (SPR), P. vivax and P. falciparum positivity rate was 21.40 %, 19.37 % and 2.03% respectively. FTA blood spotted cards were equally efficient in the blood storage for PCR and sequencing. Analysis of sequencing results of pfcrt showed only one type of haplotype SagtVMNT (AGTGTAATGAATACA) from codon 72–76 in all samples. Conclusion: The results show high prevalence of CQ resistance and AQ resistant genes. AQ is not recommended to be used as a partner drug in ACT in this locality, so as to ward off future catastrophes. PMID:26623432

  10. MOLECULAR SURVEILLANCE OF Plasmodium vivax AND Plasmodium falciparum DHFR MUTATIONS IN ISOLATES FROM SOUTHERN IRAN

    PubMed Central

    SHARIFI-SARASIABI, Khojasteh; HAGHIGHI, Ali; KAZEMI, Bahram; TAGHIPOUR, Niloofar; MOJARAD, Ehsan Nazemalhosseini; GACHKAR, Latif

    2016-01-01

    In Iran, both Plasmodium vivax and P. falciparum malaria have been detected, but P. vivax is the predominant species. Point mutations in dihydrofolate reductase (dhfr) gene in both Plasmodia are the major mechanisms of pyrimethamine resistance. From April 2007 to June 2009, a total of 134 blood samples in two endemic areas of southern Iran were collected from patients infected with P. vivax and P. falciparum. The isolates were analyzed for P. vivax dihydrofolate reductase (pvdhfr) and P. falciparum dihydrofolate reductase (pfdhfr) point mutations using various PCR-based methods. The majority of the isolates (72.9%) had wild type amino acids at five codons of pvdhfr. Amongst mutant isolates, the most common pvdhfr alleles were double mutant in 58 and 117 amino acids (58R-117N). Triple mutation in 57, 58, and 117 amino acids (57L/58R/117N) was identified for the first time in the pvdhfr gene of Iranian P. vivax isolates. All the P. falciparumsamples analyzed (n = 16) possessed a double mutant pfdhfrallele (59R/108N) and retained a wild-type mutation at position 51. This may be attributed to the fact that the falciparum malaria patients were treated using sulfadoxine-pyrimethamine (SP) in Iran. The presence of mutant haplotypes in P. vivax is worrying, but has not yet reached an alarming threshold regarding drugs such as SP. The results of this study reinforce the importance of performing a molecular surveillance by means of a continuous chemoresistance assessment. PMID:27007559

  11. K13-Propeller Polymorphisms in Plasmodium falciparum Isolates from Patients in Mayotte in 2013 and 2014

    PubMed Central

    Torrentino-Madamet, Marylin; Collet, Louis; Lepère, Jean François; Benoit, Nicolas; Amalvict, Rémy; Ménard, Didier

    2015-01-01

    Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K, and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from Grande Comore showed polymorphisms (N490H, N554K, and E596G). PMID:26416865

  12. K13-Propeller Polymorphisms in Plasmodium falciparum Isolates from Patients in Mayotte in 2013 and 2014.

    PubMed

    Torrentino-Madamet, Marylin; Collet, Louis; Lepère, Jean François; Benoit, Nicolas; Amalvict, Rémy; Ménard, Didier; Pradines, Bruno

    2015-12-01

    Plasmodium falciparum isolates were collected from 29 malaria patients treated with artemether-lumefantrine in Mayotte in 2013 and 2014. Twenty-four cases (83%) consisted of imported malaria. Seventeen percent of the isolates presented mutations in one of the six K13-propeller blades (N490H, F495L, N554H/K, and E596G). A total of 23.8% of the isolates from the Union of Comoros showed K13-propeller polymorphisms. Three of the 18 isolates (16.7%) from Grande Comore showed polymorphisms (N490H, N554K, and E596G). PMID:26416865

  13. Molecular surveillance of antimalarial drug resistance related genes in Plasmodium falciparum isolates from Eritrea.

    PubMed

    Menegon, Michela; Nurahmed, Abduselam M; Talha, Albadawi A; Nour, Bakri Y M; Severini, Carlo

    2016-05-01

    The introduction of artemisinin-based combination therapy has led to extraordinary results in malaria control, however the recent emergence of partial resistance to artemisinin therapy in Southeast Asia jeopardizes these successes. This study aimed at investigating resistance to the antimalarial drugs by evaluating the polymorphisms in the PfK13, Pfcrt and Pfmdr1 genes in Plasmodium falciparum isolates obtained from patients in Eritrea. PMID:26875763

  14. NSAIDs acutely inhibit TRPC channels in freshly isolated rat glomeruli

    SciTech Connect

    Ilatovskaya, Daria V.; Institute of Cytology RAS, St. Petersburg ; Levchenko, Vladislav; Ryan, Robert P.; Cowley, Allen W.; Staruschenko, Alexander

    2011-05-06

    Highlights: {yields} We have established a unique approach to search for physiologically relevant mechanisms of TRPC channels in podocytes. {yields} This study describes endogenous TRPC channels in the isolated decapsulated glomeruli preparation. {yields} We report for the first time that NSAIDs inhibit TRPC channels in podocytes. -- Abstract: Using a novel approach for analysis of TRPC channel activity, we report here that NSAIDs are involved into regulation of TRPC channels in the podocytes of the freshly isolated decapsulated glomeruli. Fluorescence and electron microscopy techniques confirmed the integrity of podocytes in the glomeruli. Western blotting showed that TRPC1, 3 and 6 are highly expressed in the glomeruli. Single-channel patch clamp analysis revealed cation currents with distinct TRPC properties. This is the first report describing single TRPC-like currents in glomerular podocytes. Furthermore, our data provide a novel mechanism of NSAIDs regulation of TRPC channels, which might be implicated in maintaining the glomerular filtration barrier.

  15. Full-length sequence analysis of chloroquine resistance transporter gene in Plasmodium falciparum isolates from Sabah, Malaysia.

    PubMed

    Tan, Lii Lian; Lau, Tiek Ying; Timothy, William; Prabakaran, Dhanaraj

    2014-01-01

    Chloroquine resistance (CQR) in falciparum malaria was identified to be associated with several mutations in the chloroquine resistance transporter gene (pfcrt) that encodes the transmembrane transporter in digestive vacuole membrane of the parasite. This study aimed to investigate the point mutations across the full-length pfcrt in Plasmodium falciparum isolates in Sabah, Malaysia. A total of 31 P. falciparum positive samples collected from Keningau, Kota Kinabalu, and Kudat, Sabah, were analyzed. pfcrt was PCR amplified and cloned prior to sequence analysis. This study showed that all the previously described 10 point mutations associated with CQR at codons 72, 74, 75, 76, 97, 220, 271, 326, 356, and 371 were found with different prevalence. Besides, two novel point mutations, I166V and H273N, were identified with 22.5% and 19.3%, respectively. Three haplotypes, namely, CVMNK (29%), CVIET (3.2%), and SVMNT (67.7%), were identified. High prevalence of SVMNT among P. falciparum isolates from Sabah showed that these isolates are closer to the P. falciparum isolates from Papua New Guinea rather than to the more proximal Southeast Asian CVIET haplotype. Full-length analysis of pfcrt showed that chloroquine resistant P. falciparum in Sabah is still prevalent despite the withdrawal of chloroquine usage since 1979. PMID:25574497

  16. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype.

    PubMed

    Brown, Tyler S; Jacob, Christopher G; Silva, Joana C; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V; Cummings, Michael P

    2015-03-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  17. Plasmodium falciparum field isolates from areas of repeated emergence of drug resistant malaria show no evidence of hypermutator phenotype

    PubMed Central

    Brown, Tyler S.; Jacob, Christopher G.; Silva, Joana C.; Takala-Harrison, Shannon; Djimdé, Abdoulaye; Dondorp, Arjen M.; Fukuda, Mark; Noedl, Harald; Nyunt, Myaing Myaing; Kyaw, Myat Phone; Mayxay, Mayfong; Hien, Tran Tinh; Plowe, Christopher V.; Cummings, Michael P.

    2015-01-01

    Multiple transcontinental waves of drug resistance in Plasmodium falciparum have originated in Southeast Asia before spreading westward, first into the rest of Asia and then to sub-Saharan Africa. In vitro studies have suggested that hypermutator P. falciparum parasites may exist in Southeast Asia and that an increased rate of acquisition of new mutations in these parasites may explain the repeated emergence of drug resistance in Southeast Asia. This study is the first to test the hypermutator hypothesis using field isolates. Using genome-wide SNP data from human P. falciparum infections in Southeast Asia and West Africa and a test for relative rate differences we found no evidence of increased relative substitution rates in P. falciparum isolates from Southeast Asia. Instead, we found significantly increased substitution rates in Mali and Bangladesh populations relative to those in populations from Southeast Asia. Additionally we found no association between increased relative substitution rates and parasite clearance following treatment with artemisinin derivatives. PMID:25514047

  18. No Polymorphism in Plasmodium falciparum K13 Propeller Gene in Clinical Isolates from Kolkata, India

    PubMed Central

    Chatterjee, Moytrey; Ganguly, Swagata; Saha, Pabitra; Bankura, Biswabandhu; Basu, Nandita; Das, Madhusudan; Guha, Subhasish K.; Maji, Ardhendu K.

    2015-01-01

    Molecular markers associated with artemisinin resistance in Plasmodium falciparum are yet to be well defined. Recent studies showed that polymorphisms in K13 gene are associated with artemisinin resistance. The present study was designed to know the pattern of polymorphisms in propeller region of K13 gene among the clinical isolates collected from urban Kolkata after five years of ACT implementation. We collected 59 clinical isolates from urban Kolkata and sequenced propeller region of K13 gene in 51 isolates successfully. We did not find any mutation in any isolate. All patients responded to the ACT, a combination of artesunate + sulphadoxine-pyrimethamine. The drug regimen is still effective in the study area and there is no sign of emergence of resistance against artemisinin as evidenced by wild genotype of K13 gene in all isolates studied. PMID:26688755

  19. Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen

    PubMed Central

    Al-Jasari, Adel; Sady, Hany; Dawaki, Salwa S.; Elyana, Fatin N.; Al-Areeqi, Mona A.; Nasr, Nabil A.; Abdulsalam, Awatif M.; Subramaniam, Lahvanya R.; Azzani, Meram; Ithoi, Init; Lau, Yee Ling; Surin, Johari

    2016-01-01

    Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72–76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72–76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected

  20. Different patterns of pfcrt and pfmdr1 polymorphism in Plasmodium falciparum isolates from Tehama region, Yemen.

    PubMed

    Atroosh, Wahib M; Al-Mekhlafi, Hesham M; Al-Jasari, Adel; Sady, Hany; Dawaki, Salwa S; Elyana, Fatin N; Al-Areeqi, Mona A; Nasr, Nabil A; Abdulsalam, Awatif M; Subramaniam, Lahvanya R; Azzani, Meram; Ithoi, Init; Lau, Yee Ling; Surin, Johari

    2016-01-01

    Introduction. Despite the efforts of the malaria control programme, malaria morbidity is still a common health problem in Yemen, with 60% of the population at risk. Plasmodium falciparum is responsible for 99% of malaria cases. The emergence in Yemen of parasite resistance to chloroquine (CQ) prompted the adoption of artemisinin combination therapy (ACT) in 2009, which involves the use of artesunate plus sulphadoxine-pyrimethamine (AS + SP). However, CQ was retained as the drug of choice for vivax malaria. To assess the impact of the change in the malaria treatment policy five years after its introduction, the present study investigated the mutations in the CQ resistance transporter (pfcrt) and multidrug resistance 1 (pfmdr1) genes. Method. A molecular investigation of 10 codons of pfcrt (72-76, 220, 271, 326, 356, and 371) and five codons of pfmdr1 (86, 184, 1034, 1042, and 1246) was conducted on P. falciparum isolates from districts with the highest malaria endemicity in the Hodeidah and Al-Mahwit governorates in Tehama region, Yemen. A total of 86 positive cases of falciparum monoinfection were investigated for the presence of mutations related to CQ and other antimalarials using a PCR-RFLP assay. Results. There was a wide prevalence of pfcrt gene mutations with the pfcrt 76T CQ resistance marker being predominant (97.7%). The prevalence of other pfcrt mutations varied from high (75E: 88%) to moderate (74I: 79.1%, 220S: 69.8%, 271E and 371I: 53.5%) or low (326S: 36%, 72S: 10.5%). Mutated pfcrt 72-76 amino acids haplotypes were highly prevalent (98.8%). Among these, the CVIET classic, old-world African/Southeast Asian haplotype was the most predominant, and was mostly found in the isolates from the Khamis Bani Saad district of Al-Mahwit (93.1%) and the AdDahi district of Hodeidah (88.9%). However, it was only found in 26.3% of the isolates from the Bajil district of Hodeidah. Surprisingly, the SVMNT new-world South American haplotype was exclusively detected in 9

  1. In vitro inhibition of Plasmodium falciparum by substances isolated from Amazonian antimalarial plants.

    PubMed

    de Andrade-Neto, Valter F; Pohlit, Adrian M; Pinto, Ana Cristina S; Silva, Ellen Cristina C; Nogueira, Karla L; Melo, Márcia R S; Henrique, Marycleuma C; Amorim, Rodrigo C N; Silva, Luis Francisco R; Costa, Mônica R F; Nunomura, Rita C S; Nunomura, Sergio M; Alecrim, Wilson D; Alecrim, M das Graças C; Chaves, F Célio M; Vieira, Pedro Paulo R

    2007-06-01

    In the present study, a quassinoid, neosergeolide, isolated from the roots and stems of Picrolemma sprucei (Simaroubaceae), the indole alkaloids ellipticine and aspidocarpine, isolated from the bark of Aspidosperma vargasii and A. desmanthum (Apocynaceae), respectively, and 4-nerolidylcatechol, isolated from the roots of Pothomorphe peltata (Piperaceae), all presented significant in vitro inhibition (more active than quinine and chloroquine) of the multi-drug resistant K1 strain of Plasmodium falciparum. Neosergeolide presented activity in the nanomolar range. This is the first report on the antimalarial activity of these known, natural compounds. This is also the first report on the isolation of aspidocarpine from A. desmanthum. These compounds are good candidates for pre-clinical tests as novel lead structures with the aim of finding new antimalarial prototypes and lend support to the traditional use of the plants from which these compounds are derived. PMID:17568942

  2. Genetic polymorphisms associated with sulphadoxine-pyrimethamine drug resistance among Plasmodium falciparum field isolates in malaria endemic areas of Assam

    PubMed Central

    Sharma, J; Dutta, P; Khan, SA; Soni, M; Dey, D; Mahanta, J

    2015-01-01

    Background: The emergence of antimalarial drug resistance malaria parasite is widespread in North eastern region of India. During January 2012-December 2013, we conducted active surveillance for detection of antifolate resistance-associated genetic polymorphisms in Plasmodium falciparum malaria parasite from different malaria endemic areas of Assam. Materials and Methods: A total of 281 field samples were collected from suspected malaria patients of which 106 malaria P. falciparum positive cases were detected in microscopic slide examination. A nested PCR was done for amplification of a 648 bp portion of the dhfr gene and 710 bp portion of the dhps gene. Results: Mutation analysis revealed existence of three different haplotypes of the P. falciparum dhfr gene of which ANRNI was highly prevalent (90%). Triple mutant haplotypes AIRNI (N51I + C59R + S108N) of the dhfr gene associated with pyrimethamine resistance were prevalent in Chirang district of Assam. Whereas, dhps mutation study revealed that triple mutant haplotype AGEAA (S436A + A437G + K540E) associated with Sulphadoxine resistance was found among 26% of P. falciparum field isolates. However, P. falciparum dhfr-dhps two locus mutation analysis showed that there were a total of nine dhfr-dhps genotypes. Conclusion: It was noticed that 93.62% (88/94) isolates had mutations in the sequences of both enzymes, which is an indication of prevalence of high grade of Sulphadoxine — pyrimethamine resistance in P. falciparum malaria parasites in Assam. PMID:25511211

  3. In Vitro Activity of Mirincamycin (U24729A) against Plasmodium falciparum Isolates from Gabon▿

    PubMed Central

    Held, Jana; Westerman, Richard; Kremsner, Peter G.; Mordmüller, Benjamin

    2010-01-01

    We assessed the in vitro activity of mirincamycin, a lincosamide antibiotic, against Plasmodium falciparum clinical isolates from Gabon. Growth was determined by HRP2 enzyme-linked immunosorbent assay using an adapted protocol with a prolonged incubation time (6 days) to account for antibiotic-induced delayed death. Mirincamycin's cis and trans isomers are more active (median 50% inhibitory concentrations [IC50s], 3.2 nM and 2.6 nM) than the comparator drugs clindamycin (IC50, 12 nM) and doxycycline (IC50, 720 nM), and therefore, further clinical development is promising. PMID:19841147

  4. In vitro activity of mirincamycin (U24729A) against Plasmodium falciparum isolates from Gabon.

    PubMed

    Held, Jana; Westerman, Richard; Kremsner, Peter G; Mordmüller, Benjamin

    2010-01-01

    We assessed the in vitro activity of mirincamycin, a lincosamide antibiotic, against Plasmodium falciparum clinical isolates from Gabon. Growth was determined by HRP2 enzyme-linked immunosorbent assay using an adapted protocol with a prolonged incubation time (6 days) to account for antibiotic-induced delayed death. Mirincamycin's cis and trans isomers are more active (median 50% inhibitory concentrations [IC(50)s], 3.2 nM and 2.6 nM) than the comparator drugs clindamycin (IC(50), 12 nM) and doxycycline (IC(50), 720 nM), and therefore, further clinical development is promising. PMID:19841147

  5. Molecular Analysis of Chloroquine and Sulfadoxine-Pyrimethamine Resistance-Associated Alleles in Plasmodium falciparum Isolates from Nicaragua

    PubMed Central

    Sridaran, Sankar; Rodriguez, Betzabe; Mercedes Soto, Aida; Macedo De Oliveira, Alexandre; Udhayakumar, Venkatachalam

    2014-01-01

    Chloroquine (CQ) is used as a first-line therapy for the treatment of Plasmodium falciparum malaria in Nicaragua. We investigated the prevalence of molecular markers associated with CQ and sulfadoxine-pyrimethamine (SP) resistance in P. falciparum isolates obtained from the North Atlantic Autonomous Region of Nicaragua. Blood spots for this study were made available from a CQ and SP drug efficacy trial conducted in 2005 and also from a surveillance study performed in 2011. Polymorphisms in P. falciparum CQ resistance transporter, dihydrofolate reductase, and dihydropteroate synthase gene loci that are associated with resistance to CQ, pyrimethamine, and sulfadoxine, respectively, were detected by DNA sequencing. In the 2005 dataset, only 2 of 53 isolates had a CQ resistance allele (CVIET), 2 of 52 had a pyrimethamine resistance allele, and 1 of 49 had a sulfadoxine resistance allele. In the 2011 dataset, none of 45 isolates analyzed had CQ or SP resistance alleles. PMID:24615126

  6. Plasmodium falciparum: limited genetic diversity of MSP-2 in isolates circulating in Brazilian endemic areas.

    PubMed

    Sallenave-Sales, S; Ferreira-da-Cruz, M F; Faria, C P; Cerruti, C; Daniel-Ribeiro, C T; Zalis, M G

    2003-01-01

    The genetic polymorphism of the surface merozoite protein 2 (MSP-2) was evaluated in Plasmodium falciparum isolates from individuals with uncomplicated malaria living in a Brazilian endemic area of Peixoto de Azevedo. The frequency of MSP-2 alleles and the survival of genetically different populations clones in 104 isolates were verified by Southern blot and SSCP-PCR. Single and mixed infections were observed in similar frequencies and the rate of detection of FC27 and 3D7 allelic families was equivalent. Eight alleles were identified and among them, the sequence polymorphism was mainly attributed to variations in the repetitive region. Interestingly, in three alleles nucleotide polymorphism was identical to that detected in a previous study, conducted in 1992, in a near Brazilian endemic area. This finding demonstrated the genetic similarity between two isolate groups, besides the certain temporal stability in the allelic patterns. The implications of these data for studies on the genetic diversity are also discussed. PMID:12880589

  7. Differential var gene transcription in Plasmodium falciparum isolates from patients with cerebral malaria compared to hyperparasitaemia

    PubMed Central

    Kyriacou, Helen M.; Stone, Graham N.; Challis, Richard J.; Raza, Ahmed; Lyke, Kirsten E.; Thera, Mahamadou A.; Koné, Abdoulaye K.; Doumbo, Ogobara K.; Plowe, Christopher V.; Rowe, J. Alexandra

    2006-01-01

    The Plasmodium falciparum variant erythrocyte surface antigens known as PfEMP1, encoded by the var gene family, are thought to play a crucial role in malaria pathogenesis because they mediate adhesion to host cells and immuno-modulation. Var genes have been divided into three major groups (A, B and C) and two intermediate groups (B/A and B/C) on the basis of their genomic location and upstream sequence. We analysed expressed sequence tags of the var gene DBLα domain to investigate var gene transcription in relation to disease severity in Malian children. We found that P. falciparum isolates from children with cerebral malaria (unrousable coma) predominantly transcribe var genes with DBLα1-like domains that are characteristic of Group A or B/A var genes. In contrast, isolates from children with equally high parasite burdens but no symptoms or signs of severe malaria (hyperparasitaemia patients) predominantly transcribe var genes with DBLα0-like domains that are characteristic of the B and C-related var gene groups. These results suggest that var genes with DBLα1-like domains (Group A or B/A) may be implicated in the pathogenesis of cerebral malaria, while var genes with DBLα0-like domains promote less virulent malaria infections. PMID:16996149

  8. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates

    PubMed Central

    Straimer, Judith; Gnädig, Nina F.; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D.; Urnov, Fyodor D.; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M.; Ménard, Didier; Fidock, David A.

    2015-01-01

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  9. Drug resistance. K13-propeller mutations confer artemisinin resistance in Plasmodium falciparum clinical isolates.

    PubMed

    Straimer, Judith; Gnädig, Nina F; Witkowski, Benoit; Amaratunga, Chanaki; Duru, Valentine; Ramadani, Arba Pramundita; Dacheux, Mélanie; Khim, Nimol; Zhang, Lei; Lam, Stephen; Gregory, Philip D; Urnov, Fyodor D; Mercereau-Puijalon, Odile; Benoit-Vical, Françoise; Fairhurst, Rick M; Ménard, Didier; Fidock, David A

    2015-01-23

    The emergence of artemisinin resistance in Southeast Asia imperils efforts to reduce the global malaria burden. We genetically modified the Plasmodium falciparum K13 locus using zinc-finger nucleases and measured ring-stage survival rates after drug exposure in vitro; these rates correlate with parasite clearance half-lives in artemisinin-treated patients. With isolates from Cambodia, where resistance first emerged, survival rates decreased from 13 to 49% to 0.3 to 2.4% after the removal of K13 mutations. Conversely, survival rates in wild-type parasites increased from ≤0.6% to 2 to 29% after the insertion of K13 mutations. These mutations conferred elevated resistance to recent Cambodian isolates compared with that of reference lines, suggesting a contemporary contribution of additional genetic factors. Our data provide a conclusive rationale for worldwide K13-propeller sequencing to identify and eliminate artemisinin-resistant parasites. PMID:25502314

  10. The Density of Knobs on Plasmodium falciparum-Infected Erythrocytes Depends on Developmental Age and Varies among Isolates

    PubMed Central

    Quadt, Katharina A.; Barfod, Lea; Andersen, Daniel; Bruun, Jonas; Gyan, Ben; Hassenkam, Tue; Ofori, Michael F.; Hviid, Lars

    2012-01-01

    Background The virulence of Plasmodium falciparum malaria is related to the parasite’s ability to evade host immunity through clonal antigenic variation and tissue-specific adhesion of infected erythrocytes (IEs). The P. falciparum erythrocyte membrane protein 1 (PfEMP1) family expressed on dome-shaped protrusions called knobs on the IE surface is central to both. Differences in receptor specificity and affinity of expressed PfEMP1 are important for IE adhesiveness, but it is not known whether differences in the number and size of the knobs on which the PfEMP1 proteins are expressed also play a role. Therefore, the aim of this study was to provide detailed information on isolate- and time-dependent differences in knob size and density. Methodology/Principal Findings We used atomic force microscopy to characterize knobs on the surface of P. falciparum-infected erythrocytes. Fourteen ex vivo isolates from Ghanaian children with malaria and 10 P. falciparum isolates selected in vitro for expression of a particular PfEMP1 protein (VAR2CSA) were examined. Knob density increased from ∼20 h to ∼35 h post-invasion, with significant variation among isolates. The knob density ex vivo, which was about five-fold higher than following long-term in vitro culture, started to decline within a few months of culture. Although knob diameter and height varied among isolates, we did not observe significant time-dependent variation in these dimensions. Conclusions/Significance The density of knobs on the P. falciparum-IE surface depends on time since invasion, but is also determined by the infecting isolate in a time-independent manner. This is the first study to quantitatively evaluate knob densities and dimensions on different P. falciparum isolates, to examine ex vivo isolates from humans, and to compare ex vivo and long-term in vitro-cultured isolates. Our findings contribute to the understanding of the interaction between P. falciparum parasites and the infected host. PMID

  11. Point mutations in dihydrofolate reductase and dihydropteroate synthase genes of Plasmodium falciparum isolates from Venezuela.

    PubMed

    Urdaneta, L; Plowe, C; Goldman, I; Lal, A A

    1999-09-01

    The present study was designed to characterize mutations in dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes of Plasmodium falciparum in the Bolivar region of Venezuela, where high levels of clinical resistance to sulfadoxine-pyrimethamine (SP, Fansidar; F. Hoffman-La Roche, Basel, Switzerland) has been documented. We used a nested mutation-specific polymerase chain reaction and restriction digestion methods to measure 1) the prevalence of DHFR mutations at 16, 50, 51, 59, 108, and 164 codon positions, and 2) the prevalence of mutations in the 436, 437, 581, and 613 codon sites in DHPS gene. In the case of the DHFR gene, of the 54 parasite isolates analyzed, we detected the presence of Asn-108 and Ile-51 in 96% of the isolates and Arg-50 mutation in 64% of the isolates. Each of these mutations has been associated with high level of resistance to pyrimethamine. Only 2 samples (4%) showed the wild type Ser-108 mutation and none showed Thr-108 and Val-16 mutations that are specific for resistance to cycloguanil. In the case of DHPS gene, we found a mutation at position 437 (Gly) in 100% of the isolates and Gly-581 in 96% of the isolates. The simultaneous presence of mutations Asn-108 and Ile-51 in the DHFR gene and Gly-437 and Gly-581 in the DHPS gene in 96% of the samples tested suggested that a cumulative effect of mutations could be the major mechanism conferring high SP resistance in this area. PMID:10497990

  12. Ex Vivo Activity of Endoperoxide Antimalarials, Including Artemisone and Arterolane, against Multidrug-Resistant Plasmodium falciparum Isolates from Cambodia

    PubMed Central

    Chaorattanakawee, Suwanna; Lon, Chanthap; Saunders, David L.; Rutvisuttinunt, Wiriya; Yingyuen, Kritsanai; Bathurst, Ian; Ding, Xavier C.; Tyner, Stuart D.

    2014-01-01

    Novel synthetic endoperoxides are being evaluated as new components of artemisinin combination therapies (ACTs) to treat artemisinin-resistant Plasmodium falciparum malaria. We conducted blinded ex vivo activity testing of fully synthetic (OZ78 and OZ277) and semisynthetic (artemisone, artemiside, artesunate, and dihydroartemisinin) endoperoxides in the histidine-rich protein 2 enzyme-linked immunosorbent assay against 200 P. falciparum isolates from areas of artemisinin-resistant malaria in western and northern Cambodia in 2009 and 2010. The order of potency and geometric mean (GM) 50% inhibitory concentrations (IC50s) were as follows: artemisone (2.40 nM) > artesunate (8.49 nM) > dihydroartemisinin (11.26 nM) > artemiside (15.28 nM) > OZ277 (31.25 nM) > OZ78 (755.27 nM). Ex vivo activities of test endoperoxides positively correlated with dihydroartemisinin and artesunate. The isolates were over 2-fold less susceptible to dihydroartemisinin than the artemisinin-sensitive P. falciparum W2 clone and showed sensitivity comparable to those with test endoperoxides and artesunate, with isolate/W2 IC50 susceptibility ratios of <2.0. All isolates had P. falciparum chloroquine resistance transporter mutations, with negative correlations in sensitivity to endoperoxides and chloroquine. The activities of endoperoxides (artesunate, dihydroartemisinin, OZ277, and artemisone) significantly correlated with that of the ACT partner drug, mefloquine. Isolates had mutations associated with clinical resistance to mefloquine, with 35% prevalence of P. falciparum multidrug resistance gene 1 (pfmdr1) amplification and 84.5% occurrence of the pfmdr1 Y184F mutation. GM IC50s for mefloquine, lumefantrine, and endoperoxides (artesunate, dihydroartemisinin, OZ277, OZ78, and artemisone) correlated with pfmdr1 copy number. Given that current ACTs are failing potentially from reduced sensitivity to artemisinins and partner drugs, newly identified mutations associated with artemisinin resistance

  13. Artemisinin Resistance-Associated Polymorphisms at the K13-Propeller Locus Are Absent in Plasmodium falciparum Isolates from Haiti

    PubMed Central

    Carter, Tamar E.; Boulter, Alexis; Existe, Alexandre; Romain, Jean R.; St. Victor, Jean Yves; Mulligan, Connie J.; Okech, Bernard A.

    2015-01-01

    Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations. PMID:25646258

  14. Artemisinin resistance-associated polymorphisms at the K13-propeller locus are absent in Plasmodium falciparum isolates from Haiti.

    PubMed

    Carter, Tamar E; Boulter, Alexis; Existe, Alexandre; Romain, Jean R; St Victor, Jean Yves; Mulligan, Connie J; Okech, Bernard A

    2015-03-01

    Antimalarial drugs are a key tool in malaria elimination programs. With the emergence of artemisinin resistance in southeast Asia, an effort to identify molecular markers for surveillance of resistant malaria parasites is underway. Non-synonymous mutations in the kelch propeller domain (K13-propeller) in Plasmodium falciparum have been associated with artemisinin resistance in samples from southeast Asia, but additional studies are needed to characterize this locus in other P. falciparum populations with different levels of artemisinin use. Here, we sequenced the K13-propeller locus in 82 samples from Haiti, where limited government oversight of non-governmental organizations may have resulted in low-level use of artemisinin-based combination therapies. We detected a single-nucleotide polymorphism (SNP) at nucleotide 1,359 in a single isolate. Our results contribute to our understanding of the global genomic diversity of the K13-propeller locus in P. falciparum populations. PMID:25646258

  15. Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand.

    PubMed

    Gaillard, Tiphaine; Sriprawat, Kanlaya; Briolant, Sébastien; Wangsing, Chirapat; Wurtz, Nathalie; Baragatti, Meïli; Lavina, Morgane; Pascual, Aurélie; Nosten, François; Pradines, Bruno

    2015-08-01

    Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 μM and 31.60 μM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72). PMID:26055380

  16. Molecular Markers and In Vitro Susceptibility to Doxycycline in Plasmodium falciparum Isolates from Thailand

    PubMed Central

    Gaillard, Tiphaine; Sriprawat, Kanlaya; Briolant, Sébastien; Wangsing, Chirapat; Wurtz, Nathalie; Baragatti, Meïli; Lavina, Morgane; Pascual, Aurélie; Nosten, François

    2015-01-01

    Determinations of doxycycline 50% inhibitory concentrations (IC50) for 620 isolates from northwest Thailand were performed via the isotopic method, and the data were analyzed by the Bayesian method and distributed into two populations (mean IC50s of 13.15 μM and 31.60 μM). There was no significant difference between the group with low IC50s versus the group with high IC50s with regard to copy numbers of the Plasmodium falciparum tetQ (pftetQ) gene (P = 0.11) or pfmdt gene (P = 0.87) or the number of PfTetQ KYNNNN repeats (P = 0.72). PMID:26055380

  17. Quantitative assessment of antimalarial activities from Malaysian Plasmodium falciparum isolates by modified in vitro microtechnique.

    PubMed Central

    Hoon, A H; Lam, C K; Wah, M J

    1995-01-01

    Malaysian, TGR (Thailand), and Gambian (West African) Plasmodium falciparum isolates were cultured in vitro by the candle jar method and were characterized for their susceptibilities to present antimalarial drugs by the modified in vitro microtechnique. Results showed that 93 and 47% of the Malaysian isolates were resistant at 50% inhibitory concentrations of 0.1415 to 0.7737 and 0.1025 to 0.1975 microM, respectively, while the rest were susceptible to choloroquine and cycloguanil at 0.0376 and 0.0306 to 0.0954 microM, respectively. All isolates were susceptible to mefloquine, quinine, and pyrimethamine at 0.0026 to 0.0172, 0.0062 to 0.0854, and 0.0149 to 0.0663 microM, respectively. In contrast, the Gambian isolate was susceptible to multiple drugs at 0.0024 to 0.0282 microM; TGR was resistant to chloroquine at 0.8147 microM but was susceptible to mefloquine, quinine, cycloguanil, and pyrimethamine at 0.0024, 0.0096, 0.0143, and 0.0495 microM, respectively. PMID:7793863

  18. P. falciparum isolate-specific distinct patterns of induced apoptosis in pulmonary and brain endothelial cells.

    PubMed

    N'Dilimabaka, Nadine; Taoufiq, Zacharie; Zougbédé, Sergine; Bonnefoy, Serge; Lorthiois, Audrey; Couraud, Pierre Oliver; Rebollo, Angelita; Snounou, Georges; Mazier, Dominique; Moreno Sabater, Alicia

    2014-01-01

    The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC) activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis-linked pathogenicity factors (PALPF), PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria. PMID:24686750

  19. P. falciparum Isolate-Specific Distinct Patterns of Induced Apoptosis in Pulmonary and Brain Endothelial Cells

    PubMed Central

    N'Dilimabaka, Nadine; Bonnefoy, Serge; Lorthiois, Audrey; Couraud, Pierre Oliver; Rebollo, Angelita; Snounou, Georges; Mazier, Dominique; Moreno Sabater, Alicia

    2014-01-01

    The factors implicated in the transition from uncomplicated to severe clinical malaria such as pulmonary oedema and cerebral malaria remain unclear. It is known that alterations in vascular integrity due to endothelial cell (EC) activation and death occur during severe malaria. In this study, we assessed the ability of different P. falciparum clinical isolates to induce apoptosis in ECs derived from human lung and brain. We observed that induction of EC apoptosis was sensitive to the environmental pH and required direct contact between the parasite and the cell, though it was not correlated to the ability of the parasite to cytoadhere. Moreover, the extent of induced apoptosis in the two EC types varied with the isolate. Analysis of parasite genes transcript led us to propose that the activation of different pathways, such as Plasmodium apoptosis–linked pathogenicity factors (PALPF), PALPF-2, PALPF-5 and PF11_0521, could be implied in EC death. These observations provide an experimental framework to decipher the molecular mechanism implicated in the genesis of severe malaria. PMID:24686750

  20. A YAC contig map of plasmodium falciparum chromosome 4: Characterization of a DNA amplification between two recently separated isolates

    SciTech Connect

    Rubio, J.P.; Triglia, T.; Cowman, A.F.

    1995-03-20

    We have generated a physical map of Plasmodium falciparum chromosome 4 using yeast artificial chromosomes (YACs). The map is defined by a YAC contig spanning approximately 1.05 Mb, which has been restriction mapped to a resolution of 30 kb and is punctuated by 22 sequence-tagged sites. The physical information obtained has enabled us to compare and contrast the structure of chromosome 4 in detail between FCR3 and B8, two recently separated isolates of P. falciparum, leading to characterization of a novel chromosome polymorphism occurring in a subtelomeric region. Comparison of chromosomes 4 from 10 different isolates has shown that chromosome size polymorphisms are restricted to both subtelomeric regions. These analyses provide a high-resolution physical map that will be important to complement genetic analysis of this human pathogen. 42 refs., 6 figs., 1 tab.

  1. In Vitro Activity and Interaction of Clindamycin Combined with Dihydroartemisinin against Plasmodium falciparum

    PubMed Central

    Ramharter, M.; Noedl, H.; Winkler, H.; Graninger, W.; Wernsdorfer, W. H.; Kremsner, P. G.; Winkler, S.

    2003-01-01

    Combination regimens are considered a valuable tool for the fight against drug-resistant falciparum malaria. This study was conducted to evaluate the antimalarial potential of clindamycin in combination with dihydroartemisinin in continuously cultured and in freshly isolated Plasmodium falciparum parasites, measuring the inhibition of Plasmodium falciparum histidine-rich protein II synthesis. Interaction analysis revealed a synergistic or additive mode of interaction at various concentration ratios in all continuously cultured parasites at the 50% effective concentration (EC50) level. Antagonism was not found for any of the culture-adapted parasites. In fresh P. falciparum isolates, a fixed clindamycin-dihydroartemisinin combination exhibited additive activity at the EC50 and EC90 levels. The drug mixture showed no significant activity correlation to other commonly used antimalarials. The clindamycin-dihydroartemisinin combination appears to be a promising candidate for clinical investigation. PMID:14576107

  2. Differential Association of Plasmodium falciparum Na+/H+ Exchanger Polymorphism and Quinine Responses in Field- and Culture-Adapted Isolates of Plasmodium falciparum ▿ †

    PubMed Central

    Pelleau, Stéphane; Bertaux, Lionel; Briolant, Sébastien; Ferdig, Michael T.; Sinou, Véronique; Pradines, Bruno; Parzy, Daniel; Jambou, Ronan

    2011-01-01

    Plasmodium falciparum isolates with decreased susceptibility to quinine are increasingly being found in malaria patients. Mechanisms involved in this resistance are not yet understood. Several studies claim that alongside mutations in the Pfcrt and Pfmdr1 genes, the Pfnhe-1 Na+/H+ exchanger polymorphism plays a role in decreasing susceptibility. However, conflicting results on the link between the Pfnhe-1 gene and quinine resistance arise from field- and culture-adapted isolates. We tested the association between Pfnhe-1, Pfcrt, and Pfmdr1 polymorphisms in field- and culture-adapted isolates from various countries with their in vitro susceptibility to quinine. Field isolates presented a higher diversity of the Pfnhe-1 microsatellite sequence than culture-adapted isolates. In culture-adapted isolates but not in field isolates, mutations in the Pfcrt and Pfmdr1 genes, as well as a higher number of DNNND repeats in the Pfnhe-1 gene, were associated with a higher 50% inhibitory concentration (IC50) of quinine. Furthermore, most of the culture-adapted isolates with more than one DNNND repeat in the Pfnhe-1 gene also harbored mutated Pfcrt and Pfmdr1 genes with an apparent cumulative effect on quinine susceptibility. This study supports the involvement of the Pfnhe-1 gene in the modulation of the in vitro quinine response when associated with mutated Pfcrt and Pfmdr1 genes. Culture adaptation could be responsible for selection of specific haplotypes of these three genes. Methods used for drug testing might thus influence the association between Pfnhe-1 polymorphism and quinine susceptibility. However, we do not exclude the possibility that in particular settings, Pfnhe-1 polymorphism can be used as a molecular marker for surveillance of quinine resistance. PMID:21947391

  3. Ex vivo susceptibility of Plasmodium falciparum isolates from Dakar, Senegal, to seven standard anti-malarial drugs

    PubMed Central

    2011-01-01

    Background As a result of widespread chloroquine and sulphadoxine-pyrimethamine resistance, artemisinin-based combination therapy (ACT) (which includes artemether-lumefantrine and artesunate-amodiaquine) has been recommended as a first-line anti-malarial regimen in Senegal since 2006. Since then, there have been very few reports on the ex vivo susceptibility of Plasmodium falciparum to anti-malarial drugs. To examine whether parasite susceptibility has been affected by the widespread use of ACT, the ex vivo susceptibility of local isolates was assessed at the military hospital of Dakar. Methods The ex vivo susceptibility of 93 P. falciparum isolates from Dakar was successfully determined using the Plasmodium lactate dehydrogenase (pLDH) ELISA for the following drugs: chloroquine (CQ), quinine (QN), mefloquine (MQ), monodesethylamodiaquine (MDAQ), lumefantrine (LMF), dihydroartemisinin (DHA) and doxycycline (DOX). Results After transformation of the isolate IC50 in ratio of IC50 according to the susceptibility of the 3D7 reference strain (isolate IC50/3D7 IC50), the prevalence of the in vitro resistant isolates with reduced susceptibility was 50% for MQ, 22% for CQ, 12% for DOX, 6% for both QN and MDAQ and 1% for the drugs LMF and DHA. The highest significant positive correlations were shown between responses to CQ and MDAQ (r = 0.569; P < 0.0001), LMF and QN (r = 0.511; P < 0.0001), LMF and DHA (r = 0.428; P = 0.0001), LMF and MQ (r = 0.413; P = 0.0002), QN and DHA (r = 0.402; P = 0.0003) and QN and MQ (r = 0.421; P = 0.0001). Conclusions The introduction of ACT in 2002 has not induced a decrease in P. falciparum susceptibility to the drugs DHA, MDAQ and LMF, which are common ACT components. However, the prevalence of P. falciparum isolates with reduced susceptibility has increased for both MQ and DOX. Taken together, these data suggest that intensive surveillance of the P. falciparum in vitro susceptibility to anti-malarial drugs in Senegal is required. PMID

  4. Contrasting ex vivo efficacies of "reversed chloroquine" compounds in chloroquine-resistant Plasmodium falciparum and P. vivax isolates.

    PubMed

    Wirjanata, Grennady; Sebayang, Boni F; Chalfein, Ferryanto; Prayoga; Handayuni, Irene; Noviyanti, Rintis; Kenangalem, Enny; Poespoprodjo, Jeanne Rini; Burgess, Steven J; Peyton, David H; Price, Ric N; Marfurt, Jutta

    2015-09-01

    Chloroquine (CQ) has been the mainstay of malaria treatment for more than 60 years. However, the emergence and spread of CQ resistance now restrict its use to only a few areas where malaria is endemic. The aim of the present study was to investigate whether a novel combination of a CQ-like moiety and an imipramine-like pharmacophore can reverse CQ resistance ex vivo. Between March to October 2011 and January to September 2013, two "reversed chloroquine" (RCQ) compounds (PL69 and PL106) were tested against multidrug-resistant field isolates of Plasmodium falciparum (n = 41) and Plasmodium vivax (n = 45) in Papua, Indonesia, using a modified ex vivo schizont maturation assay. The RCQ compounds showed high efficacy against both CQ-resistant P. falciparum and P. vivax field isolates. For P. falciparum, the median 50% inhibitory concentrations (IC50s) were 23.2 nM for PL69 and 26.6 nM for PL106, compared to 79.4 nM for unmodified CQ (P < 0.001 and P = 0.036, respectively). The corresponding values for P. vivax were 19.0, 60.0, and 60.9 nM (P < 0.001 and P = 0.018, respectively). There was a significant correlation between IC50s of CQ and PL69 (Spearman's rank correlation coefficient [r s] = 0.727, P < 0.001) and PL106 (rs = 0.830, P < 0.001) in P. vivax but not in P. falciparum. Both RCQs were equally active against the ring and trophozoite stages of P. falciparum, but in P. vivax, PL69 and PL106 showed less potent activity against trophozoite stages (median IC50s, 130.2 and 172.5 nM) compared to ring stages (median IC50s, 17.6 and 91.3 nM). RCQ compounds have enhanced ex vivo activity against CQ-resistant clinical isolates of P. falciparum and P. vivax, suggesting the potential use of reversal agents in antimalarial drug development. Interspecies differences in RCQ compound activity may indicate differences in CQ pharmacokinetics between the two Plasmodium species. PMID:26149984

  5. Cannabidiol induced a contrasting pro-apoptotic effect between freshly isolated and precultured human monocytes

    SciTech Connect

    Wu, Hsin-Ying; Chang, An-Chi; Wang, Chia-Chi; Kuo, Fu-Hua; Lee, Chi-Ya; Liu, Der-Zen; Jan, Tong-Rong

    2010-08-01

    It has been documented that cannabidiol (CBD) induced apoptosis in a variety of transformed cells, including lymphocytic and monocytic leukemias. In contrast, a differential sensitivity between normal lymphocytes and monocytes to CBD-mediated apoptosis has been reported. The present study investigated the pro-apoptotic effect of CBD on human peripheral monocytes that were either freshly isolated or precultured for 72 h. CBD markedly enhanced apoptosis of freshly isolated monocytes in a time- and concentration-dependent manner, whereas precultured monocytes were insensitive. By comparison, both cells were sensitive to doxorubicin-induced apoptosis. CBD significantly diminished the cellular thiols and glutathione in freshly isolated monocytes. The apoptosis induced by CBD was abrogated in the presence of N-acetyl-{sub L}-cysteine, a precursor of glutathione. In addition, precultured monocytes contained a significantly greater level of glutathione and heme oxygenase-1 (HO-1) compared to the freshly isolated cells. The HO-1 competitive inhibitor zinc protoporphyrin partially but significantly restored the sensitivity of precultured monocytes to CBD-mediated apoptosis. Collectively, our results demonstrated a contrasting pro-apoptotic effect of CBD between precultured and freshly isolated monocytes, which was closely associated with the cellular level of glutathione and the antioxidative capability of the cells.

  6. In vitro susceptibility to quinine and microsatellite variations of the Plasmodium falciparum Na+/H+ exchanger transporter (Pfnhe-1) gene in 393 isolates from Dakar, Senegal

    PubMed Central

    2013-01-01

    Background Although the World Health Organization recommends replacing quinine (QN) by artesunate due to its increased efficacy and the higher tolerance to the drug in both adults and children, QN remains a first-line treatment for severe malaria, especially in Africa. Investigations of microsatellite Pfnhe-1 ms4760 polymorphisms in culture-adapted isolates from around the world have revealed that an increase in the number of DNNND amino acid motifs was associated with decreased QN susceptibility, whereas an increase in the number of DDNHNDNHNND motifs was associated with increased QN susceptibility. Methods In this context, to further analyse associations between Pfnhe-1 ms4760 polymorphisms and QN susceptibility, 393 isolates freshly collected between October 2009 and January 2010 and July 2010 and February 2011, respectively, at the Hôpital Principal de Dakar, Senegal were assessed ex vivo for QN susceptibility, and their genes were amplified and sequenced. Results Of the 393 Plasmodium falciparum clinical isolates collected, 145 were successfully cultured. The 145 QN IC50s ranged from 2.1 to 1291 nM, and 17 isolates (11.7%) exceed the QN reduced susceptibility threshold of 611 nM. Among the 393 P. falciparum clinical isolates, 47 different alleles were observed. The three most prevalent profiles were ms4760-1 (no = 72; 18.3%), ms4760-3 (no = 65; 16.5%) and ms4760-7 (no = 40; 10.2%). There were no significant associations observed between QN IC50 values and i) the number of repeats of DNNND in block II (p = 0.0955, Kruskal-Wallis test); ii) the number of repeats of DDNHNDNHNND in block V (p = 0.1455, Kruskal-Wallis test); or iii) ms4760 profiles (p = 0.1809, Kruskal-Wallis test). Conclusions Pfnhe-1 ms4760 was highly diverse in parasite isolates from Dakar (47 different profiles). Three profiles (ms4760-1, ms4760-3 and ms4760-7) were predominant. The number of repeats for block II (DNNND) or block V (DDNHNDNHNND) was not

  7. A clonal Plasmodium falciparum population in an isolated outbreak of malaria in the Republic of Cabo Verde.

    PubMed

    Arez, A P; Snounou, G; Pinto, J; Sousa, C A; Modiano, D; Ribeiro, H; Franco, A S; Alves, J; do Rosario, V E

    1999-04-01

    We present the first parasitological, molecular and longitudinal analysis of an isolated outbreak of malaria. This outbreak occurred on Santiago Island (Republic of Cabo Verde), a region where malaria is hypoendemic and controlled, and thus the population is considered non-immune. Blood samples were collected from the inhabitants over 1 month and during cross-sectional surveys in the following year. The presence and nature of the parasites was determined by PCR. Plasmodium falciparum was the only species detected. Genetic analysis revealed that the circulating parasites were genetically homogeneous, and probably clonal. Gametocytes were found throughout this period. Our data suggest that this represented a focal outbreak, resulting in the infection of at least 40% of the villagers with a clonal parasite line. Thus, P. falciparum infections can persist for at least 1 year in a substantial proportion (10%) of the hosts. Implications for malaria control and the interpretation of epidemiological data are discussed. PMID:10340324

  8. Mycotoxigenic potential of fungi isolated from freshly harvested Argentinean blueberries.

    PubMed

    Munitz, Martin S; Resnik, Silvia L; Pacin, Ana; Salas, Paula M; Gonzalez, Hector H L; Montti, Maria I T; Drunday, Vanesa; Guillin, Eduardo A

    2014-11-01

    Alternaria alternata, A. tenuissima, Fusarium graminearum, F. semitectum, F. verticillioides, Aspergillus flavus, and Aspergillus section Nigri strains obtained from blueberries during the 2009 and 2010 harvest season from Entre Ríos, Argentina were analyzed to determine their mycotoxigenic potential. Taxonomy status at the specific level was determined both on morphological and molecular grounds. Alternariol (AOH), alternariol monomethyl ether (AME), aflatoxins (AFs), zearalenone (ZEA), fumonisins (FBs), and ochratoxin A (OTA) were analyzed by HPLC and the trichotecenes deoxynivalenol (DON), nivalenol (NIV), HT-2 toxin (HT-2), T-2 toxin (T-2), fusarenone X (FUS-X), 3-acetyl-deoxynivalenol (3-AcDON), and 15-acetyl-deoxynivalenol (15-AcDON) by GC. Twenty-five out of forty two strains were able to produce some of the mycotoxins analyzed. Fifteen strains of Aspergillus section Nigri were capable of producing Fumonisin B1 (FB1); two of them also produced Fumonisin B2 (FB2) and one Fumonisin B3 (FB3). One of the F. graminearum isolated produced ZEA, HT-2, and T-2 and the other one was capable of producing ZEA and DON. Two A. alternata isolates produced AOH and AME. Four A. tenuissima were capable of producing AOH and three of them produced AME as well. One Aspergillu flavus strain produced aflatoxin B1 (AFB1), aflatoxin B2 (AFB2), and aflatoxin G1 (AFG1). To our knowledge, this is the first report showing mycotoxigenic capacity of fungal species isolated from blueberries that include other fungi than Alternaria spp. PMID:25098914

  9. Culture-adapted Plasmodium falciparum isolates from UK travellers: in vitro drug sensitivity, clonality and drug resistance markers

    PubMed Central

    2013-01-01

    Background The screening of lead compounds against in vitro parasite cultures is an essential step in the development of novel anti-malarial drugs, but currently relies on laboratory parasite lines established in vitro during the last century. This study sought to establish in continuous culture a series of recent Plasmodium falciparum isolates to represent the current parasite populations in Africa, all of which are now exposed to artemisinin combination therapy. Methods Pre-treatment P. falciparum isolates were obtained in EDTA, and placed into continuous culture after sampling of DNA. One post-treatment blood sample was also collected for each donor to monitor parasite clonality during clearance in vivo. IC50 estimates were obtained for 11 anti-malarial compounds for each established parasite line, clonal multiplicity measured in vivo and in vitro, and polymorphic sites implicated in parasite sensitivity to drugs were investigated at the pfmdr1, pfcrt, pfdhfr, pfdhps and pfap2mu loci before and after treatment, and in the cultured lines. Results Plasmodium falciparum isolates from seven malaria patients with recent travel to three West African and two East African countries were successfully established in long-term culture. One of these, HL1211, was from a patient with recrudescent parasitaemia 14 days after a full course of artemether-lumefantrine. All established culture lines were shown to be polyclonal, reflecting the in vivo isolates from which they were derived, and at least two lines reliably produce gametocytes in vitro. Two lines displayed high chloroquine IC50 estimates, and carried the CVIET haplotype at codons 72–76, whereas the remaining five lines carried the CVMNK haplotype and were sensitive in vitro. All were sensitive to the endoperoxides dihydroartemisinin and OZ277, but IC50 estimates for lumefantrine varied, with the least sensitive parasites carrying pfmdr1 alleles encoding Asn at codon 86. Conclusions This study describes the

  10. The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture.

    PubMed

    Yeda, Redemptah; Ingasia, Luicer A; Cheruiyot, Agnes C; Okudo, Charles; Chebon, Lorna J; Cheruiyot, Jelagat; Akala, Hoseah M; Kamau, Edwin

    2016-01-01

    The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24-48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long

  11. The Genotypic and Phenotypic Stability of Plasmodium falciparum Field Isolates in Continuous In Vitro Culture

    PubMed Central

    Yeda, Redemptah; Ingasia, Luicer A.; Cheruiyot, Agnes C.; Okudo, Charles; Chebon, Lorna J.; Cheruiyot, Jelagat; Akala, Hoseah M.; Kamau, Edwin

    2016-01-01

    The Plasmodium falciparum in vitro culture system is critical for genotypic and phenotypic analyses of the parasites. For genotypic analysis, the genomic DNA can be obtained directly from the patient blood sample or from culture adapted parasites whereas for phenotypic analysis, immediate ex vivo or in vitro culture adapted parasites are used. However, parasite biology studies have not investigated whether culture adaptation process affects genotypic and/or phenotypic characteristics of the parasites in short- or long-term cultures. Here, we set out to study the dynamics and stability of parasite genetic and phenotypic profiles as field isolate parasites were adapted in continuous cultures. Parasites collected from three different patients presenting with uncomplicated malaria were adapted and maintained in drug-free continuous cultures. Aliquots from the continuous cultures were collected every 24–48 hours for analyses. Each aliquot was treated as a separate parasite sample. For genetic analysis, microsatellite (MS) typing and single nucleotide polymorphism (SNP) analyses of 23 drug resistance markers were done. The 50% inhibitory concentrations (IC50) for some of the samples were also established for four antimalarial drugs. Samples from each patient (parasite-line) were compared as they were passed through the continuous culture. Data revealed genotypic and phenotypic profiles for the three parasite-lines fluctuated from one generation to the next with no specific pattern or periodicity. With few exceptions, multilocus analysis revealed samples from each parasite-line had high genetic diversity with unique haplotypes. Interestingly, changes in MS and SNP profiles occurred simultaneously. The difference in the IC50s of samples in each parasite-line reached statistical significance. However, phenotypic changes did not correspond or correlate to genotypic changes. Our study revealed parasite genetic and phenotypic characteristics fluctuates in short- and long

  12. Temporal expression and localization patterns of variant surface antigens in clinical Plasmodium falciparum isolates during erythrocyte schizogony.

    PubMed

    Bachmann, Anna; Petter, Michaela; Tilly, Ann-Kathrin; Biller, Laura; Uliczka, Karin A; Duffy, Michael F; Tannich, Egbert; Bruchhaus, Iris

    2012-01-01

    Avoidance of antibody-mediated immune recognition allows parasites to establish chronic infections and enhances opportunities for transmission. The human malaria parasite Plasmodium falciparum possesses a number of multi-copy gene families, including var, rif, stevor and pfmc-2tm, which encode variant antigens believed to be expressed on the surfaces of infected erythrocytes. However, most studies of these antigens are based on in vitro analyses of culture-adapted isolates, most commonly the laboratory strain 3D7, and thus may not be representative of the unique challenges encountered by P. falciparum in the human host. To investigate the expression of the var, rif-A, rif-B, stevor and pfmc-2tm family genes under conditions that mimic more closely the natural course of infection, ex vivo clinical P. falciparum isolates were analyzed using a novel quantitative real-time PCR approach. Expression patterns in the clinical isolates at various time points during the first intraerythrocytic developmental cycle in vitro were compared to those of strain 3D7. In the clinical isolates, in contrast to strain 3D7, there was a peak of expression of the multi-copy gene families rif-A, stevor and pfmc-2tm at the young ring stage, in addition to the already known expression peak in trophozoites. Furthermore, most of the variant surface antigen families were overexpressed in the clinical isolates relative to 3D7, with the exception of the pfmc-2tm family, expression of which was higher in 3D7 parasites. Immunofluorescence analyses performed in parallel revealed two stage-dependent localization patterns of RIFIN, STEVOR and PfMC-2TM. Proteins were exported into the infected erythrocyte at the young trophozoite stage, whereas they remained inside the parasite membrane during schizont stage and were subsequently observed in different compartments in the merozoite. These results reveal a complex pattern of expression of P. falciparum multi-copy gene families during clinical progression

  13. Refrigeration provides a simple means to synchronize in vitro cultures of Plasmodium falciparum.

    PubMed

    Yuan, Lili; Hao, Mingming; Wu, Lanou; Zhao, Zhen; Rosenthal, Benjamin M; Li, Xiaomei; He, Yongshu; Sun, Ling; Feng, Guohua; Xiang, Zheng; Cui, Liwang; Yang, Zhaoqing

    2014-05-01

    Plasmodium falciparum is usually asynchronous during in vitro culture. Highly synchronized cultures of P. falciparum are routinely used in malaria research. Here, we describe a simple synchronization procedure for P. falciparum asexual erythrocytic culture, which involves storage at 4°C for 8-24 h followed by routine culture. When cultures with 27-60% of ring stage were synchronized using this procedure, 70-93% ring stages were obtained after 48 h of culture and relative growth synchrony remained for at least two erythrocytic cycles. To test the suitability of this procedure for subsequent work, drug sensitivity assays were performed using four laboratory strains and four freshly adapted clinical P. falciparum isolates. Parasites synchronized by sorbitol treatment or refrigeration showed similar dose-response curves and comparable IC50 values to four antimalarial drugs. The refrigeration synchronization method is simple, inexpensive, time-saving, and should be especially useful when large numbers of P. falciparum culture are handled. PMID:24632190

  14. Plasmodium falciparum Isolates in India Exhibit a Progressive Increase in Mutations Associated with Sulfadoxine-Pyrimethamine Resistance

    PubMed Central

    Ahmed, Anwar; Bararia, Deepak; Vinayak, Sumiti; Yameen, Mohammed; Biswas, Sukla; Dev, Vas; Kumar, Ashwani; Ansari, Musharraf A.; Sharma, Yagya D.

    2004-01-01

    The combination of sulfadoxine-pyrimethamine (SP) is used as a second line of therapy for the treatment of uncomplicated chloroquine-resistant Plasmodium falciparum malaria. Resistance to SP arises due to certain point mutations in the genes for the dihydrofolate reductase (DHFR) and dihydropteroate synthetase (DHPS) enzymes of the parasite. We have analyzed these mutations in 312 field isolates of P. falciparum collected from different parts of India to assess the effects of drug pressure. The rate of mutation in the gene for DHFR was found to be higher than that in the gene for DHPS, although the latter had mutations in more alleles. There was a temporal rise in the number of isolates with double dhfr mutations and single dhps mutations, resulting in an increased total number of mutations in the loci for DHFR and DHPS combined over a 5-year period. During these 5 years, the number of isolates with drug-sensitive genotypes decreased and the number of isolates with drug-resistant genotypes (double DHFR mutations and a single DHPS mutation) increased significantly. The number of isolates with the triple mutations in each of the genes for the two enzymes (for a total of six mutations), however, remained very low, coinciding with the very low rate of SP treatment failure in the country. There was a regional bias in the mutation rate, as isolates from the northeastern region (the state of Assam) showed higher rates of mutation and more complex genotypes than isolates from the other regions. It was concluded that even though SP is prescribed as a second line of treatment in India, the mutations associated with SP resistance continue to be progressively increasing. PMID:14982779

  15. Detection and isolation of Toxoplasma gondii from fresh semen of naturally infected dogs in Southern Brazil.

    PubMed

    Koch, M O; Weiss, R R; Cruz, A A; Soccol, V T; Gonçalves, K A; Bertol, Maf; Beltrame, O C; Dittrich, R L

    2016-08-01

    The aim of this study was to isolate Toxoplasma gondii and determine the viability of the parasite in fresh semen samples of clinically healthy adult dogs naturally infected. Eleven seropositive dogs with T. gondii IgG antibodies from southern Brazil were selected to confirm the presence and viability of T. gondii in fresh semen samples using in vitro isolation in Vero cell culture, polymerase chain reaction (PCR) and sequencing analysis. The presence of viable T. gondii was confirmed by in vitro isolation and PCR in five semen samples. The ITS1 region of the isolated protozoa (TG S4) was amplified and sequenced. The nucleotide sequence obtained was 99% compatible with the T. gondii DNA sequences stored in the GenBank. It has been shown that T. gondii tachyzoites may be isolated in vitro from fresh semen samples of clinically healthy dogs seropositive for T. gondii. PMID:27287987

  16. Pfhrp2 and pfhrp3 polymorphisms in Plasmodium falciparum isolates from Dakar, Senegal: impact on rapid malaria diagnostic tests

    PubMed Central

    2013-01-01

    Background An accurate diagnosis is essential for the rapid and appropriate treatment of malaria. The accuracy of the histidine-rich protein 2 (PfHRP2)-based rapid diagnostic test (RDT) Palutop+4® was assessed here. One possible factor contributing to the failure to detect malaria by this test is the diversity of the parasite PfHRP2 antigens. Methods PfHRP2 detection with the Palutop+4® RDT was carried out. The pfhrp2 and pfhrp3 genes were amplified and sequenced from 136 isolates of Plasmodium falciparum that were collected in Dakar, Senegal from 2009 to 2011. The DNA sequences were determined and statistical analyses of the variation observed between these two genes were conducted. The potential impact of PfHRP2 and PfHRP3 sequence variation on malaria diagnosis was examined. Results Seven P. falciparum isolates (5.9% of the total isolates, regardless of the parasitaemia; 10.7% of the isolates with parasitaemia ≤0.005% or ≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT. Low parasite density is not sufficient to explain the PfHRP2 detection failure. Three of these seven samples showed pfhrp2 deletion (2.4%). The pfhrp3 gene was deleted in 12.8%. Of the 122 PfHRP2 sequences, 120 unique sequences were identified. Of the 109 PfHRP3 sequences, 64 unique sequences were identified. Using the Baker’s regression model, at least 7.4% of the P. falciparum isolates in Dakar were likely to be undetected by PfHRP2 at a parasite density of ≤250 parasites/μl (slightly lower than the evaluated prevalence of 10.7%). This predictive prevalence increased significantly between 2009 and 2011 (P = 0.0046). Conclusion In the present work, 10.7% of the isolates with a parasitaemia ≤0.005% (≤250 parasites/μl) were undetected by the PfHRP2 Palutop+4® RDT (7.4% by the predictive Baker’model). In addition, all of the parasites with pfhrp2 deletion (2.4% of the total samples) and 2.1% of the parasites with parasitaemia >0.005% and presence of pfhrp2 were

  17. Genetic diversity and multiplicity of infection of Plasmodium falciparum isolates from Kolkata, West Bengal, India.

    PubMed

    Saha, Pabitra; Ganguly, Swagata; Maji, Ardhendu K

    2016-09-01

    The study of genetic diversity of Plasmodium falciparum is necessary to understand the distribution and dynamics of parasite populations. The genetic diversity of P. falciparum merozoite surface protein-1 and 2 has been extensively studied from different parts of world. However, limited data are available from India. This study was aimed to determine the genetic diversity and multiplicity of infection (MOI) of P. falciparum population in Kolkata, West Bengal, India. A total of 80day-zero blood samples from Kolkata were collected during a therapeutic efficacy study in 2008-2009. DNA was extracted; allelic frequency and diversity were investigated by PCR-genotyping method for msp1 and msp2 gene and fragment sizing was done by Bio-Rad Gel-Doc system using Image Lab (version 4.1) software. P. falciparum msp1 and msp2 markers were highly polymorphic with low allele frequencies. In Kolkata, 27 msp1 different genotypes (including 11of K1, 6 of MAD20 and 10 of Ro33 allelic families) and 30 different msp2 genotypes (of which 17 and 13 belonged to the FC27 and 3D7 allelic families, respectively) were recorded. The majority of these genotypes occurred at a frequency below 10%. The mean MOI for msp1 and msp2 gene were 2.05 and 3.72, respectively. The P. falciparum population of Kolkata was genetically diverse. As the frequencies of most of the msp1 and msp2 alleles were low, the probability of new infection with genotype identical to that in pretreatment infection was very rare. This information will serve as baseline data for evaluation of malaria control interventions as well as for monitoring the parasite population structure. PMID:27259367

  18. Rapid isolation of DNA from fresh and preserved fish scales for polymerase chain reaction.

    PubMed

    Yue, G H; Orban, L

    2001-05-01

    We developed a simple and inexpensive method to extract DNA from fresh and preserved fish scales. The procedure is based on boiling the scales in 5% Chelex 100, followed by digestion with proteinase K and subsequent absorption of genomic DNA using silica. A single fresh scale from larger species (e.g., tilapia) or a few scales from smaller species (e.g., 4 scales from zebrafish) provide over 200 ng of DNA, enough for at least 40 polymerase chain reaction amplifications. The procedure is applicable for DNA isolation not only from fresh and ethanol-preserved scales, but also from dried and formaldehyde-treated samples, and thus might be useful for investigating specimens stored in museums and other collections. Since the removal of a few scales is a gentle means of sample collection, this technique will allow analysis of genetic diversity, mating systems, and parentage in populations of endangered or ornamental fish with minimal experimental influence. PMID:14961356

  19. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India

    PubMed Central

    Rao, Pavitra N.; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K.; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P.; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C.

    2016-01-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  20. A Method for Amplicon Deep Sequencing of Drug Resistance Genes in Plasmodium falciparum Clinical Isolates from India.

    PubMed

    Rao, Pavitra N; Uplekar, Swapna; Kayal, Sriti; Mallick, Prashant K; Bandyopadhyay, Nabamita; Kale, Sonal; Singh, Om P; Mohanty, Akshaya; Mohanty, Sanjib; Wassmer, Samuel C; Carlton, Jane M

    2016-06-01

    A major challenge to global malaria control and elimination is early detection and containment of emerging drug resistance. Next-generation sequencing (NGS) methods provide the resolution, scalability, and sensitivity required for high-throughput surveillance of molecular markers of drug resistance. We have developed an amplicon sequencing method on the Ion Torrent PGM platform for targeted resequencing of a panel of six Plasmodium falciparum genes implicated in resistance to first-line antimalarial therapy, including artemisinin combination therapy, chloroquine, and sulfadoxine-pyrimethamine. The protocol was optimized using 12 geographically diverse P. falciparum reference strains and successfully applied to multiplexed sequencing of 16 clinical isolates from India. The sequencing results from the reference strains showed 100% concordance with previously reported drug resistance-associated mutations. Single-nucleotide polymorphisms (SNPs) in clinical isolates revealed a number of known resistance-associated mutations and other nonsynonymous mutations that have not been implicated in drug resistance. SNP positions containing multiple allelic variants were used to identify three clinical samples containing mixed genotypes indicative of multiclonal infections. The amplicon sequencing protocol has been designed for the benchtop Ion Torrent PGM platform and can be operated with minimal bioinformatics infrastructure, making it ideal for use in countries that are endemic for the disease to facilitate routine large-scale surveillance of the emergence of drug resistance and to ensure continued success of the malaria treatment policy. PMID:27008882

  1. Isolation and identification of Salmonella from diarrheagenic infants and young animals, sewage waste and fresh vegetables

    PubMed Central

    Nair, Amruta; Balasaravanan, T.; Malik, S. V. S; Mohan, Vysakh; Kumar, Manesh; Vergis, Jess; Rawool, Deepak B.

    2015-01-01

    Aim: This study was carried out to determine the prevalence, distribution, and identification of Salmonella serotypes in diarrheagenic infants and young animals, including sewage waste and fresh vegetables. Materials and Methods: A total of 550 samples were processed for the isolation of Salmonella spp., using standard microbiological and biochemical tests. Further polymerase chain reaction (PCR) detection of Salmonella genus was carried out using self-designed primers targeting invA gene and thereafter identification of important serotypes namely Salmonella Enterica serovar Typhimurium, Salmonella Enterica serovar Enteritidis, Salmonella Enterica serovar Typhi was performed using published standardized multiplex PCR. Results: An overall low prevalence of 2.5% (14/550) was observed. The observed prevalence of Salmonella spp. in diarrheagenic infants was 1.2% (05/400), diarrheagenic young animals 4% (02/50), sewage waste 10% (05/50), and fresh vegetables 4% (02/50), respectively. In diarrheagenic infants, of the five Salmonella isolates identified, two were Salmonella Typhimurium, two Salmonella Enteritidis, and one was unidentified and hence designated as other Salmonella serovar. All the Salmonella isolates identified from diarrheagenic young animals and sewage waste belonged to other Salmonella serovar, whereas, of the two isolates recovered from fresh vegetables, one was identified as other Salmonella serovar, and one as Salmonella Typhimurium, respectively. Conclusion: Isolation of Salmonella spp. especially from sewage waste and fresh vegetable is a matter of great concern from public health point of view because these sources can accidentally serve as a potential vehicle for transmission of Salmonella spp. to animals and human beings. PMID:27047154

  2. Prevalence and Characteristics of Salmonella Serotypes Isolated from Fresh Produce Marketed in the United States.

    PubMed

    Reddy, Shanker P; Wang, Hua; Adams, Jennifer K; Feng, Peter C H

    2016-01-01

    Salmonella continues to rank as one of the most costly foodborne pathogens, and more illnesses are now associated with the consumption of fresh produce. The U.S. Department of Agriculture Microbiological Data Program (MDP) sampled select commodities of fresh fruit and vegetables and tested them for Salmonella, pathogenic Escherichia coli, and Listeria. The Salmonella strains isolated were further characterized by serotype, antimicrobial resistance, and pulsed-field gel electrophoresis profile. This article summarizes the Salmonella data collected by the MDP between 2002 and 2012. The results show that the rates of Salmonella prevalence ranged from absent to 0.34% in cilantro. A total of 152 isolates consisting of over 50 different serotypes were isolated from the various produce types, and the top five were Salmonella enterica serotype Cubana, S. enterica subspecies arizonae (subsp. IIIa) and diarizonae (subsp. IIIb), and S. enterica serotypes Newport, Javiana, and Infantis. Among these, Salmonella serotypes Newport and Javiana are also listed among the top five Salmonella serotypes that caused most foodborne outbreaks. Other serotypes that are frequent causes of infection, such as S. enterica serotypes Typhimurium and Enteritidis, were also found in fresh produce but were not prevalent. About 25% of the MDP samples were imported produce, including 65% of green onions, 44% of tomatoes, 42% of hot peppers, and 41% of cantaloupes. However, imported produce did not show higher numbers of Salmonella-positive samples, and in some products, like cilantro, all of the Salmonella isolates were from domestic samples. About 6.5% of the Salmonella isolates were resistant to the antimicrobial compounds tested, but no single commodity or serotype was found to be the most common carrier of resistant strains or of resistance. The pulsed-field gel electrophoresis profiles of the produce isolates showed similarities with Salmonella isolates from meat samples and from outbreaks, but

  3. Emergence of Mutations in the K13 Propeller Gene of Plasmodium falciparum Isolates from Dakar, Senegal, in 2013-2014.

    PubMed

    Boussaroque, Agathe; Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Benoit, Nicolas; Fall, Mansour; Nakoulima, Aminata; Dionne, Pierre; Fall, Kadidiatou Ba; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar; Pradines, Bruno

    2016-01-01

    The kelch 13 (K13) propeller gene is associated with artemisinin resistance. In a previous work, there were no mutations found in 138 Plasmodium falciparum isolates collected in 2012 and 2013 from patients residing in Dakar, Senegal (M. Torrentino-Madamet et al., Malar J 13:472, 2014, http://dx.doi.org/10.1186/1475-2875-13-472). However, the N554H, Q613H, and V637I mutations were identified in the propeller region of K13 in 92 (5.5%) isolates in 2013 and 2014. There were five polymorphisms identified in the Plasmodium/Apicomplexa-specific domain (K123R, N137S, N142NN/NNN, T149S, and K189T/N). PMID:26503652

  4. Emergence of Mutations in the K13 Propeller Gene of Plasmodium falciparum Isolates from Dakar, Senegal, in 2013-2014

    PubMed Central

    Boussaroque, Agathe; Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Benoit, Nicolas; Fall, Mansour; Nakoulima, Aminata; Dionne, Pierre; Fall, Kadidiatou Ba; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar

    2015-01-01

    The kelch 13 (K13) propeller gene is associated with artemisinin resistance. In a previous work, there were no mutations found in 138 Plasmodium falciparum isolates collected in 2012 and 2013 from patients residing in Dakar, Senegal (M. Torrentino-Madamet et al., Malar J 13:472, 2014, http://dx.doi.org/10.1186/1475-2875-13-472). However, the N554H, Q613H, and V637I mutations were identified in the propeller region of K13 in 92 (5.5%) isolates in 2013 and 2014. There were five polymorphisms identified in the Plasmodium/Apicomplexa-specific domain (K123R, N137S, N142NN/NNN, T149S, and K189T/N). PMID:26503652

  5. Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa

    PubMed Central

    St. Laurent, Brandyce; Miller, Becky; Burton, Timothy A.; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P.; Miotto, Olivo; Gwadz, Robert W.; Anderson, Jennifer M.; Fairhurst, Rick M.

    2015-01-01

    Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread. PMID:26485448

  6. Artemisinin-resistant Plasmodium falciparum clinical isolates can infect diverse mosquito vectors of Southeast Asia and Africa.

    PubMed

    St Laurent, Brandyce; Miller, Becky; Burton, Timothy A; Amaratunga, Chanaki; Men, Sary; Sovannaroth, Siv; Fay, Michael P; Miotto, Olivo; Gwadz, Robert W; Anderson, Jennifer M; Fairhurst, Rick M

    2015-01-01

    Artemisinin-resistant Plasmodium falciparum parasites are rapidly spreading in Southeast Asia, yet nothing is known about their transmission. This knowledge gap and the possibility that these parasites will spread to Africa endanger global efforts to eliminate malaria. Here we produce gametocytes from parasite clinical isolates that displayed artemisinin resistance in patients and in vitro, and use them to infect native and non-native mosquito vectors. We show that contemporary artemisinin-resistant isolates from Cambodia develop and produce sporozoites in two Southeast Asian vectors, Anopheles dirus and Anopheles minimus, and the major African vector, Anopheles coluzzii (formerly Anopheles gambiae M). The ability of artemisinin-resistant parasites to infect such highly diverse Anopheles species, combined with their higher gametocyte prevalence in patients, may explain the rapid expansion of these parasites in Cambodia and neighbouring countries, and further compromise efforts to prevent their global spread. PMID:26485448

  7. Comparative sequence analysis of domain I of Plasmodium falciparum apical membrane antigen 1 from Saudi Arabia and worldwide isolates.

    PubMed

    Al-Qahtani, Ahmed A; Abdel-Muhsin, Abdel-Muhsin A; Bin Dajem, Saad M; AlSheikh, Adel Ali H; Bohol, Marie Fe F; Al-Ahdal, Mohammed N; Putaporntip, Chaturong; Jongwutiwes, Somchai

    2016-04-01

    The apical membrane antigen 1 of Plasmodium falciparum (PfAMA1) plays a crucial role in erythrocyte invasion and is a target of protective antibodies. Although domain I of PfAMA1 has been considered a promising vaccine component, extensive sequence diversity in this domain could compromise an effective vaccine design. To explore the extent of sequence diversity in domain I of PfAMA1, P. falciparum-infected blood samples from Saudi Arabia collected between 2007 and 2009 were analyzed and compared with those from worldwide parasite populations. Forty-six haplotypes and a novel codon change (M190V) were found among Saudi Arabian isolates. The haplotype diversity (0.948±0.004) and nucleotide diversity (0.0191±0.0008) were comparable to those from African hyperendemic countries. Positive selection in domain I of PfAMA1 among Saudi Arabian parasite population was observed because nonsynonymous nucleotide substitutions per nonsynonymous site (dN) significantly exceeded synonymous nucleotide substitutions per synonymous site (dS) and Tajima's D and its related statistics significantly deviated from neutrality in the positive direction. Despite a relatively low prevalence of malaria in Saudi Arabia, a minimum of 17 recombination events occurred in domain I. Genetic differentiation was significant between P. falciparum in Saudi Arabia and parasites from other geographic origins. Several shared or closely related haplotypes were found among parasites from different geographic areas, suggesting that vaccine derived from multiple shared epitopes could be effective across endemic countries. PMID:26867816

  8. Genome Sequence of Lactobacillus brevis Strain D6, Isolated from Smoked Fresh Cheese.

    PubMed

    Kant, Ravi; Uroić, Ksenija; Hynönen, Ulla; Kos, Blaženka; Šušković, Jagoda; Palva, Airi

    2016-01-01

    The autochthonousLactobacillus brevisstrain D6, isolated from smoked fresh cheese, carries a 45-kDa S-layer protein. Strain D6 has shown adhesion to extracellular matrix proteins and to Caco-2 intestinal epithelial cells, as well as immunomodulatory potential and beneficial milk technological properties. Hence, it could be used as a potential probiotic starter culture for cheese production. PMID:27056237

  9. Genome Sequence of Lactobacillus brevis Strain D6, Isolated from Smoked Fresh Cheese

    PubMed Central

    Uroić, Ksenija; Hynönen, Ulla; Kos, Blaženka; Šušković, Jagoda

    2016-01-01

    The autochthonous Lactobacillus brevis strain D6, isolated from smoked fresh cheese, carries a 45-kDa S-layer protein. Strain D6 has shown adhesion to extracellular matrix proteins and to Caco-2 intestinal epithelial cells, as well as immunomodulatory potential and beneficial milk technological properties. Hence, it could be used as a potential probiotic starter culture for cheese production. PMID:27056237

  10. Sodium and potassium currents in freshly isolated and in proliferating human muscle satellite cells.

    PubMed Central

    Hamann, M; Widmer, H; Baroffio, A; Aubry, J P; Krause, R M; Kaelin, A; Bader, C R

    1994-01-01

    1. Human muscle satellite cells (SC) were studied either immediately after dissociation of muscle biopsies or later, as they proliferated in culture. A purification procedure combined with clonal cultures ensured that electrophysiological recordings were done in myogenic cells. Hoechst staining for the DNA attested that cells were mononucleated. 2. The goals of this study were to examine (i) whether the electrophysiological properties of freshly isolated SC resembled those of SC that proliferated in culture for several weeks, (ii) whether freezing and thawing affected these properties, and (iii) whether SC constituted a homogeneous population. 3. We found that there were only subtle differences between the electrophysiological results obtained in freshly isolated SC and in proliferating SC with or without previous freezing and thawing. Most SC expressed two voltage-gated currents, a TTX-resistant Na+ current and a calcium-activated potassium current (IK, Ca). 4. The level of expression of the Na+ current and of IK, Ca was affected in a different way by cellular proliferation; the normalized Na+ conductance (pS pF-1) of proliferating cells resembled that of freshly isolated SC, whereas the IK, Ca conductance increased 10 times. The analysis of the amplitude distributions of the Na+ current and of IK, Ca in the various SC preparations suggested that there was only one class of SC. PMID:8021836

  11. Antimicrobial Susceptibility of Escherichia coli Isolated from Fresh-Marketed Nile Tilapia (Oreochromis niloticus)

    PubMed Central

    Rocha, Rafael dos Santos; Leite, Lana Oliveira; de Sousa, Oscarina Viana; Vieira, Regine Helena Silva dos Fernandes

    2014-01-01

    The contamination of seafood by bacteria of fecal origin, especially Escherichia coli, is a widely documented sanitary problem. The objective of the present study was to isolate E. coli strains from the gills, muscle, and body surface of farmed Nile tilapias (Oreochromis niloticus) fresh-marketed in supermarkets in Fortaleza (Ceará, Brazil), to determine their susceptibility to antibiotics of different families (amikacin, gentamicin, imipenem, cephalothin, cefotaxime, ciprofloxacin, aztreonam, ampicillin, nalidixic acid, tetracycline, and sulfametoxazol-trimetoprim), and to determine the nature of resistance by plasmid curing. Forty-four strains (body surface = 25, gills = 15, muscle = 4) were isolated, all of which were susceptible to amikacin, aztreonam, cefotaxime, ciprofloxacin, gentamicin, and imipenem. Gill and body surface samples yielded 11 isolates resistant to ampicillin, tetracycline, and sulfametoxazol-trimetoprim, 4 of which of plasmidial nature. The multiple antibiotic resistance index was higher for strains isolated from body surface than from gills. The overall high antibiotic susceptibility of E. coli strains isolated from fresh-marketed tilapia was satisfactory, although the occasional finding of plasmidial resistance points to the need for close microbiological surveillance of the farming, handling, and marketing conditions of aquaculture products. PMID:24808957

  12. Implementing Patch Clamp and Live Fluorescence Microscopy to Monitor Functional Properties of Freshly Isolated PKD Epithelium.

    PubMed

    Pavlov, Tengis S; Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Pochynyuk, Oleh; Staruschenko, Alexander

    2015-01-01

    Cyst initiation and expansion during polycystic kidney disease is a complex process characterized by abnormalities in tubular cell proliferation, luminal fluid accumulation and extracellular matrix formation. Activity of ion channels and intracellular calcium signaling are key physiologic parameters which determine functions of tubular epithelium. We developed a method suitable for real-time observation of ion channels activity with patch-clamp technique and registration of intracellular Ca2+ level in epithelial monolayers freshly isolated from renal cysts. PCK rats, a genetic model of autosomal recessive polycystic kidney disease (ARPKD), were used here for ex vivo analysis of ion channels and calcium flux. Described here is a detailed step-by-step procedure designed to isolate cystic monolayers and non-dilated tubules from PCK or normal Sprague Dawley (SD) rats, and monitor single channel activity and intracellular Ca2+ dynamics. This method does not require enzymatic processing and allows analysis in a native setting of freshly isolated epithelial monolayer. Moreover, this technique is very sensitive to intracellular calcium changes and generates high resolution images for precise measurements. Finally, isolated cystic epithelium can be further used for staining with antibodies or dyes, preparation of primary cultures and purification for various biochemical assays. PMID:26381526

  13. Different strategies of energy storage in cultured and freshly isolated Symbiodinium sp.

    PubMed

    Wang, Li-Hsueh; Chen, Hung-Kai; Jhu, Chu-Sian; Cheng, Jing-O; Fang, Lee-Shing; Chen, Chii-Shiarng

    2015-12-01

    The endosymbiotic relationship between cnidarians and Symbiodinium is critical for the survival of coral reefs. In this study, we developed a protocol to rapidly and freshly separate Symbiodinium from corals and sea anemones. Furthermore, we compared these freshly-isolated Symbiodinium with cultured Symbiodinium to investigate host and Symbiodinium interaction. Clade B Symbiodinium had higher starch content and lower lipid content than those of clades C and D in both freshly isolated and cultured forms. Clade C had the highest lipid content, particularly when associated with corals. Moreover, the coral-associated Symbiodinium had higher protein content than did cultured and sea anemone-associated Symbiodinium. Regarding fatty acid composition, cultured Symbiodinium and clades B, C, and D shared similar patterns, whereas sea anemone-associated Symbiodinium had a distinct pattern compared coral-associated Symbiodinium. Specifically, the levels of monounsaturated fatty acids were lower than those of the saturated fatty acids, and the level of polyunsaturated fatty acids (PUFAs) were the highest in all examined Symbiodinium. Furthermore, PUFAs levels were higher in coral-associated Symbiodinium than in cultured Symbiodinium. These results altogether indicated that different Symbiodinium clades used different energy storage strategies, which might be modified by hosts. PMID:26987007

  14. In vitro susceptibility of Plasmodium falciparum Welch field isolates to infusions prepared from Artemisia annua L. cultivated in the Brazilian Amazon.

    PubMed

    Silva, Luiz Francisco Rocha e; Magalhães, Pedro Melillo de; Costa, Mônica Regina Farias; Alecrim, Maria das Graças Costa; Chaves, Francisco Célio Maia; Hidalgo, Ari de Freitas; Pohlit, Adrian Martin; Vieira, Pedro Paulo Ribeiro

    2012-11-01

    Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions. PMID:23147140

  15. Isolation and functional characterization of the PfNT1 nucleoside transporter gene from Plasmodium falciparum.

    PubMed

    Carter, N S; Ben Mamoun, C; Liu, W; Silva, E O; Landfear, S M; Goldberg, D E; Ullman, B

    2000-04-01

    Plasmodium falciparum, the causative agent of the most lethal form of human malaria, is incapable of de novo purine synthesis, and thus, purine acquisition from the host is an indispensable nutritional requirement. This purine salvage process is initiated by the transport of preformed purines into the parasite. We have identified a gene encoding a nucleoside transporter from P. falciparum, PfNT1, and analyzed its function and expression during intraerythrocytic parasite development. PfNT1 predicts a polypeptide of 422 amino acids with 11 transmembrane domains that is homologous to other members of the equilibrative nucleoside transporter family. Southern analysis and BLAST searching of The Institute for Genomic Research (TIGR) malaria data base indicate that PfNT1 is a single copy gene located on chromosome 14. Northern analysis of RNA from intraerythrocytic stages of the parasite demonstrates that PfNT1 is expressed throughout the asexual life cycle but is significantly elevated during the early trophozoite stage. Functional expression of PfNT1 in Xenopus laevis oocytes significantly increases their ability to take up naturally occurring D-adenosine (K(m) = 13.2 microM) and D-inosine (K(m) = 253 microM). Significantly, PfNT1, unlike the mammalian nucleoside transporters, also has the capacity to transport the stereoisomer L-adenosine (K(m) > 500 microM). Inhibition studies with a battery of purine and pyrimidine nucleosides and bases as well as their analogs indicate that PfNT1 exhibits a broad substrate specificity for purine and pyrimidine nucleosides. These data provide compelling evidence that PfNT1 encodes a functional purine/pyrimidine nucleoside transporter whose expression is strongly developmentally regulated in the asexual stages of the P. falciparum life cycle. Moreover, the unusual ability to transport L-adenosine and the vital contribution of purine transport to parasite survival makes PfNT1 an attractive target for therapeutic evaluation. PMID

  16. Isolation and characterization of Arcobacter spp. from fresh seafood and the aquatic environment.

    PubMed

    Laishram, Martina; Rathlavath, Srinu; Lekshmi, Manjusha; Kumar, Sanath; Nayak, Binaya Bhusan

    2016-09-01

    Arcobacter is an emerging pathogen associated with foods of animal origin. Members of the genus Arcobacter are increasingly being isolated from fish, shellfish and the aquatic environment. In the present study, we analyzed fish, shellfish and water samples for the presence of Arcobacter spp. by conventional isolation as well as by direct PCR on the enrichment broth. Of 100 samples comprising of 42 finfish, 34 shellfish and 24 water samples analyzed, Arcobacter spp. was isolated from 8 (19%) finfish, 5 (14.7%) shellfish and 5 (20.8%) water samples. Arcobacter DNA was detected in 24 (24%) samples by direct PCR on the enrichment broth. Based on m-PCR specific to different Arcobacter spp. and 16S rRNA sequence analyses, majority (19) of the isolates were identified as Arcobacter butzleri, while two isolates were Arcobacter mytili. All Arcobacter butzleri isolates harbored putative virulence genes cadF, ciaB, mviN, pldA, tlyA and cj1349. The two isolates of A. mytili harbored mviN and cj1349 genes only. The study highlights emerging problem of the contamination of aquatic environment and fresh seafood with potentially pathogenic Arcobacter spp. PMID:27261768

  17. Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data.

    PubMed

    O'Brien, John D; Iqbal, Zamin; Wendler, Jason; Amenga-Etego, Lucas

    2016-06-01

    We present a rigorous statistical model that infers the structure of P. falciparum mixtures-including the number of strains present, their proportion within the samples, and the amount of unexplained mixture-using whole genome sequence (WGS) data. Applied to simulation data, artificial laboratory mixtures, and field samples, the model provides reasonable inference with as few as 10 reads or 50 SNPs and works efficiently even with much larger data sets. Source code and example data for the model are provided in an open source fashion. We discuss the possible uses of this model as a window into within-host selection for clinical and epidemiological studies. PMID:27362949

  18. Inferring Strain Mixture within Clinical Plasmodium falciparum Isolates from Genomic Sequence Data

    PubMed Central

    O’Brien, John D.; Amenga-Etego, Lucas

    2016-01-01

    We present a rigorous statistical model that infers the structure of P. falciparum mixtures—including the number of strains present, their proportion within the samples, and the amount of unexplained mixture—using whole genome sequence (WGS) data. Applied to simulation data, artificial laboratory mixtures, and field samples, the model provides reasonable inference with as few as 10 reads or 50 SNPs and works efficiently even with much larger data sets. Source code and example data for the model are provided in an open source fashion. We discuss the possible uses of this model as a window into within-host selection for clinical and epidemiological studies. PMID:27362949

  19. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance.

    PubMed

    Chaorattanakawee, Suwanna; Saunders, David L; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap; Lanteri, Charlotte A

    2015-08-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  20. Ex Vivo Drug Susceptibility Testing and Molecular Profiling of Clinical Plasmodium falciparum Isolates from Cambodia from 2008 to 2013 Suggest Emerging Piperaquine Resistance

    PubMed Central

    Chaorattanakawee, Suwanna; Saunders, David L.; Sea, Darapiseth; Chanarat, Nitima; Yingyuen, Kritsanai; Sundrakes, Siratchana; Saingam, Piyaporn; Buathong, Nillawan; Sriwichai, Sabaithip; Chann, Soklyda; Se, Youry; Yom, You; Heng, Thay Kheng; Kong, Nareth; Kuntawunginn, Worachet; Tangthongchaiwiriya, Kuntida; Jacob, Christopher; Takala-Harrison, Shannon; Plowe, Christopher; Lin, Jessica T.; Chuor, Char Meng; Prom, Satharath; Tyner, Stuart D.; Gosi, Panita; Teja-Isavadharm, Paktiya; Lon, Chanthap

    2015-01-01

    Cambodia's first-line artemisinin combination therapy, dihydroartemisinin-piperaquine (DHA-PPQ), is no longer sufficiently curative against multidrug-resistant Plasmodium falciparum malaria at some Thai-Cambodian border regions. We report recent (2008 to 2013) drug resistance trends in 753 isolates from northern, western, and southern Cambodia by surveying for ex vivo drug susceptibility and molecular drug resistance markers to guide the selection of an effective alternative to DHA-PPQ. Over the last 3 study years, PPQ susceptibility declined dramatically (geomean 50% inhibitory concentration [IC50] increased from 12.8 to 29.6 nM), while mefloquine (MQ) sensitivity doubled (67.1 to 26 nM) in northern Cambodia. These changes in drug susceptibility were significantly associated with a decreased prevalence of P. falciparum multidrug resistance 1 gene (Pfmdr1) multiple copy isolates and coincided with the timing of replacing artesunate-mefloquine (AS-MQ) with DHA-PPQ as the first-line therapy. Widespread chloroquine resistance was suggested by all isolates being of the P. falciparum chloroquine resistance transporter gene CVIET haplotype. Nearly all isolates collected from the most recent years had P. falciparum kelch13 mutations, indicative of artemisinin resistance. Ex vivo bioassay measurements of antimalarial activity in plasma indicated 20% of patients recently took antimalarials, and their plasma had activity (median of 49.8 nM DHA equivalents) suggestive of substantial in vivo drug pressure. Overall, our findings suggest DHA-PPQ failures are associated with emerging PPQ resistance in a background of artemisinin resistance. The observed connection between drug policy changes and significant reduction in PPQ susceptibility with mitigation of MQ resistance supports reintroduction of AS-MQ, in conjunction with monitoring of the P. falciparum mdr1 copy number, as a stop-gap measure in areas of DHA-PPQ failure. PMID:26014942

  1. Permissiveness of freshly isolated environmental strains of amoebae for growth of Legionella pneumophila.

    PubMed

    Dupuy, Mathieu; Binet, Marie; Bouteleux, Celine; Herbelin, Pascaline; Soreau, Sylvie; Héchard, Yann

    2016-03-01

    Legionella pneumophila is a pathogenic bacterium commonly found in water and responsible for severe pneumonia. Free-living amoebae are protozoa also found in water, which feed on bacteria by phagocytosis. Under favorable conditions, some L. pneumophila are able to resist phagocytic digestion and even multiply within amoebae. However, it is not clear whether L. pneumophila could infect at a same rate a large range of amoebae or if there is some selectivity towards specific amoebal genera or strains. Also, most studies have been performed using collection strains and not with freshly isolated strains. In our study, we assess the permissiveness of freshly isolated environmental strains of amoebae, belonging to three common genera (i.e. Acanthamoeba, Naegleria and Vermamoeba), for growth of L. pneumophila at three different temperatures. Our results indicated that all the tested strains of amoebae were permissive to L. pneumophila Lens and that there was no significant difference between the strains. Intracellular proliferation was more efficient at a temperature of 40°C. In conclusion, our work suggests that, under favorable conditions, virulent strains of L. pneumophila could equally infect a large number of isolates of common freshwater amoeba genera. PMID:26832643

  2. Antiplasmodial properties of kaempferol-3-O-rhamnoside isolated from the leaves of Schima wallichii against chloroquine-resistant Plasmodium falciparum

    PubMed Central

    BARLIANA, MELISA I.; SURADJI, EKA W.; ABDULAH, RIZKY; DIANTINI, AJENG; HATABU, TOSHIMITSU; NAKAJIMA-SHIMADA, JUNKO; SUBARNAS, ANAS; KOYAMA, HIROSHI

    2014-01-01

    Previous intervention studies have shown that the most effective agents used in the treatment of malaria were isolated from natural sources. Plants consumed by non-human primates serve as potential drug sources for human disease management due to the similarities in anatomy, physiology and disease characteristics. The present study investigated the antiplasmodial properties of the primate-consumed plant, Schima wallichii (S. wallichii) Korth. (family Theaceae), which has already been reported to have several biological activities. The ethanol extract of S. wallichii was fractionated based on polarity using n-hexane, ethyl acetate and water. The antiplasmodial activity was tested in vitro against chloroquine-resistant Plasmodium falciparum (P. falciparum) at 100 μg/ml for 72 h. The major compound of the most active ethyl acetate fraction was subsequently isolated using column chromatography and identified by nuclear magnetic resonance. The characterized compound was also tested against chloroquine-resistant P. falciparum in culture to evaluate its antiplasmodial activity. The ethanol extract of S. wallichii at 100 μg/ml exhibited a significant parasite shrinkage after 24 h of treatment. The ethyl acetate fraction at 100 μg/ml was the most active fraction against chloroquine-resistant P. falciparum. Based on the structural characterization, the major compound isolated from the ethyl acetate fraction was kaempferol-3-O-rhamnoside, which showed promising antiplasmodial activity against chloroquine-resistant P. falciparum with an IC50 of 106 μM after 24 h of treatment. The present study has provided a basis for the further investigation of kaempferol-3-O-rhamnoside as an active compound for potential antimalarial therapeutics. PMID:24944812

  3. Plasmodium falciparum and Plasmodium vivax specific lactate dehydrogenase: genetic polymorphism study from Indian isolates.

    PubMed

    Keluskar, Priyadarshan; Singh, Vineeta; Gupta, Purva; Ingle, Sanjay

    2014-08-01

    Control and eradication of malaria is hindered by the acquisition of drug resistance by Plasmodium species. This has necessitated a persistent search for novel drugs and more efficient targets. Plasmodium species specific lactate dehydrogenase is one of the potential therapeutic and diagnostic targets, because of its indispensable role in endoerythrocytic stage of the parasite. A target molecule that is highly conserved in the parasite population can be more effectively used in diagnostics and therapeutics, hence, in the present study polymorphism in PfLDH (Plasmodiumfalciparum specific LDH) and PvLDH (Plasmodiumvivax specific LDH) genes was analyzed using PCR-single strand confirmation polymorphism (PCR-SSCP) and sequencing. Forty-six P. falciparum and thirty-five P. vivax samples were screened from different states of India. Our findings have revealed presence of a single PfLDH genotype and six PvLDH genotypes among the studied samples. Interestingly, along with synonymous substitutions, nonsynonymous substitutions were reported to be present for the first time in the PvLDH genotypes. Further, through amino acid sequence alignment and homology modeling studies we observed that the catalytic residues were conserved in all PvLDH genotypes and the nonsynonymous substitutions have not altered the enzyme structure significantly. Evolutionary genetics studies have confirmed that PfLDH and PvLDH loci are under strong purifying selection. Phylogenetic analysis of the pLDH gene sequences revealed that P. falciparum compared to P. vivax, has recent origin. The study therefore supports PfLDH and PvLDH as suitable therapeutic and diagnostic targets as well as phylogenetic markers to understand the genealogy of malaria species. PMID:24953504

  4. Molecular characterization of Listeria monocytogenes isolated from fresh seafood samples in Iran

    PubMed Central

    2013-01-01

    Background Among all species of Listeria, Listeria monocytogenes (L. monocytogenes) is a major pathogenic microorganism of humans and animals and L. ivanovii is rarely pathogenic for humans. The objective of this study was to isolate and characterize Listeria species and to determine the frequencies of virulence genes in L. monocytogenes serotypes in fresh fish, shrimp, crab and lobster in Isfahan and Shahrekord, Iran. Methods From September 2010 to April 2011, a total of 300 marine food samples were purchased from supermarkets of Isfahan and Shahrekord cities, Iran. All samples were cultured and the positive samples for L. monocytogenes were analyzed for presence of serotypes and virulence genes. Results From the total 300 samples, 23 (10.45%) fresh fish and 1 (2.5%) shrimp samples were positive for Listeria spp., but there were no positive lobster and crab samples for Listeria species. Only L. monocytogenes was isolated from 17 fish (7.25%) and 1 shrimp (2.5%) samples while L. innocua, L. ivanovii and L. seeligeri only detected in fish samples (2 (0.9%), 3 (1.36%) and 1 (0.45%)), respectively. The plcA, prfA, actA, hlyA and iap virulence genes were detected in all of the 18 L. monocytogenes isolates. Totally, the 4b, 1/2a and 1/2b serotypes were detected in 66.66%, 5.55% and 27.77% bacterial isolates, respectively. Conclusions Consumption of these sea foods, either raw or undercooked, may contribute to food-borne illness due to L. monocytogenes in Iran. The hygienic quality of sea food products should be observe. Virtual slides The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/3422944359800606 PMID:24033984

  5. Histamine and cadaverine production by bacteria isolated from fresh and frozen albacore (Thunnus alalunga).

    PubMed

    Ben-Gigirey, B; Vieites Baaptista de Sousa, J M; Villa, T G; Barros-Velazquez, J

    1999-08-01

    Two hundred twenty-seven bacterial strains were isolated from fresh and frozen albacore stored either at -18 or -25 degrees C and investigated for their abilities to produce biogenic amines. As a preliminary screening, all 227 strains were tested in either Niven or Niven modified medium, which allowed the selection of 25 presumptive histamine-producing strains. High-pressure liquid chromatography revealed that only 10 of the 25 strains selected were able to produce low histamine concentrations (<25 ppm) in tryptic soy broth medium supplemented with 2% histidine. None of the 25 strains tested produced putrescine or spermine, whereas 6 strains produced spermidine. Histamine production by Stenotrophomonas maltophilia strain 25MC6 was not prevented at 4 degrees C, and the levels of this amine reached concentrations of 25.8 ppm after 6 days. Three S. maltophilia strains showed strong lysine-decarboxylating activity. Their cadaverine formation capacity was determined by high-pressure liquid chromatography in tryptic soy broth supplemented with 1% lysine; this revealed that the three S. maltophilia strains tested produced more than 700 ppm of cadaverine during the first 24 h of incubation at 37 degrees C. S. maltophilia strain 15MF, initially obtained from fresh albacore tuna, produced up to 2,399 ppm and 4,820 ppm of cadaverine after 24 and 48 h of incubation at 37 degrees C, respectively. To our knowledge, this is the first report on histamine and cadaverine production by strains of the species S. maltophilia, previously known as Pseudomonas and Xanthomonas maltophilia, isolated from fresh and frozen albacore tuna. PMID:10456749

  6. Virulence Potential of Activatable Shiga Toxin 2d–Producing Escherichia coli Isolates from Fresh Produce

    PubMed Central

    Melton-Celsa, Angela R.; O'Brien, Alison D.; Feng, Peter C. H.

    2016-01-01

    Shiga toxin (Stx)–producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named “activation.” Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  7. Virulence Potential of Activatable Shiga Toxin 2d-Producing Escherichia coli Isolates from Fresh Produce.

    PubMed

    Melton-Celsa, Angela R; O'Brien, Alison D; Feng, Peter C H

    2015-11-01

    Shiga toxin (Stx)-producing Escherichia coli (STEC) strains are food- and waterborne pathogens that are often transmitted via beef products or fresh produce. STEC strains cause both sporadic infections and outbreaks, which may result in hemorrhagic colitis and hemolytic uremic syndrome. STEC strains may elaborate Stx1, Stx2, and/or subtypes of those toxins. Epidemiological evidence indicates that STEC that produce subtypes Stx2a, Stx2c, and/or Stx2d are more often associated with serious illness. The Stx2d subtype becomes more toxic to Vero cells after incubation with intestinal mucus or elastase, a process named "activation." Stx2d is not generally found in the E. coli serotypes most commonly connected to STEC outbreaks. However, STEC strains that are stx2d positive can be isolated from foods, an occurrence that gives rise to the question of whether those food isolates are potential human pathogens. In this study, we examined 14 STEC strains from fresh produce that were stx2d positive and found that they all produced the mucus-activatable Stx2d and that a subset of the strains tested were virulent in streptomycin-treated mice. PMID:26555533

  8. An Analysis of the Binding Characteristics of a Panel of Recently Selected ICAM-1 Binding Plasmodium falciparum Patient Isolates

    PubMed Central

    Madkhali, Aymen M.; Alkurbi, Mohammed O.; Szestak, Tadge; Bengtsson, Anja; Patil, Pradeep R.; Wu, Yang; Alharthi, Saeed; Jensen, Anja T. R.; Pleass, Richard; Craig, Alister G.

    2014-01-01

    The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants. PMID:25360558

  9. An analysis of the binding characteristics of a panel of recently selected ICAM-1 binding Plasmodium falciparum patient isolates.

    PubMed

    Madkhali, Aymen M; Alkurbi, Mohammed O; Szestak, Tadge; Bengtsson, Anja; Patil, Pradeep R; Wu, Yang; Al-Harthi, Saeed A; Alharthi, Saeed; Jensen, Anja T R; Pleass, Richard; Craig, Alister G

    2014-01-01

    The basis of severe malaria pathogenesis in part includes sequestration of Plasmodium falciparum-infected erythrocytes (IE) from the peripheral circulation. This phenomenon is mediated by the interaction between several endothelial receptors and one of the main parasite-derived variant antigens (PfEMP1) expressed on the surface of the infected erythrocyte membrane. One of the commonly used host receptors is ICAM-1, and it has been suggested that ICAM-1 has a role in cerebral malaria pathology, although the evidence to support this is not conclusive. The current study examined the cytoadherence patterns of lab-adapted patient isolates after selecting on ICAM-1. We investigated the binding phenotypes using variant ICAM-1 proteins including ICAM-1Ref, ICAM-1Kilifi, ICAM-1S22/A, ICAM-1L42/A and ICAM-1L44/A using static assays. The study also examined ICAM-1 blocking by four anti-ICAM-1 monoclonal antibodies (mAb) under static conditions. We also characterised the binding phenotypes using Human Dermal Microvascular Endothelial Cells (HDMEC) under flow conditions. The results show that different isolates have variant-specific binding phenotypes under both static and flow conditions, extending our previous observations that this variation might be due to variable contact residues on ICAM-1 being used by different parasite PfEMP1 variants. PMID:25360558

  10. High prevalence of sulphadoxine-pyrimethamine resistance-associated mutations in Plasmodium falciparum field isolates from pregnant women in Brazzaville, Republic of Congo.

    PubMed

    Koukouikila-Koussounda, Felix; Bakoua, Damien; Fesser, Anna; Nkombo, Michael; Vouvoungui, Christevy; Ntoumi, Francine

    2015-07-01

    Intermittent preventive treatment during pregnancy with sulfadoxine-pyrimethamine (IPTp-SP) has not been evaluated in the Republic of Congo since its implementation in 2006 and there is no published data on molecular markers of SP resistance among Plasmodium falciparum isolates from pregnant women. This first study in this country aimed to describe the prevalence of dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) point mutations and haplotypes in P. falciparum isolates collected from pregnant women with asymptomatic infection. From March 2012 to December 2013, pregnant women attending Madibou health centre (in Southern Brazzaville) for antenatal visits were enrolled in this study after obtaining their written informed consent. Blood samples were collected and P. falciparum infections were characterized using PCR. A total of 363 pregnant women were enrolled. P. falciparum infection was detected in 67 (18.4%) samples as their PCR amplification of dhfr and dhps genes yielded bands and all the PCR products were successfully digested. Out of these 67 isolates, 59 (88%), 57 (85%) and 53 (79.1%) carried 51I, 59R and 108N dhfr mutant alleles, respectively. The prevalence of dhps 436A, 437G and 540E mutations were 67.1% (45/67), 98.5% (66/67) and 55.2% (37/67), respectively. More than one-half of the isolates carried quintuple mutations, with highly resistant haplotype dhfr51I/59R/108N + dhps437G/540E detected in 33% (22/67) whereas 25% (17/67) were found to carry sextuple mutations. We observed significantly higher frequencies of triple dhps mutations 436A/437G/540E and quintuple mutations dhfr51I/59R/108N+dhps437G/540E in isolates from women who received IPTp-SP than those who did not. Overall, this study shows high prevalence rates of SP-associated resistance mutations in P. falciparum isolates collected from pregnant women. The presence of the dhps mutant allele 540E and the high prevalence of isolates carrying quintuple dhfr/dhps mutations are here

  11. Genetic Diversity of Plasmodium falciparum Field Isolates in Central Sudan Inferred by PCR Genotyping of Merozoite Surface Protein 1 and 2

    PubMed Central

    Hamid, Muzamil M Abdel; Mohammed, Sara B; El Hassan, Ibrahim M

    2013-01-01

    Background: Characterization of Plasmodium falciparum diversity is commonly achieved by amplification of the polymorphic regions of the merozoite surface proteins 1 (MSP1) and 2 (MSP2) genes. Aims: The present study aimed to determine the allelic variants distribution of MSP1 and MSP2 and multiplicity of infection in P. falciparum field isolates from Kosti, central Sudan, an area characterized by seasonal malaria transmission. Materials and Methods: Total 121 samples (N = 121) were collected during a cross-sectional survey between March and April 2003. DNA was extracted and MSP1 and MSP2 polymorphic loci were genotyped. Results: The total number of alleles identified in MSP1 block 2 was 11, while 16 alleles were observed in MSP2 block 3. In MSP1, RO33 was found to be the predominant allelic type, carried alone or in combination with MAD20 and K1 types, whereas FC27 family was the most prevalent in MSP2. Sixty two percent of isolates had multiple genotypes and the overall mean multiplicity of infection was 1.93 (CI 95% 1.66-2.20). Age correlated with parasite density (P = 0.017). In addition, a positive correlation was observed between parasite densities and the number of alleles (P = 0.022). Conclusion: Genetic diversity in P. falciparum field isolates in central Sudan was high and consisted of multiple clones. PMID:23641369

  12. Genetic Polymorphism of msp1 and msp2 in Plasmodium falciparum Isolates from Côte d'Ivoire versus Gabon

    PubMed Central

    Yavo, William; Konaté, Abibatou; Mawili-Mboumba, Denise Patricia; Kassi, Fulgence Kondo; Tshibola Mbuyi, Marie L.; Angora, Etienne Kpongbo; Menan, Eby I. Hervé; Bouyou-Akotet, Marielle K.

    2016-01-01

    Introduction. The characterization of genetic profile of Plasmodium isolates from different areas could help in better strategies for malaria elimination. This study aimed to compare P. falciparum diversity in two African countries. Methods. Isolates collected from 100 and 73 falciparum malaria infections in sites of Côte d'Ivoire (West Africa) and Gabon (Central Africa), respectively, were analyzed by a nested PCR amplification of msp1 and msp2 genes. Results. The K1 allelic family was widespread in Côte d'Ivoire (64.6%) and in Gabon (56.6%). For msp2, the 3D7 alleles were more prevalent (>70% in both countries) compared to FC27 alleles. In Côte d'Ivoire, the frequencies of multiple infections with msp1 (45.1%) and msp2 (40.3%) were higher than those found for isolates from Gabon, that is, 30.2% with msp1 and 31.4% with msp2. The overall complexity of infection was 1.66 (SD = 0.79) in Côte d'Ivoire and 1.58 (SD = 0.83) in Gabon. It decreased with age in Côte d'Ivoire in contrast to Gabon. Conclusion. Differences observed in some allelic families and in complexity profile may suggest an impact of epidemiological facies as well as immunological response on genetic variability of P. falciparum. PMID:27110390

  13. Genetic Polymorphism of msp1 and msp2 in Plasmodium falciparum Isolates from Côte d'Ivoire versus Gabon.

    PubMed

    Yavo, William; Konaté, Abibatou; Mawili-Mboumba, Denise Patricia; Kassi, Fulgence Kondo; Tshibola Mbuyi, Marie L; Angora, Etienne Kpongbo; Menan, Eby I Hervé; Bouyou-Akotet, Marielle K

    2016-01-01

    Introduction. The characterization of genetic profile of Plasmodium isolates from different areas could help in better strategies for malaria elimination. This study aimed to compare P. falciparum diversity in two African countries. Methods. Isolates collected from 100 and 73 falciparum malaria infections in sites of Côte d'Ivoire (West Africa) and Gabon (Central Africa), respectively, were analyzed by a nested PCR amplification of msp1 and msp2 genes. Results. The K1 allelic family was widespread in Côte d'Ivoire (64.6%) and in Gabon (56.6%). For msp2, the 3D7 alleles were more prevalent (>70% in both countries) compared to FC27 alleles. In Côte d'Ivoire, the frequencies of multiple infections with msp1 (45.1%) and msp2 (40.3%) were higher than those found for isolates from Gabon, that is, 30.2% with msp1 and 31.4% with msp2. The overall complexity of infection was 1.66 (SD = 0.79) in Côte d'Ivoire and 1.58 (SD = 0.83) in Gabon. It decreased with age in Côte d'Ivoire in contrast to Gabon. Conclusion. Differences observed in some allelic families and in complexity profile may suggest an impact of epidemiological facies as well as immunological response on genetic variability of P. falciparum. PMID:27110390

  14. Abnormal PfEMP1/knob display on Plasmodium falciparum-infected erythrocytes containing hemoglobin variants: fresh insights into malaria pathogenesis and protection

    PubMed Central

    Fairhurst, Rick M.; Bess, Cameron D.; Krause, Michael A.

    2012-01-01

    Hemoglobin (Hb) variants are associated with reduced risk of life-threatening Plasmodium falciparum malaria syndromes, including cerebral malaria and severe malarial anemia. Despite decades of research, the mechanisms by which common Hb variants – sickle HbS, HbC, α-thalassemia, fetal HbF – protect African children against severe and fatal malaria have not been fully elucidated. In vitro experimental and epidemiological data have long suggested that Hb variants do not confer malaria protection by restricting the growth of parasites in red blood cells (RBCs). Recently, four Hb variants were found to impair cytoadherence, the binding of P. falciparum-infected RBCs (PfRBCs) to microvascular endothelial cells (MVECs), a centrally important event in both parasite survival and malaria pathogenesis in humans. Impaired cytoadherence is associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s major cytoadherence ligand and virulence factor, on the surface of host RBCs. We propose a model in which Hb variants allow parasites to display relatively low levels of PfEMP1, sufficient for sequestering PfRBCs in microvessels and avoiding their clearance from the bloodstream by the spleen. By preventing the display of high levels of PfEMP1, Hb variants may weaken the binding of PfRBCs to MVECs, compromising their ability to activate endothelium and initiate the downstream microvascular events that drive the pathogenesis of malaria. PMID:22634344

  15. Development of a Potential Probiotic Fresh Cheese Using Two Lactobacillus salivarius Strains Isolated from Human Milk

    PubMed Central

    Cárdenas, Nivia; Peirotén, Ángela; Rodríguez, Juan M.; Fernández, Leónides

    2014-01-01

    Cheeses have been proposed as a good alternative to other fermented milk products for the delivery of probiotic bacteria to the consumer. The objective of this study was to assess the survival of two Lactobacillus salivarius strains (CECT5713 and PS2) isolated from human milk during production and storage of fresh cheese for 28 days at 4°C. The effect of such strains on the volatile compounds profile, texture, and other sensorial properties, including an overall consumer acceptance, was also investigated. Both L. salivarius strains remained viable in the cheeses throughout the storage period and a significant reduction in their viable counts was only observed after 21 days. Globally, the addition of the L. salivarius strains did not change significantly neither the chemical composition of the cheese nor texture parameters after the storage period, although cheeses manufactured with L. salivarius CECT5713 presented significantly higher values of hardness. A total of 59 volatile compounds were identified in the headspace of experimental cheeses, and some L. salivarius-associated differences could be identified. All cheeses presented good results of acceptance after the sensory evaluation. Consequently, our results indicated that fresh cheese can be a good vehicle for the two L. salivarius strains analyzed in this study. PMID:24971351

  16. Potent Ex Vivo Activity of Naphthoquine and Methylene Blue against Drug-Resistant Clinical Isolates of Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Wirjanata, Grennady; Sebayang, Boni F; Chalfein, Ferryanto; Prayoga; Handayuni, Irene; Trianty, Leily; Kenangalem, Enny; Noviyanti, Rintis; Campo, Brice; Poespoprodjo, Jeanne Rini; Möhrle, Jörg J; Price, Ric N; Marfurt, Jutta

    2015-10-01

    The 4-aminoquinoline naphthoquine (NQ) and the thiazine dye methylene blue (MB) have potent in vitro efficacies against Plasmodium falciparum, but susceptibility data for P. vivax are limited. The species- and stage-specific ex vivo activities of NQ and MB were assessed using a modified schizont maturation assay on clinical field isolates from Papua, Indonesia, where multidrug-resistant P. falciparum and P. vivax are prevalent. Both compounds were highly active against P. falciparum (median [range] 50% inhibitory concentration [IC50]: NQ, 8.0 nM [2.6 to 71.8 nM]; and MB, 1.6 nM [0.2 to 7.0 nM]) and P. vivax (NQ, 7.8 nM [1.5 to 34.2 nM]; and MB, 1.2 nM [0.4 to 4.3 nM]). Stage-specific drug susceptibility assays revealed significantly greater IC50s in parasites exposed at the trophozoite stage than at the ring stage for NQ in P. falciparum (26.5 versus 5.1 nM, P = 0.021) and P. vivax (341.6 versus 6.5 nM, P = 0.021) and for MB in P. vivax (10.1 versus 1.6 nM, P = 0.010). The excellent ex vivo activities of NQ and MB against both P. falciparum and P. vivax highlight their potential utility for the treatment of multidrug-resistant malaria in areas where both species are endemic. PMID:26195523

  17. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca2+ concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  18. Single-channel Analysis and Calcium Imaging in the Podocytes of the Freshly Isolated Glomeruli.

    PubMed

    Ilatovskaya, Daria V; Palygin, Oleg; Levchenko, Vladislav; Staruschenko, Alexander

    2015-01-01

    Podocytes (renal glomerular epithelial cells) are known to regulate glomerular permeability and maintain glomerular structure; a key role for these cells in the pathogenesis of various renal diseases has been established since podocyte injury leads to proteinuria and foot process effacement. It was previously reported that various endogenous agents may cause a dramatic overload in intracellular Ca(2+) concentration in podocytes, presumably leading to albuminuria, and this likely occurs via calcium-conducting ion channels. Therefore, it appeared important to study calcium handling in the podocytes both under normal conditions and in various pathological states. However, available experimental approaches have remained somewhat limited to cultured and transfected cells. Although they represent a good basic model for such studies, they are essentially extracted from the native environment of the glomerulus. Here we describe the methodology of studying podocytes as a part of the freshly isolated whole glomerulus. This preparation retains the functional potential of the podocytes, which are still attached to the capillaries; therefore, podocytes remain in the environment that conserves the major parts of the glomeruli filtration apparatus. The present manuscript elaborates on two experimental approaches that allow 1) real-time detection of calcium concentration changes with the help of ratiometric confocal fluorescence microscopy, and 2) the recording of the single ion channels activity in the podocytes of the freshly isolated glomeruli. These methodologies utilize the advantages of the native environment of the glomerulus that enable researchers to resolve acute changes in the intracellular calcium handling in response to applications of various agents, measure basal concentration of calcium within the cells (for instance, to evaluate disease progression), and assess and manipulate calcium conductance at the level of single ion channels. PMID:26167808

  19. Emergence of sulfadoxine-pyrimethamine resistance in Indian isolates of Plasmodium falciparum in the last two decades.

    PubMed

    Kumar, Amit; Moirangthem, Romilla; Gahlawat, Suresh Kumar; Chandra, Jagdish; Gupta, Purva; Valecha, Neena; Anvikar, Anup; Singh, Vineeta

    2015-12-01

    Genotyping the sulfadoxine-pyrimethamine (SP) genes will help in identifying the genes under drug selection and the emergence of resistance in dhfr and dhps genes. India is an important hotspot for studying malaria due to the immense climatic diversity prevalent in the country. The central and eastern parts of the country are most vulnerable sites where malaria cases are reported throughout the year. From different regions of the country 173 field isolates were genotyped at various loci in dhfr and dhps genes collected between 1994 and 2013. This encompasses the period before antimalarial resistance emerged and the period after the use of combination therapy was made mandatory in the country. We observed the rise of resistant SP alleles from very low frequencies (in the year 1994) to steadily rising (in the year 2000) and maintaining this increasing trend subsequently (in the year 2013) as shown by the sequence analysis of dhfr and dhps genes. This study assessed the prevalence of mutations in dhfr and dhps genes associated with SP resistance in samples indicative of increase in resistance levels of Plasmodium falciparum to SP even after the change in malaria treatment policy in the country. PMID:26319997

  20. In vitro Amodiaquine Resistance and its Association with Mutations in pfcrt and pfmdr1 genes of Plasmodium falciparum isolates from Nigeria

    PubMed Central

    Folarin, O.A.; Bustamante, C; Gbotosho, G.O.; Sowunmi, A.; Zalis, M.G.; Oduola, A.M.J.; Happi, C.T.

    2011-01-01

    Amodiaquine (AQ) is currently being used as a partner drug in combination with artesunate for treatment of uncomplicated malaria in most endemic countries of Africa. In the absence of molecular markers of artemisinin resistance, molecular markers of resistance to AQ may be useful for monitoring the development and spread of parasites resistance to Artesunate-Amodiaquine combination. This study was designed to assess the potential role of polymorphisms on pfcrt and pfmdr1 genes and parasite in vitro susceptibility for epidemiological surveillance of amodiaquine resistance in Plasmodium falciparum. The modified schizont inhibition assay was used to determine in vitro susceptibility profiles of 98 patients' isolates of Plasmodium falciparum to amodiaquine. Polymorphisms on parasites pfcrt and pfmdr1 genes were determined with nested PCR followed by sequencing. The geometric mean (GM) of AQ 50% inhibitory concentration (IC-50) in the 97 P. falciparum isolates was 20.48nM (95% CI 16.53–25.36nM). Based on the cut-off value for AQ in vitro susceptibility, 87% (84) of the P. falciparum isolates were sensitive to AQ (GM IC-50= 16.32nM; 95%CI 13.3–20.04nM) while 13% were resistant to AQ in vitro (GM IC-50= 88.73nM; 95%CI 69.67–113.0nM). Molecular analysis showed presence of mutant CVIET pfcrt haplotype, mutant pfmdr1Tyr86 allele and the double mutant CVIET pfcrt haplotype+pfmdr1Tyr86 in 72%, 49% and 35% respectively. The GM IC-50 of isolates harboring the wild-type pfcrt CVMNK haplotype+ pfmdr1Asn86 allele (3.93nM; 95%CI 1.82–8.46) was significantly lower (p=0.001) than those isolates harboring the double mutant pfcrtCVIET haplotype+pfmdr1Tyr86 allele (50.40nM; 95%CI 40.17–63.24). Results from this study suggest that polymorphisms in pfcrt and pfmdr1 genes are important for AQ resistance and therefore may be useful for epidemiological surveillance of P. falciparum resistance to AQ. PMID:21920347

  1. Pyridoxal 5'-phosphate is an ATP-receptor antagonist in freshly isolated rat cardiomyocytes.

    PubMed

    Wang, X; Dakshinamurti, K; Musat, S; Dhalla, N S

    1999-05-01

    Although extracellular ATP is considered to exert a positive inotropic action on the myocardium through purinoceptors, very little information is available regarding interventions which may modify the actions of ATP on the heart. We report here that pyridoxal 5'-phosphate (PLP), an active form of vitamin B6, shows antagonism towards ATP-induced positive inotropic effect in isolated perfused rat hearts, ATP-induced increase in [Ca2+] in freshly isolated adult cardiomyocytes and ATP-binding in cardiac sarcolemma; ED50 for PLP in each of these cases varied from 10-15 microM. PLP (5-50 microM) was observed to antagonize the positive inotropic effect of ATP but did not modify the action of isoproterenol in the isolated perfused heart. Preincubation of cardiomyocytes with 1-50 microM PLP prevented the ATP-induced increase in [Ca2+]i in a concentration-dependent manner but showed no effect on the KCl-induced increase in [Ca2+]i. Creatine phosphate and Na2HPO4 as well as vitamin B6-related compounds, such as pyridoxine, pyridoxal, 4-deoxypyridoxine and isonicotinic acid hydrazide showed no effect on the ATP-induced increase in [Ca2+]i in cardiomyocytes. Furthermore, different concentrations of PLP (1-50 microM) were shown to inhibit the specific ATP gamma S binding at both the high and low affinity sites in the cardiac sarcolemmal membrane; adrenoceptor and Ca2+-channel inhibitors did not affect the ATP-binding. It is concluded that PLP may antagonize the actions of ATP on the heart in a selective manner and both pyridoxal and phosphate moieties are essential for its action. Furthermore, it is suggested that PLP may serve as a valuable tool for monitoring the role of purinoceptors in cellular function. PMID:10336844

  2. Molecular markers associated with resistance to commonly used antimalarial drugs among Plasmodium falciparum isolates from a malaria-endemic area in Taiz governorate-Yemen during the transmission season.

    PubMed

    Alareqi, Lina M Q; Mahdy, Mohammed A K; Lau, Yee-Ling; Fong, Mun-Yik; Abdul-Ghani, Rashad; Mahmud, Rohela

    2016-10-01

    Since 2005, artesunate (AS) plus sulfadoxine/pyrimethamine (SP) combination has been adopted as the first-line treatment for uncomplicated malaria in Yemen in response to the high level of Plasmodium falciparum resistance to chloroquine (CQ). Therefore, the aim of the present study was to determine the frequency distribution of molecular markers associated with resistance to CQ and AS plus SP combination among P. falciparum isolates from a malaria-endemic area in Taiz governorate, Yemen. Fifty P. falciparum isolates were collected during a cross-sectional study in Mawza district, Taiz, in the period from October 2013 to April 2014. The isolates were investigated for drug resistance-associated molecular markers in five genes, including P. falciparum CQ resistance transporter (pfcrt) 76T and P. falciparum multidrug resistance 1 (pfmdr1) 86Y as markers of resistance to CQ, mutations in the Kelch 13 (K13) propeller domain for resistance to AS, and P. falciparum dihydrofolate reductase (pfdhfr) and P. falciparum dihydropteroate synthase (pfdhps) genes for resistance to SP. Nested polymerase chain reaction was used to amplify target genes in DNA extracts of the isolates followed by restriction fragment length polymorphism for detecting 76T and 86Y mutations in pfcrt and pfmdr1, respectively, and by DNA sequencing for detecting mutations in K13, pfdhfr and pfdhps. All the investigated isolates from Mawza district were harboring the pfcrt 76T mutant and the pfmdr1 N86 wild-type alleles. The pfdhfr 51I/108N double mutant allele was found in 2.2% (1/45) of the isolates; however, no mutations were detected at codons 436, 437, 540, 581 and 613 of pfdhps. All P. falciparum isolates that were successfully sequenced (n=47) showed the K13 Y493, R539, I543 and C580 wild-type alleles. In conclusion, the pfcrt 76T mutant allele is fixed in the study area about six years after the official withdrawal of CQ, possibly indicating its over-the-counter availability and continued use as a

  3. Single-Cell Metabolomics: Changes in the Metabolome of Freshly Isolated and Cultured Neurons

    PubMed Central

    2012-01-01

    Metabolites are involved in a diverse range of intracellular processes, including a cell’s response to a changing extracellular environment. Using single-cell capillary electrophoresis coupled to electrospray ionization mass spectrometry, we investigated how placing individual identified neurons in culture affects their metabolic profile. First, glycerol-based cell stabilization was evaluated using metacerebral neurons from Aplysia californica; the measurement error was reduced from ∼24% relative standard deviation to ∼6% for glycerol-stabilized cells compared to those isolated without glycerol stabilization. In order to determine the changes induced by culturing, 14 freshly isolated and 11 overnight-cultured neurons of two metabolically distinct cell types from A. californica, the B1 and B2 buccal neurons, were characterized. Of the more than 300 distinctive cell-related signals detected, 35 compounds were selected for their known biological roles and compared among each measured cell. Unsupervised multivariate and statistical analysis revealed robust metabolic differences between these two identified neuron types. We then compared the changes induced by overnight culturing; metabolite concentrations were distinct for 26 compounds in the cultured B1 cells. In contrast, culturing had less influence on the metabolic profile of the B2 neurons, with only five compounds changing significantly. As a result of these culturing-induced changes, the metabolic composition of the B1 neurons became indistinguishable from the cultured B2 cells. This observation suggests that the two cell types differentially regulate their in vivo or in vitro metabolomes in response to a changing environment. PMID:23077722

  4. Magnetic Nanoparticle Based Nonviral MicroRNA Delivery into Freshly Isolated CD105+ hMSCs

    PubMed Central

    Schade, Anna; Müller, Paula; Delyagina, Evgenya; Voronina, Natalia; Skorska, Anna; Lux, Cornelia; Steinhoff, Gustav; David, Robert

    2014-01-01

    Genetic modifications of bone marrow derived human mesenchymal stem cells (hMSCs) using microRNAs (miRs) may be used to improve their therapeutic potential and enable innovative strategies in tissue regeneration. However, most of the studies use cultured hMSCs, although these can lose their stem cell characteristics during expansion. Therefore, we aimed to develop a nonviral miR carrier based on polyethylenimine (PEI) bound to magnetic nanoparticles (MNPs) for efficient miR delivery in freshly isolated hMSCs. MNP based transfection is preferable for genetic modifications in vivo due to improved selectivity, safety of delivery, and reduced side effects. Thus, in this study different miR/PEI and miR/PEI/MNP complex formulations were tested in vitro for uptake efficiency and cytotoxicity with respect to the influence of an external magnetic field. Afterwards, optimized magnetic complexes were selected and compared to commercially available magnetic vectors (Magnetofectamine, CombiMag). We found that all tested transfection reagents had high miR uptake rates (yielded over 60%) and no significant cytotoxic effects. Our work may become crucial for virus-free introduction of therapeutic miRs as well as other nucleic acids in vivo. Moreover, in the field of targeted stem cell therapy nucleic acid delivery prior to transplantation may allowfor initial cell modulation in vitro. PMID:24799915

  5. Emticicia aquatica sp. nov., a species of the family Cytophagaceae isolated from fresh water.

    PubMed

    Joung, Yochan; Seo, Mi-Ae; Kang, Heeyoung; Kim, Haneul; Ahn, Tae-Seok; Cho, Jang-Cheon; Joh, Kiseong

    2015-12-01

    A Gram-staining-negative, non-gliding, orange-pigmented bacterial strain, designated HMF2925T, was isolated from fresh water in Korea. The phylogenetic tree based on 16S rRNA gene sequences showed that strain HMF2925T formed a distinct lineage within the genus Emticicia. Strain HMF2925T was closely related to Emticicia oligotrophica DSM 17448T (95.5 %) and Emticicia ginsengisoli Gsoil 085T (94.1 %). The major fatty acids of strain HMF2925T were summed feature 3 (C16 : 1ω6c and/or C16 : 1ω7c), iso-C15 : 0, C16 : 1ω5c and C16 : 0.The major polar lipids of strain HMF2925T were phosphatidylethanolamine, phosphatidylinositol, diphosphatidylglycerol, phosphatidylglycerol, an unidentified glycolipid, two unidentified amino lipids and three unidentified polar lipids. The DNA G+C content of strain HMF2925T was 36.5 mol%. On the basis of the evidence presented in this study, strain HMF2925T represents a novel species of the genus Emticicia, for which the name Emticicia aquatica sp. nov. is proposed. The type strain is HMF2925T ( = KCTC 42574T = CECT 8858T). PMID:26346054

  6. Antioxidant properties in some selected cyanobacteria isolated from fresh water bodies of Sri Lanka.

    PubMed

    Hossain, Md Fuad; Ratnayake, R R; Meerajini, Kirisnashamy; Wasantha Kumara, K L

    2016-09-01

    Phytonutrients and pigments present in cyanobacteria act as antioxidants, which facilitate the formation of body's defense mechanism against free radical damage to cells. The aim of this investigation was to study the total phenolic content (TPC), total flavonoid content (TFC), antioxidant activity, phycobiliproteins (PBPs), and active compounds in four cyanobacterial species, that is, Oscillatoria sp., Lyngbya sp., Microcystis sp., and Spirulina sp. isolated from fresh water bodies of Sri Lanka. In this study, Lyngbya sp., showed highest TPC (5.02 ± 0.20 mg/g), TFC (664.07 ± 19.76 mg/g), and total PBPs (127.01 mg/g) value. The ferric reducing antioxidant power (FRAP) was recorded highest in Oscillatoria sp. (39.63 ± 7.02), whereas the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity was also reported the highest in Oscillatoria sp. (465.31 ± 25.76) followed by Lyngbya sp. (248.39 ± 11.97). In FTIR spectroscopy, Lyngbya sp. does not show any N-H stretching band which is ultimately responsible for the inhibition of antioxidant activity. The study revealed that Lyngbya sp. and Oscillatoria sp. can be an excellent source for food, pharmaceutical, and other industrial uses. PMID:27625779

  7. Uptake and metabolism of vinca alkaloids by freshly isolated human hepatocytes in suspension.

    PubMed

    Zhou, X J; Placidi, M; Rahmani, R

    1994-01-01

    A study was carried out to evaluate the uptake, release and metabolism of four currently used vinca alkaloids, including vinblastine, vincristine, vindesine and navelbine, using freshly isolated human hepatocytes in suspension. The drugs were rapidly taken up and intensely metabolised by the cells, giving a number of yet unidentified biotransformation products. Navelbine was the most rapidly and intensely accumulated drug followed by vinblastine, vindesine and vincristine. The extent of cell uptake appeared to parallel the lipophilicities of these compounds. Interestingly, we found a significant correlation between the mean uptake rates of the vinca alkaloids into the cells, which were 0.279, 0.343, 0.568 and 0.834 pmol/min/10(6) cells for vincristine, vindesine, vinblastine and navelbine, respectively, and the in vivo plasma clearances of the drugs (r = 0.9995, p < 0.001). This finding is of great importance as regards a better understanding of the structure-activity relationship among this class of antitumour drugs, as well as a reliable extrapolation of in vitro results to the in vivo situation. PMID:8074443

  8. Isolation, purification and identification of etiolation substrate from fresh-cut Chinese water-chestnut (Eleocharis tuberosa).

    PubMed

    Pan, Yong-Gui; Li, Yi-Xiao; Yuan, Meng-Qi

    2015-11-01

    Fresh cut Chinese water-chestnut is a popular ready-to-eat fresh-cut fruit in China. However, it is prone to etiolation and the chemicals responsible for this process are not known yet. To address this problem, we extracted phytochemicals from etiolated Chinese water-chestnut and separated them using MPLC and column chromatography. Four compounds were obtained and their structures were determined by interpretation of UV, TLC, HPLC and NMR spectral data and by comparison with reported data. We identified these compounds as eriodictyol, naringenin, sucrose and ethyl D-glucoside. Among those, eriodictyol and naringenin were both isolated for the first time in fresh-cut Chinese water-chestnut and are responsible for the yellowing of this fruit cutting. PMID:25976800

  9. Formation of Osteogenic Colonies on Well-Defined Adhesion Peptides by Freshly-Isolated Human Marrow Cells

    PubMed Central

    Au, Ada; Boehm, Cynthia A.; Mayes, Anne M.; Muschler, George F.; Griffith, Linda G.

    2007-01-01

    Bone graft performance can be enhanced by addition of connective tissue progenitors (CTPs) from fresh bone marrow in a manner that concentrates the CTP cell population within the graft. Here, we used small peptide adhesion ligands presented against an otherwise adhesion-resistant synthetic polymer background in order to illuminate the molecular basis for the attachment and colony formation by osteogenic CTPs from fresh human marrow, and contrast the behavior of fresh marrow to many commonly-used osteogenic cell sources. The linear GRGDSPY ligand was as effective as tissue culture polystyrene in fostering attachment of culture-expanded porcine CTPs. Although this GRGDSPY peptide was more effective than control peptides in fostering alkaline phosphatase-positive (AP) colony formation from primary human marrow in 5 of the 7 patients tested, GRGDSPY was as effective as the control glass substrate in only one patient of 7. Thus, the peptide appears capable of enabling osteoblastic development from only a subpopulation of CTPs in marrow. The bone sialoprotein-derived peptide FHRRIKA was ineffective in fostering attachment of primary culture-expanded pig CTPs, although it was as effective as GRGDSPY in fostering AP-positive colonies from fresh human marrow. This study provides insights into integrin-mediated behaviors of CTPs and highlights differences between freshly-isolated marrow and culture-expanded cells. PMID:17222453

  10. Expression of nucleoside transporter in freshly isolated neurons and astrocytes from mouse brain.

    PubMed

    Li, B; Gu, L; Hertz, L; Peng, L

    2013-11-01

    Nucleoside transporters comprise equilibrative ENT1-4 and concentrative CNT1-3. CNTs transport against an intracellular/extracellular gradient and are essential for transmitter removal, independently of metabolic need. ENT1-4 mediate transport until intracellular/extracellular equilibrium of the transported compound, but are very efficient, when the accumulated nucleoside or nucleobase is rapidly eliminated by metabolism. Most nucleoside transporters are membrane-bound, but ENT3 is mainly intracellular. This study uses freshly isolated neurons and astrocytes from two adult mouse strains. In one transgenic strain the neuronal marker Thy1 was associated with a compound fluorescing at one wavelength, and in the other the astrocytic marker GFAP was associated with a compound fluorescent at a different wavelength. Highly purified astrocytic and neuronal populations (as determined by presence/absence of cell-specific genes) were obtained from these mice by fluorescence-activated cell sorting. In each population mRNA analysis was performed by reverse-transcription polymerase chain reaction. CNT1 was absent in both cell types; all other nucleoside transporters were expressed to at least a similar degree (in relation to applied amount of RNA and to a house-keeping gene) in astrocytes as in neurons. Astrocytic ENT3 enrichment was dramatic, but it was not up-regulated after fluoxetine-mediated increase in DNA synthesis. A comparison with results obtained in cultured astrocytes shows that the latter are generally compatible with the present findings and suggests that many observations obtained in intact tissue, mainly by in situ hybridization (which also determines mRNA expression) may underestimate astrocytic nucleoside transporter expression. PMID:24026568

  11. Gender differences in methionine accumulation and metabolism in freshly isolated mouse hepatocytes: Potential roles in toxicity

    SciTech Connect

    Dever, Joseph T.; Elfarra, Adnan A.

    2009-05-01

    L-Methionine (Met) is hepatotoxic at high concentrations. Because Met toxicity in freshly isolated mouse hepatocytes is gender-dependent, the goal of this study was to assess the roles of Met accumulation and metabolism in the increased sensitivity of male hepatocytes to Met toxicity compared with female hepatocytes. Male hepatocytes incubated with Met (30 mM) at 37 {sup o}C exhibited higher levels of intracellular Met at 0.5, 1.0, and 1.5 h, respectively, compared to female hepatocytes. Conversely, female hepatocytes had higher levels of S-adenosyl-L-methionine compared to male hepatocytes. Female hepatocytes also exhibited higher L-methionine-L-sulfoxide levels relative to control hepatocytes, whereas the increases in L-methionine-D-sulfoxide (Met-D-O) levels were similar in hepatocytes of both genders. Addition of aminooxyacetic acid (AOAA), an inhibitor of Met transamination, significantly increased Met levels at 1.5 h and increased Met-D-O levels at 1.0 and 1.5 h only in Met-exposed male hepatocytes. No gender differences in cytosolic Met transamination activity by glutamine transaminase K were detected. However, female mouse liver cytosol exhibited higher methionine-DL-sulfoxide (MetO) reductase activity than male mouse liver cytosol at low (0.25 and 0.5 mM) MetO concentrations. Collectively, these results suggest that increased cellular Met accumulation, decreased Met transmethylation, and increased Met and MetO transamination in male mouse hepatocytes may be contributing to the higher sensitivity of the male mouse hepatocytes to Met toxicity in comparison with female mouse hepatocytes.

  12. Metabolism of cyclosporin A. I. Study in freshly isolated rabbit hepatocytes

    SciTech Connect

    Fabre, G.; Bertault-Peres, P.; Fabre, I.; Maurel, P.; Just, S.; Cano, J.P.

    1987-05-01

    The metabolism of cyclosporin A (CsA), a widely used immunosuppressive agent, was evaluated in freshly isolated rabbit hepatocytes by HPLC which separated CsA from its major group of derivatives, e.g. first generation metabolites (monohydroxylated and N-demethylated) and second generation derivatives (dihydroxylated and dihydroxy-N-demethylated). After exposure of hepatocytes to radiolabeled CsA (0.5 mg/liter), CsA was rapidly accumulated inside the cells and metabolized. The dihydroxylated metabolites represent the major intracellular forms after 1 hr. CsA metabolites synthesized inside the cells are then rapidly detected in the extracellular compartment. Unchanged drug and the various metabolites are concentrated inside the cells with transmembrane chemical gradients ranging between 20:1 and 40:1. Transport and metabolic processes for CsA have been evaluated over the following CsA extracellular concentration range, 0.1-10 mg/liter. Metabolism appears to be the rate-limiting step. The apparent affinity constant of CsA for the enzyme system involved in its metabolism is approximately 15 microM. Besides the lipophilicity of the molecule, which is responsible for the retention of CsA and its metabolites in the intracellular compartment, the presence of a binding component(s) in the hepatocytes was also demonstrated. CsA and its metabolites seem to have similar affinities for this binding site. These studies demonstrate that CsA is rapidly transformed inside the hepatocytes to various metabolites which may play an important role in the pharmacological activity of the drug and/or in its clinical toxicity.

  13. Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) gene and its association with in vitro drug susceptibility of parasite isolates from north-east Myanmar

    PubMed Central

    Gupta, Bhavna; Xu, Shuhui; Wang, Zenglei; Sun, Ling; Miao, Jun; Cui, Liwang; Yang, Zhaoqing

    2014-01-01

    Objectives Plasmodium falciparum multidrug resistance protein 1 (pfmrp1) has recently emerged as an important determinant of drug resistance and mutations in the gene have been associated with several drugs. The aim of this study was to understand the level of genetic diversity in pfmrp1 and to determine the association of different mutations with altered drug susceptibilities of P. falciparum. Methods We analysed 193 sequences of pfmrp1 from South-East Asia, west Asia, Africa, Oceania and South America. We measured the level of genetic diversity and determined signatures of selection on the gene. In vitro susceptibilities of 28 P. falciparum isolates from north-east Myanmar to a panel of seven commonly used antimalarials were determined. Statistical analysis was performed to determine the association of different mutations with in vitro drug susceptibilities. Results A total of 28 single nucleotide polymorphisms were identified in 193 sequences, of which 22 were non-synonymous. Whereas mutations in the pfmrp1 gene were conserved among different countries within a continent, they were different between continents. Seven non-synonymous mutations were identified in the north-east Myanmar isolates; all were relatively frequent in this region as well as in other neighbouring countries. Molecular evolutionary analysis detected signatures of positive selection on the gene. Moreover, some mutations in this gene were found to be associated with reduced susceptibilities to chloroquine, mefloquine, pyronaridine and lumefantrine. Conclusions Evidence of the positive selection of pmfrp1 and its association with the susceptibilities of parasites to multiple drugs signifies its potential as an important candidate for monitoring drug resistance. PMID:24855124

  14. Prevalence of In Vitro Resistance to Eleven Standard or New Antimalarial Drugs among Plasmodium falciparum Isolates from Pointe-Noire, Republic of the Congo

    PubMed Central

    Pradines, Bruno; Hovette, Philippe; Fusai, Thierry; Atanda, Henri Léonard; Baret, Eric; Cheval, Philippe; Mosnier, Joel; Callec, Alain; Cren, Julien; Amalvict, Rémy; Gardair, Jean Pierre; Rogier, Christophe

    2006-01-01

    We determined the level of in vitro resistance of Plasmodium falciparum parasites to standard antimalarial drugs, such as chloroquine, quinine, amodiaquine, halofantrine, mefloquine, cycloguanil, and pyrimethamine, and to new compounds, such as dihydroartemisinin, doxycycline, atovaquone, and lumefantrine. The in vitro resistance to chloroquine reached 75.5%. Twenty-eight percent of the isolates were intermediate or had reduced susceptibility to quinine. Seventy-six percent and 96% of the tested isolates showed in vitro resistance or intermediate susceptibilities to cycloguanil and pyrimethamine, respectively. Only 2% of the parasites demonstrated in vitro resistance to monodesethylamodiaquine. No resistance was shown with halofantrine, lumefantrine, dihydroartemisinin, or atovaquone. Halofantrine, mefloquine, and lumefantrine demonstrated high correlation. No cross-resistance was identified between responses to monodesethyl-amodiaquine, dihydroartemisinin, atovaquone, and cycloguanil. Since the level of chloroquine resistance in vitro exceed an unacceptable upper limit, high rates of in vitro resistance to pyrimethamine and cycloguanil and diminution of the susceptibility to quinine, antimalarial drugs used in combination, such as amodiaquine, artemisinin derivatives, mefloquine, lumefantrine, or atovaquone, seem to be appropriate alternatives for the first line of treatment of acute, uncomplicated P. falciparum malaria. PMID:16825356

  15. Prevalence of mutations associated with antimalarial drugs in Plasmodium falciparum isolates prior to the introduction of sulphadoxine-pyrimethamine as first-line treatment in Iran

    PubMed Central

    Zakeri, Sedigheh; Afsharpad, Mandana; Raeisi, Ahmad; Djadid, Navid Dinparast

    2007-01-01

    Background This work was carried out to assess the patterns and prevalence of resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP) in Iran. Methods The prevalence of pfcrt K76T, pfmdr1 N86Y, pfdhfr N51I, C59R, S108N/T and I164L and codons S436F/A, A437G, K540E, A581E, and A613S/T in pfdhps genes were genotyped by PCR/RFLP methods in 206 Plasmodium falciparum isolates from Chabahar and Sarbaz districts in Sistan and Baluchistan province, Iran, during 2003–2005. Results All P. falciparum isolates carried the 108N, while 98.5% parasite isolates carried the 59R mutation. 98.5% of patients carried both 108N and 59R. The prevalence of pfdhps 437G mutation was 17% (Chabahar) and 33% (Sarbaz) isolates. 20.4% of samples presented the pfdhfr 108N, 59R with pfdhps 437G mutations. The frequency of allele pfcrt 76T was 98%, while 41.4% (Chabahar) and 27.7% (Sarbaz) isolates carried pfmdr1 86Y allele. Eight distinct haplotypes were identified in all 206 samples, while the most prevalent haplotype was T76/N86/N51R59N108/A437 among both study areas. Conclusion Finding the fixed level of CQ resistance polymorphisms (pfcrt 76T) suggests that CQ must be withdrawn from the current treatment strategy in Iran, while SP may remain the treatment of choice for uncomplicated malaria. PMID:17999755

  16. A SYBR Green 1-based in vitro test of susceptibility of Ghanaian Plasmodium falciparum clinical isolates to a panel of anti-malarial drugs

    PubMed Central

    2013-01-01

    Background Based on report of declining efficacy of chloroquine, Ghana shifted to the use of artemisinin-based combination therapy (ACT) in 2005 as the first-line anti-malarial drug. Since then, there has not been any major evaluation of the efficacy of anti-malarial drugs in Ghana in vitro. The sensitivity of Ghanaian Plasmodium falciparum isolates to anti-malarial drugs was, therefore, assessed and the data compared with that obtained prior to the change in the malaria treatment policy. Methods A SYBR Green 1 fluorescent-based in vitro drug sensitivity assay was used to assess the susceptibility of clinical isolates of P. falciparum to a panel of 12 anti-malarial drugs in three distinct eco-epidemiological zones in Ghana. The isolates were obtained from children visiting health facilities in sentinel sites located in Hohoe, Navrongo and Cape Coast municipalities. The concentration of anti-malarial drug inhibiting parasite growth by 50% (IC50) for each drug was estimated using the online program, ICEstimator. Results Pooled results from all the sentinel sites indicated geometric mean IC50 values of 1.60, 3.80, 4.00, 4.56, 5.20, 6.11, 10.12, 28.32, 31.56, 93.60, 107.20, and 8952.50 nM for atovaquone, artesunate, dihydroartemisin, artemether, lumefantrine, amodiaquine, mefloquine, piperaquine, chloroquine, tafenoquine, quinine, and doxycycline, respectively. With reference to the literature threshold value indicative of resistance, the parasites showed resistance to all the test drugs except the artemisinin derivatives, atovaquone and to a lesser extent, lumefantrine. There was nearly a two-fold decrease in the IC50 value determined for chloroquine in this study compared to that determined in 2004 (57.56 nM). This observation is important, since it suggests a significant improvement in the efficacy of chloroquine, probably as a direct consequence of reduced drug pressure after cessation of its use. Compared to that measured prior to the change in treatment policy

  17. Identification of antibiotic resistance cassettes in class 1 integrons in Aeromonas spp. strains isolated from fresh fish (Cyprinus carpio L.).

    PubMed

    Sarria-Guzmán, Yohanna; López-Ramírez, María Patricia; Chávez-Romero, Yosef; Ruiz-Romero, Erick; Dendooven, Luc; Bello-López, Juan Manuel

    2014-05-01

    Forty-six Aeromonas spp. strains were isolated from fresh fish and investigated for their antimicrobial susceptibility, detection of Class 1 integrons by PCR, and arrangement of gene cassettes. Selected isolates were further characterized by enterobacterial repetitive intergenic consensus-PCR. Twenty isolates were found to carry Class 1 integrons. Amplification of the variable regions of the integrons revealed diverse bands ranging in size from 150 to 1,958 pb. Sequence analysis of the variable regions revealed the presence of several gene cassettes, such as adenylyl transferases (aadA2 and aadA5), dihydrofolate reductases (dfrA17 and dfrA1), chloramphenicol acetyl transferase (catB3), β-lactamase (oxa2), lincosamide nucleotidil transferase (linF), aminoglycoside-modifying enzyme (apha15), and oxacillinase (bla OXA-10). Two open reading frames with an unknown function were identified as orfC and orfD. The aadA2 cassette was the most common integron found in this study. Interestingly, five integrons were detected in the plasmids that might be involved in the transfer of resistance genes to other bacteria. This is a first report of cassette encoding for lincosamides (linF) resistance in Aeromonas spp. Implications on the incidence of integrons in isolates of Aeromonas spp. from fresh fish for human consumption, and its possible consequences to human health are discussed. PMID:24370627

  18. Glucose metabolite patterns as markers of functional differentiation in freshly isolated and cultured mouse mammary epithelial cells

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissel, M.J.

    1981-01-01

    In the mammary gland of non-ruminant animals, glucose is utilized in a characteristic and unique way during lacation. By measuring the incorporation of glucose carbon from (U-/sup 14/C)glucose into intermediary metabolitees and metabolic products in mammary epithelia cells from virgin, pregnant, and lacating mice, we domonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate are important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells isolated from pregnant mice and cultured on collagen gels have a pattern similar to that of their freshly isolated counter-parts. When isolated from lacating mice, the metabolite patterns of cells cultured on collagen gels are different from that of the cells of origin, and resembles that of freshly isolated cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  19. Ex vivo activity of the ACT new components pyronaridine and piperaquine in comparison with conventional ACT drugs against isolates of Plasmodium falciparum

    PubMed Central

    2012-01-01

    Background The aim of the present work was to assess i) ex vivo activity of pyronaridine (PND) and piperaquine (PPQ), as new components of artemisinin-based combination therapy (ACT), to define susceptibility baseline, ii) their activities compared to other partner drugs, namely monodesethylamodiaquine (MDAQ), lumefantrine (LMF), mefloquine (MQ), artesunate (AS) and dihydroartemisinin (DHA) against 181 Plasmodium falciparum isolates from African countries, India and Thailand, and iii) in vitro cross-resistance with other quinoline drugs, chloroquine (CQ) or quinine (QN). Methods The susceptibility of the 181 P. falciparum isolates to the nine anti-malarial drugs was assessed using the standard 42-hours 3H-hypoxanthine uptake inhibition method. Results The IC50 values for PND ranged from 0.55 to 80.0 nM (geometric mean = 19.9 nM) and from 11.8 to 217.3 nM for PPQ (geometric mean = 66.8 nM). A significant positive correlation was shown between responses to PPQ and PND responses (rho = 0.46) and between PPQ and MDAQ (rho = 0.30). No significant correlation was shown between PPQ IC50 and responses to other anti-malarial drugs. A significant positive correlation was shown between responses to PND and MDAQ (rho = 0.37), PND and LMF (rho = 0.28), PND and QN (rho = 0.24), PND and AS (rho = 0.19), PND and DHA (rho = 0.18) and PND and CQ (rho = 0.16). All these coefficients of correlation are too low to suggest cross-resistance between PPQ or PND and the other drugs. Conclusions In this study, the excellent anti-malarial activity of PPQ and PND was confirmed. The absence of cross-resistance with quinolines and artemisinin derivatives is consistent with the efficacy of the combinations of PPQ and DHA or PND and AS in areas where parasites are resistant to conventional anti-malarial drugs. PMID:22333675

  20. Analysis of gene mutations involved in chloroquine resistance in Plasmodium falciparum parasites isolated from patients in the southwest of Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Qahtani, Ahmed

    2010-01-01

    BACKGROUND AND OBJECTIVES: Chloroquine has been the drug of choice for the treatment of malaria for many decades. We aimed to examine the molecular basis of chloroquine resistance among Plasmodium falciparum isolates from the southwestern region of Saudi Arabia by analyzing the K76T and N86Y mutations in the PfCRT and PfMDR1 genes, respectively. PATIENTS AND METHODS: P falciparum-infected blood spot samples (n=121) were collected on filter papers. DNA was extracted and fragments from the above genes were amplified using nested PCR. The amplicons were digested by ApoI enzyme and sequenced. RESULTS: Of the 121 samples, 95 and 112 samples could be amplified for PfCRT K76T and PfMDR1 N86Y mutations, respectively. All of the samples amplified for the PfCRT K76T mutation were undigestible by ApoI, suggesting the presence of the K76T mutation. For the PfMDR1 N86Y mutation, 65/109 samples (59.6%) were digestible when treated with ApoI in a pattern, suggestive of the presence of the investigated wild allele (N86). However, 44/109 samples (40.4%) were digestible by ApoI, suggesting the presence of the mutated allele (Y) at position 86. DNA sequencing confirmed these results. CONCLUSION: Surprisingly, all isolates exhibited the mutated allele at codon 76 (K76T) of PfCRT. However, the mutated mutant allele at codon 86 (N86Y) of PfMDR1 was found in 40.4% of the samples studied. To our knowledge, this is the first study that has investigated the existence of the mutation in the PfMDR1 gene in the country. This study will contribute to the development of new strategies for therapeutic intervention against malaria in Saudi Arabia. PMID:20427933

  1. Assessment of Markers of Antimalarial Drug Resistance in Plasmodium falciparum Isolates from Pregnant Women in Lagos, Nigeria

    PubMed Central

    Agomo, Chimere Obiora; Oyibo, Wellington Aghoghovwia; Sutherland, Colin; Hallet, Rachael; Oguike, Mary

    2016-01-01

    Background The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr). Methods Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72–76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene. Results Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Yand184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/μl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and

  2. Ex vivo anti-malarial drugs sensitivity profile of Plasmodium falciparum field isolates from Burkina Faso five years after the national policy change

    PubMed Central

    2014-01-01

    Background The recent reports on the decreasing susceptibility of Plasmodium falciparum to artemisinin derivatives along the Thailand and Myanmar border are worrying. Indeed it may spread to India and then Africa, repeating the same pattern observed for chloroquine resistance. Therefore, it is essential to start monitoring P. falciparum sensitivity to artemisinin derivatives and its partner drugs in Africa. Efficacy of AL and ASAQ were tested by carrying out an in vivo drug efficacy test, with an ex vivo study against six anti-malarial drugs nested into it. Results of the latter are reported here. Methods Plasmodium falciparum ex-vivo susceptibility to chloroquine (CQ), quinine (Q), lumefantrine (Lum), monodesethylamodiaquine (MDA), piperaquine (PPQ) and dihydroartemisinin (DHA) was investigated in children (6 months – 15 years) with a parasitaemia of at least ≥4,000/μl. The modified isotopic microtest technique was used. The results of cellular proliferation were analysed using ICEstimator software to determine the 50% inhibitory concentration (IC50) values. Results DHA was the most potent among the 6 drugs tested, with IC50 values ranging from 0.8 nM to 0.9 nM (Geometric mean IC50 = 0.8 nM; 95% CI [0.8 - 0.9]). High IC50 values ranged between 0.8 nM to 166.1 nM were reported for lumefantrine (Geometric mean IC50 = 25.1 nM; 95% CI [22.4 - 28.2]). MDA and Q IC50s were significantly higher in CQ-resistant than in CQ-sensitive isolates (P = 0.0001). However, the opposite occurred for Lum and DHA (P < 0.001). No difference was observed for PPQ. Conclusion Artemisinin derivatives are still very efficacious in Burkina Faso and DHA-PPQ seems a valuable alternative ACT. The high lumefantrine IC50 found in this study is worrying as it may indicate a decreasing efficacy of one of the first-line treatments. This should be further investigated and monitored over time with large in vivo and ex vivo studies that will include also plasma drug measurements

  3. Limited artemisinin resistance-associated polymorphisms in Plasmodium falciparum K13-propeller and PfATPase6 gene isolated from Bioko Island, Equatorial Guinea

    PubMed Central

    Li, Jian; Chen, Jiangtao; Xie, Dongde; Eyi, Urbano Monsuy; Matesa, Rocio Apicante; Ondo Obono, Maximo Miko; Ehapo, Carlos Sala; Yang, Liye; Yang, Huitian; Lin, Min

    2016-01-01

    Objective With emergence and geographically expanding of antimalarial resistance worldwide, molecular markers are essential tool for surveillance of resistant Plasmodium parasites. Recently, single-nucleotide polymorphisms (SNPs) in the PF3D7_1343700 kelch propeller (K13-propeller) domain are shown to be associated with artemisinin (ART) resistance in vivo and in vitro. This study aims to investigate the ART resistance-associated polymorphisms of K13-propeller and PfATPase6 genes in Plasmodium falciparum isolates from Bioko Island, Equatorial Guinea (EG). Methods A total of 172 samples were collected from falciparum malaria patients on Bioko Island between 2013 and 2014. The polymorphisms of K13-propeller and PfATPase6 genes were analyzed by Nest-PCR and sequencing. Results Sequences of K13-propeller and PfATPase6 were obtained from 90.74% (98/108) and 91.45% (139/152) samples, respectively. The 2.04% (2/98) cases had non-synonymous K13-propeller A578S mutation but no found the mutations associated with ART resistance in Southeast Asia. For PfATPase6, the mutations were found at positions N569K and A630S with the mutation prevalence of 7.91% (11/139) and 1.44% (2/139), respectively. In addition, a sample with the mixed type at position I723V was discovered (0.72%, 1/139). Conclusions This study initially offers an insight of K13-propeller and PfATPase6 polymorphisms on Bioko Island, EG. It suggests no widespread ART resistance or tolerance in the region, and might be helpful for developing and updating guidance for the use of ART-based combination therapies (ACTs). PMID:27054064

  4. Amplification of pfmdr1, pfcrt, pvmdr1, and K13 Propeller Polymorphisms Associated with Plasmodium falciparum and Plasmodium vivax Isolates from the China-Myanmar Border

    PubMed Central

    Feng, Jun; Zhou, Daili; Lin, Yingxue; Xiao, Huihui; Yan, He

    2015-01-01

    Malaria in the China-Myanmar border region is still severe; local transmission of both falciparum and vivax malaria persists, and there is a risk of geographically expanding antimalarial resistance. In this research, the pfmdr1, pfcrt, pvmdr1, and K13-propeller genotypes were determined in 26 Plasmodium falciparum and 64 Plasmodium vivax isolates from Yingjiang county of Yunnan province. The pfmdr1 (11.5%), pfcrt (34.6%), and pvmdr1 (3.1%) mutations were prevalent at the China-Myanmar border. The indigenous samples exhibited prevalences of 14.3%, 28.6%, and 14.3% for pfmdr1 N86Y, pfcrt K76T, and pfcrt M74I, respectively, whereas the samples from Myanmar showed prevalences of 10.5%, 21.1%, and 5.3%, respectively. The most prevalent genotypes of pfmdr1 and pfcrt were Y86Y184 and M74N75T76, respectively. No pvmdr1 mutation occurred in the indigenous samples but was observed in two cases coming from Myanmar. In addition, we are the first to report on 10 patients (38.5%) with five different K13 point mutations. The F446I allele is predominant (19.2%), and its prevalence was 28.6% in the indigenous samples of Yingjiang county and 15.8% in samples from Myanmar. The present data might be helpful for enrichment of the molecular surveillance of antimalarial resistance and useful for developing and updating guidance for the use of antimalarials in this region. PMID:25691632

  5. Amplification of pfmdr1, pfcrt, pvmdr1, and K13 propeller polymorphisms associated with Plasmodium falciparum and Plasmodium vivax isolates from the China-Myanmar border.

    PubMed

    Feng, Jun; Zhou, Daili; Lin, Yingxue; Xiao, Huihui; Yan, He; Xia, Zhigui

    2015-05-01

    Malaria in the China-Myanmar border region is still severe; local transmission of both falciparum and vivax malaria persists, and there is a risk of geographically expanding antimalarial resistance. In this research, the pfmdr1, pfcrt, pvmdr1, and K13-propeller genotypes were determined in 26 Plasmodium falciparum and 64 Plasmodium vivax isolates from Yingjiang county of Yunnan province. The pfmdr1 (11.5%), pfcrt (34.6%), and pvmdr1 (3.1%) mutations were prevalent at the China-Myanmar border. The indigenous samples exhibited prevalences of 14.3%, 28.6%, and 14.3% for pfmdr1 N86Y, pfcrt K76T, and pfcrt M74I, respectively, whereas the samples from Myanmar showed prevalences of 10.5%, 21.1%, and 5.3%, respectively. The most prevalent genotypes of pfmdr1 and pfcrt were Y86Y184 and M74N75T76, respectively. No pvmdr1 mutation occurred in the indigenous samples but was observed in two cases coming from Myanmar. In addition, we are the first to report on 10 patients (38.5%) with five different K13 point mutations. The F446I allele is predominant (19.2%), and its prevalence was 28.6% in the indigenous samples of Yingjiang county and 15.8% in samples from Myanmar. The present data might be helpful for enrichment of the molecular surveillance of antimalarial resistance and useful for developing and updating guidance for the use of antimalarials in this region. PMID:25691632

  6. Pantoea sp. isolated from tropical fresh water exhibiting N-acyl homoserine lactone production.

    PubMed

    Tan, Wen-Si; Muhamad Yunos, Nina Yusrina; Tan, Pui-Wan; Mohamad, Nur Izzati; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry. PMID:25197715

  7. Pantoea sp. Isolated from Tropical Fresh Water Exhibiting N-Acyl Homoserine Lactone Production

    PubMed Central

    Tan, Wen-Si; Tan, Pui-Wan; Adrian, Tan-Guan-Sheng; Yin, Wai-Fong; Chan, Kok-Gan

    2014-01-01

    N-Acyl homoserine lactone (AHL) serves as signaling molecule for quorum sensing (QS) in Gram-negative bacteria to regulate various physiological activities including pathogenicity. With the aim of isolating freshwater-borne bacteria that can cause outbreak of disease in plants and portrayed QS properties, environmental water sampling was conducted. Here we report the preliminary screening of AHL production using Chromobacterium violaceum CV026 and Escherichia coli [pSB401] as AHL biosensors. The 16S rDNA gene sequence of isolate M009 showed the highest sequence similarity to Pantoea stewartii S9-116, which is a plant pathogen. The isolated Pantoea sp. was confirmed to produce N-3-oxohexanoyl-L-HSL (3-oxo-C6-HSL) through analysis of high resolution mass tandem mass spectrometry. PMID:25197715

  8. Insulin resistance in uremia. Characterization of lipid metabolism in freshly isolated and primary cultures of hepatocytes from chronic uremic rats.

    PubMed Central

    Caro, J F; Lanza-Jacoby, S

    1983-01-01

    We have studied the mechanism(s) of hyperlipidemia and liver insulin sensitivity in a rat model of severe chronic uremia (U). Basal lipid synthesis was decreased in freshly isolated hepatocytes from U when compared with sham-operated ad lib.-fed controls (alfC). Basal lipid synthesis in pair-fed controls (pfC) was in between U and alfC. Similarly, the activity of liver acetyl CoA carboxylase, fatty acid synthetase, citrate cleavage enzyme, malate dehydrogenase, and glucose-6-phosphate dehydrogenase was diminished in U. Muscle and adipose tissue lipoprotein lipase was also decreased. Insulin stimulated lipid synthesis in freshly isolated hepatocytes from alfC. Hepatocytes from U and pfC were resistant to this effect of insulin. To ascertain if the insulin resistance in U was due to starvation (chow intake 50% of alfC) or to uremia itself, the U and pfC were intragastrically fed an isocaloric diet via a Holter pump the last week of the experimental period. Hepatocytes from orally fed U and pfC were also cultured for 24 h in serum-free medium. While freshly isolated and cultured U hepatocytes remained insulin resistant, those from pfC normalized, in vivo and in vitro, when they were provided with enough nutrients. Conclusions: (a) Hyperlipidemia in uremia is not due to increased synthesis, but to defect(s) in clearance. (b) Insulin does not stimulate lipid synthesis in uremia. This finding, along with our recent demonstration that insulin binding and internalization are not decreased in the uremic liver, suggests that a post-binding defect(s) in the liver plays an important role in the mechanism(s) of insulin resistance in uremia. (c) Cultured hepatocytes from uremic rats remain insulin resistant. This quality renders these cells useful in studying the postinsulin binding events responsible for the insulin-resistant state in the absence of complicating hormonal and substrate changes that occur in vivo. PMID:6350367

  9. Use of the Anaerobic Pouch in Isolating Clostridium botulinum Spores from Fresh Meats

    PubMed Central

    Greenberg, Richard A.; Bladel, Bendt O.; Zingelmann, Walter J.

    1966-01-01

    The anaerobic film pouch was demonstrated to be an effective device for the primary isolation of Clostridium botulinum types A and B spores from raw pork, beef, and chicken. Optimal pasteurization of these meats (for reduction of nonspore microflora without affecting indigenous putrefactive anaerobic spore levels) was 50 min at 60 C. C. botulinum spores were recovered with good precision from meat samples inoculated with mixtures of C. botulinum and Putrefactive Anaerobe 3679 at 1:1 and at 1:99 ratios. Verification of C. botulinum isolates was accomplished by protection testing of subcultures in mice. PMID:5335387

  10. Partial characterization of low density lipoprotein preparations isolated from fresh and frozen plasma after radiolabeling by seven different methods

    SciTech Connect

    Atsma, D.E.; Kempen, H.J.; Nieuwenhuizen, W.; van 't Hooft, F.M.; Pauwels, E.K. )

    1991-01-01

    Four {sup 99m}Tc and three {sup 123}I labeling methods were evaluated for their suitability to label low density lipoproteins (LDL) for the purpose of scintigraphic biodistribution studies. For {sup 99m}Tc these methods were: direct incorporation in LDL of {sup 99m}TcO4- using sodium dithionite (dithionite method); a method using first N,N-dimethylformamide to prepare a {sup 99m}Tc-complex reacting with LDL in a subsequent step (DMF method); a technique in which {sup 99m}TcO4- is first coupled to a diamide dithiolate derivative of pentanoic acid by reduction with dithionite, followed by coupling of this ligand to LDL (N2S2 method); and a method using sodium borohydride and stannous chloride as reducing agents (borohydride method). The iodination techniques were based on oxidation of I(-)----I+, using iodine monochloride (ICl method), 1,3,4,6-tetrachloro-3,6-diphenylglycoluril (Iodogen method), and N-bromosuccinimide (NBS method) as oxidants. We studied labeling yields, modification of LDL caused by the labeling procedures using agarose-gel electrophoresis, and radiochemical stability of the labeled LDL complex upon incubation in plasma at 37 degrees C for 15 h. We used Sepharose CL6B chromatography to separate LDL from other plasma proteins. We also examined whether LDL isolated from frozen plasma (Pool-LDL) gave results similar to LDL obtained from freshly prepared plasma (Fresh-LDL). Pool-LDL radiolabeled by the dithionite, DMF, NBS, and Iodogen methods lost its label upon incubation with plasma. This also happened with Fresh-LDL when the DMF, NBS and Iodogen methods were used. Upon agarose-gel electrophoresis, no modification of LDL was observed with all methods when the radionuclide/LDL ratio was kept low.

  11. Plasmodium vivax and Plasmodium falciparum ex vivo susceptibility to anti-malarials and gene characterization in Rondônia, West Amazon, Brazil

    PubMed Central

    2014-01-01

    Background Chloroquine (CQ), a cost effective antimalarial drug with a relatively good safety profile and therapeutic index, is no longer used by itself to treat patients with Plasmodium falciparum due to CQ-resistant strains. P. vivax, representing over 90% of malaria cases in Brazil, despite reported resistance, is treated with CQ as well as with primaquine to block malaria transmission and avoid late P. vivax malaria relapses. Resistance to CQ and other antimalarial drugs influences malaria control, thus monitoring resistance phenotype by parasite genotyping is helpful in endemic areas. Methods A total of 47 P. vivax and nine P. falciparum fresh isolates were genetically characterized and tested for CQ, mefloquine (MQ) and artesunate (ART) susceptibility in vitro. The genes mdr1 and pfcrt, likely related to CQ resistance, were analyzed in all isolates. Drug susceptibility was determined using short-term parasite cultures of ring stages for 48 to 72 hour and thick blood smears counts. Each parasite isolate was tested with the antimalarials to measure the geometric mean of 50% inhibitory concentration. Results The low numbers of P. falciparum isolates reflect the species prevalence in Brazil; most displayed low sensitivity to CQ (IC50 70 nM). However, CQ resistance was rare among P. vivax isolates (IC50 of 32 nM). The majority of P. vivax and P. falciparum isolates were sensitive to ART and MQ. One hundred percent of P. falciparum isolates carried non-synonymous mutations in the pfmdr1 gene in codons 184, 1042 and 1246, 84% in codons 1034 and none in codon 86, a well-known resistance mutation. For the pfcrt gene, mutations were observed in codons 72 and 76 in all P. falciparum isolates. One P. falciparum isolate from Angola, Africa, showing sensitivity to the antimalarials, presented no mutations. In P. vivax, mutations of pvmdr1 and the multidrug resistance gene 1 marker at codon F976 were absent. Conclusion All P. falciparum Brazilian isolates showed

  12. Recombinant Scorpine Produced Using SUMO Fusion Partner in Escherichia coli Has the Activities against Clinically Isolated Bacteria and Inhibits the Plasmodium falciparum Parasitemia In Vitro

    PubMed Central

    Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel–nitrilotriacetic acid (Ni2+–NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+–NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  13. Recombinant scorpine produced using SUMO fusion partner in Escherichia coli has the activities against clinically isolated bacteria and inhibits the Plasmodium falciparum parasitemia in vitro.

    PubMed

    Zhang, Chao; He, Xinlong; Gu, Yaping; Zhou, Huayun; Cao, Jun; Gao, Qi

    2014-01-01

    Scorpine, a small cationic peptide from the venom of Pandinus imperator, which has been shown to have anti-bacterial and anti-plasmodial activities, has potential important applications in the pharmaceutical industries. However, the isolation of scorpine from natural sources is inefficient and time-consuming. Here, we first report the expression and purification of recombinant scorpine in Escherichia coli, using small ubiquitin-related modifier (SUMO) fusion partner. The fusion protein was expressed in soluble form in E. coli, and expression was verified by SDS-PAGE and western blotting analysis. The fusion protein was purified to 90% purity by nickel-nitrilotriacetic acid (Ni2+-NTA) resin chromatography. After the SUMO-scorpine fusion protein was cleaved by the SUMO protease, the cleaved sample was reapplied to a Ni2+-NTA column. Tricine/SDS-PAGE gel results indicated that Scorpine had been purified successfully to more than 95% purity. The recombinantly expressed Scorpine showed anti-bacterial activity against two standard bacteria including Staphylococcus aureus ATCC 29213 and Acinetobacter baumannii ATCC 19606, and clinically isolated bacteria including S. aureus S, S. aureus R, A. baumannii S, and A. baumannii R. It also produced 100% reduction in Plasmodium falciparum parasitemia in vitro. Thus, the expression strategy presented in this study allowed convenient high yield and easy purification of recombinant Scorpine for pharmaceutical applications in the future. PMID:25068263

  14. Helicobacter pullorum Isolated from Fresh Chicken Meat: Antibiotic Resistance and Genomic Traits of an Emerging Foodborne Pathogen

    PubMed Central

    Borges, Vítor; Santos, Andrea; Correia, Cristina Belo; Saraiva, Margarida; Ménard, Armelle; Vieira, Luís; Sampaio, Daniel A.; Pinheiro, Miguel; Gomes, João Paulo

    2015-01-01

    Meat and meat products are important sources of human intestinal infections. We report the isolation of Helicobacter pullorum strains from chicken meat. Bacteria were isolated from 4 of the 17 analyzed fresh chicken meat samples, using a membrane filter method. MIC determination revealed that the four strains showed acquired resistance to ciprofloxacin; one was also resistant to erythromycin, and another one was resistant to tetracycline. Whole-genome sequencing of the four strains and comparative genomics revealed important genetic traits within the H. pullorum species, such as 18 highly polymorphic genes (including a putative new cytotoxin gene), plasmids, prophages, and a complete type VI secretion system (T6SS). The T6SS was found in three out of the four isolates, suggesting that it may play a role in H. pullorum pathogenicity and diversity. This study suggests that the emerging pathogen H. pullorum can be transmitted to humans by chicken meat consumption/contact and constitutes an important contribution toward a better knowledge of the genetic diversity within the H. pullorum species. In addition, some genetic traits found in the four strains provide relevant clues to how this species may promote adaptation and virulence. PMID:26386065

  15. Coupling reverse osmosis with electrodialysis to isolate natural organic matter from fresh waters.

    PubMed

    Koprivnjak, J F; Perdue, E M; Pfromm, P H

    2006-10-01

    Reverse osmosis (RO) has proven to be an effective method for the concentration of natural organic matter (NOM) from fresh waters, but an undesirable consequence of this process is the co-concentration of some inorganic solutes. Accordingly, current practice yields solutions of NOM that, upon desalting and freeze-drying, are converted into dry solids containing finely dispersed sulfuric acid and silicic acid (H(4)SiO(4)). These acids will contribute to the apparent carboxylic and phenolic contents of NOM, leading to an overestimation of both. NOM may also be chemically altered by sulfuric acid, which reacts strongly with many classes of organic compounds. The sulfur content and ash content of NOM will be elevated in the presence of sulfuric acid and H(4)SiO(4). The goal of this study is to develop and test a method in which the removal of water by RO is coupled with the removal of salts by electrodialysis (ED). Like RO, ED is a relatively mild treatment that enables the desalting of NOM solutions without subjecting those samples to conditions of extremely high or low pH. The end product of the coupled process is a desalted, concentrated liquid sample from which low-ash NOM can be obtained as a freeze-dried solid material. In this study, the efficacy of ED for desalting NOM is evaluated using concentrated synthetic river waters and actual concentrated (by RO) river waters. Under optimal operating conditions, both sulfate and silica can be largely removed from RO-concentrated solutions of riverine NOM with only an average loss of 3% of total organic carbon. PMID:16952387

  16. Drancourtella massiliensis gen. nov., sp. nov. isolated from fresh healthy human faecal sample from South France.

    PubMed

    Durand, G A; Lagier, J-C; Khelaifia, S; Armstrong, N; Robert, C; Rathored, J; Fournier, P-E; Raoult, D

    2016-05-01

    Strain GD1(T) gen. nov., sp. nov., is the type strain of the newly proposed genus and species Drancourtella massiliensis, belonging to the Clostridiales order. This strain, isolated from the stool of a healthy person, is a Gram-positive rod, oxygen intolerant and nonmotile, with spore-forming activity. The features of this organism and its genome sequence are described. The draft genome is 3 057 334 bp long with 45.24% G + C content; it contains 2861 protein-coding genes and 64 RNA genes. PMID:27257490

  17. Stereospecific analysis of fatty acid esters of chloropropanediol isolated from fresh goat milk.

    PubMed

    Myher, J J; Kuksis, A; Marai, L; Cerbulis, J

    1986-05-01

    The fatty acid esters of chloropropanediol isolated from goat milk fat in small quantities were subjected to a stereospecific analysis via phospholipase C and phosphocholine esters as intermediates. Synthetic rac-1-chloro-2,3-dioleoyl-propanediol was prepared by standard methods and was used as a control. The stereospecific analyses were performed following a release of the fatty acids from the primary positions of each chloropropanediol diester with pancreatic lipase. The resulting X-1-chloro-2-acylpropanediols were then converted into the corresponding phosphocholine derivatives by a stepwise reaction with phosphorus oxychloride and choline chloride. The X-1-chloro-2-acyl-3-phosphocholinepropanediols were subjected to hydrolysis with phospholipase C (C. perfringens), which hydrolyzed 50% of the phosphatide within two min and the rest of it in two hr. From previous experience with glycerol esters, it was assumed that the more rapidly hydrolyzed molecules were the sn-1-chloro-2-acyl-propanediol derivatives and the more slowly hydrolyzed ones the sn-2-acyl-3-chloropropanediol derivatives. A hydrolysis with phospholipase A2 (Crotalus adamanteus) released 50% of the total fatty acid along with the corresponding lyso compound within 10 min, after which there was no further reaction. The hydrolysis products were assayed directly by gas liquid chromatography (GLC) or were isolated by thin layer chromatography (TLC) prior to quantitation by GLC. Both naturally occurring and synthetic chloropropanediol diesters behaved similarly on stereospecific analysis and were therefore concluded to be racemic. PMID:3724368

  18. Plasmid-Mediated Dimethoate Degradation by Bacillus licheniformis Isolated From a Fresh Water Fish Labeo rohita

    PubMed Central

    2005-01-01

    The Bacillus licheniformis strain isolated from the intestine of Labeo rohita by an enrichment technique showed capability of utilizing dimethoate as the sole source of carbon. The bacterium rapidly utilized dimethoate beyond 0.6 mg/mL and showed prolific growth in a mineral salts medium containing 0.45 mg/mL dimethoate. The isolated B licheniformis exhibited high level of tolerance of dimethoate (3.5 mg/mL) in nutrient broth, while its cured mutant did not tolerate dimethoate beyond 0.45 mg/mL and it was unable to utilize dimethoate. The wild B licheniformis strain transferred dimethoate degradation property to E coli C600 (Nar, F−) strain. The transconjugant harbored a plasmid of the same molecular size (approximately 54 kb) as that of the donor plasmid; the cured strain was plasmid less. Thus a single plasmid of approximately 54 kb was involved in dimethoate degradation. Genes encoding resistance to antibiotic and heavy metal were also located on the plasmid. PMID:16192686

  19. Var Gene promoter activation in clonal Plasmodium falciparum isolates follows a hierarchy and suggests a conserved switching program that is independent of genetic background.

    PubMed

    Enderes, Corinna; Kombila, Davy; Dal-Bianco, Matthias; Dzikowski, Ron; Kremsner, Peter; Frank, Matthias

    2011-11-15

    Antigenic variation of Plasmodium falciparum is mediated by a mutually exclusive expression mechanism that limits expression to an individual member of the multicopy var gene family. This process determines the antigenic and adhesive phenotype of the infected red blood cell. Previously, we showed that var gene switching is influenced by chromosomal position. Here, we address whether var gene transcription follows a general conserved pattern in long-term laboratory parasites and in recently culture-adapted field parasites. Activation of the var gene family was monitored in biological replicates in each parasite isolate every 3-5 generations for up to 3 months. We used transgenic parasites carrying a drug-selectable marker at a defined var locus to characterize var gene activation after the exclusive expression of the transgene. Transgenic parasites exhibited a repeatable hierarchy of var gene activation and a fluctuating transcriptional activity of the transgenic var locus. Transcriptional profiling of wild-type laboratory and field parasites showed a universal bias toward transcription of UpsC var genes and a fluctuating transcriptional activity of the dominant var promoter. The data suggest the existence of an intrinsic var gene transcription program that is independent of genetic background. PMID:21926380

  20. Antibiotic and heavy-metal resistance of Vibrio parahaemolyticus isolated from fresh shrimps in Shanghai fish markets, China.

    PubMed

    He, Yu; Jin, Lanlan; Sun, Fengjiao; Hu, Qiongxia; Chen, Lanming

    2016-08-01

    Vibrio parahaemolyticus is a causative agent of human serious seafood-borne gastroenteritis disease and even death. Shrimps, often eaten raw or undercooked, are an important reservoir of the bacterium. In this study, we isolated and characterized a total of 400 V. parahaemolyticus strains from commonly consumed fresh shrimps (Litopenaeus vannamei, Macrobrachium rosenbergii, Penaeus monodon, and Exopalaemon carinicauda) in Shanghai fish markets, China in 2013-2014. The results revealed an extremely low occurrence of pathogenic V. parahaemolyticus carrying two major toxic genes (tdh and trh, 0.0 and 0.5 %). However, high incidences of antibiotic resistance were observed among the strains against ampicillin (99 %), streptomycin (45.25 %), rifampicin (38.25 %), and spectinomycin (25.50 %). Approximately 24 % of the strains derived from the P. monodon sample displayed multidrug resistant (MDR) phenotypes, followed by 19, 12, and 6 % from the E. carinicauda, L. vannamei, and M. rosenbergii samples, respectively. Moreover, tolerance to heavy metals of Cr(3+) and Zn(2+) was observed in 90 antibiotic resistant strains, the majority of which also displayed resistance to Cu(2+) (93.3 %), Pb(2+) (87.8 %), and Cd(2+)(73.3 %). The pulsed-field gel electrophoresis (PFGE)-based genotyping of these strains revealed a total of 71 distinct pulsotypes, demonstrating a large degree of genomic variation among the isolates. The wide distribution of MDR and heavy-metal resistance isolates in the PFGE clusters suggested the co-existence of a number of resistant determinants in V. parahaemolyticus population in the detected samples. This study provided data in support of aquatic animal health management and food safety risk assessment in aquaculture industry. PMID:27083906

  1. Mechanical properties of nerve roots and rami radiculares isolated from fresh pig spinal cords

    PubMed Central

    Nishida, Norihiro; Kanchiku, Tsukasa; Ohgi, Junji; Ichihara, Kazuhiko; Chen, Xian; Taguchi, Toshihiko

    2015-01-01

    No reports have described experiments designed to determine the strength characteristics of spinal nerve roots and rami radiculares for the purpose of explaining the complexity of symptoms of medullary cone lesions and cauda equina syndrome. In this study, to explain the pathogenesis of cauda equina syndrome, monoaxial tensile tests were performed to determine the strength characteristics of spinal nerve roots and rami radiculares, and analysis was conducted to evaluate the stress-strain relationship and strength characteristics. Using the same tensile test device, the nerve root and ramus radiculares isolated from the spinal cords of pigs were subjected to the tensile test and stress relaxation test at load strain rates of 0.1, 1, 10, and 100 s-1 under identical settings. The tensile strength of the nerve root was not rate dependent, while the ramus radiculares tensile strength tended to decrease as the strain rate increased. These findings provide important insights into cauda equina symptoms, radiculopathy, and clinical symptoms of the medullary cone. PMID:26807127

  2. The nature of laboratory domestication changes in freshly isolated Escherichia coli strains.

    PubMed

    Eydallin, Gustavo; Ryall, Ben; Maharjan, Ram; Ferenci, Thomas

    2014-03-01

    Adaptation of environmental bacteria to laboratory conditions can lead to modification of important traits, what we term domestication. Little is known about the rapidity and reproducibility of domestication changes, the uniformity of these changes within a species or how diverse these are in a single culture. Here, we analysed phenotypic changes in nutrient-rich liquid media or on agar of four Escherichia coli strains newly isolated through minimal steps from different sources. The laboratory-cultured populations showed changes in metabolism, morphotype, fitness and in some phenotypes associated with the sigma factor RpoS. Domestication events and phenotypic diversity started to emerge within 2-3 days in replicate subcultures of the same ancestor. In some strains, increased amino acid usage and higher fitness under nutrient limitation resembled those in mutants with the GASP (growth advantage in stationary phase) phenotype. The domestication changes are not uniform across a species or even within a single domesticated population. However, some parallelism in adaptation within repeat cultures was observed. Differences in the laboratory environment also determine domestication effects, which differ between liquid and solid media or with extended stationary phase. Important lessons for the handling and storage of organisms can be based on these studies. PMID:23889812

  3. Molecular Epidemiology of Malaria in Cameroon. XXX. Sequence Analysis of Plasmodium falciparum ATPase 6, Dihydrofolate Reductase, and Dihydropteroate Synthase Resistance Markers in Clinical Isolates from Children Treated with an Artesunate-Sulfadoxine-Pyrimethamine Combination

    PubMed Central

    Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

    2011-01-01

    Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance. PMID:21734119

  4. Molecular epidemiology of malaria in Cameroon. XXX. sequence analysis of Plasmodium falciparum ATPase 6, dihydrofolate reductase, and dihydropteroate synthase resistance markers in clinical isolates from children treated with an artesunate-sulfadoxine-pyrimethamine combination.

    PubMed

    Menemedengue, Virginie; Sahnouni, Khalifa; Basco, Leonardo; Tahar, Rachida

    2011-07-01

    Plasmodium falciparum dihydrofolate reductase (dhfr) and dihydropteroate synthase (dhps) genes are reliable molecular markers for antifolate resistance. The P. falciparum ATPase 6 (pfatp6) gene has been proposed to be a potential marker for artemisinin resistance. In our previous clinical study, we showed that artesunate-sulfadoxine-pyrimethamine is highly effective against uncomplicated malaria in Yaoundé, Cameroon. In the present study, dhfr, dhps, and pfatp6 mutations in P. falciparum isolates obtained from children treated with artesunate-sulfadoxine-pyrimethamine were determined. All 61 isolates had wild-type Pfatp6 263, 623, and 769 alleles, and 11 (18%) had a single E431K substitution. Three additional mutations, E643Q, E432K, and E641Q, were detected. The results did not indicate any warning signal of serious concern (i.e., no parasites were seen with quintuple dhfr-dhps, DHFR Ile164Leu, or pfatp6 mutations), as confirmed by the high clinical efficacy of artesunate-sulfadoxine-pyrimethamine. Further studies are required to identify a molecular marker that reliably predicts artemisinin resistance. PMID:21734119

  5. Gram-negative bacterial isolates from fresh-cut processing plants enhance the presence of Escherichia Coli O157:H7 in dual-species biofilms

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilms formed by resident microflora may provide a microenvironment for foodborne bacterial pathogens to survive and cause cross-contamination in fresh-cut processing and handling facilities. The objective of this study is to determine the impact of individual bacteria strains isolated from two l...

  6. From In Vivo to In Vitro: Dynamic Analysis of Plasmodium falciparum var Gene Expression Patterns of Patient Isolates during Adaptation to Culture

    PubMed Central

    Huang, Yufu; Xue, Xiangyang; Yan, He; Sun, Xiaodong; Wang, Jian; McCutchan, Thomas F.; Pan, Weiqing

    2011-01-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), encoded by the var gene family, plays a crucial role in disease virulence through its involvement in binding to various host cellular receptors during infection. Growing evidence suggests that differential expression of the various var subgroups may be involved in parasite virulence. To further explore this issue, we have collected isolates from symptomatic patients in south China-Myanmar border, and characterized their sequence diversity and transcription profiles over time of var gene family, and cytoadherence properties from the time of their initial collection and extending through a two month period of adaptation to culture. Initially, we established a highly diverse, DBLα (4 cysteines) subtype-enriched, but unique local repertoire of var-DBL1α sequences by cDNA cloning and sequencing. Next we observed a rapid transcriptional decline of upsA- and upsB-subtype var genes at ring stage through qRT-PCR assays, and a switching event from initial ICAM-I binding to the CD36-binding activity during the first week of adaptive cultivation in vitro. Moreover, predominant transcription of upsA var genes was observed to be correlated with those isolates that showed a higher parasitemia at the time of collection and the ICAM-1-binding phenotype in culture. Taken together, these data indicate that the initial stage of adaptive process in vitro significantly influences the transcription of virulence-related var subtypes and expression of PfEMP1 variants. Further, the specific upregulation of the upsA var genes is likely linked to the rapid propagation of the parasite during natural infection due to the A-type PfEMP1 variant-mediated growth advantages. PMID:21674009

  7. Effects of calcium channel blockers on spontaneous electrical activity of freshly isolated three-day-old embryonic chick ventricle.

    PubMed

    Prakash, P; Meera, P; Tripathi, O

    1996-01-01

    The effects of four major types of organic Ca2+ channel blockers, verapamil, nifedipine, diltiazem and fendiline and of tetrodotoxin (TXX), a fast Na+ channel blocker, on the action potential (AP) of freshly isolated 3-day-old embryonic chick ventricle (3d ECV) were investigated to resolve the controversy about the ionic basis of upstroke. The APs were characterized by a maximum diastolic potential (MDP) of -60 mV, an overshoot (Eov) of 16 mV and a maximum upstroke velocity (+Vmax) of 42 V s-1. All four Ca2+ channel blockers (0.1-40 microM) and TTX (0.1-80 nM) produced a dose-dependent reduction in +Vmax and Eov. MDP was also reduced by Ca2+ channel blockers in a dose-dependent manner but was unaffected by TTX. A significant linear correlation between MDP and +Vmax was observed for verapamil (r = 0.99), nifedipine (r = 0.99), diltiazem (r = 0.96) and fendiline (r = 0.98). Surprisingly, all Ca2+ channel blockers produced a dose-dependent positive chronotropic effect leading to cessation of firing at high doses (20-40 microM). In preparations becoming quiescent with high doses of verapamil (20-40 microM), elevated extracellular concentrations of Ca2+ (up to 9.6 nM) and isoproterenol (0.5-40 microM) failed to restore spontaneous APs. Electrical stimulation also failed to elicit APs in preparations inhibited by verapamil, diltiazem and fendiline. The inhibition of +Vmax by TTX demonstrates that fast Na+ channels were involved in the upstroke of AP in 3d ECV. Voltage-dependent inactivation of fast Na+ channels during depolarization (reduction in MDP) by the Ca2+ channel blockers explains their inhibitory effect on +Vmax and indicates that L-type Ca2+ channels had no significant role in the upstroke. A positive chronotropic effect of the Ca2+ channel blockers further suggests that slow Ca2+ channels are not involved in automaticity in freshly isolated 3d ECV. PMID:8876052

  8. Cytotoxicity of aluminium oxide nanoparticles towards fresh water algal isolate at low exposure concentrations.

    PubMed

    Pakrashi, Sunandan; Dalai, Swayamprava; T C, Prathna; Trivedi, Shruti; Myneni, Radhika; Raichur, Ashok M; Chandrasekaran, N; Mukherjee, Amitava

    2013-05-15

    The growing commercial applications had brought aluminium oxide nanoparticles under toxicologists' purview. In the present study, the cytotoxicity of two different sized aluminium oxide nanoparticles (ANP(1), mean hydrodynamic diameter 82.6±22nm and ANP(2), mean hydrodynamic diameter 246.9±39nm) towards freshwater algal isolate Chlorella ellipsoids at low exposure levels (≤1μg/mL) using sterile lake water as the test medium was assessed. The dissolution of alumina nanoparticles and consequent contribution towards toxicity remained largely unexplored owing to its presumed insoluble nature. Herein, the leached Al(3+) ion mediated toxicity has been studied along with direct particulate toxicity to bring out the dynamics of toxicity through colloidal stability, biochemical, spectroscopic and microscopic analyses. The mean hydrodynamic diameter increased with time both for ANP(1) [82.6±22nm (0h) to 246.3±59nm (24h), to 1204±140nm (72h)] and ANP(2) [246.9±39nm (0h) to 368.28±48nm (24h), to 1225.96±186nm (72h)] signifying decreased relative abundance of submicron sized particles (<1000nm). The detailed cytotoxicity assays showed a significant reduction in the viability dependent on dose and exposure. A significant increase in ROS and LDH levels were noted for both ANPs at 1μg/mL concentration. The zeta potential and FT-IR analyses suggested surface chemical interaction between nanoparticles and algal cells. The substantial morphological changes and cell wall damage were confirmed through microscopic analyses (SEM, TEM, and CLSM). At 72h, significant Al(3+) ion release in the test medium [0.092μg/mL for ANP(1), and 0.19μg/mL for ANP(2)] was noted, and the resulting suspension containing leached ions caused significant cytotoxicity, revealing a substantial ionic contribution. This study indicates that both the nano-size and ionic dissolution play a significant role in the cytotoxicity of ANPs towards freshwater algae, and the exposure period largely determines

  9. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals

    PubMed Central

    Marcinkiewicz, Mariola M.; Baker, Sandy T.; Wu, Jichuan; Hubert, Terrence L.; Wolfson, Marla R.

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation—6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  10. A Novel Approach for Ovine Primary Alveolar Epithelial Type II Cell Isolation and Culture from Fresh and Cryopreserved Tissue Obtained from Premature and Juvenile Animals.

    PubMed

    Marcinkiewicz, Mariola M; Baker, Sandy T; Wu, Jichuan; Hubert, Terrence L; Wolfson, Marla R

    2016-01-01

    The in vivo ovine model provides a clinically relevant platform to study cardiopulmonary mechanisms and treatments of disease; however, a robust ovine primary alveolar epithelial type II (ATII) cell culture model is lacking. The objective of this study was to develop and optimize ovine lung tissue cryopreservation and primary ATII cell culture methodologies for the purposes of dissecting mechanisms at the cellular level to elucidate responses observed in vivo. To address this, we established in vitro submerged and air-liquid interface cultures of primary ovine ATII cells isolated from fresh or cryopreserved lung tissues obtained from mechanically ventilated sheep (128 days gestation-6 months of age). Presence, abundance, and mRNA expression of surfactant proteins was assessed by immunocytochemistry, Western Blot, and quantitative PCR respectively on the day of isolation, and throughout the 7 day cell culture study period. All biomarkers were significantly greater from cells isolated from fresh than cryopreserved tissue, and those cultured in air-liquid interface as compared to submerged culture conditions at all time points. Surfactant protein expression remained in the air-liquid interface culture system while that of cells cultured in the submerged system dissipated over time. Despite differences in biomarker magnitude between cells isolated from fresh and cryopreserved tissue, cells isolated from cryopreserved tissue remained metabolically active and demonstrated a similar response as cells from fresh tissue through 72 hr period of hyperoxia. These data demonstrate a cell culture methodology using fresh or cryopreserved tissue to support study of ovine primary ATII cell function and responses, to support expanded use of biobanked tissues, and to further understanding of mechanisms that contribute to in vivo function of the lung. PMID:26999050

  11. Polymorphisms of the Pfatpase 6 and Pfcrt gene and their relationship with the in vitro susceptibility to dihydroartemisinin and chloroquine of Plasmodium falciparum isolates from Abobo, Côte d'Ivoire.

    PubMed

    Bla, Brice K; Yavo, William; Trébissou, Jonhson; Kipré, Rolland G; Yapi, Félix H; N'guessan, Jean D; Djaman, Joseph A

    2014-01-01

    As a result of widespread resistance to chloroquine (CQ) and sulphadoxine-pyrimethamine (SP), artemisinin-based combination therapy (ACT) has been recommended as a first-line anti-malarial regimen in Côte d'Ivoire since 2005. A thorough understanding of the molecular bases of P. falciparum resistance to existing drugs is therefore needed. The aims of this study were to analyze the in vitro sensitivity of P. falciparum field isolates from Abobo to CQ, pyronaridine (PYR) and dihydroartemisinine (DHA), and to investigate the polymorphisms associated with drug resistance. The standard in vitro drug sensitivity microtechnique recommended by the WHO was used to assess the sensitivity of Plasmodium falciparum isolates collected in December 2006. The Pfcrt haplotype 76 was analysed by PCR-RFLP while Pfatpase 6 amplification products were sequenced. Associations between drug sensitivity and parasite gene polymorphisms were evaluated with Cohen's kappa test. The correlation between the IC50 values for different drugs was assessed by the coefficient of determination (r²). Significance was assumed at p<0.05. Of 128 in vitro tests performed, 112 (87.5%) were successful. Of the isolates, 56.2% were resistant for CQ and 48% for PYR. One isolate (3.6%) demonstrated reduced DHA sensitivity (IC50 higher than 10 nM). The mutant K76T pfcrt codon, present in 90% of DNA fragments analyzed, was associated with CQ-R (ĸ=0.76). The N669Y (16.1%), D734Y (28.6%) and D734H (1.8%) isolates were found to have mutant Pfatpase6, however, these mutations were not associated with diminished DHA sensitivity (k=0.01). These high levels of antimalarial drug resistance in Abobo (Côte d'Ivoire) demand further studies of drug efficacy across the whole country. PMID:25706423

  12. Comparative performance of isolation methods using Preston broth, Bolton broth and their modifications for the detection of Campylobacter spp. from naturally contaminated fresh and frozen raw poultry meat.

    PubMed

    Seliwiorstow, T; De Zutter, L; Houf, K; Botteldoorn, N; Baré, J; Van Damme, I

    2016-10-01

    The performance of different isolation methods was evaluated for the detection of Campylobacter from naturally contaminated raw poultry meat. Therefore, fresh and frozen poultry meat samples were analysed using the standard procedure (ISO 10272-1:2006), enrichment in Preston broth, and enrichment in modified Bolton broth (supplemented with (i) potassium clavulanate (C-BB), (ii) triclosan (T-BB), (iii) polymyxin B (P-BB)). The enrichment cultures were streaked onto both modified charcoal cefoperazone deoxycholate agar (mCCDA) and RAPID'Campylobacter agar (RCA). Moreover, direct plating on mCCDA and RCA was performed to quantify Campylobacter. In total, 33 out of 59 fresh retail meat samples (55.9%) were Campylobacter positive. For both fresh and frozen poultry meat samples, enrichment in Bolton broth (ISO 10272-1:2006) resulted in a higher number of positive samples than enrichment in Preston broth. Supplementation of Bolton broth with potassium clavulanate (C-BB) and triclosan (T-BB) enhanced the Campylobacter recovery from fresh poultry meat compared to non-supplemented Bolton broth, although the use of C-BB was less applicable than T-BB for Campylobacter recovery from frozen samples. Additionally, the use of RCA resulted in a higher isolation rate compared to mCCDA. The present study demonstrates the impact of culture medium on the recovery of Campylobacter from fresh and frozen naturally contaminated poultry meat samples and can support laboratories in choosing the most appropriate culturing method to detect Campylobacter. PMID:27391222

  13. GLUCOSE METABOLITE PATTERNS AS MARKERS OF FUNCTIONAL DIFFERENTIATION IN FRESHLY ISOLATED AND CULTURED MOUSE MAMMARY EPITHELIAL CELLS

    SciTech Connect

    Emerman, J.T.; Bartley, J.C.; Bissell, M.J.

    1980-06-01

    In the mammary gland of nonruminant animals, glucose is utilized in a characteristic and unique way during lactation. We have measured the incorporation of glucose carbon from [U-{sup 14}C] glucose into intermediary metabolites and metabolic products in mammary epithelial cells from virgin, pregnant, and lactating mice and demonstrate that glucose metabolite patterns can be used to recognize stages of differentiated function. For these cells, the rates of synthesis of glycogen and lactose, the ratio of lactate to alanine, and the ratio of citrate to malate were important parameters in identifying the degree of expression of differentiation. We further show that these patterns can be used as markers to determine the differentiated state of cultured mammary epithelial cells. Cells maintained on plastic substrates lose their distinctive glucose metabolite patterns while those on floating collagen gels do not. Cells from pregnant mice have a pattern similar to freshly isolated cells from pregnant mice. The pattern of cells from lactating mice is different from that of the cells of origin, and resembles that of the cells from pregnant mice. Our findings suggest that the floating collagen gels under the culture conditions used in these experiments provide an environment for the functional expression of the pregnant state, while additional factors are needed for the expression of the lactating state.

  14. Involvement of oxidative stress and mitochondrial/lysosomal cross-talk in olanzapine cytotoxicity in freshly isolated rat hepatocytes.

    PubMed

    Eftekhari, Aziz; Azarmi, Yadollah; Parvizpur, Alireza; Eghbal, Mohammad Ali

    2016-04-01

    1. Olanzapine (OLZ) is a widely used atypical antipsychotic agent for the treatment of schizophrenia and other disorders. Serious hepatotoxicity and elevated liver enzymes have been reported in patients receiving OLZ. However, the cellular and molecular mechanisms of the OLZ hepatotoxicity are unknown. 2. In this study, the cytotoxic effect of OLZ on freshly isolated rat hepatocytes was assessed. Our results showed that the cytotoxicity of OLZ in hepatocytes is mediated by overproduction of reactive oxygen species (ROS), mitochondrial potential collapse, lysosomal membrane leakiness, GSH depletion and lipid peroxidation preceding cell lysis. All the aforementioned OLZ-induced cellular events were significantly (p < 0.05) prevented by ROS scavengers, antioxidants, endocytosis inhibitors and adenosine triphosphate generators. Also, the present results demonstrated that CYP450 is involved in OLZ-induced oxidative stress and cytotoxicity mechanism. 3. It is concluded that OLZ hepatotoxicity is associated with both mitochondrial/lysosomal involvement following the initiation of oxidative stress in hepatocytes. PMID:26364812

  15. Characterization of Listeria monocytogenes isolated from a fresh mixed sausage processing line in Pelotas-RS by PFGE

    PubMed Central

    von Laer, Ana Eucares; de Lima, Andréia Saldanha; Trindade, Paula dos Santos; Andriguetto, Cristiano; Destro, Maria Teresa; da Silva, Wladimir Padilha

    2009-01-01

    Listeria monocytogenes is a bacterium capable to adhere to the surfaces of equipment and utensils and subsequently form biofilms. It can to persist in the food processing environmental for extended periods of time being able to contaminate the final product. The aim of this study was to trace the contamination route of L. monocytogenes on a fresh mixed sausage processing line, from raw material to the final product. The isolates obtained were characterized by serotyping and molecular typing by pulsed-field gel electrophoresis (PFGE) using the restriction enzymes ApaI and AscI. L. monocytogenes was detected in 25% of the samples. The samples of raw material were not contaminated, however, the microorganism was detected in 21% of the environmental samples (food contact and non-food contact), 20.8% of the equipments, 20% of the food worker’s hands, 40% of the mass ready to packaging and in all the final products samples, demonstrating that the contamination of final product occurred during the processing and the importance of cross contamination. PFGE yielded 22 pulsotypes wich formed 7 clusters, and serotyping yielded 3 serotypes and 1 serogroup, however, the presence of serotypes 4b and 1/2b in the final product is of great concern for public health. The tracing of contamination showed that some strains are adapted and persisted in the processing environment in this industry. PMID:24031402

  16. Transmission blocking activity of Azadirachta indica and Guiera senegalensis extracts on the sporogonic development of Plasmodium falciparum field isolates in Anopheles coluzzii mosquitoes

    PubMed Central

    2014-01-01

    Background Targeting the stages of the malaria parasites responsible for transmission from the human host to the mosquito vector is a key pharmacological strategy for malaria control. Research efforts to identify compounds that are active against these stages have significantly increased in recent years. However, at present, only two drugs are available, namely primaquine and artesunate, which reportedly act on late stage gametocytes. Methods In this study, we assessed the antiplasmodial effects of 5 extracts obtained from the neem tree Azadirachta indica and Guiera senegalensis against the early vector stages of Plasmodium falciparum, using field isolates. In an ex vivo assay gametocytaemic blood was supplemented with the plant extracts and offered to Anopheles coluzzii females by membrane feeding. Transmission blocking activity was evaluated by assessing oocyst prevalence and density on the mosquito midguts. Results Initial screening of the 5 plant extracts at 250 ppm revealed transmission blocking activity in two neem preparations. Up to a concentration of 70 ppm the commercial extract NeemAzal® completely blocked transmission and at 60 ppm mosquitoes of 4 out of 5 replicate groups remained uninfected. Mosquitoes fed on the ethyl acetate phase of neem leaves at 250 ppm showed a reduction in oocyst prevalence of 59.0% (CI95 12.0 - 79.0; p < 10-4) and in oocyst density of 90.5% (CI95 86.0 - 93.5; p < 10-4 ), while the ethanol extract from the same plant part did not exhibit any activity. No evidence of transmission blocking activity was found using G. senegalensis ethyl acetate extract from stem galls. Conclusions The results of this study highlight the potential of antimalarial plants for the discovery of novel transmission blocking molecules, and open up the potential of developing standardized transmission blocking herbal formulations as malaria control tools to complement currently used antimalarial drugs and combination treatments. PMID:24735564

  17. PKC independent inhibition of voltage gated calcium channels by volatile anesthetics in freshly isolated vascular myocytes from the aorta

    PubMed Central

    Fanchaouy, Mohammed; Cubano, Luis; Maldonado, Hector; Bychkov, Rostislav

    2013-01-01

    In this study we used barium currents through voltage gated L-type calcium channels (recorded in freshly isolated cells with a conventional patch-clamp technique) to elucidate the cellular action mechanism for volatile anesthetics. It was found that halothane and isoflurane inhibited (dose-dependently and voltage independently) Ba2+ currents through voltage gated Ca2+ channels. Half maximal inhibitions occurred at 0.64 ± 0.07 mM and 0.86 ± 0.1 mM. The Hill slope value was 2 for both volatile anesthetics, suggesting the presence of more than one interaction site. Current inhibition by volatile anesthetics was prominent over the whole voltage range without changes in the peak of the current voltage relationship. Intracellular infusion of the GDPßS (100 μM) together with staurosporine (200 nM) did not prevent the inhibitory effect of volatile anesthetics. Unlike pharmacological Ca2+ channel blockers, volatile anesthetics blocked Ca2+ channel currents at resting membrane potentials. In other words, halothane and isoflurane induced an `initial block'. After the first 4 to 7 control pulses, the cells were left unstimulated and anesthetics were applied. The first depolarization after the pause evoked a Ca2+ channel current whose amplitude was reduced to 41 ± 3.4% and to 57 ± 4.2% of control values. In an analysis of the steady-state inactivation curve for voltage dependence, volatile anesthetics induced a negative shift of the 50% inactivation of the calcium channels. By contrast, the steepness factor characterizing the voltage sensitivity of the channels was unaffected. Unitary L-type Ca2+ channels blockade occurred under cell-attached configuration, suggesting a possible action of volatile anesthetics from within the intracellular space or from the part of the channel inside the lipid bilayer. PMID:23948226

  18. Human CD34+ Progenitor Cells Freshly Isolated from Umbilical Cord Blood Attenuate Inflammatory Lung Injury following LPS Challenge

    PubMed Central

    Huang, Xiaojia; Sun, Kai; Zhao, Yidan D.; Vogel, Stephen M.; Song, Yuanling; Mahmud, Nadim; Zhao, You-Yang

    2014-01-01

    Adult stem cell-based therapy is a promising novel approach for treatment of acute lung injury. Here we investigated the therapeutic potential of freshly isolated human umbilical cord blood CD34+ progenitor cells (fCB-CD34+ cells) in a mouse model of acute lung injury. At 3 h post-lipopolysaccharide (LPS) challenge, fCB-CD34+ cells were transplanted i.v. to mice while CD34− cells or PBS were administered as controls in separate cohorts of mice. We observed that fCB-CD34+ cell treatment inhibited lung vascular injury evident by decreased lung vascular permeability. In contrast, CD34− cells had no effects on lung vascular injury. Lung inflammation determined by myeloperoxidase activity, neutrophil sequestration and expression of pro-inflammatory mediators was attenuated in fCB-CD34+ cell-treated mice at 26 h post-LPS challenge compared to PBS or CD34− cell-treated controls. Importantly, lung inflammation in fCB-CD34+ cell-treated mice was returned to normal levels as seen in basal mice at 52 h post-LPS challenge whereas PBS or CD34− cell-treated control mice exhibited persistent lung inflammation. Accordingly, fCB-CD34+ cell-treated mice exhibited a marked increase of survival rate. Employing in vivo 5-bromo-2′-deoxyuridine incorporation assay, we found a drastic induction of lung endothelial proliferation in fCB-CD34+ cell-treated mice at 52 h post-LPS compared to PBS or CD34− cell-treated controls, which contributed to restoration of vascular integrity and thereby inhibition of lung inflammation. Taken together, these data have demonstrated the protective effects of fCB-CD34+ cell on acute lung injury induced by LPS challenge, suggesting fCB-CD34+ cells are an important source of stem cells for the treatment of acute lung injury. PMID:24558433

  19. Stable CpG Hypomethylation of Adipogenic Promoters in Freshly Isolated, Cultured, and Differentiated Mesenchymal Stem Cells from Adipose Tissue

    PubMed Central

    Noer, Agate; Sørensen, Anita L.; Boquest, Andrew C.

    2006-01-01

    Mesenchymal stem cells from adipose tissue can differentiate into mesodermal lineages. Differentiation potential, however, varies between clones of adipose stem cells (ASCs), raising the hypothesis that epigenetic differences account for this variability. We report here a bisulfite sequencing analysis of CpG methylation of adipogenic (leptin [LEP], peroxisome proliferator-activated receptor gamma 2 [PPARG2], fatty acid-binding protein 4 [FABP4], and lipoprotein lipase [LPL]) promoters and of nonadipogenic (myogenin [MYOG], CD31, and GAPDH) loci in freshly isolated human ASCs and in cultured ASCs, in relation to gene expression and differentiation potential. Uncultured ASCs display hypomethylated adipogenic promoters, in contrast to myogenic and endothelial loci, which are methylated. Adipogenic promoters exhibit mosaic CpG methylation, on the basis of heterogeneous methylation between cells and of variation in the extent of methylation of a given CpG between donors, and both between and within clonal cell lines. DNA methylation reflects neither transcriptional status nor potential for gene expression upon differentiation. ASC culture preserves hypomethylation of adipogenic promoters; however, between- and within-clone mosaic methylation is detected. Adipogenic differentiation also maintains the overall CpG hypomethylation of LEP, PPARG2, FABP4, and LPL despite demethylation of specific CpGs and transcriptional induction. Furthermore, enhanced methylation at adipogenic loci in primary differentiated cells unrelated to adipogenesis argues for ASC specificity of the hypomethylated state of these loci. Therefore, mosaic hypomethylation of adipogenic promoters may constitute a molecular signature of ASCs, and DNA methylation does not seem to be a determinant of differentiation potential of these cells. PMID:16760426

  20. Comparison of Chemical Sensitivity of Fresh and Long-Stored Heat Resistant Neosartorya fischeri Environmental Isolates Using BIOLOG Phenotype MicroArray System.

    PubMed

    Panek, Jacek; Frąc, Magdalena; Bilińska-Wielgus, Nina

    2016-01-01

    Spoilage of heat processed food and beverage by heat resistant fungi (HRF) is a major problem for food industry in many countries. Neosartorya fischeri is the leading source of spoilage in thermally processed products. Its resistance to heat processing and toxigenicity makes studies about Neosartorya fischeri metabolism and chemical sensitivity essential. In this study chemical sensitivity of two environmental Neosartorya fischeri isolates were compared. One was isolated from canned apples in 1923 (DSM3700), the other from thermal processed strawberry product in 2012 (KC179765), used as long-stored and fresh isolate, respectively. The study was conducted using Biolog Phenotype MicroArray platforms of chemical sensitivity panel and traditional hole-plate method. The study allowed for obtaining data about Neosartorya fischeri growth inhibitors. The fresh isolate appeared to be much more resistant to chemical agents than the long-stored isolate. Based on phenotype microarray assay nitrogen compounds, toxic cations and membrane function compounds were the most effective in growth inhibition of N. fischeri isolates. According to the study zaragozic acid A, thallium(I) acetate and sodium selenate were potent and promising N. fischeri oriented fungicides which was confirmed by both chemical sensitivity microplates panel and traditional hole-plate methods. PMID:26815302

  1. Comparison of Chemical Sensitivity of Fresh and Long-Stored Heat Resistant Neosartorya fischeri Environmental Isolates Using BIOLOG Phenotype MicroArray System

    PubMed Central

    Panek, Jacek; Frąc, Magdalena; Bilińska-Wielgus, Nina

    2016-01-01

    Spoilage of heat processed food and beverage by heat resistant fungi (HRF) is a major problem for food industry in many countries. Neosartorya fischeri is the leading source of spoilage in thermally processed products. Its resistance to heat processing and toxigenicity makes studies about Neosartorya fischeri metabolism and chemical sensitivity essential. In this study chemical sensitivity of two environmental Neosartorya fischeri isolates were compared. One was isolated from canned apples in 1923 (DSM3700), the other from thermal processed strawberry product in 2012 (KC179765), used as long-stored and fresh isolate, respectively. The study was conducted using Biolog Phenotype MicroArray platforms of chemical sensitivity panel and traditional hole-plate method. The study allowed for obtaining data about Neosartorya fischeri growth inhibitors. The fresh isolate appeared to be much more resistant to chemical agents than the long-stored isolate. Based on phenotype microarray assay nitrogen compounds, toxic cations and membrane function compounds were the most effective in growth inhibition of N. fischeri isolates. According to the study zaragozic acid A, thallium(I) acetate and sodium selenate were potent and promising N. fischeri oriented fungicides which was confirmed by both chemical sensitivity microplates panel and traditional hole-plate methods. PMID:26815302

  2. Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy

    PubMed Central

    Ndong Ngomo, Jacques-Mari; Mawili-Mboumba, Denise Patricia; M'Bondoukwe, Noé Patrick; Nikiéma Ndong Ella, Rosalie; Bouyou Akotet, Marielle Karine

    2016-01-01

    In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon. PMID:27190671

  3. Increased Prevalence of Mutant Allele Pfdhps 437G and Pfdhfr Triple Mutation in Plasmodium falciparum Isolates from a Rural Area of Gabon, Three Years after the Change of Malaria Treatment Policy.

    PubMed

    Ndong Ngomo, Jacques-Mari; Mawili-Mboumba, Denise Patricia; M'Bondoukwe, Noé Patrick; Nikiéma Ndong Ella, Rosalie; Bouyou Akotet, Marielle Karine

    2016-01-01

    In Gabon, sulfadoxine-pyrimethamine (SP) is recommended for intermittent preventive treatment during pregnancy (IPTp-SP) and for uncomplicated malaria treatment through ACTs drug. P. falciparum strains resistant to SP are frequent in areas where this drug is highly used and is associated with the occurrence of mutations on Plasmodium falciparum dihydrofolate reductase (Pfdhfr) and dihydropteroate synthetase (Pfdhps) genes. The aim of the study was to compare the proportion of mutations on Pfdhfr and Pfdhps genes in isolates collected at Oyem in northern Gabon, in 2005 at the time of IPTp-SP introduction and three years later. Point mutations were analyzed by nested PCR-RFLP method. Among 91 isolates, more than 90% carried Pfdhfr 108N and Pfdhfr 59R alleles. Frequencies of Pfdhfr 51I (98%) and Pfdhps 437G (67.7%) mutant alleles were higher in 2008. Mutations at codons 164, 540, and 581 were not detected. The proportion of the triple Pfdhfr mutation and quadruple mutation including A437G was high: 91.9% in 2008 and 64.8% in 2008, respectively. The present study highlights an elevated frequency of Pfdhfr and Pfdhps mutant alleles, although quintuple mutations were not found in north Gabon. These data suggest the need of a continuous monitoring of SP resistance in Gabon. PMID:27190671

  4. Genetic distance in housekeeping genes between Plasmodium falciparum and Plasmodium reichenowi and within P. falciparum.

    PubMed

    Tanabe, Kazuyuki; Sakihama, Naoko; Hattori, Tetsuya; Ranford-Cartwright, Lisa; Goldman, Ira; Escalante, Ananias A; Lal, Altaf A

    2004-11-01

    The time to the most recent common ancestor of the extant populations of Plasmodium falciparum is controversial. The controversy primarily stems from the limited availability of sequences from Plasmodium reichenowi, a chimpanzee malaria parasite closely related to P. falciparum. Since the rate of nucleotide substitution differs in different loci and DNA regions, the estimation of genetic distance between P. falciparum and P. reichenowi should be performed using orthologous sequences that are evolving neutrally. Here, we obtained full-length sequences of two housekeeping genes, sarcoplasmic and endoplasmic reticulum Ca2+ -ATPase (serca) and lactate dehydrogenase (ldh), from 11 isolates of P. falciparum and 1 isolate of P. reichenowi and estimate the interspecific genetic distance (divergence) between the two species and intraspecific genetic distance (polymorphism) within P. falciparum. Interspecific distance and intraspecific distance at synonymous sites of interspecies-conserved regions of serca and ldh were 0.0672 +/- 0.0088 and 0.0011 +/- 0.0007, respectively, using the Nei and Gojobori method. Based on the ratio of interspecific distance to intraspecific distance, the time to the most recent common ancestor of P. falciparum was estimated to be (8.30 +/- 5.40) x 10(4) and (11.62 +/- 7.56) x 10(4) years ago, assuming the divergence time of the two parasite species to be 5 and 7 million years ago, respectively. PMID:15693624

  5. The MSPDBL2 codon 591 polymorphism is associated with lumefantrine in vitro drug responses in Plasmodium falciparum isolates from Kilifi, Kenya.

    PubMed

    Ochola-Oyier, Lynette Isabella; Okombo, John; Mwai, Leah; Kiara, Steven M; Pole, Lewa; Tetteh, Kevin K A; Nzila, Alexis; Marsh, Kevin

    2015-03-01

    The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). PMID:25534732

  6. The MSPDBL2 Codon 591 Polymorphism Is Associated with Lumefantrine In Vitro Drug Responses in Plasmodium falciparum Isolates from Kilifi, Kenya

    PubMed Central

    Okombo, John; Mwai, Leah; Kiara, Steven M.; Pole, Lewa; Tetteh, Kevin K. A.; Nzila, Alexis; Marsh, Kevin

    2014-01-01

    The mechanisms of drug resistance development in the Plasmodium falciparum parasite to lumefantrine (LUM), commonly used in combination with artemisinin, are still unclear. We assessed the polymorphisms of Pfmspdbl2 for associations with LUM activity in a Kenyan population. MSPDBL2 codon 591S was associated with reduced susceptibility to LUM (P = 0.04). The high frequency of Pfmspdbl2 codon 591S in Kenya may be driven by the widespread use of lumefantrine in artemisinin combination therapy (Coartem). PMID:25534732

  7. Febrile temperatures induce cytoadherence of ring-stage Plasmodium falciparum-infected erythrocytes.

    PubMed

    Udomsangpetch, Rachanee; Pipitaporn, Busaba; Silamut, Kamolrat; Pinches, Robert; Kyes, Sue; Looareesuwan, Sornchai; Newbold, Christopher; White, Nicholas J

    2002-09-01

    In falciparum malaria, the malaria parasite induces changes at the infected red blood cell surface that lead to adherence to vascular endothelium and other red blood cells. As a result, the more mature stages of Plasmodium falciparum are sequestered in the microvasculature and cause vital organ dysfunction, whereas the ring stages circulate in the blood stream. Malaria is characterized by fever. We have studied the effect of febrile temperatures on the cytoadherence in vitro of P. falciparum-infected erythrocytes. Freshly obtained ring-stage-infected red blood cells from 10 patients with acute falciparum malaria did not adhere to the principle vascular adherence receptors CD36 or intercellular adhesion molecule-1 (ICAM-1). However, after a brief period of heating to 40 degrees C, all ring-infected red blood cells adhered to CD36, and some isolates adhered to ICAM-1, whereas controls incubated at 37 degrees C did not. Heating to 40 degrees C accelerated cytoadherence and doubled the maximum cytoadherence observed (P < 0.01). Erythrocytes infected by ring-stages of the ICAM-1 binding clone A4var also did not cytoadhere at 37 degrees C, but after heating to febrile temperatures bound to both CD36 and ICAM-1. Adherence of red blood cells infected with trophozoites was also increased considerably by brief heating. The factor responsible for heat induced adherence was shown to be the parasite derived variant surface protein PfEMP-1. RNA analysis showed that levels of var mRNA did not differ between heated and unheated ring-stage parasites. Thus fever-induced adherence appeared to involve increased trafficking of PfEMP-1 to the erythrocyte membrane. Fever induced cytoadherence is likely to have important pathological consequences and may explain both clinical deterioration with fever in severe malaria and the effects of antipyretics on parasite clearance. PMID:12177447

  8. Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria

    PubMed Central

    Imwong, Mallika; Woodrow, Charles J.; Hendriksen, Ilse C. E.; Veenemans, Jacobien; Verhoef, Hans; Faiz, M. Abul; Mohanty, Sanjib; Mishra, Saroj; Mtove, George; Gesase, Samwel; Seni, Amir; Chhaganlal, Kajal D.; Day, Nicholas P. J.; Dondorp, Arjen M.; White, Nicholas J.

    2015-01-01

    In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples. PMID:25344520

  9. Artesunate Misuse and Plasmodium falciparum Malaria in Traveler Returning from Africa

    PubMed Central

    Shahinas, Dea; Lau, Rachel; Khairnar, Krishna; Hancock, David

    2010-01-01

    Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility. PMID:20875291

  10. Artesunate misuse and Plasmodium falciparum malaria in traveler returning from Africa.

    PubMed

    Shahinas, Dea; Lau, Rachel; Khairnar, Krishna; Hancock, David; Pillai, Dylan R

    2010-10-01

    Plasmodium falciparum malaria developed in an African-born traveler who returned to Canada after visiting Nigeria. While there, she took artesunate prophylactically. Isolates had an elevated 50% inhibitory concentration to artemisinin, artesunate, and artemether, compared with that of other African isolates. Inappropriate use of artemisinin derivatives can reduce P. falciparum susceptibility. PMID:20875291

  11. Purification of Immune Cell Populations from Freshly Isolated Murine Tumors and Organs by Consecutive Magnetic Cell Sorting and Multi-parameter Flow Cytometry-Based Sorting.

    PubMed

    Salvagno, Camilla; de Visser, Karin E

    2016-01-01

    It is well established that tumors evolve together with nonmalignant cells, such as fibroblasts, endothelial cells, and immune cells. These cells constantly entangle and interact with each other creating the tumor microenvironment. Immune cells can exert both tumor-promoting and tumor-protective functions. Detailed phenotypic and functional characterization of intra-tumoral immune cell subsets has become increasingly important in the field of cancer biology and cancer immunology. In this chapter, we describe a method for isolation of viable and pure immune cell subsets from freshly isolated murine solid tumors and organs. First, we describe a protocol for the generation of single-cell suspensions from tumors and organs using mechanical and enzymatic strategies. In addition, we describe how immune cell subsets can be purified by consecutive magnetic cell sorting and multi-parameter flow cytometry-based cell sorting. PMID:27581019

  12. Transfer and Detection of Freshly Isolated or Cultured Chicken (Gallus gallus) and Exotic Species’ Embryonic Gonadal Germ Stem Cells in Host Embryos

    PubMed Central

    Imus, Nastassja; Roe, Mandi; Charter, Suellen; Durrant, Barbara; Jensen, Thomas

    2015-01-01

    The management of captive avian breeding programs increasingly utilizes various artificial reproductive technologies, including in ova sexing of embryos to adjust population sex ratios. Currently, however, no attention has been given to the loss of genetic diversity following sex-selective incubation, even with respect to individuals from critically endangered species. This project evaluated the possibility of using xenotransfer of embryonic gonadal germline stem cells (GGCs) for future reintroduction of their germplasm into the gene pool. We examined and compared the host gonad colonization of freshly isolated and 3 day (3d) cultured donor GGCs from chicken and 13 species of exotic embryos. Following 3d-culture of GGCs, there was a significant increase in the percentage of stem cell marker (SSEA-1, -3, -4) positive cells. However, the percentage of positive host gonads with chicken donor-derived cells decreased from 68% (fresh) to 22% (3d), while the percentage of exotic species donor-cells positive host gonads decreased from 61% (fresh) to 49% (3d-cultured). Donor GGCs from both chicken and exotic species were localized within the caudal endoderm, including the region encompassing the gonadal ridge by 16 hours post-injection. Furthermore, donor-derived cells isolated from stage 36 host embryos were antigenic for anti SSEA-1, VASA/DDX4 and EMA-1 antibodies, presumably indicating maintenance of stem cell identity. This study demonstrates that GGCs from multiple species can migrate to the gonadal region and maintain presumed stemness following xenotransfer into a chicken host embryo, suggesting that germline stem cell migration is highly conserved in birds. PMID:24882096

  13. Antiplasmodial activity-aided isolation and identification of quercetin-4'-methyl ether in Chromolaena odorata leaf fraction with high activity against chloroquine-resistant Plasmodium falciparum.

    PubMed

    Ezenyi, I C; Salawu, O A; Kulkarni, R; Emeje, M

    2014-12-01

    The present study was undertaken to evaluate the antiplasmodial activity of Chromolaena odorata leaf extract and gradient fractions through in vivo and in vitro tests, aimed at identifying its antiplasmodial constituents. Sub-fractions obtained from the most active gradient fraction were further tested for cytotoxicity against THP-1 cells, chloroquine-sensitive (HB3) and chloroquine-resistant (FCM29) Plasmodium falciparum. Our results showed the dichloromethane gradient fraction was most effective, significantly (P < 0.05) suppressing infection by 99.46% at 100 mg/kg body weight. Amongst its 13 sub-fractions (DF1-DF13), DF11 was highly active, with IC50 of 4.8 and 6.74 μg/ml against P. falciparum HB3 and FCM29, respectively. Cytotoxicity of DF11 was estimated to be above 50 μg/ml, and its separation by column chromatography yielded a flavonoid which was characterized as 3, 5, 7, 3' tetrahydroxy-4'-methoxyflavone from its spectroscopic data. It significantly suppressed infection (65.43-81.48%) in mice at 2.5-5 mg/kg doses and compared favourably with the effects of chloroquine and artemisinin. It may therefore serve as a useful phytochemical and antiplasmodial activity marker of C. odorata leaves, which exhibit potential for development as medicine against malaria. PMID:25199554

  14. Whole-Genome Sequences of Mycobacterium bovis Strain MbURU-001, Isolated from Fresh Bovine Infected Samples

    PubMed Central

    Lasserre, Moira; Berná, Luisa; Greif, Gonzalo; Díaz-Viraqué, Florencia; Iraola, Gregorio; Naya, Hugo; Castro-Ramos, Miguel; Juambeltz, Arturo

    2015-01-01

    Bovine tuberculosis in cattle has a high incidence in Uruguay, where it is considered a disease of national importance. We present the genome sequence of Mycobacterium bovis strain MbURU-001, isolated from pectoral lymph nodes of a bovine host from a cattle farm. PMID:26543108

  15. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese.

    PubMed

    Furtado, Danielle N; Todorov, Svetoslav D; Landgraf, Mariza; Destro, Maria T; Franco, Bernadette D G M

    2015-03-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  16. Bacteriocinogenic Lactococcus lactis subsp. lactis DF04Mi isolated from goat milk: Application in the control of Listeria monocytogenes in fresh Minas-type goat cheese

    PubMed Central

    Furtado, Danielle N.; Todorov, Svetoslav D.; Landgraf, Mariza; Destro, Maria T.; Franco, Bernadette D.G.M.

    2015-01-01

    Listeria monocytogenes is a pathogen frequently found in dairy products. Its control in fresh cheeses is difficult, due to the psychrotrophic properties and salt tolerance. Bacteriocinogenic lactic acid bacteria (LAB) with proven in vitro antilisterial activity can be an innovative technological approach but their application needs to be evaluated by means of in situ tests. In this study, a novel bacteriocinogenic Lactococcus lactis strain ( Lc . lactis DF4Mi), isolated from raw goat milk, was tested for control of growth of L. monocytogenes in artificially contaminated fresh Minas type goat cheese during storage under refrigeration. A bacteriostatic effect was achieved, and counts after 10 days were 3 log lower than in control cheeses with no added LAB. However, this effect did not differ significantly from that obtained with a non-bacteriocinogenic Lc. lactis strain. Addition of nisin (12.5 mg/kg) caused a rapid decrease in the number of viable L. monocytogenes in the cheeses, suggesting that further studies with the purified bacteriocin DF4Mi may open new possibilities for this strain as biopreservative in dairy products. PMID:26221109

  17. The first evidence of paralytic shellfish toxins in the fresh water cyanobacterium Cylindrospermopsis raciborskii, isolated from Brazil.

    PubMed

    Lagos, N; Onodera, H; Zagatto, P A; Andrinolo, D; Azevedo, S M; Oshima, Y

    1999-10-01

    The blooms of toxic cyanobacteria (blue-green algae) are causing problems in many countries. During a screening of toxic freshwater cyanobacteria in Brazil, three strains isolated from the State of Sao Paulo were found toxic by the mouse bioassay. They all were identified as Cylindrospermopsis raciborskii by a close morphological examination. Extracts of cultured cells caused acute death to mice when injected intraperitoneally after developing neurotoxic symptoms which resembled to those caused by paralytic shellfish toxins. The analysis of the sample by HPLC-FLD postcolumn derivatization method for paralytic shellfish toxins resulted in the detection of several saxitoxin analogs. To avoid being misled by false peaks, the sample was reanalyzed after purification and also under the different postcolumn derivatizing conditions. Finally, the newly developed LC-MS method for paralytic shellfish toxins was applied to unambiguously identify the toxins. One isolate produced neosaxitoxin predominantly with saxitoxin as a minor component. The other two showed identical toxin profiles containing saxitoxin and gonyautoxins 2/3 isomers in the ratio of 1:9. This is the first evidence of paralytic shellfish toxins in this species and also the occurrence of the toxin producing cyanobacterium in South American countries. PMID:10414862

  18. Iodinated contrast media inhibit oxygen consumption in freshly isolated proximal tubular cells from elderly humans and diabetic rats: Influence of nitric oxide

    PubMed Central

    Liss, Per; Hansell, Peter; Fasching, Angelica; Palm, Fredrik

    2016-01-01

    Objectives Mechanisms underlying contrast medium (CM)-induced nephropathy remain elusive, but recent attention has been directed to oxygen availability. The purpose of this study was to evaluate the effect of the low-osmolar CM iopromide and the iso-osmolar CM iodixanol on oxygen consumption (QO2) in freshly isolated proximal tubular cells (PTC) from kidneys ablated from elderly humans undergoing nephrectomy for renal carcinomas and from normoglycemic or streptozotocin-diabetic rats. Materials PTC were isolated from human kidneys, or kidneys of normoglycemic or streptozotocin-diabetic rats. QO2 was measured with Clark-type microelectrodes in a gas-tight chamber with and without each CM (10 mg I/mL medium). L-NAME was used to inhibit nitric oxide (NO) production caused by nitric oxide synthase. Results Both CM reduced QO2 in human PTC (about –35%) which was prevented by L-NAME. PTC from normoglycemic rats were unaffected by iopromide, whereas iodixanol decreased QO2 (–34%). Both CM decreased QO2 in PTC from diabetic rats (–38% and –36%, respectively). L-NAME only prevented the effect of iopromide in the diabetic rat PTC. Conclusions These observations demonstrate that CM can induce NO release from isolated PTC in vitro, which affects QO2. Our results suggest that the induction of NO release and subsequent effect on the cellular oxygen metabolism are dependent on several factors, including CM type and pre-existing risk factors for the development of CM-induced nephropathy. PMID:26933994

  19. Infusion of freshly isolated autologous bone marrow derived mononuclear cells prevents endotoxin-induced lung injury in an ex-vivo perfused swine model

    PubMed Central

    2013-01-01

    Introduction The acute respiratory distress syndrome (ARDS), affects up to 150,000 patients per year in the United States. We and other groups have demonstrated that bone marrow derived mesenchymal stromal stem cells prevent ARDS induced by systemic and local administration of endotoxin (lipopolysaccharide (LPS)) in mice. Methods A study was undertaken to determine the effects of the diverse populations of bone marrow derived cells on the pathophysiology of ARDS, using a unique ex-vivo swine preparation, in which only the ventilated lung and the liver are perfused with autologous blood. Six experimental groups were designated as: 1) endotoxin alone, 2) endotoxin + total fresh whole bone marrow nuclear cells (BMC), 3) endotoxin + non-hematopoietic bone marrow cells (CD45 neg), 4) endotoxin + hematopoietic bone marrow cells (CD45 positive), 5) endotoxin + buffy coat and 6) endotoxin + in vitro expanded swine CD45 negative adherent allogeneic bone marrow cells (cultured CD45neg). We measured at different levels the biological consequences of the infusion of the different subsets of cells. The measured parameters were: pulmonary vascular resistance (PVR), gas exchange (PO2), lung edema (lung wet/dry weight), gene expression and serum concentrations of the pro-inflammatory cytokines IL-1β, TNF-α and IL-6. Results Infusion of freshly purified autologous total BMCs, as well as non-hematopoietic CD45(-) bone marrow cells significantly reduced endotoxin-induced pulmonary hypertension and hypoxemia and reduced the lung edema. Also, in the groups that received BMCs and cultured CD45neg we observed a decrease in the levels of IL-1β and TNF-α in plasma. Infusion of hematopoietic CD45(+) bone marrow cells or peripheral blood buffy coat cells did not protect against LPS-induced lung injury. Conclusions We conclude that infusion of freshly isolated autologous whole bone marrow cells and the subset of non-hematopoietic cells can suppress the acute humoral and physiologic

  20. ASIC-like Currents in Freshly Isolated Cerebral Artery Smooth Muscle Cells are Inhibited by Endogenous Oxidase Activity

    PubMed Central

    Chung, Wen-Shuo; Farley, Jerry M.; Drummond, Heather A.

    2011-01-01

    Background/Aims: The aim of this study was to determine if VSMC ASIC-like currents are regulated by oxidative state. Methods: We used whole-cell patch clamp of isolated mouse cerebral VSMCs to determine if 1) reducing agents, such as DTT and GSH, and 2) inhibition of endogenous oxidase activity from NADPH and Xanthine oxidases potentiate active currents and activate electrically silent currents. Results: Pretreatment with 2 mM DTT or GSH, increased the mean peak amplitude of ASIC-like currents evoked by pH 6.0 from 0.4 ± 0.1 to 14.9 ± 3.6 pA/pF, and from 0.9 ± 0.3 to 11.3 ± 2.4 pA/pF, respectively. Pretreatment with apocynin, a NADPH oxidase inhibitor, mimics the effect of the reducing agents, with the mean peak current amplitude increased from 0.9 ± 0.5 to 7.0 ± 2.6 pA/pF and from 0.5 ± 0.2 to 26.4 ± 6.8 pA/pF by 50 and 200 μM apocynin, respectively. Pretreatment with allopurinol, a xanthine oxidase inhibitor, also potentiates the VSMC ASIC-like activity. Conclusion: These findings suggest that VSMC ASIC-like channels are regulated by oxidative state and may be inhibited by basal endogenous oxidative sources such as NADPH and xanthine oxidase. PMID:21325830

  1. Pharmacological characterization of the P2 receptors profile in the podocytes of the freshly isolated rat glomeruli

    PubMed Central

    Ilatovskaya, Daria V.; Palygin, Oleg; Levchenko, Vladislav

    2013-01-01

    Calcium flux in the podocytes is critical for normal and pathophysiological regulation of these types of cells, and excessive calcium signaling results in podocytes damage and improper glomeruli function. Purinergic activation of P2 receptors is a powerful and rapid signaling process; however, the exact physiological identity of P2 receptors subtypes in podocytes remains essentially unknown. The goal of this study was to determine the P2 receptor profile in podocytes of the intact Sprague-Dawley rat glomeruli using available pharmacological tools. Glomeruli were isolated by differential sieving and loaded with Fluo-4/Fura Red cell permeable calcium indicators, and the purinergic response in the podocytes was analyzed with ratiometric confocal fluorescence measurements. Various P2 receptors activators were tested and compared with the effect of ATP, specifically, UDP, MRS 2365, bzATP, αβ-methylene, 2-meSADP, MRS 4062, and MRS 2768, were analyzed. Antagonists (MRS 2500, 5-BDBD, A438079, and NF 449) were tested when 10 μM ATP was applied as the EC50 for ATP activation of the calcium influx in the podocytes was determined to be 10.7 ± 1.5 μM. Several agonists including MRS 2365 and 2-meSADP caused calcium flux. Importantly, only the P2Y1-specific antagonist MRS 2500 (1 nM) precluded the effects of ATP concentrations of the physiological range. Immunohistochemical analysis confirmed that P2Y1 receptors are highly expressed in the podocytes. We conclude that P2Y1 receptor signaling is the predominant P2Y purinergic pathway in the glomeruli podocytes and P2Y1 might be involved in the pathogenesis of glomerular injury and could be a target for treatment of kidney diseases. PMID:24048730

  2. Effect of whey protein isolate-pullulan edible coatings on the quality and shelf life of freshly roasted and freeze-dried Chinese chestnut.

    PubMed

    Gounga, M E; Xu, S-Y; Wang, Z; Yang, W G

    2008-05-01

    Harvested chestnut is characterized by a short shelf life, exposing many Chinese producers to a storage problem as product losses are very high. The objective of this study was to develop a suitable technology to extend the shelf life of harvested chestnut fruits for commercial use. The effect of whey protein isolate-pullulan (WPI-Pul) coating on fresh-roasted chestnuts (FRC) and roasted freeze-dried chestnut (RFDC) quality and shelf life was studied under 2 different storage temperature (4 and 20 degrees C) conditions. Coatings were formed directly onto the surface of the fruits by dipping them into a film solution. SEM micrographs showed homogeneous WPI-Pul to cover the whole surface of chestnut with good adherence and perfect integrity. Moisture loss or gain, fruit quality, and shelf life were evaluated by weight loss or gain, surface color development, and visible decay during the storage period of 15 to 120 d at 4 and 20 degrees C, respectively. WPI-Pul coating had a low, yet significant effect on reducing moisture loss and decay incidence of FRC, hence delaying changes in their external color. The results were satisfactory when the coating was done with freeze-drying at low temperature storage, thus improving the quality and increasing the shelf life. This provides an alternative strategy to minimize the significant losses in harvested chestnut. PMID:18460124

  3. Decreased glycation and structural protection properties of γ-glutamyl-S-allyl-cysteine peptide isolated from fresh garlic scales (Allium sativum L.).

    PubMed

    Tan, Dehong; Zhang, Yao; Chen, Lulu; Liu, Ling; Zhang, Xuan; Wu, Zhaoxia; Bai, Bing; Ji, Shujuan

    2015-01-01

    The antiglycative effect of γ-glutamyl-S-allyl-cysteine (GSAC) peptide isolated from fresh garlic scales was investigated in the bovine serum albumin (BSA)/glucose system. GSAC inhibited the increase of fluorescence intensity at about 440 nm in a concentration-dependent manner and reduced reacted free lysine side chains by 10.9%, 24.7% and 37.7%, as the GSAC concentrations increased from 0.1 to 2.5 mg mL(-1). Glycation-specific decline in BSA α-helix content (from 61.3% to 55.6%) and increase in β-sheet (from 2.1% to 5.4%) were prevented by GSAC (2.5 mg mL(-1)) in vitro, implying its stabilisation effect. GSAC treatment (2.5 mg mL(-1)) suppressed protein crosslinking to form polymers. Additionally, GSAC (10, 40, and 160 μg mL(-1)) showed radical-scavenging and metal-chelating capacities. In conclusion, GSAC has an antiglycative effect, which may involve its radical-scavenging and metal-chelating capacities. PMID:25631559

  4. The use of plant cell wall-degrading enzymes from newly isolated Penicillium ochrochloron Biourge for viscosity reduction in ethanol production with fresh sweet potato tubers as feedstock.

    PubMed

    Huang, Yuhong; Jin, Yanling; Shen, Weiliang; Fang, Yang; Zhang, Guohua; Zhao, Hai

    2013-12-12

    Penicillium ochrochloron Biourge, which was isolated from rotten sweet potato, can produce plant cell wall-degrading enzymes (PCWDEs) with high viscosity reducing capability for ethanol production using fresh sweet potato tubers as feedstock. The enzyme preparation was characterized by a broad enzyme spectrum including 13 kinds of enzymes with the activity to hydrolyze cellulose, hemicellulose, pectin, starch, and protein. The maximum viscosity-reducing capability was observed when the enzyme preparation was obtained after 5 days of fermentation using 20 g/L corncob as a sole carbon source, 4.5 g/L NH4 NO3 as a sole nitrogen source, and an initial medium pH of 6.5. The sweet potato mash treated with the enzyme preparation exhibited much higher fermentation efficiency (92.58%) compared with commercial cellulase (88.06%) and control (83.5%). The enzyme production was then scaled up to 0.5, 5, and 100 L, and the viscosity-reducing rates were found to be 85%, 90%, and 91%, respectively. Thus, P. ochrochloron Biourge displays potential viscosity-reducing capability for ethanol production. PMID:24329940

  5. In vitro drug sensitivity of Plasmodium falciparum in Acre, Brazil.

    PubMed Central

    Kremsner, P. G.; Zotter, G. M.; Feldmeier, H.; Graninger, W.; Kollaritsch, M.; Wiedermann, G.; Rocha, R. M.; Wernsdorfer, W. H.

    1989-01-01

    In Acre, the westernmost state of Brazil in the Amazon region, the sensitivity of Plasmodium falciparum to chloroquine, amodiaquine, mefloquine, quinine and sulfadoxine/pyrimethamine was determined in vitro by the Rieckmann microtechnique. The study was performed between January and June 1987; the in vitro parasite responses to all antimalarial drugs were determined according to the recommendations of WHO. Of 83 isolates of P. falciparum, all were sensitive to mefloquine and of 87 isolates of P. falciparum, 84 (97%) were sensitive to quinine. The EC50 for mefloquine was 0.27 mumol/l and for quinine 4.60 mumol/l. In contrast, 65 of 89 (73%) and 70 of 83 (84%) isolates were resistant to amodiaquine and chloroquine, respectively; 11 isolates even grew at 6.4 mumol chloroquine/l. The EC50 for amodiaquine was 0.34 mumol/l and for chloroquine 0.73 mumol/l. Sulfadoxine/pyrimethamine resistance was seen in 23 of 25 (92%) cases. These data clearly indicate that in the western part of the Amazon region the 4-aminoquinolines, as well as sulfadoxine/pyrimethamine, can no longer be recommended for the treatment of P. falciparum infections. PMID:2670298

  6. Calcium-activated chloride conductance in a pancreatic adenocarcinoma cell line of ductal origin (HPAF) and in freshly isolated human pancreatic duct cells.

    PubMed

    Winpenny, J P; Harris, A; Hollingsworth, M A; Argent, B E; Gray, M A

    1998-05-01

    Using the whole-cell patch-clamp technique, a calcium-activated chloride conductance (CACC) could be elicited in HPAF cells by addition of 1 microM ionomycin to the bath solution (66 +/- 22 pA/pF;Vm + 60 mV) or by addition of 1 microM calcium to the pipette solution (136 +/- 17 pA/pF; Vm + 60 mV). Both conductances had similar biophysical characteristics, including time-dependent inactivation at hyperpolarising potentials and a linear/slightly outwardly rectifying current/voltage (I/V) curve with a reversal potential (Erev) close to the calculated chloride equilibrium potential. The anion permeability sequence obtained from shifts in Erev was I > Br >/= Cl. 4,4'-Diisothiocyanatostilbene disulphonic acid (DIDS, 500 microM) caused a 13% inhibition of the current (Vm + 60 mV) while 100 microM glibenclamide, 30 nM TS-TM-calix[4]arene and 10 microM tamoxifen, all chloride channel blockers, had no marked effects (8%, -6% and -2% inhibition respectively). Niflumic acid (100 microM) caused a voltage-dependent inhibition of the current of 48% and 17% (Vm +/- 60 mV, respectively). In freshly isolated human pancreatic duct cells (PDCs) a CACC was elicited with 1 microM calcium in the pipette solution (260 +/- 62 pA/pF; Vm + 60 mV). The presence of this CACC in human PDCs could provide a possible therapeutic pathway for treatment of pancreatic insufficiency of the human pancreas in cystic fibrosis. PMID:9518508

  7. Biochemical and functional characterization of Plasmodium falciparum GTP cyclohydrolase I

    PubMed Central

    2014-01-01

    Background Antifolates are currently in clinical use for malaria preventive therapy and treatment. The drugs kill the parasites by targeting the enzymes in the de novo folate pathway. The use of antifolates has now been limited by the spread of drug-resistant mutations. GTP cyclohydrolase I (GCH1) is the first and the rate-limiting enzyme in the folate pathway. The amplification of the gch1 gene found in certain Plasmodium falciparum isolates can cause antifolate resistance and influence the course of antifolate resistance evolution. These findings showed the importance of P. falciparum GCH1 in drug resistance intervention. However, little is known about P. falciparum GCH1 in terms of kinetic parameters and functional assays, precluding the opportunity to obtain the key information on its catalytic reaction and to eventually develop this enzyme as a drug target. Methods Plasmodium falciparum GCH1 was cloned and expressed in bacteria. Enzymatic activity was determined by the measurement of fluorescent converted neopterin with assay validation by using mutant and GTP analogue. The genetic complementation study was performed in ∆folE bacteria to functionally identify the residues and domains of P. falciparum GCH1 required for its enzymatic activity. Plasmodial GCH1 sequences were aligned and structurally modeled to reveal conserved catalytic residues. Results Kinetic parameters and optimal conditions for enzymatic reactions were determined by the fluorescence-based assay. The inhibitor test against P. falciparum GCH1 is now possible as indicated by the inhibitory effect by 8-oxo-GTP. Genetic complementation was proven to be a convenient method to study the function of P. falciparum GCH1. A series of domain truncations revealed that the conserved core domain of GCH1 is responsible for its enzymatic activity. Homology modelling fits P. falciparum GCH1 into the classic Tunnelling-fold structure with well-conserved catalytic residues at the active site. Conclusions

  8. Isoprenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Guggisberg, Ann M.; Amthor, Rachel E.

    2014-01-01

    Malaria kills nearly 1 million people each year, and the protozoan parasite Plasmodium falciparum has become increasingly resistant to current therapies. Isoprenoid synthesis via the methylerythritol phosphate (MEP) pathway represents an attractive target for the development of new antimalarials. The phosphonic acid antibiotic fosmidomycin is a specific inhibitor of isoprenoid synthesis and has been a helpful tool to outline the essential functions of isoprenoid biosynthesis in P. falciparum. Isoprenoids are a large, diverse class of hydrocarbons that function in a variety of essential cellular processes in eukaryotes. In P. falciparum, isoprenoids are used for tRNA isopentenylation and protein prenylation, as well as the synthesis of vitamin E, carotenoids, ubiquinone, and dolichols. Recently, isoprenoid synthesis in P. falciparum has been shown to be regulated by a sugar phosphatase. We outline what is known about isoprenoid function and the regulation of isoprenoid synthesis in P. falciparum, in order to identify valuable directions for future research. PMID:25217461

  9. Structure and expression of the Plasmodium falciparum SERA gene.

    PubMed

    Li, W B; Bzik, D J; Horii, T; Inselburg, J

    1989-02-01

    Plasmodium falciparum, strain FCR3, genomic DNA that encodes the SERA gene of P. falciparum was isolated and sequenced. The SERA gene coding region was interrupted by 3 introns, the largest number observed, so far, in any Plasmodium gene. Two SERA gene alleles, allele I and allele II, were identified in the FCR3 strain, while only allele I was found in the Honduras-1 strain. Allele I mRNA was abundant in vivo during the late trophozoite and schizont stages. Allele II mRNA was either not expressed, or it was labile. PMID:2651911

  10. Multiple independent introductions of Plasmodium falciparum in South America

    PubMed Central

    Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J.; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N.; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J.; Renaud, François; Prugnolle, Franck

    2012-01-01

    The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence—archeological and genetic—suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

  11. Multiple independent introductions of Plasmodium falciparum in South America.

    PubMed

    Yalcindag, Erhan; Elguero, Eric; Arnathau, Céline; Durand, Patrick; Akiana, Jean; Anderson, Timothy J; Aubouy, Agnes; Balloux, François; Besnard, Patrick; Bogreau, Hervé; Carnevale, Pierre; D'Alessandro, Umberto; Fontenille, Didier; Gamboa, Dionicia; Jombart, Thibaut; Le Mire, Jacques; Leroy, Eric; Maestre, Amanda; Mayxay, Mayfong; Ménard, Didier; Musset, Lise; Newton, Paul N; Nkoghé, Dieudonné; Noya, Oscar; Ollomo, Benjamin; Rogier, Christophe; Veron, Vincent; Wide, Albina; Zakeri, Sedigheh; Carme, Bernard; Legrand, Eric; Chevillon, Christine; Ayala, Francisco J; Renaud, François; Prugnolle, Franck

    2012-01-10

    The origin of Plasmodium falciparum in South America is controversial. Some studies suggest a recent introduction during the European colonizations and the transatlantic slave trade. Other evidence--archeological and genetic--suggests a much older origin. We collected and analyzed P. falciparum isolates from different regions of the world, encompassing the distribution range of the parasite, including populations from sub-Saharan Africa, the Middle East, Southeast Asia, and South America. Analyses of microsatellite and SNP polymorphisms show that the populations of P. falciparum in South America are subdivided in two main genetic clusters (northern and southern). Phylogenetic analyses, as well as Approximate Bayesian Computation methods suggest independent introductions of the two clusters from African sources. Our estimates of divergence time between the South American populations and their likely sources favor a likely introduction from Africa during the transatlantic slave trade. PMID:22203975

  12. Developmental changes in Ca(2+)-uptake, Na+,Ca(2+)-exchange and Ca(2+)-ATPase in freshly isolated embryonic, newborn and adult chicken heart.

    PubMed

    Prakash, P; Meera, P; Tripathi, O

    1996-01-01

    Developmental changes in cellular Ca(2+)-transport mechanisms were studied in chick heart by determining cellular Ca(2+)-uptake and Na+,Ca(2+)-exchange activity in freshly isolated ventricular tissues of embryonic (5-18 days old), newborn (1-2 days old) and young adult (90-100 days old) heart by monitoring 45Ca influx. Ca(2+)-ATPase activity was determined in microsomal fractions at different stages of development. The Ca(2+)-uptake (per g wet tissue weight) increased with the development of embryonic as well as post-hatch chick heart, reaching a maximum in the young adult chicken. The overall increase in Ca(2+)-uptake, from embryonic day 5 to young-adult stage, was more than 3 fold. The Na+,Ca(2+)-exchange activity, determined as Na(+)-gradient-induced Ca(2+)-uptake in presence of either ouabain or zero [Na+]0, showed a 6-fold increase during development of heart from the embryonic day 5 to the young adult stage. Amiloride, an inhibitor of Na+,Ca(2+)-exchange, caused a dose-dependent reduction in a ouabain-induced rise in 45Ca influx at different stages of development. The inhibitory effect of amiloride was, however, greater during later stages of development. A progressive increase in Ca(2+)-ATPase activity was also seen during development. Ca(2+)-ATPase exhibited about a 4-fold increase in activity from embryonic day 7 to the young adult. The concomitant increase in Ca(2+)-uptake, Na+,Ca(2+)-exchange and Ca(2+)-ATPase activities suggests age-dependent changes in Ca(2+)-transport and storage systems of developing heart during embryogenesis and post-embryonic life. During embryogenesis the developmental increase in Na+,Ca(2+)-exchange activity was greater than that during post-hatch development of heart. However, the increase in Ca(2+)-ATPase activity was greater during post-hatch development than during embryogenesis. It is suggested that Na+,Ca(2+)-exchange and Ca(2+)-ATPase play a prominent role in maintaining cellular Ca2+ homeostasis during embryogenesis and

  13. DNA Cloning of Plasmodium falciparum Circumsporozoite Gene: Amino Acid Sequence of Repetitive Epitope

    NASA Astrophysics Data System (ADS)

    Enea, Vincenzo; Ellis, Joan; Zavala, Fidel; Arnot, David E.; Asavanich, Achara; Masuda, Aoi; Quakyi, Isabella; Nussenzweig, Ruth S.

    1984-08-01

    A clone of complementary DNA encoding the circumsporozoite (CS) protein of the human malaria parasite Plasmodium falciparum has been isolated by screening an Escherichia coli complementary DNA library with a monoclonal antibody to the CS protein. The DNA sequence of the complementary DNA insert encodes a four-amino acid sequence: proline-asparagine-alanine-asparagine, tandemly repeated 23 times. The CS β -lactamase fusion protein specifically binds monoclonal antibodies to the CS protein and inhibits the binding of these antibodies to native Plasmodium falciparum CS protein. These findings provide a basis for the development of a vaccine against Plasmodium falciparum malaria.

  14. Plasmodium falciparum: analysis of chromosomes separated by contour-clamped homogenous electric fields.

    PubMed

    Gu, H; Inselburg, J W; Bzik, D J; Li, W B

    1990-08-01

    We have established improved conditions for separating the chromosomes of Plasmodium falciparum by pulsed field gradient gel electrophoresis (PFG) using a contour-clamped homogenous electric field (CHEF) apparatus. Thirteen clearly separable chromosomal bands were reproducibly isolated from the strain FCR3 and their sizes have been determined. Evidence that indicates one band may contain two chromosomes is presented. The relationship between the PFG separable DNA and the number of unique chromosomes in P. falciparum is considered. We have established a relationship between the maximum resolvable sizes of the chromosomes and the pulse times. The chromosomal location of twenty-seven P. falciparum DNA probes is also reported. PMID:2197113

  15. Pooled Amplicon Deep Sequencing of Candidate Plasmodium falciparum Transmission-Blocking Vaccine Antigens.

    PubMed

    Juliano, Jonathan J; Parobek, Christian M; Brazeau, Nicholas F; Ngasala, Billy; Randrianarivelojosia, Milijaona; Lon, Chanthap; Mwandagalirwa, Kashamuka; Tshefu, Antoinette; Dhar, Ravi; Das, Bidyut K; Hoffman, Irving; Martinson, Francis; Mårtensson, Andreas; Saunders, David L; Kumar, Nirbhay; Meshnick, Steven R

    2016-01-01

    Polymorphisms within Plasmodium falciparum vaccine candidate antigens have the potential to compromise vaccine efficacy. Understanding the allele frequencies of polymorphisms in critical binding regions of antigens can help in the designing of strain-transcendent vaccines. Here, we adopt a pooled deep-sequencing approach, originally designed to study P. falciparum drug resistance mutations, to study the diversity of two leading transmission-blocking vaccine candidates, Pfs25 and Pfs48/45. We sequenced 329 P. falciparum field isolates from six different geographic regions. Pfs25 showed little diversity, with only one known polymorphism identified in the region associated with binding of transmission-blocking antibodies among our isolates. However, we identified four new mutations among eight non-synonymous mutations within the presumed antibody-binding region of Pfs48/45. Pooled deep sequencing provides a scalable and cost-effective approach for the targeted study of allele frequencies of P. falciparum candidate vaccine antigens. PMID:26503281

  16. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014

    PubMed Central

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar

    2016-01-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August–December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  17. Plasmodium falciparum In Vitro Resistance to Monodesethylamodiaquine, Dakar, Senegal, 2014.

    PubMed

    Fall, Bécaye; Madamet, Marylin; Camara, Cheikhou; Amalvict, Rémy; Fall, Mansour; Nakoulima, Aminata; Diatta, Bakary; Diémé, Yaya; Wade, Boubacar; Pradines, Bruno

    2016-05-01

    We successfully cultured 36 Plasmodium falciparum isolates from blood samples of 44 malaria patients admitted to the Hôpital Principal de Dakar (Dakar, Senegal) during August-December 2014. The prevalence of isolates with in vitro reduced susceptibility was 30.6% for monodesethylamodiaquine, 52.8% for chloroquine, 44.1% for mefloquine, 16.7% for doxycycline, 11.8% for piperaquine, 8.3% for artesunate, 5.9% for pyronaridine, 2.8% for quinine and dihydroartemisinin, and 0.0% for lumefantrine. The prevalence of isolates with reduced in vitro susceptibility to the artemisinin-based combination therapy partner monodesethylamodiaquine increased from 5.6% in 2013 to 30.6% in 2014. Because of the increased prevalence of P. falciparum parasites with impaired in vitro susceptibility to monodesethylamodiaquine, the implementation of in vitro and in vivo surveillance of all artemisinin-based combination therapy partners is warranted. PMID:27088703

  18. Induction of gene amplification in Plasmodium falciparum

    SciTech Connect

    Rogers, P.L.

    1985-01-01

    Human erythrocytic in vitro cultures of Honduras I strain of the malaria parasite Plasmodium falciparum have been stressed stepwise with increasing concentrations of methotrexate (MTX), a folate antagonist. This selection has produced a strain that is 450 times more resistant to the drug than the original culture. Uptake of sublethal doses of radiolabeled MTX by infected red blood cells was 6-36 times greater in the resistant cultures than in the nonresistant controls. DNA isolated from all of the parasites was probed by hybridization with /sup 35/S-labeled DNA derived from a clone of the yeast thymidylate synthetase (TS) gene. This showed 50 to 100 times more increased hybridization of the TS probe to the DNA from the resistant parasites is direct evidence of gene amplification because DHFR and TS are actually one and the same bifunctional enzyme in P. falciparum. Hence, the evidence presented indicates that induced resistance of the malaria parasite to MTX in this case is due to overproduction of DHFR resulting from amplification of the DHFR-TS gene.

  19. A genome-wide map of diversity in Plasmodium falciparum.

    PubMed

    Volkman, Sarah K; Sabeti, Pardis C; DeCaprio, David; Neafsey, Daniel E; Schaffner, Stephen F; Milner, Danny A; Daily, Johanna P; Sarr, Ousmane; Ndiaye, Daouda; Ndir, Omar; Mboup, Soulyemane; Duraisingh, Manoj T; Lukens, Amanda; Derr, Alan; Stange-Thomann, Nicole; Waggoner, Skye; Onofrio, Robert; Ziaugra, Liuda; Mauceli, Evan; Gnerre, Sante; Jaffe, David B; Zainoun, Joanne; Wiegand, Roger C; Birren, Bruce W; Hartl, Daniel L; Galagan, James E; Lander, Eric S; Wirth, Dyann F

    2007-01-01

    Genetic variation allows the malaria parasite Plasmodium falciparum to overcome chemotherapeutic agents, vaccines and vector control strategies and remain a leading cause of global morbidity and mortality. Here we describe an initial survey of genetic variation across the P. falciparum genome. We performed extensive sequencing of 16 geographically diverse parasites and identified 46,937 SNPs, demonstrating rich diversity among P. falciparum parasites (pi = 1.16 x 10(-3)) and strong correlation with gene function. We identified multiple regions with signatures of selective sweeps in drug-resistant parasites, including a previously unidentified 160-kb region with extremely low polymorphism in pyrimethamine-resistant parasites. We further characterized 54 worldwide isolates by genotyping SNPs across 20 genomic regions. These data begin to define population structure among African, Asian and American groups and illustrate the degree of linkage disequilibrium, which extends over relatively short distances in African parasites but over longer distances in Asian parasites. We provide an initial map of genetic diversity in P. falciparum and demonstrate its potential utility in identifying genes subject to recent natural selection and in understanding the population genetics of this parasite. PMID:17159979

  20. Plasmodium falciparum genetic crosses in a humanized mouse model

    PubMed Central

    Vaughan, Ashley M.; Pinapati, Richard S.; Cheeseman, Ian H.; Camargo, Nelly; Fishbaugher, Matthew; Checkley, Lisa A.; Nair, Shalini; Hutyra, Carolyn A.; Nosten, François H.; Anderson, Timothy J. C.; Ferdig, Michael T.; Kappe, Stefan H. I.

    2015-01-01

    Genetic crosses of phenotypically distinct strains of the human malaria parasite Plasmodium falciparum are a powerful tool for identifying genes controlling drug resistance and other key phenotypes. Previous studies relied on the isolation of recombinant parasites from splenectomized chimpanzees, a research avenue that is no longer available. Here, we demonstrate that human-liver chimeric mice support recovery of recombinant progeny for the identification of genetic determinants of parasite traits and adaptations. PMID:26030447

  1. Return of chloroquine-sensitive Plasmodium falciparum parasites and emergence of chloroquine-resistant Plasmodium vivax in Ethiopia

    PubMed Central

    2014-01-01

    Background Increased resistance by Plasmodium falciparum parasites led to the withdrawal of the antimalarial drugs chloroquine and sulphadoxine-pyrimethamine in Ethiopia. Since 2004 artemether-lumefantrine has served to treat uncomplicated P. falciparum malaria. However, increasing reports on delayed parasite clearance to artemisinin opens up a new challenge in anti-malarial therapy. With the complete withdrawal of CQ for the treatment of Plasmodium falciparum malaria, this study assessed the evolution of CQ resistance by investigating the prevalence of mutant alleles in the pfmdr1 and pfcrt genes in P. falciparum and pvmdr1 gene in Plasmodium vivax in Southern and Eastern Ethiopia. Methods Of the 1,416 febrile patients attending primary health facilities in Southern Ethiopia, 329 febrile patients positive for P. falciparum or P. vivax were recruited. Similarly of the 1,304 febrile patients from Eastern Ethiopia, 81 febrile patients positive for P. falciparum or P. vivax were included in the study. Of the 410 finger prick blood samples collected from malaria patients, we used direct sequencing to investigate the prevalence of mutations in pfcrt and pfmdr1. This included determining the gene copy number in pfmdr1 in 195 P. falciparum clinical isolates, and mutations in the pvmdr1 locus in 215 P. vivax clinical isolates. Results The pfcrt K76 CQ-sensitive allele was observed in 84.1% of the investigated P.falciparum clinical isolates. The pfcrt double mutations (K76T and C72S) were observed less than 3%. The pfcrt SVMNT haplotype was also found to be present in clinical isolates from Ethiopia. The pfcrt CVMNK-sensitive haplotypes were frequently observed (95.9%). The pfmdr1 mutation N86Y was observed only in 14.9% compared to 85.1% of the clinical isolates that carried sensitive alleles. Also, the sensitive pfmdr1 Y184 allele was more common, in 94.9% of clinical isolates. None of the investigated P. falciparum clinical isolates carried S1034C, N1042D and D1246Y

  2. Isolation and characterization of starch from industrial fresh pasta by-product and its potential use in sugar-snap cookie making.

    PubMed

    Ellouzi, Soumaya Zouari; Driss, Dorra; Maktouf, Sameh; Neifar, Mohamed; Kobbi, Ameni; Kamoun, Hounaida; Chaabouni, Semia Ellouze; Ghorbel, Raoudha Ellouze

    2015-09-01

    In this paper, starch was extracted from fresh pasta by-product (PS) and its chemical composition and physical and microscopic characteristics were determined. Commercial wheat starch (CS) was used as reference. In general, purity was similar between starches studied. However, others compounds such as protein, lipid and ash were significantly different. PS starch granules had large lenticular-shape (25-33 μm) and small spherical-shape (5-8 μm). The pH and color of PS starch were similar to those reported for CS starch. On the other hand, PS had higher water absorption capacity, viscosity and cooking stability than CS. The gelatinization temperature of PS was similar to that of CS (60 and 61 °C). At high temperature (90 °C) both starches had similar rheological behavior. The results achieved suggest that PS starch has potential for application in food systems requiring high processing temperatures such the manufacture of sugar snap cookie. The effects of PS starch addition on the dough making stage and the final cookie quality were analyzed. Improvements in dough cohesiveness (24 %) and springiness (10 %) were significant relative to those of CS dough. Texture profile analysis confirmed the rheological changes. PMID:26344989

  3. Occurrence of multiple antibiotic resistance and R-plasmids in gram-negative bacteria isolated from faecally contaminated fresh-water streams in Hong Kong.

    PubMed Central

    French, G. L.; Ling, J.; Chow, K. L.; Mark, K. K.

    1987-01-01

    The bacterial populations of six freshwater streams in populated areas of the Hong Kong New Territories were studied. There is considerable faecal contamination of these streams, with coliform counts as high as 10(5) c.f.u./ml and the contaminating organisms show a high prevalence of antibiotic resistance and multiple resistance. With direct plating of water samples onto antibiotic-containing media, an average of 49% of the gram-negative bacteria were ampicillin-resistant, 3% chloramphenicol-resistant and 1% gentamicin-resistant. At individual sites resistance to these drugs was as high as 98%, 8% and 3% respectively. More than 70% of strains were resistant to two or more antibiotics, 29% to five or more and 2% to eight or more. A total of 98 patterns of antibiotic resistance were detected with no one pattern predominating. Twenty-eight gram-negative bacterial species were identified as stream contaminants. Escherichia coli was the commonest bacterial species isolated and other frequent isolates were Enterobacter sp., Klebsiella sp. and Citrobacter sp., but no enteric pathogens were detected. The greatest prevalence of resistance and multiple resistance was associated with the heaviest contamination by E. coli. Analysis of selected stream isolates revealed multiple plasmid bands arranged in many different patterns, but multiple antibiotic resistances were shown to be commonly mediated by single transferable plasmids. Faecally-contaminated freshwater streams in Hong Kong may be reservoirs of antibiotic resistance plasmids for clinically-important bacteria. Images Fig. 2 PMID:3595747

  4. Plasmodium vivax Populations Are More Genetically Diverse and Less Structured than Sympatric Plasmodium falciparum Populations

    PubMed Central

    Jennison, Charlie; Arnott, Alicia; Tessier, Natacha; Tavul, Livingstone; Koepfli, Cristian; Felger, Ingrid; Siba, Peter M.; Reeder, John C.; Bahlo, Melanie; Mueller, Ivo; Barry, Alyssa E.

    2015-01-01

    Introduction The human malaria parasite, Plasmodium vivax, is proving more difficult to control and eliminate than Plasmodium falciparum in areas of co-transmission. Comparisons of the genetic structure of sympatric parasite populations may provide insight into the mechanisms underlying the resilience of P. vivax and can help guide malaria control programs. Methodology/Principle findings P. vivax isolates representing the parasite populations of four areas on the north coast of Papua New Guinea (PNG) were genotyped using microsatellite markers and compared with previously published microsatellite data from sympatric P. falciparum isolates. The genetic diversity of P. vivax (He = 0.83–0.85) was higher than that of P. falciparum (He = 0.64–0.77) in all four populations. Moderate levels of genetic differentiation were found between P. falciparum populations, even over relatively short distances (less than 50 km), with 21–28% private alleles and clear geospatial genetic clustering. Conversely, very low population differentiation was found between P. vivax catchments, with less than 5% private alleles and no genetic clustering observed. In addition, the effective population size of P. vivax (30353; 13043–69142) was larger than that of P. falciparum (18871; 8109–42986). Conclusions/Significance Despite comparably high prevalence, P. vivax had higher diversity and a panmictic population structure compared to sympatric P. falciparum populations, which were fragmented into subpopulations. The results suggest that in comparison to P. falciparum, P. vivax has had a long-term large effective population size, consistent with more intense and stable transmission, and limited impact of past control and elimination efforts. This underlines suggestions that more intensive and sustained interventions will be needed to control and eventually eliminate P. vivax. This research clearly demonstrates how population genetic analyses can reveal deeper insight into transmission

  5. Laboratory detection of artemisinin-resistant Plasmodium falciparum.

    PubMed

    Chotivanich, Kesinee; Tripura, Rupam; Das, Debashish; Yi, Poravuth; Day, Nicholas P J; Pukrittayakamee, Sasithon; Chuor, Char Meng; Socheat, Duong; Dondorp, Arjen M; White, Nicholas J

    2014-06-01

    Conventional 48-h in vitro susceptibility tests have low sensitivity in identifying artemisinin-resistant Plasmodium falciparum, defined phenotypically by low in vivo parasite clearance rates. We hypothesized originally that this discrepancy was explained by a loss of ring-stage susceptibility and so developed a simple field-adapted 24-h trophozoite maturation inhibition (TMI) assay focusing on the ring stage and compared it to the standard 48-h schizont maturation inhibition (WHO) test. In Pailin, western Cambodia, where artemisinin-resistant P. falciparum is prevalent, the TMI test mean (95% confidence interval) 50% inhibitory concentration (IC50) for artesunate was 6.8 (5.2 to 8.3) ng/ml compared with 1.5 (1.2 to 1.8) ng/ml for the standard 48-h WHO test (P = 0.001). TMI IC50s correlated significantly with the in vivo responses to artesunate (parasite clearance time [r = 0.44, P = 0.001] and parasite clearance half-life [r = 0.46, P = 0.001]), whereas the standard 48-h test values did not. On continuous culture of two resistant isolates, the artemisinin-resistant phenotype was lost after 6 weeks (IC50s fell from 10 and 12 ng/ml to 2.7 and 3 ng/ml, respectively). Slow parasite clearance in falciparum malaria in western Cambodia results from reduced ring-stage susceptibility. PMID:24663013

  6. Efficacy of scopadulcic acid A against Plasmodium falciparum in vitro.

    PubMed

    Riel, Michael A; Kyle, Dennis E; Milhous, Wilbur K

    2002-04-01

    Scoparia dulcis is a perennial herb widely distributed in many tropical countries. It is used as an herbal remedy for gastrointestinal and many other ailments, and in Nicaragua extracts are used to treat malaria. Phytochemical screening has shown that scopadulcic acid A (SDA), scopadulcic acid B (SDB), and semisynthetic analogues are pharmacologically active compounds from S. dulcis. SDB has antiviral activity against Herpes simplex virus type 1, antitumor activity in various human cell lines, and direct inhibitory activity against porcine gastric H(+), K(+)-ATPase. A methyl ester of scopadulcic acid B showed the most potent inhibitory activity against gastric proton pumps of 30 compounds tested in one study. Compounds with antiviral, antifungal, and antitumor activity often show activity against Plasmodium falciparum. In P. falciparum, the plasma membrane and food vacuole have H(+)-ATPases and the acidocalcisome has an H(+)-Ppase. These proton pumps are potential targets for antimalarial therapy and may have their function disrupted by compounds known to inhibit gastric proton pumps. We tested pure SDA and found in vitro activity against P. falciparum with an IC(50) of 27 and 19 microM against the D6 and W2 clones, respectively. The IC(50) against the multidrug-resistant isolate, TM91C235, was 23 microM. PMID:11975516

  7. In vitro sensitivity of Plasmodium falciparum to artesunate in Thailand.

    PubMed Central

    Wongsrichanalai, C.; Wimonwattrawatee, T.; Sookto, P.; Laoboonchai, A.; Heppner, D. G.; Kyle, D. E.; Wernsdorfer, W. H.

    1999-01-01

    Reported are the in vitro susceptibilities of Plasmodium falciparum to artesunate, mefloquine, quinine and chloroquine of 86 isolates and to dihydroartemisinin of 45 isolates collected from areas of high resistance to mefloquine within Thailand near the borders with Myanmar and Cambodia, and from southern Thailand where P. falciparum is generally still sensitive to mefloquine. All the isolates were highly sensitive to artesunate, but the geometric mean IC50S were higher in isolates from the Thai-Myanmar and Thai-Cambodian borders than in those from southern Thailand. The IC50S for mefloquine and artesunate were strongly correlated (Pearson r = 0.605; n = 86; P < 0.00001). As expected, the in vitro sensitivities to dihydroartemisinin and artesunate were similar and strongly correlated (at IC50, Pearson r = 0.695; n = 45; P < 0.00002). The correlation between the activity of mefloquine and artesunate requires further investigation in order to determine the potential for development of cross-resistance in nature. Our results suggest that combination with mefloquine is not the ideal way of protecting the usefulness of artemisinin and its derivatives. A search for more suitable partner drugs to these compounds and careful regulation of their use are necessary in the interest of ensuring their long therapeutic life span. PMID:10361756

  8. Alternative anaerobic enrichments to the bacteriological analytical manual culture method for isolation of Shigella sonnei from selected types of fresh produce.

    PubMed

    Jacobson, Andrew P; Thunberg, Richard L; Johnson, Mildred L; Hammack, Thomas S; Andrews, Wallace H

    2004-01-01

    Alternative methods of reducing oxygen during anaerobic enrichment in the Bacteriological Analytical Manual (BAM) Shigella culture method were evaluated and compared to the current and less practical GasPak method. The alternative anaerobic methods included the use of reducing agents in Shigella broth and reducing culture container headspace volume to minimize atmospheric effects on oxygen concentration in Shigella broth during enrichment. The reducing agents evaluated were sodium thioglycollate, L-cystine, L-cysteine, titanium(III) citrate, and dithiothreitol, each at concentrations of 0.1, 0.05, and 0.01%. The use of Oxyrase for Broth with the enrichment medium (Shigella broth) was evaluated at concentrations of 10, 20 and 30 microL/mL. Recoveries of chill- and freeze-stressed S. sonnei strains 357 and 20143 were determined with each anaerobic method, including the GasPak method, using inoculation levels ranging from 10(0)to 10(3) cells. For each anaerobic method, strain, inoculation level, and stress type, 5 replicate enrichments were evaluated by streaking to MacConkey agar for isolation. The numbers of cultures with each method from which S. sonnei was isolated were used to compare the alternative anaerobic methods to the GasPak method. The alternative anaerobic method with which chill- and freeze-stressed S. sonnei strains 357 and 20143 were isolated most consistently was the use of Oxyrase for Broth in Shigella broth at a concentration of 20 microL/mL. This method was compared to the GasPak anaerobic method in evaluations on the recovery of S. sonnei strains 357 and 20143 from artificially contaminated test portions of parsley, cilantro, green onions, strawberries, carrots, and celery. A third anaerobic method included the use of 0.5 cm mineral oil overlay on cultures containing Oxyrase for Broth at concentrations of 20 microL/mL. Recovery rates of strain 357 were significantly greater (p < 0.05) with the GasPak method than with Oxyrase for Broth, with and

  9. Chloroquine resistance of Plasmodium falciparum is associated with severity of disease in Nigerian children.

    PubMed

    Olumese, P E; Amodu, O K; Björkman, A; Adeyemo, A A; Gbadegesin, R A; Walker, O

    2002-01-01

    Chloroquine resistance of Plasmodium falciparum in vitro was significantly higher in isolates from patients with severe malaria than those with uncomplicated disease. This association may be due to either progression of uncomplicated to severe disease following chloroquine failure or increased virulence of chloroquine-resistant parasites. The implication of this for antimalarial treatment policy is discussed. PMID:12497979

  10. Genetic structure of Plasmodium vivax and Plasmodium falciparum in the Bannu district of Pakistan

    PubMed Central

    2010-01-01

    Background Plasmodium vivax and Plasmodium falciparum are the major causative agents of malaria. While knowledge of the genetic structure of malaria parasites is useful for understanding the evolution of parasite virulence, designing anti-malarial vaccines and assessing the impact of malaria control measures, there is a paucity of information on genetic diversity of these two malaria parasites in Pakistan. This study sought to shed some light on the genetic structure of P. vivax and P. falciparum in this understudied region. Methods The genetic diversities of P. vivax and P. falciparum populations from the densely populated, malaria-endemic Bannu district of Pakistan were evaluated by analysis of their merozoite surface protein (msp) genes by PCR-RFLP. Specifically, the Pvmsp-3α and Pvmsp-3β genes of P. vivax and the Pfmsp-1 and Pfmsp-2 genes of P. falciparum were analysed. Results In P. vivax, genotyping of Pvmsp-3α and Pvmsp-3β genes showed a high level of diversity at these loci. Four distinct allele groups: A (1.9 kb), B (1.5 kb), C (1.2 kb), and D (0.3 kb) were detected for Pvmsp-3α, type A being the most prevalent (82%). Conversely, amplification of the P. vivax msp-3β locus produced two allele groups: A (1.7-2.2 kb, 62%) and B (1.4-1.5 kb, 33%), with 5% mixed-strain infections. Restriction analysis of Pvmsp-3α and Pvmsp-3β yielded 12 and 8 distinct alleles, respectively, with a combined mixed genotype prevalence of 20%. In P. falciparum, all three known genotypes of Pfmsp-1 and two of Pfmsp-2 were observed, with MAD20 occurring in 67% and 3D7/IC in 65% of the isolates, respectively. Overall, 24% P. falciparum samples exhibited mixed-strain infections. Conclusion These results indicate that both P. vivax and P. falciparum populations in Pakistan are highly diverse. PMID:20416089

  11. Molecular Investigation into a Malaria Outbreak in Cusco, Peru: Plasmodium falciparum BV1 Lineage is Linked to a Second Outbreak in Recent Times.

    PubMed

    Okoth, Sheila Akinyi; Chenet, Stella M; Arrospide, Nancy; Gutierrez, Sonia; Cabezas, Cesar; Matta, Jose Antonio; Udhayakumar, Venkatachalam

    2016-01-01

    In November 2013, a Plasmodium falciparum malaria outbreak of 11 cases occurred in Cusco, southern Peru, where falciparum malaria had not been reported since 1946. Although initial microscopic diagnosis reported only Plasmodium vivax infection in each of the specimens, subsequent examination by the national reference laboratory confirmed P. falciparum infection in all samples. Molecular typing of four available isolates revealed identity as the B-variant (BV1) strain that was responsible for a malaria outbreak in Tumbes, northern Peru, between 2010 and 2012. The P. falciparum BV1 strain is multidrug resistant, can escape detection by PfHRP2-based rapid diagnostic tests, and has contributed to two malaria outbreaks in Peru. This investigation highlights the importance of accurate species diagnosis given the potential for P. falciparum to be reintroduced to regions where it may have been absent. Similar molecular epidemiological investigations can track the probable source(s) of outbreak parasite strains for malaria surveillance and control purposes. PMID:26483121

  12. Mirincamycin, an old candidate for malaria combination treatment and prophylaxis in the 21st century: in vitro interaction profiles with potential partner drugs in continuous culture and field isolates

    PubMed Central

    2014-01-01

    Background Spreading resistance of Plasmodium falciparum to existing drugs calls for the search for novel anti-malarial drugs and combinations for the treatment of falciparum malaria. Methods In vitro and ex vivo investigations were conducted with fresh P. falciparum field isolates and culture-adapted P. falciparum clones to evaluate the anti-malarial potential of mirincamycin, a lincosamide, alone and in combination with tafenoquine (TQ), dihydroartemisinin (DHA), and chloroquine (CQ). All samples were tested in a histidine-rich protein 2 (HRP2) drug susceptibility assay. Results Interaction analysis showed additive to synergistic interaction profiles with these potential partner drugs, with an overall geometric mean fractional inhibitory concentration at 50% inhibition (FIC50) of 0.78, 0.80 and 0.80 for mirincamycin with TQ, DHA, and CQ, respectively. Antagonism was not found in any of the tested field isolates or clones. The strongest tendency toward synergy (i.e. the lowest FIC) was seen with a combination ratio of 1:0.27 to 1:7.2 (mean 1:2.7) for the combination with tafenoquine. The optimal combination ratios for DHA and CQ were 1:444.4 to 1:36,000 (mean 1:10,755.5) and 1:2.7 to 1:216 (mean 1:64.5), respectively. No evidence of an activity correlation (i.e. potential cross-resistance) with DHA, mefloquine, quinine or chloroquine was seen whereas a significant correlation with the activity of clindamycin and azithromycin was detected. Conclusions Mirincamycin combinations may be promising candidates for further clinical investigations in the therapy and prophylaxis of multidrug-resistant falciparum malaria or in combination with 4 or 8-aminoquinolines for the treatment and relapse prevention of vivax malaria. PMID:24916383

  13. Fresh SETI Strategies

    NASA Astrophysics Data System (ADS)

    Tough, A.

    If a smart probe or some other form of extraterrestrial intelligence has reached our planet, what is the next logical step in our scientific search for it? Fresh new search strategies are required. One innovative strategy, an invitation to ETI launched in 1996, was made possible by the creation of the World Wide Web. Other fresh search strategies, too, are emerging.

  14. A forward genetic screen identifies erythrocyte CD55 as essential for Plasmodium falciparum invasion **

    PubMed Central

    Egan, Elizabeth S.; Jiang, Rays H.Y.; Moechtar, Mischka A.; Barteneva, Natasha S.; Weekes, Michael P.; Nobre, Luis V.; Gygi, Steven P.; Paulo, Joao A.; Frantzreb, Charles; Tani, Yoshihiko; Takahashi, Junko; Watanabe, Seishi; Goldberg, Jonathan; Paul, Aditya S.; Brugnara, Carlo; Root, David E.; Wiegand, Roger C.; Doench, John G.; Duraisingh, Manoj T.

    2015-01-01

    Efforts to identify host determinants for malaria have been hindered by the absence of a nucleus in erythrocytes, precluding genetic manipulation in the cell where the parasite replicates. We used cultured red blood cells derived from hematopoietic stem cells to carry out a forward genetic screen for Plasmodium falciparum host determinants. We found that CD55 is an essential host factor for P. falciparum invasion. CD55-null erythrocytes were refractory to invasion by all isolates of P. falciparum because parasites failed to attach properly to the erythrocyte surface. Thus, CD55 is an attractive target for the development of malaria therapeutics. Hematopoietic stem cell-based forward genetic screens may be valuable for the identification of additional host determinants of malaria pathogenesis. PMID:25954012

  15. Field applications of agglutination and cytoadherence assays with Plasmodium falciparum from Papua New Guinea.

    PubMed

    Southwell, B R; Brown, G V; Forsyth, K P; Smith, T; Philip, G; Anders, R

    1989-01-01

    Plasmodium falciparum isolates obtained directly from patients in Papua New Guinea were tested in their first cycle of growth in vitro for adherence to melanoma cells and for susceptibility to agglutination by immune serum. Binding varied among isolates and, in many cases, increased with further rounds of replication under optimal culture conditions. Binding inhibition assays and agglutination assays demonstrated extreme heterogeneity of surface antigens; apparently none of the sera from adult patients recognized all of the variants presented. PMID:2694479

  16. Plasmodium falciparum: attenuation by irradiation

    SciTech Connect

    Waki, S.; Yonome, I.; Suzuki, M.

    1983-12-01

    The effect of irradiation on the in vitro growth of Plasmodium falciparum was investigated. The cultured malarial parasites at selected stages of development were exposed to gamma rays and the sensitivity of each stage was determined. The stages most sensitive to irradiation were the ring forms and the early trophozoites; late trophozoites were relatively insensitive. The greatest resistance was shown when parasites were irradiated at a time of transition from the late trophozoite and schizont stages to young ring forms. The characteristics of radiosensitive variation in the parasite cycle resembled that of mammalian cells. Growth curves of parasites exposed to doses of irradiation upto 150 gray had the same slope as nonirradiated controls but parasites which were exposed to 200 gray exhibited a growth curve which was less steep than that for parasites in other groups. Less than 10 organisms survived from the 10(6) parasites exposed to this high dose of irradiation; the possibility exists of obtaining radiation-attenuated P. falciparum.

  17. Exchange Transfusion in Severe Falciparum Malaria

    PubMed Central

    Khatib, Khalid Ismail

    2016-01-01

    Malaria is endemic in India with the incidence of P. falciparum Malaria increasing gradually over the last decade. Severe malaria is an acute disease, caused by P. falciparum, but increasingly also by P. vivax with major signs of organ dysfunction and/or high levels of parasitaemia (>10%) in blood smear. Use of exchange transfusion with antimalarial drug therapy as an additional modality of treatment in severe Falciparum malaria is controversial and is unclear. We report a case of severe malaria complicated by multiorgan failure and ARDS. Patient responded well to manual exchange transfusion with standard artesunate-based chemotherapy. PMID:27042503

  18. Fresh Water Life.

    ERIC Educational Resources Information Center

    Kestler, Carol Susan

    1991-01-01

    Describes methodology for a fresh water life study with elementary through college age students with suggestions for proper equipment, useful guides, and other materials. Proposes an activity for the collection and study of plankton. Includes background information.(MCO)

  19. Immunogenicity of recombinant Plasmodium falciparum SERA proteins in rodents.

    PubMed

    Barr, P J; Inselburg, J; Green, K M; Kansopon, J; Hahm, B K; Gibson, H L; Lee-Ng, C T; Bzik, D J; Li, W B; Bathurst, I C

    1991-03-01

    We have expressed defined regions of the serine-repeat antigen (SERA) of the Honduras-1 strain of Plasmodium falciparum in the yeast Saccharomyces cerevisiae. Amino-terminal domains of the natural SERA protein have been shown previously to be targets for parasite-inhibitory murine monoclonal antibodies. Two recombinant SERA antigens were selected for purification and immunological analysis. The first (SERA 1), corresponding to amino acids 24-285 of the natural SERA precursor, was expressed by the ubiquitin fusion method. This allowed for in vivo cleavage by endogenous yeast ubiquitin hydrolase, and subsequent isolation of the mature polypeptide. The second, larger protein (SERA N), encompassing amino acids 24-506, was expressed at only low levels using this system, but could be isolated in high yields when fused to human gamma-interferon (gamma-IFN). Each purified protein was used to immunize mice with either Freund's adjuvant or a muramyl tripeptide adjuvant that has been used in humans. Sera from immunized mice were shown to be capable of in vitro inhibition of invasion of erythrocytes by the Honduras-1 strain of P. falciparum. The results suggest that a recombinant SERA antigen may be an effective component of a candidate malaria vaccine. PMID:2052035

  20. Use of a colorimetric (DELI) test for the evaluation of chemoresistance of Plasmodium falciparum and Plasmodium vivax to commonly used anti-plasmodial drugs in the Brazilian Amazon

    PubMed Central

    2013-01-01

    Background The emergence and spread of Plasmodium falciparum and Plasmodium vivax resistance to available anti-malarial drugs represents a major drawback in the control of malaria and its associated morbidity and mortality. The aim of this study was to evaluate the chemoresistance profile of P. falciparum and P. vivax to commonly used anti-plasmodial drugs in a malaria-endemic area in the Brazilian Amazon. Methods The study was carried out in Manaus (Amazonas state), in the Brazilian Amazon. A total of 88 P. falciparum and 178 P. vivax isolates was collected from 2004 to 2007. The sensitivity of P. falciparum isolates was determined to chloroquine, quinine, mefloquine and artesunate and the sensitivity of P. vivax isolates was determined to chloroquine and mefloquine, by using the colorimetric DELI test. Results As expected, a high prevalence of P. falciparum isolates resistant to chloroquine (78.1%) was observed. The prevalence of isolates with profile of resistance or decreased sensitivity for quinine, mefloquine and artesunate was 12.7, 21.2 and 11.7%, respectively. In the case of P. vivax, the prevalence of isolates with profile of resistance for chloroquine and mefloquine was 9.8 and 28%, respectively. No differences in the frequencies of isolates with profile of resistance or geometric mean IC50s were seen when comparing the data obtained in 2004, 2005, 2006 and 2007, for all tested anti-malarials. Conclusions The great majority of P. falciparum isolates in the Brazilian malaria-endemic area remain resistant to chloroquine, and the decreased sensitivity to quinine, mefloquine and artesunate observed in 10–20% of the isolates must be taken with concern, especially for artesunate. Plasmodium vivax isolates also showed a significant proportion of isolates with decreased sensitivity to chloroquine (first-line drug) and mainly to mefloquine. The data presented here also confirm the usefulness of the DELI test to generate results able to impact on public health

  1. Contrasting Transmission Dynamics of Co-endemic Plasmodium vivax and P. falciparum: Implications for Malaria Control and Elimination

    PubMed Central

    Noviyanti, Rintis; Coutrier, Farah; Utami, Retno A. S.; Trimarsanto, Hidayat; Tirta, Yusrifar K.; Trianty, Leily; Kusuma, Andreas; Sutanto, Inge; Kosasih, Ayleen; Kusriastuti, Rita; Hawley, William A.; Laihad, Ferdinand; Lobo, Neil; Marfurt, Jutta; Clark, Taane G.; Price, Ric N.; Auburn, Sarah

    2015-01-01

    Background Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions. Methodology/ Principal Findings Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor). Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species. Conclusions/ Significance Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given

  2. Detectability of Plasmodium falciparum clones

    PubMed Central

    2010-01-01

    Background In areas of high transmission people often harbour multiple clones of Plasmodium falciparum, but even PCR-based diagnostic methods can only detect a fraction (the detectability, q) of all clones present in a host. Accurate measurements of detectability are desirable since it affects estimates of multiplicity of infection, prevalence, and frequency of breakthrough infections in clinical drug trials. Detectability can be estimated by typing repeated samples from the same host but it has been unclear what should be the time interval between the samples and how the data should be analysed. Methods A longitudinal molecular study was conducted in the Kassena-Nankana district in northern Ghana. From each of the 80 participants, four finger prick samples were collected over a period of 8 days, and tested for presence of different Merozoite Surface Protein (msp) 2 genotypes. Implications for estimating q were derived from these data by comparing the fit of statistical models of serial dependence and over-dispersion. Results The distribution of the frequencies of detection for msp2 genotypes was close to binomial if the time span between consecutive blood samples was at least 7 days. For shorter intervals the probabilities of detection were positively correlated, i.e. the shorter the interval between two blood collections, the more likely the diagnostic results matched for a particular genotype. Estimates of q were rather insensitive to the statistical model fitted. Conclusions A simple algorithm based on analysing blood samples collected 7 days apart is justified for generating robust estimates of detectability. The finding of positive correlation of detection probabilities for short time intervals argues against imperfect detection being directly linked to the 48-hour periodicity of P. falciparum. The results suggest that the detectability of a given parasite clone changes over time, at an unknown rate, but fast enough to regard blood samples taken one week

  3. Irradiation of Fresh and Fresh-Cut Fruits and Vegetables

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of fresh and fresh-cut fruits and vegetables in the USA has increased every year in the last decade. Unfortunately, the increasing consumption of fresh produce has been accompanied with an increase in the number of outbreaks and recalls due to contamination with human pathogens. Fresh f...

  4. Prevalence of mutation and phenotypic expression associated with sulfadoxine-pyrimethamine resistance in Plasmodium falciparum and Plasmodium vivax.

    PubMed

    Zakai, Haytham A; Khan, Wajihullah; Asma, Umme

    2013-09-01

    Therapeutic efficacy of sulfadoxine-pyrimethamine (SP), which is commonly used to treat falciparum malaria, was assessed in isolates of Plasmodium falciparum (Welch, 1897) and Plasmodium vivax (Grassi et Feletti, 1890) ofAligarh, Uttar Pradesh, North India and Taif, Saudi Arabia during 2011-2012. Both the species showed mutations in dihydrofolate reductase (DHFR) enzyme as they have common biochemical drug targets. Mutation rate for pfdhfr was higher compared to pvdhfr because the drug was mainly given to treat falciparum malaria. Since both the species coexist, P. vivax was also exposed to SP due to faulty species diagnosis or medication without specific diagnosis. Low level of mutations against SP in P. falciparum of Saudi isolates indicates that the SP combination is still effective for the treatment of falciparum malaria. Since SP is used as first-line of treatment because of high level of resistance against chloroquine (CQ), it may result in spread of higher level of mutations resulting in drug resistance and treatment failure in near future. Therefore, to avoid further higher mutations in the parasite, use of better treatment regimens such as artesunate combination therapy must be introduced against SP combination. PMID:24261139

  5. Absence of Association between Polymorphisms in the RING E3 Ubiquitin Protein Ligase Gene and Ex Vivo Susceptibility to Conventional Antimalarial Drugs in Plasmodium falciparum Isolates from Dakar, Senegal.

    PubMed

    Gendrot, Mathieu; Fall, Bécaye; Madamet, Marylin; Fall, Mansour; Wade, Khalifa Ababacar; Amalvict, Rémy; Nakoulima, Aminata; Benoit, Nicolas; Diawara, Silman; Diémé, Yaya; Diatta, Bakary; Wade, Boubacar; Pradines, Bruno

    2016-08-01

    The RING E3 ubiquitin protein ligase is crucial for facilitating the transfer of ubiquitin. The only polymorphism identified in the E3 ubiquitin protein ligase gene was the D113N mutation (62.5%) but was not significantly associated with the 50% inhibitory concentration (IC50) of conventional antimalarial drugs. However, some mutated isolates (D113N) present a trend of reduced susceptibility to piperaquine (P = 0.0938). To evaluate the association of D113N polymorphism with susceptibility to antimalarials, more isolates are necessary. PMID:27185795

  6. Polymorphisms in Anopheles gambiae Immune Genes Associated with Natural Resistance to Plasmodium falciparum

    PubMed Central

    Harris, Caroline; Lambrechts, Louis; Rousset, François; Abate, Luc; Nsango, Sandrine E.; Fontenille, Didier; Morlais, Isabelle; Cohuet, Anna

    2010-01-01

    Many genes involved in the immune response of Anopheles gambiae, the main malaria vector in Africa, have been identified, but whether naturally occurring polymorphisms in these genes underlie variation in resistance to the human malaria parasite, Plasmodium falciparum, is currently unknown. Here we carried out a candidate gene association study to identify single nucleotide polymorphisms (SNPs) associated with natural resistance to P. falciparum. A. gambiae M form mosquitoes from Cameroon were experimentally challenged with three local wild P. falciparum isolates. Statistical associations were assessed between 157 SNPs selected from a set of 67 A. gambiae immune-related genes and the level of infection. Isolate-specific associations were accounted for by including the effect of the isolate in the analysis. Five SNPs were significantly associated to the infection phenotype, located within or upstream of AgMDL1, CEC1, Sp PPO activate, Sp SNAKElike, and TOLL6. Low overall and local linkage disequilibrium indicated high specificity in the loci found. Association between infection phenotype and two SNPs was isolate-specific, providing the first evidence of vector genotype by parasite isolate interactions at the molecular level. Four SNPs were associated to either oocyst presence or load, indicating that the genetic basis of infection prevalence and intensity may differ. The validity of the approach was verified by confirming the functional role of Sp SNAKElike in gene silencing assays. These results strongly support the role of genetic variation within or near these five A. gambiae immune genes, in concert with other genes, in natural resistance to P. falciparum. They emphasize the need to distinguish between infection prevalence and intensity and to account for the genetic specificity of vector-parasite interactions in dissecting the genetic basis of Anopheles resistance to human malaria. PMID:20862317

  7. Comparative Ex Vivo Activity of Novel Endoperoxides in Multidrug-Resistant Plasmodium falciparum and P. vivax

    PubMed Central

    Chalfein, Ferryanto; Prayoga, Pak; Wabiser, Frans; Wirjanata, Grennady; Sebayang, Boni; Piera, Kim A.; Wittlin, Sergio; Haynes, Richard K.; Möhrle, Jörg J.; Anstey, Nicholas M.; Kenangalem, Enny; Price, Ric N.

    2012-01-01

    The declining efficacy of artemisinin derivatives against Plasmodium falciparum highlights the urgent need to identify alternative highly potent compounds for the treatment of malaria. In Papua Indonesia, where multidrug resistance has been documented against both P. falciparum and P. vivax malaria, comparative ex vivo antimalarial activity against Plasmodium isolates was assessed for the artemisinin derivatives artesunate (AS) and dihydroartemisinin (DHA), the synthetic peroxides OZ277 and OZ439, the semisynthetic 10-alkylaminoartemisinin derivatives artemisone and artemiside, and the conventional antimalarial drugs chloroquine (CQ), amodiaquine (AQ), and piperaquine (PIP). Ex vivo drug susceptibility was assessed in 46 field isolates (25 P. falciparum and 21 P. vivax). The novel endoperoxide compounds exhibited potent ex vivo activity against both species, but significant differences in intrinsic activity were observed. Compared to AS and its active metabolite DHA, all the novel compounds showed lower or equal 50% inhibitory concentrations (IC50s) in both species (median IC50s between 1.9 and 3.6 nM in P. falciparum and 0.7 and 4.6 nM in P. vivax). The antiplasmodial activity of novel endoperoxides showed different cross-susceptibility patterns in the two Plasmodium species: whereas their ex vivo activity correlated positively with CQ, PIP, AS, and DHA in P. falciparum, the same was not apparent in P. vivax. The current study demonstrates for the first time potent activity of novel endoperoxides against drug-resistant P. vivax. The high activity against drug-resistant strains of both Plasmodium species confirms these compounds to be promising candidates for future artemisinin-based combination therapy (ACT) regimens in regions of coendemicity. PMID:22850522

  8. Malaria vaccine candidate antigen targeting the pre-erythrocytic stage of Plasmodium falciparum produced at high level in plants.

    PubMed

    Voepel, Nadja; Boes, Alexander; Edgue, Güven; Beiss, Veronique; Kapelski, Stephanie; Reimann, Andreas; Schillberg, Stefan; Pradel, Gabriele; Fendel, Rolf; Scheuermayer, Matthias; Spiegel, Holger; Fischer, Rainer

    2014-11-01

    Plants have emerged as low-cost production platforms suitable for vaccines targeting poverty-related diseases. Besides functional efficacy, the stability, yield, and purification process determine the production costs of a vaccine and thereby the feasibility of plant-based production. We describe high-level plant production and functional characterization of a malaria vaccine candidate targeting the pre-erythrocytic stage of Plasmodium falciparum. CCT, a fusion protein composed of three sporozoite antigens (P. falciparum cell traversal protein for ookinetes and sporozoites [PfCelTOS], P. falciparum circumsporozoite protein [PfCSP], and P. falciparum thrombospondin-related adhesive protein [PfTRAP]), was transiently expressed by agroinfiltration in Nicotiana benthamiana leaves, accumulated to levels up to 2 mg/g fresh leaf weight (FLW), was thermostable up to 80°C and could be purified to >95% using a simple two-step procedure. Reactivity of sera from malaria semi-immune donors indicated the immunogenic conformation of the purified fusion protein consisting of PfCelTOS, PfCSP_TSR, PfTRAP_TSR domains (CCT) protein. Total IgG from the CCT-specific mouse immune sera specifically recognized P. falciparum sporozoites in immunofluorescence assays and induced up to 35% inhibition in hepatocyte invasion assays. Featuring domains from three promising sporozoite antigens with different roles (attachment and cell traversal) in the hepatocyte invasion process, CCT has the potential to elicit broader immune responses against the pre-erythrocytic stage of P. falciparum and represents an interesting new candidate, also as a component of multi-stage, multi-subunit malaria vaccine cocktails. PMID:25200253

  9. Shop Around for Freshness.

    ERIC Educational Resources Information Center

    Evans, George P.

    1998-01-01

    Argues that cliches make writing stale and flat, and that getting rid of them is a necessity. Lists the "top 14" cliches gathered by a Columbia Scholastic Press Association judge. Uses examples to show how cleansing a passage of its superlatives and cliches improves the writing, adding clarity, fairness, and objectivity as well as freshness and…

  10. Characterization of the 26S proteasome network in Plasmodium falciparum

    PubMed Central

    Wang, Lihui; Delahunty, Claire; Fritz-Wolf, Karin; Rahlfs, Stefan; Helena Prieto, Judith; Yates, John R.; Becker, Katja

    2015-01-01

    In eukaryotic cells, the ubiquitin-proteasome system as a key regulator of protein quality control is an excellent drug target. We therefore aimed to analyze the 26S proteasome complex in the malaria parasite Plasmodium falciparum, which still threatens almost half of the world’s population. First, we established an affinity purification protocol allowing for the isolation of functional 26S proteasome complexes from the parasite. Subunit composition of the proteasome and component stoichiometry were studied and physiologic interacting partners were identified via in situ protein crosslinking. Furthermore, intrinsic ubiquitin receptors of the plasmodial proteasome were determined and their roles in proteasomal substrate recognition were analyzed. Notably, PfUSP14 was characterized as a proteasome-associated deubiquitinase resulting in the concept that targeting proteasomal deubiquitinating activity in P. falciparum may represent a promising antimalarial strategy. The data provide insights into a profound network orchestrated by the plasmodial proteasome and identified novel drug target candidates in the ubiquitin-proteasome system. PMID:26639022

  11. Epigenetic regulation of the Plasmodium falciparum genome.

    PubMed

    Duffy, Michael F; Selvarajah, Shamista A; Josling, Gabrielle A; Petter, Michaela

    2014-05-01

    Recent research has highlighted some unique aspects of chromatin biology in the malaria parasite Plasmodium falciparum. During its erythrocytic lifecycle P. falciparum maintains its genome primarily as unstructured euchromatin. Indeed there is no clear role for chromatin-mediated silencing of the majority of the developmentally expressed genes in P. falciparum. However discontinuous stretches of heterochromatin are critical for variegated expression of contingency genes that mediate key pathogenic processes in malaria. These range from invasion of erythrocytes and antigenic variation to solute transport and growth adaptation in response to environmental changes. Despite lack of structure within euchromatin the nucleus maintains functional compartments that regulate expression of many genes at the nuclear periphery, particularly genes with clonally variant expression. The typical components of the chromatin regulatory machinery are present in P. falciparum; however, some of these appear to have evolved novel species-specific functions, e.g. the dynamic regulation of histone variants at virulence gene promoters. The parasite also appears to have repeatedly acquired chromatin regulatory proteins through lateral transfer from endosymbionts and from the host. P. falciparum chromatin regulators have been successfully targeted with multiple drugs in laboratory studies; hopefully their functional divergence from human counterparts will allow the development of parasite-specific inhibitors. PMID:24326119

  12. Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays.

    PubMed

    Cooper, Roland A; Conrad, Melissa D; Watson, Quentin D; Huezo, Stephanie J; Ninsiima, Harriet; Tumwebaze, Patrick; Nsobya, Samuel L; Rosenthal, Philip J

    2015-08-01

    We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda. PMID:26033725

  13. Lack of Artemisinin Resistance in Plasmodium falciparum in Uganda Based on Parasitological and Molecular Assays

    PubMed Central

    Conrad, Melissa D.; Watson, Quentin D.; Huezo, Stephanie J.; Ninsiima, Harriet; Tumwebaze, Patrick; Nsobya, Samuel L.; Rosenthal, Philip J.

    2015-01-01

    We evaluated markers of artemisinin resistance in Plasmodium falciparum isolated in Kampala in 2014. By standard in vitro assays, all isolates were highly sensitive to dihydroartemisinin (DHA). By the ring-stage survival assay, after a 6-h DHA pulse, parasitemia was undetectable in 40 of 43 cultures at 72 h. Two of 53 isolates had nonsynonymous K13-propeller gene polymorphisms but did not have the mutations associated with resistance in Asia. Thus, we did not see evidence for artemisinin resistance in Uganda. PMID:26033725

  14. Induction of cell death on Plasmodium falciparum asexual blood stages by Solanum nudum steroids.

    PubMed

    López, Mary Luz; Vommaro, Rossiane; Zalis, Mariano; de Souza, Wanderley; Blair, Silvia; Segura, Cesar

    2010-06-01

    Solanum nudum Dunal (Solanaceae) is a plant used in traditional medicine in Colombian Pacific Coast, from which five steroids denominated SNs have been isolated. The SNs compounds have antiplasmodial activity against asexual blood stages of Plasmodium falciparum strain 7G8 with an IC(50) between 20-87microM. However, their mode of action is unknown. Steroids regulate important cellular functions including cell growth, differentiation and death. Thus, the aim of this work was to determine the effects of S. nudum compounds on P. falciparum asexual blood stages and their association with cell death. We found that trophozoite and schizont stages were the most sensitive to SNs. By Giemsa-stained smears, induction of crisis forms was observed. Transmission electron microscopy of treated parasites showed morphological abnormalities such as a cytoplasm rich in vesicles and myelinic figures. The Mitochondria presented no morphological alterations and the nuclei showed no abnormal chromatin condensation. By the use of S. nudum compounds, cell death in P. falciparum was evident by a decrease in mitochondrial membrane potential, DNA fragmentation and cytoplasmic acidification. The asexual blood stages of P. falciparum showed some apoptotic-like and autophagic-like cell death characteristics induced by SNs treatment. PMID:20153445

  15. Genetic structure of Plasmodium falciparum populations across the Honduras-Nicaragua border

    PubMed Central

    2013-01-01

    Background The Caribbean coast of Central America remains an area of malaria transmission caused by Plasmodium falciparum despite the fact that morbidity has been reduced in recent years. Parasite populations in that region show interesting characteristics such as chloroquine susceptibility and low mortality rates. Genetic structure and diversity of P. falciparum populations in the Honduras-Nicaragua border were analysed in this study. Methods Seven neutral microsatellite loci were analysed in 110 P. falciparum isolates from endemic areas of Honduras (n = 77) and Nicaragua (n = 33), mostly from the border region called the Moskitia. Several analyses concerning the genetic diversity, linkage disequilibrium, population structure, molecular variance, and haplotype clustering were conducted. Results There was a low level of genetic diversity in P. falciparum populations from Honduras and Nicaragua. Expected heterozigosity (He) results were similarly low for both populations. A moderate differentiation was revealed by the FST index between both populations, and two putative clusters were defined through a structure analysis. The main cluster grouped most of samples from Honduras and Nicaragua, while the second cluster was smaller and included all the samples from the Siuna community in Nicaragua. This result could partially explain the stronger linkage disequilibrium (LD) in the parasite population from that country. These findings are congruent with the decreasing rates of malaria endemicity in Central America. PMID:24093629

  16. Neutralizing Antibodies against Plasmodium falciparum Associated with Successful Cure after Drug Therapy

    PubMed Central

    Goh, Yun Shan; Peng, Kaitian; Chia, Wan Ni; Siau, Anthony; Chotivanich, Kesinee; Gruner, Anne-Charlotte; Preiser, Peter; Mayxay, Mayfong; Pukrittayakamee, Sasithon; Sriprawat, Kanlaya; Nosten, Francois; White, Nicholas J.

    2016-01-01

    An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection. PMID:27427762

  17. Neutralizing Antibodies against Plasmodium falciparum Associated with Successful Cure after Drug Therapy.

    PubMed

    Goh, Yun Shan; Peng, Kaitian; Chia, Wan Ni; Siau, Anthony; Chotivanich, Kesinee; Gruner, Anne-Charlotte; Preiser, Peter; Mayxay, Mayfong; Pukrittayakamee, Sasithon; Sriprawat, Kanlaya; Nosten, Francois; White, Nicholas J; Renia, Laurent

    2016-01-01

    An effective antibody response can assist drug treatment to contribute to better parasite clearance in malaria patients. To examine this, sera were obtained from two groups of adult patients with acute falciparum malaria, prior to drug treatment: patients who (1) have subsequent recrudescent infection, or (2) were cured by Day 28 following treatment. Using a Plasmodium falciparum antigen library, we examined the antibody specificities in these sera. While the antibody repertoire of both sera groups was extremely broad and varied, there was a differential antibody profile between the two groups of sera. The proportion of cured patients with antibodies against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 was higher than the proportion of patients with recrudescent infection. The presence of these antibodies was associated with higher odds of treatment cure. Sera containing all six antibodies impaired the invasion of P. falciparum clinical isolates into erythrocytes. These results suggest that antibodies specific against EXP1, MSP3, GLURP, RAMA, SEA and EBA181 in P. falciparum infections could assist anti-malarial drug treatment and contribute to the resolution of the malarial infection. PMID:27427762

  18. Anaemia of Plasmodium falciparum malaria.

    PubMed

    Phillips, R E; Pasvol, G

    1992-04-01

    The pathophysiology of the anaemia of falciparum malaria is both complex and multifactorial, and results in a condition which is a major cause of mortality and morbidity in patients, especially children and pregnant women, living in malarial endemic areas. The importance of anaemia as a cause of death in malaria may well be underestimated because of difficulty in diagnosis, especially where parasitaemia may be low and the clinical picture may be confused with other causes of anaemia. Two clinical presentations predominate: severe acute malaria in which anaemia supervenes, and severe anaemia in patients in whom there have been repeated attacks of malaria. The major mechanisms are those of red cell destruction and decreased red cell production. Potential causes of haemolysis include loss of infected cells by rupture or phagocytosis, removal of uninfected cells due to antibody sensitization or other physicochemical membrane changes, and increased reticuloendothelial activity, particularly in organs such as the spleen. Decreased production results from marrow hypoplasia seen in acute infections, and dyserythropoiesis, a morphological appearance, which in functional terms results in ineffective erythropoiesis. The role of parvovirus B19 as a possible cause of bone marrow aplasia in a few cases is postulated. Finally, there is now evidence which points to genetic factors, HLA associated, which may protect against the development of malarial anaemia and which has become common in areas endemic for malaria. PMID:1511178

  19. Plasmodium falciparum in Haiti: susceptibility to pyrimethamine and sulfadoxine-pyrimethamine

    PubMed Central

    Nguyen-Dinh, Phuc; Zevallos-Ipenza, Arturo; Magloire, Roc

    1984-01-01

    Eighteen patients with Plasmodium falciparum infection were studied in Port-au-Prince, Haiti, to monitor the response of the malaria parasite to sulfadoxine-pyrimethamine. In all infections the parasitaemia was cleared rapidly following treatment with standard dose of the drug combination; no recrudescence was observed during follow-up periods of 1 week (4 patients) and 4 weeks (14 patients). Parallel in vitro tests indicated that 5 of the 16 isolates successfully tested were resistant to pyrimethamine alone. PMID:6386210

  20. In Vitro Generation of Plasmodium falciparum Ookinetes

    PubMed Central

    Bounkeua, Viengngeun; Li, Fengwu; Vinetz, Joseph M.

    2010-01-01

    Plasmodium transmission from the human host to the mosquito depends on the ability of gametocytes to differentiate into ookinetes, the invasive form of the parasite that invades and establishes infection in the mosquito midgut. The biology of P. falciparum ookinetes is poorly understood, because sufficient quantities of this stage of this parasite species have not been obtained for detailed study. This report details methods to optimize production of P. falciparum sexual stage parasites, including ookinetes. Flow cytometric sorting was used to separate diploid/tetraploid zygotes and ookinetes from haploid gametetocytes and unfertilized gametes based on DNA content. Consistent production of 106–107 P. falciparum ookinetes per 10 mL culture was observed, with ookinete transformation present in 10–40% of all parasite forms. Transmission electron micrographs of cultured parasites confirmed ookinete development. PMID:21118920

  1. Variation in Plasmodium falciparum Histidine-Rich Protein 2 (Pfhrp2) and Plasmodium falciparum Histidine-Rich Protein 3 (Pfhrp3) Gene Deletions in Guyana and Suriname

    PubMed Central

    Akinyi Okoth, Sheila; Abdallah, Joseph F.; Ceron, Nicolas; Adhin, Malti R.; Chandrabose, Javin; Krishnalall, Karanchand; Huber, Curtis S.; Goldman, Ira F.; Macedo de Oliveira, Alexandre; Barnwell, John W.; Udhayakumar, Venkatachalam

    2015-01-01

    Guyana and Suriname have made important progress in reducing the burden of malaria. While both countries use microscopy as the primary tool for clinical diagnosis, malaria rapid diagnostic tests (RDTs) are useful in remote areas of the interior where laboratory support may be limited or unavailable. Recent reports indicate that histidine-rich protein 2 (PfHRP2)-based diagnostic tests specific for detection of P. falciparum may provide false negative results in some parts of South America due to the emergence of P. falciparum parasites that lack the pfhrp2 gene, and thus produce no PfHRP2 antigen. Pfhrp2 and pfhrp3 genes were amplified in parasite isolates collected from Guyana and Suriname to determine if there were circulating isolates with deletions in these genes. Pfhrp3 deletions were monitored because some monoclonal antibodies utilized in PfHRP2-based RDTs cross-react with the PfHRP3 protein. We found that all 97 isolates from Guyana that met the inclusion criteria were both pfhrp2- and pfhrp3-positive. In Suriname (N = 78), 14% of the samples tested were pfhrp2-negative while 4% were pfhrp3-negative. Furthermore, analysis of the genomic region proximal to pfhrp2 and pfhrp3 revealed that genomic deletions extended to the flanking genes. We also investigated the population substructure of the isolates collected to determine if the parasites that had deletions of pfhrp2 and pfhrp3 belonged to any genetic subtypes. Cluster analysis revealed that there was no predominant P. falciparum population substructure among the isolates from either country, an indication of genetic admixture among the parasite populations. Furthermore, the pfhrp2-deleted parasites from Suriname did not appear to share a single, unique genetic background. PMID:25978499

  2. Toward a high-resolution Plasmodium falciparum linkage map: Polymorphic markers from hundreds of simple sequence repeats

    SciTech Connect

    Su, Xin-Zhuan; Wellems, T.E.

    1996-05-01

    A total of 5.7 simple sequence repeats (SSRs or {open_quotes}microsatellites{close_quotes}) were identified from Plasmodium falciparum sequences in GenBank and from inserts in a genomic DNA library. Oligonucleotide primers from sequences that flank 224 of these SSRs were synthesized and used in PCR assays to test for simple sequence length polymorphisms (SSLPs). Of the 224 SSRs, 188 showed SSLPs were assigned to chromosome linkage groups by physical mapping and by comparing their inheritance patterns against those of restriction fragment length polymorphism markers in a genetic cross (HB3XDd2). The predominant SSLPs in P. falciparum were found to contain [TA]{sub n}, and [TAA]{sub n}, a feature that is reminiscent of plant genomes and is consistent with the proposed algal-like origin of malaria parasites. Since such SSLPs are abundant and readily isolated, they are a powerful resource for genetic analysis of P. falciparum. 38 refs., 2 figs., 2 tabs.

  3. Fucosylated Chondroitin Sulfate Inhibits Plasmodium falciparum Cytoadhesion and Merozoite Invasion

    PubMed Central

    Bastos, Marcele F.; Albrecht, Letusa; Kozlowski, Eliene O.; Lopes, Stefanie C. P.; Blanco, Yara C.; Carlos, Bianca C.; Castiñeiras, Catarina; Vicente, Cristina P.; Werneck, Claudio C.; Wunderlich, Gerhard; Ferreira, Marcelo U.; Marinho, Claudio R. F.; Mourão, Paulo A. S.; Pavão, Mauro S. G.

    2014-01-01

    Sequestration of Plasmodium falciparum-infected erythrocytes (Pf-iEs) in the microvasculature of vital organs plays a key role in the pathogenesis of life-threatening malaria complications, such as cerebral malaria and malaria in pregnancy. This phenomenon is marked by the cytoadhesion of Pf-iEs to host receptors on the surfaces of endothelial cells, on noninfected erythrocytes, and in the placental trophoblast; therefore, these sites are potential targets for antiadhesion therapies. In this context, glycosaminoglycans (GAGs), including heparin, have shown the ability to inhibit Pf-iE cytoadherence and growth. Nevertheless, the use of heparin was discontinued due to serious side effects, such as bleeding. Other GAG-based therapies were hampered due to the potential risk of contamination with prions and viruses, as some GAGs are isolated from mammals. In this context, we investigated the effects and mechanism of action of fucosylated chondroitin sulfate (FucCS), a unique and highly sulfated GAG isolated from the sea cucumber, with respect to P. falciparum cytoadhesion and development. FucCS was effective in inhibiting the cytoadherence of Pf-iEs to human lung endothelial cells and placenta cryosections under static and flow conditions. Removal of the sulfated fucose branches of the FucCS structure virtually abolished the inhibitory effects of FucCS. Importantly, FucCS rapidly disrupted rosettes at high levels, and it was also able to block parasite development by interfering with merozoite invasion. Collectively, these findings highlight the potential of FucCS as a candidate for adjunct therapy against severe malaria. PMID:24395239

  4. Plasmodium falciparum Genotype Diversity in Artemisinin Derivatives Treatment Failure Patients along the Thai-Myanmar Border

    PubMed Central

    Hoonchaiyapoom, Thirasak; Inorn, Kornnarin

    2014-01-01

    Genetic characteristics of Plasmodium falciparum may play a role in the treatment outcome of malaria infection. We have studied the association between diversity at the merozoite surface protein-1 (msp-1), msp-2, and glutamate-rich protein (glurp) loci and the treatment outcome of uncomplicated falciparum malaria patients along the Thai-Myanmar border who were treated with artemisinin derivatives combination therapy. P. falciparum isolates were collected prior to treatment from 3 groups of patients; 50 cases of treatment failures, 50 recrudescences, and 56 successful treatments. Genotyping of the 3 polymorphic markers was analyzed by nested PCR. The distribution of msp-1 alleles was significantly different among the 3 groups of patients but not the msp-2 and glurp alleles. The allelic frequencies of K1 and MAD20 alleles of msp1 gene were higher while RO33 allele was significantly lower in the successful treatment group. Treatment failure samples had a higher median number of alleles as compared to the successful treatment group. Specific genotypes of msp-1, msp-2, and glurp were significantly associated with the treatment outcomes. Three allelic size variants were significantly higher among the isolates from the treatment failure groups, i.e., K1270-290, 3D7610-630, G650-690, while 2 variants, K1150-170, and 3D7670-690 were significantly lower. In conclusion, the present study reports the differences in multiplicity of infection and distribution of specific alleles of msp-1, msp-2, and glurp genes in P. falciparum isolates obtained from treatment failure and successful treatment patients following artemisinin derivatives combination therapy. PMID:25548414

  5. Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

    PubMed Central

    Juliano, Jonathan J; Randrianarivelojosia, Milijaona; Ramarosandratana, Benjamin; Ariey, Frédéric; Mwapasa, Victor; Meshnick, Steven R

    2009-01-01

    Background Strains of Plasmodium falciparum genetically resistant to chloroquine (CQ) due to the presence of pfcrt 76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant P. falciparum isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect pfcrt 76T-bearing P. falciparum minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings. Methods This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect pfcrt 76T-bearing minority variant P. falciparum. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples. Results Thirty one clinical P. falciparum isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the pfcrt K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant P. falciparum strains after therapy as detected by HTA. RFLP analysis failed to detect any pfcrt K76T-bearing isolates. Conclusion These findings indicate that genetically CQ-resistant P. falciparum are more common than previously thought in Madagascar even though the fitness of the minority variant pfcrt 76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the

  6. Parity and Placental Infection Affect Antibody Responses against Plasmodium falciparum during Pregnancy▿ †

    PubMed Central

    Mayor, Alfredo; Rovira-Vallbona, Eduard; Machevo, Sonia; Bassat, Quique; Aguilar, Ruth; Quintó, Llorenç; Jiménez, Alfons; Sigauque, Betuel; Dobaño, Carlota; Kumar, Sanjeev; Singh, Bijender; Gupta, Puneet; Chauhan, Virander S.; Chitnis, Chetan E.; Alonso, Pedro L.; Menéndez, Clara

    2011-01-01

    Women are at higher risk of Plasmodium falciparum infection when pregnant. The decreasing risk of malaria with subsequent pregnancies is attributed to parity-dependent acquisition of antibodies against placental parasites expressing variant surface antigens, VAR2CSA, that mediate placental sequestration through adhesion to chondroitin sulfate A (CSA). However, modulation of immunity during pregnancy may also contribute to increase the risk of malaria. We compared antibody responses among 30 Mozambican primigravidae and 60 multigravidae at delivery, 40 men, and 40 children. IgG levels were measured against the surface antigens of erythrocytes infected with P. falciparum isolated from 12 pregnant women (4 placental and 8 peripheral blood isolates) and 26 nonpregnant hosts. We also measured IgG levels against merozoite recombinant antigens and total IgG. Placental P. falciparum infection was associated with increased levels of total IgG as well as IgG levels against merozoite antigens and parasite isolates from pregnant and nonpregnant hosts. We therefore stratified comparisons of antibody levels by placental infection. Compared to multigravidae, uninfected primigravidae had lower total IgG as well as lower levels of IgGs against peripheral blood isolates from both pregnant and nonpregnant hosts. These differences were not explained by use of bed nets, season at delivery, neighborhood of residence, or age. Compared to men, infected primigravidae had higher levels of IgGs against isolates from pregnant women and CSA-binding lines but not against other isolates, supporting the concept of a pregnancy-specific development of immunity to these parasite variants. Results of this study show that parity and placental infection can modulate immune responses during pregnancy against malaria parasites. PMID:21300778

  7. Population Genetics of GYPB and Association Study between GYPB*S/s Polymorphism and Susceptibility to P. falciparum Infection in the Brazilian Amazon

    PubMed Central

    Amaral, Daphne R. T.; Costa, Daiane C.; Furlani, Natália G.; Zuccherato, Luciana W.; Machado, Moara; Reid, Marion E.; Zalis, Mariano G.; Rossit, Andréa R.; Santos, Sidney E. B.; Machado, Ricardo L.; Lustigman, Sara

    2011-01-01

    Background Merozoites of Plasmodium falciparum invade through several pathways using different RBC receptors. Field isolates appear to use a greater variability of these receptors than laboratory isolates. Brazilian field isolates were shown to mostly utilize glycophorin A-independent invasion pathways via glycophorin B (GPB) and/or other receptors. The Brazilian population exhibits extensive polymorphism in blood group antigens, however, no studies have been done to relate the prevalence of the antigens that function as receptors for P. falciparum and the ability of the parasite to invade. Our study aimed to establish whether variation in the GYPB*S/s alleles influences susceptibility to infection with P. falciparum in the admixed population of Brazil. Methods Two groups of Brazilian Amazonians from Porto Velho were studied: P. falciparum infected individuals (cases); and uninfected individuals who were born and/or have lived in the same endemic region for over ten years, were exposed to infection but have not had malaria over the study period (controls). The GPB Ss phenotype and GYPB*S/s alleles were determined by standard methods. Sixty two Ancestry Informative Markers were genotyped on each individual to estimate admixture and control its potential effect on the association between frequency of GYPB*S and malaria infection. Results GYPB*S is associated with host susceptibility to infection with P. falciparum; GYPB*S/GYPB*S and GYPB*S/GYPB*s were significantly more prevalent in the in the P. falciparum infected individuals than in the controls (69.87% vs. 49.75%; P<0.02). Moreover, population genetics tests applied on the GYPB exon sequencing data suggest that natural selection shaped the observed pattern of nucleotide diversity. Conclusion Epidemiological and evolutionary approaches suggest an important role for the GPB receptor in RBC invasion by P. falciparum in Brazilian Amazons. Moreover, an increased susceptibility to infection by this parasite is

  8. Expression of Plasmodium falciparum lactate dehydrogenase in Escherichia coli.

    PubMed

    Bzik, D J; Fox, B A; Gonyer, K

    1993-05-01

    A Plasmodium falciparum gene is described which encodes lactate dehydrogenase activity (P. falciparum LDH). The P. falciparum LDH gene contains no introns and is present in a single copy on chromosome 13. P. falciparum LDH was expressed in all asexual blood stages as a 1.6-kb mRNA. The predicted 316 amino acid protein coding region of P. falciparum LDH was inserted into the prokaryotic expression vector pKK223-3 and a 33-kDa protein having LDH activity was synthesized in Escherichia coli. P. falciparum LDH primary structure displays high amino acid similarity (50-57%) to vertebrate and bacterial LDH, but lacks the amino terminal extension observed in all vertebrate LDH. The majority of amino acid residues implicated in substrate and coenzyme binding and catalysis of other LDH are well conserved in P. falciparum LDH. However, several notable differences in amino acid composition were observed. P. falciparum LDH contained several distinctive single amino acid insertions and deletions compared to other LDH enzymes, and most remarkably, it contained a novel insertion of 5 amino acids within the conserved mobile loop region near arginine residue 109, a residue which is known to make contact with pyruvate in the ternary complex of other LDH. These results suggest that novel features of P. falciparum LDH primary structure may be correlated with previously characterized and distinctive kinetic, biochemical, immunochemical, and electrophoretic properties of P. falciparum LDH. PMID:8515777

  9. Genetic Structure of Plasmodium falciparum and Elimination of Malaria, Comoros Archipelago

    PubMed Central

    Rebaudet, Stanislas; Bogreau, Hervé; Silaï, Rahamatou; Lepère, Jean-François; Bertaux, Lionel; Pradines, Bruno; Delmont, Jean; Gautret, Philippe; Parola, Philippe

    2010-01-01

    The efficacy of malaria control and elimination on islands may depend on the intensity of new parasite inflow. On the Comoros archipelago, where falciparum malaria remains a major public health problem because of spread of drug resistance and insufficient malaria control, recent interventions for malaria elimination were planned on Moheli, 1 of 4 islands in the Comoros archipelago. To assess the relevance of such a local strategy, we performed a population genetics analysis by using multilocus microsatellite and resistance genotyping of Plasmodium falciparum sampled from each island of the archipelago. We found a contrasted population genetic structure explained by geographic isolation, human migration, malaria transmission, and drug selective pressure. Our findings suggest that malaria elimination interventions should be implemented simultaneously on the entire archipelago rather than restricted to 1 island and demonstrate the necessity for specific chemoresistance surveillance on each of the 4 Comorian islands. PMID:21029525

  10. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    PubMed Central

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-01-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes. PMID:2825189

  11. Molecular cloning and sequence analysis of the Plasmodium falciparum dihydrofolate reductase-thymidylate synthase gene.

    PubMed

    Bzik, D J; Li, W B; Horii, T; Inselburg, J

    1987-12-01

    Genomic DNA clones that coded for the bifunctional dihydrofolate reductase (DHFR) and thymidylate synthase (TS) (DHFR-TS) activities from a pyrimethamine-sensitive strain of Plasmodium falciparum were isolated and sequenced. The deduced DHFR-TS protein contained 608 amino acids (71,682 Da). The coding region for DHFR-TS contained no intervening sequences and had a high A + T content (75%). The DHFR domain, in the amino-terminal portion of the protein, was joined by a 94-amino acid junction sequence to the TS domain in the carboxyl-terminal portion of the protein. The TS domain was more conserved than the DHFR domain and both P. falciparum domains were more homologous to eukaryotic than to prokaryotic forms of the enzymes. Predicted secondary structures of the DHFR and TS domains were nearly identical to the structures identified in other DHFR and TS enzymes. PMID:2825189

  12. A Unique Plasmodium falciparum K13 Gene Mutation in Northwest Ethiopia.

    PubMed

    Bayih, Abebe Genetu; Getnet, Gebeyaw; Alemu, Abebe; Getie, Sisay; Mohon, Abu Naser; Pillai, Dylan R

    2016-01-01

    Artemisinin combination therapy (ACT) is the first line to treat uncomplicated Plasmodium falciparum malaria worldwide. Artemisinin treatment failures are on the rise in southeast Asia. Delayed parasite clearance after ACT is associated with mutations of the P. falciparum kelch 13 gene. Patients (N = 148) in five districts of northwest Ethiopia were enrolled in a 28-day ACT trial. We identified a unique kelch 13 mutation (R622I) in 3/125 (2.4%) samples. The three isolates with R622I were from Negade-Bahir and Aykel districts close to the Ethiopia-Sudan border. One of three patients with the mutant strain was parasitemic at day 3; however, all patients cleared parasites by day 28. Correlation between kelch 13 mutations and parasite clearance was not possible due to the low frequency of mutations in this study. PMID:26483118

  13. Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth.

    PubMed

    Divo, A A; Geary, T G; Davis, N L; Jensen, J B

    1985-02-01

    Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640. PMID:3886898

  14. Identification of two integral membrane proteins of Plasmodium falciparum

    SciTech Connect

    Smythe, J.A.; Coppel, R.L.; Brown, G.V.; Ramasamy, R.; Kemp, D.J.; Anders, R.F. )

    1988-07-01

    The authors describe the isolation and cloning of two integral membrane protein antigens of Plasmodium falciparum. The antigens were isolated by Triton X-114 temperature-dependent phase separation, electrophoretically transferred to nitrocellulose, and used to affinity-purify monospecific human antibodies. These antibodies were used to isolate the corresponding cDNA clones from a phage {lambda}gt11-Amp3 cDNA expression library. Clone Ag512 corresponds to a M{sub r} 55,000 merozoite rhoptry antigen, and clone Ag513 corresponds to a M{sub r} 45,000 merozoite surface antigen. Both proteins can be biosynthetically labeled with ({sup 3}H)glucosamine and ({sup 3}H)myristic acid, suggesting that they may be anchored in membranes via a glycosylphosphatidylinositol moiety. Similarities in the C-terminal sequences of the M{sub r} 45,000 merozoite surface antigen and the Trypanosoma brucei variant surface glycoproteins provides further evidence that this antigen has a glycosylphosphatidylinositol anchor.

  15. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes.

    PubMed

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A; Sauerwein, Robert W

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3-5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  16. Variation in susceptibility of African Plasmodium falciparum malaria parasites to TEP1 mediated killing in Anopheles gambiae mosquitoes

    PubMed Central

    Eldering, Maarten; Morlais, Isabelle; van Gemert, Geert-Jan; van de Vegte-Bolmer, Marga; Graumans, Wouter; Siebelink-Stoter, Rianne; Vos, Martijn; Abate, Luc; Roeffen, Will; Bousema, Teun; Levashina, Elena A.; Sauerwein, Robert W.

    2016-01-01

    Anopheles gambiae s.s. mosquitoes are efficient vectors for Plasmodium falciparum, although variation exists in their susceptibility to infection. This variation depends partly on the thioester-containing protein 1 (TEP1) and TEP depletion results in significantly elevated numbers of oocysts in susceptible and resistant mosquitoes. Polymorphism in the Plasmodium gene coding for the surface protein Pfs47 modulates resistance of some parasite laboratory strains to TEP1-mediated killing. Here, we examined resistance of P. falciparum isolates of African origin (NF54, NF165 and NF166) to TEP1-mediated killing in a susceptible Ngousso and a refractory L3–5 strain of A. gambiae. All parasite clones successfully developed in susceptible mosquitoes with limited evidence for an impact of TEP1 on transmission efficiency. In contrast, NF166 and NF165 oocyst densities were strongly reduced in refractory mosquitoes and TEP1 silencing significantly increased oocyst densities. Our results reveal differences between African P. falciparum strains in their capacity to evade TEP1-mediated killing in resistant mosquitoes. There was no significant correlation between Pfs47 genotype and resistance of a given P. falciparum isolate for TEP1 killing. These data suggest that polymorphisms in this locus are not the sole mediators of immune evasion of African malaria parasites. PMID:26861587

  17. Genetic diversity in the block 2 region of the merozoite surface protein-1 of Plasmodium falciparum in central India

    PubMed Central

    2012-01-01

    Background Malaria continues to be a significant health problem in India. Several of the intended Plasmodium falciparum vaccine candidate antigens are highly polymorphic. The genetic diversity of P. falciparum merozoite surface protein-1 (MSP-1) has been extensively studied from various parts of the world. However, limited data are available from India. The aim of the present study was a molecular characterization of block 2 region of MSP-1 gene from the tribal-dominated, forested region of Madhya Pradesh. Methods DNA sequencing analysis was carried out in 71 field isolates collected between July 2005 to November 2005 and in 98 field isolates collected from July 2009 to December 2009. Alleles identified by DNA sequencing were aligned with the strain 3D7 and polymorphism analysis was done by using Edit Sequence tool (DNASTAR). Results The malaria positivity was 26% in 2005, which rose to 29% in 2009 and P. falciparum prevalence was also increased from 72% in 2005 to 81% in 2009. The overall allelic prevalence was higher in K1 (51%) followed by MAD20 (28%) and RO33 (21%) in 2005 while in 2009, RO33 was highest (40%) followed by K1 (36%) and MAD20 (24%). Conclusions The present study reports extensive genetic variations and dynamic evolution of block 2 region of MSP-1 in central India. Characterization of antigenic diversity in vaccine candidate antigens are valuable for future vaccine trials as well as understanding the population dynamics of P. falciparum parasites in this area. PMID:22439658

  18. Regular production of infective sporozoites of Plasmodium falciparum and P. vivax in laboratory-bred Anopheles albimanus.

    PubMed

    Hurtado, S; Salas, M L; Romero, J F; Zapata, J C; Ortiz, H; Arevalo-Herrera, M; Herrera, S

    1997-01-01

    One of the major constraints for studies on the sporogonic cycle of the parasites causing human malaria, and on the protective efficacy of pre-erythrocytic vaccines, is the scarcity of laboratory-reared Anopheles mosquitoes as a source of infective sporozoites. The aim of the present study was to reproduce the life-cycles of Plasmodium falciparum and P. vivax in the laboratory and so develop the ability to produce infective sporozoites of these two species regularly under laboratory conditions. Colonized Anopheles albimanus, of Buenaventura and Tecojate strains, were infected by feeding either on Plasmodium-infected blood, from human patients or experimentally inoculated Aotus monkeys, or on gametocytes of the P. falciparum NF-54 isolate grown in vitro. The monkeys were infected with the blood stages of a Colombian P. vivax isolate and then, after recovery, with the Santa Lucia strain of P. falciparum from El Salvador. Although both of the mosquito strains used were successfully infected with both parasite species, the Buenaventura strain of mosquito was generally more susceptible to infection than the Tecojate strain, and particularly to infection with the parasites from the patients, who lived where this strain of mosquitoes was originally isolated. Monkeys injected intravenously with the P. vivax sporozoites produced in the mosquitoes developed patent sexual and asexual parasitaemias; the gametocytes that developed could then be used to infect mosquitoes, allowing the development of more sporozoites. However, experimental infections failed to establish after the P. falciparum sporozoites were used to inoculate monkeys. The ability to reproduce the complete life cycle of P. vivax in the laboratory, from human to mosquito and then to monkey, should greatly facilitate many studies on vivax malaria and on the efficacy of candidate malaria vaccines. The availability of the sporogonic cycles of P. falciparum from three different sources should also permit a variety of

  19. Fresh, Rayed Impact Crater

    NASA Technical Reports Server (NTRS)

    2003-01-01

    MGS MOC Release No. MOC2-416, 9 July 2003

    This Mars Global Surveyor (MGS) Mars Orbiter Camera (MOC) image shows a fresh, young meteor impact crater on the martian surface. It is less than 400 meters (less than 400 yards) across. While there is no way to know the exact age of this or any other martian surface feature, the rays are very well preserved. On a planet where wind can modify surface features at the present time, a crater with rayed ejecta patterns must be very young indeed. Despite its apparent youth, the crater could still be many hundreds of thousands, if not several million, of years old. This impact scar is located within the much larger Crommelin Crater, near 5.6oN, 10.0oW. Sunlight illuminates the scene from the left.

  20. Genome scanning of Amazonian Plasmodium falciparum shows subtelomeric instability and clindamycin-resistant parasites

    PubMed Central

    Dharia, Neekesh V.; Plouffe, David; Bopp, Selina E.R.; González-Páez, Gonzalo E.; Lucas, Carmen; Salas, Carola; Soberon, Valeria; Bursulaya, Badry; Kochel, Tadeusz J.; Bacon, David J.; Winzeler, Elizabeth A.

    2010-01-01

    Here, we fully characterize the genomes of 14 Plasmodium falciparum patient isolates taken recently from the Iquitos region using genome scanning, a microarray-based technique that delineates the majority of single-base changes, indels, and copy number variants distinguishing the coding regions of two clones. We show that the parasite population in the Peruvian Amazon bears a limited number of genotypes and low recombination frequencies. Despite the essentially clonal nature of some isolates, we see high frequencies of mutations in subtelomeric highly variable genes and internal var genes, indicating mutations arising during self-mating or mitotic replication. The data also reveal that one or two meioses separate different isolates, showing that P. falciparum clones isolated from different individuals in defined geographical regions could be useful in linkage analyses or quantitative trait locus studies. Through pairwise comparisons of different isolates we discovered point mutations in the apicoplast genome that are close to known mutations that confer clindamycin resistance in other species, but which were hitherto unknown in malaria parasites. Subsequent drug sensitivity testing revealed over 100-fold increase of clindamycin EC50 in strains harboring one of these mutations. This evidence of clindamycin-resistant parasites in the Amazon suggests that a shift should be made in health policy away from quinine + clindamycin therapy for malaria in pregnant women and infants, and that the development of new lincosamide antibiotics for malaria should be reconsidered. PMID:20829224

  1. Targeting the gyrase of Plasmodium falciparum with topoisomerase poisons.

    PubMed

    Tang Girdwood, Sonya C; Nenortas, Elizabeth; Shapiro, Theresa A

    2015-06-15

    Drug-resistant malaria poses a major public health problem throughout the world and the need for new antimalarial drugs is growing. The apicoplast, a chloroplast-like organelle essential for malaria parasite survival and with no counterpart in humans, offers an attractive target for selectively toxic new therapies. The apicoplast genome (plDNA) is a 35 kb circular DNA that is served by gyrase, a prokaryotic type II topoisomerase. Gyrase is poisoned by fluoroquinolone antibacterials that stabilize a catalytically inert ternary complex of enzyme, its plDNA substrate, and inhibitor. We used fluoroquinolones to study the gyrase and plDNA of Plasmodium falciparum. New methods for isolating and separating plDNA reveal four topologically different forms and permit a quantitative exam of perturbations that result from gyrase poisoning. In keeping with its role in DNA replication, gyrase is most abundant in late stages of the parasite lifecycle, but several lines of evidence indicate that even in these cells the enzyme is present in relatively low abundance: about 1 enzyme for every two plDNAs or a ratio of 1 gyrase: 70 kb DNA. For a spectrum of quinolones, correlation was generally good between antimalarial activity and gyrase poisoning, the putative molecular mechanism of drug action. However, in P. falciparum there is evidence for off-target toxicity, particularly for ciprofloxacin. These studies highlight the utility of the new methods and of fluoroquinolones as a tool for studying the in situ workings of gyrase and its plDNA substrate. PMID:25881748

  2. Plasmodium falciparum: multifaceted resistance to artemisinins.

    PubMed

    Paloque, Lucie; Ramadani, Arba P; Mercereau-Puijalon, Odile; Augereau, Jean-Michel; Benoit-Vical, Françoise

    2016-01-01

    Plasmodium falciparum resistance to artemisinins, the most potent and fastest acting anti-malarials, threatens malaria elimination strategies. Artemisinin resistance is due to mutation of the PfK13 propeller domain and involves an unconventional mechanism based on a quiescence state leading to parasite recrudescence as soon as drug pressure is removed. The enhanced P. falciparum quiescence capacity of artemisinin-resistant parasites results from an increased ability to manage oxidative damage and an altered cell cycle gene regulation within a complex network involving the unfolded protein response, the PI3K/PI3P/AKT pathway, the PfPK4/eIF2α cascade and yet unidentified transcription factor(s), with minimal energetic requirements and fatty acid metabolism maintained in the mitochondrion and apicoplast. The detailed study of these mechanisms offers a way forward for identifying future intervention targets to fend off established artemisinin resistance. PMID:26955948

  3. Artemisinin Action and Resistance in Plasmodium falciparum.

    PubMed

    Tilley, Leann; Straimer, Judith; Gnädig, Nina F; Ralph, Stuart A; Fidock, David A

    2016-09-01

    The worldwide use of artemisinin-based combination therapies (ACTs) has contributed in recent years to a substantial reduction in deaths resulting from Plasmodium falciparum malaria. Resistance to artemisinins, however, has emerged in Southeast Asia. Clinically, resistance is defined as a slower rate of parasite clearance in patients treated with an artemisinin derivative or an ACT. These slow clearance rates associate with enhanced survival rates of ring-stage parasites briefly exposed in vitro to dihydroartemisinin. We describe recent progress made in defining the molecular basis of artemisinin resistance, which has identified a primary role for the P. falciparum K13 protein. Using K13 mutations as molecular markers, epidemiological studies are now tracking the emergence and spread of artemisinin resistance. Mechanistic studies suggest potential ways to overcome resistance. PMID:27289273

  4. Acute renal failure due to falciparum malaria.

    PubMed

    Habte, B

    1990-01-01

    Seventy-two patients with severe falciparum malaria are described. Twenty-four (33.3%) were complicated by acute renal failure. Comparing patients with renal failure and those without, statistically significant differences occurred regarding presence of cerebral malaria (83% vs 46%), jaundice (92% vs 33%), and death (54% vs 17%). A significantly higher number of patients with renal failure were nonimmune visitors to malaria endemic regions. Renal failure was oliguric in 45% of cases. Dialysis was indicated in 38%, 29% died in early renal failure, and 33% recovered spontaneously. It is concluded that falciparum malaria is frequently complicated by cerebral malaria and renal failure. As nonimmune individuals are prone to develop serious complications, malaria prophylaxis and vigorous treatment of cases is mandatory. PMID:2236718

  5. The paradoxical population genetics of Plasmodium falciparum.

    PubMed

    Hartl, Daniel L; Volkman, Sarah K; Nielsen, Kaare M; Barry, Alyssa E; Day, Karen P; Wirth, Dyann F; Winzeler, Elizabeth A

    2002-06-01

    Among the leading causes of death in African children is cerebral malaria caused by the parasitic protozoan Plasmodium falciparum. Endemic forms of this disease are thought to have originated in central Africa 5000-10000 years ago, coincident with the innovation of slash-and-burn agriculture and the diversification of the Anopheles gambiae complex of mosquito vectors. Population genetic studies of P. falciparum have yielded conflicting results. Some evidence suggests that today's population includes multiple ancient lineages pre-dating human speciation. Other evidence suggests that today's population derives from only one, or a small number, of these ancient lineages. Resolution of this issue is important for the evaluation of the long-term efficacy of drug and immunological control strategies. PMID:12036741

  6. UvrD helicase of Plasmodium falciparum.

    PubMed

    Shankar, Jay; Tuteja, Renu

    2008-03-15

    Malaria caused by the mosquito-transmitted parasite Plasmodium is the cause of enormous number of deaths every year in the tropical and subtropical areas of the world. Among four species of Plasmodium, Plasmodium falciparum causes most fatal form of malaria. With time, the parasite has developed insecticide and drug resistance. Newer strategies and advent of novel drug targets are required so as to combat the deadly form of malaria. Helicases is one such class of enzymes which has previously been suggested as potential antiviral and anticancer targets. These enzymes play an essential role in nearly all the nucleic acid metabolic processes, catalyzing the transient opening of the duplex nucleic acids in an NTP-dependent manner. DNA helicases from the PcrA/UvrD/Rep subfamily are important for the survival of the various organisms. Members from this subfamily can be targeted and inhibited by a variety of synthetic compounds. UvrD from this subfamily is the only member present in the P. falciparum genome, which shows no homology with UvrD from human and thus can be considered as a strong potential drug target. In this manuscript we provide an overview of UvrD family of helicases and bioinformatics analysis of UvrD from P. falciparum. PMID:18242886

  7. Fresh Veggies from Space

    NASA Technical Reports Server (NTRS)

    2001-01-01

    Professor Marc Anderson of the University of Wisconsin-Madison developed a technology for use in plant-growth experiments aboard the Space Shuttle. Anderson's research and WCSAR's technology were funded by NASA and resulted in a joint technology licensed to KES Science and Technology, Inc. This transfer of space-age technology resulted in the creation of a new plant-saving product, an ethylene scrubber for plant growth chambers. This innovation presents commercial benefits for the food industry in the form of a new device, named Bio-KES. Bio-KES removes ethylene and helps to prevent spoilage. Ethylene accounts for up to 10 percent of produce losses and 5 percent of flower losses. Using Bio-KES in storage rooms and displays will increase the shelf life of perishable foods by more than one week, drastically reducing the costs associated with discarded rotten foods and flowers. The savings could potentially be passed on to consumers. For NASA, the device means that astronauts can conduct commercial agricultural research in space. Eventually, it may also help to grow food in space and keep it fresh longer. This could lead to less packaged food being taken aboard missions since it could be cultivated in an ethylene-free environment.

  8. Characterization of the gene encoding the largest subunit of Plasmodium falciparum RNA polymerase III.

    PubMed

    Li, W B; Bzik, D J; Tanaka, M; Gu, H M; Fox, B A; Inselburg, J

    1991-06-01

    We report here the isolation, sequence analysis, structure, and expression of the gene encoding the largest subunit of RNA polymerase III (RPIII) from Plasmodium falciparum. The P. falciparum RPIII gene consists of 5 exons and 4 introns, is expressed in all of the asexual erythrocytic stages of the parasite as a 8.5-kb mRNA, and is present in a single copy on chromosome 13. The predicted 2339 amino acid residue RPIII subunit contained 5 regions that were conserved between different eukaryotic RPIII subunits, and 4 variable regions that separated the conserved regions. Three of the variable regions were greatly enlarged in comparison to the corresponding variable regions in other RPIII subunits. Variable region C' represented nearly one-third of the P. falciparum RPIII subunit (750 amino acid residues), included a unique repeated decapeptide sequence, and had some homology with yeast DNA topoisomerase II. Noteworthy amino acid sequences and structures were identified in both the conserved regions and in the enlarged variable regions, and their possible role(s) as domains that regulate RPIII enzyme activity is discussed. PMID:1656254

  9. Deformability based sorting of red blood cells improves diagnostic sensitivity for malaria caused by Plasmodium falciparum.

    PubMed

    Guo, Quan; Duffy, Simon P; Matthews, Kerryn; Deng, Xiaoyan; Santoso, Aline T; Islamzada, Emel; Ma, Hongshen

    2016-02-21

    The loss of red blood cell (RBC) deformability is part of the pathology of many diseases. In malaria caused by Plasmodium falciparum infection, metabolism of hemoglobin by the parasite results in progressive reduction in RBC deformability that is directly correlated with the growth and development of the parasite. The ability to sort RBCs based on deformability therefore provides a means to isolate pathological cells and to study biochemical events associated with disease progression. Existing methods have not been able to sort RBCs based on deformability or to effectively enrich for P. falciparum infected RBCs at clinically relevant concentrations. Here, we develop a method to sort RBCs based on deformability and demonstrate the ability to enrich the concentration of ring-stage P. falciparum infected RBCs (Pf-iRBCs) by >100× from clinically relevant parasitemia (<0.01%). Deformability based sorting of RBCs is accomplished using ratchet transport through asymmetrical constrictions using oscillatory flow. This mechanism provides dramatically improved selectivity over previous biophysical methods by preventing the accumulation of cells in the filter microstructure to ensure that consistent filtration forces are applied to each cell. We show that our approach dramatically improves the sensitivity of malaria diagnosis performed using both microscopy and rapid diagnostic test by converting samples with difficult-to-detect parasitemia (<0.01%) into samples with easily detectable parasitemia (>0.1%). PMID:26768227

  10. Microbial safety of fresh produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The book entitled “Microbial Safety of Fresh Produce” with 23 chapters is divided into following six sections: Microbial contamination of fresh produce, Pre-harvest strategies, post-harvest interventions, Produce safety during processing and handling, Public, legal, and economic Perspectives, and Re...

  11. Effect of Fluorescent Dyes on In Vitro-Differentiated, Late-Stage Plasmodium falciparum Gametocytes

    PubMed Central

    Gebru, Tamirat; Mordmüller, Benjamin

    2014-01-01

    Plasmodium falciparum gametocytes are not associated with clinical symptoms, but they are responsible for transmitting the pathogen to mosquitoes. Therefore, gametocytocidal interventions are important for malaria control and resistance containment. Currently available drugs and vaccines are not well suited for that purpose. Several dyes have potent antimicrobial activity, but their use against gametocytes has not been investigated systematically. The gametocytocidal activity of nine synthetic dyes and four control compounds was tested against stage V gametocytes of the laboratory strain 3D7 and three clinical isolates of P. falciparum with a bioluminescence assay. Five of the fluorescent dyes had submicromolar 50% inhibitory concentration (IC50) values against mature gametocytes. Three mitochondrial dyes, MitoRed, dihexyloxacarbocyanine iodide (DiOC6), and rhodamine B, were highly active (IC50s < 200 nM). MitoRed showed the highest activity against gametocytes, with IC50s of 70 nM against 3D7 and 120 to 210 nM against clinical isolates. All compounds were more active against the laboratory strain 3D7 than against clinical isolates. In particular, the endoperoxides artesunate and dihydroartemisinin showed a 10-fold higher activity against 3D7 than against clinical isolates. In contrast to all clinically used antimalarials, several fluorescent dyes had surprisingly high in vitro activity against late-stage gametocytes. Since they also act against asexual blood stages, they shall be considered starting points for the development of new antimalarial lead compounds. PMID:25267675

  12. Population genetic structure of Plasmodium falciparum across a region of diverse endemicity in West Africa

    PubMed Central

    2012-01-01

    Background Malaria parasite population genetic structure varies among areas of differing endemicity, but this has not been systematically studied across Plasmodium falciparum populations in Africa where most infections occur. Methods Ten polymorphic P. falciparum microsatellite loci were genotyped in 268 infections from eight locations in four West African countries (Republic of Guinea, Guinea Bissau, The Gambia and Senegal), spanning a highly endemic forested region in the south to a low endemic Sahelian region in the north. Analysis was performed on proportions of mixed genotype infections, genotypic diversity among isolates, multilocus standardized index of association, and inter-population differentiation. Results Each location had similar levels of pairwise genotypic diversity among isolates, although there were many more mixed parasite genotype infections in the south. Apart from a few isolates that were virtually identical, the multilocus index of association was not significant in any population. Genetic differentiation between populations was low (most pairwise FST values < 0.03), and an overall test for isolation by distance was not significant. Conclusions Although proportions of mixed genotype infections varied with endemicity as expected, population genetic structure was similar across the diverse sites. Very substantial reduction in transmission would be needed to cause fragmented or epidemic sub-structure in this region. PMID:22759447

  13. Lack of artemisinin resistance in Plasmodium falciparum in northwest Benin after 10 years of use of artemisinin-based combination therapy

    PubMed Central

    Ogouyèmi-Hounto, Aurore; Damien, Georgia; Deme, Awa Bineta; Ndam, Nicaise T.; Assohou, Constance; Tchonlin, Didier; Mama, Atika; Hounkpe, Virgile Olivier; Moutouama, Jules Doumitou; Remoué, Franck; Ndiaye, Daouda; Gazard, Dorothée Kinde

    2016-01-01

    Aim: In Benin, artemisinin-based combination therapy (ACT) has been recommended as the first-line treatment for uncomplicated Plasmodium falciparum malaria since 2004. The emergence in Southeast Asia of parasites that are resistant to artemisinins poses a serious threat to global control of this disease. The presence of artemisinin resistance genotypes in parasite populations in Benin is currently unknown. The present study investigated the prevalence of relevant K13-propeller gene polymorphisms in parasite isolates from the north-western region of Benin. Method: Plasmodium falciparum isolates were collected from children with a confirmed diagnosis of malaria aged 6 months to 5 years in two towns, Cobly and Djougou, in the north-western part of Benin. The study was conducted during the rainy season from July to November 2014 in local health facilities. The K13-propeller gene was amplified in parasite isolates using nested PCR and subsequently sequenced. Results: A total of 108 children were recruited into the study. The efficiency of amplification reactions was 72% (78/108). The propeller domain of the K13 gene was successfully sequenced in 78 P. falciparum isolates; all of them were wild type with no polymorphisms detectable. Conclusion: The absence of mutations in the K13 gene indicates that P. falciparum parasite populations in the study area are still fully susceptible to artemisinins. PMID:27443837

  14. Tracing the origins and signatures of selection of antifolate resistance in island populations of Plasmodium falciparum

    PubMed Central

    2010-01-01

    Background Resistance of the malaria parasite Plasmodium falciparum to sulfadoxine-pyrimethamine (SP) has evolved worldwide. In the archipelago of São Tomé and Principe (STP), West Africa, although SP resistance is highly prevalent the drug is still in use in particular circumstances. To address the evolutionary origins of SP resistance in these islands, we genotyped point mutations at P. falciparum dhfr and dhps genes and analysed microsatellites flanking those genes. Methods Blood samples were collected in July and December 2004 in three localities of São Tomé Island and one in Principe Island. Species-specific nested-PCR was used to identify P. falciparum infected samples. Subsequently, SNPs at the dhfr and dhps genes were identified through PCR-RFLP. Isolates were also analysed for three microsatellite loci flanking the dhfr gene, three loci flanking dhps and four loci located at putative neutral genomic regions. Results An increase of resistance-associated mutations at dhfr and dhps was observed, in particular for the dhfr/dhps quintuple mutant, associated with clinical SP failure. Analysis of flanking microsatellites suggests multiple independent introductions for dhfr and dhps mutant haplotypes, possibly from West Africa. A reduced genetic diversity and increased differentiation at flanking microsatellites when compared to neutral loci is consistent with a selective sweep for resistant alleles at both loci. Conclusions This study provides additional evidence for the crucial role of gene flow and drug selective pressures in the rapid spread of SP resistance in P. falciparum populations, from only a few mutation events giving rise to resistance-associated mutants. It also highlights the importance of human migration in the spread of drug resistant malaria parasites, as the distance between the islands and mainland is not consistent with mosquito-mediated parasite dispersal. PMID:20534146

  15. Sterile Protective Immunity to Malaria is Associated with a Panel of Novel P. falciparum Antigens*

    PubMed Central

    Trieu, Angela; Kayala, Matthew A.; Burk, Chad; Molina, Douglas M.; Freilich, Daniel A.; Richie, Thomas L.; Baldi, Pierre; Felgner, Philip L.; Doolan, Denise L.

    2011-01-01

    The development of an effective malaria vaccine remains a global public health priority. Less than 0.5% of the Plasmodium falciparum genome has been assessed as potential vaccine targets and candidate vaccines have been based almost exclusively on single antigens. It is possible that the failure to develop a malaria vaccine despite decades of effort might be attributed to this historic focus. To advance malaria vaccine development, we have fabricated protein microarrays representing 23% of the entire P. falciparum proteome and have probed these arrays with plasma from subjects with sterile protection or no protection after experimental immunization with radiation attenuated P. falciparum sporozoites. A panel of 19 pre-erythrocytic stage antigens was identified as strongly associated with sporozoite-induced protective immunity; 16 of these antigens were novel and 85% have been independently identified in sporozoite and/or liver stage proteomic or transcriptomic data sets. Reactivity to any individual antigen did not correlate with protection but there was a highly significant difference in the cumulative signal intensity between protected and not protected individuals. Functional annotation indicates that most of these signature proteins are involved in cell cycle/DNA processing and protein synthesis. In addition, 21 novel blood-stage specific antigens were identified. Our data provide the first evidence that sterile protective immunity against malaria is directed against a panel of novel P. falciparum antigens rather than one antigen in isolation. These results have important implications for vaccine development, suggesting that an efficacious malaria vaccine should be multivalent and targeted at a select panel of key antigens, many of which have not been previously characterized. PMID:21628511

  16. Enhancing Blockade of Plasmodium falciparum Erythrocyte Invasion: Assessing Combinations of Antibodies against PfRH5 and Other Merozoite Antigens

    PubMed Central

    Miura, Kazutoyo; Illingworth, Joseph J.; Choudhary, Prateek; Murungi, Linda M.; Furze, Julie M.; Diouf, Ababacar; Miotto, Olivo; Crosnier, Cécile; Wright, Gavin J.; Kwiatkowski, Dominic P.; Fairhurst, Rick M.; Long, Carole A.; Draper, Simon J.

    2012-01-01

    No vaccine has yet proven effective against the blood-stages of Plasmodium falciparum, which cause the symptoms and severe manifestations of malaria. We recently found that PfRH5, a P. falciparum-specific protein expressed in merozoites, is efficiently targeted by broadly-neutralizing, vaccine-induced antibodies. Here we show that antibodies against PfRH5 efficiently inhibit the in vitro growth of short-term-adapted parasite isolates from Cambodia, and that the EC50 values of antigen-specific antibodies against PfRH5 are lower than those against PfAMA1. Since antibody responses elicited by multiple antigens are speculated to improve the efficacy of blood-stage vaccines, we conducted detailed assessments of parasite growth inhibition by antibodies against PfRH5 in combination with antibodies against seven other merozoite antigens. We found that antibodies against PfRH5 act synergistically with antibodies against certain other merozoite antigens, most notably with antibodies against other erythrocyte-binding antigens such as PfRH4, to inhibit the growth of a homologous P. falciparum clone. A combination of antibodies against PfRH4 and basigin, the erythrocyte receptor for PfRH5, also potently inhibited parasite growth. This methodology provides the first quantitative evidence that polyclonal vaccine-induced antibodies can act synergistically against P. falciparum antigens and should help to guide the rational development of future multi-antigen vaccines. PMID:23144611

  17. A 75 kd merozoite surface protein of Plasmodium falciparum which is related to the 70 kd heat-shock proteins.

    PubMed Central

    Ardeshir, F; Flint, J E; Richman, S J; Reese, R T

    1987-01-01

    Proteins on the merozoite surface of the human malarial parasite Plasmodium falciparum are targets of the host's immune response. The merozoite surface location of p75, a 75 kd P. falciparum protein, was established by immunoelectron microscopy using antisera raised to the expressed product of a cDNA clone. Immunoprecipitation from protein extracts biosynthetically labeled during different periods of the asexual cycle showed that p75 is made continuously, although ring-stage parasites appear to synthesize larger quantities. p75 is conserved and invariant in size in eight isolates of P. falciparum. The 880 bp cDNA sequence encoding part of p75 reveals one open reading frame containing a repetitive sequence unit of four amino acids. The predicted reading frame is correct since antisera to a synthetic peptide corresponding to the repetitive region recognize p75 in immunoblots. The sequence of p75 is homologous with the sequences of proteins from the ubiquitous, highly conserved family of 70 kd heat-shock proteins, suggesting an important physiological function for p75. The cDNA fragment encoding part of p75 hybridizes with multiple genomic fragments, whose sizes are identical in DNA from nine P. falciparum strains, suggesting that the gene for p75 is well conserved and may be part of a gene family. Images Fig. 1. Fig. 2. Fig. 4. Fig. 5. Fig. 6. PMID:3556166

  18. Antibody recognition of Plasmodium falciparum infected red blood cells by symptomatic and asymptomatic individuals in the Brazilian Amazon

    PubMed Central

    Fratus, Alessandra Sampaio Bassi; Cabral, Fernanda Janku; Fotoran, Wesley Luzetti; Medeiros, Márcia Melo; Carlos, Bianca Cechetto; Martha, Rosimeire dalla; da Silva, Luiz Hildebrando Pereira; Lopes, Stefanie Costa Pinto; Costa, Fabio Trindade Maranhão; Wunderlich, Gerhard

    2014-01-01

    In the Amazon Region, there is a virtual absence of severe malaria and few fatal cases of naturally occurring Plasmodium falciparum infections; this presents an intriguing and underexplored area of research. In addition to the rapid access of infected persons to effective treatment, one cause of this phenomenon might be the recognition of cytoadherent variant proteins on the infected red blood cell (IRBC) surface, including the var gene encoded P. falciparum erythrocyte membrane protein 1. In order to establish a link between cytoadherence, IRBC surface antibody recognition and the presence or absence of malaria symptoms, we phenotype-selected four Amazonian P. falciparum isolates and the laboratory strain 3D7 for their cytoadherence to CD36 and ICAM1 expressed on CHO cells. We then mapped the dominantly expressed var transcripts and tested whether antibodies from symptomatic or asymptomatic infections showed a differential recognition of the IRBC surface. As controls, the 3D7 lineages expressing severe disease-associated phenotypes were used. We showed that there was no profound difference between the frequency and intensity of antibody recognition of the IRBC-exposed P. falciparum proteins in symptomatic vs. asymptomatic infections. The 3D7 lineages, which expressed severe malaria-associated phenotypes, were strongly recognised by most, but not all plasmas, meaning that the recognition of these phenotypes is frequent in asymptomatic carriers, but is not necessarily a prerequisite to staying free of symptoms. PMID:25099336

  19. Chloroquine-Resistant Haplotype Plasmodium falciparum Parasites, Haiti

    PubMed Central

    Londono, Berlin L.; Eisele, Thomas P.; Keating, Joseph; Bennett, Adam; Chattopadhyay, Chandon; Heyliger, Gaetan; Mack, Brian; Rawson, Ian; Vely, Jean-Francois; Désinor, Olbeg

    2009-01-01

    Plasmodium falciparum parasites have been endemic to Haiti for >40 years without evidence of chloroquine (CQ) resistance. In 2006 and 2007, we obtained blood smears for rapid diagnostic tests (RDTs) and filter paper blots of blood from 821 persons by passive and active case detection. P. falciparum infections diagnosed for 79 persons by blood smear or RDT were confirmed by PCR for the small subunit rRNA gene of P. falciparum. Amplification of the P. falciparum CQ resistance transporter (pfcrt) gene yielded 10 samples with amplicons resistant to cleavage by ApoI. A total of 5 of 9 samples had threonine at position 76 of pfcrt, which is consistent with CQ resistance (haplotypes at positions 72–76 were CVIET [n = 4] and CVMNT [n = 1]); 4 had only the wild-type haplotype associated with CQ susceptibility (CVMNK). These results indicate that CQ-resistant haplotype P. falciparum malaria parasites are present in Haiti. PMID:19402959

  20. Cryo-electron microscopy of extracellular vesicles in fresh plasma

    PubMed Central

    Yuana, Yuana; Koning, Roman I.; Kuil, Maxim E.; Rensen, Patrick C. N.; Koster, Abraham J.; Bertina, Rogier M; Osanto, Susanne

    2013-01-01

    Introduction Extracellular vesicles (EV) are phospholipid bilayer-enclosed vesicles recognized as new mediators in intercellular communication and potential biomarkers of disease. They are found in many body fluids and mainly studied in fractions isolated from blood plasma in view of their potential in medicine. Due to the limitations of available analytical methods, morphological information on EV in fresh plasma is still rather limited. Objectives To image EV and determine the morphology, structure and size distribution in fresh plasma by cryo-electron microscopy (cryo-EM). Methods Fresh citrate- and ethylenediaminetetraacetic acid (EDTA)-anticoagulated plasma or EV isolated from these plasmas were rapidly cryo-immobilized by vitrification and visualized by cryo-EM. Results EV isolated from fresh plasma were highly heterogeneous in morphology and size and mostly contain a discernible lipid bilayer (lipid vesicles). In fresh plasma there were 2 types of particles with a median diameter of 30 nm (25–260 nm). The majority of these particles are electron dense particles which most likely represent lipoproteins. The minority are lipid vesicles, either electron dense or electron lucent, which most likely represent EV. Lipid vesicles were occasionally observed in close proximity of platelets in citrate and EDTA-anticoagulated platelet-rich plasma. Cryo-electron tomography (cryo-ET) was employed to determine the 3D structure of platelet secretory granules. Conclusions Cryo-EM is a powerful technique that enables the characterization of EV in fresh plasma revealing structural details and considerable morphological heterogeneity. Only a small proportion of the submicron structures in fresh plasma are lipid vesicles representing EV. PMID:24455109

  1. A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras.

    PubMed

    Fontecha, Gustavo A; Sanchez, Ana L; Mendoza, Meisy; Banegas, Engels; Mejía-Torres, Rosa E

    2014-07-01

    Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt "CVMNK" genotype in codons 72-76. PMID:25075788

  2. A four-year surveillance program for detection of Plasmodium falciparum chloroquine resistance in Honduras

    PubMed Central

    Fontecha, Gustavo A; Sanchez, Ana L; Mendoza, Meisy; Banegas, Engels; Mejía-Torres, Rosa E

    2014-01-01

    Countries could use the monitoring of drug resistance in malaria parasites as an effective early warning system to develop the timely response mechanisms that are required to avert the further spread of malaria. Drug resistance surveillance is essential in areas where no drug resistance has been reported, especially if neighbouring countries have previously reported resistance. Here, we present the results of a four-year surveillance program based on the sequencing of the pfcrt gene of Plasmodium falciparum populations from endemic areas of Honduras. All isolates were susceptible to chloroquine, as revealed by the pfcrt “CVMNK” genotype in codons 72-76. PMID:25075788

  3. Steroidal saponins from fresh stems of Dracaena angustifolia

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Six new steroidal saponins (1-6), angudracanosides A-F, were isolated from fresh stems of Dracaena angustifolia, together with eight known compounds. The structures of compounds 1-6 were determined by detailed spectroscopic analyses and chemical methods. Antifungal testing of all compounds showed th...

  4. Hypervariability within the Rifin, Stevor and Pfmc-2TM superfamilies in Plasmodium falciparum

    PubMed Central

    Lavazec, Catherine; Sanyal, Sohini; Templeton, Thomas J.

    2006-01-01

    The human malaria parasite, Plasmodium falciparum, possesses a broad repertoire of proteins that are proposed to be trafficked to the erythrocyte cytoplasm or surface, based upon the presence within these proteins of a Pexel/VTS erythrocyte-trafficking motif. This catalog includes large families of predicted 2 transmembrane (2TM) proteins, including the Rifin, Stevor and Pfmc-2TM superfamilies, of which each possesses a region of extensive sequence diversity across paralogs and between isolates that is confined to a proposed surface-exposed loop on the infected erythrocyte. Here we express epitope-tagged versions of the 2TM proteins in transgenic NF54 parasites and present evidence that the Stevor and Pfmc-2TM families are exported to the erythrocyte membrane, thus supporting the hypothesis that host immune pressure drives antigenic diversity within the loop. An examination of multiple P.falciparum isolates demonstrates that the hypervariable loop within Stevor and Pfmc-2TM proteins possesses sequence diversity across isolate boundaries. The Pfmc-2TM genes are encoded within large amplified loci that share profound nucleotide identity, which in turn highlight the divergences observed within the hypervariable loop. The majority of Pexel/VTS proteins are organized together within sub-telomeric genome neighborhoods, and a mechanism must therefore exist to differentially generate sequence diversity within select genes, as well as within highly defined regions within these genes. PMID:17148488

  5. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut processing plant

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Biofilm forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been ...

  6. Foods - fresh vs. frozen or canned

    MedlinePlus

    Frozen foods vs. fresh or canned; Fresh foods vs. frozen or canned; Frozen vegetables versus fresh ... a well-balanced diet. Many people wonder if frozen and canned vegetables are as healthy for you ...

  7. Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax

    PubMed Central

    Talundzic, Eldin; Chenet, Stella M.; Goldman, Ira F.; Patel, Dhruviben S.; Nelson, Julia A.; Plucinski, Mateusz M.; Barnwell, John W.; Udhayakumar, Venkatachalam

    2015-01-01

    Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species. PMID:26292024

  8. Genetic Analysis and Species Specific Amplification of the Artemisinin Resistance-Associated Kelch Propeller Domain in P. falciparum and P. vivax.

    PubMed

    Talundzic, Eldin; Chenet, Stella M; Goldman, Ira F; Patel, Dhruviben S; Nelson, Julia A; Plucinski, Mateusz M; Barnwell, John W; Udhayakumar, Venkatachalam

    2015-01-01

    Plasmodium falciparum resistance to artemisinin has emerged in the Greater Mekong Subregion and now poses a threat to malaria control and prevention. Recent work has identified mutations in the kelch propeller domain of the P. falciparum K13 gene to be associated artemisinin resistance as defined by delayed parasite clearance and ex vivo ring stage survival assays. Species specific primers for the two most prevalent human malaria species, P. falciparum and P. vivax, were designed and tested on multiple parasite isolates including human, rodent, and non- humans primate Plasmodium species. The new protocol described here using the species specific primers only amplified their respective species, P. falciparum and P. vivax, and did not cross react with any of the other human malaria Plasmodium species. We provide an improved species specific PCR and sequencing protocol that could be effectively used in areas where both P. falciparum and P. vivax are circulating. To design this improved protocol, the kelch gene was analyzed and compared among different species of Plasmodium. The kelch propeller domain was found to be highly conserved across the mammalian Plasmodium species. PMID:26292024

  9. In vitro susceptibility to pyrimethamine of DHFR I164L single mutant Plasmodium falciparum

    PubMed Central

    2011-01-01

    Background Recently, Plasmodium falciparum parasites bearing Pfdhfr I164L single mutation were found in Madagascar. These new mutants may challenge the use of antifolates for the intermittent preventive treatment of malaria during pregnancy (IPTp). Assays with transgenic bacteria suggested that I164L parasites have a wild-type phenotype for pyrimethamine but it had to be confirmed by testing the parasites themselves. Methods Thirty Plasmodium falciparum clinical isolates were collected in 2008 in the south-east of Madagascar. A part of Pfdhfr gene encompassing codons 6 to 206 was amplified by PCR and the determination of the presence of single nucleotide polymorphisms was performed by DNA sequencing. The multiplicity of infection was estimated by using an allelic family-specific nested PCR. Isolates that appeared monoclonal were submitted to culture adaptation. Determination of IC50s to pyrimethamine was performed on adapted isolates. Results Four different Pfdhfr alleles were found: the 164L single mutant-type (N = 13), the wild-type (N = 7), the triple mutant-type 51I/59R/108N (N = 9) and the double mutant-type 108N/164L (N = 1). Eleven out 30 (36.7%) of P. falciparum isolates were considered as monoclonal infection. Among them, five isolates were successfully adapted in culture and tested for pyrimethamine in vitro susceptibility. The wild-type allele was the most susceptible with a 50% inhibitory concentration (IC50) < 10 nM. The geometric mean of IC50 of the three I164L mutant isolates was 6-fold higher than the wild-type with 61.3 nM (SD = 3.2 nM, CI95%: 53.9-69.7 nM). These values remained largely below the IC50 of the triple mutant parasite (13,804 nM). Conclusion The IC50s of the I164L mutant isolates were significantly higher than those of the wild-type (6-fold higher) and close from those usually reported for simple mutants S108N (roughly10-fold higher than wild type). Given the observed values, the determination of IC50s directly on parasites did not

  10. Malarial parasite diversity in chimpanzees: the value of comparative approaches to ascertain the evolution of Plasmodium falciparum antigens

    PubMed Central

    2013-01-01

    Background Plasmodium falciparum shares its most recent common ancestor with parasites found in African apes; these species constitute the so-called Laverania clade. In this investigation, the evolutionary history of Plasmodium lineages found in chimpanzees (Pan troglodytes) was explored. Methods Here, the remainders of 74 blood samples collected as part of the chimpanzees’ routine health examinations were studied. For all positive samples with parasite lineages belonging to the Laverania clade, the complete mitochondrial genome (mtDNA), the gene encoding dihydrofolate reductase-thymidylate synthase (dhfr-ts), the chloroquine resistance transporter (Pfcrt), the circumsporozoite protein (csp), merozoite surface protein 2 (msp2), and the DBL-1 domain from var2CSA were amplified, cloned, and sequenced. Other Plasmodium species were included in the mtDNA, dhfr-ts, and csp analyses. Phylogenetic and evolutionary genetic analyses were performed, including molecular clock analyses on the mtDNA. Results/Conclusions Nine chimpanzees were malaria positive (12.2%); four of those infections were identified as P. falciparum, two as a Plasmodium reichenowi-like parasite or Plasmodium sp., one as Plasmodium gaboni, and two as Plasmodium malariae. All P. falciparum isolates were resistant to chloroquine indicating that the chimpanzees acquired such infections from humans in recent times. Such findings, however, are not sufficient for implicating chimpanzees as an animal reservoir for P. falciparum. Timing estimates support that the Laverania clade has co-existed with hominids for a long-period of time. The proposed species P. gaboni, Plasmodium billbrayi, and Plasmodium billcollinsi are monophyletic groups supporting that they are indeed different species. An expanded CSP phylogeny is presented, including all the Laverania species and other malarial parasites. Contrasting with other Plasmodium, the Laverania csp exhibits great conservation at the central tandem repeat region

  11. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    NASA Astrophysics Data System (ADS)

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2014-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (`K13-propeller') with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread.

  12. The gene encoding topoisomerase II from Plasmodium falciparum.

    PubMed Central

    Cheesman, S; McAleese, S; Goman, M; Johnson, D; Horrocks, P; Ridley, R G; Kilbey, B J

    1994-01-01

    The gene for topoisomerase II has been isolated from genomic libraries of strain K1 of the human malarial parasite, Plasmodium falciparum. The sequence reveals an open reading frame of 4194 nucleotides which predicts a polypeptide of 1398 amino acids. There are apparently no introns. The sequence is present as a single copy which has an identity of 47.4% and a similarity of 65.4% with its human homologue. Sequences conserved in topoisomerase II from other species are present in Pftopoisomerase II but in addition it has two adjacent asparagine-rich insertions which are unique to it. We have also detected asparagine-rich regions in the gene for PfDNA polymerase alpha. The gene for Pftopoisomerase II has been localised to chromosome 14 and northern analysis reveals a transcript of 5.8 kb. Two independent antisera raised in mice against glutathione-S-transferase fusion proteins containing the amino terminal portion of the malarial protein detect a weak band on western blots at about 160kDa, the expected size of the protein. Use of the same antisera for immunofluorescence analysis suggests that the protein is present at all stages of intraerythrocytic growth of the parasite. Images PMID:8041616

  13. Polycyclic amines as chloroquine resistance modulating agents in Plasmodium falciparum.

    PubMed

    Joubert, Jacques; Kapp, Erika; Taylor, Dale; Smith, Peter J; Malan, Sarel F

    2016-02-15

    Pentacycloundecylamines (PCUs) and adamantane amines, such as NGP1-01 (1) and amantadine, have shown significant channel blocking activities. They are postulated to act as chemosensitizers and circumvent the resistance of the plasmodia parasite against chloroquine (CQ) by inhibiting the p-glycoprotein efflux pump and enabling the accumulation of CQ inside the parasite digestive vacuole. Twelve polycyclic amines containing either a PCU or adamantane amine moiety conjugated to different aromatic functionalities through various tethered linkers were selected based on their channel blocking abilities and evaluated as potential chemosensitizers. Compounds 2, 4, 5 and 10 showed significant voltage-gated calcium channel (VGCC) blocking ability (IC50=0.27-35 μM) and were able to alter the CQ IC50 in differing degrees (45-81%) in the multidrug resistant Plasmodium falciparum Dd2 isolate. Among them, the PCU-dansyl amine compound (4) displayed the best potential to act as a chemosensitizer against the Dd2 strain at a 1 μM concentration (RMI=0.19) while displaying moderate antiplasmodial activity (Dd2 IC50=6.25 μM) and low in vitro cytotoxicity against a mammalian cell line (CHO, IC50=119 μM). Compounds 2 and 10 also showed some promising chemosensitizing abilities (RMI=0.36 and 0.35 respectively). A direct correlation was found between the VGCC blocking ability of these polycyclic amines and their capacity to act as CQ resistance modulating agents. PMID:26832222

  14. A molecular marker of artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Ariey, Frédéric; Witkowski, Benoit; Amaratunga, Chanaki; Beghain, Johann; Langlois, Anne-Claire; Khim, Nimol; Kim, Saorin; Duru, Valentine; Bouchier, Christiane; Ma, Laurence; Lim, Pharath; Leang, Rithea; Duong, Socheat; Sreng, Sokunthea; Suon, Seila; Chuor, Char Meng; Bout, Denis Mey; Ménard, Sandie; Rogers, William O.; Genton, Blaise; Fandeur, Thierry; Miotto, Olivo; Ringwald, Pascal; Le Bras, Jacques; Berry, Antoine; Barale, Jean-Christophe; Fairhurst, Rick M.; Benoit-Vical, Françoise; Mercereau-Puijalon, Odile; Ménard, Didier

    2016-01-01

    Plasmodium falciparum resistance to artemisinin derivatives in southeast Asia threatens malaria control and elimination activities worldwide. To monitor the spread of artemisinin resistance, a molecular marker is urgently needed. Here, using whole-genome sequencing of an artemisinin-resistant parasite line from Africa and clinical parasite isolates from Cambodia, we associate mutations in the PF3D7_1343700 kelch propeller domain (‘K13-propeller’) with artemisinin resistance in vitro and in vivo. Mutant K13-propeller alleles cluster in Cambodian provinces where resistance is prevalent, and the increasing frequency of a dominant mutant K13-propeller allele correlates with the recent spread of resistance in western Cambodia. Strong correlations between the presence of a mutant allele, in vitro parasite survival rates and in vivo parasite clearance rates indicate that K13-propeller mutations are important determinants of artemisinin resistance. K13-propeller polymorphism constitutes a useful molecular marker for large-scale surveillance efforts to contain artemisinin resistance in the Greater Mekong Subregion and prevent its global spread. PMID:24352242

  15. Genetic Diversity of Plasmodium falciparum Populations in Malaria Declining Areas of Sabah, East Malaysia

    PubMed Central

    Mohd Abd Razak, Mohd Ridzuan; Sastu, Umi Rubiah; Norahmad, Nor Azrina; Abdul-Karim, Abass; Muhammad, Amirrudin; Muniandy, Prem Kumar; Jelip, Jenarun; Rundi, Christina; Imwong, Mallika; Mudin, Rose Nani; Abdullah, Noor Rain

    2016-01-01

    Malaysia has a national goal to eliminate malaria by 2020. Understanding the genetic diversity of malaria parasites in residual transmission foci can provide invaluable information which may inform the intervention strategies used to reach elimination targets. This study was conducted to determine the genetic diversity level of P. falciparum isolates in malaria residual foci areas of Sabah. Malaria active case detection was conducted in Kalabakan and Kota Marudu. All individuals in the study sites were screened for malaria infection by rapid diagnostic test. Blood from P. falciparum-infected individuals were collected on filter paper prior to DNA extraction. Genotyping was performed using merozoite surface protein-1 (MSP-1), merozoite surface protein-2 (MSP-2), glutamate rich protein (GLURP) and 10 neutral microsatellite loci markers. The size of alleles, multiplicity of infection (MOI), mean number of alleles (Na), expected heterozygosity (He), linkage disequilibrium (LD) and genetic differentiation (FST) were determined. In Kalabakan, the MSP-1 and MSP-2 alleles were predominantly K1 and FC27 family types, respectively. The GLURP genotype VI (751–800 bp) was predominant. The MOI for MSP-1 and MSP-2 were 1.65 and 1.20, respectively. The Na per microsatellite locus was 1.70. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.17, 0.37, 0.70 and 0.33, respectively. In Kota Marudu, the MSP-1 and MSP-2 alleles were predominantly MAD20 and 3D7 family types, respectively. The GLURP genotype IV (651–700 bp) was predominant. The MOI for both MSP-1 and MSP-2 was 1.05. The Na per microsatellite locus was 3.60. The He values for MSP-1, MSP-2, GLURP and neutral microsatellites were 0.24, 0.25, 0.69 and 0.30, respectively. A significant LD was observed in Kalabakan (0.495, p<0.01) and Kota Marudu P. falciparum populations (0.601, p<0.01). High genetic differentiation between Kalabakan and Kota Marudu P. falciparum populations was observed (FST = 0

  16. Protein profiling of mefloquine resistant Plasmodium falciparum using mass spectrometry-based proteomics

    PubMed Central

    Reamtong, Onrapak; Srimuang, Krongkan; Saralamba, Naowarat; Sangvanich, Polkit; Day, Nicholas P.J.; White, Nicholas J.; Imwong, Mallika

    2015-01-01

    Malaria is a mosquito borne infectious disease caused by protozoa of genus Plasmodium. There are five species of Plasmodium that are found to infect humans. Plasmodium falciparum can cause severe malaria leading to higher morbidity and mortality of malaria than the other four species. Antimalarial resistance is the major obstacle to control malaria. Mefloquine was used in combination with Artesunate for uncomplicated P. falciparum in South East Asia and it has developed and established mefloquine resistance in this region. Here, gel-enhanced liquid chromatography/tandem mass spectrometry (GeLC–MS/MS)-based proteomics and label-free quantification were used to explore the protein profiles of mefloquine-sensitive and -induced resistant P. falciparum. A Thai P. falciparum isolate (S066) was used as a model in this research. Our data revealed for the first time that 69 proteins exhibited at least 2-fold differences in their expression levels between the two parasite lines. Of these, 36 were up-regulated and 33 were down-regulated in the mefloquine-resistant line compared with the mefloquine-sensitive line. These findings are consistent with those of past studies, where the multidrug resistance protein Pgh1 showed an up-regulation pattern consistent with that expected from its average 3-copy pfmdr1 gene number. Pgh1 and eight other up-regulated proteins (i.e., histo-aspartyl protease protein, exportin 1, eukaryotic translation initiation factor 3 subunit 8, peptidyl-prolyl cis-trans isomerase, serine rich protein homologue, exported protein 1, ATP synthase beta chain and phospholipid scramblase 1) were further validated for their expression levels using reverse transcriptase quantitative real-time PCR. The data support the up-regulation status in the mefloquine-resistant parasite line of all the candidate genes referred to above. Therefore, GeLC–MS/MS-based proteomics combined with label-free quantification is a reliable approach for exploring mefloquine resistance

  17. Artemisinins target the SERCA of Plasmodium falciparum.

    PubMed

    Eckstein-Ludwig, U; Webb, R J; Van Goethem, I D A; East, J M; Lee, A G; Kimura, M; O'Neill, P M; Bray, P G; Ward, S A; Krishna, S

    2003-08-21

    Artemisinins are extracted from sweet wormwood (Artemisia annua) and are the most potent antimalarials available, rapidly killing all asexual stages of Plasmodium falciparum. Artemisinins are sesquiterpene lactones widely used to treat multidrug-resistant malaria, a disease that annually claims 1 million lives. Despite extensive clinical and laboratory experience their molecular target is not yet identified. Activated artemisinins form adducts with a variety of biological macromolecules, including haem, translationally controlled tumour protein (TCTP) and other higher-molecular-weight proteins. Here we show that artemisinins, but not quinine or chloroquine, inhibit the SERCA orthologue (PfATP6) of Plasmodium falciparum in Xenopus oocytes with similar potency to thapsigargin (another sesquiterpene lactone and highly specific SERCA inhibitor). As predicted, thapsigargin also antagonizes the parasiticidal activity of artemisinin. Desoxyartemisinin lacks an endoperoxide bridge and is ineffective both as an inhibitor of PfATP6 and as an antimalarial. Chelation of iron by desferrioxamine abrogates the antiparasitic activity of artemisinins and correspondingly attenuates inhibition of PfATP6. Imaging of parasites with BODIPY-thapsigargin labels the cytosolic compartment and is competed by artemisinin. Fluorescent artemisinin labels parasites similarly and irreversibly in an Fe2+-dependent manner. These data provide compelling evidence that artemisinins act by inhibiting PfATP6 outside the food vacuole after activation by iron. PMID:12931192

  18. Artesunate-amodiaquine efficacy in Congolese children with acute uncomplicated falciparum malaria in Brazzaville

    PubMed Central

    2013-01-01

    Background Congo-Brazzaville adopted artemisinin-based combination therapy (ACT) in 2006. Artesunate-amodiaquine (AS + AQ) and artemether-lumefantrine are the first-line and second-line anti-malarial drugs to treat uncomplicated Plasmodium falciparum malaria, respectively. The baseline efficacy of AS + AQ was evaluated from February to August 2005 in patients living in Brazzaville, the capital city of the Republic of Congo. Methods One hundred and ninety-seven patients (96 ≤5 years old and 101 >5 years old, including adults) were recruited in a non-randomized study, treated under supervision with AS + AQ, and were followed up for 28 days in accordance with the 2003 World Health Organization protocol. Plasmodium falciparum recrudescent isolates from day 7 to day 28 were compared to pretreatment isolates by polymerase chain reaction (PCR) to distinguish between re-infection and recrudescence. Results The overall efficacy of AS + AQ after PCR correction on day 28 was 94.4%. An adequate clinical and parasitological response was observed in 94.3% and 94.4% of children aged ≤5 years old and those aged >5 years old (including adults), respectively. The main reported adverse events were dizziness, vomiting, diarrhoea, pruritus, headache, anorexia, and abdominal pain. Conclusion This study has shown the high efficacy of AS + AQ in Congolese patients of all ages with acute uncomplicated falciparum malaria and serves as the baseline efficacy and tolerance of this ACT in Brazzaville. PMID:23384005

  19. Acute Pancreatitis in a Patient with Complicated Falciparum Malaria

    PubMed Central

    Bhattacharya, Prasanta Kumar; Lynrah, Kryshan G; Ete, Tony; Issar, Neel Kanth

    2016-01-01

    Malaria is one of the most common protozoan diseases, especially in tropical countries. The clinical manifestation of malaria, especially falciparum malaria varies from mild acute febrile illness to life threatening severe systemic complications involving one or more organ systems. We would like to report a case of complicated falciparum malaria involving cerebral, renal, hepatic system along with acute pancreatitis. The patient was successfully treated with anti malarial and other supportive treatment. To the best of our knowledge there are very few reports of acute pancreatitis due to malaria. Falciparum malaria therefore should be added to the list of infectious agents causing acute pancreatitis especially in areas where malaria is endemic. PMID:26894117

  20. Postharvest treatments of fresh produce.

    PubMed

    Mahajan, P V; Caleb, O J; Singh, Z; Watkins, C B; Geyer, M

    2014-06-13

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  1. Postharvest treatments of fresh produce

    PubMed Central

    Mahajan, P. V.; Caleb, O. J.; Singh, Z.; Watkins, C. B.; Geyer, M.

    2014-01-01

    Postharvest technologies have allowed horticultural industries to meet the global demands of local and large-scale production and intercontinental distribution of fresh produce that have high nutritional and sensory quality. Harvested products are metabolically active, undergoing ripening and senescence processes that must be controlled to prolong postharvest quality. Inadequate management of these processes can result in major losses in nutritional and quality attributes, outbreaks of foodborne pathogens and financial loss for all players along the supply chain, from growers to consumers. Optimal postharvest treatments for fresh produce seek to slow down physiological processes of senescence and maturation, reduce/inhibit development of physiological disorders and minimize the risk of microbial growth and contamination. In addition to basic postharvest technologies of temperature management, an array of others have been developed including various physical (heat, irradiation and edible coatings), chemical (antimicrobials, antioxidants and anti-browning) and gaseous treatments. This article examines the current status on postharvest treatments of fresh produce and emerging technologies, such as plasma and ozone, that can be used to maintain quality, reduce losses and waste of fresh produce. It also highlights further research needed to increase our understanding of the dynamic response of fresh produce to various postharvest treatments. PMID:24797137

  2. Allelic family-specific humoral responses to merozoite surface protein 2 (MSP2) in Gabonese residents with Plasmodium falciparum infections

    PubMed Central

    EKALA, M-T; JOUIN, H; LEKOULOU, F; MERCEREAU-PUIJALON, O; NTOUMI, F

    2002-01-01

    Merozoite surface protein 2 (MSP2) expressed by Plasmodium falciparum asexual blood stages has been identified as a promising vaccine candidate. In order to explore allelic family-specific humoral responses which may be responsible for parasite neutralization during natural infections, isolates from individuals with either asymptomatic infections or uncomplicated malaria and residing in a Central African area where Plasmodium transmission is high and perennial, were analysed using MSP2 as polymorphic marker. The family-specific antibody responses were assessed by ELISA using MSP2 synthetic peptides. We observed an age-dependence of P. falciparum infection complexity. The decrease of infection complexity around 15 years of age was observed simultaneously with an increase in the mean number of MSP2 variants recognized. No significant difference in the P. falciparum genetic diversity and infection complexity was found in isolates from asymptomatic subjects and patients with uncomplicated malaria. The longitudinal follow-up showed a rapid development of immune responses to various regions of MSP2 variants within one week. Comparing humoral responses obtained with the other major antigen on the merozoite surface, MSP1, our findings suggest that different pathways of responsiveness are involved in antibody production to merozoite surface antigens. PMID:12165090

  3. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    PubMed Central

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M; Fox, B A; Inselburg, J

    1989-01-01

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension. Images PMID:2690004

  4. An enlarged largest subunit of Plasmodium falciparum RNA polymerase II defines conserved and variable RNA polymerase domains.

    PubMed

    Li, W B; Bzik, D J; Gu, H M; Tanaka, M; Fox, B A; Inselburg, J

    1989-12-11

    We have isolated the gene encoding the largest subunit of RNA polymerase II from Plasmodium falciparum. The RPII gene is expressed in the asexual erythrocytic stages of the parasite as a 9 kb mRNA, and is present as a single copy gene located on chromosome 3. The P. falciparum RPII subunit is the largest (2452 amino acids) eukaryotic RPII subunit, and it contains enlarged variable regions that clearly separate and define five conserved regions of the eukaryotic RPII largest subunits. A distinctive carboxyl-terminal domain contains a short highly conserved heptapeptide repeat domain which is bounded on its 5' side by a highly diverged heptapeptide repeat domain, and is bounded on its 3' side by a long carboxyl-terminal extension. PMID:2690004

  5. Development of the human immune response against the major surface protein (gp190) of Plasmodium falciparum.

    PubMed Central

    Müller, H M; Früh, K; von Brunn, A; Esposito, F; Lombardi, S; Crisanti, A; Bujard, H

    1989-01-01

    The 190-kilodalton glycoprotein (gp190) of Plasmodium falciparum, the precursor of the major surface proteins of merozoites, is considered a promising candidate for a blood stage malaria vaccine. DNA sequences specific for the gp190 of the two isolates K1 and MAD20 were subcloned and expressed in Escherichia coli. The panel of fusion proteins obtained represents about 80% of the polymorphic sequences observed so far within various isolates of P. falciparum. Sera from individuals living in a malaria-endemic area of West Africa were tested in immunoblots against the gp190 fusion proteins, and antibody reactivity was mapped to defined regions of the gp190. Depending on the age of the individual and on the presence of parasites in the blood, distinct regions of gp190 were differentially recognized by the respective antibodies. Similarly, the analysis of sera from German patients with acute malaria revealed a distinct pattern. When grouped according to age and to parasitemia, the reactivity of the sera of people living in malaria-endemic areas may indicate a correlation between certain gp190 regions and protective immune response. Images PMID:2680981

  6. Historical review: does stress provoke Plasmodium falciparum recrudescence?

    PubMed

    Shanks, G Dennis

    2015-06-01

    Plasmodium falciparum, unlike P. vivax, must maintain infection in the blood/bone marrow over many months/years in order to bridge periods between transmission periods. Asymptomatic parasitemia at very low concentrations is now known to be quite common due to molecular detection methods. Old tropical medicine texts commonly list many stressful events stated to provoke recrudescent falciparum parasitemia such as fatigue, heat/chill, trauma/surgery, famine/war, transit between areas and other febrile illness. The older literature is reviewed to discover the factual basis of such varied reports since they have not been recently confirmed. It seems likely that human stress sometimes induces falciparum recrudescence of an otherwise asymptomatic infection. Reproducing such observations today has been radically altered as malaria chemotherapy has evolved from suppressive quinine to curative artemisinin combinations. Host stress-provoked recrudescence may be part of P. falciparum's survival strategy. PMID:25918217

  7. Chloroquine Clinical Failures in P. falciparum Malaria Are Associated with Mutant Pfmdr-1, Not Pfcrt in Madagascar

    PubMed Central

    Andriantsoanirina, Valérie; Ratsimbasoa, Arsène; Bouchier, Christiane; Tichit, Magali; Jahevitra, Martial; Rabearimanana, Stéphane; Raherinjafy, Rogelin; Mercereau-Puijalon, Odile; Durand, Rémy; Ménard, Didier

    2010-01-01

    Molecular studies have demonstrated that mutations in the Plasmodium falciparum chloroquine resistance transporter gene (Pfcrt) play a major role in chloroquine resistance, while mutations in P. falciparum multidrug resistance gene (Pfmdr-1) act as modulator. In Madagascar, the high rate of chloroquine treatment failure (44%) appears disconnected from the overall level of in vitro CQ susceptibility (prevalence of CQ-resistant parasites <5%) or Pfcrt mutant isolates (<1%), strongly contrasting with sub-Saharan African countries. Previous studies showed a high frequency of Pfmdr-1 mutant parasites (>60% of isolates), but did not explore their association with P. falciparum chloroquine resistance. To document the association of Pfmdr-1 alleles with chloroquine resistance in Madagascar, 249 P. falciparum samples collected from patients enrolled in a chloroquine in vivo efficacy study were genotyped in Pfcrt/Pfmdr-1 genes as well as the estimation of the Pfmdr-1 copy number. Except 2 isolates, all samples displayed a wild-type Pfcrt allele without Pfmdr-1 amplification. Chloroquine treatment failures were significantly associated with Pfmdr-1 86Y mutant codon (OR = 4.6). The cumulative incidence of recurrence of patients carrying the Pfmdr-1 86Y mutation at day 0 (21 days) was shorter than patients carrying Pfmdr-1 86N wild type codon (28 days). In an independent set of 90 selected isolates, in vitro susceptibility to chloroquine was not associated with Pfmdr-1 polymorphisms. Analysis of two microsatellites flanking Pfmdr-1 allele showed that mutations occurred on multiple genetic backgrounds. In Madagascar, Pfmdr-1 polymorphism is associated with late chloroquine clinical failures and unrelated with in vitro susceptibility or Pfcrt genotype. These results highlight the limits of the current in vitro tests routinely used to monitor CQ drug resistance in this unique context. Gaining insight about the mechanisms that regulate polymorphism in Pfmdr1 remains important

  8. Spatial and temporal distribution of falciparum malaria in China

    PubMed Central

    Lin, Hualiang; Lu, Liang; Tian, Linwei; Zhou, Shuisen; Wu, Haixia; Bi, Yan; Ho, Suzanne C; Liu, Qiyong

    2009-01-01

    Background Falciparum malaria is the most deadly among the four main types of human malaria. Although great success has been achieved since the launch of the National Malaria Control Programme in 1955, malaria remains a serious public health problem in China. This paper aimed to analyse the geographic distribution, demographic patterns and time trends of falciparum malaria in China. Methods The annual numbers of falciparum malaria cases during 1992–2003 and the individual case reports of each clinical falciparum malaria during 2004–2005 were extracted from communicable disease information systems in China Center for Diseases Control and Prevention. The annual number of cases and the annual incidence were mapped by matching them to corresponding province- and county-level administrative units in a geographic information system. The distribution of falciparum malaria by age, gender and origin of infection was analysed. Time-series analysis was conducted to investigate the relationship between the falciparum malaria in the endemic provinces and the imported falciparum malaria in non-endemic provinces. Results Falciparum malaria was endemic in two provinces of China during 2004–05. Imported malaria was reported in 26 non-endemic provinces. Annual incidence of falciparum malaria was mapped at county level in the two endemic provinces of China: Yunnan and Hainan. The sex ratio (male vs. female) for the number of cases in Yunnan was 1.6 in the children of 0–15 years and it reached 5.7 in the adults over 15 years of age. The number of malaria cases in Yunnan was positively correlated with the imported malaria of concurrent months in the non-endemic provinces. Conclusion The endemic area of falciparum malaria in China has remained restricted to two provinces, Yunnan and Hainan. Stable transmission occurs in the bordering region of Yunnan and the hilly-forested south of Hainan. The age and gender distribution in the endemic area is characterized by the predominance

  9. Genes necessary for expression of a virulence determinant and for transmission of Plasmodium falciparum are located on a 0.3-megabase region of chromosome 9.

    PubMed Central

    Day, K P; Karamalis, F; Thompson, J; Barnes, D A; Peterson, C; Brown, H; Brown, G V; Kemp, D J

    1993-01-01

    Virulence of the human malaria parasite Plasmodium falciparum is believed to relate to adhesion of parasitized erythrocytes to postcapillary venular endothelium (asexual cytoadherence). Transmission of malaria to the mosquito vector involves a switch from asexual to sexual development (gametocytogenesis). Continuous in vitro culture of P. falciparum frequently results in irreversible loss of asexual cytoadherence and gametocytogenesis. Field isolates and cloned lines differing in expression of these phenotypes were karyotyped by pulse-field gel electrophoresis. This analysis showed that expression of both phenotypes mapped to a 0.3-Mb subtelomeric deletion of chromosome 9. This deletion frequently occurs during adaptation of parasite isolates to in vitro culture. Parasites with this deletion did not express the variant surface agglutination phenotype and the putative asexual cytoadherence ligand designated P. falciparum erythrocyte membrane protein 1, which has recently been shown to undergo antigenic variation. The syntenic relationship between asexual cytoadherence and gametocytogenesis suggests that expression of these phenotypes is genetically linked. One explanation for this linkage is that both developmental pathways share a common cytoadherence mechanism. This proposed biological and genetic linkage between a virulence factor (asexual cytoadherence) and transmissibility (gametocytogenesis) would help explain why a high degree of virulence has evolved and been maintained in falciparum malaria. Images Fig. 1 Fig. 2 Fig. 3 PMID:8367496

  10. Hemoglobinopathies: slicing the Gordian knot of Plasmodium falciparum malaria pathogenesis.

    PubMed

    Taylor, Steve M; Cerami, Carla; Fairhurst, Rick M

    2013-01-01

    Plasmodium falciparum malaria kills over 500,000 children every year and has been a scourge of humans for millennia. Owing to the co-evolution of humans and P. falciparum parasites, the human genome is imprinted with polymorphisms that not only confer innate resistance to falciparum malaria, but also cause hemoglobinopathies. These genetic traits--including hemoglobin S (HbS), hemoglobin C (HbC), and α-thalassemia--are the most common monogenic human disorders and can confer remarkable degrees of protection from severe, life-threatening falciparum malaria in African children: the risk is reduced 70% by homozygous HbC and 90% by heterozygous HbS (sickle-cell trait). Importantly, this protection is principally present for severe disease and largely absent for P. falciparum infection, suggesting that these hemoglobinopathies specifically neutralize the parasite's in vivo mechanisms of pathogenesis. These hemoglobin variants thus represent a "natural experiment" to identify the cellular and molecular mechanisms by which P. falciparum produces clinical morbidity, which remain partially obscured due to the complexity of interactions between this parasite and its human host. Multiple lines of evidence support a restriction of parasite growth by various hemoglobinopathies, and recent data suggest this phenomenon may result from host microRNA interference with parasite metabolism. Multiple hemoglobinopathies mitigate the pathogenic potential of parasites by interfering with the export of P. falciparum erythrocyte membrane protein 1 (PfEMP1) to the surface of the host red blood cell. Few studies have investigated their effects upon the activation of the innate and adaptive immune systems, although recent murine studies suggest a role for heme oxygenase-1 in protection. Ultimately, the identification of mechanisms of protection and pathogenesis can inform future therapeutics and preventive measures. Hemoglobinopathies slice the "Gordian knot" of host and parasite

  11. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2012 CFR

    2012-04-01

    ... 21 Food and Drugs 2 2012-04-01 2012-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen... frozen,” “fresh frozen,” “frozen fresh.” The terms defined in this section may be used on the label or in... state and has not been frozen or subjected to any form of thermal processing or any other form...

  12. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2014 CFR

    2014-04-01

    ... 21 Food and Drugs 2 2014-04-01 2014-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen... frozen,” “fresh frozen,” “frozen fresh.” The terms defined in this section may be used on the label or in... state and has not been frozen or subjected to any form of thermal processing or any other form...

  13. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2013 CFR

    2013-04-01

    ... 21 Food and Drugs 2 2013-04-01 2013-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen... frozen,” “fresh frozen,” “frozen fresh.” The terms defined in this section may be used on the label or in... state and has not been frozen or subjected to any form of thermal processing or any other form...

  14. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2011 CFR

    2011-04-01

    ... 21 Food and Drugs 2 2011-04-01 2011-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen... frozen,” “fresh frozen,” “frozen fresh.” The terms defined in this section may be used on the label or in... state and has not been frozen or subjected to any form of thermal processing or any other form...

  15. Plasmodium falciparum RuvB proteins

    PubMed Central

    Ahmad, Moaz; Tuteja, Renu

    2012-01-01

    The urgent requirement of next generation antimalarials has been of recent interest due to the emergence of drug-resistant parasite. The genome-wide analysis of Plasmodium falciparum helicases revealed three RuvB proteins. Due to the presence of helicase motif I and II in PfRuvBs, there is a high probability that they contain ATPase and possibly helicase activity. The Plasmodium database has homologs of several key proteins that interact with RuvBs and are most likely involved in the cell cycle progression, chromatin remodeling, and other cellular activities. Phylogenetically PfRuvBs are closely related to Saccharomyces cerevisiae RuvB, which is essential for cell cycle progression and survival of yeast. Thus PfRuvBs can serve as potential drug target if they show an essential role in the survival of parasite. PMID:23060959

  16. The Dynamics of Natural Plasmodium falciparum Infections

    PubMed Central

    Felger, Ingrid; Maire, Martin; Bretscher, Michael T.; Falk, Nicole; Tiaden, André; Sama, Wilson; Beck, Hans-Peter; Owusu-Agyei, Seth; Smith, Thomas A.

    2012-01-01

    Background Natural immunity to Plasmodium falciparum has been widely studied, but its effects on parasite dynamics are poorly understood. Acquisition and clearance rates of untreated infections are key elements of the dynamics of malaria, but estimating these parameters is challenging because of frequent super-infection and imperfect detectability of parasites. Consequently, information on effects of host immune status or age on infection dynamics is fragmentary. Methods An age-stratified cohort of 347 individuals from Northern Ghana was sampled six times at 2 month intervals. High-throughput capillary electrophoresis was used to genotype the msp-2 locus of all P. falciparum infections detected by PCR. Force of infection (FOI) and duration were estimated for each age group using an immigration-death model that allows for imperfect detection of circulating parasites. Results Allowing for imperfect detection substantially increased estimates of FOI and duration. Effects of naturally acquired immunity on the FOI and duration would be reflected in age dependence in these indices, but in our cohort data FOI tended to increase with age in children. Persistence of individual parasite clones was characteristic of all age-groups. Duration peaked in 5–9 year old children (average duration 319 days, 95% confidence interval 318;320). Conclusions The main age-dependence is on parasite densities, with only small age-variations in the FOI and persistence of infections. This supports the hypothesis that acquired immunity controls transmission mainly by limiting blood-stage parasite densities rather than changing rates of acquisition or clearance of infections. PMID:23029082

  17. Genetic diversity of Plasmodium falciparum histidine-rich protein 2 in the China-Myanmar border area

    PubMed Central

    Li, Peipei; Xing, Hua; Zhao, Zhenjun; Yang, Zhaoqing; Cao, Yaming; Yan, Guiyun; Sattabongkot, Jetsumon; Cui, Liwang; Fan, Qi

    2016-01-01

    Deletion of the Plasmodium falciparum histidine-rich protein 2 (pfhrp2) gene may affect the performance of PfHRP2-based rapid diagnostic tests (RDTs). Here we investigated the genetic diversity of the pfhrp2 gene in clinical parasite isolates collected in recent years from the China-Myanmar border area. Deletion of pfhrp2 has been identified in 4 out of 97 parasite isolates. Sequencing of the pfhrp2 exon 2 from 67 isolates revealed a high level of genetic diversity in pfhrp2, which is reflected in the presence of many repeat types and their variants, as well as variable copy numbers and different arrangements of these repeats in parasite isolates. In addition, we observed pfhrp3 deletion in three of the four parasites harboring pfhrp2 deletion, suggesting of double deletions of both genes in these three isolates. Analysis of two cases, which were P. falciparum-positive by microscopy and PCR but failed by two PfHRP2-based RDTs, did not find pfhrp2 deletion. Further correlational studies of pfhrp2 polymorphisms with detection sensitivity are needed to identify factors influencing the performance of RDTs in malaria-endemic areas. PMID:26297799

  18. Molecular Epidemiology of Plasmodium falciparum Malaria Outbreak, Tumbes, Peru, 2010–2012

    PubMed Central

    Okoth, Sheila Akinyi; Arrospide, Nancy; Gonzalez, Rommell V.; Sánchez, Juan F.; Macedo, Silvia; Conde, Silvia; Tapia, L. Lorena; Salas, Carola; Gamboa, Dionicia; Herrera, Yeni; Edgel, Kimberly A.; Udhayakumar, Venkatachalam; Lescano, Andrés G.

    2015-01-01

    During 2010–2012, an outbreak of 210 cases of malaria occurred in Tumbes, in the northern coast of Peru, where no Plasmodium falciparum malaria case had been reported since 2006. To identify the source of the parasite causing this outbreak, we conducted a molecular epidemiology investigation. Microsatellite typing showed an identical genotype in all 54 available isolates. This genotype was also identical to that of parasites isolated in 2010 in the Loreto region of the Peruvian Amazon and closely related to clonet B, a parasite lineage previously reported in the Amazon during 1998–2000. These findings are consistent with travel history of index case-patients. DNA sequencing revealed mutations in the Pfdhfr, Pfdhps, Pfcrt, and Pfmdr1 loci, which are strongly associated with resistance to chloroquine and sulfadoxine/pyrimethamine, and deletion of the Pfhrp2 gene. These results highlight the need for timely molecular epidemiology investigations to trace the parasite source during malaria reintroduction events. PMID:25897626

  19. Effects of environmental parameters on the dual-species biofilms formed by Escherichia coli O157:H7 and Ralstonia insidiosa, a strong biofilm producer isolated from a fresh-cut produce processing plant.

    PubMed

    Liu, Nancy T; Nou, Xiangwu; Bauchan, Gary R; Murphy, Charles; Lefcourt, Alan M; Shelton, Daniel R; Lo, Y Martin

    2015-01-01

    Biofilm-forming bacteria resident to food processing facilities are a food safety concern due to the potential of biofilms to harbor foodborne bacterial pathogens. When cultured together, Ralstonia insidiosa, a strong biofilm former frequently isolated from produce processing environments, has been shown to promote the incorporation of Escherichia coli O157:H7 into dual-species biofilms. In this study, interactions between E. coli O157:H7 and R. insidiosa were examined under different incubating conditions. Under static culture conditions, the incorporation of E. coli O157:H7 into biofilms with R. insidiosa was not significantly affected by either low incubating temperature (10°C) or by limited nutrient availability. Greater enhancement of E. coli O157:H7 incorporation in dual-species biofilms was observed by using a continuous culture system with limited nutrient availability. Under the continuous culture conditions used in this study, E coli O157:H7 cells showed a strong tendency of colocalizing with R. insidiosa on a glass surface at the early stage of biofilm formation. As the biofilms matured, E coli O157:H7 cells were mostly found at the bottom layer of the dual-species biofilms, suggesting an effective protection by R. insidiosa in the mature biofilms. PMID:25581186

  20. Effect of cooking on physical and sensory properties of fresh yellow alkaline noodles prepared by partial substitution of wheat flour with soy protein isolate and treated with cross-linking agents.

    PubMed

    Yeoh, Shin-Yong; Alkarkhi, Abbas F M; Ramli, Saifullah Bin; Easa, Azhar Mat

    2011-06-01

    Yellow alkaline noodles (YAN) prepared by partial substitution of wheat flour with soy protein isolate and treated with microbial transglutaminase (MTG) and ribose were investigated during cooking. Cooking caused an increase in lightness but a decrease in redness and yellowness, pH, tensile strength and elasticity values of noodles. The extents of these changes were influenced by formulation and cross-linking treatments. The pH and lightness for YAN-ribose were lowest but the yellowness and redness were the highest whilst the tensile strength and elasticity values remained moderate. For YAN-MTG, the color and pH values were moderate, but tensile strength and elasticity values were the highest. YAN prepared with both cross-linking agents had physical values between YAN-ribose and YAN-MTG. Although certain sensory parameters showed differences in score, the overall acceptability of all 10-min-cooked YAN was similar. It is possible to employ cross-linking agents to improve physical properties of cooked YAN. PMID:21306189

  1. Assessment of competitive and mechanism-based inhibition by clarithromycin: use of domperidone as a CYP3A probe-drug substrate and various enzymatic sources including a new cell-based assay with freshly isolated human hepatocytes.

    PubMed

    Michaud, Veronique; Turgeon, Jacques

    2010-04-01

    Clarithromycin is involved in a large number of clinically relevant drug-drug interactions. Discrepancies are observed between the magnitude of drug interactions predicted from in vitro competitive inhibition studies and changes observed clinically in the plasma levels of affected CYP3A substrates. The formation of metabolic-intermediate complexes has been proposed to explain these differences. The objectives of our study were: 1) to determine the competitive inhibition potency of clarithromycin on the metabolism of domperidone as a CYP3A probe drug using human recombinant CYP3A4 and CYP3A5 isoenzymes, human liver microsomes and cultured human hepatocytes; 2) to establish the modulatory role of cytochrome b5 on the competitive inhibition potency of clarithromycin; 3) to demonstrate the clarithromycin-induced formation of CYP450 metabolic-intermediate complexes in human liver microsomes; and 4) to determine the extent of CYP3A inhibition due to metabolic-intermediate complex formation using human liver microsomes and cultured human hepatocytes. At high concentrations (100 µM), clarithromycin had weak competitive inhibition potency towards CYP3A4 and CYP3A5. Inhibition potency was further decreased by the addition of cytochrome b5 (9-19%). Clarithromycin-induced metabolic-intermediate complexes were revealed by spectrophotometry analysis using human liver microsomes while time- and concentration-dependent mechanism-based inhibitions were quantified using isolated hepatocytes. These results indicate that mechanism-based but not competitive inhibition of CYP3As is the major underlying mechanism of drug-drug interactions observed clinically with clarithromycin. Drug interactions between clarithromycin and several CYP3A substrates are predicted to be insidious; the risk of severe adverse events should increase over time and persist for a few days after cessation of the drug. PMID:20446912

  2. Functional Analysis of Sirtuin Genes in Multiple Plasmodium falciparum Strains

    PubMed Central

    Merrick, Catherine J.; Jiang, Rays H. Y.; Skillman, Kristen M.; Samarakoon, Upeka; Moore, Rachel M.; Dzikowski, Ron; Ferdig, Michael T.; Duraisingh, Manoj T.

    2015-01-01

    Plasmodium falciparum, the causative agent of severe human malaria, employs antigenic variation to avoid host immunity. Antigenic variation is achieved by transcriptional switching amongst polymorphic var genes, enforced by epigenetic modification of chromatin. The histone-modifying ‘sirtuin’ enzymes PfSir2a and PfSir2b have been implicated in this process. Disparate patterns of var expression have been reported in patient isolates as well as in cultured strains. We examined var expression in three commonly used laboratory strains (3D7, NF54 and FCR-3) in parallel. NF54 parasites express significantly lower levels of var genes compared to 3D7, despite the fact that 3D7 was originally a clone of the NF54 strain. To investigate whether this was linked to the expression of sirtuins, genetic disruption of both sirtuins was attempted in all three strains. No dramatic changes in var gene expression occurred in NF54 or FCR-3 following PfSir2b disruption, contrasting with previous observations in 3D7. In 3D7, complementation of the PfSir2a genetic disruption resulted in a significant decrease in previously-elevated var gene expression levels, but with the continued expression of multiple var genes. Finally, rearranged chromosomes were observed in the 3D7 PfSir2a knockout line. Our results focus on the potential for parasite genetic background to contribute to sirtuin function in regulating virulence gene expression and suggest a potential role for sirtuins in maintaining genome integrity. PMID:25780929

  3. Calmidazolium evokes high calcium fluctuations in Plasmodium falciparum.

    PubMed

    Budu, Alexandre; Gomes, Mayrim M; Melo, Pollyana M; El Chamy Maluf, Sarah; Bagnaresi, Piero; Azevedo, Mauro F; Carmona, Adriana K; Gazarini, Marcos L

    2016-03-01

    Calcium and calmodulin (CaM) are important players in eukaryote cell signaling. In the present study, by using a knockin approach, we demonstrated the expression and localization of CaM in all erythrocytic stages of Plasmodium falciparum. Under extracellular Ca(2+)-free conditions, calmidazolium (CZ), a potent CaM inhibitor, promoted a transient cytosolic calcium ([Ca(2+)]cyt) increase in isolated trophozoites, indicating that CZ mobilizes intracellular sources of calcium. In the same extracellular Ca(2+)-free conditions, the [Ca(2+)]cyt rise elicited by CZ treatment was ~3.5 fold higher when the endoplasmic reticulum (ER) calcium store was previously depleted ruling out the mobilization of calcium from the ER by CZ. The effects of the Ca(2+)/H(+) ionophore ionomycin (ION) and the Na(+)/H(+) ionophore monensin (MON) suggest that the [Ca(2+)]cyt-increasing effect of CZ is driven by the removal of Ca(2+) from at least one Ca(2+)-CaM-related (CaMR) protein as well as by the mobilization of Ca(2+) from intracellular acidic calcium stores. Moreover, we showed that the mitochondrion participates in the sequestration of the cytosolic Ca(2+) elicited by CZ. Finally, the modulation of membrane Ca(2+) channels by CZ and thapsigargin (THG) was demonstrated. The opened channels were blocked by the unspecific calcium channel blocker Co(2+) but not by 2-APB (capacitative calcium entry inhibitor) or nifedipine (L-type Ca(2+) channel inhibitor). Taken together, the results suggested that one CaMR protein is an important modulator of calcium signaling and homeostasis during the Plasmodium intraerythrocytic cell cycle, working as a relevant intracellular Ca(2+) reservoir in the parasite. PMID:26689736

  4. Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model.

    PubMed

    Obaldía, Nicanor; Dow, Geoffrey S; Gerena, Lucia; Kyle, Dennis; Otero, William; Mantel, Pierre-Yves; Baro, Nicholas; Daniels, Rachel; Mukherjee, Angana; Childs, Lauren M; Buckee, Caroline; Duraisingh, Manoj T; Volkman, Sarah K; Wirth, Dyann F; Marti, Matthias

    2016-01-01

    Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model. PMID:26880111

  5. The primary structure of Plasmodium falciparum DNA polymerase delta is similar to drug sensitive delta-like viral DNA polymerases.

    PubMed

    Fox, B A; Bzik, D J

    1991-12-01

    We report the isolation and sequencing of genomic DNA clones that encode the 1094-amino acid catalytic subunit of DNA polymerase delta from the human malaria parasite Plasmodium falciparum. Protein sequence comparison to other DNA polymerases revealed the presence of six highly conserved regions found in alpha-like DNA polymerases from different prokaryotic, viral, and eukaryotic sources. Five additional regions of amino acid sequence similarity that are only conserved in delta and delta-like DNA polymerases, so far, were present in P. falciparum DNA polymerase delta. P. falciparum DNA polymerase delta was highly similar to both Saccharomyces cerevisiae DNA polymerase delta (DNA polymerase III; CDC2) and Epstein-Barr virus DNA polymerase at the amino acid sequence, and the predicted protein secondary structure levels. The gene that encodes DNA polymerase delta resides as a single copy on chromosome 10, and is expressed as a 4.5-kb mRNA during the trophozoite and schizont stages when parasite chromosomal DNA synthesis is active. PMID:1775172

  6. Use of the in vitro microtechnique for the assessment of drug sensitivity of Plasmodium falciparum in Sennar, Sudan

    PubMed Central

    Kouznetsov, R. L.; Rooney, W.; Wernsdorfer, W. H.; El Gaddal, A. A.; Payne, D.; Abdalla, R. E.

    1980-01-01

    In 1978, studies on the chloroquine sensitivity of Plasmodium falciparum were carried out in the district of Sennar, Sudan. The results of the in vivo tests showed parasites resistant at the RI level only, but the mean clearance time of trophozoites from the blood was higher than for strains found in many other areas of tropical Africa. The in vitro tests, using the microtechnique, indicated a lower sensitivity to chloroquine in the local P. falciparum isolates than in those of most other African countries. However, similar results have been reported from Ethiopia. The chloroquine sensitivity of P. falciparum from Sennar is close to the critical level of resistance. The in vitro microtechnique was also used to test for the sensitivity to Dabequin, 4-aminobenzo-quinoline, and was generally found to be a suitable and reproducible method, with a greater potential than the standard macro method. At parasite densities of over 100 000 asexual parasites per microlitre of blood the effect of a given concentration of chloroquine was related to the parasite density owing to the selective uptake of the compound by the parasitized cells. PMID:7028301

  7. Altered drug susceptibility during host adaptation of a Plasmodium falciparum strain in a non-human primate model

    PubMed Central

    Obaldía III, Nicanor; Dow, Geoffrey S.; Gerena, Lucia; Kyle, Dennis; Otero, William; Mantel, Pierre-Yves; Baro, Nicholas; Daniels, Rachel; Mukherjee, Angana; Childs, Lauren M.; Buckee, Caroline; Duraisingh, Manoj T.; Volkman, Sarah K.; Wirth, Dyann F.; Marti, Matthias

    2016-01-01

    Infections with Plasmodium falciparum, the most pathogenic of the Plasmodium species affecting man, have been reduced in part due to artemisinin-based combination therapies. However, artemisinin resistant parasites have recently emerged in South-East Asia. Novel intervention strategies are therefore urgently needed to maintain the current momentum for control and elimination of this disease. In the present study we characterize the phenotypic and genetic properties of the multi drug resistant (MDR) P. falciparum Thai C2A parasite strain in the non-human Aotus primate model, and across multiple passages. Aotus infections with C2A failed to clear upon oral artesunate and mefloquine treatment alone or in combination, and ex vivo drug assays demonstrated reduction in drug susceptibility profiles in later Aotus passages. Further analysis revealed mutations in the pfcrt and pfdhfr loci and increased parasite multiplication rate (PMR) across passages, despite elevated pfmdr1 copy number. Altogether our experiments suggest alterations in parasite population structure and increased fitness during Aotus adaptation. We also present data of early treatment failures with an oral artemisinin combination therapy in a pre-artemisinin resistant P. falciparum Thai isolate in this animal model. PMID:26880111

  8. Identification and deconvolution of cross-resistance signals from antimalarial compounds using multidrug-resistant Plasmodium falciparum strains.

    PubMed

    Chugh, Monika; Scheurer, Christian; Sax, Sibylle; Bilsland, Elizabeth; van Schalkwyk, Donelly A; Wicht, Kathryn J; Hofmann, Natalie; Sharma, Anil; Bashyam, Sridevi; Singh, Shivendra; Oliver, Stephen G; Egan, Timothy J; Malhotra, Pawan; Sutherland, Colin J; Beck, Hans-Peter; Wittlin, Sergio; Spangenberg, Thomas; Ding, Xavier C

    2015-02-01

    Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs. PMID:25487796

  9. Identification and Deconvolution of Cross-Resistance Signals from Antimalarial Compounds Using Multidrug-Resistant Plasmodium falciparum Strains

    PubMed Central

    Chugh, Monika; Scheurer, Christian; Sax, Sibylle; Bilsland, Elizabeth; van Schalkwyk, Donelly A.; Wicht, Kathryn J.; Hofmann, Natalie; Sharma, Anil; Bashyam, Sridevi; Singh, Shivendra; Oliver, Stephen G.; Egan, Timothy J.; Malhotra, Pawan; Sutherland, Colin J.; Beck, Hans-Peter; Wittlin, Sergio; Spangenberg, Thomas

    2014-01-01

    Plasmodium falciparum, the most deadly agent of malaria, displays a wide variety of resistance mechanisms in the field. The ability of antimalarial compounds in development to overcome these must therefore be carefully evaluated to ensure uncompromised activity against real-life parasites. We report here on the selection and phenotypic as well as genotypic characterization of a panel of sensitive and multidrug-resistant P. falciparum strains that can be used to optimally identify and deconvolute the cross-resistance signals from an extended panel of investigational antimalarials. As a case study, the effectiveness of the selected panel of strains was demonstrated using the 1,2,4-oxadiazole series, a newly identified antimalarial series of compounds with in vitro activity against P. falciparum at nanomolar concentrations. This series of compounds was to be found inactive against several multidrug-resistant strains, and the deconvolution of this signal implicated pfcrt, the genetic determinant of chloroquine resistance. Targeted mode-of-action studies further suggested that this new chemical series might act as falcipain 2 inhibitors, substantiating the suggestion that these compounds have a site of action similar to that of chloroquine but a distinct mode of action. New antimalarials must overcome existing resistance and, ideally, prevent its de novo appearance. The panel of strains reported here, which includes recently collected as well as standard laboratory-adapted field isolates, is able to efficiently detect and precisely characterize cross-resistance and, as such, can contribute to the faster development of new, effective antimalarial drugs. PMID:25487796

  10. Steroidal Saponins from Fresh Stems of Dracaena angustifolia

    PubMed Central

    Min, Xu; Ying-Jun, Zhang; Xing-Cong, Li; Jacob, Melissa R.; Chong-Ren, Yang

    2010-01-01

    Six new steroidal saponins (1-6), angudracanosides A-F, were isolated from fresh stems of Dracaena angustifolia (Agavaceae), together with eight known compounds. The structures of compounds 1-6 were determined by detailed spectroscopic analyses and chemical methods. Antifungal testing of all compounds showed that 6 and 7 were active against Cryptococcus neoformans with IC50s of 9.5 and 20.0 μg/mL, respectively. PMID:20718450

  11. α-Thalassemia Impairs the Cytoadherence of Plasmodium falciparum-Infected Erythrocytes

    PubMed Central

    Krause, Michael A.; Diakite, Seidina A. S.; Lopera-Mesa, Tatiana M.; Amaratunga, Chanaki; Arie, Takayuki; Traore, Karim; Doumbia, Saibou; Konate, Drissa; Keefer, Jeffrey R.; Diakite, Mahamadou; Fairhurst, Rick M.

    2012-01-01

    Background α-thalassemia results from decreased production of α-globin chains that make up part of hemoglobin tetramers (Hb; α2β2) and affects up to 50% of individuals in some regions of sub-Saharan Africa. Heterozygous (−α/αα) and homozygous (−α/−α) genotypes are associated with reduced risk of severe Plasmodium falciparum malaria, but the mechanism of this protection remains obscure. We hypothesized that α-thalassemia impairs the adherence of parasitized red blood cells (RBCs) to microvascular endothelial cells (MVECs) and monocytes – two interactions that are centrally involved in the pathogenesis of severe disease. Methods and Findings We obtained P. falciparum isolates directly from Malian children with malaria and used them to infect αα/αα (normal), −α/αα and −α/−α RBCs. We also used laboratory-adapted P. falciparum clones to infect −/−α RBCs obtained from patients with HbH disease. Following a single cycle of parasite invasion and maturation to the trophozoite stage, we tested the ability of parasitized RBCs to bind MVECs and monocytes. Compared to parasitized αα/αα RBCs, we found that parasitized −α/αα, −α/−α and −/−α RBCs showed, respectively, 22%, 43% and 63% reductions in binding to MVECs and 13%, 33% and 63% reductions in binding to monocytes. α-thalassemia was associated with abnormal display of P. falciparum erythrocyte membrane protein 1 (PfEMP1), the parasite’s main cytoadherence ligand and virulence factor, on the surface of parasitized RBCs. Conclusions Parasitized α-thalassemic RBCs show PfEMP1 display abnormalities that are reminiscent of those on the surface of parasitized sickle HbS and HbC RBCs. Our data suggest a model of malaria protection in which α-thalassemia ameliorates the pro-inflammatory effects of cytoadherence. Our findings also raise the possibility that other unstable hemoglobins such as HbE and unpaired α-globin chains (in the case of β-thalassemia) protect against

  12. Comparative Assessment of Transmission-Blocking Vaccine Candidates against Plasmodium falciparum

    PubMed Central

    Kapulu, M. C.; Da, D. F.; Miura, K.; Li, Y; Blagborough, A. M.; Churcher, T. S.; Nikolaeva, D.; Williams, A. R.; Goodman, A. L.; Sangare, I.; Turner, A. V.; Cottingham, M. G.; Nicosia, A.; Straschil, U.; Tsuboi, T.; Gilbert, S. C.; Long, Carole A.; Sinden, R. E.; Draper, S. J.; Hill, A. V. S.; Cohuet, A.; Biswas, S.

    2015-01-01

    Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63, and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25, and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species, and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform. PMID:26063320

  13. Genetic diversity and population structure of Plasmodium falciparum over space and time in an African archipelago.

    PubMed

    Salgueiro, Patrícia; Vicente, José Luís; Figueiredo, Rita Carrilho; Pinto, João

    2016-09-01

    The archipelago of São Tomé and Principe (STP), West Africa, has suffered the heavy burden of malaria since the 16th century. Until the last decade, when after a successful control program STP has become a low transmission country and one of the few nations with decreases of more than 90% in malaria admission and death rates. We carried out a longitudinal study to determine the genetic structure of STP parasite populations over time and space. Twelve microsatellite loci were genotyped in Plasmodium falciparum samples from two islands collected in 1997, 2000 and 2004. Analysis was performed on proportions of mixed genotype infections, allelic diversity, population differentiation, effective population size and bottleneck effects. We have found high levels of genetic diversity and minimal inter-population genetic differentiation typical of African continental regions with intense and stable malaria transmission. We detected significant differences between the years, with special emphasis for 1997 that showed the highest proportion of samples infected with P. falciparum and the highest mean number of haplotypes per isolate. This study establishes a comprehensive genetic data baseline of a pre-intervention scenario for future studies; taking into account the most recent and successful control intervention on the territory. PMID:27262356

  14. Comparative assessment of transmission-blocking vaccine candidates against Plasmodium falciparum.

    PubMed

    Kapulu, M C; Da, D F; Miura, K; Li, Y; Blagborough, A M; Churcher, T S; Nikolaeva, D; Williams, A R; Goodman, A L; Sangare, I; Turner, A V; Cottingham, M G; Nicosia, A; Straschil, U; Tsuboi, T; Gilbert, S C; Long, Carole A; Sinden, R E; Draper, S J; Hill, A V S; Cohuet, A; Biswas, S

    2015-01-01

    Malaria transmission-blocking vaccines (TBVs) target the development of Plasmodium parasites within the mosquito, with the aim of preventing malaria transmission from one infected individual to another. Different vaccine platforms, mainly protein-in-adjuvant formulations delivering the leading candidate antigens, have been developed independently and have reported varied transmission-blocking activities (TBA). Here, recombinant chimpanzee adenovirus 63, ChAd63, and modified vaccinia virus Ankara, MVA, expressing AgAPN1, Pfs230-C, Pfs25, and Pfs48/45 were generated. Antibody responses primed individually against all antigens by ChAd63 immunization in BALB/c mice were boosted by the administration of MVA expressing the same antigen. These antibodies exhibited a hierarchy of inhibitory activity against the NF54 laboratory strain of P. falciparum in Anopheles stephensi mosquitoes using the standard membrane feeding assay (SMFA), with anti-Pfs230-C and anti-Pfs25 antibodies giving complete blockade. The observed rank order of inhibition was replicated against P. falciparum African field isolates in A. gambiae in direct membrane feeding assays (DMFA). TBA achieved was IgG concentration dependent. This study provides the first head-to-head comparative analysis of leading antigens using two different parasite sources in two different vector species, and can be used to guide selection of TBVs for future clinical development using the viral-vectored delivery platform. PMID:26063320

  15. A microfluidic system to study cytoadhesion of Plasmodium falciparum infected erythrocytes to primary brain microvascularendothelial cells.

    PubMed

    Herricks, Thurston; Seydel, Karl B; Turner, George; Molyneux, Malcolm; Heyderman, Robert; Taylor, Terrie; Rathod, Pradipsinh K

    2011-09-01

    The cellular events leading to severe and complicated malaria in some Plasmodium falciparum infections are poorly understood. Additional tools are required to better understand the pathogenesis of this disease. In this technical report, we describe a microfluidic culture system and image processing algorithms that were developed to observe cytoadhesion interactions of P. falciparum parasitized erythrocytes rolling on primary brain microvascularendothelial cells. We isolated and cultured human primary microvascular brain endothelial cells in a closed loop microfluidic culture system where a peristaltic pump and media reservoirs were integrated onto a microscope stage insert. We developed image processing methods to enhance contrast of rolling parasitized erythrocytes on endothelial cells and to estimate the local wall shear stress. The velocity of parasitized erythrocytes rolling on primary brain microvascularendothelial cells was then measured under physiologically relevant wall shear stresses. Finally, we deployed this method successfully at a field site in Blantyre, Malawi. The method is a promising new tool for the investigation of the pathogenesis of severe malaria. PMID:21743938

  16. Survey of chloroquine-resistant mutations in the Plasmodium falciparum pfcrt and pfmdr-1 genes in Hadhramout, Yemen.

    PubMed

    Bamaga, Omar A A; Mahdy, Mohammed A K; Lim, Yvonne A L

    2015-09-01

    Malaria is still a major public health problem in Yemen. More than 95% of the malaria cases are due to Plasmodium ‎falciparum‎. Recently in Yemen, the antimalarial treatment policy was changed from chloroquine (CQ) to artemisinin combination therapy (ACTs). However, CQ is still available and prescribed in the Yemeni market. The persistence of CQ resistance will be prolonged if the shift to ACT and the simultaneous withdrawal of CQ are not rigorously implemented. The aim of the current survey is to detect chloroquine-resistant mutations in P. falciparum chloroquine-resistance transporter (pfcrt) and P. falciparum multi-drug resistance-1 (pfmdr1) genes. These data will be important for future monitoring and assessment of antimalarial drug policy in Yemen. Blood specimens were collected from 735 individuals from different districts of the Hadhramout province, Yemen by house-to-house visit. Mutation-specific nested polymerase chain reaction (PCR) and restriction fragment length polymorphism (PCR-RFLP) methods were used to investigate the mutations in the pfmdr1(codons 86 and 1246) and pfcrt (codons 76, 271, 326, 356 and 371) genes. The overall prevalence of pfcrt mutations at codons 76, 271, 326 and 371 were 50.4%, 58.7%, 54.3% and 44.9%, respectively. All isolates had wild-type pfcrt 356 allele. The majority of pfmdr1 86 alleles (83.3%) and all pfmdr1 1246 alleles were wild type. There was no association between pfcrt mutations and symptomatology, gender and age groups. In conclusion, point mutations in codons 76, 271, 326 and 371 of pfcrt of P. falciparum are high suggesting a sustained high CQ resistance even after 4 years of shifting to ACTs. These findings warrant complete withdrawal of CQ use from the Yemeni market for P. falciparum and careful usage of CQ for treating Plasmodium vivax. PMID:26001972

  17. Detection of single-nucleotide polymorphisms in Plasmodium falciparum by PCR primer extension and lateral flow immunoassay.

    PubMed

    Moers, A P H A; Hallett, R L; Burrow, R; Schallig, H D F H; Sutherland, C J; van Amerongen, A

    2015-01-01

    The resistance of Plasmodium falciparum to some antimalarial drugs is linked to single-nucleotide polymorphisms (SNPs). Currently, there are no methods for the identification of resistant parasites that are sufficiently simple, cheap, and fast enough to be performed at point-of-care, i.e., in local hospitals where drugs are prescribed. Primer extension methods (PEXT) were developed to identify 4 SNPs in P. falciparum positioned at amino acids 86, 184, and 1246 of the P. falciparum multidrug resistance 1 gene (pfmdr1) and amino acid 76 of the chloroquine resistance transporter gene (pfcrt). The PEXT products were visualized by a nucleic acid lateral flow immunoassay (NALFIA) with carbon nanoparticles as the detection labels. PCR-PEXT-NALFIAs showed good correlation to the reference methods, quantitative PCR (qPCR) or direct amplicon sequence analysis, in an initial open-label evaluation with 17 field samples. The tests were further evaluated in a blind study design in a set of 150 patient isolates. High specificities of 98 to 100% were found for all 4 PCR-PEXT genotyping assays. The sensitivities ranged from 75% to 100% when all PEXT-positive tests were considered. A number of samples with a low parasite density were successfully characterized by the reference methods but failed to generate a result in the PCR-PEXT-NALFIA, particularly those samples with microscopy-negative subpatent infections. This proof-of principle study validates the use of PCR-PEXT-NALFIA for the detection of resistance-associated mutations in P. falciparum, particularly for microscopy-positive infections. Although it requires a standard thermal cycler, the procedure is cheap and rapid and thus a potentially valuable tool for point-of-care detection in developing countries. PMID:25367901

  18. [Molecular epidemiology of imported malaria in Italy: the use of genetic markers and in vitro sensitivity test in a study of chloroquine resistance in Plasmodium falciparum].

    PubMed

    Menegon, Michela; Sannella, Anna Rosa; Severini, Carlo; Paglia, Maria Grazia; Matteelli, Alberto; Caramello, Pietro; Severini, Francesco; Taramelli, Donatella; Majori, Giancarlo

    2006-01-01

    The emergence of Plasmodium falciparum drug-resistance, especially chloroquine resistance, represents one of the main obstacles to the control of malaria. Several studies have shown that in P. falciparum the mechanism of chloroquine resistance is linked to specific point mutations in the pfcrt gene of the parasite. In the present study we have analyzed 120 Italian imported malaria cases to evaluate the prevalence of 76T and 220S mutantions in the pfcrt gene. Moreover, the correlation between the presence of pfcrt point mutations and in vitro chloroquine resistance has been evaluated on 25 plasmodial isolates. The results showed a high prevalence of the pfcrt point mutations in isolates analyzed and a significant association between point mutations and in vitro chloroquine resistance. Molecular screening on imported malaria cases can be a useful tool to be employed in surveillance activity and also in monitoring the development and spread of drug resistance in endemic areas. PMID:17033142

  19. Eleganolone, a Diterpene from the French Marine Alga Bifurcaria bifurcata Inhibits Growth of the Human Pathogens Trypanosoma brucei and Plasmodium falciparum

    PubMed Central

    Gallé, Jean-Baptiste; Attioua, Barthélémy; Kaiser, Marcel; Rusig, Anne-Marie; Lobstein, Annelise; Vonthron-Sénécheau, Catherine

    2013-01-01

    Organic extracts of 20 species of French seaweed have been screened against Trypanosoma brucei rhodesiense trypomastigotes, the parasite responsible for sleeping sickness. These extracts have previously shown potent antiprotozoal activities in vitro against Plasmodium falciparum and Leishmania donovani. The selectivity of the extracts was also evaluated by testing cytotoxicity on a mammalian L6 cell line. The ethyl acetate extract of the brown seaweed, Bifurcaria bifurcata, showed strong trypanocidal activity with a mild selectivity index (IC50 = 0.53 µg/mL; selectivity index (SI) = 11.6). Bio-guided fractionation led to the isolation of eleganolone, the main diterpenoid isolated from this species. Eleganolone contributes only mildly to the trypanocidal activity of the ethyl acetate extract (IC50 = 45.0 µM, SI = 4.0). However, a selective activity against P. falciparum erythrocytic stages in vitro has been highlighted (IC50 = 7.9 µM, SI = 21.6). PMID:23442789

  20. Submicroscopic Plasmodium falciparum infections in pregnancy in Ghana.

    PubMed

    Mockenhaupt, F P; Rong, B; Till, H; Eggelte, T A; Beck, S; Gyasi-Sarpong, C; Thompson, W N; Bienzle, U

    2000-03-01

    Malarial parasitaemia below the threshold of microscopy but detectable by polymerase chain reaction (PCR) assays is common in endemic regions. This study was conducted to examine prevalence, predictors, and effects of submicroscopic Plasmodium falciparum infections in pregnancy. In a cross-sectional study among 530 pregnant women in Ghana, plasmodial infections were assessed by microscopy and PCR assays. Concentrations of haemoglobin and C-reactive protein (CRP) were measured and antimalarial drugs (chloroquine, pyrimethamine) in urine were demonstrated by ELISA dipsticks. By microscopy, 32% of the women were found to harbour malaria parasites. This rate increased to 63% adding the results of the parasite-specific PCR. P. falciparum was present in all but one infection. With increasing gravidity, infection rates and parasite densities decreased and the proportions of submicroscopic parasitaemia among infected women grew. Correspondingly, anaemia, fever and evidence of inflammation (CRP > 0.6 mg/dl) were more frequent in primigravidae than in multigravidae. Antimalarial drugs were detected in 65% of the women and were associated with a reduced prevalence of P. falciparum infections and a raised proportion of submicroscopic parasitaemia. Both gravidity and antimalarial drug use were independent predictors of submicroscopic P. falciparum infections. These infections caused a slight reduction of Hb levels and considerably increased serum concentrations of CRP. Conventional microscopy underestimates the actual extent of malarial infections in pregnancy in endemic regions. Submicroscopic P. falciparum infections are frequent and may contribute to mild anaemia and inflammation in seemingly aparasitaemic pregnant women. PMID:10747278

  1. Expression of Plasmodium falciparum surface antigens in Escherichia coli.

    PubMed Central

    Ardeshir, F; Flint, J E; Reese, R T

    1985-01-01

    The asexual blood stages of the human malarial parasite Plasmodium falciparum produce many antigens, only some of which are important for protective immunity. Most of the putative protective antigens are believed to be expressed in schizonts and merozoites, the late stages of the asexual cycle. With the aim of cloning and characterizing genes for important parasite antigens, we used late-stage P. falciparum mRNA to construct a library of cDNA sequences inserted in the Escherichia coli expression vector pUC8. Nine thousand clones from the expression library were immunologically screened in situ with serum from Aotus monkeys immune to P. falciparum, and 95 clones expressing parasite antigens were identified. Mice were immunized with lysates from 49 of the bacterial clones that reacted with Aotus sera, and the mouse sera were tested for their reactivity with parasite antigens by indirect immunofluorescence, immunoprecipitation, and immunoblotting assays. Several different P. falciparum antigens were identified by these assays. Indirect immunofluorescence studies of extracellular merozoites showed that three of these antigens appear to be located on the merozoite surface. Thus, we have identified cDNA clones to three different P. falciparum antigens that may be important in protective immunity. Images PMID:3887406

  2. Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

    PubMed Central

    Malik, A; Egan, J E; Houghten, R A; Sadoff, J C; Hoffman, S L

    1991-01-01

    Cytotoxic T lymphocytes (CTL) against the circumsporozoite (CS) protein of malaria sporozoites protect against malaria in rodents. Although there is interest in developing human vaccines that induce CTL against the Plasmodium falciparum CS protein, humans have never been shown to produce CTL against any Plasmodium species protein or other parasite protein. We report that when peripheral blood mononuclear cells (PBMC) from three of four volunteers immunized with irradiated P. falciparum sporozoites were stimulated in vitro with a recombinant vaccinia virus expressing the P. falciparum CS protein or a peptide including only amino acids 368-390 of the P. falciparum CS protein [CS-(368-390)], the PBMC lysed autologous Epstein-Barr virus-transformed B cells transfected with the P. falciparum CS protein gene or incubated with CS-(368-390) tricosapeptide. Activity was antigen specific, genetically restricted, and dependent on CD8+ T cells. In one volunteer, seven peptides reflecting amino acids 311-400 were tested, and, as in B10.BR mice, CTL activity was only associated with the CS-(368-390) peptide. Development of an assay for studying human CTL against the CS and other malaria proteins and a method for constructing target cells by direct gene transfection provide a foundation for studying the role of CTL in protection against malaria. PMID:1707538

  3. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites

    PubMed Central

    Lee, Andrew H.; Fidock, David A.

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or “Accelerated Resistance to Multiple Drugs” (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  4. Evidence of a Mild Mutator Phenotype in Cambodian Plasmodium falciparum Malaria Parasites.

    PubMed

    Lee, Andrew H; Fidock, David A

    2016-01-01

    Malaria control efforts have been continuously stymied by drug-resistant strains of Plasmodium falciparum, which typically originate in Southeast Asia prior to spreading into high-transmission settings in Africa. One earlier proposed explanation for Southeast Asia being a hotbed of resistance has been the hypermutability or "Accelerated Resistance to Multiple Drugs" (ARMD) phenotype, whereby multidrug-resistant Southeast Asian parasites were reported to exhibit 1,000-fold higher rates of resistance to unrelated antimalarial agents when compared to drug-sensitive parasites. However, three recent studies do not recapitulate this hypermutability phenotype. Intriguingly, genome sequencing of recently derived multidrug-resistant Cambodian isolates has identified a high proportion of DNA repair gene mutations in multidrug-resistant parasites, suggesting their potential role in shaping local parasite evolution. By adapting fluctuation assays for use in P. falciparum, we have examined the in vitro mutation rates of five recent Cambodian isolates and three reference laboratory strains. For these studies we also generated a knockout parasite line lacking the DNA repair factor Exonuclease I. In these assays, parasites were typed for their ability to acquire resistance to KAE609, currently in advanced clinical trials, yielding 13 novel mutations in the Na+/H+-ATPase PfATP4, the primary resistance determinant. We observed no evidence of hypermutability. Instead, we found evidence of a mild mutator (up to a 3.4-fold increase in mutation rate) phenotype in two artemisinin-resistant Cambodian isolates, which carry DNA repair gene mutations. We observed that one such mutation in the Mismatch Repair protein Mlh1 contributes to the mild mutator phenotype when modeled in yeast (scmlh1-P157S). Compared to basal rates of mutation, a mild mutator phenotype may provide a greater overall benefit for parasites in Southeast Asia in terms of generating drug resistance without incurring

  5. 21 CFR 101.95 - “Fresh,” “freshly frozen,” “fresh frozen,” “frozen fresh.”

    Code of Federal Regulations, 2010 CFR

    2010-04-01

    ... 21 Food and Drugs 2 2010-04-01 2010-04-01 false âFresh,â âfreshly frozen,â âfresh frozen,â âfrozen fresh.â 101.95 Section 101.95 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) FOOD FOR HUMAN CONSUMPTION FOOD LABELING Specific Requirements...

  6. Antibody Responses to a Novel Plasmodium falciparum Merozoite Surface Protein Vaccine Correlate with Protection against Experimental Malaria Infection in Aotus Monkeys

    PubMed Central

    Cavanagh, David R.; Kocken, Clemens H. M.; White, John H.; Cowan, Graeme J. M.; Samuel, Kay; Dubbeld, Martin A.; der Wel, Annemarie Voorberg-van; Thomas, Alan W.; McBride, Jana S.; Arnot, David E.

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  7. Antibody responses to a novel Plasmodium falciparum merozoite surface protein vaccine correlate with protection against experimental malaria infection in Aotus monkeys.

    PubMed

    Cavanagh, David R; Kocken, Clemens H M; White, John H; Cowan, Graeme J M; Samuel, Kay; Dubbeld, Martin A; Voorberg-van der Wel, Annemarie; Thomas, Alan W; McBride, Jana S; Arnot, David E

    2014-01-01

    The Block 2 region of the merozoite surface protein-1 (MSP-1) of Plasmodium falciparum has been identified as a target of protective immunity by a combination of seroepidemiology and parasite population genetics. Immunogenicity studies in small animals and Aotus monkeys were used to determine the efficacy of recombinant antigens derived from this region of MSP-1 as a potential vaccine antigen. Aotus lemurinus griseimembra monkeys were immunized three times with a recombinant antigen derived from the Block 2 region of MSP-1 of the monkey-adapted challenge strain, FVO of Plasmodium falciparum, using an adjuvant suitable for use in humans. Immunofluorescent antibody assays (IFA) against erythrocytes infected with P. falciparum using sera from the immunized monkeys showed that the MSP-1 Block 2 antigen induced significant antibody responses to whole malaria parasites. MSP-1 Block 2 antigen-specific enzyme-linked immunosorbent assays (ELISA) showed no significant differences in antibody titers between immunized animals. Immunized animals were challenged with the virulent P. falciparum FVO isolate and monitored for 21 days. Two out of four immunized animals were able to control their parasitaemia during the follow-up period, whereas two out of two controls developed fulminating parasitemia. Parasite-specific serum antibody titers measured by IFA were four-fold higher in protected animals than in unprotected animals. In addition, peptide-based epitope mapping of serum antibodies from immunized Aotus showed distinct differences in epitope specificities between protected and unprotected animals. PMID:24421900

  8. Serological Conservation of Parasite-Infected Erythrocytes Predicts Plasmodium falciparum Erythrocyte Membrane Protein 1 Gene Expression but Not Severity of Childhood Malaria.

    PubMed

    Warimwe, George M; Abdi, Abdirahman I; Muthui, Michelle; Fegan, Gregory; Musyoki, Jennifer N; Marsh, Kevin; Bull, Peter C

    2016-05-01

    Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1), expressed on P. falciparum-infected erythrocytes, is a major family of clonally variant targets of naturally acquired immunity to malaria. Previous studies have demonstrated that in areas where malaria is endemic, antibodies to infected erythrocytes from children with severe malaria tend to be more seroprevalent than antibodies to infected erythrocytes from children with nonsevere malaria. These data have led to a working hypothesis that PfEMP1 variants associated with parasite virulence are relatively conserved in structure. However, the longevity of such serologically conserved variants in the parasite population is unknown. Here, using infected erythrocytes from recently sampled clinical P. falciparum samples, we measured serological conservation using pools of antibodies in sera that had been sampled 10 to 12 years earlier. The serological conservation of infected erythrocytes strongly correlated with the expression of specific PfEMP1 subsets previously found to be associated with severe malaria. However, we found no association between serological conservation per se and disease severity within these data. This contrasts with the simple hypothesis that P. falciparum isolates with a serologically conserved group of PfEMP1 variants cause severe malaria. The data are instead consistent with periodic turnover of the immunodominant epitopes of PfEMP1 associated with severe malaria. PMID:26883585

  9. Five-year tracking of Plasmodium falciparum allele frequencies in a holoendemic area with indistinct seasonal transitions

    PubMed Central

    Akala, Hoseah M; Achieng, Angela O; Eyase, Fredrick L; Juma, Dennis W; Ingasia, Luiser; Cheruiyot, Agnes C; Okello, Charles; Omariba, Duke; Owiti, Eunice A; Muriuki, Catherine; Yeda, Redemptah; Andagalu, Ben; Johnson, Jacob D; Kamau, Edwin

    2014-01-01

    Background The renewed malaria eradication efforts require an understanding of the seasonal patterns of frequency of polymorphic variants in order to focus limited funds productively. Although cross-sectional studies in holoendemic areas spanning a single year could be useful in describing parasite genotype status at a given point, such information is inadequate in describing temporal trends in genotype polymorphisms. For Plasmodium falciparum isolates from Kisumu District Hospital, Plasmodium falciparum chloroquine-resistance transporter gene (Pfcrt-K76T) and P. falciparum multidrug resistance gene 1 (PfMDR1-N86Y), were analyzed for polymorphisms and parasitemia changes in the 53 months from March 2008 to August 2012. Observations were compared with prevailing climatic factors, including humidity, rainfall, and temperature. Methods Parasitemia (the percentage of infected red blood cells per total red blood cells) was established by microscopy for P. falciparum malaria-positive samples. P. falciparum DNA was extracted from whole blood using a Qiagen DNA Blood Mini Kit. Single nucleotide polymorphism identification at positions Pfcrt-K76T and PfMDR1-N86Y was performed using real-time polymerase chain reaction and/or sequencing. Data on climatic variables were obtained from http://www.tutiempo.net/en/. Results A total of 895 field isolates from 2008 (n=169), 2009 (n=161), 2010 (n=216), 2011 (n=223), and 2012 (n=126) showed large variations in monthly frequency of PfMDR1-N86Y and Pfcrt-K76T as the mutant genotypes decreased from 68.4%±15% and 38.1%±13% to 29.8%±18% and 13.3%±9%, respectively. The mean percentage of parasitemia was 2.61%±1.01% (coefficient of variation 115.86%; n=895). There was no correlation between genotype or parasitemia and climatic factors. Conclusion This study shows variability in the frequency of Pfcrt-K76T and PfMDR1-N86Y polymorphisms during the study period, bringing into focus the role of cross-sectional studies in describing temporal

  10. Plasmodium falciparum Secretome in Erythrocyte and Beyond.

    PubMed

    Soni, Rani; Sharma, Drista; Bhatt, Tarun K

    2016-01-01

    Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies. PMID:26925057

  11. Plasmodium falciparum Secretome in Erythrocyte and Beyond

    PubMed Central

    Soni, Rani; Sharma, Drista; Bhatt, Tarun K.

    2016-01-01

    Plasmodium falciparum is the causative agent of deadly malaria disease. It is an intracellular eukaryote and completes its multi-stage life cycle spanning the two hosts viz, mosquito and human. In order to habituate within host environment, parasite conform several strategies to evade host immune responses such as surface antigen polymorphism or modulation of host immune system and it is mediated by secretion of proteins from parasite to the host erythrocyte and beyond, collectively known as, malaria secretome. In this review, we will discuss about the deployment of parasitic secretory protein in mechanism implicated for immune evasion, protein trafficking, providing virulence, changing permeability and cyto-adherence of infected erythrocyte. We will be covering the possibilities of developing malaria secretome as a drug/vaccine target. This gathered information will be worthwhile in depicting a well-organized picture for host-pathogen interplay during the malaria infection and may also provide some clues for the development of novel anti-malarial therapies. PMID:26925057

  12. Antimicrobial packaging for fresh-cut fruits

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fresh-cut fruits are minimally processed produce which are consumed directly at their fresh stage without any further kill step. Microbiological quality and safety are major challenges to fresh-cut fruits. Antimicrobial packaging is one of the innovative food packaging systems that is able to kill o...

  13. Hiding in Fresh Fruits and Vegetables: Opportunistic Pathogens May Cross Geographical Barriers

    PubMed Central

    Al-Kharousi, Zahra S.; Al-Sadi, Abdullah M.; Al-Bulushi, Ismail M.; Shaharoona, Baby

    2016-01-01

    Different microbial groups of the microbiome of fresh produce can have diverse effects on human health. This study was aimed at identifying some microbial communities of fresh produce by analyzing 105 samples of imported fresh fruits and vegetables originated from different countries in the world including local samples (Oman) for aerobic plate count and the counts of Enterobacteriaceae, Enterococcus, and Staphylococcus aureus. The isolated bacteria were identified by molecular (PCR) and biochemical methods (VITEK 2). Enterobacteriaceae occurred in 60% of fruits and 91% of vegetables. Enterococcus was isolated from 20% of fruits and 42% of vegetables. E. coli and S. aureus were isolated from 22% and 7% of vegetables, respectively. Ninety-seven bacteria comprising 21 species were similarly identified by VITEK 2 and PCR to species level. E. coli, Klebsiella pneumoniae, Enterococcus casseliflavus, and Enterobacter cloacae were the most abundant species; many are known as opportunistic pathogens which may raise concern to improve the microbial quality of fresh produce. Phylogenetic trees showed no relationship between clustering of the isolates based on the 16S rRNA gene and the original countries of fresh produce. Intercountry passage of opportunistic pathogens in fresh produce cannot be ruled out, which requires better management. PMID:26989419

  14. Possible association of the Plasmodium falciparum T1526C resa2 gene mutation with severe malaria

    PubMed Central

    2012-01-01

    Background Plasmodium falciparum exports proteins that remodel the erythrocyte membrane. One such protein, called Pf155/RESA (RESA1) contributes to parasite fitness, optimizing parasite survival during febrile episodes. Resa1 gene is a member of a small family comprising three highly related genes. Preliminary evidence led to a search for clues indicating the involvement of RESA2 protein in the pathophysiology of malaria. In the present study, cDNA sequence of resa2 gene was obtained from two different strains. The proportion of P. falciparum isolates having a non-stop T1526C mutation in resa2 gene was evaluated and the association of this genotype with severity of malaria was investigated. Methods Resa2 cDNAs of two different strains (a patient isolate and K1 culture adapted strain) was obtained by RT-PCR and DNA sequencing was performed to confirm its gene structure. The proportion of isolates having a T1526C mutation was evaluated using a PCR-RFLP methodology on groups of severe malaria and uncomplicated patients recruited in 1991–1994 in Senegal and in 2009 in Benin. Results A unique ORF with an internal translation stop was found in the patient isolate (Genbank access number : JN183870), while the K1 strain harboured the T1526C mutation (Genbank access number : JN183869) which affects the internal stop codon and restores a full length coding sequence. About 14% of isolates obtained from Senegal and Benin harboured mutant T1526C parasites. Some isolates had both wild and mutant resa alleles. The analysis excluding those mixed isolates showed that the resa2 T1526C mutation was found more frequently in severe malaria cases than in uncomplicated cases (p = 0.008). The association of the presence of the mutant allele and parasitaemia >4% was shown in multivariate analysis (p = 0.03) in the group of Beninese children. Conclusions All T1526C mutant parasites theoretically have the ability to give rise to a full-length RESA2 protein. This study raises the

  15. Functional genomics of Plasmodium falciparum using metabolic modelling and analysis

    PubMed Central

    Oppenheim, Rebecca D.; Soldati-Favre, Dominique; Hatzimanikatis, Vassily

    2013-01-01

    Plasmodium falciparum is an obligate intracellular parasite and the leading cause of severe malaria responsible for tremendous morbidity and mortality particularly in sub-Saharan Africa. Successful completion of the P. falciparum genome sequencing project in 2002 provided a comprehensive foundation for functional genomic studies on this pathogen in the following decade. Over this period, a large spectrum of experimental approaches has been deployed to improve and expand the scope of functionally annotated genes. Meanwhile, rapidly evolving methods of systems biology have also begun to contribute to a more global understanding of various aspects of the biology and pathogenesis of malaria. Herein we provide an overview on metabolic modelling, which has the capability to integrate information from functional genomics studies in P. falciparum and guide future malaria research efforts towards the identification of novel candidate drug targets. PMID:23793264

  16. Nitric oxide inhibits falcipain, the Plasmodium falciparum trophozoite cysteine protease.

    PubMed

    Venturini, G; Colasanti, M; Salvati, L; Gradoni, L; Ascenzi, P

    2000-01-01

    Nitric oxide (NO) is a pluripotent regulatory molecule possessing, among others, an antiparasitic activity. In the present study, the inhibitory effect of NO on the catalytic activity of falcipain, the papain-like cysteine protease involved in Plasmodium falciparum trophozoite hemoglobin degradation, is reported. In particular, NO donors S-nitrosoglutathione (GSNO), (+/-)-(E)-p6ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexenami de (NOR-3), 3-morpholinosydnonimine (SIN-1), and sodium nitroprusside (SNP) inhibit dose-dependently the falcipain activity present in the P. falciparum trophozoite extract, this effect likely attributable to S-nitrosylation of the Cys25 catalytic residue. The results represent a new insight into the modulation mechanism of falcipain activity, thereby being relevant in developing new strategies for inhibition of the P. falciparum life cycle. PMID:10623597

  17. Monkey-derived monoclonal antibodies against Plasmodium falciparum.

    PubMed Central

    Stanley, H A; Reese, R T

    1985-01-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a Mr 95,000 antigen. Images PMID:3898084

  18. Plasmodium falciparum: growth response to potassium channel blocking compounds.

    PubMed

    Waller, Karena L; Kim, Kami; McDonald, Thomas V

    2008-11-01

    Potassium channels are essential for cell survival and regulate the cell membrane potential and electrochemical gradient. During its lifecycle, Plasmodium falciparum parasites must rapidly adapt to dramatically variant ionic conditions within the mosquito mid-gut, the hepatocyte and red blood cell (RBC) cytosols, and the human circulatory system. To probe the participation of K(+) channels in parasite viability, growth response assays were performed in which asexual stage P. falciparum parasites were cultured in the presence of various Ca(2+)-activated K(+) channel blocking compounds. These data describe the novel anti-malarial effects of bicuculline methiodide and tubocurarine chloride and the novel lack of effect of apamine and verruculogen. Taken together, the data herein imply the presence of K(+) channels, or other parasite-specific targets, in P. falciparum-infected RBCs that are sensitive to blockade with Ca(2+)-activated K(+) channel blocking compounds. PMID:18703053

  19. [Research Progress on Artemisinin Resistance in Plasmodium falciparum].

    PubMed

    Zhang, Yi-long; Pan, Wei-qing

    2015-12-01

    Artemisinin (ART) is a novel and effective antimalarial drug discovered in China. As recommended by the World Health Organization, the ART-based combination therapies (ACTs) have become the first-line drugs for the treatment of falciparum malaria. ART and its derivatives have contributed greatly to the effective control of malaria globally, leading to yearly decrease of malaria morbidity and mortality. However, there have recently been several reports on the resistance of Plasmodium falciparum to ART in Southeast Asia. This is deemed a serious threat to the global malaria control programs. In this paper, we reviewed recent research progress on ART resistance to P. falciparum, including new tools for resistance measurement, resistance-associated molecular markers, and the origin and spread of the ART-resistant parasite strains. PMID:27089770

  20. Active case detection, treatment of falciparum malaria with combined chloroquine and sulphadoxine/pyrimethamine and vivax malaria with chloroquine and molecular markers of anti-malarial resistance in the Republic of Vanuatu

    PubMed Central

    2010-01-01

    Background Chloroquine-resistant Plasmodium falciparum was first described in the Republic of Vanuatu in the early 1980s. In 1991, the Vanuatu Ministry of Health instituted new treatment guidelines for uncomplicated P. falciparum infection consisting of chloroquine/sulphadoxine-pyrimethamine combination therapy. Chloroquine remains the recommended treatment for Plasmodium vivax. Methods In 2005, cross-sectional blood surveys at 45 sites on Malo Island were conducted and 4,060 adults and children screened for malaria. Of those screened, 203 volunteer study subjects without malaria at the time of screening were followed for 13 weeks to observe peak seasonal incidence of infection. Another 54 subjects with malaria were followed over a 28-day period to determine efficacy of anti-malarial therapy; chloroquine alone for P. vivax and chloroquine/sulphadoxine-pyrimethamine for P. falciparum infections. Results The overall prevalence of parasitaemia by mass blood screening was 6%, equally divided between P. falciparum and P. vivax. Twenty percent and 23% of participants with patent P. vivax and P. falciparum parasitaemia, respectively, were febrile at the time of screening. In the incidence study cohort, after 2,303 person-weeks of follow-up, the incidence density of malaria was 1.3 cases per person-year with P. vivax predominating. Among individuals participating in the clinical trial, the 28-day chloroquine P. vivax cure rate was 100%. The 28-day chloroquine/sulphadoxine-pyrimethamine P. falciparum cure rate was 97%. The single treatment failure, confirmed by merozoite surface protein-2 genotyping, was classified as a day 28 late parasitological treatment failure. All P. falciparum isolates carried the Thr-76 pfcrt mutant allele and the double Asn-108 + Arg-59 dhfr mutant alleles. Dhps mutant alleles were not detected in the study sample. Conclusion Peak seasonal malaria prevalence on Malo Island reached hypoendemic levels during the study observation period. The only in

  1. Plasmodium falciparum Drug Resistance in Madagascar: Facing the Spread of Unusual pfdhfr and pfmdr-1 Haplotypes and the Decrease of Dihydroartemisinin Susceptibility▿

    PubMed Central

    Andriantsoanirina, Valérie; Ratsimbasoa, Arsène; Bouchier, Christiane; Jahevitra, Martial; Rabearimanana, Stéphane; Radrianjafy, Rogelin; Andrianaranjaka, Voahangy; Randriantsoa, Tantely; Rason, Marie Ange; Tichit, Magali; Rabarijaona, Léon Paul; Mercereau-Puijalon, Odile; Durand, Rémy; Ménard, Didier

    2009-01-01

    The aim of this study was to provide the first comprehensive spatiotemporal picture of Plasmodium falciparum resistance in various geographic areas in Madagascar. Additional data about the antimalarial resistance in the neighboring islands of the Comoros archipelago were also collected. We assessed the prevalence of pfcrt, pfmdr-1, pfdhfr, and pfdhps mutations and the pfmdr-1 gene copy number in 1,596 P. falciparum isolates collected in 26 health centers (20 in Madagascar and 6 in the Comoros Islands) from 2006 to 2008. The in vitro responses to a panel of drugs by 373 of the parasite isolates were determined. The results showed (i) unusual profiles of chloroquine susceptibility in Madagascar, (ii) a rapid rise in the frequency of parasites with both the pfdhfr and the pfdhps mutations, (iii) the alarming emergence of the single pfdhfr 164L genotype, and (iv) the progressive loss of the most susceptible isolates to artemisinin derivatives. In the context of the implementation of the new national policy for the fight against malaria, continued surveillance for the detection of P. falciparum resistance in the future is required. PMID:19704124

  2. RIFINs are adhesins implicated in severe Plasmodium falciparum malaria.

    PubMed

    Goel, Suchi; Palmkvist, Mia; Moll, Kirsten; Joannin, Nicolas; Lara, Patricia; Akhouri, Reetesh R; Moradi, Nasim; Öjemalm, Karin; Westman, Mattias; Angeletti, Davide; Kjellin, Hanna; Lehtiö, Janne; Blixt, Ola; Ideström, Lars; Gahmberg, Carl G; Storry, Jill R; Hult, Annika K; Olsson, Martin L; von Heijne, Gunnar; Nilsson, IngMarie; Wahlgren, Mats

    2015-04-01

    Rosetting is a virulent Plasmodium falciparum phenomenon associated with severe malaria. Here we demonstrate that P. falciparum-encoded repetitive interspersed families of polypeptides (RIFINs) are expressed on the surface of infected red blood cells (iRBCs), where they bind to RBCs--preferentially of blood group A--to form large rosettes and mediate microvascular binding of iRBCs. We suggest that RIFINs have a fundamental role in the development of severe malaria and thereby contribute to the varying global distribution of ABO blood groups in the human population. PMID:25751816

  3. A rapid and simple DNA extraction procedure to detect Salmonella spp. and Listeria monocytogenes from fresh produce using real-time PCR

    Technology Transfer Automated Retrieval System (TEKTRAN)

    DNA isolation procedures significantly influence the outcome of PCR-based detection of human pathogens. Unlike clinical samples, DNA isolation from food samples such as fresh and fresh-cut produce has remained a formidable task and has hampered the sensitivity and accuracy of molecular methods. We...

  4. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains.

    PubMed

    Preston, Mark D; Campino, Susana; Assefa, Samuel A; Echeverry, Diego F; Ocholla, Harold; Amambua-Ngwa, Alfred; Stewart, Lindsay B; Conway, David J; Borrmann, Steffen; Michon, Pascal; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A; Doumbo, Ogobara K; Nosten, Francois; Pain, Arnab; Bousema, Teun; Drakeley, Chris J; Fairhurst, Rick M; Sutherland, Colin J; Roper, Cally; Clark, Taane G

    2014-01-01

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. PMID:24923250

  5. Potential antimalarial candidates from African plants: and in vitro approach using Plasmodium falciparum.

    PubMed

    Khalid, S A; Farouk, A; Geary, T G; Jensen, J B

    1986-02-01

    Twenty-one compounds isolated from nine medicinal plants used in traditional medicine in the Sudan and other African countries were examined in vitro for antimalarial activity against Plasmodium falciparum, the major human malaria parasite. Compounds tested include alkaloids, lignans, triterpenes, coumarins, limonoids and flavonoids. Most were relatively inactive; one limonoid, gedunin, had an IC50 value of about 1 microM after 48 h exposure (0.3 microM after 96 h), roughly equivalent to quinine. In this protocol, the flavonoid quercetin purified from Diosma pilosa was found to have the same activity as a commercially obtained preparation. Simple radiometric assays for antimalarial activity can thus be used to rapidly screen purified plant material or secondary plant metabolites. The high potency and efficacy of quinine and the Chinese herbal antimalarial quinghaosu (artemisinine) illustrate the merit of this approach. PMID:3520157

  6. A barcode of organellar genome polymorphisms identifies the geographic origin of Plasmodium falciparum strains

    PubMed Central

    Preston, Mark D.; Campino, Susana; Assefa, Samuel A.; Echeverry, Diego F.; Ocholla, Harold; Amambua-Ngwa, Alfred; Stewart, Lindsay B.; Conway, David J.; Borrmann, Steffen; Michon, Pascal; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A.; Doumbo, Ogobara K.; Nosten, Francois; Pain, Arnab; Bousema, Teun; Drakeley, Chris J.; Fairhurst, Rick M.; Sutherland, Colin J.; Roper, Cally; Clark, Taane G.

    2014-01-01

    Malaria is a major public health problem that is actively being addressed in a global eradication campaign. Increased population mobility through international air travel has elevated the risk of re-introducing parasites to elimination areas and dispersing drug-resistant parasites to new regions. A simple genetic marker that quickly and accurately identifies the geographic origin of infections would be a valuable public health tool for locating the source of imported outbreaks. Here we analyse the mitochondrion and apicoplast genomes of 711 Plasmodium falciparum isolates from 14 countries, and find evidence that they are non-recombining and co-inherited. The high degree of linkage produces a panel of relatively few single-nucleotide polymorphisms (SNPs) that is geographically informative. We design a 23-SNP barcode that is highly predictive (~92%) and easily adapted to aid case management in the field and survey parasite migration worldwide. PMID:24923250

  7. Large-scale growth of the Plasmodium falciparum malaria parasite in a wave bioreactor.

    PubMed

    Dalton, John P; Demanga, Corine G; Reiling, Sarah J; Wunderlich, Juliane; Eng, Jenny W L; Rohrbach, Petra

    2012-01-01

    We describe methods for the large-scale in vitro culturing of synchronous and asynchronous blood-stage Plasmodium falciparum parasites in sterile disposable plastic bioreactors controlled by wave-induced motion (wave bioreactor). These cultures perform better than static flask cultures in terms of preserving parasite cell cycle synchronicity and reducing the number of multiple-infected erythrocytes. The straight-forward methods described here will facilitate the large scale production of malaria parasites for antigen and organelle isolation and characterisation, for the high throughput screening of compound libraries with whole cells or extracts, and the development of live- or whole-cell malaria vaccines under good manufacturing practice compliant standards. PMID:22326740

  8. Artemisinin-resistant Plasmodium falciparum malaria

    PubMed Central

    Fairhurst, Rick M.; Dondorp, Arjen M.

    2016-01-01

    For more than five decades, Southeast Asia (SEA) has been fertile ground for the emergence of drug-resistant Plasmodium falciparum malaria. After generating parasites resistant to chloroquine, sulfadoxine, pyrimethamine, quinine, and mefloquine, this region has now spawned parasites resistant to artemisinins – the world's most potent antimalarial drugs. In areas where artemisinin resistance is prevalent, artemisinin combination therapies (ACTs) – the first-line treatments for malaria – are failing fast. This worrisome development threatens to make malaria practically untreatable in SEA, and threatens to compromise global endeavors to eliminate this disease. A recent series of clinical, in-vitro, genomics, and transcriptomics studies in SEA have defined in-vivo and in-vitro phenotypes of artemisinin resistance; identified its causal genetic determinant; explored its molecular mechanism; and assessed its clinical impact. Specifically, these studies have established that artemisinin resistance manifests as slow parasite clearance in patients and increased survival of early ring-stage parasites in vitro; is caused by single nucleotide polymorphisms in the parasite's ‘K13’ gene; is associated with an upregulated “unfolded protein response” pathway that may antagonize the pro-oxidant activity of artemisinins; and selects for partner drug resistance that rapidly leads to ACT failures. In SEA, clinical studies are urgently needed to monitor ACT efficacy where K13 mutations are prevalent; test whether new combinations of currently-available drugs cure ACT failures; and advance new antimalarial compounds through preclinical pipelines and into clinical trials. Intensifying these efforts should help to forestall the spread of artemisinin and partner drug resistance from SEA to Sub-Saharan Africa, where the world's malaria transmission, morbidity, and mortality rates are highest. PMID:27337450

  9. Genetic diversity of Plasmodium falciparum among school-aged children from the Man region, western Côte d’Ivoire

    PubMed Central

    2013-01-01

    Background The genetic diversity of Plasmodium falciparum allows the molecular discrimination of otherwise microscopically identical parasites and the identification of individual clones in multiple infections. The study reported here investigated the P. falciparum multiplicity of infection (MOI) and genetic diversity among school-aged children in the Man region, western Côte d’Ivoire. Methods Blood samples from 292 children aged seven to 15 years were collected in four nearby villages located at altitudes ranging from 340 to 883 m above sea level. Giemsa-stained thick and thin blood films were prepared and examined under a microscope for P. falciparum prevalence and parasitaemia. MOI and genetic diversity of the parasite populations were investigated using msp2 typing by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Results Plasmodium falciparum prevalence and parasitaemia were both found to be significantly lower in the highest altitude village. Genotyping of the isolates revealed 25 potentially new msp2 alleles. MOI varied significantly across villages but did not correlate with altitude nor children’s age, and only to a limited extent with parasitaemia. An analysis of molecular variance (AMOVA) indicated that a small, but close to statistical significance (p = 0.07), fraction of variance occurs specifically between villages of low and high altitudes. Conclusions Higher altitude was associated with lower prevalence of P. falciparum but not with reduced MOI, suggesting that, in this setting, MOI is not a good proxy for transmission. The evidence for partially parted parasite populations suggests the existence of local geographical barriers that should be taken into account when deploying anti-malarial interventions. PMID:24228865

  10. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border.

    PubMed

    Ye, Run; Hu, Dongwei; Zhang, Yilong; Huang, Yufu; Sun, Xiaodong; Wang, Jian; Chen, Xuedi; Zhou, Hongning; Zhang, Dongmei; Mungthin, Mathirut; Pan, Weiqing

    2016-01-01

    The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0-3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced. PMID:26831371

  11. Distinctive origin of artemisinin-resistant Plasmodium falciparum on the China-Myanmar border

    PubMed Central

    Ye, Run; Hu, Dongwei; Zhang, Yilong; Huang, Yufu; Sun, Xiaodong; Wang, Jian; Chen, Xuedi; Zhou, Hongning; Zhang, Dongmei; Mungthin, Mathirut; Pan, Weiqing

    2016-01-01

    The artemisinin (ART), discovered in China, has been widely used against malaria in China over the last 30 years. Understanding the emergence and origin of ART resistance in China is therefore of great interest. We analyzed 111 culture-adapted isolates of P. falciparum from China-Myanmar (CM) border for their susceptibility to dihydroartemisinin using the ring stage survival assay (RSA0−3h) and genotyped their K13 genes. Of the isolates, 59 had a wild type of the K13 marker and a median ring survival rate of 0.26% (P95 = 1.005%). Among the remaining isolates harboring single mutations in the K13 marker, 26 survived at >P95(median survival rate = 2.95%). Further, we genotyped the K13 gene of 693 isolates collected from different regions in China and China-Myanmar/Thai-Cambodia/Thai-Myanmar (CM/TC/TM) borders, 308 (44.4%) had K13 mutations and marked differences in the patterns of K13 mutations were observed between the CM and the TC/TM borders. A network diagram showed that majority of the K13 mutant alleles from the CM border clustered together including those harboring the high resistant-associated R539T mutations. The resistant parasites carrying distinct halplotypes suggested the multiple indigenous origins of the resistant alleles, which highlight the importance of surveillance of resistance in all malaria endemic areas where ART has been introduced. PMID:26831371

  12. Salmonella surveillance on fresh produce in retail in Turkey.

    PubMed

    Gunel, Elif; Polat Kilic, Gozde; Bulut, Ece; Durul, Bora; Acar, Sinem; Alpas, Hami; Soyer, Yeşim

    2015-04-16

    Although Turkey is one of the major producers of fruits and vegetables in the world, there has been no information available on the prevalence of pathogens in fresh produce. To fill this gap, we collected 503 fresh produce samples including tomato, parsley, iceberg lettuce, green-leaf lettuce and five different fresh pepper varieties (i.e., green, kapya, bell, mazamort and Charleston) from 3 major districts within 9 supermarkets and 3 bazaars in Ankara, Turkey to investigate the presence of Salmonella. Salmonella was detected in 0.8% (4/503) of samples by conventional culturing method with molecular confirmation conducted through polymerase chain reaction (PCR). For further characterization of isolates, serotyping, antimicrobial susceptibility testing, multi-locus sequence typing (MLST; aroC, thrA, purE, sucA, hisD, hemD and dnaN) and pulsed-field gel electrophoresis (PFGE) were performed. Salmonella enterica subsp. enterica serotypes Anatum, Charity, Enteritidis and Mikawasima were isolated from two parsley, one pepper and one lettuce samples, respectively. MLST resulted in 4 sequence types (STs) for each serotype, including one novel ST for serotype Mikawasima. Similarly, PFGE revealed four different XbaI PFGE patterns. The results of this survey, obtained by the most common subtyping methods (i.e. serotyping, MLST and PFGE) worldwide, contributes to the development of a national database in Turkey, which is essential for investigating the evolutionary pathways, geographical distribution and genetic diversity of Salmonella strains. PMID:25643853

  13. Surface binding properties of aged and fresh (recently excreted) Toxoplasma gondii oocysts.

    PubMed

    Harito, Jemere Bekele; Campbell, Andrew T; Prestrud, Kristin W; Dubey, J P; Robertson, Lucy J

    2016-06-01

    The surfaces of aged (10 years) and fresh (recently excreted) oocysts of Toxoplasma gondii were investigated using monoclonal antibody (mAb) and lectin-binding assays. Fresh oocysts bound a wall-specific mAb labelled with fluorescein isothiocyanate while aged oocysts did not. In contrast, the walls of aged oocysts bound a lectin (wheat germ agglutinin, WGA), but not the walls of fresh oocysts. Exposure of oocysts to detergent solutions or trypsin did not affect the binding properties of the walls of the oocysts. However, exposure of fresh oocysts to acidified pepsin enabled labelling of the walls with WGA, presumably due to the relevant moieties on the oocyst walls becoming exposed. WGA binding, but not mAb binding, was partially abrogated with periodate exposure. These findings reveal a significant difference in the binding properties of oocyst walls from "aged" and "fresh" oocysts. The results are of relevance when considering technologies for isolating or detecting T. gondii oocysts in environmental samples based on oocyst surface properties, as used for other protozoan parasites. Our results suggest the possibility of developing a WGA-based separation procedure for isolating Toxoplasma oocysts from environmental matrices, in which pepsin pre-treatment would be included to ensure that both fresh and aged oocysts were isolated. PMID:27003461

  14. Dissecting the role of glutathione biosynthesis in Plasmodium falciparum

    PubMed Central

    Patzewitz, Eva-Maria; Wong, Eleanor H; Müller, Sylke

    2012-01-01

    Glutathione (γ-glutamylcysteinyl-glycine, GSH) has vital functions as thiol redox buffer and cofactor of antioxidant and detoxification enzymes. Plasmodium falciparum possesses a functional GSH biosynthesis pathway and contains mM concentrations of the tripeptide. It was impossible to delete in P. falciparum the genes encoding γ-glutamylcysteine synthetase (γGCS) or glutathione synthetase (GS), the two enzymes synthesizing GSH, although both gene loci were not refractory to recombination. Our data show that the parasites cannot compensate for the loss of GSH biosynthesis via GSH uptake. This suggests an important if not essential function of GSH biosynthesis pathway for the parasites. Treatment with the irreversible inhibitor of γGCS L-buthionine sulfoximine (BSO) reduced intracellular GSH levels in P. falciparum and was lethal for their intra-erythrocytic development, corroborating the suggestion that GSH biosynthesis is important for parasite survival. Episomal expression of γgcs in P. falciparum increased tolerance to BSO attributable to increased levels of γGCS. Concomitantly expression of glutathione reductase was reduced leading to an increased GSH efflux. Together these data indicate that GSH levels are tightly regulated by a functional GSH biosynthesis and the reduction of GSSG. PMID:22151036

  15. Antimalarial Benzoxaboroles Target Plasmodium falciparum Leucyl-tRNA Synthetase.

    PubMed

    Sonoiki, Ebere; Palencia, Andres; Guo, Denghui; Ahyong, Vida; Dong, Chen; Li, Xianfeng; Hernandez, Vincent S; Zhang, Yong-Kang; Choi, Wai; Gut, Jiri; Legac, Jennifer; Cooper, Roland; Alley, M R K; Freund, Yvonne R; DeRisi, Joseph; Cusack, Stephen; Rosenthal, Philip J

    2016-08-01

    There is a need for new antimalarials, ideally with novel mechanisms of action. Benzoxaboroles have been shown to be active against bacteria, fungi, and trypanosomes. Therefore, we investigated the antimalarial activity and mechanism of action of 3-aminomethyl benzoxaboroles against Plasmodium falciparum Two 3-aminomethyl compounds, AN6426 and AN8432, demonstrated good potency against cultured multidrug-resistant (W2 strain) P. falciparum (50% inhibitory concentration [IC50] of 310 nM and 490 nM, respectively) and efficacy against murine Plasmodium berghei infection when administered orally once daily for 4 days (90% effective dose [ED90], 7.4 and 16.2 mg/kg of body weight, respectively). To characterize mechanisms of action, we selected parasites with decreased drug sensitivity by culturing with stepwise increases in concentration of AN6426. Resistant clones were characterized by whole-genome sequencing. Three generations of resistant parasites had polymorphisms in the predicted editing domain of the gene encoding a P. falciparum leucyl-tRNA synthetase (LeuRS; PF3D7_0622800) and in another gene (PF3D7_1218100), which encodes a protein of unknown function. Solution of the structure of the P. falciparum LeuRS editing domain suggested key roles for mutated residues in LeuRS editing. Short incubations with AN6426 and AN8432, unlike artemisinin, caused dose-dependent inhibition of [(14)C]leucine incorporation by cultured wild-type, but not resistant, parasites. The growth of resistant, but not wild-type, parasites was impaired in the presence of the unnatural amino acid norvaline, consistent with a loss of LeuRS editing activity in resistant parasites. In summary, the benzoxaboroles AN6426 and AN8432 offer effective antimalarial activity and act, at least in part, against a novel target, the editing domain of P. falciparum LeuRS. PMID:27270277

  16. [From malaria parasite point of view--Plasmodium falciparum evolution].

    PubMed

    Zerka, Agata; Kaczmarek, Radosław; Jaśkiewicz, Ewa

    2015-01-01

    Malaria is caused by infection with protozoan parasites belonging to the genus Plasmodium, which have arguably exerted the greatest selection pressure on humans in the history of our species. Besides humans, different Plasmodium parasites infect a wide range of animal hosts, from marine invertebrates to primates. On the other hand, individual Plasmodium species show high host specificity. The extraordinary evolution of Plasmodium probably began when a free-living red algae turned parasitic, and culminated with its ability to thrive inside a human red blood cell. Studies on the African apes generated new data on the evolution of malaria parasites in general and the deadliest human-specific species, Plasmodium falciparum, in particular. Initially, it was hypothesized that P. falciparum descended from the chimpanzee malaria parasite P. reichenowi, after the human and the chimp lineage diverged about 6 million years ago. However, a recently identified new species infecting gorillas, unexpectedly showed similarity to P. falciparum and was therefore named P. praefalciparum. That finding spurred an alternative hypothesis, which proposes that P. falciparum descended from its gorilla rather than chimp counterpart. In addition, the gorilla-to-human host shift may have occurred more recently (about 10 thousand years ago) than the theoretical P. falciparum-P. reichenowi split. One of the key aims of the studies on Plasmodium evolution is to elucidate the mechanisms that allow the incessant host shifting and retaining the host specificity, especially in the case of human-specific species. Thorough understanding of these phenomena will be necessary to design effective malaria treatment and prevention strategies. PMID:27259224

  17. Synthesis, Biological Evaluation and Structure-Activity Relationships of N-Benzoyl-2-hydroxybenzamides as Agents Active against P. falciparum (K1 strain), Trypanosomes, and Leishmania

    PubMed Central

    Stec, Jozef; Huang, Qingqing; Pieroni, Marco; Kaiser, Marcel; Fomovska, Alina; Mui, Ernest; Witola, William H.; Bettis, Samuel; McLeod, Rima; Brun, Reto; Kozikowski, Alan P.

    2012-01-01

    In our efforts to identify novel chemical scaffolds for the development of new antiprotozoal drugs, a compound library was screened against T. gondii tachyzoites with activity discovered for N-(4-ethylbenzoyl)-2-hydroxybenzamide 1a against T. gondii as described elsewhere.1 Synthesis of a compound set was guided by T. gondii SAR with 1r found to be superior for T. gondii, also active against Thai and Sierra Leone strains of P. falciparum, and with superior ADMET properties as described elsewhere.1 Herein, synthesis methods and details of the chemical analysis of the compounds in this series are described. Further, this series of N-benzoyl-2-hydroxybenzamides was re-purposed for testing against four other protozoan parasites: T. b. rhodesiense, T. cruzi, L. donovani, and P. falciparum (K1 isolate). Structure-activity analyses led to the identification of compounds in this set with excellent anti-leishmanial activity (compound 1d). Overall, compound 1r was the best and had activity 21-fold superior to that of the standard anti-malarial drug chloroquine against the K1 P. falciparum isolate. PMID:22352841

  18. Cytoadherence of Plasmodium falciparum to intercellular adhesion molecule 1 and chondroitin-4-sulfate expressed by the syncytiotrophoblast in the human placenta.

    PubMed Central

    Maubert, B; Guilbert, L J; Deloron, P

    1997-01-01

    Late stages of Plasmodium falciparum-infected erythrocytes (IRBCs) frequently sequester in the placentas of pregnant women, a phenomenon associated with low birth weight of the offspring. To investigate the physiological mechanism of this sequestration, we developed an in vitro assay for studying the cytoadherence of IRBCs to cultured term human trophoblasts. The capacity for binding to the syncytiotrophoblast varied greatly among P. falciparum isolates and was mediated by intercellular adhesion molecule 1 (ICAM-1), as binding was totally inhibited by 84H10, a monoclonal antibody specific for ICAM-1. Binding of the P. falciparum line RP5 to the syncytiotrophoblast involves chondroitin-4-sulfate (CSA), as this binding was dramatically impaired by addition of free CSA to the binding medium or by preincubation of the syncytiotrophoblast with chondroitinase ABC. ICAM-1 and CSA were visualized on the syncytiotrophoblast by immunofluorescence, while CD36, E-selectin, and vascular cell adhesion molecule 1 were not expressed even on tumor necrosis factor alpha (TNF-alpha)-stimulated syncytiotrophoblast tissue, and monoclonal antibodies against these cell adhesion molecules did not inhibit cytoadherence. ICAM-1 expression and cytoadherence of wild isolates was upregulated by TNF-alpha, a cytokine that can be secreted by the numerous mononuclear phagocytes present in malaria-infected placentas. These results suggest that cytoadherence may be involved in the placental sequestration and broaden the understanding of the physiopathology of the malaria-infected placenta. PMID:9119459

  19. Systematic Study of the Content of Phytochemicals in Fresh and Fresh-Cut Vegetables

    PubMed Central

    Alarcón-Flores, María Isabel; Romero-González, Roberto; Martínez Vidal, José Luis; Garrido Frenich, Antonia

    2015-01-01

    Vegetables and fruits have beneficial properties for human health, because of the presence of phytochemicals, but their concentration can fluctuate throughout the year. A systematic study of the phytochemical content in tomato, eggplant, carrot, broccoli and grape (fresh and fresh-cut) has been performed at different seasons, using liquid chromatography coupled to triple quadrupole mass spectrometry. It was observed that phenolic acids (the predominant group in carrot, eggplant and tomato) were found at higher concentrations in fresh carrot than in fresh-cut carrot. However, in the case of eggplant, they were detected at a higher content in fresh-cut than in fresh samples. Regarding tomato, the differences in the content of phenolic acids between fresh and fresh-cut were lower than in other matrices, except in winter sampling, where this family was detected at the highest concentration in fresh tomato. In grape, the flavonols content (predominant group) was higher in fresh grape than in fresh-cut during all samplings. The content of glucosinolates was lower in fresh-cut broccoli than in fresh samples in winter and spring sampling, although this trend changes in summer and autumn. In summary, phytochemical concentration did show significant differences during one-year monitoring, and the families of phytochemicals presented different behaviors depending on the matrix studied. PMID:26783709

  20. Systematic Study of the Content of Phytochemicals in Fresh and Fresh-Cut Vegetables.

    PubMed

    Alarcón-Flores, María Isabel; Romero-González, Roberto; Vidal, José Luis Martínez; Frenich, Antonia Garrido

    2015-01-01

    Vegetables and fruits have beneficial properties for human health, because of the presence of phytochemicals, but their concentration can fluctuate throughout the year. A systematic study of the phytochemical content in tomato, eggplant, carrot, broccoli and grape (fresh and fresh-cut) has been performed at different seasons, using liquid chromatography coupled to triple quadrupole mass spectrometry. It was observed that phenolic acids (the predominant group in carrot, eggplant and tomato) were found at higher concentrations in fresh carrot than in fresh-cut carrot. However, in the case of eggplant, they were detected at a higher content in fresh-cut than in fresh samples. Regarding tomato, the differences in the content of phenolic acids between fresh and fresh-cut were lower than in other matrices, except in winter sampling, where this family was detected at the highest concentration in fresh tomato. In grape, the flavonols content (predominant group) was higher in fresh grape than in fresh-cut during all samplings. The content of glucosinolates was lower in fresh-cut broccoli than in fresh samples in winter and spring sampling, although this trend changes in summer and autumn. In summary, phytochemical concentration did show significant differences during one-year monitoring, and the families of phytochemicals presented different behaviors depending on the matrix studied. PMID:26783709

  1. A Sequence in Subdomain 2 of DBL1α of Plasmodium falciparum Erythrocyte Membrane Protein 1 Induces Strain Transcending Antibodies

    PubMed Central

    Blomqvist, Karin; Albrecht, Letusa; Quintana, Maria del Pilar; Angeletti, Davide; Joannin, Nicolas; Chêne, Arnaud; Moll, Kirsten; Wahlgren, Mats

    2013-01-01

    Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1α-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. PMID:23335956

  2. Polymorphism in the gene encoding the apical membrane antigen-1 (AMA-1) of Plasmodium falciparum. X. Asembo Bay Cohort Project.

    PubMed

    Escalante, A A; Grebert, H M; Chaiyaroj, S C; Magris, M; Biswas, S; Nahlen, B L; Lal, A A

    2001-04-01

    We have investigated the genetic diversity of the gene encoding the apical membrane antigen-1 (AMA-1) in natural populations of Plasmodium falciparum from western Kenya and compared it with parasite populations from other geographic regions. A total of 28 complete sequences from Kenya, Thailand, India, and Venezuela field isolates were obtained. The genetic polymorphism is not evenly distributed across the gene, which is in agreement with the pattern reported in earlier studies. The alleles from Kenya exhibit 20 and 30% more polymorphism than that found in Southeast Asia and Venezuelan alleles, respectively. Based on the gene genealogies derived from sequencing data, no evidence for allele families was found. We have found evidence supporting limited gene flow between the parasite populations, specifically, between the Southeast Asian and Venezuelan isolates; however, no alleles could be linked to a specific geographic region. This study reveals that positive natural selection is an important factor in the maintenance of genetic diversity for AMA-1. We did not find conclusive evidence indicating intragenic recombination is important in the generation of the AMA-1 allelic diversity. The study provides information on the genetic diversity of the AMA-1 gene that would be useful in vaccine development and testing, as well as in assessing factors that are involved in the generation and maintenance of the genetic diversity in P. falciparum. PMID:11295182

  3. A sequence in subdomain 2 of DBL1α of Plasmodium falciparum erythrocyte membrane protein 1 induces strain transcending antibodies.

    PubMed

    Blomqvist, Karin; Albrecht, Letusa; Quintana, Maria del Pilar; Angeletti, Davide; Joannin, Nicolas; Chêne, Arnaud; Moll, Kirsten; Wahlgren, Mats

    2013-01-01

    Immunity to severe malaria is the first level of immunity acquired to Plasmodium falciparum. Antibodies to the variant antigen PfEMP1 (P. falciparum erythrocyte membrane protein 1) present at the surface of the parasitized red blood cell (pRBC) confer protection by blocking microvascular sequestration. Here we have generated antibodies to peptide sequences of subdomain 2 of PfEMP1-DBL1α previously identified to be associated with severe or mild malaria. A set of sera generated to the amino acid sequence KLQTLTLHQVREYWWALNRKEVWKA, containing the motif ALNRKE, stained the live pRBC. 50% of parasites tested (7/14) were positive both in flow cytometry and immunofluorescence assays with live pRBCs including both laboratory strains and in vitro adapted clinical isolates. Antibodies that reacted selectively with the sequence REYWWALNRKEVWKA in a 15-mer peptide array of DBL1α-domains were also found to react with the pRBC surface. By utilizing a peptide array to map the binding properties of the elicited anti-DBL1α antibodies, the amino acids WxxNRx were found essential for antibody binding. Complementary experiments using 135 degenerate RDSM peptide sequences obtained from 93 Ugandan patient-isolates showed that antibody binding occurred when the amino acids WxLNRKE/D were present in the peptide. The data suggests that the ALNRKE sequence motif, associated with severe malaria, induces strain-transcending antibodies that react with the pRBC surface. PMID:23335956

  4. Occurrence of Bacillus thuringiensis in fresh waters of Japan.

    PubMed

    Ichimatsu, T; Mizuki, E; Nishimura, K; Akao, T; Saitoh, H; Higuchi, K; Ohba, M

    2000-04-01

    Bacillus thuringiensis was recovered at a relatively high frequency from both running and still fresh waters in natural environments of Kyushu, Japan. Of 107 water samples examined, 53 (49.5%) contained this organism. The frequency of B. thuringiensis colonies was 4.4% among 4414 colonies of the Bacillus cereus/B. thuringiensis group. The density of this bacterium in fresh waters averaged 0.45 cfu/ml. Serologically, B. thuringiensis isolates were assigned to 26 H serotypes. Of these, H14/36 (H serovar israelensis/malaysiensis) was the predominant, followed by the serotypes H3abc (kurstaki), H27 (mexicanensis), H3ad (sumiyoshiensis), and H35 (seoulensis). Of 195 isolates, 52 (26.7%) exhibited larvicidal activity against aquatic Diptera; 21 killed Culex pipiens molestus (Culicidae) only, and 31 were active on both the culicine mosquito and the moth-fly, Clogmia albipunctata (Psychodidae). The Diptera-toxic isolates produced spherical or irregularly pointed parasporal inclusions. PMID:10688688

  5. Reduced artemisinin susceptibility of Plasmodium falciparum ring stages in western Cambodia.

    PubMed

    Witkowski, Benoit; Khim, Nimol; Chim, Pheaktra; Kim, Saorin; Ke, Sopheakvatey; Kloeung, Nimol; Chy, Sophy; Duong, Socheat; Leang, Rithea; Ringwald, Pascal; Dondorp, Arjen M; Tripura, Rupam; Benoit-Vical, Françoise; Berry, Antoine; Gorgette, Olivier; Ariey, Frédéric; Barale, Jean-Christophe; Mercereau-Puijalon, Odile; Menard, Didier

    2013-02-01

    The declining efficacy of artemisinin derivatives against Plasmodium falciparum in western Cambodia is a major concern. The knowledge gap in the understanding of the mechanisms involved hampers designing monitoring tools. Here, we culture-adapted 20 isolates from Pailin and Ratanakiri (areas of artemisinin resistance and susceptibility in western and eastern Cambodia, respectively) and studied their in vitro response to dihydroartemisinin. No significant difference between the two sets of isolates was observed in the classical isotopic test. However, a 6-h pulse exposure to 700 nM dihydroartemisinin (ring-stage survival assay -RSA]) revealed a clear-cut geographic dichotomy. The survival rate of exposed ring-stage parasites (ring stages) was 17-fold higher in isolates from Pailin (median, 13.5%) than in those from Ratanakiri (median, 0.8%), while exposed mature stages were equally and highly susceptible (0.6% and 0.7%, respectively). Ring stages survived drug exposure by cell cycle arrest and resumed growth upon drug withdrawal. The reduced susceptibility to artemisinin in Pailin appears to be associated with an altered in vitro phenotype of ring stages from Pailin in the RSA. PMID:23208708

  6. Host genotype by parasite genotype interactions underlying the resistance of anopheline mosquitoes to Plasmodium falciparum

    PubMed Central

    Lambrechts, Louis; Halbert, Jean; Durand, Patrick; Gouagna, Louis C; Koella, Jacob C

    2005-01-01

    Background Most studies on the resistance of mosquitoes to their malaria parasites focus on the response of a mosquito line or colony against a single parasite genotype. In natural situations, however, it may be expected that mosquito-malaria relationships are based, as are many other host-parasite systems, on host genotype by parasite genotype interactions. In such systems, certain hosts are resistant to one subset of the parasite's genotypes, while other hosts are resistant to a different subset. Methods To test for genotype by genotype interactions between malaria parasites and their anopheline vectors, different genetic backgrounds (families consisting of the F1 offspring of individual females) of the major African vector Anopheles gambiae were challenged with several isolates of the human malaria parasite Plasmodium falciparum (obtained from naturally infected children in Kenya). Results Averaged across all parasites, the proportion of infected mosquitoes and the number of oocysts found in their midguts were similar in all mosquito families. Both indices of resistance, however, differed considerably among isolates of the parasite. In particular, no mosquito family was most resistant to all parasites, and no parasite isolate was most infectious to all mosquitoes. Conclusions These results suggest that the level of mosquito resistance depends on the interaction between its own and the parasite's genotype. This finding thus emphasizes the need to take into account the range of genetic diversity exhibited by mosquito and malaria field populations in ideas and studies concerning the control of malaria. PMID:15644136

  7. Development of a selective agar plate for the detection of Campylobacter spp. in fresh produce.

    PubMed

    Yoo, Jin-Hee; Choi, Na-Young; Bae, Young-Min; Lee, Jung-Su; Lee, Sun-Young

    2014-10-17

    This study was conducted to develop a selective medium for the detection of Campylobacter spp. in fresh produce. Campylobacter spp. (n=4), non-Campylobacter (showing positive results on Campylobacter selective agar) strains (n=49) isolated from fresh produce, indicator bacteria (n=13), and spoilage bacteria isolated from fresh produce (n=15) were plated on four Campylobacter selective media. Bolton agar and modified charcoal cefoperazone deoxycholate agar (mCCDA) exhibited higher sensitivity for Campylobacter spp. than did Preston agar and Hunt agar, although certain non-Campylobacter strains isolated from fresh produce by using a selective agar isolation method, were still able to grow on Bolton agar and mCCDA. To inhibit the growth of non-Campylobacter strains, Bolton agar and mCCDA were supplemented with 5 antibiotics (rifampicin, polymyxin B, sodium metabisulfite, sodium pyruvate, ferrous sulfate) and the growth of Campylobacter spp. (n=7) and non-Campylobacter strains (n=44) was evaluated. Although Bolton agar supplemented with rifampicin (BR agar) exhibited a higher selectivity for Campylobacter spp. than did mCCDA supplemented with antibiotics, certain non-Campylobacter strains were still able to grow on BR agar (18.8%). When BR agar with various concentrations of sulfamethoxazole-trimethoprim were tested with Campylobacter spp. (n=8) and non-Campylobacter (n=7), sulfamethoxazole-trimethoprim was inhibitory against 3 of 7 non-Campylobacter strains. Finally, we validated the use of BR agar containing 50mg/L sulfamethoxazole (BRS agar) or 0.5mg/L ciprofloxacin (BRCS agar) and other selective agars for the detection of Campylobacter spp. in chicken and fresh produce. All chicken samples were positive for Campylobacter spp. when tested on mCCDA, BR agar, and BRS agar. In fresh produce samples, BRS agar exhibited the highest selectivity for Campylobacter spp., demonstrating its suitability for the detection of Campylobacter spp. in fresh produce. PMID:25126968

  8. Genetic diversity of the Plasmodium falciparum apical membrane antigen I gene in parasite population from the China-Myanmar border area.

    PubMed

    Zhu, Xiaotong; Zhao, Zhenjun; Feng, Yonghui; Li, Peipei; Liu, Fei; Liu, Jun; Yang, Zhaoqing; Yan, Guiyun; Fan, Qi; Cao, Yaming; Cui, Liwang

    2016-04-01

    To investigate the genetic diversity of the Plasmodium falciparum apical membrane antigen 1 (PfAMA1) gene in Southeast Asia, we determined PfAMA1 sequences from 135 field isolates collected from the China-Myanmar border area and compared them with 956 publically available PfAMA1 sequences from seven global P. falciparum populations. This analysis revealed high genetic diversity of PfAMA1 in global P. falciparum populations with a total of 229 haplotypes identified. The genetic diversity of PfAMA1 gene from the China-Myanmar border is not evenly distributed in the different domains of this gene. Sequence diversity in PfAMA1 from the China-Myanmar border is lower than that observed in Thai, African and Oceanian populations, but higher than that in the South American population. This appeared to correlate well with the levels of endemicity of different malaria-endemic regions, where hyperendemic regions favor genetic cross of the parasite isolates and generation of higher genetic diversity. Neutrality tests show significant departure from neutrality in the entire ectodomain and Domain I of PfAMA1 in the China-Myanmar border parasite population. We found evidence supporting a substantial continent-wise genetic structure among P. falciparum populations, with the highest genetic differentiation detected between the China-Myanmar border and the South American populations. Whereas no alleles were unique to a specific region, there were considerable geographical differences in major alleles and their frequencies, highlighting further necessity to include more PfAMA1 alleles in vaccine designs. PMID:26825252

  9. Reduced polymorphism in the Kelch propeller domain in Plasmodium vivax isolates from Cambodia.

    PubMed

    Popovici, Jean; Kao, Sokheng; Eal, Leanghor; Bin, Sophalai; Kim, Saorin; Ménard, Didier

    2015-01-01

    Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism. PMID:25385109

  10. Reduced Polymorphism in the Kelch Propeller Domain in Plasmodium vivax Isolates from Cambodia

    PubMed Central

    Popovici, Jean; Kao, Sokheng; Eal, Leanghor; Bin, Sophalai; Kim, Saorin

    2014-01-01

    Polymorphism in the ortholog gene of the Plasmodium falciparum K13 gene was investigated in Plasmodium vivax isolates collected in Cambodia. All of them were Sal-1 wild-type alleles except two (2/284, 0.7%), and P. vivax K12 polymorphism was reduced compared to that of the P. falciparum K13 gene. Both mutant allele isolates had the same nonsynonymous mutation at codon 552 (V552I) and were from Ratanak Kiri province. These preliminary data should encourage additional studies for associating artemisinin or chloroquine resistance and K12 polymorphism. PMID:25385109

  11. Striving for safety in fresh and fresh-cut fruits and vegetables

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Consumption of fresh produce is a central component of a healthy diet. However, contamination of leafy greens, tomatoes, cantaloupes and other fresh and fresh-cut fruits and vegetables with human pathogens is a source of ongoing concern for consumers. Industry and regulators have worked together to ...

  12. DIETARY FIBER CONTENT IN FRESH CITRUS

    Technology Transfer Automated Retrieval System (TEKTRAN)

    There is a wide variation in the reported values for pectin and dietary fiber content in the edible portions of fresh orange and grapefruit. Two studies done by the Produce Marketing Association in 1990 reported 4.0 g dietary fiber/ 100 g of fresh edible grapefruit and 4.4 g dietary fiber / 100 g f...

  13. DISPERSIBILITY OF CRUDE OIL IN FRESH WATER

    EPA Science Inventory

    The effects of surfactant composition on the ability of chemical dispersants to disperse crude oil in fresh water were investigated. The objective of this research was to determine whether effective fresh water dispersants can be designed in case this technology is ever consider...

  14. What determines fresh fish consumption in Croatia?

    PubMed

    Tomić, Marina; Matulić, Daniel; Jelić, Margareta

    2016-11-01

    Although fresh fish is widely available, consumption still remains below the recommended intake levels among the majority of European consumers. The economic crisis affects consumer food behaviour, therefore fresh fish is perceived as healthy but expensive food product. The aim of this study was to determine the factors influencing fresh fish consumption using an expanded Theory of Planned Behaviour (Ajzen, 1991) as a theoretical framework. The survey was conducted on a heterogeneous sample of 1151 Croatian fresh fish consumers. The study investigated the relationship between attitudes, perceived behavioural control, subjective norm, moral obligation, involvement in health, availability, intention and consumption of fresh fish. Structural Equation Modeling by Partial Least Squares was used to analyse the collected data. The results indicated that attitudes are the strongest positive predictor of the intention to consume fresh fish. Other significant predictors of the intention to consume fresh fish were perceived behavioural control, subjective norm, health involvement and moral obligation. The intention to consume fresh fish showed a strong positive correlation with behaviour. This survey provides valuable information for food marketing professionals and for the food industry in general. PMID:26721719

  15. Microbial Safety of Fresh Produce - Preface

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fresh produce has been the source of recent outbreaks of foodborne illness which have caused sickness, hospitalizations and deaths of consumers, as well as serious adverse economic impact on growers and processors. The preface for the book entitled “Microbial Safety of Fresh Produce” discusses possi...

  16. Plasmodium falciparum malaria: Convergent evolutionary trajectories towards delayed clearance following artemisinin treatment.

    PubMed

    Wilairat, Prapon; Kümpornsin, Krittikorn; Chookajorn, Thanat

    2016-05-01

    Malaria is a major global health challenge with 300million new cases every year. The most effective regimen for treating Plasmodium falciparum malaria is based on artemisinin and its derivatives. The drugs are highly effective, resulting in rapid clearance of parasites even in severe P. falciparum malaria patients. During the last five years, artemisinin-resistant parasites have begun to emerge first in Cambodia and now in Thailand and Myanmar. At present, the level of artemisinin resistance is relatively low with clinical presentation of delayed artemisinin clearance (a longer time to reduce parasite load) and a small decrease in artemisinin sensitivity in cultured isolates. Nevertheless, multiple genetic loci associated with delayed parasite clearance have been reported, but they cannot account for a large portion of cases. Even the most well-studied kelch 13 propeller mutations cannot always predict the outcome of artemisinin treatment in vitro and in vivo. Here we propose that delayed clearance by artemisinin could be the result of convergent evolution, driven by multiple trajectories to overcome artemisinin-induced stress, but precluded to become full blown resistance by high fitness cost. Genetic association studies by several genome-wide approaches reveal linkage disequilibrium between multiple loci and delayed parasite clearance. Genetic manipulations at some of these loci already have resulted in loss in artemisinin sensitivity. The notion presented here is by itself consistent with existing evidence on artemisinin resistance and has the potential to be explored using available genomic data. Most important of all, molecular surveillance of artemisinin resistance based on multi-genic markers could be more informative than relying on any one particular molecular marker. PMID:27063079

  17. Targeting the cell stress response of Plasmodium falciparum to overcome artemisinin resistance.

    PubMed

    Dogovski, Con; Xie, Stanley C; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A; Simpson, Julie A; Dondorp, Arjen M; Foote, Simon; Klonis, Nectarios; Tilley, Leann

    2015-04-01

    Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART

  18. Targeting the Cell Stress Response of Plasmodium falciparum to Overcome Artemisinin Resistance

    PubMed Central

    Dogovski, Con; Xie, Stanley C.; Burgio, Gaetan; Bridgford, Jess; Mok, Sachel; McCaw, James M.; Chotivanich, Kesinee; Kenny, Shannon; Gnädig, Nina; Straimer, Judith; Bozdech, Zbynek; Fidock, David A.; Simpson, Julie A.; Dondorp, Arjen M.; Foote, Simon; Klonis, Nectarios; Tilley, Leann

    2015-01-01

    Successful control of falciparum malaria depends greatly on treatment with artemisinin combination therapies. Thus, reports that resistance to artemisinins (ARTs) has emerged, and that the prevalence of this resistance is increasing, are alarming. ART resistance has recently been linked to mutations in the K13 propeller protein. We undertook a detailed kinetic analysis of the drug responses of K13 wild-type and mutant isolates of Plasmodium falciparum sourced from a region in Cambodia (Pailin). We demonstrate that ART treatment induces growth retardation and an accumulation of ubiquitinated proteins, indicative of a cellular stress response that engages the ubiquitin/proteasome system. We show that resistant parasites exhibit lower levels of ubiquitinated proteins and delayed onset of cell death, indicating an enhanced cell stress response. We found that the stress response can be targeted by inhibiting the proteasome. Accordingly, clinically used proteasome inhibitors strongly synergize ART activity against both sensitive and resistant parasites, including isogenic lines expressing mutant or wild-type K13. Synergy is also observed against Plasmodium berghei in vivo. We developed a detailed model of parasite responses that enables us to infer, for the first time, in vivo parasite clearance profiles from in vitro assessments of ART sensitivity. We provide evidence that the clinical marker of resistance (delayed parasite clearance) is an indirect measure of drug efficacy because of the persistence of unviable parasites with unchanged morphology in the circulation, and we suggest alternative approaches for the direct measurement of viability. Our model predicts that extending current three-day ART treatment courses to four days, or splitting the doses, will efficiently clear resistant parasite infections. This work provides a rationale for improving the detection of ART resistance in the field and for treatment strategies that can be employed in areas with ART

  19. Artemisinin resistance in Plasmodium falciparum is associated with an altered temporal pattern of transcription

    PubMed Central

    2011-01-01

    Background Artemisinin resistance in Plasmodium falciparum malaria has emerged in Western Cambodia. This is a major threat to global plans to control and eliminate malaria as the artemisinins are a key component of antimalarial treatment throughout the world. To identify key features associated with the delayed parasite clearance phenotype, we employed DNA microarrays to profile the physiological gene expression pattern of the resistant isolates. Results In the ring and trophozoite stages, we observed reduced expression of many basic metabolic and cellular pathways which suggests a slower growth and maturation of these parasites during the first half of the asexual intraerythrocytic developmental cycle (IDC). In the schizont stage, there is an increased expression of essentially all functionalities associated with protein metabolism which indicates the prolonged and thus increased capacity of protein synthesis during the second half of the resistant parasite IDC. This modulation of the P. falciparum intraerythrocytic transcriptome may result from differential expression of regulatory proteins such as transcription factors or chromatin remodeling associated proteins. In addition, there is a unique and uniform copy number variation pattern in the Cambodian parasites which may represent an underlying genetic background that contributes to the resistance phenotype. Conclusions The decreased metabolic activities in the ring stages are consistent with previous suggestions of higher resilience of the early developmental stages to artemisinin. Moreover, the increased capacity of protein synthesis and protein turnover in the schizont stage may contribute to artemisinin resistance by counteracting the protein damage caused by the oxidative stress and/or protein alkylation effect of this drug. This study reports the first global transcriptional survey of artemisinin resistant parasites and provides insight to the complexities of the molecular basis of pathogens with drug

  20. Natural selection of K13 mutants of Plasmodium falciparum in response to artemisinin combination therapies in Thailand.

    PubMed

    Putaporntip, C; Kuamsab, N; Kosuwin, R; Tantiwattanasub, W; Vejakama, P; Sueblinvong, T; Seethamchai, S; Jongwutiwes, S; Hughes, A L

    2016-03-01

    Resistance of Plasmodium falciparum to artemisinin combination therapy (ACT) in Southeast Asia can have a devastating impact on chemotherapy and control measures. In this study, the evolution of artemisinin-resistant P. falciparum in Thailand was assessed by exploring mutations in the K13 locus believed to confer drug resistance phenotype. P. falciparum-infected blood samples were obtained from patients in eight provinces of Thailand over two decades (1991-2014; n = 904). Analysis of the K13 gene was performed by either sequencing the complete coding region (n = 259) or mutation-specific PCR-restriction fragment length polymorphism method (n = 645). K13 mutations related to artesunate resistance were detected in isolates from Trat province bordering Cambodia in 1991, about 4 years preceding widespread deployment of ACT in Thailand and increased in frequency over time. Nonsynonymous nucleotide diversity exceeded synonymous nucleotide diversity in the propeller region of the K13 gene, supporting the hypothesis that this diversity was driven by natural selection. No single mutant appeared to be favoured in every population, and propeller-region mutants were rarely observed in linkage with each other in the same haplotype. On the other hand, there was a highly significant association between the occurrence of a propeller mutant and the insertion of two or three asparagines after residue 139 of K13. Whether this insertion plays a compensatory role for deleterious effects of propeller mutants on the function of the K13 protein requires further investigation. However, modification of duration of ACT from 2-day to 3-day regimens in 2008 throughout the country does not halt the increase in frequency of mutants conferring artemisinin resistance phenotype. PMID:26548510

  1. Genetic variability and population structure of Plasmodium falciparum parasite populations from different malaria ecological regions of Kenya.

    PubMed

    Ingasia, Luicer A; Cheruiyot, Jelagat; Okoth, Sheila Akinyi; Andagalu, Ben; Kamau, Edwin

    2016-04-01

    Transmission intensity, movement of human and vector hosts, biogeographical features, and malaria control measures are some of the important factors that determine Plasmodium falciparum parasite genetic variability and population structure. Kenya has different malaria ecologies which might require different disease intervention methods. Refined parasite population genetic studies are critical for informing malaria control and elimination strategies. This study describes the genetic diversity and population structure of P. falciparum parasites from the different malaria ecological zones in Kenya. Twelve multi-locus microsatellite (MS) loci previously described were genotyped in 225 P. falciparum isolates collected between 2012 and 2013 from five sites; three in lowland endemic regions (Kisumu, Kombewa, and Malindi) and two in highland, epidemic regions (Kisii and Kericho). Parasites from the lowland endemic and highland epidemic regions of western Kenya had high genetic diversity compared to coastal lowland endemic region of Kenya [Malindi]. The Kenyan parasites had a mean genetic differentiation index (FST) of 0.072 (p=0.011). The multi-locus genetic analysis of the 12 MS revealed all the parasites had unique haplotypes. Significant linkage disequilibrium (LD) was observed in all the five parasite populations. Kisumu had the most significant index of association values (0.16; p<0.0001) whereas Kisii had the least significant index of association values (0.03; p<0.0001). Our data suggest high genetic diversity in Kenyan parasite population with the exception of parasite from Malindi where malaria has been on the decline. The presence of significant LD suggests that there is occurrence of inbreeding in the parasite population. Parasite populations from Kisii showed the strongest evidence for epidemic population structure whereas the rest of the regions showed panmixia. Defining the genetic diversity of the parasites in different ecological regions of Kenya after

  2. An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand.

    PubMed

    Imwong, Mallika; Jindakhad, Thantip; Kunasol, Chanon; Sutawong, Kreepol; Vejakama, Phisitt; Dondorp, Arjen M

    2015-01-01

    Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak. PMID:26616851

  3. How specific is Plasmodium falciparum adherence to chondroitin 4-sulfate?

    PubMed Central

    Goel, Suchi; Gowda, D. Channe

    2011-01-01

    Plasmodium falciparum infection during pregnancy results in the sequestration of infected red blood cells (IRBCs) in the placenta, contributing to pregnancy associated malaria (PAM). IRBC adherence is mediated by the binding of a variant Plasmodium falciparum erythrocyte binding protein 1 named VAR2CSA to the low sulfated chondroitin 4-sulfate (C4S) proteoglycan (CSPG) present predominantly in the intervillous space of the placenta. IRBC binding is highly specific to the level and distribution of 4-sulfate groups in C4S. Given the strict specificity of IRBC-C4S interactions, it is better to use either placental CSPG or CSPGs bearing structurally similar C4S chains in defining VAR2CSA structural architecture that interact with C4S, evaluating VAR2CSA constructs for vaccine development or studying structure-based inhibitors as therapeutics for PAM. PMID:21507719

  4. Symmetrical peripheral gangrene due to Plasmodium falciparum malaria

    PubMed Central

    Abdali, Nasar; Malik, Azharuddin Mohammed; Kamal, Athar; Ahmad, Mehtab

    2014-01-01

    A 45-year-old man presented with a 4-day history of high-grade fever with rigours and a 2-day history of painful bluish black discolouration of extremities (acrocyanosis). He was haemodynamically stable and all peripheral pulses palpable, but the extremities were cold with gangrene involving bilateral fingers and toes. Mild splenomegaly was present on abdominal examination but rest of the physical examinations were normal. On investigating he was found to have anaemia, thrombocytopaenia with gametocytes of Plasmodium falciparum on peripheral blood smear. His blood was uncoagulable during performance of prothrombin time with a raised D-dimer. Oxygen saturation was normal and the arterial Doppler test showed reduced blood flow to the extremities. A diagnosis of complicated P. falciparum malaria with disseminated intravascular coagulation (DIC) leading to symmetrical peripheral gangrene was performed. Artemisinin combination therapy was started and heparin was given for DIC. A final line of demarcation of gangrene started forming by 12th day. PMID:24862424

  5. Erythrocyte invasion receptors for Plasmodium falciparum: new and old.

    PubMed

    Satchwell, T J

    2016-04-01

    Understanding the complex process by which the invasive form of the Plasmodium falciparum parasite, the merozoite, attaches to and invades erythrocytes as part of its blood stage life cycle represents a key area of research in the battle to combat malaria. Central to this are efforts to determine the identity of receptors on the host cell surface, their corresponding merozoite-binding proteins and the functional relevance of these binding events as part of the invasion process. This review will provide an updated summary of studies identifying receptor interactions essential for or implicated in P. falciparum merozoite invasion of human erythrocytes, highlighting the recent identification of new receptors using groundbreaking high throughput approaches and with particular focus on the properties and putative involvement of the erythrocyte proteins targeted by these invasion pathways. PMID:26862042

  6. An outbreak of artemisinin resistant falciparum malaria in Eastern Thailand

    PubMed Central

    Imwong, Mallika; Jindakhad, Thantip; Kunasol, Chanon; Sutawong, Kreepol; Vejakama, Phisitt; Dondorp, Arjen M.

    2015-01-01

    Artemisinin resistant falciparum malaria is an increasing problem in Southeast Asia, but has not been associated with increased transmission of the disease, yet. During a recent outbreak in 2014 in Ubon Ratchatani, Eastern Thailand, parasites from 101 patients with falciparum malaria were genotyped for antimalarial drug resistance markers. Mutations in the Kelch13 marker for artemisinin resistance were present in 93% of samples, mainly C580Y from 2 major clusters as identified by microsatellite typing. Resistance markers for antifolates and chloroquine were also highly prevalent. Most strains (91%) carried single copy number PfMDR1, suggesting sustained sensitivity to mefloquine, the partner drug in the local first-line artemisinin combination therapy (ACT). The high prevalence of artemisinin resistance in this recent malaria outbreak suggests but does not prove a causative role in increased transmission. Careful monitoring of ACT efficacy and additional genetic epidemiological studies are warranted to guide the public health response to the outbreak. PMID:26616851

  7. Studies on serum requirements for the cultivation of Plasmodium falciparum

    PubMed Central

    Divo, A. A.; Jensen, J. B.

    1982-01-01

    Previous experiments using RPMI 1640 medium have indicated that the dialysis of human serum removes components of low relative molecular mass (6000-8000 RMM) that are essential for continuous cultivation of Plasmodium falciparum. To determine which low-RMM components are important for parasite development, we compared growth in normal serum to that in dialysed serum using a number of other commercially available media, which we considered to be richer than RPMI 1640. Through these comparisons, we determined that hypoxanthine was the major dialysable nutrient required for parasite development. High quality bovine serum requires 3 - 12 × 10-5 mol/litre of hypoxanthine as a supplement to support continuous cultures of P. falciparum. Thus far we have been unable to attain parasite growth in medium containing supplemented bovine serum that is as good as growth in medium containing human serum. PMID:6754122

  8. Squalestatin Is an Inhibitor of Carotenoid Biosynthesis in Plasmodium falciparum

    PubMed Central

    Gabriel, Heloisa B.; Silva, Marcia F.; Kimura, Emília A.; Wunderlich, Gerhard

    2015-01-01

    The increasing resistance of malaria parasites to almost all available drugs calls for the characterization of novel targets and the identification of new compounds. Carotenoids are polyisoprenoids from plants, algae, and some bacteria, and they are biosynthesized by Plasmodium falciparum but not by mammalian cells. Biochemical and reverse genetics approaches were applied to demonstrate that phytoene synthase (PSY) is a key enzyme for carotenoid biosynthesis in P. falciparum and is essential for intraerythrocytic growth. The known PSY inhibitor squalestatin reduces biosynthesis of phytoene and kills parasites during the intraerythrocytic cycle. PSY-overexpressing parasites showed increased biosynthesis of phytoene and its derived product phytofluene and presented a squalestatin-resistant phenotype, suggesting that this enzyme is the primary target of action of this drug in the parasite. PMID:25779575

  9. Modeling Combinations of Pre-erythrocytic Plasmodium falciparum Malaria Vaccines.

    PubMed

    Walker, Andrew S; Lourenço, José; Hill, Adrian V S; Gupta, Sunetra

    2015-12-01

    Despite substantial progress in the control of Plasmodium falciparum infection due to the widespread deployment of insecticide-treated bed nets and artemisinin combination therapies, malaria remains a prolific killer, with over half a million deaths estimated to have occurred in 2013 alone. Recent evidence of the development of resistance to treatments in both parasites and their mosquito vectors has underscored the need for a vaccine. Here, we use a mathematical model of the within-host dynamics of P. falciparum infection, fit to data from controlled human malaria infection clinical trials, to predict the efficacy of co-administering the two most promising subunit vaccines, RTS,S/AS01 and ChAd63-MVA ME-TRAP. We conclude that currently available technologies could be combined to induce very high levels of sterile efficacy, even in immune-naive individuals. PMID:26503278

  10. Genotype comparison of Plasmodium vivax and Plasmodium falciparum clones from pregnant and non-pregnant populations in North-west Colombia

    PubMed Central

    2012-01-01

    Background Placental malaria is the predominant pathology secondary to malaria in pregnancy, causing substantial maternal and infant morbidity and mortality in tropical areas. While it is clear that placental parasites are phenotypically different from those in the peripheral circulation, it is not known whether unique genotypes are associated specifically with placental infection or perhaps more generally with pregnancy. In this study, genetic analysis was performed on Plasmodium vivax and Plasmodium falciparum parasites isolated from peripheral and placental blood in pregnant women living in North-west Colombia, and compared with parasites causing acute malaria in non-pregnant populations. Methods A total of 57 pregnant women at delivery with malaria infection confirmed by real-time PCR in peripheral or placental blood were included, as well as 50 pregnant women in antenatal care and 80 men or non-pregnant women with acute malaria confirmed by a positive thick smear for P. vivax or P. falciparum. Five molecular markers per species were genotyped by nested PCR and capillary electrophoresis. Genetic diversity and the fixation index FST per species and study group were calculated and compared. Results Almost all infections at delivery were asymptomatic with significantly lower levels of infection compared with the groups with acute malaria. Expected heterozygosity for P. vivax molecular markers ranged from 0.765 to 0.928 and for P. falciparum markers ranged from 0.331 to 0.604. For P. vivax infections, the genetic diversity was similar amongst the four study groups and the fixation index from each pairwise comparison failed to show significant genetic differentiation. For P. falciparum, no genetic differentiation was observed between placental and peripheral parasites from the same woman at delivery, but the parasites isolated at delivery showed significant genetic differentiation compared with parasites isolated from subjects with acute malaria. Conclusions In

  11. Heterologous Protection against Malaria after Immunization with Plasmodium falciparum Sporozoites

    PubMed Central

    van Gemert, Geert-Jan; Graumans, Wouter; van de Vegte-Bolmer, Marga; van Lieshout, Lisette; Haks, Mariëlle C.; Hermsen, Cornelus C.; Scholzen, Anja; Visser, Leo G.; Sauerwein, Robert W.

    2015-01-01

    Background Sterile protection in >90% of volunteers against homologous Plasmodium falciparum infection has been achieved only using the controlled human malaria infection (CHMI) model. This efficient model involves whole parasite immunizations under chloroquine prophylaxis (CPS-immunization), requiring only 30–45 mosquitoes bites infected with P. falciparum-sporozoites. Given the large diversity of P. falciparum parasites, it is essential to assess protection against heterologous parasite strains. Methods In an open-label follow-up study, 16 volunteers previously CPS-immunized and challenged with P. falciparum NF54 (West-Africa) in a dose de-escalation and challenge trial were re-challenged with clone NF135.C10 (Cambodia) at 14 months after the last immunization (NCT01660854). Results Two out of thirteen NF54 protected volunteers previously fully protected against NF54 were also fully protected against NF135.C10, while 11/13 showed a delayed patency (median prepatent period of 10.5 days (range 9.0–15.5) versus 8.5 days in 5 malaria-naïve controls (p = 0.0005). Analysis of patency by qPCR indicated a 91 to >99% estimated reduction of liver parasite load in 7/11 partially protected subjects. Three volunteers previously not protected against NF54, were also not protected against NF135.C10. Conclusion This study shows that CPS-immunization can induce heterologous protection for a period of more than one year, which is a further impetus for clinical development of whole parasite vaccines. Trial Registration Clinicaltrials.gov NCT01660854 PMID:25933168

  12. The Fragmented Mitochondrial Ribosomal RNAs of Plasmodium falciparum

    PubMed Central

    Feagin, Jean E.; Harrell, Maria Isabel; Lee, Jung C.; Coe, Kevin J.; Sands, Bryan H.; Cannone, Jamie J.; Tami, Germaine; Schnare, Murray N.; Gutell, Robin R.

    2012-01-01

    Background The mitochondrial genome in the human malaria parasite Plasmodium falciparum is most unusual. Over half the genome is composed of the genes for three classic mitochondrial proteins: cytochrome oxidase subunits I and III and apocytochrome b. The remainder encodes numerous small RNAs, ranging in size from 23 to 190 nt. Previous analysis revealed that some of these transcripts have significant sequence identity with highly conserved regions of large and small subunit rRNAs, and can form the expected secondary structures. However, these rRNA fragments are not encoded in linear order; instead, they are intermixed with one another and the protein coding genes, and are coded on both strands of the genome. This unorthodox arrangement hindered the identification of transcripts corresponding to other regions of rRNA that are highly conserved and/or are known to participate directly in protein synthesis. Principal Findings The identification of 14 additional small mitochondrial transcripts from P. falcipaurm and the assignment of 27 small RNAs (12 SSU RNAs totaling 804 nt, 15 LSU RNAs totaling 1233 nt) to specific regions of rRNA are supported by multiple lines of evidence. The regions now represented are highly similar to those of the small but contiguous mitochondrial rRNAs of Caenorhabditis elegans. The P. falciparum rRNA fragments cluster on the interfaces of the two ribosomal subunits in the three-dimensional structure of the ribosome. Significance All of the rRNA fragments are now presumed to have been identified with experimental methods, and nearly all of these have been mapped onto the SSU and LSU rRNAs. Conversely, all regions of the rRNAs that are known to be directly associated with protein synthesis have been identified in the P. falciparum mitochondrial genome and RNA transcripts. The fragmentation of the rRNA in the P. falciparum mitochondrion is the most extreme example of any rRNA fragmentation discovered. PMID:22761677

  13. Correlation between 'H' blood group antigen and Plasmodium falciparum invasion.

    PubMed

    Pathak, Vrushali; Colah, Roshan; Ghosh, Kanjaksha

    2016-06-01

    The ABO blood group system is the most important blood group system in clinical practice. The relationship between Plasmodium falciparum and ABO blood groups has been studied for many years. This study was undertaken to investigate the abilities of different blood group erythrocytes to support in vitro growth of P. falciparum parasites. P. falciparum parasites of four different strains (3D7, 7G8, Dd2 and RKL9) were co-cultured with erythrocytes of blood group 'A', 'B', 'O' (n = 10 for each) and 'O(h)' (Bombay group) (n = 7) for 5 days. Statistically significant differences were observed on the fourth day among the mean percent parasitemias of 'O', non-'O' ('A' and 'B') and 'O(h)' group cultures. The parasitemias of four strains ranged from 12.23 to 14.66, 11.68 to 13.24, 16.89 to 22.3, and 7.37 to 11.27 % in 'A', 'B', 'O' and Bombay group cultures, respectively. As the expression of H antigen decreased from 'O' blood group to 'A' and 'B' and then to Bombay blood group, parasite invasion (percent parasitemia) also decreased significantly (p < 0.01) and concomitantly, indicating the association of parasite invasion with the amount of H antigen present on the surface of erythrocyte. Thus, the question arises, could H antigen be involved in P. falciparum invasion? To evaluate erythrocyte invasion inhibition, 'O' group erythrocytes were virtually converted to Bombay group-like erythrocytes by the treatment of anti-H lectins extracted from Ulex europaeus seeds. Mean percent parasitemia of lectin-treated cultures on the fourth day was significantly lower (p < 0.05) than that of non-treated cultures and was found to be similar with the mean percent parasitemia demonstrated by the Bombay group erythrocyte cultures, thus further strengthening the hypothesis. PMID:27071756

  14. Structure of Plasmodium falciparum ADP-ribosylation factor 1

    SciTech Connect

    Cook, William J.; Smith, Craig D.; Senkovich, Olga; Holder, Anthony A.; Chattopadhyay, Debasish

    2011-09-26

    Vesicular trafficking may play a crucial role in the pathogenesis and survival of the malaria parasite. ADP-ribosylation factors (ARFs) are among the major components of vesicular trafficking pathways in eukaryotes. The crystal structure of ARF1 GTPase from Plasmodium falciparum has been determined in the GDP-bound conformation at 2.5 {angstrom} resolution and is compared with the structures of mammalian ARF1s.

  15. Population structure and recent evolution of Plasmodium falciparum

    PubMed Central

    Rich, Stephen M.; Ayala, Francisco J.

    2000-01-01

    Plasmodium falciparum is the agent of malignant malaria, one of mankind's most severe maladies. The parasite exhibits antigenic polymorphisms that have been postulated to be ancient. We have proposed that the extant world populations of P. falciparum have derived from one single parasite, a cenancestor, within the last 5,000–50,000 years. This inference derives from the virtual or complete absence of synonymous nucleotide polymorphisms at genes not involved in immune or drug responses. Seeking to conciliate this claim with extensive antigenic polymorphism, we first note that allele substitutions or polymorphisms can arise very rapidly, even in a single generation, in large populations subject to strong natural selection. Second, new alleles can arise not only by single-nucleotide mutations, but also by duplication/deletion of short simple-repeat DNA sequences, a process several orders of magnitude faster than single-nucleotide mutation. We analyze three antigenic genes known to be extremely polymorphic: Csp, Msp-1, and Msp-2. We identify regions consisting of tandem or proximally repetitive short DNA sequences, including some previously unnoticed. We conclude that the antigenic polymorphisms are consistent with the recent origin of the world populations of P. falciparum inferred from the analysis of nonantigenic genes. PMID:10860962

  16. Intrarectal quinine for treating Plasmodium falciparum malaria: a systematic review

    PubMed Central

    Eisenhut, Michael; Omari, Aika; MacLehose, Harriet G

    2005-01-01

    Background In children with malaria caused by Plasmodium falciparum, quinine administered rectally may be easier to use and less painful than intramuscular or intravenous administration. The objective of this review was to compare the effectiveness of intrarectal with intravenous or intramuscular quinine for treating falciparum malaria. Methods All randomized and quasi-randomized controlled trials comparing intrarectal with intramuscular or intravenous quinine for treating people with falciparum malaria located through the following sources were included: Cochrane Infectious Diseases Group Specialized Register, CENTRAL, MEDLINE, EMBASE, LILACS and CINAHL. Trial quality was assessed and data, including adverse event data, were extracted. Dichotomous data were analysed using odds ratios and continuous data using weighted mean difference. Results Eight randomized controlled trials (1,247 children) fulfilled the inclusion criteria. The same principal investigator led seven of the trials. Five compared intrarectal with intravenous quinine, and six compared intrarectal with intramuscular treatment. No statistically significant difference was detected for death, parasite clearance by 48 hours and seven days, parasite and fever clearance time, coma recovery time, duration of hospitalization and time before drinking began. One trial (898 children) reported that intrarectal was less painful than intramuscular administration. Conclusion No difference in the effect on parasites and clinical illness was detected for the use of intrarectal quinine compared with other routes, but most trials were small. Pain during application may be less with intrarectal quinine. Further larger trials, in patients with severe malaria and in adults, are required before the intrarectal route could be recommended. PMID:15904520

  17. Monkey-derived monoclonal antibodies against Plasmodium falciparum

    SciTech Connect

    Stanley, H.A.; Reese, R.T.

    1985-09-01

    A system has been developed that allows efficient production of monkey monoclonal antibodies from owl monkeys. Splenocytes or peripheral blood lymphocytes from monkeys immune to the human malarial parasite, Plasmodium falciparum, were fused with P3X63 Ag8.653 mouse myelomas. The resulting hybridomas were screened by an indirect fluorescent antibody test for the production of monkey monoclonal antibodies (mAb) reactive with P. falciparum. Most of the mAb reacted with the P. falciparum merozoites and immunoprecipitated a parasite-derived glycoprotein having a relative molecular weight of 185,000. These mAb gave a minimum of five different immunoprecipitation patterns, thus demonstrating that a large number of polypeptides obtained when parasitized erythrocytes are solubilized share epitopes with this large glycoprotein. In addition, mAb were obtained that reacted with antigens associated with the infected erythrocyte membrane. One of these mAb bound a M/sub r/ 95,000 antigen. Radioimmunoprecipitation assays using /sup 125/T-antibodies were done.

  18. Outbreak of betanodavirus infection in tilapia, Oreochromis niloticus (L.), in fresh water.

    PubMed

    Bigarré, L; Cabon, J; Baud, M; Heimann, M; Body, A; Lieffrig, F; Castric, J

    2009-08-01

    A betanodavirus associated with a massive mortality was isolated from larvae of tilapia, Oreochromis niloticus, maintained in fresh water at 30 degrees C. Histopathology revealed vacuolation of the nervous system, suggesting an infection by a betanodavirus. The virus was identified by indirect fluorescent antibody test in the SSN1 cell line and further characterized by sequencing of a PCR product. Sequencing of the T4 region of the coat protein gene indicated a phylogenetic clustering of this isolate within the red-spotted grouper nervous necrosis virus type. However, the tilapia isolate formed a unique branch distinct from other betanodavirus isolates. The disease was experimentally reproduced by bath infection of young tilapia at 30 degrees C. The reservoir of virus at the origin of the outbreak remains unidentified. To our knowledge, this is the first report of natural nodavirus infection in tilapia reared in fresh water. PMID:19500206

  19. HS-GC-MS volatile compounds recovered in freshly pressed 'Wonderful' cultivar and commercial pomegranate juices.

    PubMed

    Beaulieu, John C; Stein-Chisholm, R E

    2016-01-01

    Consumption and production of superfruits has been increasing. Highly colored fruits often have bitter and astringent components that may make them undesirable, especially when processed. Many pomegranate volatile reports involved commercial samples, complicated isolation methods, or blending and concentrating that were from arils only, cultivars other than 'Wonderful', or mixed cultivars. Solid phase microextraction (SPME), polydimethylsiloxane stir bar sorptive extraction, and Tenax adsorption were performed with freshly pressed 'Wonderful' juices, commercial juices and concentrates. Using SPME, 36 compounds were isolated in whole pressed 'Wonderful' juices, including 18 of the 21 consensus compounds. In arils-only juices, 41 compounds were isolated by SPME, including 17 of the consensus volatiles. Dramatic variation existed in volatiles recovered in commercial juices and isolation of consensus compounds was sporadic. This article and summary of the literature serves to possibly deliver an improved volatile data set via a rapid method for fresh and partially processed (pressed) pomegranates. PMID:26213022

  20. Numbers of fecal streptococci and Escherichia coli in fresh and dry cattle, horse, and sheep manure.

    PubMed

    Weaver, R W; Entry, J A; Graves, Alexandria

    2005-10-01

    Livestock are known contributors to stream pollution. Numbers of fecal streptococci and Escherichia coli in manure naturally deposited by livestock in the field are needed for activities related to bacterial source tracking and determining maximum daily bacterial loading of streams. We measured populations of fecal streptococci and E. coli in fresh and dry manure from cattle (Bos taurus L.), horses (Equus caballus L.), and sheep (Ovis aires L.) on farms in southern Idaho. Populations of indicator bacteria in dry manure were often as high as that in fresh manure from horse and sheep. There was a 2 log10 drop in the population of fecal coliform numbers in dry cattle manure from cattle in pastures but not from cattle in pens. Bacterial isolates used in source tracking should include isolates from both fresh and dry manure to better represent the bacterial source loading of streams. PMID:16333344

  1. Prospective Genotyping of Mycobacterium tuberculosis from Fresh Clinical Samples

    PubMed Central

    Bidovec-Stojkovič, Urška; Seme, Katja; Žolnir-Dovč, Manca; Supply, Philip

    2014-01-01

    Shorter time-to-result is key for improving molecular-guided epidemiological investigation of tuberculosis (TB) cases. We performed a prospective study to evaluate the use of standardized MIRU-VNTR (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat) typing of Mycobacterium tuberculosis directly on 79 fresh clinical samples from 26 TB patients consecutively enrolled over a 17-month period. Overall, complete 24-locus types were obtained for 18 out of the 26 (69.2%) patients and 14 of the 16 grade 3+ and grade 2+ samples (87.5%). The degree of completion of the genotypes obtained significantly correlated with smear microscopy grade both for 26 first samples (p = 0.0003) and for 53 follow-up samples (p = 0.002). For 20 of the 26 patients for whom complete or even incomplete M. tuberculosis isolate genotypes were obtained, typing applied to the clinical samples allowed the same unambiguous conclusions regarding case clustering or uniqueness as those that could have been drawn based on the corresponding cultured isolates. Standard 24 locus MIRU-VNTR typing of M. tuberculosis can be applied directly to fresh clinical samples, with typeability depending on the bacterial load in the sample. PMID:25313883

  2. Primary structure of the 175K Plasmodium falciparum erythrocyte binding antigen and identification of a peptide which elicits antibodies that inhibit malaria merozoite invasion.

    PubMed

    Sim, B K; Orlandi, P A; Haynes, J D; Klotz, F W; Carter, J M; Camus, D; Zegans, M E; Chulay, J D

    1990-11-01

    The Plasmodium falciparum gene encoding erythrocyte binding antigen-175 (EBA-175), a putative receptor for red cell invasion (Camus, D., and T. J. Hadley. 1985. Science (Wash. DC). 230:553-556.), has been isolated and characterized. DNA sequencing demonstrated a single open reading frame encoding a translation product of 1,435 amino acid residues. Peptides corresponding to regions on the deduced amino acid sequence predicted to be B cell epitopes were assessed for immunogenicity. Immunization of mice and rabbits with EBA-peptide 4, a synthetic peptide encompassing amino acid residues 1,062-1,103, produced antibodies that recognized P. falciparum merozoites in an indirect fluorescent antibody assay. When compared to sera from rabbits immunized with the same adjuvant and carrier protein, sera from rabbits immunized with EBA-peptide 4 inhibited merozoite invasion of erythrocytes in vitro by 80% at a 1:5 dilution. Furthermore, these sera inhibited the binding of purified, authentic EBA-175 to erythrocytes, suggesting that their activity in inhibiting merozoite invasion of erythrocytes is mediated by blocking the binding of EBA-175 to erythrocytes. Since the nucleotide sequence of EBA-peptide 4 is conserved among seven strains of P. falciparum from throughout the world (Sim, B. K. L. 1990. Mol. Biochem. Parasitol. 41:293-296.), these data identify a region of the protein that should be a focus of vaccine development efforts. PMID:2229177

  3. Clonal Variants of Plasmodium falciparum Exhibit a Narrow Range of Rolling Velocities to Host Receptor CD36 under Dynamic Flow Conditions

    PubMed Central

    Herricks, Thurston; Avril, Marion; Janes, Joel; Smith, Joseph D.

    2013-01-01

    Cytoadhesion of Plasmodium falciparum parasitized red blood cells (pRBCs) has been implicated in the virulence of malaria infection. Cytoadhesive interactions are mediated by the protein family of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1). The PfEMP1 family is under strong antibody and binding selection, resulting in extensive sequence and size variation of the extracellular domains. Here, we investigated cytoadhesion of pRBCs to CD36, a common receptor of P. falciparum field isolates, under dynamic flow conditions. Isogeneic parasites, predominantly expressing single PfEMP1 variants, were evaluated for binding to recombinant CD36 under dynamic flow conditions using microfluidic devices. We tested if PfEMP1 size (number of extracellular domains) or sequence variation affected the pRBC-CD36 interaction. Our analysis showed that clonal parasite variants varied ∼5-fold in CD36 rolling velocity despite extensive PfEMP1 sequence polymorphism. In addition, adherent pRBCs exhibited a characteristic hysteresis in rolling velocity at microvascular flow rates, which was accompanied by changes in pRBC shape and may represent important adaptations that favor stable binding. PMID:24014767

  4. Native microflora in fresh-cut processing plants and their potentials of biofilm formation

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Representative food contact and non-food contact surfaces in two mid-sized fresh cut processing facilities were sampled for microbiological analyses post routine daily sanitization. Mesophilic and psychrotrophic bacteria on the sampled surfaces were isolated by plating on non-selective bacterial med...

  5. Caspar Controls Resistance to Plasmodium falciparum in Diverse Anopheline Species

    PubMed Central

    Garver, Lindsey S.; Dong, Yuemei; Dimopoulos, George

    2009-01-01

    Immune responses mounted by the malaria vector Anopheles gambiae are largely regulated by the Toll and Imd (immune deficiency) pathways via the NF-kappaB transcription factors Rel1 and Rel2, which are controlled by the negative regulators Cactus and Caspar, respectively. Rel1- and Rel2-dependent transcription in A. gambiae has been shown to be particularly critical to the mosquito's ability to manage infection with the rodent malaria parasite Plasmodium berghei. Using RNA interference to deplete the negative regulators of these pathways, we found that Rel2 controls resistance of A. gambiae to the human malaria parasite Plasmodium falciparum, whereas Rel 1 activation reduced infection levels. The universal relevance of this defense system across Anopheles species was established by showing that caspar silencing also prevents the development of P. falciparum in the major malaria vectors of Asia and South America, A. stephensi and A. albimanus, respectively. Parallel studies suggest that while Imd pathway activation is most effective against P. falciparum, the Toll pathway is most efficient against P. berghei, highlighting a significant discrepancy between the human pathogen and its rodent model. High throughput gene expression analyses identified a plethora of genes regulated by the activation of the two Rel factors and revealed that the Toll pathway played a more diverse role in mosquito biology than the Imd pathway, which was more immunity-specific. Further analyses of key anti-Plasmodium factors suggest they may be responsible for the Imd pathway–mediated resistance phenotype. Additionally, we found that the fitness cost caused by Rel2 activation through caspar gene silencing was undetectable in sugar-fed, blood-fed, and P. falciparum-infected female A. gambiae, while activation of the Toll pathway's Rel1 had a major impact. This study describes for the first time a single gene that influences an immune mechanism that is able to abort development of P

  6. Foods - fresh vs. frozen or canned

    MedlinePlus

    ... canned vegetables. Try to buy those without added salt and don't overcook any vegetable, whether fresh, frozen, or canned. Instead of boiling them in water for longer periods of time, they should be ...

  7. Whole transcriptome expression analysis and comparison of two different strains of Plasmodium falciparum using RNA-Seq.

    PubMed

    Antony, Hiasindh Ashmi; Pathak, Vrushali; Parija, Subhash Chandra; Ghosh, Kanjaksha; Bhattacherjee, Amrita

    2016-06-01

    The emergence and distribution of drug resistance in malaria are serious public health concerns in tropical and subtropical regions of the world. However, the molecular mechanism of drug resistance remains unclear. In the present study, we performed a high-throughput RNA-Seq to identify and characterize the differentially expressed genes between the chloroquine (CQ) sensitive (3D7) and resistant (Dd2) strains of Plasmodium falciparum. The parasite cells were cultured in the presence and absence of CQ by in vitro method. Total RNA was isolated from the harvested parasite cells using TRIzol, and RNA-Seq was conducted using an Illumina HiSeq 2500 sequencing platform with paired-end reads and annotated using Tophat. The transcriptome analysis of P. falciparum revealed the expression of ~ 5000 genes, in which ~ 60% of the genes have unknown function. Cuffdiff program was used to identify the differentially expressed genes between the CQ-sensitive and resistant strains. Here, we furnish a detailed description of the experimental design, procedure, and analysis of the transcriptome sequencing data, that have been deposited in the National Center for Biotechnology Information (accession nos. PRJNA308455 and GSE77499). PMID:27222812

  8. Plasmodium falciparum parasites causing cerebral malaria share variant surface antigens, but are they specific?

    PubMed Central

    2010-01-01

    Background Variant surface antigens (VSA) expressed on the surface of Plasmodium falciparum-infected red blood cells constitute a key for parasite sequestration and immune evasion. In distinct malaria pathologies, such as placental malaria, specific antibody response against VSA provides protection. This study investigated the antibody response specifically directed against VSA expressed by parasites isolated from individuals presenting a given type of clinical presentation. Methods Plasma and isolates were obtained from four groups of Beninese subjects: healthy adults, patients presenting uncomplicated malaria (UM), cerebral malaria (CM), or pregnancy-associated malaria (PAM). The reactivity of plasma samples from each clinical group was measured by flow cytometry against parasites isolated from individuals from each clinical group. Results Antibody responses against VSAUM were predominant in CM, UM and HA plasmas. When analysed according to age in all plasma groups, anti-VSACM and -VSAUM antibody levels were similar until six years of age. In older groups (6-18 and >19 years of age), VSAUM antibody levels were higher than VSACM antibody levels (P = .01, P = .0008, respectively). Mean MFI values, measured in all plasmas groups except the PAM plasmas, remained low for anti-VSAPAM antibodies and did not vary with age. One month after infection the level of anti-VSA antibodies able to recognize heterologous VSACM variants was increased in CM patients. In UM patients, antibody levels directed against heterologous VSAUM were similar, both during the infection and one month later. Conclusions In conclusion, this study suggests the existence of serologically distinct VSACM and VSAUM. CM isolates were shown to share common epitopes. Specific antibody response to VSAUM was predominant, suggesting a relative low diversity of VSAUM in the study area. PMID:20663188

  9. Hazardous materials in Fresh Kills landfill

    SciTech Connect

    Hirschhorn, J.S.

    1997-12-31

    No environmental monitoring and corrective action programs can pinpoint multiple locations of hazardous materials the total amount of them in a large landfill. Yet the consequences of hazardous materials in MSW landfills are considerable, in terms of public health concerns, environmental damage, and cleanup costs. In this paper a rough estimation is made of how much hazardous material may have been disposed in Fresh Kills landfill in Staten Island, New York. The logic and methods could be used for other MSW landfills. Fresh Kills has frequently been described as the world`s largest MSW landfill. While records of hazardous waste disposal at Fresh Kills over nearly 50 years of operation certainly do not exist, no reasonable person would argue with the conclusion that large quantities of hazardous waste surely have been disposed at Fresh Kills, both legally and illegally. This study found that at least 2 million tons of hazardous wastes and substances have been disposed at Fresh Kills since 1948. Major sources are: household hazardous waste, commercial RCRA hazardous waste, incinerator ash, and commercial non-RCRA hazardous waste, governmental RCRA hazardous waste. Illegal disposal of hazardous waste surely has contributed even more. This is a sufficient amount to cause serious environmental contamination and releases, especially from such a landfill without an engineered liner system, for example. This figure is roughly 1% of the total amount of waste disposed in Fresh Kills since 1948, probably at least 200 million tons.

  10. Amplification of a Gene Related to Mammalian mdr Genes in Drug-Resistant Plasmodium falciparum

    NASA Astrophysics Data System (ADS)

    Wilson, Craig M.; Serrano, Adelfa E.; Wasley, Annemarie; Bogenschutz, Michael P.; Shankar, Anuraj H.; Wirth, Dyann F.

    1989-06-01

    The malaria parasite Plasmodium falciparum contains at least two genes related to the mammalian multiple drug resistance genes, and at least one of the P. falciparum genes is expressed at a higher level and is present in higher copy number in a strain that is resistant to multiple drugs than in a strain that is sensitive to the drugs.

  11. Possible Clinical Failure of Artemether-Lumefantrine in an Italian Traveler with Uncomplicated Falciparum Malaria.

    PubMed Central

    Repetto, Ernestina C.; Traverso, Antonio; Giacomazzi, Claudio G.

    2011-01-01

    Artemisinin-combination therapies (ACTs) are recommended for the treatment of uncomplicated malaria in endemic areas with multidrug resistant Plasmodium falciparum. We report a case of possible artemether-lumefantrine clinical failure in an Italian traveler with uncomplicated P. falciparum malaria imported from Democratic Republic of Congo. PMID:22084655

  12. A Cost-Effectiveness Analysis of Plasmodium falciparum Malaria Elimination in Hainan Province, 2002-2012.

    PubMed

    Sun, Ding-Wei; Du, Jian-Wei; Wang, Guang-Ze; Li, Yu-Chun; He, Chang-Hua; Xue, Rui-De; Wang, Shan-Qing; Hu, Xi-Min

    2015-12-01

    In Hainan Province, China, great achievements in elimination of falciparum malaria have been made since 2010. There have been no locally acquired falciparum malaria cases since that time. The cost-effectiveness of elimination of falciparum malaria has been analyzed in Hainan Province. There were 4,422 falciparum malaria cases reported from 2002 to 2012, more cases occurred in males than in females. From 2002 to 2012, a total of 98.5 disability-adjusted life years (DALYs) were reported because of falciparum malaria. Populations in the age ranges of 15-25 and 30-44 years had higher incidences and DALYs than other age groups. From 2002 to 2012, malaria-related costs for salaries of staff, funds from the provincial government, national government, and the GFATM were US$3.02, US$2.24, US$1.44, and US$5.08 million, respectively. An estimated 9,504 falciparum malaria cases were averted during the period 2003-2012. The estimated cost per falciparum malaria case averted was US$116.5. The falciparum malaria elimination program in Hainan was highly effective and successful. However, funding for maintenance is still needed because of imported cases. PMID:26438030

  13. Repeat polymorphisms in the low-complexity regions of Plasmodium falciparum ABC transporters and associations with in vitro antimalarial responses.

    PubMed

    Okombo, John; Abdi, Abdirahman I; Kiara, Steven M; Mwai, Leah; Pole, Lewa; Sutherland, Colin J; Nzila, Alexis; Ochola-Oyier, Lynette Isabella

    2013-12-01

    The Plasmodium falciparum genome is rich in regions of low amino acid complexity which evolve with few constraints on size. To explore the extent of diversity in these loci, we sequenced repeat regions in pfmdr1, pfmdr5, pfmdr6, pfmrp2, and the antigenic locus pfmsp8 in laboratory and cultured-adapted clinical isolates. We further assessed associations between the repeats and parasite in vitro responses to 7 antimalarials to determine possible adaptive roles of these repeats in drug tolerance. Our results show extensive repeat variations in the reference and clinical isolates in all loci. We also observed a modest increase in dihydroartemisinin activity in parasites harboring the pfmdr1 sequence profile 7-2-10 (reflecting the number of asparagine repeats, number of aspartate repeats, and number of asparagine repeats in the final series of the gene product) (P = 0.0321) and reduced sensitivity to chloroquine, mefloquine, quinine, and dihydroartemisinin in those with the 7-2-11 profile (P = 0.0051, 0.0068, 0.0011, and 0.0052, respectively). Interestingly, we noted an inverse association between two drugs whereby isolates with 6 asparagine repeats encoded by pfmdr6 were significantly more susceptible to piperaquine than those with 8 (P = 0.0057). Against lumefantrine, those with 8 repeats were, however, more sensitive (P = 0.0144). In pfmrp2, the 7-DNNNTS/NNNNTS (number of DNNNTS or NNNNTS motifs; underlining indicates dimorphism) repeat group was significantly associated with a higher lumefantrine 50% inhibitory concentration (IC50) (P = 0.008) than in those without. No associations were observed with pfmsp8. These results hint at the probable utility of some repeat conformations as markers of in vitro antimalarial response; hence, biochemical functional studies to ascertain their role in P. falciparum are required. PMID:24080667

  14. Efficacy of Chloroquine for the Treatment of Uncomplicated Plasmodium falciparum Malaria in Honduras

    PubMed Central

    Torres, Rosa Elena Mejia; Banegas, Engels Ilich; Mendoza, Meisy; Diaz, Cesar; Bucheli, Sandra Tamara Mancero; Fontecha, Gustavo A.; Alam, Md Tauqeer; Goldman, Ira; Udhayakumar, Venkatachalam; Zambrano, Jose Orlinder Nicolas

    2013-01-01

    Chloroquine (CQ) is officially used for the primary treatment of Plasmodium falciparum malaria in Honduras. In this study, the therapeutic efficacy of CQ for the treatment of uncomplicated P. falciparum malaria in the municipality of Puerto Lempira, Gracias a Dios, Honduras was evaluated using the Pan American Health Organization—World Health Organization protocol with a follow-up of 28 days. Sixty-eight patients from 6 months to 60 years of age microscopically diagnosed with uncomplicated P. falciparum malaria were included in the final analysis. All patients who were treated with CQ (25 mg/kg over 3 days) cleared parasitemia by day 3 and acquired no new P. falciparum infection within 28 days of follow-up. All the parasite samples sequenced for CQ resistance mutations (pfcrt) showed only the CQ-sensitive genotype (CVMNK). This finding shows that CQ remains highly efficacious for the treatment of uncomplicated P. falciparum malaria in Gracias a Dios, Honduras. PMID:23458957

  15. Microbes Associated with Freshly Prepared Juices of Citrus and Carrots

    PubMed Central

    Aneja, Kamal Rai; Dhiman, Romika; Aggarwal, Neeraj Kumar; Kumar, Vikas; Kaur, Manpreeet

    2014-01-01

    Fruit juices are popular drinks as they contain antioxidants, vitamins, and minerals that are essential for human being and play important role in the prevention of heart diseases, cancer, and diabetes. They contain essential nutrients which support the growth of acid tolerant bacteria, yeasts, and moulds. In the present study, we have conducted a microbiological examination of freshly prepared juices (sweet lime, orange, and carrot) by serial dilution agar plate technique. A total of 30 juice samples were examined for their microbiological quality. Twenty-five microbial species including 9 bacterial isolates, 5 yeast isolates, and 11 mould isolates were isolated from juices. Yeasts and moulds were the main cause of spoilage of juices. Aspergillus flavus and Rhodotorula mucilaginosa were observed in the maximum number of juice samples. Among bacteria Bacillus cereus and Serratia were dominant. Escherichia coli and Staphylococcus aureus were detected in few samples. Candida sp., Curvularia, Colletotrichum, and Acetobacter were observed only in citrus juice samples. Alternaria, Aspergillus terreus, A. niger, Cladosporium, and Fusarium were also observed in tested juice samples. Some of the microorganisms detected in these juice samples can cause disease in human beings, so there is need for some guidelines that can improve the quality of fruit juices. PMID:26904628

  16. Drug-Resistant Genotypes and Multi-Clonality in Plasmodium falciparum Analysed by Direct Genome Sequencing from Peripheral Blood of Malaria Patients

    PubMed Central

    Auburn, Sarah; Assefa, Samuel A.; Polley, Spencer D.; Manske, Magnus; MacInnis, Bronwyn; Rockett, Kirk A.; Maslen, Gareth L.; Sanders, Mandy; Quail, Michael A.; Chiodini, Peter L.; Kwiatkowski, Dominic P.; Clark, Taane G.; Sutherland, Colin J.

    2011-01-01

    Naturally acquired blood-stage infections of the malaria parasite Plasmodium falciparum typically harbour multiple haploid clones. The apparent number of clones observed in any single infection depends on the diversity of the polymorphic markers used for the analysis, and the relative abundance of rare clones, which frequently fail to be detected among PCR products derived from numerically dominant clones. However, minority clones are of clinical interest as they may harbour genes conferring drug resistance, leading to enhanced survival after treatment and the possibility of subsequent therapeutic failure. We deployed new generation sequencing to derive genome data for five non-propagated parasite isolates taken directly from 4 different patients treated for clinical malaria in a UK hospital. Analysis of depth of coverage and length of sequence intervals between paired reads identified both previously described and novel gene deletions and amplifications. Full-length sequence data was extracted for 6 loci considered to be under selection by antimalarial drugs, and both known and previously unknown amino acid substitutions were identified. Full mitochondrial genomes were extracted from the sequencing data for each isolate, and these are compared against a panel of polymorphic sites derived from published or unpublished but publicly available data. Finally, genome-wide analysis of clone multiplicity was performed, and the number of infecting parasite clones estimated for each isolate. Each patient harboured at least 3 clones of P. falciparum by this analysis, consistent with results obtained with conventional PCR analysis of polymorphic merozoite antigen loci. We conclude that genome sequencing of peripheral blood P. falciparum taken directly from malaria patients provides high quality data useful for drug resistance studies, genomic structural analyses and population genetics, and also robustly represents clonal multiplicity. PMID:21853089

  17. Global sequence variation in the histidine-rich proteins 2 and 3 of Plasmodium falciparum: implications for the performance of malaria rapid diagnostic tests

    PubMed Central

    2010-01-01

    Background Accurate diagnosis is essential for prompt and appropriate treatment of malaria. While rapid diagnostic tests (RDTs) offer great potential to improve malaria diagnosis, the sensitivity of RDTs has been reported to be highly variable. One possible factor contributing to variable test performance is the diversity of parasite antigens. This is of particular concern for Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-detecting RDTs since PfHRP2 has been reported to be highly variable in isolates of the Asia-Pacific region. Methods The pfhrp2 exon 2 fragment from 458 isolates of P. falciparum collected from 38 countries was amplified and sequenced. For a subset of 80 isolates, the exon 2 fragment of histidine-rich protein 3 (pfhrp3) was also amplified and sequenced. DNA sequence and statistical analysis of the variation observed in these genes was conducted. The potential impact of the pfhrp2 variation on RDT detection rates was examined by analysing the relationship between sequence characteristics of this gene and the results of the WHO product testing of malaria RDTs: Round 1 (2008), for 34 PfHRP2-detecting RDTs. Results Sequence analysis revealed extensive variations in the number and arrangement of various repeats encoded by the genes in parasite populations world-wide. However, no statistically robust correlation between gene structure and RDT detection rate for P. falciparum parasites at 200 parasites per microlitre was identified. Conclusions The results suggest that despite extreme sequence variation, diversity of PfHRP2 does not appear to be a major cause of RDT sensitivity variation. PMID:20470441

  18. The Limits and Intensity of Plasmodium falciparum Transmission: Implications for Malaria Control and Elimination Worldwide

    PubMed Central

    Guerra, Carlos A; Gikandi, Priscilla W; Tatem, Andrew J; Noor, Abdisalan M; Smith, Dave L; Hay, Simon I; Snow, Robert W

    2008-01-01

    Background The efficient allocation of financial resources for malaria control using appropriate combinations of interventions requires accurate information on the geographic distribution of malaria risk. An evidence-based description of the global range of Plasmodium falciparum malaria and its endemicity has not been assembled in almost 40 y. This paper aims to define the global geographic distribution of P. falciparum malaria in 2007 and to provide a preliminary description of its transmission intensity within this range. Methods and Findings The global spatial distribution of P. falciparum malaria was generated using nationally reported case-incidence data, medical intelligence, and biological rules of transmission exclusion, using temperature and aridity limits informed by the bionomics of dominant Anopheles vector species. A total of 4,278 spatially unique cross-sectional survey estimates of P. falciparum parasite rates were assembled. Extractions from a population surface showed that 2.37 billion people lived in areas at any risk of P. falciparum transmission in 2007. Globally, almost 1 billion people lived under unstable, or extremely low, malaria risk. Almost all P. falciparum parasite rates above 50% were reported in Africa in a latitude band consistent with the distribution of Anopheles gambiae s.s. Conditions of low parasite prevalence were also common in Africa, however. Outside of Africa, P. falciparum malaria prevalence is largely hypoendemic (less than 10%), with the median below 5% in the areas surveyed. Conclusions This new map is a plausible representation of the current extent of P. falciparum risk and the most contemporary summary of the population at risk of P. falciparum malaria within these limits. For 1 billion people at risk of unstable malaria transmission, elimination is epidemiologically feasible, and large areas of Africa are more amenable to control than appreciated previously. The release of this information in the public domain will

  19. Establishing the Global Fresh Water Sensor Web

    NASA Technical Reports Server (NTRS)

    Hildebrand, Peter H.

    2005-01-01

    This paper presents an approach to measuring the major components of the water cycle from space using the concept of a sensor-web of satellites that are linked to a data assimilation system. This topic is of increasing importance, due to the need for fresh water to support the growing human population, coupled with climate variability and change. The net effect is that water is an increasingly valuable commodity. The distribution of fresh water is highly uneven over the Earth, with both strong latitudinal distributions due to the atmospheric general circulation, and even larger variability due to landforms and the interaction of land with global weather systems. The annual global fresh water budget is largely a balance between evaporation, atmospheric transport, precipitation and runoff. Although the available volume of fresh water on land is small, the short residence time of water in these fresh water reservoirs causes the flux of fresh water - through evaporation, atmospheric transport, precipitation and runoff - to be large. With a total atmospheric water store of approx. 13 x 10(exp 12)cu m, and an annual flux of approx. 460 x 10(exp 12)cu m/y, the mean atmospheric residence time of water is approx. 10 days. River residence times are similar, biological are approx. 1 week, soil moisture is approx. 2 months, and lakes and aquifers are highly variable, extending from weeks to years. The hypothesized potential for redistribution and acceleration of the global hydrological cycle is therefore of concern. This hypothesized speed-up - thought to be associated with global warming - adds to the pressure placed upon water resources by the burgeoning human population, the variability of weather and climate, and concerns about anthropogenic impacts on global fresh water availability.

  20. A World Malaria Map: Plasmodium falciparum Endemicity in 2007

    PubMed Central

    Hay, Simon I; Guerra, Carlos A; Gething, Peter W; Patil, Anand P; Tatem, Andrew J; Noor, Abdisalan M; Kabaria, Caroline W; Manh, Bui H; Elyazar, Iqbal R. F; Brooker, Simon; Smith, David L; Moyeed, Rana A; Snow, Robert W

    2009-01-01

    Background Efficient allocation of resources to intervene against malaria requires a detailed understanding of the contemporary spatial distribution of malaria risk. It is exactly 40 y since the last global map of malaria endemicity was published. This paper describes the generation of a new world map of Plasmodium falciparum malaria endemicity for the year 2007. Methods and Findings A total of 8,938 P. falciparum parasite rate (PfPR) surveys were identified using a variety of exhaustive search strategies. Of these, 7,953 passed strict data fidelity tests for inclusion into a global database of PfPR data, age-standardized to 2–10 y for endemicity mapping. A model-based geostatistical procedure was used to create a continuous surface of malaria endemicity within previously defined stable spatial limits of P. falciparum transmission. These procedures were implemented within a Bayesian statistical framework so that the uncertainty of these predictions could be evaluated robustly. The uncertainty was expressed as the probability of predicting correctly one of three endemicity classes; previously stratified to be an informative guide for malaria control. Population at risk estimates, adjusted for the transmission modifying effects of urbanization in Africa, were then derived with reference to human population surfaces in 2007. Of the 1.38 billion people at risk of stable P. falciparum malaria, 0.69 billion were found in Central and South East Asia (CSE Asia), 0.66 billion in Africa, Yemen, and Saudi Arabia (Africa+), and 0.04 billion in the Americas. All those exposed to stable risk in the Americas were in the lowest endemicity class (PfPR2−10 ≤ 5%). The vast majority (88%) of those living under stable risk in CSE Asia were also in this low endemicity class; a small remainder (11%) were in the intermediate endemicity class (PfPR2−10 > 5 to < 40%); and the remaining fraction (1%) in high endemicity (PfPR2−10 ≥ 40%) areas. High endemicity was widespread in the

  1. [Plasmodium falciparum malaria: evaluation of three imported cases].

    PubMed

    İnkaya, Ahmet Çağkan; Kaya, Filiz; Yıldız, İrem; Uzun, Ömrüm; Ergüven, Sibel

    2016-04-01

    Among Plasmodium species the causative agent of malaria in Turkey is P.vivax, however the incidence of imported falciparum malaria cases is steadily increasing. P.falciparum may cause severe malaria with the involvement of central nervous system, acute renal failure, severe anemia or acute respiratory distress syndrome. Furhermore most of the casualties due to malaria are related with P.falciparum. There is recently, a considerable increase in malaria infections especially in tropical areas. In this report, three cases, who have admitted to our hospital with three different clinical presentations of falciparum malaria, and all shared common history of travelling to Africa were presented. First case was a 27 years old, male patient who returned from Malawi seven days ago where he stayed for two weeks. He admitted to our hospital with the complaints of sensation of cold, shivering and fever. In physical examination his body temperature was 37.9°C, C-reactive protein level was high, and the other systemic results were normal. The second case was a 25 years old, male patient who returned from Gambia two weeks ago. He was suffering from fever, headache, shivering and unable to maintain his balance. The patient's body temperature was 38°C. Laboratory tests revealed hyperbilirubinemia and thrombocytopenia. Parasitological examination of the Giemsa-stained peripheral blood smear of these two patients demonstrated ring forms compatible with P.falciparum. Treatment was commenced with arthemeter plus lumefantrine, resulting with complete cure. Third case was a 46 years old, male patient who had been working in Uganda, and returned to Turkey two weeks ago. He had sudden onset of fever, headache, nausea and vomiting and impaired consciousness. His peripheral blood smear revealed ring-formed trophozoites and banana-shaped gametocytes of P.falciparum. Arthemeter plus lumefantrine therapy was started, however, he developed severe thrombocytopenia and jaundice under treatment

  2. Amodiaquine failure associated with erythrocytic glutathione in Plasmodium falciparum malaria

    PubMed Central

    Zuluaga, Lina; Pabón, Adriana; López, Carlos; Ochoa, Aleida; Blair, Silvia

    2007-01-01

    Objective To establish the relationship between production of glutathione and the therapeutic response to amodiaquine (AQ) monotherapy in Plasmodium falciparum non-complicated malaria patients. Methodology Therapeutic response to AQ was evaluated in 32 patients with falciparum malaria in two townships of Antioquia, Colombia, and followed-up for 28 days. For every patient, total glutathione and enzymatic activity (glutathione reductase, GR, and γ-glutamylcysteine synthetase, γ-GCS) were determined in parasitized erythrocytes, non-infected erythrocytes and free parasites, on the starting day (day zero, before ingestion of AQ) and on the day of failure (in case of occurrence). Results There was found an AQ failure of 31.25%. Independent of the therapeutic response, on the starting day and on the day of failure, lower total glutathione concentration and higher GR activities in parasitized erythrocytes were found, compared with non-infected erythrocytes (p < 0.003). In addition, only on the day of failure, γ-GCS activity of parasitized erythrocytes was higher, compared with that of healthy erythrocytes (p = 0.01). Parasitized and non-parasitized erythrocytes in therapeutic failure patients (TF) had higher total glutathione on the starting day compared with those of adequate clinical response (ACR) (p < 0.02). Parasitized erythrocytes of TF patients showed lower total glutathione on the failure day, compared with starting day (p = 0.017). No differences was seen in the GR and γ-GCS activities by compartment, neither between the two therapeutic response groups nor between the two treatment days. Conclusion This study is a first approach to explaining P. falciparum therapeutic failure in humans through differences in glutathione metabolism in TF and ACR patients. These results suggest a role for glutathione in the therapeutic failure to antimalarials. PMID:17451604

  3. pfmdr2 confers heavy metal resistance to Plasmodium falciparum.

    PubMed

    Rosenberg, Elli; Litus, Ilena; Schwarzfuchs, Nurit; Sinay, Rosa; Schlesinger, Pnina; Golenser, Jacob; Baumeister, Stefan; Lingelbach, Klaus; Pollack, Yaakov

    2006-09-15

    Heavy metals are required by all organisms for normal function, but high levels of heavy metals are toxic. Therefore, homeostasis of these metals is crucial. In the human malaria-causing agent Plasmodium falciparum, the mechanisms of heavy metal transport have yet to be characterized. We have developed a P. falciparum line resistant to heavy metals from a wild-type line sensitive to heavy metals. A molecular and biochemical analysis of the involvement of the P. falciparum multidrug resistance 2 (pfmdr2) gene, an ABC-type transporter, in heavy metal homeostasis was studied. Using a novel uptake assay applied on these two strains, it was demonstrated that, when exposed to heavy metals, the sensitive line accumulates metal, whereas no accumulation was observed in the resistant line. The accumulation occurs within the parasite itself and not in the cytoplasm of the red blood cell. This difference in the accumulation pattern is not a result of amplification of the pfmdr2 gene or of a change in the expression pattern of the gene in the two lines. Sequencing of the gene from both lines revealed a major difference; a stop codon is found in the sensitive line upstream of the normal termination, resulting in a truncated protein that lacks 188 amino acids that contain a portion of the essential cytoplasmatic transporter domain, thereby rendering it inactive. In contrast, the resistant line harbors a full-length, active protein. These findings strongly suggest that the PFMDR2 protein acts as an efflux pump of heavy metals. PMID:16849328

  4. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum.

    PubMed

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M; Holder, Anthony A

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  5. Alternative Protein Secretion in the Malaria Parasite Plasmodium falciparum

    PubMed Central

    Thavayogarajah, Thuvaraka; Gangopadhyay, Preetish; Rahlfs, Stefan; Becker, Katja; Lingelbach, Klaus; Przyborski, Jude M.; Holder, Anthony A.

    2015-01-01

    Plasmodium falciparum invades human red blood cells, residing in a parasitophorous vacuole (PV), with a parasitophorous vacuole membrane (PVM) separating the PV from the host cell cytoplasm. Here we have investigated the role of N-myristoylation and two other N-terminal motifs, a cysteine potential S-palmitoylation site and a stretch of basic residues, as the driving force for protein targeting to the parasite plasma membrane (PPM) and subsequent translocation across this membrane. Plasmodium falciparum adenylate kinase 2 (Pf AK2) contains these three motifs, and was previously proposed to be targeted beyond the parasite to the PVM, despite the absence of a signal peptide for entry into the classical secretory pathway. Biochemical and microscopy analyses of PfAK2 variants tagged with green fluorescent protein (GFP) showed that these three motifs are involved in targeting the protein to the PPM and translocation across the PPM to the PV. It was shown that the N-terminal 37 amino acids of PfAK2 alone are sufficient to target and translocate GFP across the PPM. As a control we examined the N-myristoylated P. falciparum ADP-ribosylation factor 1 (PfARF1). PfARF1 was found to co-localise with a Golgi marker. To determine whether or not the putative palmitoylation and the cluster of lysine residues from the N-terminus of PfAK2 would modulate the subcellular localization of PfARF1, a chimeric fusion protein containing the N-terminus of PfARF1 and the two additional PfAK2 motifs was analysed. This chimeric protein was targeted to the PPM, but not translocated across the membrane into the PV, indicating that other features of the N-terminus of PfAK2 also play a role in the secretion process. PMID:25909331

  6. Fresh fruit: microstructure, texture, and quality

    NASA Astrophysics Data System (ADS)

    Wood, Delilah F.; Imam, Syed H.; Orts, William J.; Glenn, Gregory M.

    2009-05-01

    Fresh-cut produce has a huge following in today's supermarkets. The trend follows the need to decrease preparation time as well as the desire to follow the current health guidelines for consumption of more whole "heart-healthy" foods. Additionally, consumers are able to enjoy a variety of fresh produce regardless of the local season because produce is now shipped world-wide. However, most fruits decompose rapidly once their natural packaging has been disrupted by cutting. In addition, some intact fruits have limited shelf-life which, in turn, limits shipping and storage. Therefore, a basic understanding of how produce microstructure relates to texture and how microstructure changes as quality deteriorates is needed to ensure the best quality in the both the fresh-cut and the fresh produce markets. Similarities between different types of produce include desiccation intolerance which produces wrinkling of the outer layers, cracking of the cuticle and increased susceptibility to pathogen invasion. Specific examples of fresh produce and their corresponding ripening and storage issues, and degradation are shown in scanning electron micrographs.

  7. Immunoregulatory alterations in Plasmodium falciparum and Plasmodium vivax infections.

    PubMed

    Merino, F; Layrisse, Z; Godoy, G; Volcán, G

    1986-09-01

    Studies on the immune function of patients with acute Plasmodium vivax or P. falciparum infections were performed. All subjects were residing in recent malaria endemic areas of Venezuela. Lymphopenia, reduction of peripheral blood T-lymphocytes positive for monoclonal antibody OKT4 (T helper) a decrease of in vitro mitogenic proliferative response and natural killer cell activity were observed. Serum lymphocytotoxic antibodies reactive at 37 degrees C were detected in both groups of patients as well as serum autoantibodies. The possible role of lymphocytotoxic autoantibodies in the etiology of the T-lymphocyte depletion and acquired immunological perturbations in human malaria is discussed. PMID:2947313

  8. Unraveling the 'DEAD-box' helicases of Plasmodium falciparum.

    PubMed

    Tuteja, Renu; Pradhan, Arun

    2006-07-01

    The causative agent for the most fatal form of malaria, Plasmodium falciparum, has developed insecticide and drug resistance with time. Therefore combating this disease is becoming increasingly difficult and this calls for finding alternate ways to control malaria. One of the feasible ways could be to find out inhibitors/drugs specific for the indispensable enzymes of malaria parasite such as helicases. These helicases, which contain intrinsic nucleic acid-dependent ATPase activity, are capable of enzymatically unwinding energetically stable duplex nucleic acids into single-stranded templates and are required for all the nucleic acid transactions. Most of the helicases contain a set of nine extremely conserved amino acid sequences, which are called 'helicase motifs'. Due to the presence of the DEAD (Asp-Glu-Ala-Asp) in one of the conserved motifs, this family is also known as the 'DEAD-box' family. In this review, using bioinformatic approach, we describe the 'DEAD-box' helicases of malaria parasite P. falciparum. An in depth analysis shows that the parasite contains 22 full-length genes, some of which are homologues of well-characterized helicases of this family from other organisms. Recently we have cloned and characterized the first member of this family, which is a homologue of p68 and is expressed during the schizont stage of the development of the parasite [Pradhan, A., Chauhan, V.S., Tuteja, R., 2005a. A novel 'DEAD-box' DNA helicase from Plasmodium falciparum is homologous to p68. Mol. Biochem. Parasitol. 140, 55-60.; Pradhan A., Chauhan V.S., Tuteja R., 2005b. Plasmodium falciparum DNA helicase 60 is a schizont stage specific, bipolar and dual helicase stimulated by PKC phosphorylation. Mol. Biochem. Parasitol. 144, 133-141.]. It will be really interesting to clone and characterize other members of the 'DEAD-box' family and understand their role in the replication and transmission of the parasite. These detailed studies may help to identify a parasite

  9. Spread of Artemisinin Resistance in Plasmodium falciparum Malaria

    PubMed Central

    Ashley, E.A.; Dhorda, M.; Fairhurst, R.M.; Amaratunga, C.; Lim, P.; Suon, S.; Sreng, S.; Anderson, J.M.; Mao, S.; Sam, B.; Sopha, C.; Chuor, C.M.; Nguon, C.; Sovannaroth, S.; Pukrittayakamee, S.; Jittamala, P.; Chotivanich, K.; Chutasmit, K.; Suchatsoonthorn, C.; Runcharoen, R.; Hien, T.T.; Thuy-Nhien, N.T.; Thanh, N.V.; Phu, N.H.; Htut, Y.; Han, K-T.; Aye, K.H.; Mokuolu, O.A.; Olaosebikan, R.R.; Folaranmi, O.O.; Mayxay, M.; Khanthavong, M.; Hongvanthong, B.; Newton, P.N.; Onyamboko, M.A.; Fanello, C.I.; Tshefu, A.K.; Mishra, N.; Valecha, N.; Phyo, A.P.; Nosten, F.; Yi, P.; Tripura, R.; Borrmann, S.; Bashraheil, M.; Peshu, J.; Faiz, M.A.; Ghose, A.; Hossain, M.A.; Samad, R.; Rahman, M.R.; Hasan, M.M.; Islam, A.; Miotto, O.; Amato, R.; MacInnis, B.; Stalker, J.; Kwiatkowski, D.P.; Bozdech, Z.; Jeeyapant, A.; Cheah, P.Y.; Sakulthaew, T.; Chalk, J.; Intharabut, B.; Silamut, K.; Lee, S.J.; Vihokhern, B.; Kunasol, C.; Imwong, M.; Tarning, J.; Taylor, W.J.; Yeung, S.; Woodrow, C.J.; Flegg, J.A.; Das, D.; Smith, J.; Venkatesan, M.; Plowe, C.V.; Stepniewska, K.; Guerin, P.J.; Dondorp, A.M.; Day, N.P.; White, N.J.

    2014-01-01

    BACKGROUND Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand–Cambodia border. Slowly clearing in fections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the “propeller” region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of

  10. Replication and maintenance of the Plasmodium falciparum apicoplast genome.

    PubMed

    Milton, Morgan E; Nelson, Scott W

    2016-08-01

    Members of the phylum Apicomplexa are responsible for many devastating diseases including malaria (Plasmodium spp.), toxoplasmosis (Toxoplasma gondii), babesiosis (Babesia bovis), and cyclosporiasis (Cyclospora cayetanensis). Most Apicomplexans contain a unique and essential organelle called the apicoplast. Derived from an ancient chloroplast, the apicoplast replicates and maintains a 35 kilobase (kb) circular genome. Due to its essential nature within the parasite, drugs targeted to proteins involved in DNA replication and repair of the apicoplast should be potent and specific. This review summarizes the current knowledge surrounding the replication and repair of the Plasmodium falciparum apicoplast genome and identifies several putative proteins involved in replication and repair pathways. PMID:27338018

  11. [Artemisinin resistance in Plasmodium falciparum: global status and basic research].

    PubMed

    Zhao, Shao-min; Wang, Man-yuan

    2014-10-01

    Artemisinin-resistant Plasmodium falciparum has been identified by WHO in the Greater Mekong subregion. While there is no report on artemisinin resistance in Africa and South America by now, related surveillance measures have been taken place. The genes related artemisinin-resistance has been identified and the molecular markers will be used for large-scale surveillance efforts to contain artemisinin resistance. The emergence and spread of artemisinin resistance worldwide is a present danger and needs more attention. This article reviews the progress of artemisininresistance malaria parasites and artemisinin-based combination therapies. PMID:25726605

  12. Discrete-Event Simulation Models of Plasmodium falciparum Malaria

    PubMed Central

    McKenzie, F. Ellis; Wong, Roger C.; Bossert, William H.

    2008-01-01

    We develop discrete-event simulation models using a single “timeline” variable to represent the Plasmodium falciparum lifecycle in individual hosts and vectors within interacting host and vector populations. Where they are comparable our conclusions regarding the relative importance of vector mortality and the durations of host immunity and parasite development are congruent with those of classic differential-equation models of malaria, epidemiology. However, our results also imply that in regions with intense perennial transmission, the influence of mosquito mortality on malaria prevalence in humans may be rivaled by that of the duration of host infectivity. PMID:18668185

  13. Decontamination of fresh and fresh-cut fruits and vegetables with cold plasma technology

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Contamination of fresh and fresh-cut fruits and vegetables by foodborne pathogens has prompted research into novel interventions. Cold plasma is a nonthermal food processing technology which uses energetic, reactive gases to inactivate contaminating microbes. This flexible sanitizing method uses ele...

  14. What is a fresh scent in perfumery? Perceptual freshness is correlated with substantivity.

    PubMed

    Zarzo, Manuel

    2013-01-01

    Perfumes are manufactured by mixing odorous materials with different volatilities. The parameter that measures the lasting property of a material when applied on the skin is called substantivity or tenacity. It is well known by perfumers that citrus and green notes are perceived as fresh and they tend to evaporate quickly, while odors most dissimilar to 'fresh' (e.g., oriental, powdery, erogenic and animalic scents) are tenacious. However, studies aimed at quantifying the relationship between fresh odor quality and substantivity have not received much attention. In this work, perceptual olfactory ratings on a fresh scale, estimated in a previous study, were compared with substantivity parameters and antierogenic ratings from the literature. It was found that the correlation between fresh odor character and odorant substantivity is quite strong (r = -0.85). 'Fresh' is sometimes interpreted in perfumery as 'cool' and the opposite of 'warm'. This association suggests that odor freshness might be somehow related to temperature. Assuming that odor perception space was shaped throughout evolution in temperate climates, results reported here are consistent with the hypothesis that 'fresh' evokes scents typically encountered in the cool season, while 'warm' would be evoked by odors found in nature during summer. This hypothesis is rather simplistic but it may provide a new insight to better understand the perceptual space of scents. PMID:23275083

  15. Consumer's Fresh Produce Food Safety Practices: Outcomes of a Fresh Produce Safety Education Program

    ERIC Educational Resources Information Center

    Scott, Amanda R.; Pope, Paul E.; Thompson, Britta M.

    2009-01-01

    The Centers for Disease Control and Prevention estimate that there are 76 million cases of foodborne disease annually. Foodborne disease is usually associated with beef, poultry, and seafood. However, there is an increasing number of foodborne disease cases related to fresh produce. Consumers may not associate fresh produce with foodborne disease…

  16. Irradiation of fresh and fresh-cut fruits and vegetables: quality and shelf-life

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The effects of ionizing radiation on each quality parameters (appearance, texture, flavor/taste and nutritional values) of fresh produce are reviewed with a focus on recent developments on fresh-cut fruits and vegetables. Changes in respiration rate, ethylene production and enzymatic activities are ...

  17. A fresh fruit and vegetable program improves high school students' consumption of fresh produce

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Low fruit and vegetable intake may be associated with overweight. The United States Department of Agriculture implemented the Fresh Fruit and Vegetable Program in 2006-2007. One Houston-area high school was selected and received funding to provide baskets of fresh fruits and vegetables daily for eac...

  18. Critical Issues in Maintaining Fresh and Fresh-cut Produce Safety

    Technology Transfer Automated Retrieval System (TEKTRAN)

    An increasing number of food-borne illness outbreaks have been associated with the consumption of fresh and fresh-cut produce contaminated with human pathogens. Produce grows in the natural environment and undergoes much handling on its journey from farm to table, making it vulnerable to human path...

  19. Polyamine quinoline rhodium complexes: synthesis and pharmacological evaluation as antiparasitic agents against Plasmodium falciparum and Trichomonas vaginalis.

    PubMed

    Stringer, Tameryn; Taylor, Dale; Guzgay, Hajira; Shokar, Ajit; Au, Aaron; Smith, Peter J; Hendricks, Denver T; Land, Kirkwood M; Egan, Timothy J; Smith, Gregory S

    2015-09-01

    A series of mono- and bis-salicylaldimine ligands and their corresponding Rh(i) complexes were prepared. The compounds were characterised using standard spectroscopic techniques including NMR, IR spectroscopy and mass spectrometry. The salicylaldimine ligands and complexes were screened for antiparasitic activity against two strains of Plasmodium falciparum i.e. the NF54 CQ-sensitive and K1 CQ-resistant strain as well as against the G3 isolate of Trichomonas vaginalis. The monomeric salicylaldimine quinolines exhibited good activity against the NF54 strain and the dimeric salicylaldimine quinolines exhibited no cross resistance across the two strains. The binuclear 5-chloro Rh(i) complex displayed the best activity against the Trichomonas vaginalis parasite, possibly a consequence of its enhanced lipophilicity. The compounds were also screened for cytotoxicity in vitro against WHCO1 oesophageal cancer cells. The monomeric salicylaldimine quinolines exhibited high selectivity towards malaria parasites compared to cancer cells, while the dimeric compounds were less selective. PMID:26226082

  20. Synthesis of novel guttiferone A derivatives: in-vitro evaluation toward Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani.

    PubMed

    Fromentin, Yann; Gaboriaud-Kolar, Nicolas; Lenta, Bruno Ndjakou; Wansi, Jean Duplex; Buisson, Didier; Mouray, Elisabeth; Grellier, Philippe; Loiseau, Philippe M; Lallemand, Marie-Christine; Michel, Sylvie

    2013-07-01

    The catechol pharmacomodulation of the natural product guttiferone A, isolated from the Symphonia globulifera tree, led to the semisynthesis of a collection of twenty derivatives. The ester and ether derivatives of guttiferone A were evaluated for their anti-plasmodial, trypanocidal and anti-leishmanial activities. Some compounds described below have shown potent antiparasitic activity against Plasmodium falciparum, Trypanosoma brucei and Leishmania donovani in a range from 1 to 5 μM. The evaluation of guttiferone A derivatives against VERO cells highlighted catechol modulations as an interesting tool to decrease the toxicity and keep the activity of this natural compound. The current study revealed new molecules as promising new antiparasitic drug candidates. PMID:23727538

  1. Inheritance of fresh-cut fruit quality attributes in Capsicum

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The fresh-cut fruit and vegetable industry has expanded rapidly during the past decade, due to freshness, convenience and the high nutrition that fresh-cut produce offers to consumers. The current report evaluates the inheritance of postharvest attributes that contribute to pepper fresh-cut product...

  2. 9 CFR 319.141 - Fresh pork sausage.

    Code of Federal Regulations, 2010 CFR

    2010-01-01

    ... 9 Animals and Animal Products 2 2010-01-01 2010-01-01 false Fresh pork sausage. 319.141 Section... Sausage § 319.141 Fresh pork sausage. “Fresh Pork Sausage” is sausage prepared with fresh pork or frozen pork or both, but not including pork byproducts, and may contain Mechanically Separated (Species)...

  3. Modified atmosphere packaging for fresh-cut fruits and vegetables

    Technology Transfer Automated Retrieval System (TEKTRAN)

    The latest development in and different aspects of modified atmosphere packaging for fresh-cut fruits and vegetables are reviewed in the book. This book provides all readers, including fresh-cut academic researchers, fresh-cut R&D personnel, and fresh-cut processing engineers, with unique, essential...

  4. Distribution of Drug Resistance Genotypes in Plasmodium falciparum in an Area of Limited Parasite Diversity in Saudi Arabia

    PubMed Central

    Bin Dajem, Saad M.; Al-Farsi, Hissa M.; Al-Hashami, Zainab S.; Al-Sheikh, Adel Ali H.; Al-Qahtani, Ahmed; Babiker, Hamza A.

    2012-01-01

    Two hundred and three Plasmodium falciparum isolates from Jazan area, southwest Saudi Arabia, were typed for Pfcrt, Pfmdr1, dhps, and dhfr mutations associated with resistance to chloroquine, mefloquine, halofantrine, artemisinin, sulfadoxine-pyrimethamine, and the neutral polymorphic gene Pfg377. A large proportion (33%) of isolates harbored double mutant dhfr genotype (51I,59C,108N). However, only one isolate contained mutation dhps-437G. For Pfcrt, almost all examined isolates (163; 99%) harbored the mutant genotype (72C,73V,74I,75E,76T), whereas only 49 (31%) contained the mutant Pfmdr1 genotype (86Y,184F,1034S,1042N), 109 (66%) harbored the single mutant genotype (86N,184F,1034S,1042N), and no mutations were seen in codons 1034, 1042, and 1246. Nonetheless, three new single-nucleotide polymorphisms were detected at codons 182, 192, and 102. No differences were seen in distribution of drug resistance genes among Saudis and expatriates. There was a limited multiplicity (5%), mean number of clones (1.05), and two dominant multilocus genotypes among infected individuals in Jazan. A pattern consistent with limited cross-mating and recombination among local parasite was apparent. PMID:22556074

  5. Role of Pfmdr1 in In Vitro Plasmodium falciparum Susceptibility to Chloroquine, Quinine, Monodesethylamodiaquine, Mefloquine, Lumefantrine, and Dihydroartemisinin

    PubMed Central

    Wurtz, Nathalie; Fall, Bécaye; Pascual, Aurélie; Fall, Mansour; Baret, Eric; Camara, Cheikhou; Nakoulima, Aminata; Diatta, Bakary; Fall, Khadidiatou Ba; Mbaye, Pape Saliou; Diémé, Yaya; Bercion, Raymond; Wade, Boubacar

    2014-01-01

    The involvement of Pfmdr1 (Plasmodium falciparum multidrug resistance 1) polymorphisms in antimalarial drug resistance is still debated. Here, we evaluate the association between polymorphisms in Pfmdr1 (N86Y, Y184F, S1034C, N1042D, and D1246Y) and Pfcrt (K76T) and in vitro responses to chloroquine (CQ), mefloquine (MQ), lumefantrine (LMF), quinine (QN), monodesethylamodiaquine (MDAQ), and dihydroartemisinin (DHA) in 174 Plasmodium falciparum isolates from Dakar, Senegal. The Pfmdr1 86Y mutation was identified in 14.9% of the samples, and the 184F mutation was identified in 71.8% of the isolates. No 1034C, 1042N, or 1246Y mutations were detected. The Pfmdr1 86Y mutation was significantly associated with increased susceptibility to MDAQ (P = 0.0023), LMF (P = 0.0001), DHA (P = 0.0387), and MQ (P = 0.00002). The N86Y mutation was not associated with CQ (P = 0.214) or QN (P = 0.287) responses. The Pfmdr1 184F mutation was not associated with various susceptibility responses to the 6 antimalarial drugs (P = 0.168 for CQ, 0.778 for MDAQ, 0.324 for LMF, 0.961 for DHA, 0.084 for QN, and 0.298 for MQ). The Pfmdr1 86Y-Y184 haplotype was significantly associated with increased susceptibility to MDAQ (P = 0.0136), LMF (P = 0.0019), and MQ (P = 0.0001). The additional Pfmdr1 86Y mutation increased significantly the in vitro susceptibility to MDAQ (P < 0.0001), LMF (P < 0.0001), MQ (P < 0.0001), and QN (P = 0.0026) in wild-type Pfcrt K76 parasites. The additional Pfmdr1 86Y mutation significantly increased the in vitro susceptibility to CQ (P = 0.0179) in Pfcrt 76T CQ-resistant parasites. PMID:25199781

  6. Global distribution of polymorphisms associated with delayed Plasmodium falciparum parasite clearance following artemisinin treatment: genotyping of archive blood samples.

    PubMed

    Murai, Kenji; Culleton, Richard; Hisaoka, Teruhiko; Endo, Hiroyoshi; Mita, Toshihiro

    2015-06-01

    The recent emergence and spread of artemisinin-resistant Plasmodium falciparum isolates is a growing concern for global malaria-control efforts. A recent genome-wide analysis study identified two SNPs at genomic positions MAL10-688956 and MAL13-1718319, which are linked to delayed clearance of parasites following artemisinin combination therapy (ACT). It is expected that continuous artemisinin pressure will affect the distribution of these SNPs. Here, we investigate the worldwide distribution of these SNPs using a large number of archived samples in order to generate baseline data from the period before the emergence of ACT resistance. The presence of SNPs in MAL10-688956 and MAL13-1718319 was assessed by nested PCR RFLP and direct DNA sequencing using 653 global P. falciparum samples obtained before the reported emergence of ACT resistance. SNPs at MAL10-688956 and MAL13-1718319 associated with delayed parasite clearance following ACT administration were observed in 8% and 3% of parasites, respectively, mostly in Cambodia and Thailand. Parasites harbouring both SNPs were found in only eight (1%) isolates, all of which were from Cambodia and Thailand. Linkage disequilibrium was detected between MAL10-688956 and MAL13-1718319, suggesting that this SNP combination may have been selected by ACT drug pressure. Neither of the SNPs associated with delayed parasite clearance were observed in samples from Africa or South America. Baseline information of the geographical difference of MAL10-688956 and MAL13-1718319 SNPs provides a solid basis for assessing whether these SNPs are selected by artemisinin-based combination therapies. PMID:25449286

  7. Immune Characterization of Plasmodium falciparum Parasites with a Shared Genetic Signature in a Region of Decreasing Transmission

    PubMed Central

    Bei, Amy K.; Diouf, Ababacar; Miura, Kazutoyo; Larremore, Daniel B.; Ribacke, Ulf; Tullo, Gregory; Moss, Eli L.; Neafsey, Daniel E.; Daniels, Rachel F.; Zeituni, Amir E.; Nosamiefan, Iguosadolo; Volkman, Sarah K.; Ahouidi, Ambroise D.; Ndiaye, Daouda; Dieye, Tandakha; Mboup, Souleymane; Buckee, Caroline O.; Long, Carole A.

    2014-01-01

    As the intensity of malaria transmission has declined, Plasmodium falciparum parasite populations have displayed decreased clonal diversity resulting from the emergence of many parasites with common genetic signatures (CGS). We have monitored such CGS parasite clusters from 2006 to 2013 in Thiès, Senegal, using the molecular barcode. The first, and one of the largest observed clusters of CGS parasites, was present in 24% of clinical isolates in 2008, declined to 3.4% of clinical isolates in 2009, and then disappeared. To begin to explore the relationship between the immune responses of the population and the emergence and decline of specific parasite genotypes, we have determined whether antibodies to CGS parasites correlate with their prevalence. We measured (i) antibodies capable of inhibiting parasite growth in culture and (ii) antibodies recognizing the surfaces of infected erythrocytes (RBCs). IgG obtained from volunteers in 2009 showed increased reactivity to the surfaces of CGS-parasitized erythrocytes over IgG from 2008. Since P. falciparum EMP-1 (PfEMP-1) is a major variant surface antigen, we used var Ups quantitative reverse transcription-PCR (qRT-PCR) and sequencing with degenerate DBL1α domain primers to characterize the var genes expressed by CGS parasites after short-term in vitro culture. CGS parasites show upregulation of UpsA var genes and 2-cysteine-containing PfEMP-1 molecules and express the same dominant var transcript. Our work indicates that the CGS parasites in this cluster express similar var genes, more than would be expected by chance in the population, and that there is year-to-year variation in immune recognition of surface antigens on CGS parasite-infected erythrocytes. This study lays the groundwork for detailed investigations of the mechanisms driving the expansion or contraction of specific parasite clones in the population. PMID:25368109

  8. Hot water, fresh beer, and salt

    NASA Astrophysics Data System (ADS)

    Crawford, Frank S.

    1990-11-01

    In the ``hot chocolate effect'' the best musical scales (those with the finest tone quality, largest range, and best tempo) are obtained by adding salt to a glass of hot water supersaturated with air. Good scales can also be obtained by adding salt to a glass of freshly opened beer (supersaturated with CO2) provided you first (a) get rid of much of the excess CO2 so as to produce smaller, hence slower, rising bubbles, and (b) get rid of the head of foam, which damps the standing wave and ruins the tone quality. Finally the old question, ``Do ionizing particles produce bubbles in fresh beer?'' is answered experimentally.

  9. Characterization of promoters and stable transfection by homologous and nonhomologous recombination in Plasmodium falciparum.

    PubMed Central

    Crabb, B S; Cowman, A F

    1996-01-01

    Genetic studies of the protozoan parasite Plasmodium falciparum have been severely limited by the inability to introduce or modify genes. In this paper we describe a system of stable transfection of P. falciparum using a Toxoplasma gondii dihydrofolate reductase-thymidylate synthase gene, modified to confer resistance to pyrimethamine, as a selectable marker. This gene was placed under the transcriptional control of the P. falciparum calmodulin gene flanking sequences. Transfected parasites generally maintained plasmids episomally while under selection; however, parasite clones containing integrated forms of the plasmid were obtained. Integration occurred by both homologous and nonhomologous recombination. In addition to the flanking sequence of the P. falciparum calmodulin gene, the 5' sequences of the P. falciparum and P. chabaudi dihydrofolate reductase-thymidylate synthase genes were also shown to be transcriptionally active in P. falciparum. The minimal 5' sequence that possessed significant transcriptional activity was determined for each gene and short sequences containing important transcriptional control elements were identified. These sequences will provide considerable flexibility in the future construction of plasmid vectors to be used for the expression of foreign genes or for the deletion or modification of P. falciparum genes of interest. Images Fig. 4 Fig. 5 PMID:8692985

  10. As Leaching into Fresh Water from Highly Contaminated Hawaiian Soils

    NASA Astrophysics Data System (ADS)

    Niklis, N. J.; Rubin, K. H.; El-Kadi, A. I.

    2009-12-01

    Arsenic contamination of current and former agricultural soils in Hawaii is an unfortunate legacy of plantation era agricultural practices. Here, we report an investigation of As mobility in fresh water from highly contaminated (0.8 % As) A-zone Hawaiian andisols from the Hamakua Coast of the Island of Hawai’i. Aliquots of the same acidic soil (pH= 5.0) were exposed to fresh water for varying lengths of time and analyzed to quantify the fraction of As and other elements leached from the soil relative to concentrations determined by total digestion. A maximum of 0.04% of As and 0.05% of Fe were removed from the soils in initial rinses and multi-day leaches using 18 megaohm Millipore water, in experiments lasting up to 35 days. Arsenic concentrations were highest in initial soil rinses, indicating that a small fraction of the total As in the soil is either loosely bound or present as a fine-grained, soluble As-bearing phase. During subsequent leaching experiments, arsenic and most other inorganic ions that we analyzed for reached equilibrium after 3 days; Fe reached equilibrium concentrations after 10 days. All soil solutions contained As levels that exceeded the EPA acceptable drinking water limit of 0.01 ppm. However, contaminant transport modeling suggests that As contaminated leachates would not migrate substantially from this site, so that local isolation and storage of contaminated soils would likely be an acceptable containment method.

  11. Glutathione transferase from Plasmodium falciparum--interaction with malagashanine and selected plant natural products.

    PubMed

    Mangoyi, Rumbidzai; Hayeshi, Rose; Ngadjui, Bonventure; Ngandeu, Francois; Bezabih, Merhatibebe; Abegaz, Berhanu; Razafimahefa, Solofoniaina; Rasoanaivo, Philippe; Mukanganyama, Stanley

    2010-12-01

    A glutathione transferase (PfGST) isolated from Plasmodium falciparum has been associated with chloroquine resistance. A range of natural products including malagashanine (MG) were screened for inhibition of PfGST by a GST assay with 1-chloro-2,4-dinitrobenzene as a substrate. Only the sesquiterpene (JBC 42C), the bicoumarin (Tral-1), ellagic acid and curcumin, were shown to be potent inhibitors of PfGST with IC(50) values of 8.5, 12, 50 and 69 μM, respectively. Kinetic studies were performed on PfGST using ellagic acid as an inhibitor. Uncompetitive and mixed types of inhibition were obtained for glutathione (GSH) and 1-chloro-2, 4-dinitrobenzene (CDNB). The K(i) for GSH and CDNB were -0.015 μM and 0.011 μM, respectively. Malagashanine (100 µM) only reduced the activity of PfGST to 80% but showed a time-dependent inactivation of PfGST with a t(1/2) of 34 minutes compared to >120 minutes in the absence of MG or in the presence of 5 mM GSH. This work facilitates the understanding of the interaction of PfGST with some plant derived compounds. PMID:20521884

  12. PlasmoView: A Web-based Resource to Visualise Global Plasmodium falciparum Genomic Variation

    PubMed Central

    Preston, Mark D.; Assefa, Samuel A.; Ocholla, Harold; Sutherland, Colin J.; Borrmann, Steffen; Nzila, Alexis; Michon, Pascal; Hien, Tran Tinh; Bousema, Teun; Drakeley, Christopher J.; Zongo, Issaka; Ouédraogo, Jean-Bosco; Djimde, Abdoulaye A.; Doumbo, Ogobara K.; Nosten, Francois; Fairhurst, Rick M.; Conway, David J.; Roper, Cally; Clark, Taane G.

    2014-01-01

    Malaria is a global public health challenge, with drug resistance a major barrier to disease control and elimination. To meet the urgent need for better treatments and vaccines, a deeper knowledge of Plasmodium biology and malaria epidemiology is required. An improved understanding of the genomic variation of malaria parasites, especially the most virulent Plasmodium falciparum (Pf) species, has the potential to yield new insights in these areas. High-throughput sequencing and genotyping is generating large amounts of genomic data across multiple parasite populations. The resulting ability to identify informative variants, particularly single-nucleotide polymorphisms (SNPs), will lead to the discovery of intra- and inter-population differences and thus enable the development of genetic barcodes for diagnostic assays and clinical studies. Knowledge of genetic variability underlying drug resistance and other differential phenotypes will also facilitate the identification of novel mutations and contribute to surveillance and stratified medicine applications. The PlasmoView interactive web-browsing tool enables the research community to visualise genomic variation and annotation (eg, biological function) in a geographic setting. The first release contains over 600 000 high-quality SNPs in 631 Pf isolates from laboratory strains and four malaria-endemic regions (West Africa, East Africa, Southeast Asia and Oceania). PMID:24338354

  13. The effect of ascaridole on the in vitro development of Plasmodium falciparum.

    PubMed

    Pollack, Y; Segal, R; Golenser, J

    1990-01-01

    Ascaridole is a terpene isolated from the plant Chenopodium ambrosioides (American wormseed); it is one of the few naturally occurring endoperoxidases. Artemisinin, which also belongs to this group, is a potent antimalarial. We therefore undertook a study to determine the effect of ascaridole, a known anthelmintic, on the in vitro development of Plasmodium falciparum. Ascaridole was found to be a potent inhibitor of plasmodial growth; after 3 days, development was arrested by a drug concentration of 0.05 microM, and at 0.1 microM no parasites were visible in the culture. At lower concentrations the effect was observed mainly at the trophozoite stage, whereas the ring stage was marginally affected. However, even at these lower concentrations, the ring culture could not continue normal development and ceased to grow at a later stage. The peroxide group is essential for the antimalarial activity of ascaridole, as judged from the fact that cineol, which bears an epoxide group instead of the peroxide group found in ascaridole, was totally inactive at identical concentrations. PMID:2217117

  14. High levels of Plasmodium falciparum rosetting in all clinical forms of severe malaria in African children.

    PubMed

    Doumbo, Ogobara K; Thera, Mahamadou A; Koné, Abdoulaye K; Raza, Ahmed; Tempest, Louisa J; Lyke, Kirsten E; Plowe, Christopher V; Rowe, J Alexandra

    2009-12-01

    Plasmodium falciparum rosetting (the spontaneous binding of infected erythrocytes to uninfected erythrocytes) is a well-recognized parasite virulence factor. However, it is currently unclear whether rosetting is associated with all clinical forms of severe malaria, or only with specific